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Sample records for allele-specific virulence attenuation

  1. Allele specific expression and methylation in the bumblebee, Bombus terrestris

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    Zoë Lonsdale

    2017-09-01

    Full Text Available The social hymenoptera are emerging as models for epigenetics. DNA methylation, the addition of a methyl group, is a common epigenetic marker. In mammals and flowering plants methylation affects allele specific expression. There is contradictory evidence for the role of methylation on allele specific expression in social insects. The aim of this paper is to investigate allele specific expression and monoallelic methylation in the bumblebee, Bombus terrestris. We found nineteen genes that were both monoallelically methylated and monoallelically expressed in a single bee. Fourteen of these genes express the hypermethylated allele, while the other five express the hypomethylated allele. We also searched for allele specific expression in twenty-nine published RNA-seq libraries. We found 555 loci with allele-specific expression. We discuss our results with reference to the functional role of methylation in gene expression in insects and in the as yet unquantified role of genetic cis effects in insect allele specific methylation and expression.

  2. Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa.

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    Kumar, Lokender; Chhibber, Sanjay; Kumar, Rajnish; Kumar, Manoj; Harjai, Kusum

    2015-04-01

    Quorum sensing in Pseudomonas aeruginosa plays an imperative role in virulence factor, biofilm formation and antimicrobial resistance. Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. Previously, our lab proved zingerone as potent anti-biofilm agent hence; further its anti-virulent and anti-quorum activities were evaluated. Zingerone, besides decreasing swimming, swarming and twitching phenotypes of P. aeruginosa PAO1, reduced biofilm forming capacity and production of virulence factors including rhamnolipid, elastase, protease, pyocyanin, cell free and cell bound hemolysin (pquorum sensing signal molecules by clinical isolates of P. aeruginosa but also showed significant interference with the activation of QS reporter strains. To study the mechanism of blocking quorum sensing cascade, in silico analysis was carried out. Anti-QS activity was attributed to interference with the ligand receptor interaction of zingerone with QS receptors (TraR, LasR, RhlR and PqsR). Zingerone showed a good comparative docking score to respective autoinducer molecules which was even higher than that of vanillin, a proven anti-quorum sensing phytochemical. The results of the present study revealed the anti-quorum sensing activity of zingerone targeting ligand-receptor interaction, hence proposing zingerone as a suitable anti-virulent drug candidate against P. aeruginosa infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Genome-wide analysis of peptidase content and expression in a virulent and attenuated Babesia bovis strain pair.

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    Mesplet, Maria; Palmer, Guy H; Pedroni, Monica J; Echaide, Ignacio; Florin-Christensen, Monica; Schnittger, Leonhard; Lau, Audrey O T

    2011-10-01

    Identifying virulence determinants in Apicomplexan parasites remains a major gap in knowledge for members within this phylum. We hypothesized that peptidases would segregate with virulence between a virulent parent Babesia bovis strain and an attenuated daughter strain derived by rapid in vivo passage. Using the complete genome sequence of the virulent T2Bo strain, 66 peptidases were identified and active sites confirmed. The presence, sequence identity and expression levels were tested for each of the 66 peptidases in the virulent parent and attenuated daughter T2Bo strains using whole genome, targeted sequencing approaches and microarrays analyses. Quantitative PCR revealed that there was no significant difference in peptidase expression between the virulent and attenuated strains. We conclude that while peptidases may well play a required role in B. bovis pathogenesis, neither loss of peptidase gene content nor reduced gene expression underlies the loss of virulence associated with in vivo passage and attenuation. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium.

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    Tago, Damian; Meyer, Damien F

    2016-01-01

    Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria.

  5. Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

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    Basombrío Miguel Ángel

    2000-01-01

    Full Text Available Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL. The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.

  6. Genomic comparison between attenuated Chinese equine infectious anemia virus vaccine strains and their parental virulent strains.

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    Wang, Xuefeng; Wang, Shuai; Lin, Yuezhi; Jiang, Chenggang; Ma, Jian; Zhao, Liping; Lv, Xiaoling; Wang, Fenglong; Shen, Rongxian; Kong, Xiangang; Zhou, Jianhua

    2011-02-01

    A lentiviral vaccine, live attenuated equine infectious anemia virus (EIAV) vaccine, was developed in the 1970s, and this has made tremendous contributions to the control of equine infectious anemia (EIA) in China. Four key virus strains were generated during the attenuation of the EIAV vaccine: the original Liao-Ning strain (EIAV(LN40)), a donkey-adapted virulent strain (EIAV(DV117)), a donkey-leukocyte-attenuated vaccine strain (EIAV(DLV121)), and a fetal donkey dermal cell (FDD)-adapted vaccine strain (EIAV(FDDV13)). In this study, we analyzed the proviral genomes of these four EIAV strains and found a series of consensus substitutions among these strains. These mutations provide useful information for understanding the genetic basis of EIAV attenuation. Our results suggest that multiple mutations in a variety of genes in our attenuated EIAV vaccines not only provide a basis for virulence attenuation and induction of protective immunity but also greatly reduce the risk of reversion to virulence.

  7. Long terminal repeat sequences from virulent and attenuated equine infectious anemia virus demonstrate distinct promoter activities.

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    Zhou, Tao; Yuan, Xiu-Fang; Hou, Shao-Hua; Tu, Ya-Bin; Peng, Jin-Mei; Wen, Jian-Xin; Qiu, Hua-Ji; Wu, Dong-Lai; Chen, Huan-Chun; Wang, Xiao-Jun; Tong, Guang-Zhi

    2007-09-01

    In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV(DLA) into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV(DLA) showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.

  8. Alteration in apyrase enzyme attenuated virulence of Shigella flexneri.

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    BangaSingh, Kirnpal Kaur; Nisha, Mehru; Lau, Hut Yee; Ravichandran, Manickam; Salleh, Mohd Zaki

    2016-02-01

    Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing mitochondrial damage and host cell death. In this study we constructed an apy mutant of Shigella flexneri by insertional activation using a kanamycin resistant gene cassette. The wild type apy gene of S. flexneri 2a was PCR amplified, cloned and mutated with insertion of kanamycin resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type S. flexneri 2a by conjugation with Escherichia coli. After undergoing homologous recombination, the wild apy gene was deleted from the construct using the sucrose selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the guinea pig keratoconjunctivitis model. The strain was Sereny negative and it elicited a significant protection following challenge with wild S. flexneri strain. This apy mutant strain will form a base for the development of a vaccine target for shigellosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Allele-specific KRT1 expression is a complex trait.

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    Heng Tao

    2006-06-01

    Full Text Available The differential expression of alleles occurs commonly in humans and is likely an important genetic factor underlying heritable differences in phenotypic traits. Understanding the molecular basis of allelic expression differences is thus an important challenge. Although many genes have been shown to display differential allelic expression, this is the first study to examine in detail the cumulative effects of multiple cis-regulatory polymorphisms responsible for allele-specific expression differences. We have used a variety of experimental approaches to identify and characterize cis-regulatory polymorphisms responsible for the extreme allele-specific expression differences of keratin-1 (KRT1 in human white blood cells. The combined data from our analyses provide strong evidence that the KRT1 allelic expression differences result from the haplotypic combinations and interactions of five cis-regulatory single nucleotide polymorphisms (SNPs whose alleles differ in their affinity to bind transcription factors and modulate KRT1 promoter activity. Two of these cis-regulatory SNPs bind transcriptional activators with the alleles on the high-expressing KRT1 haplotype pattern having a higher affinity than the alleles on the low-expressing haplotype pattern. In contrast, the other three cis-regulatory SNPs bind transcriptional inhibitors with the alleles on the low-expressing haplotype pattern having a higher affinity than the alleles on the high-expressing haplotype pattern. Our study provides important new insights into the degree of complexity that the cis-regulatory sequences responsible for allele-specific transcriptional regulation have. These data suggest that allelic expression differences result from the cumulative contribution of multiple DNA sequence polymorphisms, with each having a small effect, and that allele-specific expression can thus be viewed as a complex trait.

  10. Statin Concentrations Below the Minimum Inhibitory Concentration Attenuate the Virulence of Rhizopus oryzae.

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    Bellanger, Anne-Pauline; Tatara, Alexander M; Shirazi, Fazal; Gebremariam, Teclegiorgis; Albert, Nathaniel D; Lewis, Russell E; Ibrahim, Ashraf S; Kontoyiannis, Dimitrios P

    2016-07-01

    Mucormycosis is a destructive invasive mold infection afflicting patients with diabetes and hematologic malignancies. Patients with diabetes are often treated with statins, which have been shown to have antifungal properties. We sought to examine the effects of statins on Rhizopus oryzae, a common cause of mucormycosis. Clinical strains of R. oryzae were exposed to lovastatin, atorvastatin, and simvastatin and the minimum inhibitory concentrations (MICs) were determined. R. oryzae germination, DNA fragmentation, susceptibility to oxidative stress, and ability to damage endothelial cells were assessed. We further investigated the impact of exposure to lovastatin on the virulence of R. oryzae  All statins had MICs of >64 µg/mL against R. oryzae Exposure of R. oryzae to statins decreased germling formation, induced DNA fragmentation, and attenuated damage to endothelial cells independently of the expression of GRP78 and CotH. Additionally, R. oryzae exposed to lovastatin showed macroscopic loss of melanin, yielded increased susceptibility to the oxidative agent peroxide, and had attenuated virulence in both fly and mouse models of mucormycosis. Exposure of R. oryzae to statins at concentrations below their MICs decreased virulence both in vitro and in vivo. Further investigation is warranted into the use of statins as adjunctive therapy in mucormycosis. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  11. Azithromycin and Doxycycline Attenuation of Acanthamoeba Virulence in a Human Corneal Tissue Model.

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    Purssell, Andrew; Lau, Rachel; Boggild, Andrea K

    2017-04-15

    Amoebic keratitis is a potentially blinding eye infection caused by ubiquitous, free-living, environmental acanthamoebae, which are known to harbor bacterial endosymbionts. A Chlamydia-like endosymbiont has previously enhanced Acanthamoeba virulence in vitro. We investigated the potential effect of Acanthamoeba-endosymbiont coinfection in a human corneal tissue model representing clinical amoebic keratitis infection. Environmental and corneal Acanthamoeba isolates from the American Type Culture Collection were screened for endosymbionts by amplifying and sequencing bacterial 16S as well as Chlamydiales-specific DNA. Each Acanthamoeba isolate was used to infect EpiCorneal cells, a 3-dimensional human corneal tissue model. EpiCorneal cells were then treated with azithromycin, doxycycline, or control medium to determine whether antibiotics targeting common classes of bacterial endosymbionts attenuated Acanthamoeba virulence, as indicated by decreased observed cytopathic effect and inflammatory biomarker production. A novel endosymbiont closely related to Mycobacterium spp. was identified in Acanthamoeba polyphaga 50495. Infection of EpiCorneal cells with Acanthamoeba castellanii 50493 and A. polyphaga 50372 led to increased production of inflammatory cytokines and cytopathic effects visible under microscopy. These increases were attenuated by azithromycin and doxycycline. Our findings suggest that azithromycin and doxycycline may be effective adjuvants to standard antiacanthamoebal chemotherapy by potentially abrogating virulence-enhancing properties of bacterial endosymbionts. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  12. Attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein.

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    Préhaud, Christophe; Wolff, Nicolas; Terrien, Elouan; Lafage, Mireille; Mégret, Françoise; Babault, Nicolas; Cordier, Florence; Tan, Gene S; Maitrepierre, Elodie; Ménager, Pauline; Chopy, Damien; Hoos, Sylviane; England, Patrick; Delepierre, Muriel; Schnell, Matthias J; Buc, Henri; Lafon, Monique

    2010-01-19

    The capacity of a rabies virus to promote neuronal survival (a signature of virulence) or death (a marker of attenuation) depends on the cellular partners recruited by the PDZ-binding site (PDZ-BS) of its envelope glycoprotein (G). Neuronal survival requires the selective association of the PDZ-BS of G with the PDZ domains of two closely related serine-threonine kinases, MAST1 and MAST2. Here, we found that a single amino acid change in the PDZ-BS triggered the apoptotic death of infected neurons and enabled G to interact with additional PDZ partners, in particular the tyrosine phosphatase PTPN4. Knockdown of PTPN4 abrogated virus-mediated apoptosis. Thus, we propose that attenuation of rabies virus requires expansion of the set of host PDZ proteins with which G interacts, which interferes with the finely tuned homeostasis required for survival of the infected neuron.

  13. Non-thermal Plasma Exposure Rapidly Attenuates Bacterial AHL-Dependent Quorum Sensing and Virulence

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    Flynn, Padrig B.; Busetti, Alessandro; Wielogorska, Ewa; Chevallier, Olivier P.; Elliott, Christopher T.; Laverty, Garry; Gorman, Sean P.; Graham, William G.; Gilmore, Brendan F.

    2016-01-01

    The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30–60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyryl-homoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa. PMID:27242335

  14. Key hub and bottleneck genes differentiate the macrophage response to virulent and attenuated Mycobacterium bovis

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    Kate E. Killick

    2014-10-01

    Full Text Available Mycobacterium bovis is an intracellular pathogen that causes tuberculosis in cattle. Following infection, the pathogen resides and persists inside host macrophages by subverting host immune responses via a diverse range of mechanisms. Here, a high-density bovine microarray platform was used to examine the bovine monocyte-derived macrophage transcriptome response to M. bovis infection relative to infection with the attenuated vaccine strain, M. bovis Bacille Calmette–Guérin. Differentially expressed genes were identified (adjusted P-value  0.01 and interaction networks generated across an infection time course of 2, 6 and 24 h. The largest number of biological interactions was observed in the 24 h network, which exhibited scale-free network properties. The 24 h network featured a small number of key hub and bottleneck gene nodes, including IKBKE, MYC, NFKB1 and EGR1 that differentiated the macrophage response to virulent and attenuated M. bovis strains, possibly via the modulation of host cell death mechanisms. These hub and bottleneck genes represent possible targets for immunomodulation of host macrophages by virulent mycobacterial species that enable their survival within a hostile environment.

  15. Reverse genetics of rabies virus: new strategies to attenuate virus virulence for vaccine development.

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    Zhu, Shimao; Li, Hui; Wang, Chunhua; Luo, Farui; Guo, Caiping

    2015-08-01

    Rabies is an ancient neurological disease that is almost invariably fatal once the clinical symptoms develop. Currently, prompt wound cleansing after exposing to a potentially rabid animal and vaccination using rabies vaccine combined with administration of rabies immune globulin are the only effective methods for post-exposure prophylaxis against rabies. Reverse genetic technique is a novel approach to investigate the function of a specific gene by analyzing the phenotypic effects through directly manipulating the gene sequences. It has revolutionized and provided a powerful tool to study the molecular biology of RNA viruses and has been widely used in rabies virus research. The attenuation of rabies virus virulence is the prerequisite for rabies vaccine development. Given the current challenge that sufficient and affordable high-quality vaccines are limited and lacking for global rabies prevention and control, highly cell-adapted, stable, and attenuated rabies viruses with broad cross-reactivity against different viral variants are ideal candidates for consideration to meet the need for human rabies control in the future. A number of approaches have been pursued to reduce the virulence of the virus and improve the safety of rabies vaccines. The application of reverse genetic technique has greatly advanced the engineering of rabies virus and paves the avenue for utilizing rabies virus for vaccine against rabies, viral vectors for exogenous antigen expression, and gene therapy in the future.

  16. Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements.

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    Marschalek, Adriane; Finke, Stefan; Schwemmle, Martin; Mayer, Daniel; Heimrich, Bernd; Stitz, Lothar; Conzelmann, Karl-Klaus

    2009-02-01

    Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and relies on the canonical 5'-end-dependent translation of capped viral mRNAs. Here, we have used internal ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2 IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-beta) promoter, and increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2 IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-alpha receptor (IFNAR(-/-)). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demonstrated only in newborn IFNAR(-/-) mice. Translational control of individual genes is a promising strategy to attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study the function of IRES elements in detail.

  17. Trophozoites of Entamoeba histolytica epigenetically silenced in several genes are virulence-attenuated

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    Mirelman D.

    2008-09-01

    Full Text Available The human intestinal parasite Entamoeba histolytica causes amoebic colitis and amoebic liver abscesses. Three classes of amoebic molecules have been identified as the major virulence factors, the Gal/GalNAc inhibitable lectin that mediates adherence to mammalian cells, the amoebapores which cause the formation of membrane ion channels in the target cells and the cysteine proteinases which degrade the matrix proteins, the intestinal mucus and secretory IgA. Transcriptional silencing of the amoebapore (Ehap-a gene occurred after transfection of trophozoites with a plasmid containing a segment of the 5’ upstream region of the gene. Transcriptional silencing of the Ehap-a gene continued even after the removal of the plasmid and the cloned amoebae were termed G3. Transfection of G3 trophozoites with a plasmid construct containing the cysteine proteinase (EhCP-5 gene and the light subunit of the Gal- lectin (Ehlgl1 gene, each under the 5’ upstream sequences of the amoebapore gene, caused the simultaneous epigenetic silencing of expression of these two genes. The resulting trophozoites, termed RB-9, were cured from the plasmid and they do not express the three types of virulent genes. The RB-9 amoeba are virulence attenuated and are incapable of killing mammalian cells, they can not induce the formation of liver abscesses and they do not cause ulcerations in the cecum of experimental animals. The gene-silenced amoebae express the same surface antigens which are present in virulent strains and following intra peritoneal inoculation of live trophozoites into hamsters they evoked a protective immune response. Further studies are needed to find out if RB-9 trophozoites could be used for vaccination against amoebaisis.

  18. Host behaviour manipulation as an evolutionary route towards attenuation of parasitoid virulence.

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    Maure, F; Doyon, J; Thomas, F; Brodeur, J

    2014-12-01

    By definition, insect parasitoids kill their host during their development. Data are presented showing that ladybirds not only can survive parasitism by Dinocampus coccinellae, but also can retain their capacity to reproduce following parasitoid emergence. We hypothesize that host behaviour manipulation constitutes a preadaptation leading to the attenuation of parasitoid virulence. Following larval development, the parasitoid egresses from the host and spins a cocoon between the ladybird's legs. Throughout parasitoid pupation, the manipulated host acts as a bodyguard to protect the parasitoid cocoon from predation. The parasitoid has evolved mechanisms to avoid killing the host prematurely so that its own survival is not compromised. Bodyguard manipulation may thus constitute a selective trait for the evolution of true parasitism in some host-parasitoid associations. © 2014 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.

  19. Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene rosa in virulent and attenuated strains of Renibacterium salmoninarum

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    O'Farrell, C. L.; Strom, M.S.

    1999-01-01

    Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5, to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of rosa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization, and total p57 expression between 33209 anti MT 239 are not due to differences in rosa sequence or differences in steady state transcript levels.

  20. Absence of Protoheme IX Farnesyltransferase CtaB Causes Virulence Attenuation but Enhances Pigment Production and Persister Survival in MRSA.

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    Xu, Tao; Han, Jian; Zhang, Jia; Chen, Jiazhen; Wu, Nan; Zhang, Wenhong; Zhang, Ying

    2016-01-01

    The membrane protein CtaB in S. aureus is a protoheme IX farnesyltransferase involved in the synthesis of the heme containing terminal oxidases of bacterial respiratory chain. In this study, to assess the role of CtaB in S. aureus virulence, pigment production, and persister formation, we constructed a ctaB mutant in the methicillin-resistant Staphylococcus aureus (MRSA) strain USA500. We found that deletion of ctaB attenuated growth and virulence in mice but enhanced pigment production and formation of quinolone tolerant persister cells in stationary phase. RNA-seq analysis showed that deletion of ctaB caused decreased transcription of several virulence genes including RNAIII which is consistent with its virulence attenuation. In addition, transcription of 20 ribosomal genes and 24 genes involved in amino acid biosynthesis was significantly down-regulated in the ctaB knockout mutant compared with the parent strain. These findings suggest the importance of heme biosynthesis in virulence and persister formation of S. aureus .

  1. Effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen.

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    Zhao, Shanshan; Gao, Qi; Qin, Tao; Yin, Yinyan; Lin, Jian; Yu, Qinghua; Yang, Qian

    2014-06-25

    Virulent transmissible gastroenteritis virus (TGEV) results in an acute, severe pathology and high mortality in piglets, while attenuated TGEV only causes moderate clinical reactions. Dendritic cells (DCs), through uptake and presentation of antigens to T cells, initiate distinct immune responses to different infections. In this study, an attenuated TGEV (STC3) and a virulent TGEV (SHXB) were used to determine whether porcine DCs play an important role in pathogenetic differences between these two TGEVs. Our results showed that immature and mature monocyte-derived dendritic cells (Mo-DCs) were susceptible to infection with SHXB and STC3. However, only SHXB inhibited Mo-DCs to activate T-cell proliferation by down-regulating the expression of cell-surface markers and the secretion of cytokines in vitro. In addition, after 48 h of SHXB infection, there was the impairment in the ability of porcine intestinal DCs to sample the antigen, to migrate from the villi to the lamina propria and to activate T-cell proliferation in vivo. In contrast, these abilities of intestinal DCs were enhanced in STC3-infected piglets. In conclusion, our results show that SHXB significantly impaired the functions of Mo-DCs and intestinal DCs in vitro and in vivo, while STC3 had the opposite effect. These differences may underlie the pathogenesis of virulent and attenuated TGEV in piglets, and could help us to develop a better strategy to prevent virulent TGEV infection. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Isocitrate dehydrogenase mutation in Vibrio anguillarum results in virulence attenuation and immunoprotection in rainbow trout (Oncorhynchus mykiss).

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    Mou, Xiangyu; Spinard, Edward J; Hillman, Shelby L; Nelson, David R

    2017-11-14

    Vibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum. Seven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout. The data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain.

  3. Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris

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    Harindra E. Amarasinghe

    2015-07-01

    Full Text Available Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes.

  4. Comparative genomic analysis of Brucella abortus vaccine strain 104M reveals a set of candidate genes associated with its virulence attenuation.

    Science.gov (United States)

    Yu, Dong; Hui, Yiming; Zai, Xiaodong; Xu, Junjie; Liang, Long; Wang, Bingxiang; Yue, Junjie; Li, Shanhu

    2015-01-01

    The Brucella abortus strain 104M, a spontaneously attenuated strain, has been used as a vaccine strain in humans against brucellosis for 6 decades in China. Despite many studies, the molecular mechanisms that cause the attenuation are still unclear. Here, we determined the whole-genome sequence of 104M and conducted a comprehensive comparative analysis against the whole genome sequences of the virulent strain, A13334, and other reference strains. This analysis revealed a highly similar genome structure between 104M and A13334. The further comparative genomic analysis between 104M and A13334 revealed a set of genes missing in 104M. Some of these genes were identified to be directly or indirectly associated with virulence. Similarly, a set of mutations in the virulence-related genes was also identified, which may be related to virulence alteration. This study provides a set of candidate genes associated with virulence attenuation in B.abortus vaccine strain 104M.

  5. Mutations within ICP4 acquired during in vitro attenuation do not alter virulence of recombinant Marek's disease viruses in vivo

    Directory of Open Access Journals (Sweden)

    Evin Hildebrandt

    2015-12-01

    Full Text Available Marek's disease (MD is a T-cell lymphoma of chickens caused by the oncogenic Marek's disease virus (MDV. MD is primarily controlled by live-attenuated vaccines generated by repeated in vitro serial passage. Previous efforts to characterize attenuated MDVs identified numerous mutations, particularly a convergence of high-frequency mutations around amino acids 60–63 within ICP4 (RS1, therefore, ICP4 was considered a candidate gene deserving further characterization. Recombinant MDVs were generated containing a single Q63H mutation or double Q63H + S1630P mutations. Despite the repetitive nature of mutations within ICP4, neither recombinant virus decreased virulence, although one mutant reduced in vivo replication and failed to transmit horizontally. Our results indicate that these mutations are insufficient to reduce disease incidence in infected birds, and suggest that variants in ICP4 do not directly alter virulence, but rather may enhance MDV replication rates in vitro, offering an explanation for the widespread occurrence of ICP4 mutations in a variety of attenuated herpesviruses.

  6. Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

    Directory of Open Access Journals (Sweden)

    SAG Macoris

    2006-05-01

    Full Text Available Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.

  7. Streptococcus iniae M-like protein contributes to virulence in fish and is a target for live attenuated vaccine development.

    Directory of Open Access Journals (Sweden)

    Jeffrey B Locke

    2008-07-01

    Full Text Available Streptococcus iniae is a significant pathogen in finfish aquaculture, though knowledge of virulence determinants is lacking. Through pyrosequencing of the S. iniae genome we have identified two gene homologues to classical surface-anchored streptococcal virulence factors: M-like protein (simA and C5a peptidase (scpI.S. iniae possesses a Mga-like locus containing simA and a divergently transcribed putative mga-like regulatory gene, mgx. In contrast to the Mga locus of group A Streptococcus (GAS, S. pyogenes, scpI is located distally in the chromosome. Comparative sequence analysis of the Mgx locus revealed only one significant variant, a strain with an insertion frameshift mutation in simA and a deletion mutation in a region downstream of mgx, generating an ORF which may encode a second putative mga-like gene, mgx2. Allelic exchange mutagenesis of simA and scpI was employed to investigate the potential role of these genes in S. iniae virulence. Our hybrid striped bass (HSB and zebrafish models of infection revealed that M-like protein contributes significantly to S. iniae pathogenesis whereas C5a peptidase-like protein does not. Further, in vitro cell-based analyses indicate that SiMA, like other M family proteins, contributes to cellular adherence and invasion and provides resistance to phagocytic killing. Attenuation in our virulence models was also observed in the S. iniae isolate possessing a natural simA mutation. Vaccination of HSB with the Delta simA mutant provided 100% protection against subsequent challenge with a lethal dose of wild-type (WT S. iniae after 1,400 degree days, and shows promise as a target for live attenuated vaccine development.Analysis of M-like protein and C5a peptidase through allelic replacement revealed that M-like protein plays a significant role in S. iniae virulence, and the Mga-like locus, which may regulate expression of this gene, has an unusual arrangement. The M-like protein mutant created in this research

  8. Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice.

    Science.gov (United States)

    Gromowski, Gregory D; Firestone, Cai-Yen; Whitehead, Stephen S

    2015-06-01

    The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 (E) protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, the nucleotide substitution T39A in the 5' UTR (5'-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5'-UTR-T39A and C-L66S mutations, individually, were somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety. The live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed >25 years ago by passaging wild-type JEV strain SA14 in tissue

  9. Attenuation of Pseudomonas aeruginosa virulence by marine invertebrate–derived Streptomyces sp.

    Digital Repository Service at National Institute of Oceanography (India)

    Naik, D.N.; Wahidullah, S.; Meena, R.M.

    -mediated virulence factors like swarming, biofilm formation, pyocyanin, rhamnolipid and LasA production in Ps. aeruginosa ATCC 27853. The isolates NIO 10068, NIO 10058 and NIO 10090 exhibited very good anti-QS activity, with NIO 10068 being the most promising one...

  10. Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling.

    Science.gov (United States)

    Witsø, Ingun Lund; Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

    2016-01-01

    Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli.

  11. Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling

    Science.gov (United States)

    Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

    2016-01-01

    Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli. PMID:27309855

  12. Virulence attenuation of Babesia gibsoni by serial passages in vitro and assessment of the protection provided by the immunization against the passaged isolate in dogs.

    Science.gov (United States)

    Sunaga, Fujiko; Taharaguchi, Satoshi; Arai, Sachiko; Itoh, Seigo; Kanno, Yasunori

    2013-11-08

    The virulence of the Babesia gibsoni Oita isolate was attenuated by serial passages in vitro by using the microaerophilus stationary phase (MASP) technique. After 400 serial passages, the virulence of the isolate was found to be attenuated. This was evidenced by the response of two dogs inoculated intravenously with 10(9)B. gibsoni passaged isolate. Specific antibodies were produced at a titer of 1:20,480, as detected by the fluorescent antibody test (IFAT). These results suggested that the serial passages of B. gibsoni reduced its virulence while retaining its antigenicity. The dogs that were inoculated with the attenuated isolate (1 and 2) and two naïve dogs (3 and 4) were challenged by intravenous inoculation of 2×10(8) infected erythrocytes of the virulent Oita isolate. Protection afforded by exposure to the attenuated isolate was evidenced by a lower parasitemia in dogs 1 and 2 with a rapid decrease to nondetectable levels, accompanied by a slight decrease in the PCV that returned to normal values. Dogs 3 and 4 developed typical acute clinical signs, including severe anemia and hyperthermia. These results suggested that the attenuated isolate was a candidate for live vaccine. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. [Quercetin attenuates Staphylococcus aureus virulence by reducing alpha-toxin secretion].

    Science.gov (United States)

    Carrada López, Gabriela; Castañón Sánchez, Carlos A

    2017-10-31

    Alpha toxin, a pore-forming protein with cytotoxic activity, is one of the major virulence factors secreted by most strains of Staphylococcus aureus. The relevance of this protein in the pathogenesis of pneumonia associated with S.aureus infections has already been established. Therefore, inhibiting alpha toxin secretion can be an alternative for controlling these infections. This study shows that quercetin, a naturally occurring flavonoid, inhibits hemolytic activity in a dose-dependent manner and reduces alpha toxin secretion in culture supernatants of methicillin-sensitive and methicillin-resistant S.aureus. Furthermore, quercetin significantly prevents damage to human alveolar cells when co-cultured with S.aureus. Our results suggest that quercetin can reduce S.aureus virulence by affecting alpha-toxin secretion. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  14. Allele Workbench: transcriptome pipeline and interactive graphics for allele-specific expression.

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    Carol A Soderlund

    Full Text Available Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor, where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense, and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available

  15. Deletion of luxS further attenuates the virulence of the avian pathogenic Escherichia coli aroA mutant.

    Science.gov (United States)

    Han, Xiangan; Bai, Hao; Tu, Jian; Yang, Lijun; Xu, Da; Wang, Shaohui; Qi, Kezong; Fan, Guobo; Zhang, Yuxi; Zuo, Jiakun; Tian, Mingxing; Ding, Chan; Yu, Shengqing

    2015-11-01

    In this study, an aroA-deletion avian pathogenic Escherichia coli (APEC) mutant (strain DE17ΔaroA) and aroA and luxS double deletion APEC mutant (strain DE17ΔluxSΔaroA) were constructed from the APEC DE17 strain. The results showed that as compared to DE17ΔaroA, the virulence of DE17ΔluxSΔaroA was further attenuated by 200- and 31.7-fold, respectively, in ducklings based on the 50% lethal dose. The adherence and invasion abilities of DE17ΔluxSΔaroA and DE17ΔaroA were reduced by 36.5%/42.5% and 25.8%/29.3%, respectively, as compared to the wild-type strain DE17 (p avian colibacillosis than DE17ΔaroA. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Attenuation of quorum sensing controlled virulence factors and biofilm formation in Pseudomonas aeruginosa by pentacyclic triterpenes, betulin and betulinic acid.

    Science.gov (United States)

    Rajkumari, Jobina; Borkotoky, Subhomoi; Murali, Ayaluru; Suchiang, Kitlangki; Mohanty, Saswat Kumar; Busi, Siddhardha

    2018-03-08

    The production of virulence determinants and biofilm formation in numerous pathogens is regulated by the cell-density-dependent phenomenon, Quorum sensing (QS). The QS system in multidrug resistant opportunistic pathogen, P. aeruginosa constitutes of three main regulatory circuits namely Las, Rhl, and Pqs which are closely linked to its pathogenicity and establishment of chronic infections. In spite intensive antibiotic therapy, P. aeruginosa continue to be an important cause of nosocomial infections and also the major cause of mortality in Cystic Fibrosis patients with 80% of the adults suffering from chronic P. aeruginosa infection. Hence, targeting QS circuit offers an effective intervention to the ever increasing problem of drug resistant pathogens. In the present study, the pentacyclic triterpenes i.e. Betulin (BT) and Betulinic acid (BA) exhibited significant attenuation in production of QS-regulated virulence factors and biofilm formation in P. aeruginosa, at the sub-lethal concentration. The test compound remarkably interfered in initial stages of biofilm development by decreasing the exopolysaccharide production and cell surface hydrophobicity. Based on the in vivo studies, the test compounds notably enhanced the survival of Caenorhabditis elegans infected with P. aeruginosa. Furthermore, molecular docking analysis revealed that BT and BA can act as a strong competitive inhibitor for QS receptors, LasR and RhlR. The findings suggest that BT and BA can serve as potential anti-infectives in the controlling chronic infection of P. aeruginosa. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Retigeric acid B attenuates the virulence of Candida albicans via inhibiting adenylyl cyclase activity targeted by enhanced farnesol production.

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    Wenqiang Chang

    Full Text Available Candida albicans, the most prevalent fungal pathogen, undergoes yeast-to-hyphal switch which has long been identified as a key fungal virulence factor. We showed here that the lichen-derived small molecule retigeric acid B (RAB acted as an inhibitor that significantly inhibited the filamentation of C. albicans, leading to the prolonged survival of nematodes infected by C. albicans. Quantitative real-time PCR analysis and intracellular cAMP measurement revealed RAB regulated the Ras1-cAMP-Efg1 pathway by reducing cAMP level to inhibit the hyphae formation. Confocal microscopic observation showed RAB induced the expression of Dpp3, synthesizing more farnesol, which was confirmed by gas chromatography-mass spectroscopy detection. An adenylyl cyclase activity assay demonstrated RAB could repress the activity of Cdc35 through stimulating farnesol synthesis, thus causing a decrease in cAMP synthesis, leading to retarded yeast-to-hyphal transition. Moreover, reduced levels of intracellular cAMP resulted in the inhibition of downstream adhesins. Together, these findings indicate that RAB stimulates farnesol production that directly inhibits the Cdc35 activity, reducing the synthesis of cAMP and thereby causing the disruption of the morphologic transition and attenuating the virulence of C. albicans. Our work illustrates the underlying mechanism of RAB-dependent inhibition of the yeast-to-hyphal switch and provides a potential application in treating the infection of C. albicans.

  18. Morin hydrate attenuates Staphylococcus aureus virulence by inhibiting the self-assembly of α-hemolysin.

    Science.gov (United States)

    Wang, J; Zhou, X; Liu, S; Li, G; Shi, L; Dong, J; Li, W; Deng, X; Niu, X

    2015-03-01

    To investigate the mechanism by which morin hydrate inhibits the haemolytic activity of α-hemolysin (Hla), a channel-forming toxin that is important for the pathogenesis of disease in experimental animals, and its therapeutic effect against Staphylococcus aureus pneumonia in a mouse model. The results from the in vitro (haemolysis, western blot and cytotoxicity assays) and in vivo (mouse model of intranasal lung infection) experiments indicated that morin hydrate, a natural compound with little anti-Staph. aureus activity, could effectively antagonize the cytolytic activity of Hla, alleviate human lung cell injury, and protect against mortality of Staph. aureus pneumonia in a mouse model of infection. Molecular dynamics simulations, free energy calculations and mutagenesis assays were further employed to determine the catalytic mechanism of inhibition, which indicated that a direct binding of morin to the 'Stem' domain of Hla (residues I107 and T109) and the concomitant change in conformation led to the inhibition of the self-assembly of the heptameric transmembrane pore, thus inhibiting the biological activity of Hla for cell lysis. Morin inhibited Staph. aureus virulence via inhibiting the haemolytic activity of α-hemolysin. These findings suggested that morin is a promising candidate for the development of anti-virulence therapeutic agents for the treatment of Staph. aureus infections. © 2015 The Society for Applied Microbiology.

  19. Attenuated Virulence and Biofilm Formation in Staphylococcus aureus following Sublethal Exposure to Triclosan

    Science.gov (United States)

    Latimer, Joe; Forbes, Sarah

    2012-01-01

    Subeffective exposure of Staphylococcus aureus to the biocide triclosan can reportedly induce a small-colony variant (SCV) phenotype. S. aureus SCVs are characterized by low growth rates, reduced pigmentation, and lowered antimicrobial susceptibility. While they may exhibit enhanced intracellular survival, there are conflicting reports regarding their pathogenicity. The current study reports the characteristics of an SCV-like strain of S. aureus created by repeated passage on sublethal triclosan concentrations. S. aureus ATCC 6538 (the passage 0 [P0] strain) was serially exposed 10 times to concentration gradients of triclosan to generate strain P10. This strain was then further passaged 10 times on triclosan-free medium (designated strain ×10). The MICs and minimum bactericidal concentrations of triclosan for P0, P10, and ×10 were determined, and growth rates in biofilm and planktonic cultures were measured. Hemolysin, DNase, and coagulase activities were measured, and virulence was determined using a Galleria mellonella pathogenicity model. Strain P10 exhibited decreased susceptibility to triclosan and characteristics of an SCV phenotype, including a considerably reduced growth rate and the formation of pinpoint colonies. However, this strain also had delayed coagulase production, had impaired hemolysis (P triclosan may result in an SCV-like phenotype, this is not necessarily associated with increased virulence and adapted bacteria may exhibit other functional deficiencies. PMID:22430975

  20. ACNE: a summarization method to estimate allele-specific copy numbers for Affymetrix SNP arrays.

    Science.gov (United States)

    Ortiz-Estevez, Maria; Bengtsson, Henrik; Rubio, Angel

    2010-08-01

    Current algorithms for estimating DNA copy numbers (CNs) borrow concepts from gene expression analysis methods. However, single nucleotide polymorphism (SNP) arrays have special characteristics that, if taken into account, can improve the overall performance. For example, cross hybridization between alleles occurs in SNP probe pairs. In addition, most of the current CN methods are focused on total CNs, while it has been shown that allele-specific CNs are of paramount importance for some studies. Therefore, we have developed a summarization method that estimates high-quality allele-specific CNs. The proposed method estimates the allele-specific DNA CNs for all Affymetrix SNP arrays dealing directly with the cross hybridization between probes within SNP probesets. This algorithm outperforms (or at least it performs as well as) other state-of-the-art algorithms for computing DNA CNs. It better discerns an aberration from a normal state and it also gives more precise allele-specific CNs. The method is available in the open-source R package ACNE, which also includes an add on to the aroma.affymetrix framework (http://www.aroma-project.org/).

  1. Simultaneous SNP identification and assessment of allele-specific bias from ChIP-seq data

    Directory of Open Access Journals (Sweden)

    Ni Yunyun

    2012-09-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs have been associated with many aspects of human development and disease, and many non-coding SNPs associated with disease risk are presumed to affect gene regulation. We have previously shown that SNPs within transcription factor binding sites can affect transcription factor binding in an allele-specific and heritable manner. However, such analysis has relied on prior whole-genome genotypes provided by large external projects such as HapMap and the 1000 Genomes Project. This requirement limits the study of allele-specific effects of SNPs in primary patient samples from diseases of interest, where complete genotypes are not readily available. Results In this study, we show that we are able to identify SNPs de novo and accurately from ChIP-seq data generated in the ENCODE Project. Our de novo identified SNPs from ChIP-seq data are highly concordant with published genotypes. Independent experimental verification of more than 100 sites estimates our false discovery rate at less than 5%. Analysis of transcription factor binding at de novo identified SNPs revealed widespread heritable allele-specific binding, confirming previous observations. SNPs identified from ChIP-seq datasets were significantly enriched for disease-associated variants, and we identified dozens of allele-specific binding events in non-coding regions that could distinguish between disease and normal haplotypes. Conclusions Our approach combines SNP discovery, genotyping and allele-specific analysis, but is selectively focused on functional regulatory elements occupied by transcription factors or epigenetic marks, and will therefore be valuable for identifying the functional regulatory consequences of non-coding SNPs in primary disease samples.

  2. Simultaneous SNP identification and assessment of allele-specific bias from ChIP-seq data

    Science.gov (United States)

    2012-01-01

    Background Single nucleotide polymorphisms (SNPs) have been associated with many aspects of human development and disease, and many non-coding SNPs associated with disease risk are presumed to affect gene regulation. We have previously shown that SNPs within transcription factor binding sites can affect transcription factor binding in an allele-specific and heritable manner. However, such analysis has relied on prior whole-genome genotypes provided by large external projects such as HapMap and the 1000 Genomes Project. This requirement limits the study of allele-specific effects of SNPs in primary patient samples from diseases of interest, where complete genotypes are not readily available. Results In this study, we show that we are able to identify SNPs de novo and accurately from ChIP-seq data generated in the ENCODE Project. Our de novo identified SNPs from ChIP-seq data are highly concordant with published genotypes. Independent experimental verification of more than 100 sites estimates our false discovery rate at less than 5%. Analysis of transcription factor binding at de novo identified SNPs revealed widespread heritable allele-specific binding, confirming previous observations. SNPs identified from ChIP-seq datasets were significantly enriched for disease-associated variants, and we identified dozens of allele-specific binding events in non-coding regions that could distinguish between disease and normal haplotypes. Conclusions Our approach combines SNP discovery, genotyping and allele-specific analysis, but is selectively focused on functional regulatory elements occupied by transcription factors or epigenetic marks, and will therefore be valuable for identifying the functional regulatory consequences of non-coding SNPs in primary disease samples. PMID:22950704

  3. Chalcone AttenuatesStaphylococcus aureusVirulence by Targeting Sortase A and Alpha-Hemolysin.

    Science.gov (United States)

    Zhang, Bing; Teng, Zihao; Li, Xianhe; Lu, Gejin; Deng, Xuming; Niu, Xiaodi; Wang, Jianfeng

    2017-01-01

    Staphylococcus aureus ( S .aureus) resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA), an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla), a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S. aureus infection. In this study, we discovered that chalcone, a natural small compound with little anti- S. aureus activity, could significantly inhibit SrtA activity with an IC 50 of 53.15 μM and Hla hemolysis activity with an IC 50 of 17.63 μM using a fluorescence resonance energy transfer (FRET) assay and a hemolysis assay, respectively. In addition, chalcone was proven to reduce protein A (SpA) display in intact bacteria, binding to fibronectin, formation of biofilm and S. aureus invasion. Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene, thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury; more importantly, chalcone could also reduce mortality in infected mice. Additionally, molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA, which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168, Ile182, and Arg197. Taken together, the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S. aureus infection via targeting SrtA and Hla.

  4. Chalcone Attenuates Staphylococcus aureus Virulence by Targeting Sortase A and Alpha-Hemolysin

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2017-09-01

    Full Text Available Staphylococcus aureus (S.aureus resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA, an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla, a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S. aureus infection. In this study, we discovered that chalcone, a natural small compound with little anti-S. aureus activity, could significantly inhibit SrtA activity with an IC50 of 53.15 μM and Hla hemolysis activity with an IC50 of 17.63 μM using a fluorescence resonance energy transfer (FRET assay and a hemolysis assay, respectively. In addition, chalcone was proven to reduce protein A (SpA display in intact bacteria, binding to fibronectin, formation of biofilm and S. aureus invasion. Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene, thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury; more importantly, chalcone could also reduce mortality in infected mice. Additionally, molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA, which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168, Ile182, and Arg197. Taken together, the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S. aureus infection via targeting SrtA and Hla.

  5. Listeria monocytogenes Isolates Carrying Virulence-Attenuating Mutations in Internalin A Are Commonly Isolated from Ready-to-Eat Food Processing Plant and Retail Environments.

    Science.gov (United States)

    VAN Stelten, A; Roberts, A R; Manuel, C S; Nightingale, K K

    2016-10-01

    Listeria monocytogenes is a human foodborne pathogen that may cause an invasive disease known as listeriosis in susceptible individuals. Internalin A (InlA; encoded by inlA) is a virulence factor that facilitates crossing of host cell barriers by L. monocytogenes . At least 19 single nucleotide polymorphisms (SNPs) in inlA that result in a premature stop codon (PMSC) have been described worldwide. SNPs leading to a PMSC in inlA have been shown to be causally associated with attenuated virulence. L. monocytogenes pathogens carrying virulence-attenuating (VA) mutations in inlA have been commonly isolated from ready-to-eat (RTE) foods but rarely have been associated with human disease. This study was conducted to determine the prevalence of VA SNPs in inlA among L. monocytogenes from environments associated with RTE food production and handling. More than 700 L. monocytogenes isolates from RTE food processing plant (n = 409) and retail (n = 319) environments were screened for the presence of VA SNPs in inlA. Overall, 26.4% of isolates from RTE food processing plant and 32.6% of isolates from retail environments carried a VA mutation in inlA. Food contact surfaces sampled at retail establishments were significantly (P food production and handling environments have reduced virulence. These data will be useful in the revision of current and the development of future risk assessments that incorporate strain-specific virulence parameters.

  6. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    Energy Technology Data Exchange (ETDEWEB)

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. (Beckman Research Institute of the City of Hope, Duarte, CA (USA))

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  7. Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans

    Science.gov (United States)

    Barbosa, Júnia Oliveira; Rossoni, Rodnei Dennis; Vilela, Simone Furgeri Godinho; de Alvarenga, Janaína Araújo; Velloso, Marisol dos Santos; Prata, Márcia Cristina de Azevedo; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2016-01-01

    Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model. PMID:26934196

  8. Contrasting transcriptional responses of a virulent and an attenuated strain of Mycobacterium tuberculosis infecting macrophages.

    Directory of Open Access Journals (Sweden)

    Alice H Li

    2010-06-01

    Full Text Available H37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes.In this study, the transcriptomes of the two strains during axenic growth in broth and during intracellular growth within murine bone-marrow macrophages were compared by whole genome expression profiling. We identified and compared adaptations of either strain upon encountering an intracellular environment, and also contrasted the transcriptomes of the two strains while inside macrophages. In the former comparison, both strains induced genes that would facilitate intracellular survival including those involved in mycobactin synthesis and fatty acid metabolism. However, this response was stronger and more extensive for H37Rv than for H37Ra. This was manifested as the differential expression of a greater number of genes and an increased magnitude of expression for these genes in H37Rv. In comparing intracellular transcriptional signatures, fifty genes were found to be differentially expressed between the strains. Of these fifty, twelve were under control of the PhoPR regulon. Further differences between strains included genes whose products were members of the ESAT-6 family of proteins, or were associated with their secretion.Along with the recent identification of single nucleotide polymorphisms in H37Ra when compared to H37Rv, our demonstration of differential expression of PhoP-regulated and ESX-1 region-related genes during macrophage infection further highlights the significance of these genes in the attenuation of H37Ra.

  9. [Study of the correlation between the plasma viral load and protective immunity induced by the equine infectious anemia attenuated vaccine and its parental virulent strain].

    Science.gov (United States)

    Cao, Xue-Zhi; Lin, Yue-Zhi; Li, Li; Jiang, Cheng-Gang; Zhao, Li-Ping; Lv, Xiao-Ling; Zhou, Jian-Hua

    2010-03-01

    The threshold hypothesis of attenuated lentiviral vaccine considers that the type of host response to infections of lentiviruses depends on the viral load. To evaluate the correlation between viral loads of the attenuated vaccine strain of equine infectious anemia virus (EIAV) and their effects to induce protective immunity, longitudinal plasma viral loads in groups of horses inoculated with either an attenuated EIAV vaccine strain (EIAV(DLV125)) or sub-lethal dose of an EIAV virulent strain (EIAV(LN40)) were compared. Similar levels of plasma viral loads ranging from 10(3)-10(5) copies/mL were detected from samples of these two groups of animals (P > 0.05) during 23 weeks post the inoculation. However, different responses to the challenge performed thereafter with lethal dose of the EIAV virulent strain were observed from the groups of horses inoculated with either EIAV(DLV125) or sub-lethal dose of EIAV(LN40). The protective efficiency was 67% (3 of 4 cases) and 0 (none of 2 cases), respectively. Our results implicate that the viral load of EIAV attenuated vaccine is not the primary factor, or at least not the solo primary factor, to determine the establishment of immune protection.

  10. Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV).

    Science.gov (United States)

    Dulwich, Katherine L; Giotis, Efstathios S; Gray, Alice; Nair, Venugopal; Skinner, Michael A; Broadbent, Andrew J

    2017-12-01

    Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B-cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B-cell activation and signalling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

  11. Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

    Science.gov (United States)

    Taira, Chiaki; Matsuda, Kazuyuki; Takeichi, Naoya; Furukawa, Satomi; Sugano, Mitsutoshi; Uehara, Takeshi; Okumura, Nobuo; Honda, Takayuki

    2018-01-01

    ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care. © 2017 Wiley Periodicals, Inc.

  12. Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes.

    Science.gov (United States)

    Scheen, Anne-Cathrine; Pfeil, Bernard E; Petri, Anna; Heidari, Nahid; Nylinder, Stephan; Oxelman, Bengt

    2012-01-01

    Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing. © 2011 Blackwell Publishing Ltd.

  13. Complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein 3b of US strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus.

    Science.gov (United States)

    Zhang, Xinsheng; Hasoksuz, Mustafa; Spiro, David; Halpin, Rebecca; Wang, Shiliang; Stollar, Sarah; Janies, Daniel; Hadya, Nagesh; Tang, Yuxin; Ghedin, Elodie; Saif, Linda

    2007-02-20

    Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60 and virulent TGEV Purdue and attenuated TGEV Purdue P115, together with the ISU-1 strain of porcine respiratory coronavirus (PRCV-ISU-1), a naturally occurring TGEV deletion mutant with an altered respiratory tropism and reduced virulence. Pairwise comparison at both the nucleotide (nt) and amino acid (aa) levels between virulent and attenuated TGEV strains identified a common change in nt 1753 of the spike gene, resulting in a serine to alanine mutation at aa position 585 of the spike proteins of the attenuated TGEV strains. Alanine was also present in this protein in PRCV-ISU-1. Particularly noteworthy, the serine to alanine mutation resides in the region of the major antigenic site A/B (aa 506-706) that elicits neutralizing antibodies and within the domain mediating the cell surface receptor aminopeptidase N binding (aa 522-744). Comparison of the predicted polypeptide products of ORF3b showed significant deletions in the naturally attenuated PRCV-ISU-1 and TGEV Miller M60; these deletions occurred at a common break point, suggesting a related mechanism of recombination that may affect viral virulence or tropism. Sequence comparisons at both genomic and protein levels indicated that PRCV-ISU-1 had a closer relationship with TGEV Miller strains than Purdue strains. Phylogenetic analyses showed that virulence is an evolutionarily labile trait in TGEV and that TGEV strains as a group share a common ancestor with PRCV.

  14. Loss of RNA expression and allele-specific expression associated with congenital heart disease

    Science.gov (United States)

    McKean, David M.; Homsy, Jason; Wakimoto, Hiroko; Patel, Neil; Gorham, Joshua; DePalma, Steven R.; Ware, James S.; Zaidi, Samir; Ma, Wenji; Patel, Nihir; Lifton, Richard P.; Chung, Wendy K.; Kim, Richard; Shen, Yufeng; Brueckner, Martina; Goldmuntz, Elizabeth; Sharp, Andrew J.; Seidman, Christine E.; Gelb, Bruce D.; Seidman, J. G.

    2016-01-01

    Congenital heart disease (CHD), a prevalent birth defect occurring in 1% of newborns, likely results from aberrant expression of cardiac developmental genes. Mutations in a variety of cardiac transcription factors, developmental signalling molecules and molecules that modify chromatin cause at least 20% of disease, but most CHD remains unexplained. We employ RNAseq analyses to assess allele-specific expression (ASE) and biallelic loss-of-expression (LOE) in 172 tissue samples from 144 surgically repaired CHD subjects. Here we show that only 5% of known imprinted genes with paternal allele silencing are monoallelic versus 56% with paternal allele expression—this cardiac-specific phenomenon seems unrelated to CHD. Further, compared with control subjects, CHD subjects have a significant burden of both LOE genes and ASE events associated with altered gene expression. These studies identify FGFBP2, LBH, RBFOX2, SGSM1 and ZBTB16 as candidate CHD genes because of significantly altered transcriptional expression. PMID:27670201

  15. High-throughput genotyping with infrared fluorescence allele specific hybridization (iFLASH): a simple, reliable and low-cost alternative.

    NARCIS (Netherlands)

    Himbergen, T. van; Voorbij, H.A.; Barendrecht, A.D.; Rijn, B.B. van; Brambilla, R.; Tits, L.J.H. van; Roest, M.

    2006-01-01

    OBJECTIVES: To develop and validate a novel genotyping approach, named infrared Fluorescence Allele Specific Hybridization (iFLASH), which combines the principles of allele specific oligonucleotide (ASO) hybridization with the advanced possibilities of infrared imaging. DESIGN AND METHODS: As an

  16. Breast Cancer Family History and Allele-Specific DNA Methylation in the Legacy Girls Study.

    Science.gov (United States)

    Wu, Hui-Chen; Do, Catherine; Andrulis, Irene L; John, Esther M; Daly, Mary B; Buys, Saundra S; Chung, Wendy K; Knight, Julia A; Bradbury, Angela R; Keegan, Theresa H M; Schwartz, Lisa; Krupska, Izabela; Miller, Rachel L; Santella, Regina M; Tycko, Benjamin; Terry, Mary Beth

    2018-02-13

    Family history, a well-established risk factor for breast cancer, can have both genetic and environmental contributions. Shared environment in families as well as epigenetic changes that also may be influenced by shared genetics and environment may also explain familial clustering of cancers. Epigenetic regulation, such as DNA methylation, can change the activity of a DNA segment without a change in the sequence; environmental exposures experienced across the life course can induce such changes. However, genetic-epigenetic interactions, detected as methylation quantitative trait loci (mQTLs; a.k.a. meQTLs) and haplotype-dependent allele-specific methylation (hap-ASM), can also contribute to inter-individual differences in DNA methylation patterns. To identify differentially methylated regions (DMRs) associated with breast cancer susceptibility, we examined differences in white blood cell DNA methylation in 29 candidate genes in 426 girls (ages 6-13 years) from the LEGACY Girls Study, 239 with and 187 without a breast cancer family history (BCFH). We measured methylation by targeted massively parallel bisulfite sequencing (bis-seq) and observed BCFH DMRs in two genes: ESR1 (Δ 4.9%, P = 0.003) and SEC16B (Δ 3.6%, P = 0.026), each of which has been previously implicated in breast cancer susceptibility and pubertal development. These DMRs showed high inter-individual variability in methylation, suggesting the presence of mQTLs/hap-ASM. Using single nucleotide polymorphisms data in the bis-seq amplicon, we found strong hap-ASM in SEC16B (with allele specific-differences ranging from 42% to 74%). These findings suggest that differential methylation in genes relevant to breast cancer susceptibility may be present early in life, and that inherited genetic factors underlie some of these epigenetic differences.

  17. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus.

    Science.gov (United States)

    Gaupp, Rosmarie; Wirf, Jessica; Wonnenberg, B; Biegel, Tanja; Eisenbeis, J; Graham, J; Herrmann, M; Lee, C Y; Beisswenger, C; Wolz, C; Tschernig, T; Bischoff, M; Somerville, G A

    2016-07-01

    In Staphylococcus aureus, metabolism is intimately linked with virulence determinant biosynthesis, and several metabolite-responsive regulators have been reported to mediate this linkage. S. aureus possesses at least three members of the RpiR family of transcriptional regulators. Of the three RpiR homologs, RpiRc is a potential regulator of the pentose phosphate pathway, which also regulates RNAIII levels. RNAIII is the regulatory RNA of the agr quorum-sensing system that controls virulence determinant synthesis. The effect of RpiRc on RNAIII likely involves other regulators, as the regulators that bind the RNAIII promoter have been intensely studied. To determine which regulators might bridge the gap between RpiRc and RNAIII, sarA, sigB, mgrA, and acnA mutations were introduced into an rpiRc mutant background, and the effects on RNAIII were determined. Additionally, phenotypic and genotypic differences were examined in the single and double mutant strains, and the virulence of select strains was examined using two different murine infection models. The data suggest that RpiRc affects RNAIII transcription and the synthesis of virulence determinants in concert with σ(B), SarA, and the bacterial metabolic status to negatively affect virulence. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Immunization of African Indigenous Pigs with Attenuated Genotype I African Swine Fever Virus OURT88/3 Induces Protection Against Challenge with Virulent Strains of Genotype I.

    Science.gov (United States)

    Mulumba-Mfumu, L K; Goatley, L C; Saegerman, C; Takamatsu, H-H; Dixon, L K

    2016-10-01

    The attenuated African swine fever virus genotype I strain OURT88/3 has previously been shown to induce protection of European breeds of domestic pigs against challenge with virulent isolates. To determine whether protective immune responses could also be induced in indigenous breeds of pigs from the Kinshassa region in Democratic Republic of Congo, we immunized a group of eight pigs with OURT88/3 strain and challenged the pigs 3 weeks later with virulent genotype I strain OURT88/1. Four of the pigs were protected against challenge. Three of the eight pigs died from African swine fever virus and a fourth from an unknown cause. The remaining four pigs all survived challenge with a recent virulent genotype I strain from the Democratic Republic of Congo, DRC 085/10. Control groups of non-immune pigs challenged with OURT88/1 or DRC 085/10 developed signs of acute ASFV as expected and had high levels of virus genome in blood. © 2015 Blackwell Verlag GmbH.

  20. Reverse mutation of the virulence-associated S2 gene does not cause an attenuated equine infectious anemia virus strain to revert to pathogenicity.

    Science.gov (United States)

    Gao, Xu; Jiang, Cheng-Gang; Wang, Xue-Feng; Lin, Yue-Zhi; Ma, Jian; Han, Xiu-E; Zhao, Li-Ping; Shen, Rong-Xian; Xiang, Wen-Hua; Zhou, Jian-Hua

    2013-09-01

    The contribution of S2 accessory gene of equine infectious anemia virus (EIAV) to the virulence of pathogenic strains was investigated in the present study by reverse mutation of all four consensus S2 mutation sites in an attenuated EIAV proviral strain, FDDV3-8, to the corresponding sequences of a highly pathogenic strain DV117. The S2 reverse-mutated recombinant strain FDDVS2r1-2-3-4 replicated with similar kinetics to FDDV3-8 in cultivated target cells. In contrast to the results of other studies of EIAV with dysfunctional S2, reverse mutation of S2 only transiently and moderately increased the plasma viral load of inoculated horses, and induction of transient immunosuppression did not boost viral pathogenicity. In addition, inoculation of FDDVS2r1-2-3-4 induced partial protection to a challenge pathogenic virus. These results suggest that the attenuated EIAV vaccine strain with multiple mutations in multiple genes will not easily revert to a virulent phenotype. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution.

    Science.gov (United States)

    McGranahan, Nicholas; Rosenthal, Rachel; Hiley, Crispin T; Rowan, Andrew J; Watkins, Thomas B K; Wilson, Gareth A; Birkbak, Nicolai J; Veeriah, Selvaraju; Van Loo, Peter; Herrero, Javier; Swanton, Charles

    2017-11-30

    Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. VIDEO ABSTRACT. Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

  2. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    Science.gov (United States)

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described.

  3. Allele-specific marker generation and linkage mapping on the Xiphophorus sex chromosomes.

    Science.gov (United States)

    Woolcock, B; Kazianis, S; Lucito, R; Walter, R B; Kallman, K D; Morizot, D C; Vielkind, J R

    2006-01-01

    There is great interest in the sex chromosomes of Xiphophorus fishes because both WY/YY and XX/XY sex-determining mechanisms function in these species, with at least one taxon possessing all three types of sex chromosomes, and because in certain interspecific hybrids melanoma arises as a consequence of inheritance of the sex-linked macromelanophore determining locus (MDL). Representational difference analysis (RDA) has been used to clone two sequences from the sex-determining region of X. maculatus, including a cholinergic receptor, nicotinic, delta polypeptide (CHRND) orthologue. Allele-specific assays for these sequences, as well as for the sex-linked XMRK1 and XMRK2 genes, were developed to distinguish W, X, and Y chromosomes derived from a X. maculatus (XX/XY) strain and a X. helleri (WY/YY) strain. Linkage mapping localized these markers to linkage group (LG) 24. No recombinants were observed between XMRK2 and MDL, confirming a role for XMRK2 in macromelanophore development. Although the master sex-determining (SD) locus certainly resides on Xiphophorus LG 24, autosomal loci are probably involved in sex determination as well, as indicated by the abnormal sex ratios in the backcross hybrids that contrast theoretical predictions based on LG 24 genotyping. Marker development and allelic discrimination on the Xiphophorus sex chromosomes should prove highly useful for studies that utilize this genus as an animal model.

  4. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish

    Directory of Open Access Journals (Sweden)

    Samuel J. Capon

    2017-01-01

    Full Text Available The advent of genome editing has significantly altered genetic research, including research using the zebrafish model. To better understand the selectivity of the commonly used CRISPR/Cas9 system, we investigated single base pair mismatches in target sites and examined how they affect genome editing in the zebrafish model. Using two different zebrafish strains that have been deep sequenced, CRISPR/Cas9 target sites containing polymorphisms between the two strains were identified. These strains were crossed (creating heterozygotes at polymorphic sites and CRISPR/Cas9 complexes that perfectly complement one strain injected. Sequencing of targeted sites showed biased, allele-specific editing for the perfectly complementary sequence in the majority of cases (14/19. To test utility, we examined whether phenotypes generated by F0 injection could be internally controlled with such polymorphisms. Targeting of genes bmp7a and chordin showed reduction in the frequency of phenotypes in injected ‘heterozygotes’ compared with injecting the strain with perfect complementarity. Next, injecting CRISPR/Cas9 complexes targeting two separate sites created deletions, but deletions were biased to selected chromosomes when one CRISPR/Cas9 target contained a polymorphism. Finally, integration of loxP sequences occurred preferentially in alleles with perfect complementarity. These experiments demonstrate that single nucleotide polymorphisms (SNPs present throughout the genome can be utilised to increase the efficiency of in cis genome editing using CRISPR/Cas9 in the zebrafish model.

  5. Allele-specific gene expression is widespread across the genome and biological processes.

    Directory of Open Access Journals (Sweden)

    Ricardo Palacios

    Full Text Available Allelic specific gene expression (ASGE appears to be an important factor in human phenotypic variability and as a consequence, for the development of complex traits and diseases. In order to study ASGE across the human genome, we have performed a study in which genotyping was coupled with an analysis of ASGE by screening 11,500 SNPs using the Mapping 10 K Array to identify differential allelic expression. We found that from the 5,133 SNPs that were suitable for analysis (heterozygous in our sample and expressed in peripheral blood mononuclear cells, 2,934 (57% SNPs had differential allelic expression. Such SNPs were equally distributed along human chromosomes and biological processes. We validated the presence or absence of ASGE in 18 out 20 SNPs (90% randomly selected by real time PCR in 48 human subjects. In addition, we observed that SNPs close to -but not included in- segmental duplications had increased levels of ASGE. Finally, we found that transcripts of unknown function or non-coding RNAs, also display ASGE: from a total of 2,308 intronic SNPs, 1510 (65% SNPs underwent differential allelic expression. In summary, ASGE is a widespread mechanism in the human genome whose regulation seems to be far more complex than expected.

  6. DNA Methylation Maintains Allele-specific KIR Gene Expression in Human Natural Killer Cells

    Science.gov (United States)

    Chan, Huei-Wei; Kurago, Zoya B.; Stewart, C. Andrew; Wilson, Michael J.; Martin, Maureen P.; Mace, Brian E.; Carrington, Mary; Trowsdale, John; Lutz, Charles T.

    2003-01-01

    Killer immunoglobulin-like receptors (KIR) bind self–major histocompatibility complex class I molecules, allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes, but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression, 3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5′ gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor, 5-aza-2′-deoxycytidine, induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus, NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles. PMID:12538663

  7. Allele specific LAMP- gold nanoparticle for characterization of single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Fábio Ferreira Carlos

    2017-12-01

    Full Text Available Due to their relevance as disease biomarkers and for diagnostics, screening of single nucleotide polymorphism (SNPs requires simple and straightforward strategies capable to provide results in medium throughput settings. Suitable approaches relying on isothermal amplification techniques have been evolving to substitute the cumbersome and highly specialized PCR amplification detection schemes. Nonetheless, identification of an individual’s genotype still requires sophisticated equipment and laborious methods.Here, we present a low-cost and reliable approach based on the allele specific loop-mediated isothermal amplification (AS-LAMP coupled to ssDNA functionalized gold nanoparticle (Au-nanoprobe colorimetric sequence discrimination. The Au-nanoprobe integration allows for the colorimetric detection of AS-LAMP amplification product that can be easily interpreted in less than 15 min. We targeted a clinical relevant SNP responsible for lactose intolerance (-13910C/T dbSNP rs#: 4988235 to demonstrate its proof of concept and full potential of this novel approach. Keywords: SNP, Isothermal amplification, Gold nanoparticles, Gold nanoprobes, Lactose intolerance

  8. Analysis of BDNF Val66Met allele-specific mRNA levels in bipolar disorder.

    Science.gov (United States)

    De Luca, Vincenzo; Strauss, John; Semeralul, Mawahib; Huang, Sheeda; Li, Peter P; Warsh, Jerry J; Kennedy, James L; Wong, Albert H C

    2008-08-22

    We have previously reported an association between the BDNF Val66Met polymorphism and bipolar disorder (BD). However, the possibility that genomic imprinting in BDNF gene affects risk for BD has not been investigated. To examine the possibility of genomic imprinting in the BDNF gene in BD, we analyzed the parent-of-origin effect (POE) and differential expression of the BDNF Val66Met alleles in BD. We performed a family-based association study and ETDT analyses of the Val66Met polymorphism in 312 BD nuclear families, and compared allele-specific mRNA levels in both post-mortem brain samples and B lymphoblasts from BD patients and controls. The BDNF Val66 allele was transmitted significantly more often to patients with BD (maternal transmissions: 46/22, p=0.003; paternal transmissions: 55/30, p=0.006). There was no significant difference between maternal and paternal transmission ratios. There was no significant difference in the ratio of Val/Met-specific mRNA expression between BD and controls, in either brain or B lymphoblasts. The Val/Met ratio was much lower in the brain vs. B lymphoblasts. These data do not support a role for genomic imprinting as a modifier of the contribution of BDNF gene to risk of susceptibility to BD.

  9. Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

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    Carolina De Marco Verissimo

    2013-11-01

    Full Text Available Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

  10. Mechanisms and Disease Associations of Haplotype-Dependent Allele-Specific DNA Methylation

    Science.gov (United States)

    Do, Catherine; Lang, Charles F.; Lin, John; Darbary, Huferesh; Krupska, Izabela; Gaba, Aulona; Petukhova, Lynn; Vonsattel, Jean-Paul; Gallagher, Mary P.; Goland, Robin S.; Clynes, Raphael A.; Dwork, Andrew; Kral, John G.; Monk, Catherine; Christiano, Angela M.; Tycko, Benjamin

    2016-01-01

    Haplotype-dependent allele-specific methylation (hap-ASM) can impact disease susceptibility, but maps of this phenomenon using stringent criteria in disease-relevant tissues remain sparse. Here we apply array-based and Methyl-Seq approaches to multiple human tissues and cell types, including brain, purified neurons and glia, T lymphocytes, and placenta, and identify 795 hap-ASM differentially methylated regions (DMRs) and 3,082 strong methylation quantitative trait loci (mQTLs), most not previously reported. More than half of these DMRs have cell type-restricted ASM, and among them are 188 hap-ASM DMRs and 933 mQTLs located near GWAS signals for immune and neurological disorders. Targeted bis-seq confirmed hap-ASM in 12/13 loci tested, including CCDC155, CD69, FRMD1, IRF1, KBTBD11, and S100A∗-ILF2, associated with immune phenotypes, MYT1L, PTPRN2, CMTM8 and CELF2, associated with neurological disorders, NGFR and HLA-DRB6, associated with both immunological and brain disorders, and ZFP57, a trans-acting regulator of genomic imprinting. Polymorphic CTCF and transcription factor (TF) binding sites were over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to macaques, indicated that CTCF and TF binding likelihood predicts the strength and direction of the allelic methylation asymmetry. These results show that hap-ASM is highly tissue specific; an important trans-acting regulator of genomic imprinting is regulated by this phenomenon; and variation in CTCF and TF binding sites is an underlying mechanism, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supra- and sub-threshold GWAS peaks in immunological and neurological disorders. PMID:27153397

  11. An informational view of accession rarity and allele specificity in germplasm banks for management and conservation.

    Science.gov (United States)

    Reyes-Valdés, M Humberto; Burgueño, Juan; Singh, Sukhwinder; Martínez, Octavio; Sansaloni, Carolina Paola

    2018-01-01

    Germplasm banks are growing in their importance, number of accessions and amount of characterization data, with a large emphasis on molecular genetic markers. In this work, we offer an integrated view of accessions and marker data in an information theory framework. The basis of this development is the mutual information between accessions and allele frequencies for molecular marker loci, which can be decomposed in allele specificities, as well as in rarity and divergence of accessions. In this way, formulas are provided to calculate the specificity of the different marker alleles with reference to their distribution across accessions, accession rarity, defined as the weighted average of the specificity of its alleles, and divergence, defined by the Kullback-Leibler formula. Albeit being different measures, it is demonstrated that average rarity and divergence are equal for any collection. These parameters can contribute to the knowledge of the structure of a germplasm collection and to make decisions about the preservation of rare variants. The concepts herein developed served as the basis for a strategy for core subset selection called HCore, implemented in a publicly available R script. As a proof of concept, the mathematical view and tools developed in this research were applied to a large collection of Mexican wheat accessions, widely characterized by SNP markers. The most specific alleles were found to be private of a single accession, and the distribution of this parameter had its highest frequencies at low levels of specificity. Accession rarity and divergence had largely symmetrical distributions, and had a positive, albeit non-strictly linear relationship. Comparison of the HCore approach for core subset selection, with three state-of-the-art methods, showed it to be superior for average divergence and rarity, mean genetic distance and diversity. The proposed approach can be used for knowledge extraction and decision making in germplasm collections of

  12. Molecular Basis of Allele-Specific Efficacy of a Blood-Stage Malaria Vaccine: Vaccine Development Implications

    Science.gov (United States)

    Ouattara, Amed; Takala-Harrison, Shannon; Thera, Mahamadou A.; Coulibaly, Drissa; Niangaly, Amadou; Saye, Renion; Tolo, Youssouf; Dutta, Sheetij; Heppner, D. Gray; Soisson, Lorraine; Diggs, Carter L.; Vekemans, Johan; Cohen, Joe; Blackwelder, William C.; Dube, Tina; Laurens, Matthew B.; Doumbo, Ogobara K.; Plowe, Christopher V.

    2013-01-01

    The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02A, a blood-stage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02A had the highest efficacy against AMA1 alleles that were identical to the 3D7 vaccine-type allele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alleles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of allele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alleles of this extremely polymorphic antigen. PMID:23204168

  13. Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus.

    Science.gov (United States)

    Li, J; Kasper, D L; Ausubel, F M; Rosner, B; Michel, J L

    1997-11-25

    The alpha C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The alpha C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the alpha C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the alpha C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the alpha antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of alpha-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, alpha antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of alpha-specific antibodies. In addition, antibodies to the alpha C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the alpha antigen as expressed from the bca gene. Therefore, the alpha C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

  14. Vitamin A deficiency impairs adaptive B and T cell responses to a prototype monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model.

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    Kuldeep S Chattha

    Full Text Available Rotaviruses (RV are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12 and 2-3 fold lower anti-inflammatory (IL10 cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented pigs had significantly higher serum IL12 (PID2 and IFNγ (PID6 compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more

  15. Mutation of the maturase lipoprotein attenuates the virulence of Streptococcus equi to a greater extent than does loss of general lipoprotein lipidation.

    Science.gov (United States)

    Hamilton, Andrea; Robinson, Carl; Sutcliffe, Iain C; Slater, Josh; Maskell, Duncan J; Davis-Poynter, Nick; Smith, Ken; Waller, Andrew; Harrington, Dean J

    2006-12-01

    Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (DeltaprtM(138-213), with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Deltalgt(190-685)). Moreover, mucus production was significantly greater in both wild-type-infected and Deltalgt(190-685)-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Deltalgt(190-685) mutant did still exhibit signs of disease. In contrast, only the DeltaprtM(138-213) mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Deltalgt(190-685) mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

  16. Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation▿

    Science.gov (United States)

    Hamilton, Andrea; Robinson, Carl; Sutcliffe, Iain C.; Slater, Josh; Maskell, Duncan J.; Davis-Poynter, Nick; Smith, Ken; Waller, Andrew; Harrington, Dean J.

    2006-01-01

    Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt190-685 mutant did still exhibit signs of disease. In contrast, only the ΔprtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted. PMID:17015455

  17. [The difference in specific humoral immune responses induced with the attenuated equine infectious anemia vaccine strain and virulent strain.].

    Science.gov (United States)

    Zhu, Zhen-Ying; Lin, Yue-Zhi; Wang, Yu-Hong; Zhao, Li-Ping; Zhu, Yuan-Mao; Zhou, Jian-Hua

    2009-12-01

    To disclose the potential roles of humoral immune response in the EIAV vaccine-induced protective immunity. In this study, major parameters of humoral immunity be compared between horses inoculated with the EIAV vaccine strain and the pathogenic virulent strain. Experimental horses were randomly assigned into the group inoculated with the vaccine strain EIAV(DLV); (the vaccinated group) and the group inoculated with sub-morbigenous dose of virulent strain EIAV(Liao); (the inapparent infection group). Humoral immunity parameters, including binding endpoint titer and avidity index of antibodies to the envelop protein (Env) and the capsid protein (P26), and the conformation-dependent index of the Env antibody, were assayed and compared between these two groups by using ELISA. Neutralizing antibodies to the EIAV vaccine strain and a pathogenic strain were simultaneously detected by using plaque forming unite assay (PFU) and reverse transcriptase activity assay, respectively. In general, all humoral parameters increased with a time-dependent manner in both the vaccinated and the inapparent infection group. However, significantly higher antibody activities for P26 binding endpoint titer and Env avidity index were detected in the vaccinated group within 2 months post infection (Pinfection group throughout the entire observation period (Pinfected horses (P<0.01 for EIAV(FDDV); and P<0.05 for EIAV(DLV34);). Statistically significant differences in EIAV-specific binding antibodies and the neutralizing antibody are detected between animals induced with the EIAV vaccine strain and the virulent strain. Importantly, the significantly early and strong responses in the neutralizing antibody and the conformation-dependent Env antibody induced by the vaccine implicate special roles these antibodies playing in EIAV vaccine-induced immune protection.

  18. Attenuation of quorum-sensing-dependent virulence factors and biofilm formation by medicinal plants against antibiotic resistant Pseudomonas aeruginosa

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    P. Sankar Ganesh

    2018-01-01

    Full Text Available Pseudomonas aeruginosa use small signaling molecules such as acyl homoserine lactones (AHLs, which play an important role in release virulence factors and toxin for further establishment of host infection. Thus, involving with the QS system would provide alternative ways of preventing the pathogenicity. In the present study, totally six medicinal plants (Terminalia bellerica, Celastrus paniculatus, Kingiodendron pinnatum, Schleichera oleosa, Melastoma malabathricum, Garcinia gummi-gutta were screened for anti-QS activity using biomonitor strain of Chromobacterium violaceum CV12472. The primary screening of antimicrobial activity of all the plant extracts have inhibited the growth of tested bacterial species. Of these at the sub-minimum inhibitory concentration the methanol extract of T. bellerica (0.0625–0.5 mg/ml has significantly inhibited violacein production (20.07–66.22% in C. violaceum (CV12472. Consequently, the extract of T. bellerica has reduced the production of pyocyanin, exopolysaccharide and biofilm formation in P. aeruginosa strains. Fluorescence and scanning electron microscopy analysis confirmed the reduction of biofilm formation in P. aeruginosa strains when treated with T. bellerica. GC–MS analysis showed the active compounds inhibited the production of virulence factors of P. aeruginosa. The results suggest the possible use of this T. bellerica as an anti-QS and anti-biofilm agent to control Pseudomonas infection. Interference of QS provides an important means for the inhibition of bacterial virulence and thus aids in treatment strategies.

  19. Ellagic acid derivatives from Terminalia chebula Retz. downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa PAO1 virulence.

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    Sajal Sarabhai

    Full Text Available BACKGROUND: Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors. METHODS AND RESULTS: Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7, obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001 in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05 reduced with enhanced (20% susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI and their cognate receptor (lasR and rhlR genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12HSL and C(4HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C(4HSL. F7 also showed antagonistic activity against 3-oxo-C(12HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract. CONCLUSIONS: This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors

  20. Lateral flow strip for visual detection of K-ras mutations based on allele-specific PCR.

    Science.gov (United States)

    Wang, Cong; Chen, Xiaomin; Wu, Yuying; Li, Hao; Wang, Yu; Pan, Xiaofu; Tang, Tingting; Liu, Ziying; Li, Xiaokun

    2016-10-01

    To develop a convenient and sensitive point-of-care test for detecting gene mutations based on allele-specific PCR. To develop a lateral flow strip for visual detection of K-ras mutations based on a modified PCR, a specific DNA tag was covalently linked to the 5'-end of each primer by a nine-carbon linker to produce a sticky end. One of the sticky ends of the PCR products bound to gold nano-particles, while the other sticky end was captured onto a nitrocellulose membrane of lateral flow strips. The lateral flow strip showed a great sensitivity, which detected mutations in as low as 10 tumor cells. The positive rate and accuracy of the lateral flow strip for blood samples were over 92 and 96 %, respectively. The lateral flow strip provides an easy method for sensitive detection of gene mutations based on allele specific-PCR.

  1. Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge

    Science.gov (United States)

    Franzoni, Giulia; Kurkure, Nitin V.; Edgar, Daniel S.; Everett, Helen E.; Gerner, Wilhelm; Bodman-Smith, Kikki B.; Crooke, Helen R.

    2013-01-01

    Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3+ CD4− CD8hi T cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3+ CD4+ CD8α+) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44hi CD62L− and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ+ CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines. PMID:23966552

  2. Immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (TGEV) and recombinant TGEV spike protein vaccines and protection of their suckling pigs against virulent TGEV challenge exposure.

    Science.gov (United States)

    Park, S; Sestak, K; Hodgins, D C; Shoup, D I; Ward, L A; Jackwood, D J; Saif, L J

    1998-08-01

    To compare recombinant transmissible gastroenteritis virus (TGEV) spike protein, (SP) R2-2, with attenuated live virus (ALV) vaccine in sows during late pregnancy. 13 TGEV-seronegative sows and their pigs. At prepartum weeks (PPW) 6 and 4, sows of groups 1 and 2 received ALV via the oral/intranasal (O/IN) route. At PPW 2, group-1 sows received ALV IM and group-2 sows received SPR2-2 IM. Group-3 sows received SPR2-2 IM at PPW 4 and ALV O/IN at PPW 2. Sows of group 4 (negative controls) were inoculated O/IN with mock-infected ST cell fluids at PPW 6 and 4 and IM with Sf9 cell lysates at PPW2 (n = 2), or IM with Sf9 cell lysates at PPW4 and O/IN with mock-infected ST cell fluids at PPW2 (2). Serum, colostrum, and milk samples were tested for antibody to TGEV, and a lymphoproliferative (LP) assay was done on blood mononuclear cells. Suckling pigs were challenge exposed with virulent TGEV. Sows of groups 1 and 2 had higher IgG and significantly higher antibody titers in colostrum; their pigs had significantly higher serum antibody titer. At challenge exposure of their pigs, LP responses of group-2 sows were significantly higher than those of sows in the other 3 groups. Mean pig mortality ranged from 43 (group 2) to 92% (group 4). Significant negative correlations were observed among litter mortality and sow LP response, colostral titer, and pig serum titer at time of challenge exposure. In sows vaccinated twice with attenuated live TGEV, the recombinant SPR2-2 administered IM may be comparable to ALV administered IM as a booster. Vaccination failed to provide complete protection to suckling pigs after challenge exposure.

  3. Attenuation of Quorum Sensing Regulated Virulence of Pectobacterium carotovorum subsp. carotovorum through an AHL Lactonase Produced by Lysinibacillus sp. Gs50.

    Science.gov (United States)

    Garge, Sneha S; Nerurkar, Anuradha S

    2016-01-01

    Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved "HXHXDH" motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent.

  4. Attenuation of Quorum Sensing Regulated Virulence of Pectobacterium carotovorum subsp. carotovorum through an AHL Lactonase Produced by Lysinibacillus sp. Gs50.

    Directory of Open Access Journals (Sweden)

    Sneha S Garge

    Full Text Available Quorum sensing (QS is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ, is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved "HXHXDH" motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc, a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent.

  5. Construction of an attenuated Salmonella delivery system harboring genes encoding various virulence factors of avian pathogenic Escherichia coli and its potential as a candidate vaccine for chicken colibacillosis.

    Science.gov (United States)

    Chaudhari, Atul A; Matsuda, Kiku; Lee, John Hwa

    2013-03-01

    An attenuated Salmonella (deltalon, deltacpxR, and deltaasdA16) delivery system containing the genes encoding P-fimbriae (papa and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of avian pathogenic Escherichia coli (APEC) was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. The birds were divided into three groups designated group A (nonvaccinated control), group B (given a single immunization), and group C (administered prime and boost immunizations). Prime and booster vaccinations with the constructions were administered to 1-day-old and 14-day-old birds, respectively. Immune responses were measured postimmunization, and the birds were challenged via an intra-air sac route with a virulent APEC strain at the second, third, and fourth weeks of age. Group B birds were partially protected against the challenge and showed increased levels of plasma immunoglobulin (Ig)G, mucosal IgA antibodies, and lymphocyte proliferation. Group C birds showed greater protection against the challenge, with significantly stronger immune responses compared with the birds in the other groups. Overall, our data suggest that the Salmonella delivery system with recombinant constructs is capable of inducing robust immune responses and induces effective protection against colibacillosis caused by APEC.

  6. Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data

    OpenAIRE

    Degner, Jacob F.; Marioni, John C.; Pai, Athma A.; Pickrell, Joseph K.; Nkadori, Everlyne; Gilad, Yoav; Pritchard, Jonathan K.

    2009-01-01

    Motivation: Next-generation sequencing has become an important tool for genome-wide quantification of DNA and RNA. However, a major technical hurdle lies in the need to map short sequence reads back to their correct locations in a reference genome. Here, we investigate the impact of SNP variation on the reliability of read-mapping in the context of detecting allele-specific expression (ASE). Results: We generated 16 million 35 bp reads from mRNA of each of two HapMap Yoruba individuals. When ...

  7. Reversion to virulence and efficacy of an attenuated canarypox vaccine in Hawai'i 'Amakihi (Hemignathus Virens)

    Science.gov (United States)

    Atkinson, Carter T.; Wiegand, Kimberly C.; Triglia, Dennis; Jarvi, Susan I.

    2012-01-01

    Vaccines may be effective tools for protecting small populations of highly susceptible endangered, captive-reared, or translocated Hawaiian honeycreepers from introduced Avipoxvirus, but their efficacy has not been evaluated. An attenuated Canarypox vaccine that is genetically similar to one of two passerine Avipoxvirus isolates from Hawai‘i and distinct from Fowlpox was tested to evaluate whether Hawai‘i ‘Amakihi (Hemignathus virens) can be protected from wild isolates of Avipoxvirus from the Hawaiian Islands. Thirty-one (31) Hawai‘i ‘Amakihi were collected from high-elevation habitats on Mauna Kea Volcano, where pox transmission is rare, and randomly divided into two groups. One group was vaccinated with Poximune C®, whereas the other group received a sham vaccination with sterile water. Four of 15 (27%) vaccinated birds developed life-threatening disseminated lesions or lesions of unusually long duration, whereas one bird never developed a vaccine-associated lesion or “take.” After vaccine lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox (FP) from Hawai‘i, a Hawai‘i ‘Amakihi isolate of a Canarypox-like virus (PV1), or a Hawai‘i ‘Amakihi isolate of a related, but distinct, passerine Avipoxvirus (PV2). Similarly, three random groups of five unvaccinated ‘Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated ‘Amakihi challenged with FP had transient infections with no clinical signs of infection. Mortality in vaccinated ‘Amakihi challenged with PV1 and PV2 ranged from 0% (0/5) for PV1 to 60% (3/5) for PV2. Mortality in unvaccinated ‘Amakihi ranged from 40% (2/5) for PV1 to 100% (5/5) for PV2. Although the vaccine provided some protection against PV1, both potential for vaccine reversion and low efficacy against PV2 preclude its use in captive or wild honeycreepers.

  8. Brucella abortusΔcydCΔcydD and ΔcydCΔpurD double-mutants are highly attenuated and confer long-term protective immunity against virulent Brucella abortus.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Kim, Kiju; Hahn, Tae-Wook

    2016-01-04

    We constructed double deletion (ΔcydCΔcydD and ΔcydCΔpurD) mutants from virulent Brucella abortus biovar 1 field isolate (BA15) by deleting the genes encoding an ATP-binding cassette-type transporter (cydC and cydD genes) and a phosphoribosylamine-glycine ligase (purD). Both BA15ΔcydCΔcydD and BA15ΔcydCΔpurD double-mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. Both double-mutants were readily cleared from spleens by 4 weeks post-inoculation even when inoculated at the dose of 10(8) CFU per mouse. Moreover, the inoculated mice showed no splenomegaly, which indicates that the mutants are highly attenuated. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of these mutants to confer long-term protective immunity in mice against challenge with B. abortus strain 2308. Vaccination of mice with either mutant induced humoral and cell-mediated immune responses, and provided significantly better protection than commercial B. abortus strain RB51 vaccine. These results suggest that highly attenuated BA15ΔcydCΔcydD and BA15ΔcydCΔpurD mutants can be used effectively as potential live vaccine candidates against bovine brucellosis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription.

    Science.gov (United States)

    Xu, Xingli; Fan, Shengtao; Zhou, Jienan; Zhang, Ying; Che, Yanchun; Cai, Hongzhi; Wang, Lichun; Guo, Lei; Liu, Longding; Li, Qihan

    2016-09-13

    UL7, a tegument protein of Herpes Simplex Virus type I (HSV-1), is highly conserved in viral infection and proliferation and has an unknown mechanism of action. A HSV-1 UL7 mutant (UL7-MU) was constructed using the CRISPR-cas9 system. The replication rate and plaque morphology were used to analyze the biological characteristics of the wild-type (WT), UL7-MU and MU-complemented P1 viruses. The virulence of the viruses was evaluated in mice. Real-time RT-qPCR and ChIP assays were used to determine the expression levels of relevant genes. The replication capacity of a recombinant virus (UL7-MU strain) was 10-fold lower than that of the WT strain. The neurovirulence and pathologic effect of the UL7-MU strain were attenuated in infected mice compared with the WT strain. In the latency model, the expression of latency-associated transcript (LAT) in the central nervous system (CNS) and trigeminal nerve was lower in UL7-MU-infected mice than in WT strain-infected mice. The transcription level of the immediate-early gene α-4 in UL7-MU-infected cells was reduced by approximately 2-fold compared with the clear transcriptional peak identified in WT strain-infected Vero cells within 7 h post-infection (p.i.). By modulating the transcription of the α-4 gene, UL7 may be involved in transcriptional regulation through its interaction with the transcript complex structure of the viral genome during HSV-1 infection.

  10. A Live Attenuated Equine Infectious Anemia Virus Proviral Vaccine with a Modified S2 Gene Provides Protection from Detectable Infection by Intravenous Virulent Virus Challenge of Experimentally Inoculated Horses

    Science.gov (United States)

    Li, Feng; Craigo, Jodi K.; Howe, Laryssa; Steckbeck, Jonathan D.; Cook, Sheila; Issel, Charles; Montelaro, Ronald C.

    2003-01-01

    Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAVUKΔS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAVUKΔS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAVPV. This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAVPV by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID50], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID50. In contrast, naïve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure. PMID:12805423

  11. A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses.

    Science.gov (United States)

    Li, Feng; Craigo, Jodi K; Howe, Laryssa; Steckbeck, Jonathan D; Cook, Sheila; Issel, Charles; Montelaro, Ronald C

    2003-07-01

    Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, naïve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.

  12. 2-Furaldehyde diethyl acetal from tender coconut water (Cocos nucifera) attenuates biofilm formation and quorum sensing-mediated virulence of Chromobacterium violaceum and Pseudomonas aeruginosa.

    Science.gov (United States)

    Sethupathy, Sivasamy; Nithya, Chari; Pandian, Shunmugiah Karutha

    2015-01-01

    The aim of this study was to evaluate the anti-biofilm and quorum sensing inhibitory (QSI) potential of tender coconut water (TCW) against Chromobacterium violaceum and Pseudomonas aeruginosa. TCW significantly inhibited the QS regulated violacein, virulence factors and biofilm production without affecting their growth. qRT-PCR analysis revealed the down-regulation of autoinducer synthase, transcriptional regulator and virulence genes. Mass-spectrometric analysis of a petroleum ether extract of the TCW hydrolyte revealed that 2-furaldehyde diethyl acetal (2FDA) and palmitic acid (PA) are the major compounds. In vitro bioassays confirmed the ability of 2FDA to inhibit the biofilm formation and virulence factors. In addition, the combination of PA with 2FDA resulted in potent inhibition of biofilm formation and virulence factors. The results obtained strongly suggest that TCW can be exploited as a base for designing a novel antipathogenic drug formulation to treat biofilm mediated infections caused by P. aeruginosa.

  13. [Identification of Panax ginseng, P. notoginseng and P. quinquefolius admixture by multiplex allele-specific polymerase chain reaction].

    Science.gov (United States)

    Jiang, Chao; Luo, Yu-Qing; Yuan, Yuan; Huang, Lu-Qi; Jin, Yan; Zhao, Yu-Yang

    2017-04-01

    To achieve a molecular method to identify Panax ginseng, P. notoginseng,P. quinquefolius and their admixture. The ITS,18S and matK sequences of Panax genus were analyzed to develop species-specific SNP marker. Three pairs of species-specific primers were designed to establish a multiplex allele-specific polymerase chain reaction (MAS-PCR) and the samples from different region were tested. The results showed that when the annealing temperature was 60 ℃ and the cycle number was 35, approximately 250, 500,1 000 bp specific band were obtained from P. ginseng, P. notoginseng and P. quinquefolius obtain, respectively. This method could also be used to authentificate admixture samples and could detect 0.5% percent of P. notoginseng or P. quinquefolius adulterated in P. ginseng, or 0.5% percent of P. ginseng or P. quinquefolius adulterated in P. notoginseng. The detect limit of P. ginseng in P. quinquefolius was 0.5% and P. notoginseng in P. quinquefolius was 1%. This results showed that the present method could be used as a promise method to identify Panax ginseng, P. notoginseng, P. quinquefolius and their admixture. Copyright© by the Chinese Pharmaceutical Association.

  14. Simple and sensitive method for identification of human DNA by allele-specific polymerase chain reaction of FOXP2.

    Science.gov (United States)

    Hiroshige, Kenichi; Soejima, Mikiko; Nishioka, Tomoki; Kamimura, Shigeo; Koda, Yoshiro

    2009-07-01

    The forkhead box P2 (FOXP2) gene is specifically involved in speech and language development in humans. The sequence is well conserved among many vertebrate species but has accumulated amino acid changes in the human lineage. The aim of this study was to develop a simple method to discriminate between human and nonhuman vertebrate DNA in forensic specimens by amplification of a human-specific genomic region. In the present study, we designed an allele-specific polymerase chain reaction (PCR) using primers to amplify smaller than 70-bp regions of FOXP2 to identify DNA as being of human or nonhuman, including ape, origin. PCR amplification was also successfully performed using fluorescence-labeled primers, and this method allows a single PCR reaction with a genomic DNA sample as small as 0.01 ng. This system also identified the presence of human DNA in two blood stains stored for 20 and 38 years. The results suggested the potential usefulness of FOXP2 as an identifier of human DNA in forensic samples.

  15. Allele-specific Gene Silencing of Mutant mRNA Restores Cellular Function in Ullrich Congenital Muscular Dystrophy Fibroblasts

    Directory of Open Access Journals (Sweden)

    Satoru Noguchi

    2014-01-01

    Full Text Available Ullrich congenital muscular dystrophy (UCMD is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.

  16. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    Science.gov (United States)

    Skotte, Niels H; Southwell, Amber L; Østergaard, Michael E; Carroll, Jeffrey B; Warby, Simon C; Doty, Crystal N; Petoukhov, Eugenia; Vaid, Kuljeet; Kordasiewicz, Holly; Watt, Andrew T; Freier, Susan M; Hung, Gene; Seth, Punit P; Bennett, C Frank; Swayze, Eric E; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  17. Allele-specific expression at the androgen receptor alpha gene in a hybrid unisexual fish, the Amazon molly (Poecilia formosa.

    Directory of Open Access Journals (Sweden)

    Fangjun Zhu

    Full Text Available The all-female Amazon molly (Poecilia formosa is the result of a hybridization of the Atlantic molly (P. mexicana and the sailfin molly (P. latipinna approximately 120,000 years ago. As a gynogenetic species, P. formosa needs to copulate with heterospecific males including males from one of its bisexual ancestral species. However, the sperm only triggers embryogenesis of the diploid eggs. The genetic information of the sperm donor typically will not contribute to the next generation of P. formosa. Hence, P. formosa possesses generally one allele from each of its ancestral species at any genetic locus. This raises the question whether both ancestral alleles are equally expressed in P. formosa. Allele-specific expression (ASE has been previously assessed in various organisms, e.g., human and fish, and ASE was found to be important in the context of phenotypic variability and disease. In this study, we utilized Real-Time PCR techniques to estimate ASE of the androgen receptor alpha (arα gene in several distinct tissues of Amazon mollies. We found an allelic bias favoring the maternal ancestor (P. mexicana allele in ovarian tissue. This allelic bias was not observed in the gill or the brain tissue. Sequencing of the promoter regions of both alleles revealed an association between an Indel in a known CpG island and differential expression. Future studies may reveal whether our observed cis-regulatory divergence is caused by an ovary-specific trans-regulatory element, preferentially activating the allele of the maternal ancestor.

  18. Genetic prevention of hepatitis C virus-induced liver fibrosis by allele-specific downregulation of MERTK.

    Science.gov (United States)

    Cavalli, Marco; Pan, Gang; Nord, Helena; Wallén Arzt, Emelie; Wallerman, Ola; Wadelius, Claes

    2017-07-01

    Infection by hepatitis C virus (HCV) can result in the development of liver fibrosis and may eventually progress into cirrhosis and hepatocellular carcinoma. However, the molecular mechanisms for this process are not fully known. Several genome-wide association studies have been carried out to pinpoint causative variants in HCV-infected patient cohorts, but these variants are usually not the functional ones. The aim of this study was to identify the regulatory single nucleotide polymorphism associated with the risk of HCV-induced liver fibrosis and elucidate its molecular mechanism. We utilized a bioinformatics approach to identify a non-coding regulatory variant, located in an intron of the MERTK gene, based on differential transcription factor binding between the alleles. We validated the results using expression reporter assays and electrophoresis mobility shift assays. Chromatin immunoprecipitation sequencing indicated that transcription factor(s) bind stronger to the A allele of rs6726639. Electrophoresis mobility shift assays supported these findings and suggested that the transcription factor is interferon regulatory factor 1 (IRF1). Luciferase report assays showed lower enhancer activity from the A allele and that IRF1 may act as a repressor. Treatment of hepatitis C with interferon-α results in increased IRF1 levels and our data suggest that this leads to an allele-specific downregulation of MERTK mediated by an allelic effect on the regulatory element containing the functional rs6726639. This variant also shows the hallmarks for being the driver of the genome-wide association studies for reduced risk of liver fibrosis and non-alcoholic fatty liver disease at MERTK. © 2016 The Authors. Hepatology Research published by John Wiley & Sons Australia, Ltd on behalf of Japan Society of Hepatology.

  19. Imprinted chromosomal domains revealed by allele-specific replication timing of the GABRB3 and GABRA5 genes

    Energy Technology Data Exchange (ETDEWEB)

    LaSalle, J.; Flint, A.; Lalande, M. [Harvard Medical School, Boston, MA (United States)] [and others

    1994-09-01

    The GABRB3 and GABRA5 genes are organized as a cluster in chromosome 15q11-q13. The genes are separated by around 100 kb and arranged in opposite transcriptional orientations. The GABA{sub A} receptor cluster lies near the Angelman and Prader-Willi loci and displays asynchronous DNA replication, suggesting that this region is subject to parental imprinting. In order to further study the association between DNA replication and imprinting, allele-specific replication was assayed by fluorescence in situ hybridization with {lambda}-phage probes from the GABRB3/A5 region and a D15Z1 satellite probe to identify the parental origin of each chromosome. The replication kinetics of each allele was determined by using a flow sorter to fractionate mitogen-stimulated lymphocytes on the basis of cell cycle progression prior to FISH analysis. These kinetic studies reveal a 50-150 kb chromosomal domain extending from the middle of the GABRB3/A5 intergenic region into the GABRA5 5{prime}-UTR which displays maternal replication in early S with paternal replication delayed until the end of S. In contrast, genomic regions on either side of this maternal early replication domain exhibit the opposite pattern with paternal before maternal replication and both alleles replicating in the latter half of S. These results indicate that the GABRB3/A5 region is divided into domains in which replication timing is determined by parental origin. In addition to a loss of asynchronous replication, organization into replication timing domains is also lost in lymphocytes from maternal and paternal uniparental disomy 15 patients suggesting that a chromosome contribution from both parents is required for the establishment of the imprinted replication domains.

  20. An integrative approach to assess X-chromosome inactivation using allele-specific expression with applications to epithelial ovarian cancer.

    Science.gov (United States)

    Larson, Nicholas B; Fogarty, Zachary C; Larson, Melissa C; Kalli, Kimberly R; Lawrenson, Kate; Gayther, Simon; Fridley, Brooke L; Goode, Ellen L; Winham, Stacey J

    2017-12-01

    X-chromosome inactivation (XCI) epigenetically silences transcription of an X chromosome in females; patterns of XCI are thought to be aberrant in women's cancers, but are understudied due to statistical challenges. We develop a two-stage statistical framework to assess skewed XCI and evaluate gene-level patterns of XCI for an individual sample by integration of RNA sequence, copy number alteration, and genotype data. Our method relies on allele-specific expression (ASE) to directly measure XCI and does not rely on male samples or paired normal tissue for comparison. We model ASE using a two-component mixture of beta distributions, allowing estimation for a given sample of the degree of skewness (based on a composite likelihood ratio test) and the posterior probability that a given gene escapes XCI (using a Bayesian beta-binomial mixture model). To illustrate the utility of our approach, we applied these methods to data from tumors of ovarian cancer patients. Among 99 patients, 45 tumors were informative for analysis and showed evidence of XCI skewed toward a particular parental chromosome. For 397 X-linked genes, we observed tumor XCI patterns largely consistent with previously identified consensus states based on multiple normal tissue types. However, 37 genes differed in XCI state between ovarian tumors and the consensus state; 17 genes aberrantly escaped XCI in ovarian tumors (including many oncogenes), whereas 20 genes were unexpectedly inactivated in ovarian tumors (including many tumor suppressor genes). These results provide evidence of the importance of XCI in ovarian cancer and demonstrate the utility of our two-stage analysis. © 2017 WILEY PERIODICALS, INC.

  1. Novel method for analysis of allele specific expression in triploid Oryzias latipes reveals consistent pattern of allele exclusion.

    Directory of Open Access Journals (Sweden)

    Tzintzuni I Garcia

    Full Text Available Assessing allele-specific gene expression (ASE on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types and diseased tissues (trisomies, non-disjunction events, cancerous tissues. In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82% shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18% displayed a wide range of ASE levels. Interestingly the majority of genes (78% displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression.

  2. Identification of transcriptome SNPs for assessing allele-specific gene expression in a super-hybrid rice Xieyou9308.

    Directory of Open Access Journals (Sweden)

    Rongrong Zhai

    Full Text Available Hybridization, a common process in nature, can give rise to a vast reservoir of allelic variants. Combination of these allelic variants may result in novel patterns of gene action and is thought to contribute to heterosis. In this study, we analyzed genome-wide allele-specific gene expression (ASGE in the super-hybrid rice variety Xieyou9308 using RNA sequencing technology (RNA-Seq. We identified 9325 reliable single nucleotide polymorphisms (SNPs distributed throughout the genome. Nearly 68% of the identified polymorphisms were CT and GA SNPs between R9308 and Xieqingzao B, suggesting the existence of DNA methylation, a heritable epigenetic mark, in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17% showed significant allelic biases during tillering and/or heading stages, implying that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed R9308 allelic expression biases at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed R9308 allelic expression biases. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play important roles in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study may provide valuable insights into molecular mechanisms of heterosis.

  3. Bamgineer: Introduction of simulated allele-specific copy number variants into exome and targeted sequence data sets.

    Science.gov (United States)

    Samadian, Soroush; Bruce, Jeff P; Pugh, Trevor J

    2018-03-01

    Somatic copy number variations (CNVs) play a crucial role in development of many human cancers. The broad availability of next-generation sequencing data has enabled the development of algorithms to computationally infer CNV profiles from a variety of data types including exome and targeted sequence data; currently the most prevalent types of cancer genomics data. However, systemic evaluation and comparison of these tools remains challenging due to a lack of ground truth reference sets. To address this need, we have developed Bamgineer, a tool written in Python to introduce user-defined haplotype-phased allele-specific copy number events into an existing Binary Alignment Mapping (BAM) file, with a focus on targeted and exome sequencing experiments. As input, this tool requires a read alignment file (BAM format), lists of non-overlapping genome coordinates for introduction of gains and losses (bed file), and an optional file defining known haplotypes (vcf format). To improve runtime performance, Bamgineer introduces the desired CNVs in parallel using queuing and parallel processing on a local machine or on a high-performance computing cluster. As proof-of-principle, we applied Bamgineer to a single high-coverage (mean: 220X) exome sequence file from a blood sample to simulate copy number profiles of 3 exemplar tumors from each of 10 tumor types at 5 tumor cellularity levels (20-100%, 150 BAM files in total). To demonstrate feasibility beyond exome data, we introduced read alignments to a targeted 5-gene cell-free DNA sequencing library to simulate EGFR amplifications at frequencies consistent with circulating tumor DNA (10, 1, 0.1 and 0.01%) while retaining the multimodal insert size distribution of the original data. We expect Bamgineer to be of use for development and systematic benchmarking of CNV calling algorithms by users using locally-generated data for a variety of applications. The source code is freely available at http://github.com/pughlab/bamgineer.

  4. Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data.

    Science.gov (United States)

    Degner, Jacob F; Marioni, John C; Pai, Athma A; Pickrell, Joseph K; Nkadori, Everlyne; Gilad, Yoav; Pritchard, Jonathan K

    2009-12-15

    Next-generation sequencing has become an important tool for genome-wide quantification of DNA and RNA. However, a major technical hurdle lies in the need to map short sequence reads back to their correct locations in a reference genome. Here, we investigate the impact of SNP variation on the reliability of read-mapping in the context of detecting allele-specific expression (ASE). We generated 16 million 35 bp reads from mRNA of each of two HapMap Yoruba individuals. When we mapped these reads to the human genome we found that, at heterozygous SNPs, there was a significant bias toward higher mapping rates of the allele in the reference sequence, compared with the alternative allele. Masking known SNP positions in the genome sequence eliminated the reference bias but, surprisingly, did not lead to more reliable results overall. We find that even after masking, approximately 5-10% of SNPs still have an inherent bias toward more effective mapping of one allele. Filtering out inherently biased SNPs removes 40% of the top signals of ASE. The remaining SNPs showing ASE are enriched in genes previously known to harbor cis-regulatory variation or known to show uniparental imprinting. Our results have implications for a variety of applications involving detection of alternate alleles from short-read sequence data. Scripts, written in Perl and R, for simulating short reads, masking SNP variation in a reference genome and analyzing the simulation output are available upon request from JFD. Raw short read data were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE18156. jdegner@uchicago.edu; marioni@uchicago.edu; gilad@uchicago.edu; pritch@uchicago.edu Supplementary data are available at Bioinformatics online.

  5. Infrequent detection of germline allele-specific expression of TGFBR1 in lymphoblasts and tissues of colon cancer patients.

    LENUS (Irish Health Repository)

    Guda, Kishore

    2009-06-15

    Recently, germline allele-specific expression (ASE) of the gene encoding for transforming growth factor-beta type I receptor (TGFBR1) has been proposed to be a major risk factor for cancer predisposition in the colon. Germline ASE results in a lowered expression of one of the TGFBR1 alleles (>1.5-fold), and was shown to occur in approximately 20% of informative familial and sporadic colorectal cancer (CRC) cases. In the present study, using the highly quantitative pyrosequencing technique, we estimated the frequency of ASE in TGFBR1 in a cohort of affected individuals from familial clusters of advanced colon neoplasias (cancers and adenomas with high-grade dysplasia), and also from a cohort of individuals with sporadic CRCs. Cases were considered positive for the presence of ASE if demonstrating an allelic expression ratio <0.67 or >1.5. Using RNA derived from lymphoblastoid cell lines, we find that of 46 informative Caucasian advanced colon neoplasia cases with a family history, only 2 individuals display a modest ASE, with allelic ratios of 1.65 and 1.73, respectively. Given that ASE of TGFBR1, if present, would likely be more pronounced in the colon compared with other tissues, we additionally determined the allele ratios of TGFBR1 in the RNA derived from normal-appearing colonic mucosa of sporadic CRC cases. We, however, found no evidence of ASE in any of 44 informative sporadic cases analyzed. Taken together, we find that germline ASE of TGFBR1, as assayed in lymphoblastoid and colon epithelial cells of colon cancer patients, is a relatively rare event.

  6. Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    DEFF Research Database (Denmark)

    Milani, Lili; Lundmark, Anders; Nordlund, Jessica

    2008-01-01

    To identify genes that are regulated by cis-acting functional elements in acute lymphoblastic leukemia (ALL) we determined the allele-specific expression (ASE) levels of 2, 529 genes by genotyping a genome-wide panel of single nucleotide polymorphisms in RNA and DNA from bone marrow and blood...

  7. Development of Allele-Specific Primer PCR for a Swine TLR2 SNP and Comparison of the Frequency among Several Pig Breeds of Japan and the Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Muneta, Y.; Minagawa, Y.; Kusumoto, M.; Shinkai, H.; Uenishi, H.; Šplíchal, Igor

    2012-01-01

    Roč. 74, č. 5 (2012), s. 553-559 ISSN 0916-7250 R&D Projects: GA ČR GA524/09/0365 Institutional support: RVO:61388971 Keywords : allele-specific PCR * Mycoplasma hyopneumoniae * single nucleotide polymorphism Subject RIV: EC - Immunology Impact factor: 0.876, year: 2012

  8. Mutations within ICP4 acquired during in vitro attenuation do not alter virulence of recombinant Marek’s disease viruses in vivo

    Science.gov (United States)

    Marek's disease (MD) is a T-cell lymphoma of chickens caused by the oncogenic Marek's disease virus (MDV). MD is primarily controlled by live-attenuated vaccines generated by repeated in vitro serial passage. Previous efforts to characterize attenuated MDVs identified numerous mutations, particularl...

  9. Impact of pre-existing MSP142-allele specific immunity on potency of an erythrocytic Plasmodium falciparum vaccine

    Directory of Open Access Journals (Sweden)

    Bergmann-Leitner Elke S

    2012-09-01

    Full Text Available Abstract Background MSP1 is the major surface protein on merozoites and a prime candidate for a blood stage malaria vaccine. Preclinical and seroepidemiological studies have implicated antibodies to MSP1 in protection against blood stage parasitaemia and/or reduced parasite densities, respectively. Malaria endemic areas have multiple strains of Plasmodium falciparum circulating at any given time, giving rise to complex immune responses, an issue which is generally not addressed in clinical trials conducted in non-endemic areas. A lack of understanding of the effect of pre-existing immunity to heterologous parasite strains may significantly contribute to vaccine failure in the field. The purpose of this study was to model the effect of pre-existing immunity to MSP142 on the immunogenicity of blood-stage malaria vaccines based on alternative MSP1 alleles. Methods Inbred and outbred mice were immunized with various recombinant P. falciparum MSP142 proteins that represent the two major alleles of MSP142, MAD20 (3D7 and Wellcome (K1, FVO. Humoral immune responses were analysed by ELISA and LuminexTM, and functional activity of induced MSP142-specific antibodies was assessed by growth inhibition assays. T-cell responses were characterized using ex vivo ELISpot assays. Results Analysis of the immune responses induced by various immunization regimens demonstrated a strong allele-specific response at the T cell level in both inbred and outbred mice. The success of heterologous regimens depended on the degree of homology of the N-terminal p33 portion of the MSP142, likely due to the fact that most T cell epitopes reside in this part of the molecule. Analysis of humoral immune responses revealed a marked cross-reactivity between the alleles. Functional analyses showed that some of the heterologous regimens induced antibodies with improved growth inhibitory activities. Conclusion The development of a more broadly efficacious MSP1 based vaccine may be

  10. WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations

    Directory of Open Access Journals (Sweden)

    Assawamakin Anunchai

    2007-08-01

    Full Text Available Abstract Background Allele-specific (AS Polymerase Chain Reaction is a convenient and inexpensive method for genotyping Single Nucleotide Polymorphisms (SNPs and mutations. It is applied in many recent studies including population genetics, molecular genetics and pharmacogenomics. Using known AS primer design tools to create primers leads to cumbersome process to inexperience users since information about SNP/mutation must be acquired from public databases prior to the design. Furthermore, most of these tools do not offer the mismatch enhancement to designed primers. The available web applications do not provide user-friendly graphical input interface and intuitive visualization of their primer results. Results This work presents a web-based AS primer design application called WASP. This tool can efficiently design AS primers for human SNPs as well as mutations. To assist scientists with collecting necessary information about target polymorphisms, this tool provides a local SNP database containing over 10 million SNPs of various populations from public domain databases, namely NCBI dbSNP, HapMap and JSNP respectively. This database is tightly integrated with the tool so that users can perform the design for existing SNPs without going off the site. To guarantee specificity of AS primers, the proposed system incorporates a primer specificity enhancement technique widely used in experiment protocol. In particular, WASP makes use of different destabilizing effects by introducing one deliberate 'mismatch' at the penultimate (second to last of the 3'-end base of AS primers to improve the resulting AS primers. Furthermore, WASP offers graphical user interface through scalable vector graphic (SVG draw that allow users to select SNPs and graphically visualize designed primers and their conditions. Conclusion WASP offers a tool for designing AS primers for both SNPs and mutations. By integrating the database for known SNPs (using gene ID or rs number

  11. Identifying breast cancer risk loci by global differential allele-specific expression (DASE analysis in mammary epithelial transcriptome

    Directory of Open Access Journals (Sweden)

    Gao Chuan

    2012-10-01

    Full Text Available Abstract Background The significant mortality associated with breast cancer (BCa suggests a need to improve current research strategies to identify new genes that predispose women to breast cancer. Differential allele-specific expression (DASE has been shown to contribute to phenotypic variables in humans and recently to the pathogenesis of cancer. We previously reported that nonsense-mediated mRNA decay (NMD could lead to DASE of BRCA1/2, which is associated with elevated susceptibility to breast cancer. In addition to truncation mutations, multiple genetic and epigenetic factors can contribute to DASE, and we propose that DASE is a functional index for cis-acting regulatory variants and pathogenic mutations, and that global analysis of DASE in breast cancer precursor tissues can be used to identify novel causative alleles for breast cancer susceptibility. Results To test our hypothesis, we employed the Illumina® Omni1-Quad BeadChip in paired genomic DNA (gDNA and double-stranded cDNA (ds-cDNA samples prepared from eight BCa patient-derived normal mammary epithelial lines (HMEC. We filtered original array data according to heterozygous genotype calls and calculated DASE values using the Log ratio of cDNA allele intensity, which was normalized to the corresponding gDNA. We developed two statistical methods, SNP- and gene-based approaches, which allowed us to identify a list of 60 candidate DASE loci (DASE ≥ 2.00, P ≤ 0.01, FDR ≤ 0.05 by both methods. Ingenuity Pathway Analysis of DASE loci revealed one major breast cancer-relevant interaction network, which includes two known cancer causative genes, ZNF331 (DASE = 2.31, P = 0.0018, FDR = 0.040 and USP6 (DASE = 4.80, P = 0.0013, FDR = 0.013, and a breast cancer causative gene, DMBT1 (DASE=2.03, P = 0.0017, FDR = 0.014. Sequence analysis of a 5′ RACE product of DMBT1 demonstrated that rs2981745, a putative breast cancer risk locus, appears to be one of the causal variants leading to DASE

  12. Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Kim, Kiju; Park, Bo-Kyoung; Hahn, Tae-Wook

    2015-11-01

    Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

  13. Mechanisms Controlling Virulence Thresholds of Mixed Viral Populations ▿

    Science.gov (United States)

    Lancaster, Karen Z.; Pfeiffer, Julie K.

    2011-01-01

    The propensity of RNA viruses to revert attenuating mutations contributes to disease and complicates vaccine development. Despite the presence of virulent revertant viruses in some live-attenuated vaccines, disease from vaccination is rare. This suggests that in mixed viral populations, attenuated viruses may limit the pathogenesis of virulent viruses, thus establishing a virulence threshold. Here we examined virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that a 1,000-fold excess of the attenuated Sabin strain of poliovirus was protective against disease induced by the virulent Mahoney strain. Protection was induced locally, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo, suggesting that one mechanism controlling virulence thresholds may be competition for a viral receptor. Additionally, the type I interferon response reduces poliovirus pathogenesis; therefore, we examined virulence thresholds in mice lacking the alpha/beta interferon receptor. We found that the attenuated virus was virulent in immunodeficient mice due to the enhanced replication and reversion of attenuating mutations. Therefore, while the type I interferon response limits the virulence of the attenuated strain by reducing replication, protection from disease conferred by the attenuated strain in immunocompetent mice can occur independently of replication. Our results identified mechanisms controlling the virulence of mixed viral populations and indicate that live-attenuated vaccines containing virulent virus may be safe, as long as virulent viruses are present at levels below a critical threshold. PMID:21795346

  14. Rift valley fever virus lacking the NSs and NSm genes is highly attenuated, confers protective immunity from virulent virus challenge, and allows for differential identification of infected and vaccinated animals.

    Science.gov (United States)

    Bird, Brian H; Albariño, César G; Hartman, Amy L; Erickson, Bobbie Rae; Ksiazek, Thomas G; Nichol, Stuart T

    2008-03-01

    Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping "abortion storms" and high mortality among young animals. Human infection results in self-limiting febrile disease that in approximately 1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 x 10(4) PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, approximately 1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas.

  15. A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

    Science.gov (United States)

    Moleleki, Lucy Novungayo; Pretorius, Rudolph Gustav; Tanui, Collins Kipngetich; Mosina, Gabolwelwe; Theron, Jacques

    2017-01-01

    Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain. © 2016 BSPP and John Wiley & Sons Ltd.

  16. Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence.

    OpenAIRE

    Hube, B; Sanglard, D; Odds, F C; Hess, D; Monod, M; Schäfer, W; Brown, A J; Gow, N A

    1997-01-01

    Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null muta...

  17. Genome-wide allele-specific expression analysis using Massively Parallel Signature Sequencing (MPSS) reveals cis- and trans-effects on gene expression in maize hybrid meristem tissue.

    Science.gov (United States)

    Guo, Mei; Yang, Sean; Rupe, Mary; Hu, Bin; Bickel, David R; Arthur, Lane; Smith, Oscar

    2008-03-01

    Allelic differences in expression are important genetic factors contributing to quantitative trait variation in various organisms. However, the extent of genome-wide allele-specific expression by different modes of gene regulation has not been well characterized in plants. In this study we developed a new methodology for allele-specific expression analysis by applying Massively Parallel Signature Sequencing (MPSS), an open ended and sequencing based mRNA profiling technology. This methodology enabled a genome-wide evaluation of cis- and trans-effects on allelic expression in six meristem stages of the maize hybrid. Summarization of data from nearly 400 pairs of MPSS allelic signature tags showed that 60% of the genes in the hybrid meristems exhibited differential allelic expression. Because both alleles are subjected to the same trans-acting factors in the hybrid, the data suggest the abundance of cis-regulatory differences in the genome. Comparing the same allele expressed in the hybrid versus its inbred parents showed that 40% of the genes were differentially expressed, suggesting different trans-acting effects present in different genotypes. Such trans-acting effects may result in gene expression in the hybrid different from allelic additive expression. With this approach we quantified gene expression in the hybrid relative to its inbred parents at the allele-specific level. As compared to measuring total transcript levels, this study provides a new level of understanding of different modes of gene regulation in the hybrid and the molecular basis of heterosis.

  18. A genome-wide screen in human embryonic stem cells reveals novel sites of allele-specific histone modification associated with known disease loci

    LENUS (Irish Health Repository)

    Prendergast, James G D

    2012-05-19

    AbstractBackgroundChromatin structure at a given site can differ between chromosome copies in a cell, and such imbalances in chromatin structure have been shown to be important in understanding the molecular mechanisms controlling several disease loci. Human genetic variation, DNA methylation, and disease have been intensely studied, uncovering many sites of allele-specific DNA methylation (ASM). However, little is known about the genome-wide occurrence of sites of allele-specific histone modification (ASHM) and their relationship to human disease. The aim of this study was to investigate the extent and characteristics of sites of ASHM in human embryonic stem cells (hESCs).ResultsUsing a statistically rigorous protocol, we investigated the genomic distribution of ASHM in hESCs, and their relationship to sites of allele-specific expression (ASE) and DNA methylation. We found that, although they were rare, sites of ASHM were substantially enriched at loci displaying ASE. Many were also found at known imprinted regions, hence sites of ASHM are likely to be better markers of imprinted regions than sites of ASM. We also found that sites of ASHM and ASE in hESCs colocalize at risk loci for developmental syndromes mediated by deletions, providing insights into the etiology of these disorders.ConclusionThese results demonstrate the potential importance of ASHM patterns in the interpretation of disease loci, and the protocol described provides a basis for similar studies of ASHM in other cell types to further our understanding of human disease susceptibility.

  19. Detection of „Hotspot Mutations in Catalytic Subunit of Phosphatidylinositol 3-Kinase (Pik3ca by Allele-Specific Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    A. Mendelova

    2014-09-01

    Full Text Available The phosphatidylinositol 3-kinases (PI3Ks are a family of proteins involved in the regulation of cell survival, growth, metabolism, and glucose homeostasis. Increased PI3K activity is associated with many cancers. PIK3CA gene (encoding p110 , the catalytic subunit of PI3K is commonly mutated in breast cancer. In our study we focused on the detection of “hotspot” mutations in exons 9 and 20 of the PIK3CA gene in paraffin-embedded tissue of patients with breast cancer. We optimized conditions of allele specific polymerase chain reaction (PCR and we used direct sequencing to verify our results. Overall, three “hotspot” mutations in PIK3CA gene in paraffin-embadded tissue from breast cancer were detected by allele-specific PCR. All results were verified by direct sequencing of PCR products and we observed 100% agreement between those two methods. We confirmed that allele-specific PCR assay is low cost method usefull for accurate detection of PIK3CA mutations.

  20. Systematic evaluation of the impact of ChIP-seq read designs on genome coverage, peak identification, and allele-specific binding detection.

    Science.gov (United States)

    Zhang, Qi; Zeng, Xin; Younkin, Sam; Kawli, Trupti; Snyder, Michael P; Keleş, Sündüz

    2016-02-24

    Chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments revolutionized genome-wide profiling of transcription factors and histone modifications. Although maturing sequencing technologies allow these experiments to be carried out with short (36-50 bps), long (75-100 bps), single-end, or paired-end reads, the impact of these read parameters on the downstream data analysis are not well understood. In this paper, we evaluate the effects of different read parameters on genome sequence alignment, coverage of different classes of genomic features, peak identification, and allele-specific binding detection. We generated 101 bps paired-end ChIP-seq data for many transcription factors from human GM12878 and MCF7 cell lines. Systematic evaluations using in silico variations of these data as well as fully simulated data, revealed complex interplay between the sequencing parameters and analysis tools, and indicated clear advantages of paired-end designs in several aspects such as alignment accuracy, peak resolution, and most notably, allele-specific binding detection. Our work elucidates the effect of design on the downstream analysis and provides insights to investigators in deciding sequencing parameters in ChIP-seq experiments. We present the first systematic evaluation of the impact of ChIP-seq designs on allele-specific binding detection and highlights the power of pair-end designs in such studies.

  1. Lactobacillus bulgaricus, Lactobacillus rhamnosus and Lactobacillus paracasei Attenuate Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Typhimurium Colonization and Virulence Gene Expression In Vitro.

    Science.gov (United States)

    Muyyarikkandy, Muhammed Shafeekh; Amalaradjou, Mary Anne

    2017-11-09

    Salmonella Enteritidis (SE), Salmonella Typhimurium (ST), and Salmonella Heidelberg (SH) have been responsible for numerous outbreaks associated with the consumption of poultry meat and eggs. Salmonella colonization in chicken is characterized by initial attachment to the cecal epithelial cells (CEC) followed by dissemination to the liver, spleen, and oviduct. Since cecal colonization is critical to Salmonella transmission along the food chain continuum, reducing this intestinal association could potentially decrease poultry meat and egg contamination. Hence, this study investigated the efficacy of Lactobacillus delbreuckii sub species bulgaricus (NRRL B548; LD), Lactobacillus paracasei (DUP-13076; LP), and Lactobacillus rhamnosus (NRRL B442; LR) in reducing SE, ST, and SH colonization in CEC and survival in chicken macrophages. Additionally, their effect on expression of Salmonella virulence genes essential for cecal colonization and survival in macrophages was evaluated. All three probiotics significantly reduced Salmonella adhesion and invasion in CEC and survival in chicken macrophages ( p < 0.05). Further, the probiotic treatment led to a significant reduction in Salmonella virulence gene expression ( p < 0.05). Results of the study indicate that LD, LP, and LR could potentially be used to control SE, ST, and SH colonization in chicken. However, these observations warrant further in vivo validation.

  2. Lactobacillus bulgaricus, Lactobacillus rhamnosus and Lactobacillus paracasei Attenuate Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Typhimurium Colonization and Virulence Gene Expression In Vitro

    Directory of Open Access Journals (Sweden)

    Muhammed Shafeekh Muyyarikkandy

    2017-11-01

    Full Text Available Salmonella Enteritidis (SE, Salmonella Typhimurium (ST, and Salmonella Heidelberg (SH have been responsible for numerous outbreaks associated with the consumption of poultry meat and eggs. Salmonella colonization in chicken is characterized by initial attachment to the cecal epithelial cells (CEC followed by dissemination to the liver, spleen, and oviduct. Since cecal colonization is critical to Salmonella transmission along the food chain continuum, reducing this intestinal association could potentially decrease poultry meat and egg contamination. Hence, this study investigated the efficacy of Lactobacillus delbreuckii sub species bulgaricus (NRRL B548; LD, Lactobacillus paracasei (DUP-13076; LP, and Lactobacillus rhamnosus (NRRL B442; LR in reducing SE, ST, and SH colonization in CEC and survival in chicken macrophages. Additionally, their effect on expression of Salmonella virulence genes essential for cecal colonization and survival in macrophages was evaluated. All three probiotics significantly reduced Salmonella adhesion and invasion in CEC and survival in chicken macrophages (p < 0.05. Further, the probiotic treatment led to a significant reduction in Salmonella virulence gene expression (p < 0.05. Results of the study indicate that LD, LP, and LR could potentially be used to control SE, ST, and SH colonization in chicken. However, these observations warrant further in vivo validation.

  3. The supposedly attenuated Hy-HK variant of highly virulent Hypr strain of Tick-borne encephalitis virus is obviously a strain of Langat virus

    Czech Academy of Sciences Publication Activity Database

    Růžek, Daniel; Štěrba, Ján; Kopecký, Jan; Grubhoffer, Libor

    2006-01-01

    Roč. 50, č. 4 (2006), s. 277-278 ISSN 0001-723X R&D Projects: GA ČR(CZ) GA524/06/1479 Grant - others:Grant Agency of the University of South Bohemia(CZ) 35/2005/P-BF Institutional research plan: CEZ:AV0Z60220518 Keywords : TBE virus * Langat virus * Hy-HK attenuated variant Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 0.788, year: 2006

  4. The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Máté Manczinger

    2015-01-01

    Full Text Available To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis.

  5. Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain.

    Science.gov (United States)

    Ma, Jian; Wang, Shan-Shan; Lin, Yue-Zhi; Liu, Hai-Fang; Liu, Qiang; Wei, Hua-Mian; Wang, Xue-Feng; Wang, Yu-Hong; Du, Cheng; Kong, Xian-Gang; Zhou, Jian-Hua; Wang, Xiaojun

    2014-08-09

    The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAVFDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAVUK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon β (IFNβ) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAVFDDV13 and was correlated with enhanced TLR3, sELR1 and IFNβ expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNβ and lowered the level of infection resistance induced by EIAVFDDV13. These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNβ, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

  6. New prediction model for probe specificity in an allele-specific extension reaction for haplotype-specific extraction (HSE of Y chromosome mixtures.

    Directory of Open Access Journals (Sweden)

    Jessica Rothe

    Full Text Available Allele-specific extension reactions (ASERs use 3' terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3' terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3' terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.

  7. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    Science.gov (United States)

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  8. siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Véronique Bolduc

    2014-01-01

    Full Text Available Congenital muscular dystrophy type Ullrich (UCMD is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies.

  9. Allele-specific wild-type TP53 expression in the unaffected carrier parent of children with Li-Fraumeni syndrome.

    Science.gov (United States)

    Buzby, Jeffrey S; Williams, Shirley A; Schaffer, Lana; Head, Steven R; Nugent, Diane J

    2017-02-01

    Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is passed from parent to child. Tumor protein p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Paradoxically, some mutant TP53 carriers remain unaffected, while their children develop cancer within the first few years of life. To address this paradox, response to UV stress was compared in dermal fibroblasts (dFb) from an affected LFS patient vs. their unaffected carrier parent. UV induction of CDKN1A/p21, a regulatory target of p53, in LFS patient dFb was significantly reduced compared to the unaffected parent. UV exposure also induced significantly greater p53[Ser15]-phosphorylation in LFS patient dFb, a reported property of some mutant p53 variants. Taken together, these results suggested that unaffected parental dFb may express an increased proportion of wild-type vs. mutant p53. Indeed, a significantly increased ratio of wild-type to mutant TP53 allele-specific expression in the unaffected parent dFb was confirmed by RT-PCR-RFLP and RNA-seq analysis. Hence, allele-specific expression of wild-type TP53 may allow an unaffected parent to mount a response to genotoxic stress more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Assembly of a phased diploid Candida albicans genome facilitates allele-specific measurements and provides a simple model for repeat and indel structure.

    Science.gov (United States)

    Muzzey, Dale; Schwartz, Katja; Weissman, Jonathan S; Sherlock, Gavin

    2013-01-01

    Candida albicans is a ubiquitous opportunistic fungal pathogen that afflicts immunocompromised human hosts. With rare and transient exceptions the yeast is diploid, yet despite its clinical relevance the respective sequences of its two homologous chromosomes have not been completely resolved. We construct a phased diploid genome assembly by deep sequencing a standard laboratory wild-type strain and a panel of strains homozygous for particular chromosomes. The assembly has 700-fold coverage on average,allowing extensive revision and expansion of the number of known SNPs and indels. This phased genome significantly enhances the sensitivity and specificity of allele-specific expression measurements by enabling pooling and cross-validation of signal across multiple polymorphic sites. Additionally, the diploid assembly reveals pervasive and unexpected patterns in allelic differences between homologous chromosomes. Firstly, we see striking clustering of indels, concentrated primarily in the repeat sequences in promoters. Secondly, both indels and their repeat-sequence substrate are enriched near replication origins. Finally, we reveal an intimate link between repeat sequences and indels, which argues that repeat length is under selective pressure for most eukaryotes. This connection is described by a concise one-parameter model that explains repeat-sequence abundance in C. albicans as a function of the indel rate,and provides a general framework to interpret repeat abundance in species ranging from bacteria to humans. The phased genome assembly and insights into repeat plasticity will be valuable for better understanding allele-specific phenomena and genome evolution.

  11. MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma.

    Science.gov (United States)

    Martikainen, Miika; Niittykoski, Minna; von und zu Fraunberg, Mikael; Immonen, Arto; Koponen, Susanna; van Geenen, Maartje; Vähä-Koskela, Markus; Ylösmäki, Erkko; Jääskeläinen, Juha E; Saksela, Kalle; Hinkkanen, Ari

    2015-10-01

    Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem that severely restricts viral replication and oncolysis. We show here that the Semliki Forest virus (SFV) strain SFV4, which causes lethal encephalitis in mice, is able to infect and replicate independent of the IFN-I defense in mouse glioblastoma cells and cell lines originating from primary human glioblastoma patient samples. The ability to tolerate IFN-I was retained in SFV4-miRT124 cells, a derivative cell line of strain SFV4 with a restricted capacity to replicate in neurons due to insertion of target sites for neuronal microRNA 124. The IFN-I tolerance was associated with the viral nsp3-nsp4 gene region and distinct from the genetic loci responsible for SFV neurovirulence. In contrast to the naturally attenuated strain SFV A7(74) and its derivatives, SFV4-miRT124 displayed increased oncolytic potency in CT-2A murine astrocytoma cells and in the human glioblastoma cell lines pretreated with IFN-I. Following a single intraperitoneal injection of SFV4-miRT124 into C57BL/6 mice bearing CT-2A orthotopic gliomas, the virus homed to the brain and was amplified in the tumor, resulting in significant tumor growth inhibition and improved survival. Although progress has been made in development of replicative oncolytic viruses, information regarding their overall therapeutic potency in a clinical setting is still lacking. This could be at least partially dependent on the IFN-I sensitivity of the viruses used. Here, we show that the conditionally replicating SFV4-miRT124 virus shares the IFN-I tolerance of the pathogenic wild-type SFV, thereby allowing efficient targeting of a glioma that is refractory

  12. Efficacy of HVT-IBD vector vaccine compared to attenuated live vaccine using in-ovo vaccination against a Korean very virulent IBDV in commercial broiler chickens.

    Science.gov (United States)

    Roh, J-H; Kang, M; Wei, B; Yoon, R-H; Seo, H-S; Bahng, J-Y; Kwon, J-T; Cha, S-Y; Jang, H-K

    2016-05-01

    The production performance, efficacy, and safety of two types of vaccines for infectious bursal disease virus (IBDV) were compared with in-ovo vaccination of Cobb 500 broiler chickens for gross and microscopic examination of the bursa of Fabricius, bursa/body weight (b/B) ratio, flow cytometry, and serologic response to Newcastle disease virus (NDV) vaccination. One vaccine was a recombinant HVT-IBD vector vaccine (HVT as for herpesvirus of turkeys) and the other was an intermediate plus live IBDV vaccine. A significant difference was detected at 21 d. Eight of 10 chickens that received the IBDV live vaccine had severe bursal lesions and a relatively low b/B ratio of 0.95, and an inhibited NDV vaccine response. On the other hand, the HVT-IBD vector vaccine resulted in mild bursal lesions and a b/B ratio of 1.89. Therefore, the live vaccine had lower safety than that of the HVT-IBD vector vaccine. To determine the protective efficacy, chickens were intraocularly challenged at 24 d. Eight of 10 chickens in the IBDV live vaccination group showed gross and histological lesions characterized by hemorrhage, cyst formation, lymphocytic depletion, and a decreased b/B ratio. In contrast, the HVT-IBD vector vaccinated chickens showed mild gross and histological lesions in three of 10 chickens with a b/B ratio of 1.36, which was similar to that of the unchallenged controls. Vaccinated chickens showed a significant increase in IBDV antibody titers, regardless of the type of vaccine used. In addition, significantly better broiler flock performance was observed with the HVT-IBD vector vaccine compared to that of the live vaccine. Our results revealed that the HVT-IBD vector vaccine could be used as an alternative vaccine to increase efficacy, and to have an improved safety profile compared with the IBDV live vaccine using in-ovo vaccination against the Korean very virulent IBDV in commercial broiler chickens. © 2016 Poultry Science Association Inc.

  13. An extensive polymerase chain reaction-allele-specific polymorphism strategy for clinical ABO blood group genotyping that avoids potential errors caused by null, subgroup, and hybrid alleles.

    Science.gov (United States)

    Hosseini-Maaf, Bahram; Hellberg, Asa; Chester, M Alan; Olsson, Martin L

    2007-11-01

    ABO genotyping is complicated by the remarkable diversity at the ABO locus. Recombination or gene conversion between common alleles may lead to hybrids resulting in unexpected ABO phenotypes. Furthermore, numerous mutations associated with weak subgroups and nondeletional null alleles should be considered. All known ABO genotyping methods, however, risk incorrect phenotype predictions if any such alleles are present. An extensive set of allele-specific primers was designed to accomplish hybrid-proof multiplex polymerase chain reaction (PCR) amplification of DNA fragments for detection of ABO alleles. Results were compared with serologic findings and ABO genotypes defined by previously published PCR-restriction fragment length polymorphism/PCR-allele-specific polymorphism (ASP) methods or DNA sequencing. Phenotypically well-characterized samples from blood donors with common blood groups and rare-subgroup families were analyzed. In addition to the commonly encountered alleles (A1, A1(467C>T), A2, B, O1, O1v, and O2), the new method can detect hybrid alleles thanks to long-range amplification across intron 6. Four of 12 PCR-ASP procedures are used to screen for multiple infrequent subgroup and null alleles. This concept allows for a low-resolution typing format in which the presence of, for example, a weak subgroup or cis-AB/B(A) is indicated but not further defined. In an optional high-resolution step, more detailed genotype information is obtained. A new genotyping approach has been developed and evaluated that can correctly identify ABO alleles including nondeletional null alleles, subgroups, and hybrids resulting from recombinational crossing-over events between exons 6 and 7. This approach is clinically applicable and decreases the risk for erroneous ABO phenotype prediction compared to previously published methods.

  14. Simultaneous detection of major drug resistance mutations in the protease and reverse transcriptase genes for HIV-1 subtype C by use of a multiplex allele-specific assay.

    Science.gov (United States)

    Zhang, Guoqing; Cai, Fangping; Zhou, Zhiyong; DeVos, Joshua; Wagar, Nick; Diallo, Karidia; Zulu, Isaac; Wadonda-Kabondo, Nellie; Stringer, Jeffrey S A; Weidle, Paul J; Ndongmo, Clement B; Sikazwe, Izukanji; Sarr, Abdoulaye; Kagoli, Matthew; Nkengasong, John; Gao, Feng; Yang, Chunfu

    2013-11-01

    High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5' end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.

  15. Management of the virulent influenza virus infection by oral formulation of nonhydrolized carnosine and isopeptide of carnosine attenuating proinflammatory cytokine-induced nitric oxide production.

    Science.gov (United States)

    Babizhayev, Mark A; Deyev, Anatoly I

    2012-01-01

    important factors of natural immunity in controlling the initial stages of influenza A virus infection (inhibition of virus replication) and virus-induced regulation of cytokine gene expression. The protective effects of orally applied nonhydrolized formulated species of carnosine include at least direct interaction with nitric oxide, inhibition of cytotoxic NO-induced proinflammatory condition, and attenuation of the effects of cytokines and chemokines that can exert profound effects on inflammatory cells. These data are consistent with the hypothesis that natural products, such as chicken soup and chicken breast extracts rich in carnosine and its derivative anserine (beta-alanyl-1-methyl-L-histidine) could contribute to the pathogenesis and prevention of influenza virus infections and cold but have a limitation due to susceptibility to enzymatic hydrolysis of dipeptides with serum carnosinase and urine excretion after oral ingestion of a commercial chicken extract. The developed and patented by the authors formulations of nonhydrolized in digestive tract and blood natural carnosine peptide and isopeptide (gamma-glutamyl-carnosine) products have a promise in the Influenza A (H1N1) virus infection disease control and prevention.

  16. Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi.

    Directory of Open Access Journals (Sweden)

    Yusuke Ohnishi

    Full Text Available Allele-specific gene silencing by RNA interference (RNAi is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi, the design and assessment of small interfering RNA (siRNA duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs against mutant alleles of the human Prion Protein (PRNP gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs, of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense

  17. Quantification of classical HLA class I mRNA by allele-specific, real-time polymerase chain reaction for most Han individuals.

    Science.gov (United States)

    Pan, N; Lu, S; Wang, W; Miao, F; Sun, H; Wu, S; Nan, D; Qiu, J; Xu, J; Zhang, J

    2018-02-01

    Recent studies have shown that expression levels of different alleles at the same HLA class I locus can vary dramatically, which might have a broad influence on human disease. However, precise quantification of the relative expression level of each HLA allele is challenging, because distinguishing different alleles on the same locus is difficult. Here, we developed a series of allele-specific, real-time polymerase chain reaction assays for quantifying HLA class I allele mRNA in most Han individuals. The alleles of almost all heterozygous genotypes with a frequency higher than 0.5% in our population (78 alleles on HLA-A locus, 124 alleles on HLA-B locus, and 74 alleles on HLA-C locus) were specifically amplified. The specificity of the amplification was strictly validated by setting the corresponding negative control for each allele of each genotype. The amplification efficiency of each reaction was determined, and the slopes of the reactions were compared. This study provides a tool for detecting the comprehensive expression profile of HLA class I alleles and will be useful not only for the investigation of the molecular mechanism underlying HLA allele expression regulation but also for exploration of immunological mechanisms involving HLA expression in the fields of tumour immune evasion, viral infection, auto-immune disorders, and graft vs host disease after haematopoietic stem cell transplantation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. A Generalized Linear Model for Decomposing Cis-regulatory, Parent-of-Origin, and Maternal Effects on Allele-Specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Yasuaki Takada

    2017-07-01

    Full Text Available Joint quantification of genetic and epigenetic effects on gene expression is important for understanding the establishment of complex gene regulation systems in living organisms. In particular, genomic imprinting and maternal effects play important roles in the developmental process of mammals and flowering plants. However, the influence of these effects on gene expression are difficult to quantify because they act simultaneously with cis-regulatory mutations. Here we propose a simple method to decompose cis-regulatory (i.e., allelic genotype, genomic imprinting [i.e., parent-of-origin (PO], and maternal [i.e., maternal genotype (MG] effects on allele-specific gene expression using RNA-seq data obtained from reciprocal crosses. We evaluated the efficiency of method using a simulated dataset and applied the method to whole-body Drosophila and mouse trophoblast stem cell (TSC and liver RNA-seq data. Consistent with previous studies, we found little evidence of PO and MG effects in adult Drosophila samples. In contrast, we identified dozens and hundreds of mouse genes with significant PO and MG effects, respectively. Interestingly, a similar number of genes with significant PO effect were detect in mouse TSCs and livers, whereas more genes with significant MG effect were observed in livers. Further application of this method will clarify how these three effects influence gene expression levels in different tissues and developmental stages, and provide novel insight into the evolution of gene expression regulation.

  19. A Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) for the Detection of Factor V Leiden and Prothrombin G20210A

    Science.gov (United States)

    Bagheri, Morteza; Rad, Isa Abdi

    2011-01-01

    ABSTRACT Introduction: In order to determine the frequencies of factor V Leiden and prothrombin G20210A point mutations in the Iranian population with Azeri Turkish origin. Material and methods: 120 unrelated individuals from general population randomly selected and were examined for factor V Leiden and prothrombin G20210A mutations using a multiplex allele specific polymerase chain reaction (MAS-PCR) assay Outcomes: The frequency of prothrombin G20210A mutation was 2.08%, which means 5 chromosomes out of 240 chromosomes had prothrombin G20210A mutation. The distribution of prothrombin 20210 GG, GA, AA genotypes and prothrombin 20210A allele were 37(92.5%), 3(7.5%), 0(0%) and 3(3.75%) in males and 78(97.5%), 2(2.5%), 0(0%) and 2(1.25%) in females, respectively. Factor V Leiden was not found in our tested group (zero chromosomes out of 240 chromosomes). Analysis of the observed frequencies in the studied groups indicates that there is no statistically significant difference between females and males, regarding prothrombin G20210A mutation (p value>0.05). Conclusions: This is the first study in its own kind in this population and implies that the frequency of Factor V Leiden G1691A (R506Q, FV-Leiden) allele is extremely low but the prothrombin G20210A mutation is more frequent in the tested group. PMID:21977183

  20. A and MdMYB1 allele-specific markers controlling apple (Malus x domestica Borkh.) skin color and suitability for marker-assisted selection.

    Science.gov (United States)

    Zhang, X J; Wang, L X; Chen, X X; Liu, Y L; Meng, R; Wang, Y J; Zhao, Z Y

    2014-10-31

    Pre-selection for fruit skin color at the seedling stage would be highly advantageous, with marker-assisted selection offering a potential method for apple pre-selection. A and MdMYB1 alleles are allele-specific DNA markers that are potentially associated with apple skin color, and co-segregate with the Rf and Rni loci, respectively. Here, we assessed the potential application of these 2 alleles for marker-assisted breeding across 30 diverse cultivars and 2 apple seedling progenies. The red skin color phenotype was usually associated with the MdMYB1-1 allele and A(1) allele, respectively, while the 2 molecular markers provided approximately 91% predictability in the 'Fuji' x 'Cripps Pink' and 'Fuji' x 'Gala' progenies. The results obtained from the 30 cultivars and 2 progenies were consistent for the 2 molecular markers. Hence, the results supported that Rf and Rni could be located in a gene cluster, or even correspond to alleles of the same gene. Our results are consistent with the hypothesis that red/yellow dimorphism is controlled by a monogenic system, with the presence of the red anthocyanin pigmentation being dominant. In addition, our results supported that the practical utilization of the 2 function markers to efficiently and accurately select red-skinned apple cultivars in apple scion breeding programs.

  1. A Generalized Linear Model for DecomposingCis-regulatory, Parent-of-Origin, and Maternal Effects on Allele-Specific Gene Expression.

    Science.gov (United States)

    Takada, Yasuaki; Miyagi, Ryutaro; Takahashi, Aya; Endo, Toshinori; Osada, Naoki

    2017-07-05

    Joint quantification of genetic and epigenetic effects on gene expression is important for understanding the establishment of complex gene regulation systems in living organisms. In particular, genomic imprinting and maternal effects play important roles in the developmental process of mammals and flowering plants. However, the influence of these effects on gene expression are difficult to quantify because they act simultaneously with cis -regulatory mutations. Here we propose a simple method to decompose cis -regulatory ( i.e. , allelic genotype), genomic imprinting [ i.e. , parent-of-origin (PO)], and maternal [ i.e. , maternal genotype (MG)] effects on allele-specific gene expression using RNA-seq data obtained from reciprocal crosses. We evaluated the efficiency of method using a simulated dataset and applied the method to whole-body Drosophila and mouse trophoblast stem cell (TSC) and liver RNA-seq data. Consistent with previous studies, we found little evidence of PO and MG effects in adult Drosophila samples. In contrast, we identified dozens and hundreds of mouse genes with significant PO and MG effects, respectively. Interestingly, a similar number of genes with significant PO effect were detect in mouse TSCs and livers, whereas more genes with significant MG effect were observed in livers. Further application of this method will clarify how these three effects influence gene expression levels in different tissues and developmental stages, and provide novel insight into the evolution of gene expression regulation. Copyright © 2017 Takada et al.

  2. Prenatal diagnosis of a Japanese family at risk for Tay-Sachs disease. Application of a fluorescent competitive allele-specific polymerase chain reaction (PCR) method.

    Science.gov (United States)

    Tamasu, S; Nishio, H; Ayaki, H; Lee, M J; Mizutori, M; Takeshima, Y; Nakamura, H; Matsuo, M; Maruo, T; Sumino, K

    1999-12-01

    Tay-Sachs disease (TSD) is caused by mutation of the HEXA gene, which results in a deficiency of the alpha-subunit of hexosaminidase A. The major mutation in Japanese TSD is a G-to-T transversion at the 3'-splice site of intron 5. We established a fluorescent competitive allele-specific polymerase chain reaction (FCAS-PCR) method for detection of the mutation and applied it to prenatal diagnosis of a Japanese TSD family. FCAS-PCR distinguished the wild and mutant alleles clearly, with broad ranges in the amount of template DNA, the dNTP concentration, the MgCl2 concentration and the number of PCR cycles. After obtaining ethics committee approval and informed consent from the parents in the index family, chorionic villus sampling was performed. FCAS-PCR analysis using chorionic villus DNA disclosed that the fetus was homozygous for the mutation. To confirm the diagnosis, direct sequencing analysis of the genomic PCR fragment was performed, and showed the same results as those of the FCAS-PCR analysis. FCAS-PCR proved to be helpful for carrier screening and prenatal diagnosis in TSD families in the Japanese population. It would also be a useful DNA-diagnostic method for many other inherited disorders.

  3. Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls.

    Science.gov (United States)

    Parenti, Ilaria; Rovina, Davide; Masciadri, Maura; Cereda, Anna; Azzollini, Jacopo; Picinelli, Chiara; Limongelli, Giuseppe; Finelli, Palma; Selicorni, Angelo; Russo, Silvia; Gervasini, Cristina; Larizza, Lidia

    2014-07-01

    Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS.

  4. Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways1[OPEN

    Science.gov (United States)

    Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Doubský, Jan; Schneeberger, Korbinian; Schulze-Lefert, Paul

    2015-01-01

    Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes. PMID:26023163

  5. Genome-wide identification and quantification of cis- and trans-regulated genes responding to Marek’s disease virus infection via analysis of allele-specific expression

    Directory of Open Access Journals (Sweden)

    Sean eMaceachern

    2012-01-01

    Full Text Available Marek’s disease (MD is a commercially important neoplastic disease of chickens caused by Marek’s disease virus (MDV, an oncogenic alphaherpesvirus. Selecting for increased genetic resistance to MD is a control strategy that can augment vaccinal control measures. To identify high-confidence candidate MD resistance genes, we conducted a genome-wide screen for allele-specific expression (ASE amongst F1 progeny of two inbred chicken lines that differ in MD resistance. High throughput sequencing was used to profile transcriptomes from pools of uninfected and infected individuals at 4 days post-infection to identify any genes showing ASE in response to MDV infection. RNA sequencing identified 22,655 single nucleotide polymorphisms (SNPs of which 5,360 in 3,773 genes exhibited significant allelic imbalance. Illumina GoldenGate assays were subsequently used to quantify regulatory variation controlled at the gene (cis and elsewhere in the genome (trans by examining differences in expression between F1 individuals and artificial F1 RNA pools over 6 time periods in 1,536 of the most significant SNPs identified by RNA sequencing. Allelic imbalance as a result of cis-regulatory changes was confirmed in 861 of the 1,233 GoldenGate assays successfully examined. Furthermore we have identified 7 genes that display trans-regulation only in infected animals and approximately 500 SNP that show a complex interaction between cis- and trans-regulatory changes. Our results indicate ASE analyses are a powerful approach to identify regulatory variation responsible for differences in transcript abundance in genes underlying complex traits. And the genes with SNPs exhibiting ASE provide a strong foundation to further investigate the causative polymorphisms and genetic mechanisms for MD resistance. Finally, the methods used here for identifying specific genes and SNPs may have practical implications for applying marker-assisted selection to complex traits that are

  6. Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

    Science.gov (United States)

    Barbaro, Vanessa; Nasti, Annamaria A; Del Vecchio, Claudia; Ferrari, Stefano; Migliorati, Angelo; Raffa, Paolo; Lariccia, Vincenzo; Nespeca, Patrizia; Biasolo, Mariangela; Willoughby, Colin E; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

    2016-06-01

    Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600. © 2016 AlphaMed Press.

  7. CGmapTools improves the precision of heterozygous SNV calls and supports allele-specific methylation detection and visualization in bisulfite-sequencing data.

    Science.gov (United States)

    Guo, Weilong; Zhu, Ping; Pellegrini, Matteo; Zhang, Michael Q; Wang, Xiangfeng; Ni, Zhongfu

    2018-02-01

    DNA methylation is important for gene silencing and imprinting in both plants and animals. Recent advances in bisulfite sequencing allow detection of single nucleotide variations (SNVs) achieving high sensitivity, but accurately identifying heterozygous SNVs from partially C-to-T converted sequences remains challenging. We designed two methods, BayesWC and BinomWC, that substantially improved the precision of heterozygous SNV calls from ∼80% to 99% while retaining comparable recalls. With these SNV calls, we provided functions for allele-specific DNA methylation (ASM) analysis and visualizing the methylation status on reads. Applying ASM analysis to a previous dataset, we found that an average of 1.5% of investigated regions showed allelic methylation, which were significantly enriched in transposon elements and likely to be shared by the same cell-type. A dynamic fragment strategy was utilized for DMR analysis in low-coverage data and was able to find differentially methylated regions (DMRs) related to key genes involved in tumorigenesis using a public cancer dataset. Finally, we integrated 40 applications into the software package CGmapTools to analyze DNA methylomes. This package uses CGmap as the format interface, and designs binary formats to reduce the file size and support fast data retrieval, and can be applied for context-wise, gene-wise, bin-wise, region-wise and sample-wise analyses and visualizations. The CGmapTools software is freely available at https://cgmaptools.github.io/. guoweilong@cau.edu.cn. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  8. Nonfluorescent denaturing HPLC-based primer-extension method for allele-specific expression: application to analysis of mismatch repair genes.

    Science.gov (United States)

    Aceto, Gitana M; De Lellis, Laura; Catalano, Teresa; Veschi, Serena; Radice, Paolo; Di Iorio, Angelo; Mariani-Costantini, Renato; Cama, Alessandro; Curia, Maria Cristina

    2009-09-01

    Altered germline expression of genes may represent a powerful marker of genetic or epigenetic predisposition to cancer or other diseases. We developed and validated a method of nonfluorescent primer extension that uses a single dideoxynucleotide and denaturing HPLC (DHPLC) to analyze the relative allele expression. We devised 5 independent assays for measuring allele-specific expression (ASE) to exploit different markers of mismatch repair genes MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)]. We initially confirmed method reproducibility with genomic DNA (gDNA) from individuals heterozygous for a frequent single-nucleotide polymorphism in the MLH1 gene. After this preliminary validation with gDNA, we confirmed assay reproducibility with cDNA templates from control individuals. Relative allele expression was estimated by comparing the heights of the peaks corresponding to the 2 alleles. Results obtained with gDNA templates were used to normalize cDNA results. With these DHPLC-based primer-extension assays, we detected and confirmed a 5-fold imbalance in MLH1 allele expression in a mutation-negative patient with hereditary nonpolyposis colorectal cancer and in another patient with a modest degree of imbalance in MLH1 expression. Among control individuals, the relative expression of MLH1 alleles displayed a narrow range of variation. Independent DHPLC-based primer-extension assays for measuring and confirming ASE can be developed for different sequence variants of interest. This DHPLC application provides a cost-effective method for detecting ASE in cases for which conventional screening fails to detect pathogenic mutations in candidate genes and may be applicable for confirming ASE revealed by other methods, such as those used for transcriptome-wide analyses. .

  9. Minority drug-resistant HIV-1 variants in treatment naïve East-African and Caucasian patients detected by allele-specific real-time PCR.

    Directory of Open Access Journals (Sweden)

    Halime Ekici

    Full Text Available To assess the presence of two major non-nucleoside reverse transcriptase inhibitors (NNRTI drug resistance mutations (DRMs, Y181C and K103N, in minor viral quasispecies of treatment naïve HIV-1 infected East-African and Swedish patients by allele-specific polymerase chain reaction (AS-PCR.Treatment naïve adults (n=191 with three epidemiological backgrounds were included: 92 Ethiopians living in Ethiopia; 55 East-Africans who had migrated to Sweden; and 44 Caucasians living in Sweden. The pol gene was analysed by standard population sequencing and by AS-PCR for the detection of Y181C and K103N.The Y181C was detected in the minority quasispecies of six Ethiopians (6.5%, in two Caucasians (4.5%, and in one East-African (1.8%. The K103N was detected in one East- African (1.8%, by both methods. The proportion of mutants ranged from 0.25% to 17.5%. Additional DRMs were found in all three treatment naïve patient groups by population sequencing.Major NNRTI mutations can be found by AS-PCR in minor quasispecies of treatment naïve HIV-1 infected Ethiopians living in Ethiopia, in East-African and Caucasian patients living in Sweden in whom population sequencing reveal wild-type virus only. Surveys with standard sequencing are likely to underestimate transmitted drug resistance and the presence of resistant minor quasispecies in treatment naïve patients should be topic for future large scale studies.

  10. TumorBoost: Normalization of allele-specific tumor copy numbers from a single pair of tumor-normal genotyping microarrays

    Directory of Open Access Journals (Sweden)

    Neuvial Pierre

    2010-05-01

    Full Text Available Abstract Background High-throughput genotyping microarrays assess both total DNA copy number and allelic composition, which makes them a tool of choice for copy number studies in cancer, including total copy number and loss of heterozygosity (LOH analyses. Even after state of the art preprocessing methods, allelic signal estimates from genotyping arrays still suffer from systematic effects that make them difficult to use effectively for such downstream analyses. Results We propose a method, TumorBoost, for normalizing allelic estimates of one tumor sample based on estimates from a single matched normal. The method applies to any paired tumor-normal estimates from any microarray-based technology, combined with any preprocessing method. We demonstrate that it increases the signal-to-noise ratio of allelic signals, making it significantly easier to detect allelic imbalances. Conclusions TumorBoost increases the power to detect somatic copy-number events (including copy-neutral LOH in the tumor from allelic signals of Affymetrix or Illumina origin. We also conclude that high-precision allelic estimates can be obtained from a single pair of tumor-normal hybridizations, if TumorBoost is combined with single-array preprocessing methods such as (allele-specific CRMA v2 for Affymetrix or BeadStudio's (proprietary XY-normalization method for Illumina. A bounded-memory implementation is available in the open-source and cross-platform R package aroma.cn, which is part of the Aroma Project (http://www.aroma-project.org/.

  11. Allele-specific primer polymerase chain reaction for a single nucleotide polymorphism (C1205T) of swine Toll-like receptor 5 and comparison of the allelic frequency among several pig breeds in Japan and the Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Muneta, Y.; Minagawa, Y.; Kusumoto, M.; Shinkai, H.; Uenishi, H.; Šplíchal, Igor

    2012-01-01

    Roč. 56, č. 6 (2012), s. 385-391 ISSN 0385-5600 R&D Projects: GA ČR GA524/09/0365 Institutional support: RVO:61388971 Keywords : allele-specific PCR * Salmonella enterica serovar Choleraesuis * single nucleotide polymorphism Subject RIV: EC - Immunology Impact factor: 1.545, year: 2012

  12. Inter- and intra-individual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology.

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    Nahid Turan

    2010-07-01

    Full Text Available Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART, suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.

  13. Fluorescent duplex allele-specific PCR and amplicon melting for rapid homogeneous mtDNA haplogroup H screening and sensitive mixture detection.

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    Harald Niederstätter

    Full Text Available BACKGROUND: For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA, sequence variation in particular mt haplogroups (hgs and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS assay were scrutinized in the light of these requirements. METHODOLOGY/PRINCIPAL FINDINGS: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples or the derived 7028C allele (hg H samples in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA. The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification. CONCLUSIONS: Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the

  14. Establishment of real time allele specific locked nucleic acid quantitative PCR for detection of HBV YIDD (ATT mutation and evaluation of its application.

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    Yongbin Zeng

    Full Text Available BACKGROUND: Long-term use of nucleos(tide analogues can increase risk of HBV drug-resistance mutations. The rtM204I (ATT coding for isoleucine is one of the most important resistance mutation sites. Establishing a simple, rapid, reliable and highly sensitive assay to detect the resistant mutants as early as possible is of great clinical significance. METHODS: Recombinant plasmids for HBV YMDD (tyrosine-methionine-aspartate-aspartate and YIDD (tyrosine-isoleucine-aspartate-aspartate were constructed by TA cloning. Real time allele specific locked nucleic acid quantitative PCR (RT-AS-LNA-qPCR with SYBR Green I was established by LNA-modified primers and evaluated with standard recombinant plasmids, clinical templates (the clinical wild type and mutant HBV DNA mixture and 102 serum samples from nucleos(tide analogues-experienced patients. The serum samples from a chronic hepatitis B (CHB patient firstly received LMV mono therapy and then switched to LMV + ADV combined therapy were also dynamically analyzed for 10 times. RESULTS: The linear range of the assay was between 1×10(9 copies/μl and 1 × 10(2 copies/μl. The low detection limit was 1 × 10(1 copies/μl. Sensitivity of the assay were 10(-6, 10(-4 and 10(-2 in the wild-type background of 1 × 10(9 copies/μl, 1 × 10(7 copies/μl and 1 × 10(5 copies/μl, respectively. The sensitivity of the assay in detection of clinical samples was 0.03%. The complete coincidence rate between RT-AS-LNA-qPCR and direct sequencing was 91.2% (93/102, partial coincidence rate was 8.8% (9/102, and no complete discordance was observed. The two assays showed a high concordance (Kappa = 0.676, P = 0.000. Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level. CONCLUSIONS: A rapid, cost-effective, high sensitive, specific and reliable method of RT-AS-LNA-qPCR with SYBR Green I for early and absolute quantification of HBV YIDD (ATT coding for isoleucine

  15. Isolation of rifampicin resistant Flavobacterium psychrophilum strains and their potential as live attenuated vaccine candidates

    Science.gov (United States)

    Previous studies have demonstrated that passage of pathogenic bacteria on increasing concentrations of the antibiotic rifampicin leads to the attenuation of virulence and these resistant strains may serve as live attenuated vaccines. Two rifampicin resistant strains of Flavobacterium psychrophilum,...

  16. A synthetic peptide selectively kills only virulent Paracoccidioides brasiliensis yeasts.

    Science.gov (United States)

    Kioshima, Erika Seki; Aliperti, Fabiana; Maricato, Juliana Terzi; Mortara, Renato Arruda; Bagagli, Eduardo; Mariano, Mario; Lopes, José Daniel

    2011-03-01

    This work was conducted to identify virulence biomarkers for Paracoccidioides brasiliensis (Pb), the fungus responsible for Paracoccidioidomycosis (PCM), a systemic disease endemic in Latin America. Measurement of mortality showed that all B10.A mice were killed after 250 days by the virulent Pb18 isolate while only one of the mice that received the attenuated counterpart died. Also, number of lung CFUs from virulent Pb18 inoculated mice were much higher when these isolates were compared. Phage display methodology allowed selection of three phages that specifically bound to virulent Pb18. Variability of p04 phage binding to different Pb isolates were examples of variability of expression by the fungus of its binding molecule, strongly suggesting p04 as a biomarker of virulence. In vitro, its derived peptide pep04 killed only virulent fungi, and confocal microscopy showed that it was internalized only by the virulent isolate. Pep04 blocked establishment of Pb infection in mice and virulent Pb18 pre-incubated with p04 showed significantly inhibited lung infection. Furthermore, infected mice treated with p04 showed highly significant reduction in lung CFUs. These findings firmly establish p04 as a biomarker of Pb virulence. Therefore, after proper peptide engineering, p04 may become a useful adjuvant for the distressing treatment of PCM. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  17. Lipoprotein(a) and HIV: Allele-Specific Apolipoprotein(a) Levels Predict Carotid Intima-Media Thickness in HIV-Infected Young Women in the Women's Interagency HIV Study.

    Science.gov (United States)

    Enkhmaa, Byambaa; Anuurad, Erdembileg; Zhang, Wei; Li, Chin-Shang; Kaplan, Robert; Lazar, Jason; Merenstein, Dan; Karim, Roksana; Aouizerat, Brad; Cohen, Mardge; Butler, Kenneth; Pahwa, Savita; Ofotokun, Igho; Adimora, Adaora A; Golub, Elizabeth; Berglund, Lars

    2017-05-01

    In the general population, lipoprotein(a) [Lp(a)] has been established as an independent causal risk factor for cardiovascular disease. Lp(a) levels are to a major extent regulated by a size polymorphism in the apolipoprotein(a) [apo(a)] gene. The roles of Lp(a)/apo(a) in human immunodeficiency virus (HIV)-related elevated cardiovascular disease risk remain unclear. The associations between total plasma Lp(a) level, allele-specific apo(a) level, an Lp(a) level carried by individual apo(a) alleles, and common carotid artery intima-media thickness were assessed in 150 HIV-infected and 100 HIV-uninfected women in the WIHS (Women's Interagency HIV Study). Linear regression analyses with and without adjustments were used. The cohort was young (mean age, ≈31 years), with the majority being Blacks (≈70%). The prevalence of a small size apo(a) (≤22 Kringle repeats) or a high Lp(a) level (≥30 mg/dL) was similar by HIV status. Total plasma Lp(a) level ( P =0.029) and allele-specific apo(a) level carried by the smaller apo(a) sizes ( P =0.022) were significantly associated with carotid artery intima-media thickness in the HIV-infected women only. After accounting for confounders (age, race, smoking, body mass index, blood pressure, hepatitis C virus coinfection, menopause, plasma lipids, treatment status, CD4 + T cell count, and HIV/RNA viral load), the association remained significant for both Lp(a) ( P =0.035) and allele-specific apo(a) level carried by the smaller apo(a) sizes ( P =0.010) in the HIV-infected women. Notably, none of the other lipids/lipoproteins was associated with carotid artery intima-media thickness. Lp(a) and allele-specific apo(a) levels predict carotid artery intima-media thickness in HIV-infected young women. Further research is needed to identify underlying mechanisms of an increased Lp(a) atherogenicity in HIV infection. © 2017 American Heart Association, Inc.

  18. Development of an allele-specific PCR assay for simultaneous sero-typing of avian pathogenic Escherichia coli predominant O1, O2, O18 and O78 strains.

    Science.gov (United States)

    Wang, Shaohui; Meng, Qingmei; Dai, Jianjun; Han, Xiangan; Han, Yue; Ding, Chan; Liu, Haiwen; Yu, Shengqing

    2014-01-01

    Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR) assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs) bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development.

  19. Development of an allele-specific PCR assay for simultaneous sero-typing of avian pathogenic Escherichia coli predominant O1, O2, O18 and O78 strains.

    Directory of Open Access Journals (Sweden)

    Shaohui Wang

    Full Text Available Systemic infections by avian pathogenic Escherichia coli (APEC are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development.

  20. Allele-Specific Droplet Digital PCR Combined with a Next-Generation Sequencing-Based Algorithm for Diagnostic Copy Number Analysis in Genes with High Homology: Proof of Concept Using Stereocilin.

    Science.gov (United States)

    Amr, Sami S; Murphy, Elissa; Duffy, Elizabeth; Niazi, Rojeen; Balciuniene, Jorune; Luo, Minjie; Rehm, Heidi L; Abou Tayoun, Ahmad N

    2018-04-01

    Copy number variants (CNVs) can substantially contribute to the pathogenic variant spectrum in several disease genes. The detection of this type of variant is complicated in genes with high homology to other genomic sequences, yet such genomics regions are more likely to lead to CNVs, making it critical to address detection in these settings. We developed a copy number analysis approach for high homology genes/regions that consisted of next-generation sequencing (NGS)-based dosage analysis accompanied by allele-specific droplet digital PCR (ddPCR) confirmatory testing. We applied this approach to copy number analysis in STRC , a gene with 98.9% homology to a nonfunctional pseudogene, pSTRC , and characterized its accuracy in detecting different copy number states by use of known samples. Using a cohort of 517 patients with hearing loss, we prospectively demonstrated the clinical utility of the approach, which contributed 30 of the 122 total positives (6%) to the diagnostic yield, increasing the overall yield from 17.6% to 23.6%. Positive STRC genotypes included homozygous (n = 15) or compound heterozygous (n = 8) deletions, or heterozygous deletions in trans with pathogenic sequence variants (n = 7). Finally, this approach limited ddPCR testing to cases with NGS copy number findings, thus markedly reducing the number of costly and laborious, albeit specific, ddPCR tests. NGS-based CNV detection followed by allele-specific ddPCR confirmatory testing is a reliable and affordable approach for copy number analysis in medically relevant genes with homology issues. © 2017 American Association for Clinical Chemistry.

  1. Virulence Factors Associated with Enterococcus Faecalis Infective Endocarditis

    DEFF Research Database (Denmark)

    Madsen, Kristian T; Skov, Marianne N; Gill, Sabine

    2017-01-01

    been associated with E. faecalis infective endocarditis. Absence of these factors entailed attenuation of strains in both mixed- and mono-bacterial infection endocarditis models as well as in in vitro and ex vivo assays when compared to their virulence factor expressing parental strains. PATHOGENESIS......INTRODUCTION: The enterococci are accountable for up to 20% of all cases of infective endocarditis, with Enterococcus faecalis being the primary causative isolate. Infective endocarditis is a life-threatening infection of the endocardium that results in the formation of vegetations. Based...... on a literature review, this paper provides an overview of the virulence factors associated with E. faecalis infective endocarditis. Furthermore, it reports the effects of active or passive immunization against some of these involved factors. INDIVIDUAL VIRULENCE FACTORS: Nine virulence factors have in particular...

  2. Anti-quorum Sensing and Anti-biofilm Activity ofDelftia tsuruhatensisExtract by Attenuating the Quorum Sensing-Controlled Virulence Factor Production inPseudomonas aeruginosa.

    Science.gov (United States)

    Singh, Vijay K; Mishra, Avinash; Jha, Bhavanath

    2017-01-01

    Multidrug-resistance bacteria commonly use cell-to-cell communication that leads to biofilm formation as one of the mechanisms for developing resistance. Quorum sensing inhibition (QSI) is an effective approach for the prevention of biofilm formation. A Gram-negative bacterium, Delftia tsuruhatensis SJ01, was isolated from the rhizosphere of a species of sedge ( Cyperus laevigatus ) grown along the coastal-saline area. The isolate SJ01 culture and bacterial crude extract showed QSI activity in the biosensor plate containing the reference strain Chromobacterium violaceum CV026. A decrease in the violacein production of approximately 98% was detected with the reference strain C. violaceum CV026. The bacterial extract (strain SJ01) exhibited anti-quorum sensing activity and inhibited the biofilm formation of clinical isolates wild-type Pseudomonas aeruginosa PAO1 and P. aeruginosa PAH. A non-toxic effect of the bacterial extract (SJ01) was detected on the cell growth of the reference strains as P. aeruginosa viable cells were present within the biofilm. It is hypothesized that the extract (SJ01) may change the topography of the biofilm and thus prevent bacterial adherence on the biofilm surface. The extract also inhibits the motility, virulence factors (pyocyanin and rhamnolipid) and activity (elastase and protease) in P. aeruginosa treated with SJ01 extract. The potential active compound present was identified as 1,2-benzenedicarboxylic acid, diisooctyl ester. Microarray and transcript expression analysis unveiled differential expression of quorum sensing regulatory genes. The key regulatory genes, LasI, LasR, RhlI , and RhlR were down-regulated in the P. aeruginosa analyzed by quantitative RT-PCR. A hypothetical model was generated of the transcriptional regulatory mechanism inferred in P. aeruginosa for quorum sensing, which will provide useful insight to develop preventive strategies against the biofilm formation. The potential active compound identified, 1

  3. Cryptosporidium Pathogenicity and Virulence

    Science.gov (United States)

    Bouzid, Maha; Chalmers, Rachel M.; Tyler, Kevin M.

    2013-01-01

    Cryptosporidium is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts. Several studies have reported different degrees of pathogenicity and virulence among Cryptosporidium species and isolates of the same species as well as evidence of variation in host susceptibility to infection. The identification and validation of Cryptosporidium virulence factors have been hindered by the renowned difficulties pertaining to the in vitro culture and genetic manipulation of this parasite. Nevertheless, substantial progress has been made in identifying putative virulence factors for Cryptosporidium. This progress has been accelerated since the publication of the Cryptosporidium parvum and C. hominis genomes, with the characterization of over 25 putative virulence factors identified by using a variety of immunological and molecular techniques and which are proposed to be involved in aspects of host-pathogen interactions from adhesion and locomotion to invasion and proliferation. Progress has also been made in the contribution of host factors that are associated with variations in both the severity and risk of infection. Here we provide a review comprised of the current state of knowledge on Cryptosporidium infectivity, pathogenesis, and transmissibility in light of our contemporary understanding of microbial virulence. PMID:23297262

  4. Identification of Burkholderia cenocepacia strain H111 virulence factors using nonmammalian infection hosts

    DEFF Research Database (Denmark)

    Schwager, Stephan; Agnoli, Kirsty; Köthe, Manuela

    2013-01-01

    or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in the C. elegans infection model were also tested in the Drosophila melanogaster pricking model, and those also attenuated in this model were further tested in Galleria mellonella. Six...... of the 22 mutants were attenuated in D. melanogaster, and five of these were less pathogenic in the G. mellonella model. We show that genes encoding enzymes of the purine, pyrimidine, and shikimate biosynthesis pathways are critical for virulence in multiple host models of infection....

  5. The − 5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Starska, Katarzyna, E-mail: katarzyna.starska@umed.lodz.pl [I Department of Otolaryngology and Laryngological Oncology, Medical University of Łódź, Kopcinskiego 22, 90-153 Łódź (Poland); Krześlak, Anna; Forma, Ewa [Department of Cytobiochemistry, University of Łódź, Pomorska 142/143, 90-236 Łódź (Poland); Olszewski, Jurek [II Department of Otolaryngology and Laryngological Oncology, Medical University of Łódź, Żeromskiego 113, 90-549 Łódź (Poland); Morawiec-Sztandera, Alina [Department of Head and Neck Surgery, Medical University of Łódź, Paderewskiego 4, 93-509 Łódź (Poland); Aleksandrowicz, Paweł [Department of Otolaryngology and Laryngological Oncology, Medical University of Lublin, Jaczewskiego 8, 20-954 Lublin (Poland); Lewy-Trenda, Iwona [Department of Pathology, Medical University of Łódź, Pomorska 251, 92-213 Łódź (Poland); and others

    2014-10-15

    Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the − 5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region − 5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the − 5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that − 5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer. - Highlights: • MT2A gene expression and metal content in laryngeal cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn and Cu levels • Negative correlation between MT2A gene expression and Cd, Zn and Cu levels.

  6. Analysis of the contribution of bacteriophage ST64B to in vitro virulence traits of Salmonella enterica serovar Typhimurium

    DEFF Research Database (Denmark)

    Fresno, Ana Herrero; Leekitcharoenphon, Pimlapas; Hendriksen, Rene S.

    2014-01-01

    Comparison of the publicly available genomes of the virulent Salmonella enterica serovar Typhimurium (S. Typhimurium) strains SL1344, 14028s and D23580 to that of the virulence-attenuated isolate LT2 revealed the absence of a full sequence of bacteriophage ST64B in the latter. Four selected ST64B...

  7. Attenuation of monkeypox virus by deletion of genomic regions

    Science.gov (United States)

    Lopera, Juan G.; Falendysz, Elizabeth A.; Rocke, Tonie E.; Osorio, Jorge E.

    2015-01-01

    Monkeypox virus (MPXV) is an emerging pathogen from Africa that causes disease similar to smallpox. Two clades with different geographic distributions and virulence have been described. Here, we utilized bioinformatic tools to identify genomic regions in MPXV containing multiple virulence genes and explored their roles in pathogenicity; two selected regions were then deleted singularly or in combination. In vitro and in vivostudies indicated that these regions play a significant role in MPXV replication, tissue spread, and mortality in mice. Interestingly, while deletion of either region led to decreased virulence in mice, one region had no effect on in vitro replication. Deletion of both regions simultaneously also reduced cell culture replication and significantly increased the attenuation in vivo over either single deletion. Attenuated MPXV with genomic deletions present a safe and efficacious tool in the study of MPX pathogenesis and in the identification of genetic factors associated with virulence.

  8. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell......Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing...... signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...

  9. Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions

    NARCIS (Netherlands)

    Manoussaka, Maria S.; Berry, Neil; Ferguson, Deborah; Stebbings, Richard; Robinson, Mark; Ham, Claire; Page, Mark; Li, Bo; Das, Atze T.; Berkhout, Ben; Almond, Neil; Cranage, Martin P.

    2013-01-01

    Live attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV. Using a novel, doxycycline dependent,

  10. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

    DEFF Research Database (Denmark)

    Hentzer, Morten; Wu, H.; Andersen, Jens Bo

    2003-01-01

    afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate...

  11. Aureusimines in Staphylococcus aureus are not involved in virulence.

    Directory of Open Access Journals (Sweden)

    Fei Sun

    2010-12-01

    Full Text Available Recently, dipeptide aureusimines were reported to activate expression of staphylococcal virulence genes, such as alpha-hemolysin, and increase S. aureus virulence. Surprisingly, most of the virulence genes affected by aureusimines form part of the regulon of the SaeRS two component system (TCS, raising the possibility that SaeRS might be directly or indirectly involved in the aureusimine-dependent signaling process.Using HPLC analyses, we confirmed that a transposon mutant of ausA, the gene encoding the aureusimine dipeptide synthesis enzyme, does not produce dipeptides. However, the transposon mutant showed normal hemolysis activity and alpha-hemolysin/SaeP production. Furthermore, the P1 promoter of the sae operon, one of the targets of the SaeRS TCS, showed normal transcription activity. Moreover, in contrast to the original report, the ausA transposon mutant did not exhibit attenuated virulence in an animal infection model. DNA sequencing revealed that the ausA deletion mutant used in the original study has an 83 nt-duplication in saeS. Hemolysis activity of the original mutant was restored by a plasmid carrying the sae operon. A mutant of the sae operon showed elevated resistance to chloramphenicol and erythromycin, two antibiotics widely used during staphylococcal mutagenesis. At 43°C in the presence of erythromycin and aeration, the conditions typically employed for staphylococcal mutagenesis, an saeR transposon mutant grew much faster than a control mutant and the saeR mutant was highly enriched in a mixed culture experiment.Our results show that the previously reported roles of aureusimines in staphylococcal gene regulation and virulence were due to an unintended mutation in saeS, which was likely selected due to elevated resistance of the mutant to environmental stresses. Thus, there is no evidence indicating that the dipeptide aureusimines play a role in sae-mediated virulence factor production or contribute to staphylococcal

  12. Attenuated Phenotype of a Recent House Finch-Associated Mycoplasma gallisepticum Isolate in Domestic Poultry.

    Science.gov (United States)

    Pflaum, K; Tulman, E R; Beaudet, J; Liao, X; Dhondt, K V; Dhondt, A A; Hawley, D M; Ley, D H; Kerr, K M; Geary, S J

    2017-06-01

    Mycoplasma gallisepticum , known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch ( Haemorhous mexicanus ) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host. Copyright © 2017 American Society for Microbiology.

  13. Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

    Science.gov (United States)

    Hauser, Andrea; Kuecherer, Claudia; Kunz, Andrea; Dabrowski, Piotr Wojtek; Radonić, Aleksandar; Nitsche, Andreas; Theuring, Stefanie; Bannert, Norbert; Sewangi, Julius; Mbezi, Paulina; Dugange, Festo; Harms, Gundel; Meixenberger, Karolin

    2015-01-01

    Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

  14. Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

    Directory of Open Access Journals (Sweden)

    Andrea Hauser

    Full Text Available Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS and allele-specific real-time PCR (ASPCR for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT.Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F. In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.Drug-resistant HIV-variants were identified in 69% (20/29 of women by UDS and in 45% (13/29 by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24. By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41. The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml, resulting in missing or insufficient sequence coverage.Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

  15. Brucella, nitrogen and virulence.

    Science.gov (United States)

    Ronneau, Severin; Moussa, Simon; Barbier, Thibault; Conde-Álvarez, Raquel; Zuniga-Ripa, Amaia; Moriyon, Ignacio; Letesson, Jean-Jacques

    2016-08-01

    The brucellae are α-Proteobacteria causing brucellosis, an important zoonosis. Although multiplying in endoplasmic reticulum-derived vacuoles, they cause no cell death, suggesting subtle but efficient use of host resources. Brucellae are amino-acid prototrophs able to grow with ammonium or use glutamate as the sole carbon-nitrogen source in vitro. They contain more than twice amino acid/peptide/polyamine uptake genes than the amino-acid auxotroph Legionella pneumophila, which multiplies in a similar vacuole, suggesting a different nutritional strategy. During these two last decades, many mutants of key actors in nitrogen metabolism (transporters, enzymes, regulators, etc.) have been described to be essential for full virulence of brucellae. Here, we review the genomic and experimental data on Brucella nitrogen metabolism and its connection with virulence. An analysis of various aspects of this metabolism (transport, assimilation, biosynthesis, catabolism, respiration and regulation) has highlighted differences and similarities in nitrogen metabolism with other α-Proteobacteria. Together, these data suggest that, during their intracellular life cycle, the brucellae use various nitrogen sources for biosynthesis, catabolism and respiration following a strategy that requires prototrophy and a tight regulation of nitrogen use.

  16. Principles underlying rational design of live attenuated influenza vaccines

    OpenAIRE

    Jang, Yo Han; Seong, Baik-Lin

    2012-01-01

    Despite recent innovative advances in molecular virology and the developments of vaccines, influenza virus remains a serious burden for human health. Vaccination has been considered a primary countermeasure for prevention of influenza infection. Live attenuated influenza vaccines (LAIVs) are particularly attracting attention as an effective strategy due to several advantages over inactivated vaccines. Cold-adaptation, as a classical means for attenuating viral virulence, has been successfully...

  17. Life history trade-offs and relaxed selection can decrease bacterial virulence in environmental reservoirs.

    Directory of Open Access Journals (Sweden)

    Lauri Mikonranta

    Full Text Available Pathogen virulence is usually thought to evolve in reciprocal selection with the host. While this might be true for obligate pathogens, the life histories of opportunistic pathogens typically alternate between within-host and outside-host environments during the infection-transmission cycle. As a result, opportunistic pathogens are likely to experience conflicting selection pressures across different environments, and this could affect their virulence through life-history trait correlations. We studied these correlations experimentally by exposing an opportunistic bacterial pathogen Serratia marcescens to its natural protist predator Tetrahymena thermophila for 13 weeks, after which we measured changes in bacterial traits related to both anti-predator defence and virulence. We found that anti-predator adaptation (producing predator-resistant biofilm caused a correlative attenuation in virulence. Even though the direct mechanism was not found, reduction in virulence was most clearly connected to a predator-driven loss of a red bacterial pigment, prodigiosin. Moreover, life-history trait evolution was more divergent among replicate populations in the absence of predation, leading also to lowered virulence in some of the 'predator absent' selection lines. Together these findings suggest that the virulence of non-obligatory, opportunistic bacterial pathogens can decrease in environmental reservoirs through life history trade-offs, or random accumulation of mutations that impair virulence traits under relaxed selection.

  18. Development of a human live attenuated West Nile infectious DNA vaccine: Suitability of attenuating mutations found in SA14-14-2 for WN vaccine design

    International Nuclear Information System (INIS)

    Yamshchikov, Vladimir; Manuvakhova, Marina; Rodriguez, Efrain

    2016-01-01

    Direct attenuation of West Nile (WN) virus strain NY99 for the purpose of vaccine development is not feasible due to its high virulence and pathogenicity. Instead, we created highly attenuated chimeric virus W1806 with the serological identity of NY99. To further attenuate W1806, we investigated effects of mutations found in Japanese encephalitis virus vaccine SA14-14-2. WN viruses carrying all attenuating mutations lost infectivity in mammalian, but not in mosquito cells. No single reversion restored infectivity in mammalian cells, although increased infectivity in mosquito cells was observed. To identify a subset of mutations suitable for further attenuation of W1806, we analyzed effects of E 138 K and K 279 M changes on virulence, growth properties, and immunogenicity of derivatized W956, from which chimeric W1806 inherited its biological properties and attenuation profile. Despite strong dominant attenuating effect, introduction of only two mutations was not sufficient for attenuating W1806 to the safety level acceptable for human use. - Highlights: • Further attenuation of a WN vaccine precursor is outlined. • Effect of SA14-14-2 attenuating mutations is tested. • Mechanism of attenuation is proposed and illustrated. • The need for additional attenuating mutations is justified.

  19. Development of a human live attenuated West Nile infectious DNA vaccine: Suitability of attenuating mutations found in SA14-14-2 for WN vaccine design

    Energy Technology Data Exchange (ETDEWEB)

    Yamshchikov, Vladimir, E-mail: yaximik@gmail.com; Manuvakhova, Marina; Rodriguez, Efrain

    2016-01-15

    Direct attenuation of West Nile (WN) virus strain NY99 for the purpose of vaccine development is not feasible due to its high virulence and pathogenicity. Instead, we created highly attenuated chimeric virus W1806 with the serological identity of NY99. To further attenuate W1806, we investigated effects of mutations found in Japanese encephalitis virus vaccine SA14-14-2. WN viruses carrying all attenuating mutations lost infectivity in mammalian, but not in mosquito cells. No single reversion restored infectivity in mammalian cells, although increased infectivity in mosquito cells was observed. To identify a subset of mutations suitable for further attenuation of W1806, we analyzed effects of E{sub 138}K and K{sub 279}M changes on virulence, growth properties, and immunogenicity of derivatized W956, from which chimeric W1806 inherited its biological properties and attenuation profile. Despite strong dominant attenuating effect, introduction of only two mutations was not sufficient for attenuating W1806 to the safety level acceptable for human use. - Highlights: • Further attenuation of a WN vaccine precursor is outlined. • Effect of SA14-14-2 attenuating mutations is tested. • Mechanism of attenuation is proposed and illustrated. • The need for additional attenuating mutations is justified.

  20. Molecular Characterization of Putative Virulence Determinants in Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Suat Moi Puah

    2014-01-01

    Full Text Available The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P=0.049 at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

  1. Metals in fungal virulence.

    Science.gov (United States)

    Gerwien, Franziska; Skrahina, Volha; Kasper, Lydia; Hube, Bernhard; Brunke, Sascha

    2018-01-01

    Metals are essential for life, and they play a central role in the struggle between infecting microbes and their hosts. In fact, an important aspect of microbial pathogenesis is the 'nutritional immunity', in which metals are actively restricted (or, in an extended definition of the term, locally enriched) by the host to hinder microbial growth and virulence. Consequently, fungi have evolved often complex regulatory networks, uptake and detoxification systems for essential metals such as iron, zinc, copper, nickel and manganese. These systems often differ fundamentally from their bacterial counterparts, but even within the fungal pathogens we can find common and unique solutions to maintain metal homeostasis. Thus, we here compare the common and species-specific mechanisms used for different metals among different fungal species-focusing on important human pathogens such as Candida albicans, Aspergillus fumigatus or Cryptococcus neoformans, but also looking at model fungi such as Saccharomyces cerevisiae or A. nidulans as well-studied examples for the underlying principles. These direct comparisons of our current knowledge reveal that we have a good understanding how model fungal pathogens take up iron or zinc, but that much is still to learn about other metals and specific adaptations of individual species-not the least to exploit this knowledge for new antifungal strategies. © FEMS 2017.

  2. Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus).

    Science.gov (United States)

    Elsworth, Peter; Cooke, Brian D; Kovaliski, John; Sinclair, Ronald; Holmes, Edward C; Strive, Tanja

    2014-09-01

    The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes.

    Science.gov (United States)

    Maury, Mylène M; Chenal-Francisque, Viviane; Bracq-Dieye, Hélène; Han, Lei; Leclercq, Alexandre; Vales, Guillaume; Moura, Alexandra; Gouin, Edith; Scortti, Mariela; Disson, Olivier; Vázquez-Boland, José A; Lecuit, Marc

    2017-11-01

    The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene ( hly ) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA - /LLO - ) mutants belonged to phylogenetically diverse clades of L. monocytogenes , and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA - /LLO - mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. Copyright © 2017 Maury et al.

  4. A novel anti-virulence gene revealed by proteomic analysis in Shigella flexneri 2a

    Directory of Open Access Journals (Sweden)

    Ying Tianyi

    2010-06-01

    Full Text Available Abstract Background Shigella flexneri is a gram-negative, facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. The virulence factors of S. flexneri have been shown to be produced at 37 degrees C but not at 30 degrees C. To discover potential, novel virulence-related proteins of S. flexneri, we performed differential in-gel electrophoresis (DIGE analysis to measure changes in the expression profile that are induced by a temperature increase. Results The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic E. coli did not show this differential expression as in S. flexneri, which suggested that argT might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with argT mutants were performed, and the results indicated that the over-expression of ArgTY225D would attenuate the virulence of S. flexneri. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in S. flexneri at the molecular level. We show that HtrA is differentially expressed among different derivative strains. Conclusion Gene argT is a novel anti-virulence gene that may interfere with the virulence of S. flexneri via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.

  5. Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

    Science.gov (United States)

    Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

    2014-01-01

    ABSTRACT Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3′ consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCE Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its

  6. Effect of Dietary Minerals on Virulence Attributes of Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    Varunkumar Bhattaram

    2017-05-01

    Full Text Available Vibrio cholerae is a water-borne pathogen responsible for causing a toxin-mediated profuse diarrhea in humans, leading to severe dehydration and death in unattended patients. With increasing reports of antibiotic resistance in V. cholerae, there is a need for alternate interventional strategies for controlling cholera. A potential new strategy for treating infectious diseases involves targeting bacterial virulence rather than growth, where a pathogen’s specific mechanisms critical for causing infection in hosts are inhibited. Since bacterial motility, intestinal colonization and cholera toxin are critical components in V. cholerae pathogenesis, attenuating these virulence factors could potentially control cholera in humans. In this study, the efficacy of sub-inhibitory concentration (SIC, highest concentration not inhibiting bacterial growth of essential minerals, zinc (Zn, selenium (Se, and manganese (Mn in reducing V. cholerae motility and adhesion to intestinal epithelial cells (Caco-2, cholera toxin production, and toxin binding to the ganglioside receptor (GM1 was investigated. Additionally, V. cholerae attachment and toxin production in an ex vivo mouse intestine model was determined. Further, the effect of Zn, Se and Mn on V. cholerae virulence genes, ctxAB (toxin production, fliA (motility, tcpA (intestinal colonization, and toxR (master regulon was determined using real-time quantitative PCR. All three minerals significantly reduced V. cholerae motility, adhesion to Caco-2 cells, and cholera toxin production in vitro, and decreased adhesion and toxin production in mouse intestine ex vivo (P < 0.05. In addition, Zn, Se, and Mn down-regulated the transcription of virulence genes, ctxAB, fliA, and toxR. Results suggest that Zn, Se, and Mn could be potentially used to reduce V. cholerae virulence. However, in vivo studies in an animal model are necessary to validate these results.

  7. Genomic Analysis of Attenuation in Pandemic Vibrio parahaemolyticus

    Science.gov (United States)

    Pinnell, L. J.; Tallman, J. J., III; Turner, J.

    2016-02-01

    A critical problem in the prevention and treatment of infectious disease is the ability to differentiate virulent from avirulent bacterial strains. The distinction is commonly based on the presence or absence of specific virulence-associated genes. Alternately, serotypic or phylogenetic typing can accurately differentiate virulent from avirulent strains. When these approaches fail, more discriminatory analysis is needed. Pandemic Vibiro parahaemolyticus, distinguishable by genotyping (thermostable direct hemolysin or tdh), serotyping (O3:K6) and multilocus sequence typing (ST3), is regarded as a highly virulent clonal complex. We have previously shown, through population genetics and cytotoxicity testing, that some pandemic strains isolated from environmental sources are avirulent. To investigate the basis for attenuation, we sequenced the draft genomes of 10 pandemic V. parahaemolyticus isolates originating from environmental (N = 7) and clinical sources (N = 3). Genomic comparison of these 10 draft genomes, and the pandemic type strain (RIMD2210633), revealed a large core genome (5,158,719 bp) and a much smaller accessory genome (141,403 bp). The accessory genome was largely comprised of hypothetical proteins; however, several genes encoded phage-related proteins. Phylogenetic analysis, based on 2,902 single nucleotide polymorphisms in the core genome, did not reveal a discernable pattern. Current efforts are focused on the identification of insertions, deletions and point mutations that may alter protein expression or protein function. Preliminary results show that attenuated strains lack the virulence-associated vacB gene (VP1890). This gene encodes a 741 amino acid exoribonuclease homologous to exoribonucleases known to modulate virulence in Salmonella enterica and Helicobacter pylori. The correlation between attenuation and the absence of this gene, suggests that VP1890 plays an important role in human pathogenesis.

  8. [Virulence factors of Candida albicans].

    Science.gov (United States)

    Staniszewska, Monika; Bondaryk, Małgorzata; Piłat, Joanna; Siennicka, Katarzyna; Magda, Urszula; Kurzatkowski, Wiesław

    2012-01-01

    Candida albicans is the most common etiological factor of opportunistic human fungal infections. In this review, we focus on the major virulence factors that mediate the pathogenesis of C. albicans. Among these virulence factors, secreted aspartyl proteases, adherence, pleomorphism are the most important features of C. albicans infections. Ability to exist as different pleomorphic forms is defined as pleomorphism. A number of quorum sensing (QS) molecules have been described which affect morphogenesis process in C. albicans. Furthermore, the morphological transition of C. albicans in response to changing environmental conditions represent a means by which the strain adapts to different biological niches. Furthermore, every morphotype has own virulence profile and each pleomorphic form provide critical functions required for pathogenesis. Candida albicans is a producer of extracellular hydrolytic enzymes. Among them lipases, phospholipases and secreted aspartyl proteinases (Sap) are most significant in virulence. Sap proteins contribute to pathogenesis by digestion of host cell membranes and molecules of the host immune system to avoid antimicrobial attack by the host. One of the key features in the development of candidiasis is adhesion ofC. albicans to buccal and vaginal epithelial cells. The adhesion to host cells represents the first step in the internalization process which involves adhesins. Knowledge of the role of the various C. albicans' virulence factors during in vivo infections is still incomplete, therefore further studies including quantification of genes expression and histopathological examination of tissues damage are required to fully understand pathogenesis of this opportunistic pathogen.

  9. Virulence Markers of Dengue Viruses

    Science.gov (United States)

    1990-02-20

    pathogenetic mechanism from dengue-2 and dengue-4 viruses . Additional detailed epidemiological, virological and clinical evaluation on dengue-1 and...Soawy Ca saoouj Virulence Markers of Dengue Viruses (U) 12. PCIRSONAL AUTHORS) James L. Hardy, Ph.D. and Srisakul C. Kliks, Ph.D. 13a. TYPE Of REPORT...17. COSATI COOLS I& S UBiJECT TERMS0,G ’-mPJ!’ iwin.. - fl OV nu0a mef) FIELD I GROUP SUS-GROUIP Dengue viruses , dengue hemorrhagic fever, virulence

  10. Molecular determinants of the virulence and infectivity of California serogroup bunyaviruses.

    Science.gov (United States)

    Griot, C; Gonzalez-Scarano, F; Nathanson, N

    1993-01-01

    California bunyaviruses cause encephalitis in mammalian hosts after peripheral infection. The virulence of these viruses is determined by their ability to replicate sequentially in striated muscle, cause viremia, and invade and replicate in the central nervous system. These viruses are also able to infect vector mosquitoes following ingestion of a blood meal containing virus. Bunyaviruses are negative stranded RNA viruses with a trisegmented genome, and the large, medium, and small RNA segments encode the polymerase, the glycoproteins, and the nucleoprotein, respectively. Reassortants between virulent and avirulent virus clones have been used to map virulence determinants in mice as well as determinants of infectivity in mosquitoes. Attenuation in mice and infectivity in mosquitoes of some virus clones maps to the medium RNA segment, implying that the virus glycoproteins, which are involved in virus entry, play a role in virulence. Attenuation in mice and mosquito infectivity of other clones maps to the large RNA segment, suggesting that cell-specific differences in the function of the viral polymerase can also determine virulence and host range.

  11. Functional dissection of Streptococcus pyogenes M5 protein: the hypervariable region is essential for virulence.

    Directory of Open Access Journals (Sweden)

    Johan Waldemarsson

    Full Text Available The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

  12. Elongation factor P mediates a novel post-transcriptional regulatory pathway critical for bacterial virulence

    DEFF Research Database (Denmark)

    Zou, S Betty; Roy, Hervé; Ibba, Michael

    2012-01-01

    of the pathogen to respond to external cues are typically attenuating. Here we discuss our recent discovery of a novel post-transcriptional regulatory pathway critical for Salmonella virulence and stress resistance. The enzymes PoxA and YjeK coordinately attach a unique beta-amino acid onto a highly conserved......Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability...... changes in the translation machinery during stress adaptation, indicating that the role of these factors in physiology may be broadly conserved....

  13. Elongation factor P mediates a novel post-transcriptional regulatory pathway critical for bacterial virulence

    DEFF Research Database (Denmark)

    Zou, S Betty; Roy, Hervé; Ibba, Michael

    2012-01-01

    Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability...... of the pathogen to respond to external cues are typically attenuating. Here we discuss our recent discovery of a novel post-transcriptional regulatory pathway critical for Salmonella virulence and stress resistance. The enzymes PoxA and YjeK coordinately attach a unique beta-amino acid onto a highly conserved...

  14. cipC is important for Aspergillus fumigatus virulence.

    Science.gov (United States)

    Canela, Heliara Maria Spina; Takami, Luciano Akira; da Silva Ferreira, Márcia Eliana

    2017-02-01

    Aspergillus fumigatus is the main causative agent of invasive aspergillosis, a disease that affects immunocompromised patients and has a high mortality rate. We previously observed that the transcription of a cipC-like gene was increased when A. fumigatus encountered an increased CO 2 concentration, as occurs during the infection process. CipC is a protein of unknown function that might be associated with fungal pathogenicity. In this study, the cipC gene was disrupted in A. fumigatus to evaluate its importance for fungal pathogenicity. The gene was replaced, and the germination, growth phenotype, stress responses, and virulence of the resultant mutant were assessed. Although cipC was not essential, its deletion attenuated A. fumigatus virulence in a low-dose murine infection model, suggesting the involvement of the cipC gene in the virulence of this fungus. This study is the first to disrupt the cipC gene in A. fumigatus. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  15. Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain

    Science.gov (United States)

    2013-01-01

    Background Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), a mild, chronic pneumonia of swine. Despite presenting with low direct mortality, EP is responsible for major economic losses in the pig industry. To identify the virulence-associated determinants of M. hyopneumoniae, we determined the whole genome sequence of M. hyopneumoniae strain 168 and its attenuated high-passage strain 168-L and carried out comparative genomic analyses. Results We performed the first comprehensive analysis of M. hyopneumoniae strain 168 and its attenuated strain and made a preliminary survey of coding sequences (CDSs) that may be related to virulence. The 168-L genome has a highly similar gene content and order to that of 168, but is 4,483 bp smaller because there are 60 insertions and 43 deletions in 168-L. Besides these indels, 227 single nucleotide variations (SNVs) were identified. We further investigated the variants that affected CDSs, and compared them to reported virulence determinants. Notably, almost all of the reported virulence determinants are included in these variants affected CDSs. In addition to variations previously described in mycoplasma adhesins (P97, P102, P146, P159, P216, and LppT), cell envelope proteins (P95), cell surface antigens (P36), secreted proteins and chaperone protein (DnaK), mutations in genes related to metabolism and growth may also contribute to the attenuated virulence in 168-L. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for virulence. Conclusions We studied the virulence attenuation mechanism of M. hyopneumoniae by comparative genomic analysis of virulent strain 168 and its attenuated high-passage strain 168-L. Our findings provide a preliminary survey of CDSs that may be related to virulence. While these include reported virulence-related genes, other novel virulence determinants were also detected. This new information will form

  16. TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa.

    Science.gov (United States)

    Neidig, Anke; Yeung, Amy T Y; Rosay, Thibaut; Tettmann, Beatrix; Strempel, Nikola; Rueger, Martina; Lesouhaitier, Olivier; Overhage, Joerg

    2013-04-09

    Pseudomonas aeruginosa is an important opportunistic human pathogen and is extremely difficult to treat due to its high intrinsic and adaptive antibiotic resistance, ability to form biofilms in chronic infections and broad arsenal of virulence factors, which are finely regulated. TypA is a GTPase that has recently been identified to modulate virulence in enteric Gram-negative pathogens. Here, we demonstrate that mutation of typA in P. aeruginosa resulted in reduced virulence in phagocytic amoebae and human macrophage models of infection. In addition, the typA mutant was attenuated in rapid cell attachment to surfaces and biofilm formation, and exhibited reduced antibiotic resistance to ß-lactam, tetracycline and antimicrobial peptide antibiotics. Quantitative RT-PCR revealed the down-regulation, in a typA mutant, of important virulence-related genes such as those involved in regulation and assembly of the Type III secretion system, consistent with the observed phenotypes and role in virulence of P. aeruginosa. These data suggest that TypA is a newly identified modulator of pathogenesis in P. aeruginosa and is involved in multiple virulence-related characteristics.

  17. Gluconate metabolism is required for virulence of the soft-rot pathogen Pectobacterium carotovorum.

    Science.gov (United States)

    Mole, Beth; Habibi, Sohrab; Dangl, Jeffery L; Grant, Sarah R

    2010-10-01

    Pectobacterium carotovorum is a ubiquitous soft rot pathogen that uses global virulence regulators to coordinate pathogenesis in response to undefined environmental conditions. We characterize an operon in P. carotovorum required for gluconate metabolism and virulence. The operon contains four genes that are highly conserved among proteobacteria (initially annotated ygbJKLM), one of which was misassigned as a type III secreted effector, (ygbK, originally known as hopAN1). A mutant with a deletion-insertion within this operon is unable to metabolize gluconate, a precursor for the pentose phosphate pathway. The mutant exhibits attenuated growth on the leaves of its host of isolation, potato, and those of Arabidopsis thaliana. Notably, the mutant hypermacerates potato tubers and is deficient in motility. Global virulence regulators that are responsive to cell wall pectin breakdown products and other undefined environmental signals, KdgR and FlhD, respectively, are misregulated in the mutant. The alteration of virulence mediated via changes in transcription of known global virulence regulators in our ygbJ-M operon mutant suggests a role for host-derived catabolic intermediates in P. carotovorum pathogenesis. Thus, we rename this operon in P. carotovorum vguABCD for virulence and gluconate metabolism.

  18. Pressure surge attenuator

    Science.gov (United States)

    Christie, Alan M.; Snyder, Kurt I.

    1985-01-01

    A pressure surge attenuation system for pipes having a fluted region opposite crushable metal foam. As adapted for nuclear reactor vessels and heads, crushable metal foam is disposed to attenuate pressure surges.

  19. Campylobacter virulence and survival factors.

    Science.gov (United States)

    Bolton, Declan J

    2015-06-01

    Despite over 30 years of research, campylobacteriosis is the most prevalent foodborne bacterial infection in many countries including in the European Union and the United States of America. However, relatively little is known about the virulence factors in Campylobacter or how an apparently fragile organism can survive in the food chain, often with enhanced pathogenicity. This review collates information on the virulence and survival determinants including motility, chemotaxis, adhesion, invasion, multidrug resistance, bile resistance and stress response factors. It discusses their function in transition through the food processing environment and human infection. In doing so it provides a fundamental understanding of Campylobacter, critical for improved diagnosis, surveillance and control. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica

    DEFF Research Database (Denmark)

    Navarre, William Wiley; Zou, S Betty; Roy, Hervé

    2010-01-01

    phenotypic pleiotropy, including attenuated virulence in mice, an increased ability to respire under nutrient-limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors, and altered expression of multiple proteins, including several encoded on the SPI-1 pathogenicity island. Pox...

  1. Development of a human live attenuated West Nile infectious DNA vaccine: Suitability of attenuating mutations found in SA14-14-2 for WN vaccine design.

    Science.gov (United States)

    Yamshchikov, Vladimir; Manuvakhova, Marina; Rodriguez, Efrain

    2016-01-01

    Direct attenuation of West Nile (WN) virus strain NY99 for the purpose of vaccine development is not feasible due to its high virulence and pathogenicity. Instead, we created highly attenuated chimeric virus W1806 with the serological identity of NY99. To further attenuate W1806, we investigated effects of mutations found in Japanese encephalitis virus vaccine SA14-14-2. WN viruses carrying all attenuating mutations lost infectivity in mammalian, but not in mosquito cells. No single reversion restored infectivity in mammalian cells, although increased infectivity in mosquito cells was observed. To identify a subset of mutations suitable for further attenuation of W1806, we analyzed effects of E138K and K279M changes on virulence, growth properties, and immunogenicity of derivatized W956, from which chimeric W1806 inherited its biological properties and attenuation profile. Despite strong dominant attenuating effect, introduction of only two mutations was not sufficient for attenuating W1806 to the safety level acceptable for human use. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Molecular Mechanism of Quorum-Sensing in Enterococcus faecalis: Its Role in Virulence and Therapeutic Approaches.

    Science.gov (United States)

    Ali, Liaqat; Goraya, Mohsan Ullah; Arafat, Yasir; Ajmal, Muhammad; Chen, Ji-Long; Yu, Daojin

    2017-05-03

    Quorum-sensing systems control major virulence determinants in Enterococcus faecalis , which causes nosocomial infections. The E . faecalis quorum-sensing systems include several virulence factors that are regulated by the cytolysin operon, which encodes the cytolysin toxin. In addition, the E . faecalis Fsr regulator system controls the expression of gelatinase, serine protease, and enterocin O16. The cytolysin and Fsr virulence factor systems are linked to enterococcal diseases that affect the health of humans and other host models. Therefore, there is substantial interest in understanding and targeting these regulatory pathways to develop novel therapies for enterococcal infection control. Quorum-sensing inhibitors could be potential therapeutic agents for attenuating the pathogenic effects of E . faecalis . Here, we discuss the regulation of cytolysin, the LuxS system, and the Fsr system, their role in E . faecalis -mediated infections, and possible therapeutic approaches to prevent E . faecalis infection.

  3. Salmonella-secreted Virulence Factors

    Energy Technology Data Exchange (ETDEWEB)

    Heffron, Fred; Niemann, George; Yoon, Hyunjin; Kidwai, Afshan S.; Brown, Roslyn N.; McDermott, Jason E.; Smith, Richard D.; Adkins, Joshua N.

    2011-05-01

    In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellent reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.

  4. Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence

    Directory of Open Access Journals (Sweden)

    Orihuela Carlos J

    2007-08-01

    Full Text Available Abstract Background Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. Previously, using comparative genomic analyses, 13 regions of genomic plasticity have been identified in the S. pneumoniae genome. These "Regions of Diversity" (RDs accounted for half the genomic variation observed amongst all pneumococci tested, moreover, were determined to encode a variety of putative virulence factors. To date, genes within 5 RDs have been unequivocally demonstrated to contribute to S. pneumoniae virulence. It is unknown if the remaining RDs also contribute to virulence. Results Using allelic exchange, we created S. pneumoniae mutants that were deficient in RD2, 5, 7, 8, 9, 12 and 13. Mutants deficient in RD8, 9 and 13 were attenuated in a mouse model of disease. RD8 is 40,358 nucleotides in length and encodes 37 genes. Using a panel of isogenic mutants, we determined that RD8b3 is the operon within RD8 that is responsible for virulence. Mice infected with mutants deficient in RD8, RD8b3, RD9 and RD13 had significantly less bacteria in the blood two days after intranasal challenge and improved survival over time versus mice infected with wild type. In all instances mutants colonized the nasopharynx at levels equivalent to wild type. Conclusion Genes within RD1, 3, 4, 6, and 10 have previously been shown to contribute to virulence. This study demonstrates that genes within RD8, 9 and 13 also contribute to virulence. The ability of mutants deficient in RD2, 5, 7, 8, 9, 12, and 13 to colonize the nasopharynx indicates that genes within these RDs are not required for asymptomatic carriage. Nonetheless, the observation that mutants deficient in RD8b3, 9 and 13 are attenuated indicates that genes within these loci are necessary for spread of the bacteria beyond the nasopharynx to normally sterile sites.

  5. Vitamin B6 Is Required for Full Motility and Virulence in Helicobacter pylori

    OpenAIRE

    Grubman, Alexandra; Phillips, Alexandra; Thibonnier, Marie; Kaparakis-Liaskos, Maria; Johnson, Chad; Thiberge, Jean-Michel; Radcliff, Fiona J.; Ecobichon, Chantal; Labigne, Agn?s; de Reuse, Hilde; Mendz, George L.; Ferrero, Richard L.

    2010-01-01

    Despite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. To identify new virulence factors in H.?pylori, we generated low-infectivity variants of a mouse-colonizing H.?pylori strain using the classical technique of in vitro attenuation. The resulting variants and their highly infectious progenitor bacteria were then analyzed by global gene expression profiling. ...

  6. Loss of Lipid Virulence Factors Reduces the Efficacy of the BCG Vaccine

    Science.gov (United States)

    Tran, Vanessa; Ahn, Sang Kyun; Ng, Mark; Li, Ming; Liu, Jun

    2016-01-01

    Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. BCG comprises a number of substrains that exhibit genetic and biochemical differences. Whether and how these differences affect BCG efficacy remain unknown. Compared to other BCG strains, BCG-Japan, -Moreau, and -Glaxo are defective in the production of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), two lipid virulence factors. To determine if the loss of PDIMs/PGLs affects BCG efficacy, we constructed a PDIM/PGL-deficient strain of BCG-Pasteur by deleting fadD28, and compared virulence, immunogenicity, and protective efficacy in animal models. SCID mouse infection experiments showed that ∆fadD28 was more attenuated than wild type (WT). The ∆fadD28 and WT strains induced equivalent levels of antigen specific IFN-γ by CD4+ and CD8+ T cells; however, ∆fadD28 was less effective against Mycobacterium tuberculosis challenge in both BALB/c mice and guinea pigs. These results indicate that the loss of PIDMs/PGLs reduces the virulence and protective efficacy of BCG. Since the loss of PDIMs/PGLs occurs naturally in a subset of BCG strains, it also suggests that these strains may have been over-attenuated, which compromises their effectiveness. Our finding has important implications for current BCG programs and future vaccine development. PMID:27357109

  7. Virulence-associated plasmids in Rhodococcus equi.

    Science.gov (United States)

    Takai, S; Watanabe, Y; Ikeda, T; Ozawa, T; Matsukura, S; Tamada, Y; Tsubaki, S; Sekizaki, T

    1993-01-01

    Twenty-three strains of Rhodococcus equi from independent clinical cases were analyzed for the presence of virulence plasmid DNA. Of the clinical isolates, 19 contained an 85-kb plasmid and the remaining 4 contained a 90-kb plasmid. All of the isolates expressed 15- to 17-kDa antigens and were virulent in mice. Restriction enzyme and Southern blot analyses showed large regions of DNA homology between the 85- and 90-kb virulence plasmids. It was concluded tentatively that there are at least two virulence plasmids in R. equi and that they have a common origin. Images PMID:8349748

  8. Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity.

    Science.gov (United States)

    Park, Youngjin; Herbert, Erin E; Cowles, Charles E; Cowles, Kimberly N; Menard, Megan L; Orchard, Samantha S; Goodrich-Blair, Heidi

    2007-03-01

    Virulence of the insect pathogen Xenorhabdus nematophila is attributed in part to its ability to suppress immunity. For example, X. nematophila suppresses transcripts encoding several antimicrobial proteins, even in the presence of Salmonella enterica, an inducer of these transcripts. We show here that virulence and immune suppression phenotypes can be lost in a subpopulation of X. nematophila. Cells that have undergone 'virulence modulation' (vmo) have attenuated virulence and fail to suppress antimicrobial transcript levels, haemocyte aggregation and nodulation in Manduca sexta insects. When plated on certain media, vmo cells have a higher proportion of translucent (versus opaque) colonies compared with non-vmo cells. Like vmo strains, translucent colony isolates are defective in virulence and immune suppression. The X. nematophila genome encodes two 'opacity' genes with similarity to the Ail/PagC/Rck family of outer membrane proteins involved in adherence, invasion and serum resistance. Quantitative polymerase chain reaction analysis shows that RNA levels of one of these opacity genes, opaB, are higher in opaque relative to translucent colonies. We propose that in X. nematophila opaB may be one of several factors involved in immune suppression during infection, and expression of these factors can be co-ordinately eliminated in a subpopulation, possibly through a phase variation mechanism.

  9. Had1 Is Required for Cell Wall Integrity and Fungal Virulence in Cryptococcus neoformans

    Directory of Open Access Journals (Sweden)

    Won-Hee Jung

    2018-02-01

    Full Text Available Calcineurin modulates environmental stress survival and virulence of the human fungal pathogen Cryptococcus neoformans. Previously, we identified 44 putative calcineurin substrates, and proposed that the calcineurin pathway is branched to regulate targets including Crz1, Pbp1, and Puf4 in C. neoformans. In this study, we characterized Had1, which is one of the putative calcineurin substrates belonging to the ubiquitously conserved haloacid dehalogenase β-phosphoglucomutase protein superfamily. Growth of the had1∆ mutant was found to be compromised at 38° or higher. In addition, the had1∆ mutant exhibited increased sensitivity to cell wall perturbing agents, including Congo Red and Calcofluor White, and to an endoplasmic reticulum stress inducer dithiothreitol. Virulence studies revealed that the had1 mutation results in attenuated virulence compared to the wild-type strain in a murine inhalation infection model. Genetic epistasis analysis revealed that Had1 and the zinc finger transcription factor Crz1 play roles in parallel pathways that orchestrate stress survival and fungal virulence. Overall, our results demonstrate that Had1 is a key regulator of thermotolerance, cell wall integrity, and virulence of C. neoformans.

  10. Variable Virulence and Efficacy of BCG Vaccine Strains in Mice and Correlation With Genome Polymorphisms

    Science.gov (United States)

    Zhang, Lu; Ru, Huan-wei; Chen, Fu-zeng; Jin, Chun-yan; Sun, Rui-feng; Fan, Xiao-yong; Guo, Ming; Mai, Jun-tao; Xu, Wen-xi; Lin, Qing-xia; Liu, Jun

    2016-01-01

    Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development. PMID:26643797

  11. Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis

    Science.gov (United States)

    Lopez-Medina, Eduardo; Fan, Di; Coughlin, Laura A.; Ho, Evi X.; Lamont, Iain L.; Reimmann, Cornelia; Hooper, Lora V.; Koh, Andrew Y.

    2015-01-01

    Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa’s ability to colonize the GI tract but does decrease P. aeruginosa’s cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease. PMID:26313907

  12. Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1

    DEFF Research Database (Denmark)

    Wallrodt, Inke; Jelsbak, Lotte; Thomsen, Line E.

    2014-01-01

    , we investigated the contribution of individual psp genes to virulence of S. Typhimurium. Interestingly, deletion of the whole pspA-D set of genes caused attenuation in both NRAMP1(+) and NRAMP1(-) mice, indicating that one or more of the psp genes contribute to virulence independently of NRAMP1......IV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA....

  13. Genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains

    NARCIS (Netherlands)

    Berkhout, B.; Verhoef, K.; van Wamel, J. L.; Back, N. K.

    1999-01-01

    Live, attenuated viruses have been the most successful vaccines in monkey models of human immunodeficiency virus type 1 (HIV-1) infection. However, there are several safety concerns about using such an anti-HIV vaccine in humans, including reversion of the vaccine strain to virulence and

  14. An allele-specific polymerase chain reaction assay for the ...

    Indian Academy of Sciences (India)

    Unknown

    designated as species A, B (Green and Miles 1980), C. (Subbarao et al 1983), ... 3 | September 2004. O P Singh et al. 276 able to distinguish species A from species B/C when sin- gle mosquito-extract was diluted to 1/200. However such hybridization assay .... in Rameshwaram Island and Sri Lanka only) are not pre- sent.

  15. Allele specific gene expression on chromosome 7 in human tumorigenesis

    NARCIS (Netherlands)

    Boot, A.

    2017-01-01

    Both copy number losses and homozygosity of chromosome 7 are extremely rare events in many tumor types, indicating that the retention of both the maternal and paternal copies of chromosome 7 is essential for the tumor cells. This thesis compiles our research into the driving force that is behind the

  16. An allele-specific polymerase chain reaction assay for the ...

    Indian Academy of Sciences (India)

    Unknown

    Krzywinski J and Besansky N J 2003 Molecular systematics of. Anopheles: from subgenera to subpopulations; Annu. Rev. Entomol. 48 111–139. Litvaitis M K, Nunn G, Thomas W K and Kocher T D 1994 A molecular approach for the identification of Meiofaunal tur- bellarians (Platyhelminthes, Turbellaria); Marine Biol. 120.

  17. Anaerobiosis induced virulence of Salmonella typhi

    DEFF Research Database (Denmark)

    Kapoor, Sarika; Singh, R D; Sharma, P C

    2002-01-01

    , we examined the effect of anaerobiosis on the virulence of Salmonella Typhi, a Gram negative bacteria which invades through the gut mucosa and is responsible for typhoid fever. METHODS: Salmonella Typhi (ty2) was cultured in aerobic and anaerobic conditions to compare its virulence by rabbit ileal...

  18. Anaerobiosis induced virulence of Salmonella typhi

    DEFF Research Database (Denmark)

    Kapoor, Sarika; Singh, R D; Sharma, P C

    2002-01-01

    BACKGROUND & OBJECTIVES: Anaerobic conditions are frequently encountered by pathogens invading the gastrointestinal tract due to low/limiting oxygen conditions prevalent in the small intestine. This anaerobic stress has been suggested to enhance the virulence of gut pathogens. In the present stud...... dismutase (SOD) and catalase. INTERPRETATION & CONCLUSION: Our results suggest that exposure of S. Typhi to anaerobic conditions enhances its virulence....

  19. Evolution of viral virulence: empirical studies

    Science.gov (United States)

    Kurath, Gael; Wargo, Andrew R.

    2016-01-01

    The concept of virulence as a pathogen trait that can evolve in response to selection has led to a large body of virulence evolution theory developed in the 1980-1990s. Various aspects of this theory predict increased or decreased virulence in response to a complex array of selection pressures including mode of transmission, changes in host, mixed infection, vector-borne transmission, environmental changes, host vaccination, host resistance, and co-evolution of virus and host. A fundamental concept is prediction of trade-offs between the costs and benefits associated with higher virulence, leading to selection of optimal virulence levels. Through a combination of observational and experimental studies, including experimental evolution of viruses during serial passage, many of these predictions have now been explored in systems ranging from bacteriophage to viruses of plants, invertebrates, and vertebrate hosts. This chapter summarizes empirical studies of viral virulence evolution in numerous diverse systems, including the classic models myxomavirus in rabbits, Marek's disease virus in chickens, and HIV in humans. Collectively these studies support some aspects of virulence evolution theory, suggest modifications for other aspects, and show that predictions may apply in some virus:host interactions but not in others. Finally, we consider how virulence evolution theory applies to disease management in the field.

  20. Evolution, Clonality and Some Virulence Characteristics of ...

    African Journals Online (AJOL)

    All these changes together with other factors which may be genetic in origin have resulted in evolution, the existence of clones and the occurrence and acquisition of virulence characteristics of enterohaemorrhagic Escherichia coli. Keywords: Evolution, clonality, enterohaemorrhagic E. coli, virulence characteristics.

  1. Virulence, serotype and phylogenetic groups of diarrhoeagenic ...

    African Journals Online (AJOL)

    Dr DADIE Thomas

    2014-02-17

    Feb 17, 2014 ... The virulence, serotype and phylogenetic traits of diarrhoeagenic Escherichia coli were detected in 502 strains isolated during digestive infections. Molecular detection of the target virulence genes, rfb gene of operon O and phylogenetic grouping genes Chua, yjaA and TSPE4.C2 was performed.

  2. Stilbenes reduce Staphylococcus aureus hemolysis, biofilm formation, and virulence.

    Science.gov (United States)

    Lee, Kayeon; Lee, Jin-Hyung; Ryu, Shi Yong; Cho, Moo Hwan; Lee, Jintae

    2014-09-01

    Stilbenoids have a broad range of beneficial health effects. On the other hand, the emergence of antibiotic-resistant Staphylococcus aureus presents a worldwide problem that requires new antibiotics or nonantibiotic strategies. S. aureus produces α-hemolysin (a pore-forming cytotoxin) that has been implicated in the pathogenesis of sepsis and pneumonia. Furthermore, the biofilms formed by S. aureus constitute a mechanism of antimicrobial resistance. In this study, we investigated the hemolytic and antibiofilm activities of 10 stilbene-related compounds against S. aureus. trans-Stilbene and resveratrol at 10 μg/mL were found to markedly inhibit human blood hemolysis by S. aureus, and trans-stilbene also inhibited S. aureus biofilm formation without affecting its bacterial growth. Furthermore, trans-stilbene and resveratrol attenuated S. aureus virulence in vivo in the nematode Caenorhabditis elegans, which is normally killed by S. aureus. Transcriptional analysis showed that trans-stilbene repressed the α-hemolysin hla gene and the intercellular adhesion locus (icaA and icaD) in S. aureus, and this finding was in line with observed reductions in virulence and biofilm formation. In addition, vitisin B, a stilbenoid tetramer, at 1 μg/mL was observed to significantly inhibit human blood hemolysis by S. aureus.

  3. Klebsiella pneumoniae FimK Promotes Virulence in Murine Pneumonia.

    Science.gov (United States)

    Rosen, David A; Hilliard, Julia K; Tiemann, Kristin M; Todd, Elizabeth M; Morley, S Celeste; Hunstad, David A

    2016-02-15

    Klebsiella pneumoniae, a chief cause of nosocomial pneumonia, is a versatile and commonly multidrug-resistant human pathogen for which further insight into pathogenesis is needed. We show that the pilus regulatory gene fimK promotes the virulence of K. pneumoniae strain TOP52 in murine pneumonia. This contrasts with the attenuating effect of fimK on urinary tract virulence, illustrating that a single factor may exert opposing effects on pathogenesis in distinct host niches. Loss of fimK in TOP52 pneumonia was associated with diminished lung bacterial burden, limited innate responses within the lung, and improved host survival. FimK expression was shown to promote serum resistance, capsule production, and protection from phagocytosis by host immune cells. Finally, while the widely used K. pneumoniae model strain 43816 produces rapid dissemination and death in mice, TOP52 caused largely localized pneumonia with limited lethality, thereby providing an alternative tool for studying K. pneumoniae pathogenesis and control within the lung. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  4. Virulence of Fusarium species to alfalfa seedlings

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    Krnjaja Vesna

    2005-01-01

    Full Text Available In in vitro conditions, virulence of 91 isolates of species Fusarium genus (F. oxysporum, F. solani, F. acuminatum, F. equiseti, F. arthrosporioides, F. prolifera- tum, F. avenaceum, F. semitectum, F. tricinctum, F. sporotrichioides and F. graminearum towards alfalfa seedlings was investigated. Isolates of investigated species originated from diseased alfalfa plants collected at four locations in Serbia based on symptoms of wilting caused by Fusarium and root rotting. Pathogenicity and virulence of investigated isolates of Fusarium spp. were determined by visual evaluation of inoculated seedlings of cultivar K28 in laboratory conditions. All isolated of investigated species had pathogenic effect on alfalfa seedlings which expressed symptoms such as necrosis of root, moist rotting and "melting of seedlings". Colour of necrotic root tissue varied from light brown, brown lipstick red to explicit black, depending on the Fusarium species. Strong virulence was established in 48 isolates, medium virulence in 31 and weak virulence in 12 isolates.

  5. Virulence of Fusarium species to alfalfa seedlings

    Directory of Open Access Journals (Sweden)

    Krnjaja Vesna

    2005-01-01

    Full Text Available In in vitro conditions, virulence of 91 isolates of species Fusarium genus (F. oxysporum, F. solani, F. acuminatum, F. equiseti, F. arthrosporioides, F. proliferatum, F. avenaceum, F. semitectum, F. tricinctum, F. sporotrichioides and F. graminearum towards alfalfa seedlings was investigated. Isolates of investigated species originated from diseased alfalfa plants collected on four locations in Serbia based on symptoms of wilting caused by fusarium and root rotting. Pathogenicity and virulence of investigated isolates of Fusarium spp. were determined by visual evaluation of inoculated seedlings of cultivars K28 in laboratory conditions. All isolated of investigated species had pathogenic effect on alfalfa seedlings, which expressed symptoms such as necrosis of root, moist rotting and "melting of seedlings". Colour of necrotic root tissue varied from light brown, brown, lipstick red to explicit black, depending on the Fusarium species. Strong virulence was established in 48 isolates, medium virulence in 31 and weak virulence in 12 isolates.

  6. The Role of TonB Gene in Edwardsiella ictaluri Virulence

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    Hossam Abdelhamed

    2017-12-01

    Full Text Available Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen that causes enteric septicemia in catfish (ESC. Stress factors including poor water quality, poor diet, rough handling, overcrowding, and water temperature fluctuations increase fish susceptibility to ESC. The TonB energy transducing system (TonB-ExbB-ExbD and TonB-dependent transporters of Gram-negative bacteria support active transport of scarce resources including iron, an essential micronutrient for bacterial virulence. Deletion of the tonB gene attenuates virulence in several pathogenic bacteria. In the current study, the role of TonB (NT01EI_RS07425 in iron acquisition and E. ictaluri virulence were investigated. To accomplish this, the E. ictaluri tonB gene was in-frame deleted. Growth kinetics, iron utilization, and virulence of the EiΔtonB mutant were determined. Loss of TonB caused a significant reduction in bacterial growth in iron-depleted medium (p > 0.05. The EiΔtonB mutant grew similarly to wild-type E. ictaluri when ferric iron was added to the iron-depleted medium. The EiΔtonB mutant was significantly attenuated in catfish compared with the parent strain (21.69 vs. 46.91% mortality. Catfish surviving infection with EiΔtonB had significant protection against ESC compared with naïve fish (100 vs. 40.47% survival. These findings indicate that TonB participates in pathogenesis of ESC and is an important E. ictaluri virulence factor.

  7. Xenorhabdus bovienii CS03, the bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran insects.

    Science.gov (United States)

    Bisch, Gaëlle; Pagès, Sylvie; McMullen, John G; Stock, S Patricia; Duvic, Bernard; Givaudan, Alain; Gaudriault, Sophie

    2015-01-01

    Xenorhabdus bacteria (γ-proteobacteria: Enterobacteriaceae) have dual lifestyles. They have a mutualistic relationship with Steinernema nematodes (Nematoda: Steinernematidae) and are pathogenic to a wide range of insects. Each Steinernema nematode associates with a specific Xenorhabdus species. However, a Xenorhabdus species can have multiple nematode hosts. For example, Xenorhabdus bovienii (Xb) colonizes at least nine Steinernema species from two different phylogenetic clades. The Steinernema-Xb partnership has been found in association with different insect hosts. Biological and molecular data on the Steinernema jollieti-Xb strain SS-2004 pair have recently been described. In particular, the Xb SS-2004 bacteria are virulent alone after direct injection into insect, making this strain a model for studying Xb virulence. In this study, we searched for Xb strains attenuated in virulence. For this purpose, we underwent infection assays with five Steinernema spp.-Xb pairs with two insects, Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). The S. weiseri-Xb CS03 pair showed attenuated virulence and lower fitness in S. littoralis in comparison to the other nematode-bacteria pairs. Furthermore, when injected alone into the hemolymph of G. mellonella or S. littoralis, the Xb CS03 bacterial strain was the only non-virulent strain. By comparison with the virulent Xb SS-2004 strain, Xb CS03 showed an increased sensitivity to the insect antimicrobial peptides, suggesting an attenuated response to the insect humoral immunity. To our current knowledge, Xb CS03 is the first non-virulent Xb strain identified. We propose this strain as a new model for studying the Xenorhabdus virulence. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. An attemp at reversibility and increase of the virulence of axenic strains of Entamoeba histolytica Tentativa de reversibilidade e aumento de virulência de cepas axônicas de Entamoeba histolytica

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    Maria Aparecida Gomes

    1993-12-01

    Full Text Available In this study we have tried to verify whether the interaction "in vitro" with bacteria or small pieces of normal hamster liver would modify the pathogenic behavior of axenic strains of E. histolytica: avirulent ones (ICB-32 and ICB-RPS, of attenuated virulence (ICB-CSP and HM1 and of mean virulence (ICB-462. Every attempt to render virulent, recover or increase the virulence of axenic strains of E. histolytica has failedNeste trabalho procuramos verificar se a interação "in vitro" com bactérias e fragmentos de fígado de hamster normal, modificaria o comportamento patogênico de cepas axênicas de E. histolytica avirulentas (ICB-32 e ICB-RPS; virulentas, porém atenuadas (ICB-CSP e HM1 e de média virulência (ICB-462. Todas as tentativas de tornar virulentas, restabelecer ou aumentar a virulência das cepas axênicas de E. histolytica utilizadas fracassaram

  9. Landing gear noise attenuation

    Science.gov (United States)

    Moe, Jeffrey W. (Inventor); Whitmire, Julia (Inventor); Kwan, Hwa-Wan (Inventor); Abeysinghe, Amal (Inventor)

    2011-01-01

    A landing gear noise attenuator mitigates noise generated by airframe deployable landing gear. The noise attenuator can have a first position when the landing gear is in its deployed or down position, and a second position when the landing gear is in its up or stowed position. The noise attenuator may be an inflatable fairing that does not compromise limited space constraints associated with landing gear retraction and stowage. A truck fairing mounted under a truck beam can have a compliant edge to allow for non-destructive impingement of a deflected fire during certain conditions.

  10. Effects of Elevated CO₂and Temperature on Pathogenicity Determinants and Virulence of Potato virus X/Potyvirus-Associated Synergism.

    Science.gov (United States)

    Aguilar, Emmanuel; Allende, Lucía; Del Toro, Francisco J; Chung, Bong-Nam; Canto, Tomás; Tenllado, Francisco

    2015-12-01

    Infections of plants by multiple viruses are common in nature and may result in synergisms in pathologies. Several environmental factors influence plant-virus interactions and act on virulence and host defense responses. Mixed viral infections may be more frequent under environmental conditions associated with global warming. Here, we address how changes in the two main parameters behind global warming, carbon dioxide concentrations ([CO₂]) and temperature, may affect virulence of Potato virus X (PVX)/potyvirus-associated synergism compared with single infections in Nicotiana benthamiana. Elevated [CO₂] resulted in attenuated virulence of single infection by PVX, which correlated with a lower accumulation of virus. In contrast, virulence of PVX/potyvirus-associated synergism was maintained at elevated [CO₂]. On the other hand, elevated temperature decreased markedly both virulence and virus titers in the synergistic infection. We also show that the HR-like response elicited by transient coexpression of PVX P25 together with the potyviral helper component-proteinase protein was significantly enhanced by elevated temperature, whereas it was reduced by elevated [CO₂]. Both proteins are main pathogenicity determinants in PVX-associated synergisms. These findings indicate that, under environmental conditions associated with global warming, virulence of PVX/potyvirus-associated synergisms is expected to vary relative to single infections and, thus, may have pathological consequences in the future.

  11. Fis is a global regulator critical for modulation of virulence factor production and pathogenicity of Dickeya zeae.

    Science.gov (United States)

    Lv, Mingfa; Chen, Yufan; Liao, Lisheng; Liang, Zhibin; Shi, Zurong; Tang, Yingxin; Ye, Sixuan; Zhou, Jianuan; Zhang, Lianhui

    2018-01-10

    Dickeya zeae is the causal agent of rice foot rot disease, which has recently become a great threat to rice planting countries and regions. The pathogen produces a family of phytotoxins named zeamines that is critical for bacterial virulence, but little is known about the signaling pathways and regulatory mechanisms that govern zeamine production. In this study, we showed that a conserved transcriptional regulator Fis is involved in the regulation of zeamine production in D. zeae strain EC1. Deletion mutants were markedly attenuated in the virulence against rice seed germination. Transcriptome and phenotype analyses showed that Fis is a potent global transcriptional regulator modulating various virulence traits, including production of extracellular enzymes and exopolysaccharides, swimming and swarming motility, biofilm formation and cell aggregation. DNA gel retardation analysis showed that Fis directly regulates the transcription of key virulence genes and the genes encoding Vfm quorum sensing system through DNA/protein interaction. Our findings unveil a key regulator associated with the virulence of D. zeae EC1, and present useful clues for further elucidation of the regulatory complex and signaling pathways which govern the virulence of this important pathogen.

  12. Staphylococcus aureus virulence factors identified by using a high-throughput Caenorhabditis elegans-killing model.

    Science.gov (United States)

    Begun, Jakob; Sifri, Costi D; Goldman, Samuel; Calderwood, Stephen B; Ausubel, Frederick M

    2005-02-01

    Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.

  13. Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei that Provides Partial Protection against Inhalational Glanders in Mice

    Science.gov (United States)

    2016-02-26

    in vitro and in vivo phenotypes, we explored the use of 1TssN as a candidate live -attenuated vaccine . Mice immunized with aerosolized 1TssN showed a... vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating ...acute infection in inhalational glanders. Keywords: Burkholderia mallei, virulence factor, live -attenuated vaccine , glanders, aerosol Bozue et al

  14. Discerning an Effective Balance between Equine Infectious Anemia Virus Attenuation and Vaccine Efficacy

    Science.gov (United States)

    Craigo, Jodi K.; Li, Feng; Steckbeck, Jonathan D.; Durkin, Shannon; Howe, Laryssa; Cook, Sheila J.; Issel, Charles; Montelaro, Ronald C.

    2005-01-01

    Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAVUKΔS2 derived from a virulent proviral clone, EIAVUKΔS2/DU containing a second gene mutation in the virulent proviral clone, and EIAVPRΔS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 102 to 103 RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAVPV strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity. PMID:15708986

  15. Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy.

    Science.gov (United States)

    Craigo, Jodi K; Li, Feng; Steckbeck, Jonathan D; Durkin, Shannon; Howe, Laryssa; Cook, Sheila J; Issel, Charles; Montelaro, Ronald C

    2005-03-01

    Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAV(UK)DeltaS2 derived from a virulent proviral clone, EIAV(UK)DeltaS2/DU containing a second gene mutation in the virulent proviral clone, and EIAV(PR)DeltaS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 10(2) to 10(3) RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAV(PV) strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity.

  16. Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response.

    Science.gov (United States)

    Reis, Ana Luisa; Abrams, Charles C; Goatley, Lynnette C; Netherton, Chris; Chapman, Dave G; Sanchez-Cordon, Pedro; Dixon, Linda K

    2016-09-07

    African swine fever virus (ASFV) encodes multiple copies of MGF360 and MGF530/505 gene families. These genes have been implicated in the modulation of the type I interferon (IFN) response. We investigated the effect of modulating the IFN response on virus attenuation and induction of protective immunity by deleting genes MGF360 (MGF360-10L, 11L, 12L, 13L, 14L) and MGF530/505 (MGF530/505-1R, 2R and 3R) and interrupting genes (MGF360-9L and MGF530/505-4R) in the genome of the virulent ASFV isolate Benin 97/1. Replication of this deletion mutant, BeninΔMGF, in porcine macrophages in vitro was similar to that of the parental virulent virus Benin 97/1 and the natural attenuated isolate OURT88/3, which has a similar deletion of MGF360 and 530/505 genes. Levels of IFN-β mRNA in macrophages infected with virulent Benin 97/1 isolate were barely detectable but high levels were detected in macrophages infected with OURT88/3 and intermediate levels in macrophages infected with BeninΔMGF. The data confirms that these MGF360 and MGF530/505 genes have roles in suppressing induction of type I IFN. Immunisation and boost of pigs with BeninΔMGF showed that the virus was attenuated and all pigs (5/5) were protected against challenge with a lethal dose of virulent Benin 97/1. A short transient fever was observed at day 5 or 6 post-immunisation but no other clinical signs. Following immunisation and boost with the OURT88/3 isolate 3 of 4 pigs were protected against challenge. Differences were observed in the cellular and antibody responses in pigs immunised with BeninΔMGF compared to OURT88/3. Deletion of IFN modulators is a promising route for construction of rationally attenuated ASFV candidate vaccine strains. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Two Major Inositol Transporters and Their Role in Cryptococcal Virulence ▿ †

    Science.gov (United States)

    Wang, Yina; Liu, Tong-bao; Delmas, Guillaume; Park, Steven; Perlin, David; Xue, Chaoyang

    2011-01-01

    Cryptococcus neoformans is an AIDS-associated human fungal pathogen and the most common cause of fungal meningitis, with a mortality rate over 40% in AIDS patients. Significant advances have been achieved in understanding its disease mechanisms. Yet the underlying mechanism of a high frequency of cryptococcal meningitis remains unclear. The existence of high inositol concentrations in brain and our earlier discovery of a large inositol transporter (ITR) gene family in C. neoformans led us to investigate the potential role of inositol in Cryptococcus-host interactions. In this study, we focus on functional analyses of two major ITR genes to understand their role in virulence of C. neoformans. Our results show that ITR1A and ITR3C are the only two ITR genes among 10 candidates that can complement the growth defect of a Saccharomyces cerevisiae strain lacking inositol transporters. Both S. cerevisiae strains heterologously expressing ITR1A or ITR3C showed high inositol uptake activity, an indication that they are major inositol transporters. Significantly, itr1a itr3c double mutants showed significant virulence attenuation in murine infection models. Mutating both ITR1A and ITR3C in an ino1 mutant background activates the expression of several remaining ITR candidates and does not show more severe virulence attenuation, suggesting that both inositol uptake and biosynthetic pathways are important for inositol acquisition. Overall, our study provides evidence that host inositol and fungal inositol transporters are important for Cryptococcus pathogenicity. PMID:21398509

  18. Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model.

    Directory of Open Access Journals (Sweden)

    Rhonda L Feinbaum

    Full Text Available Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700 were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

  19. Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model.

    Science.gov (United States)

    Feinbaum, Rhonda L; Urbach, Jonathan M; Liberati, Nicole T; Djonovic, Slavica; Adonizio, Allison; Carvunis, Anne-Ruxandra; Ausubel, Frederick M

    2012-01-01

    Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

  20. Lability of the pAA Virulence Plasmid in Escherichia coli O104:H4: Implications for Virulence in Humans.

    Directory of Open Access Journals (Sweden)

    Wenlan Zhang

    Full Text Available Escherichia coli O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC and enteroaggregative (EAEC E. coli. The strain displays "stacked-brick" aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I encoded on the pAA plasmid. The AAF/I-mediated augmented intestinal adherence might facilitate systemic absorption of Shiga toxin, the major virulence factor of EHEC, presumably enhancing virulence of the outbreak strain. However, the stability of pAA in the outbreak strain is unknown. We therefore tested outbreak isolates for pAA, monitored pAA loss during infection, and determined the impact of pAA loss on adherence and clinical outcome of infection.E. coli O104:H4 outbreak isolates from 170 patients (128 with hemolytic uremic syndrome [HUS] and 42 with diarrhea without HUS were tested for pAA using polymerase chain reaction and plasmid profiling. pAA-harboring bacteria in stool samples were quantified using colony blot hybridization, and adherence to HCT-8 cells was determined. Isolates from 12 (7.1% patients lacked pAA. Analyses of sequential stool samples demonstrated that the percentages of pAA-positive populations in the initial stools were significantly higher than those in the follow-up stools collected two to eight days later in disease (P≤0.01. This indicates a rapid loss of pAA during infections of humans. The pAA loss was associated with loss of the aggregative adherence phenotype and significantly reduced correlation with HUS (P  = 0.001.The pAA plasmid can be lost by E. coli O104:H4 outbreak strain in the human gut in the course of disease. pAA loss might attenuate virulence and diminish the ability to cause HUS. The pAA instability has clinical, diagnostic, epidemiologic, and evolutionary implications.

  1. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

    KAUST Repository

    Houben, Diane

    2012-05-08

    Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

  2. AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

    Science.gov (United States)

    Guiton, Pascale S.; Barnes, Aaron M. T.; Manias, Dawn A.; Chuang-Smith, Olivia N.; Kohler, Petra L.; Spaulding, Adam R.; Hultgren, Scott J.; Schlievert, Patrick M.; Dunny, Gary M.

    2013-01-01

    Enterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species. PMID:23460519

  3. Multiple roles of putrescine and spermidine in stress resistance and virulence of Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Espinel, Irene Cartas; Guerra, Priscila Regina; Jelsbak, Lotte

    2016-06-01

    Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present in most living cells. In recent years they have been linked to virulence of several human pathogens including Shigella spp and Salmonella enterica serovar Typhimurium (S. Typhimurium). Central to S. Typhimurium virulence is the ability to survive and replicate inside macrophages and resisting the antimicrobial attacks in the form of oxidative and nitrosative stress elicited from these cells. In the present study, we have investigated the role of polyamines in intracellular survival and systemic infections of mice. Using a S. Typhimurium mutant defective for putrescine and spermidine biosynthesis, we show that polyamines are essential for coping with reactive nitrogen species, possibly linking polyamines to increased intracellular stress resistance. However, using a mouse model defective for nitric oxide production, we find that polyamines are required for systemic infections independently of host-produced reactive nitrogen species. To distinguish between the physiological roles of putrescine and spermidine, we constructed a strain deficient for spermidine biosynthesis and uptake, but with retained ability to produce and import putrescine. Interestingly, in this mutant we observe a strong attenuation of virulence during infection of mice proficient and deficient for nitric oxide production suggesting that spermidine, specifically, is essential for virulence of S. Typhimurium. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Oxidoreductases that act as conditional virulence suppressors in Salmonella enterica serovar Typhimurium.

    Directory of Open Access Journals (Sweden)

    Naeem Anwar

    Full Text Available In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium.

  5. The novel polysaccharide deacetylase homologue Pdi contributes to virulence of the aquatic pathogen Streptococcus iniae

    Science.gov (United States)

    Milani, Carlo J. E.; Aziz, Ramy K.; Locke, Jeffrey B.; Dahesh, Samira; Nizet, Victor; Buchanan, John T.

    2010-01-01

    The aquatic zoonotic pathogen Streptococcus iniae represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of S. iniae pathogenesis or virulence factors, we established a high-throughput system combining whole-genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of S. iniae, that we describe here. Using bioinformatics tools, we identified a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A Δpdi mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing or resistance to cationic antimicrobial peptides between S. iniae wild-type and Δpdi. In conclusion, we have demonstrated that pdi is involved in S. iniae adherence and invasion, lysozyme resistance and survival in fish blood, and have shown that pdi plays a role in the pathogenesis of S. iniae. Identification of Pdi and other S. iniae virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economic losses caused by this pathogen. PMID:19762441

  6. Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis

    KAUST Repository

    Ates, Louis S.

    2018-01-12

    Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems1,2. The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus3,4. However, the role of these proteins remains largely elusive1. Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion5. Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the ‘modern’ Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution6,7.

  7. Virulence Factors IN Fungi OF Systemic Mycoses

    Directory of Open Access Journals (Sweden)

    KUROKAWA Cilmery Suemi

    1998-01-01

    Full Text Available Pathogenic fungi that cause systemic mycoses retain several factors which allow their growth in adverse conditions provided by the host, leading to the establishment of the parasitic relationship and contributing to disease development. These factors are known as virulence factors which favor the infection process and the pathogenesis of the mycoses. The present study evaluates the virulence factors of pathogenic fungi such as Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Paracoccidioides brasiliensis in terms of thermotolerance, dimorphism, capsule or cell wall components as well as enzyme production. Virulence factors favor fungal adhesion, colonization, dissemination and the ability to survive in hostile environments and elude the immune response mechanisms of the host. Both the virulence factors presented by different fungi and the defense mechanisms provided by the host require action and interaction of complex processes whose knowledge allows a better understanding of the pathogenesis of systemic mycoses.

  8. The evolution of virulence and emerging diseases

    OpenAIRE

    Ewald, Paul W.

    1998-01-01

    Insights into the evolution of virulence may aid efforts to control or even prevent emerging diseases. Specifically, dangerous pathogens can be distinguished from those that pose relatively little threat by identifying characteristics that favor intense exploitation of hosts by pathogens, hence causing high virulence. Studies to date have implicated several such characteristics, including transmission by vectors, attendants, water, and durable propagules. These insights may improve the return...

  9. Rapid Identification of Bacterial Virulence Factors

    Science.gov (United States)

    2014-04-15

    anti-5. melitensis antibodies. In Brucella this protein shares homology with that of plant pathogen Agrobacterium tumefaciens wherein it is involved...Brucella suis homologue of the Agrobacterium tumefaciens chromosomal virulence operon ChvE is essential for sugar utilization but not for survival in...Phosphoenolpyruvate Carboxykinase Is an Acid-Induced, Chromosomally Encoded Virulence Factor in Agrobacterium tumefaciens . J. Bacteriol 187:6039-6045. Martin-Martin

  10. Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1.

    Science.gov (United States)

    Wallrodt, Inke; Jelsbak, Lotte; Thomsen, Line E; Brix, Lena; Lemire, Sébastien; Gautier, Laurent; Nielsen, Dennis S; Jovanovic, Goran; Buck, Martin; Olsen, John E

    2014-06-01

    The phage-shock protein (Psp) system is believed to manage membrane stress in all Enterobacteriaceae and has recently emerged as being important for virulence in several pathogenic species of this phylum. The core of the Psp system consists of the pspA-D operon and the distantly located pspG gene. In Salmonella enterica serovar Typhimurium (S. Typhimurium), it has recently been reported that PspA is essential for systemic infection of mice, but only in NRAMP1(+) mice, signifying that attenuation is related to coping with divalent cation starvation in the intracellular environment. In the present study, we investigated the contribution of individual psp genes to virulence of S. Typhimurium. Interestingly, deletion of the whole pspA-D set of genes caused attenuation in both NRAMP1(+) and NRAMP1(-) mice, indicating that one or more of the psp genes contribute to virulence independently of NRAMP1 expression in the host. Investigations of single gene mutants showed that knock out of pspB reduced virulence in both types of mice, while deletion of pspA only caused attenuation in NRAMP1(+) mice, and deletion of pspD had a minor effect in NRAMP1(-) mice, while deletions of either pspC or pspG did not affect virulence. Experiments addressed at elucidating the role of PspB in virulence revealed that PspB is dispensable for uptake to and intracellular replication in cultured macrophages and resistance to complement-induced killing. Furthermore, the Psp system of S. Typhimurium was dispensable during pIV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA. © 2014 The Authors.

  11. A multi-omic analysis reveals the role of fumarate in regulating the virulence of enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    Kuo, Cheng-Ju; Wang, Sin-Tian; Lin, Chia-Mei; Chiu, Hao-Chieh; Huang, Cheng-Rung; Lee, Der-Yen; Chang, Geen-Dong; Chou, Ting-Chen; Chen, Jenn-Wei; Chen, Chang-Shi

    2018-03-07

    The enteric pathogen enterohemorrhagic Escherichia coli (EHEC) is responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Several molecular mechanisms have been described for the pathogenicity of EHEC; however, the role of bacterial metabolism in the virulence of EHEC during infection in vivo remains unclear. Here we show that aerobic metabolism plays an important role in the regulation of EHEC virulence in Caenorhabditis elegans. Our functional genomic analyses showed that disruption of the genes encoding the succinate dehydrogenase complex (Sdh) of EHEC, including the sdhA gene, attenuated its toxicity toward C. elegans animals. Sdh converts succinate to fumarate and links the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) simultaneously. Succinate accumulation and fumarate depletion in the EHEC sdhA mutant cells were also demonstrated to be concomitant by metabolomic analyses. Moreover, fumarate replenishment to the sdhA mutant significantly increased its virulence toward C. elegans. These results suggest that the TCA cycle, ETC, and alteration in metabolome all account for the attenuated toxicity of the sdhA mutant, and Sdh catabolite fumarate in particular plays a critical role in the regulation of EHEC virulence. In addition, we identified the tryptophanase (TnaA) as a downstream virulence determinant of SdhA using a label-free proteomic method. We demonstrated that expression of tnaA is regulated by fumarate in EHEC. Taken together, our multi-omic analyses demonstrate that sdhA is required for the virulence of EHEC, and aerobic metabolism plays important roles in the pathogenicity of EHEC infection in C. elegans. Moreover, our study highlights the potential targeting of SdhA, if druggable, as alternative preventive or therapeutic strategies by which to combat EHEC infection.

  12. Patterns of variation at Ustilago maydis virulence clusters 2A and 19A largely reflect the demographic history of its populations.

    Directory of Open Access Journals (Sweden)

    Ronny Kellner

    Full Text Available The maintenance of an intimate interaction between plant-biotrophic fungi and their hosts over evolutionary times involves strong selection and adaptative evolution of virulence-related genes. The highly specialised maize pathogen Ustilago maydis is assigned with a high evolutionary capability to overcome host resistances due to its high rates of sexual recombination, large population sizes and long distance dispersal. Unlike most studied fungus-plant interactions, the U. maydis - Zea mays pathosystem lacks a typical gene-for-gene interaction. It exerts a large set of secreted fungal virulence factors that are mostly organised in gene clusters. Their contribution to virulence has been experimentally demonstrated but their genetic diversity within U. maydis remains poorly understood. Here, we report on the intraspecific diversity of 34 potential virulence factor genes of U. maydis. We analysed their sequence polymorphisms in 17 isolates of U. maydis from Europe, North and Latin America. We focused on gene cluster 2A, associated with virulence attenuation, cluster 19A that is crucial for virulence, and the cluster-independent effector gene pep1. Although higher compared to four house-keeping genes, the overall levels of intraspecific genetic variation of virulence clusters 2A and 19A, and pep1 are remarkably low and commensurate to the levels of 14 studied non-virulence genes. In addition, each gene is present in all studied isolates and synteny in cluster 2A is conserved. Furthermore, 7 out of 34 virulence genes contain either no polymorphisms or only synonymous substitutions among all isolates. However, genetic variation of clusters 2A and 19A each resolve the large scale population structure of U. maydis indicating subpopulations with decreased gene flow. Hence, the genetic diversity of these virulence-related genes largely reflect the demographic history of U. maydis populations.

  13. Host range, growth property, and virulence of the smallpox vaccine: Vaccinia virus Tian Tan strain

    International Nuclear Information System (INIS)

    Fang Qing; Yang Lin; Zhu Weijun; Liu Li; Wang Haibo; Yu Wenbo; Xiao Genfu; Tien Po; Zhang Linqi; Chen Zhiwei

    2005-01-01

    Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene. Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens

  14. Radiofrequency attenuator and method

    Science.gov (United States)

    Warner, Benjamin P [Los Alamos, NM; McCleskey, T Mark [Los Alamos, NM; Burrell, Anthony K [Los Alamos, NM; Agrawal, Anoop [Tucson, AZ; Hall, Simon B [Palmerston North, NZ

    2009-01-20

    Radiofrequency attenuator and method. The attenuator includes a pair of transparent windows. A chamber between the windows is filled with molten salt. Preferred molten salts include quarternary ammonium cations and fluorine-containing anions such as tetrafluoroborate (BF.sub.4.sup.-), hexafluorophosphate (PF.sub.6.sup.-), hexafluoroarsenate (AsF.sub.6.sup.-), trifluoromethylsulfonate (CF.sub.3SO.sub.3.sup.-), bis(trifluoromethylsulfonyl)imide ((CF.sub.3SO.sub.2).sub.2N.sup.-), bis(perfluoroethylsulfonyl)imide ((CF.sub.3CF.sub.2SO.sub.2).sub.2N.sup.-) and tris(trifluoromethylsulfonyl)methide ((CF.sub.3SO.sub.2).sub.3C.sup.-). Radicals or radical cations may be added to or electrochemically generated in the molten salt to enhance the RF attenuation.

  15. Riemerella anatipestifer Type IX Secretion System Is Required for Virulence and Gelatinase Secretion

    Directory of Open Access Journals (Sweden)

    Yunqing Guo

    2017-12-01

    Full Text Available Riemerella anatipestifer (RA, a major causative agent of septicemia anserum exsudativa in domesticated ducklings, has a protein secretion system known as the type IX secretion system (T9SS. It is unknown whether the T9SS contributes to the virulence of RA through secretion of factors associated with pathogenesis. To answer this question, we constructed an RA mutant deficient in sprT, which encodes a core protein of the T9SS. Deletion of sprT yielded cells that failed to digest gelatin, an effect that was rescued via complementation by a plasmid encoding wild-type sprT. Complement-mediated killing was significantly increased in the deletion mutant, suggesting that proteins secreted by the T9SS are necessary for complement evasion in RA. Liquid chromatography-tandem mass spectrometry analysis revealed that RAYM_01812 and RAYM_04099 proteins containing a subtilisin-like serine protease domain and exhibiting extracellular gelatinase activity were secreted by the T9SS. Animal experiments demonstrated that the virulence of mutant strain ΔsprT strain was attenuated by 42,000-fold relative to wild-type RA-YM. Immunization with the ΔsprT protected ducks from challenge with RA-YM, suggesting that the former can be used as a live attenuated vaccine. These results indicate that the T9SS is functional in RA and contributes to its virulence by exporting key proteins. In addition, subtilisin-like serine proteases which are important virulence factors that interact with complement proteins may enable RA to evade immune surveillance in the avian innate immune system.

  16. ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

    Directory of Open Access Journals (Sweden)

    Dawoon Chung

    2014-11-01

    Full Text Available The Aspergillus fumigatus sterol regulatory element binding protein (SREBP SrbA belongs to the basic Helix-Loop-Helix (bHLH family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA. How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

  17. Non-hydrolyzed in digestive tract and blood natural L-carnosine peptide ("bioactivated Jewish penicillin") as a panacea of tomorrow for various flu ailments: signaling activity attenuating nitric oxide (NO) production, cytostasis, and NO-dependent inhibition of influenza virus replication in macrophages in the human body infected with the virulent swine influenza A (H1N1) virus.

    Science.gov (United States)

    Babizhayev, Mark A; Deyev, Anatoliy I; Yegorov, Yegor E

    2013-01-01

    in excessive amounts mediate the overreaction of the host's immune response against the organs or tissues in which viruses are replicating, and this may explain the mechanism of tissue injuries observed in influenza virus infection of various types. In this article, the types of protection of carnosine in its bioavailable non-hydrolyzed forms in formulations are considered against reactive oxygen radical species-dependent injury, peroxynitrite damage, and other types of viral injuries in which impaired immune responses to viral pathogens are usually involved. Carnosine (β-alanyl-L-histidine) shows the pharmacological intracellular correction of NO release, which might be one of the important factors of natural immunity in controlling the initial stages of influenza A virus infection (inhibition of virus replication) and virus-induced regulation of cytokine gene expression. The protective effects of orally applied non-hydrolyzed formulated species of carnosine include at least the direct interaction with NO, inhibition of cytotoxic NO-induced proinflammatory condition, and attenuation of the effects of cytokines and chemokines that can exert profound effects on inflammatory cells. These data are consistent with the hypothesis that natural products, such as chicken soup and chicken breast extracts rich in carnosine and its derivative anserine (β-alanyl-1-methyl-L-histidine), could contribute to the pathogenesis and prevention of influenza virus infections and cold but have a limitation due to the susceptibility to enzymatic hydrolysis of dipeptides with serum carnosinase and urine excretion after oral ingestion of a commercial chicken extract. The formulations of non-hydrolyzed in digestive tract and blood natural carnosine peptide and isopeptide (γ-glutamyl-carnosine) products, manufactured at the cGMP-certified facility and patented by the authors, have promise in the control and prevention of influenza A (H1N1) virus infection, cough, and cold.

  18. Comparison of the nucleotide sequence of wild-type hepatitis - A virus and its attenuated candidate vaccine derivative

    International Nuclear Information System (INIS)

    Cohen, J.I.; Rosenblum, B.; Ticehurst, J.R.; Daemer, R.; Feinstone, S.; Purcell, R.H.

    1987-01-01

    Development of attenuated mutants for use as vaccines is in progress for other viruses, including influenza, rotavirus, varicella-zoster, cytomegalovirus, and hepatitis-A virus (HAV). Attenuated viruses may be derived from naturally occurring mutants that infect human or nonhuman hosts. Alternatively, attenuated mutants may be generated by passage of wild-type virus in cell culture. Production of attenuated viruses in cell culture is a laborious and empiric process. Despite previous empiric successes, understanding the molecular basis for attenuation of vaccine viruses could facilitate future development and use of live-virus vaccines. Comparison of the complete nucleotide sequences of wild-type (virulent) and vaccine (attenuated) viruses has been reported for polioviruses and yellow fever virus. Here, the authors compare the nucleotide sequence of wild-type HAV HM-175 with that of a candidate vaccine derivative

  19. Brunenders: a partially attenuated historic poliovirus type I vaccine strain.

    Science.gov (United States)

    Sanders, Barbara P; Liu, Ying; Brandjes, Alies; van Hoek, Vladimir; de Los Rios Oakes, Isabel; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2015-09-01

    Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.

  20. Generation of growth arrested Leishmania amastigotes: a tool to develop live attenuated vaccine candidates against visceral leishmaniasis.

    Science.gov (United States)

    Selvapandiyan, Angamuthu; Dey, Ranadhir; Gannavaram, Sreenivas; Solanki, Sumit; Salotra, Poonam; Nakhasi, Hira L

    2014-06-30

    Visceral leishmaniasis (VL) is fatal if not treated and is prevalent widely in the tropical and sub-tropical regions of world. VL is caused by the protozoan parasite Leishmania donovani or Leishmania infantum. Although several second generation vaccines have been licensed to protect dogs against VL, there are no effective vaccines against human VL [1]. Since people cured of leishmaniasis develop lifelong protection, development of live attenuated Leishmania parasites as vaccines, which can have controlled infection, may be a close surrogate to leishmanization. This can be achieved by deletion of genes involved in the regulation of growth and/or virulence of the parasite. Such mutant parasites generally do not revert to virulence in animal models even under conditions of induced immune suppression due to complete deletion of the essential gene(s). In the Leishmania life cycle, the intracellular amastigote form is the virulent form and causes disease in the mammalian hosts. We developed centrin gene deleted L. donovani parasites that displayed attenuated growth only in the amastigote stage and were found safe and efficacious against virulent challenge in the experimental animal models. Thus, targeting genes differentially expressed in the amastigote stage would potentially attenuate only the amastigote stage and hence controlled infectivity may be effective in developing immunity. This review lays out the strategies for attenuation of the growth of the amastigote form of Leishmania for use as live vaccine against leishmaniasis, with a focus on visceral leishmaniasis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Intraspecific bovine herpesvirus 1 recombinants carrying glycoprotein E deletion as a vaccine marker are virulent in cattle.

    Science.gov (United States)

    Muylkens, Benoît; Meurens, François; Schynts, Frédéric; Farnir, Frédéric; Pourchet, Aldo; Bardiau, Marjorie; Gogev, Sacha; Thiry, Julien; Cuisenaire, Adeline; Vanderplasschen, Alain; Thiry, Etienne

    2006-08-01

    Vaccines used in control programmes of Bovine herpesvirus 1 (BoHV-1) utilize highly attenuated BoHV-1 strains marked by a deletion of the glycoprotein E (gE) gene. Since BoHV-1 recombinants are obtained at high frequency in experimentally coinfected cattle, the consequences of recombination on the virulence of gE-negative BoHV-1 were investigated. Thus, gE-negative BoHV-1 recombinants were generated in vitro from several virulent BoHV-1 and one mutant BoHV-1 deleted in the gC and gE genes. Four gE-negative recombinants were tested in the natural host. All the recombinants were more virulent than the gE-negative BoHV-1 vaccine and the gC- and gE-negative parental BoHV-1. The gE-negative recombinant isolated from a BoHV-1 field strain induced the highest severe clinical score. Latency and reactivation studies showed that three of the recombinants were reexcreted. Recombination can therefore restore virulence of gE-negative BoHV-1 by introducing the gE deletion into a different virulence background.

  2. The RpoE Stress Response Pathway Mediates Reduction of the Virulence of Enteropathogenic Escherichia coli by Zinc.

    Science.gov (United States)

    Xue, Yuan; Osborn, Jossef; Panchal, Anand; Mellies, Jay L

    2015-06-01

    Zinc supplements are an effective clinical treatment for infantile diarrheal disease caused by enteric pathogens. Previous studies demonstrated that zinc acts on enteropathogenic Escherichia coli (EPEC) bacteria directly to suppress several virulence-related genes at a concentration that can be achieved by oral delivery of dietary zinc supplements. Our in vitro studies showed that a micromolar concentration of zinc induced the envelope stress response and suppressed virulence in EPEC, providing a possible mechanistic explanation for zinc's therapeutic action. In this report, we investigated the molecular and physiological changes in EPEC induced by zinc. We found that micromolar concentrations of zinc reduced the bacterial growth rate without affecting viability. We observed increased membrane permeability caused by zinc. Zinc upregulated the RpoE-dependent envelope stress response pathway and suppressed EPEC virulence gene expression. RpoE alone was sufficient to inhibit virulence factor expression and to attenuate attaching and effacing lesion formation on human host cells. By mutational analysis we demonstrate that the DNA-binding motif of RpoE is necessary for suppression of the LEE1, but not the LEE4, operon. Predictably, inhibition of the RpoE-mediated envelope stress response in combination with micromolar concentrations of zinc reduced EPEC viability. In conclusion, zinc induces the RpoE and stress response pathways in EPEC, and the alternate sigma factor RpoE downregulates EPEC LEE and non-LEE virulence genes by multiple mechanisms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence.

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    Carlos J Sanchez

    Full Text Available We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4 mutants expressing the capsule types 6A (+6A, 7F (+7F and 23F (+23F as well as a deleted and restored serotype 4 (+4 control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA, Choline binding protein A (CbpA, and Pneumococcal serine-rich repeat protein (PsrP changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.

  4. The Ebola Virus Glycoprotein Contributes to but Is Not Sufficient for Virulence In Vivo

    Science.gov (United States)

    Groseth, Allison; Marzi, Andrea; Hoenen, Thomas; Herwig, Astrid; Gardner, Don; Becker, Stephan; Ebihara, Hideki; Feldmann, Heinz

    2012-01-01

    Among the Ebola viruses most species cause severe hemorrhagic fever in humans; however, Reston ebolavirus (REBOV) has not been associated with human disease despite numerous documented infections. While the molecular basis for this difference remains unclear, in vitro evidence has suggested a role for the glycoprotein (GP) as a major filovirus pathogenicity factor, but direct evidence for such a role in the context of virus infection has been notably lacking. In order to assess the role of GP in EBOV virulence, we have developed a novel reverse genetics system for REBOV, which we report here. Together with a previously published full-length clone for Zaire ebolavirus (ZEBOV), this provides a unique possibility to directly investigate the role of an entire filovirus protein in pathogenesis. To this end we have generated recombinant ZEBOV (rZEBOV) and REBOV (rREBOV), as well as chimeric viruses in which the glycoproteins from these two virus species have been exchanged (rZEBOV-RGP and rREBOV-ZGP). All of these viruses could be rescued and the chimeras replicated with kinetics similar to their parent virus in tissue culture, indicating that the exchange of GP in these chimeric viruses is well tolerated. However, in a mouse model of infection rZEBOV-RGP demonstrated markedly decreased lethality and prolonged time to death when compared to rZEBOV, confirming that GP does indeed contribute to the full expression of virulence by ZEBOV. In contrast, rREBOV-ZGP did not show any signs of virulence, and was in fact slightly attenuated compared to rREBOV, demonstrating that GP alone is not sufficient to confer a lethal phenotype or exacerbate disease in this model. Thus, while these findings provide direct evidence that GP contributes to filovirus virulence in vivo, they also clearly indicate that other factors are needed for the acquisition of full virulence. PMID:22876185

  5. Amino Acid Permeases and Virulence in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Kevin Felipe Cruz Martho

    Full Text Available Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis, where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work

  6. Virulence factors of the Mycobacterium tuberculosis complex

    Science.gov (United States)

    Forrellad, Marina A.; Klepp, Laura I.; Gioffré, Andrea; Sabio y García, Julia; Morbidoni, Hector R.; Santangelo, María de la Paz; Cataldi, Angel A.; Bigi, Fabiana

    2013-01-01

    The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. PMID:23076359

  7. Potential drivers of virulence evolution in aquaculture

    Science.gov (United States)

    Kennedy, David A.; Kurath, Gael; Brito, Ilana L.; Purcell, Maureen K.; Read, Andrew F.; Winton, James R.; Wargo, Andrew R.

    2016-01-01

    Infectious diseases are economically detrimental to aquaculture, and with continued expansion and intensification of aquaculture, the importance of managing infectious diseases will likely increase in the future. Here, we use evolution of virulence theory, along with examples, to identify aquaculture practices that might lead to the evolution of increased pathogen virulence. We identify eight practices common in aquaculture that theory predicts may favor evolution toward higher pathogen virulence. Four are related to intensive aquaculture operations, and four others are related specifically to infectious disease control. Our intention is to make aquaculture managers aware of these risks, such that with increased vigilance, they might be able to detect and prevent the emergence and spread of increasingly troublesome pathogen strains in the future.

  8. Glucose starvation boosts Entamoeba histolytica virulence.

    Directory of Open Access Journals (Sweden)

    Ayala Tovy

    2011-08-01

    Full Text Available The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS. The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP, a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1 which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A and cysteine proteinase A5 (CP-A5, two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon.

  9. Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

    Directory of Open Access Journals (Sweden)

    Andrey A Filippov

    Full Text Available BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

  10. DFVF: database of fungal virulence factors

    Science.gov (United States)

    Lu, Tao; Yao, Bo; Zhang, Chi

    2012-01-01

    Fungal pathogens cause various diseases for plant and animal hosts. Despite the extensive impact of fungi on human health and life, the threats posed by emerging fungal pathogens are poorly understood. Specifically, there exist few fungal virulence gene databases, which prevent effective bioinformatics studies on fungal pathogens. Therefore, we constructed a comprehensive online database of known fungal virulence factors, which collected 2058 pathogenic genes produced by 228 fungal strains from 85 genera. This database creates a pivotal platform capable of stimulating and facilitating further bench studies on fungal pathogens. Database URL: http://sysbio.unl.edu/DFVF/ PMID:23092926

  11. Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis

    Directory of Open Access Journals (Sweden)

    Michell Stephen L

    2011-01-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella infection models. Results B. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis. Conclusions We have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.

  12. Control algorithms for dynamic attenuators

    International Nuclear Information System (INIS)

    Hsieh, Scott S.; Pelc, Norbert J.

    2014-01-01

    Purpose: The authors describe algorithms to control dynamic attenuators in CT and compare their performance using simulated scans. Dynamic attenuators are prepatient beam shaping filters that modulate the distribution of x-ray fluence incident on the patient on a view-by-view basis. These attenuators can reduce dose while improving key image quality metrics such as peak or mean variance. In each view, the attenuator presents several degrees of freedom which may be individually adjusted. The total number of degrees of freedom across all views is very large, making many optimization techniques impractical. The authors develop a theory for optimally controlling these attenuators. Special attention is paid to a theoretically perfect attenuator which controls the fluence for each ray individually, but the authors also investigate and compare three other, practical attenuator designs which have been previously proposed: the piecewise-linear attenuator, the translating attenuator, and the double wedge attenuator. Methods: The authors pose and solve the optimization problems of minimizing the mean and peak variance subject to a fixed dose limit. For a perfect attenuator and mean variance minimization, this problem can be solved in simple, closed form. For other attenuator designs, the problem can be decomposed into separate problems for each view to greatly reduce the computational complexity. Peak variance minimization can be approximately solved using iterated, weighted mean variance (WMV) minimization. Also, the authors develop heuristics for the perfect and piecewise-linear attenuators which do not requirea priori knowledge of the patient anatomy. The authors compare these control algorithms on different types of dynamic attenuators using simulated raw data from forward projected DICOM files of a thorax and an abdomen. Results: The translating and double wedge attenuators reduce dose by an average of 30% relative to current techniques (bowtie filter with tube current

  13. Side effects of immunization with liver attenuated Trypanosoma cruzi in mice and rabbits.

    OpenAIRE

    Basombrío, M A; Besuschio, S; Cossio, P M

    1982-01-01

    Immunity against lethal, bloodstream forms of Trypanosoma cruzi was achieved in mice by preinoculation of approximately equal to 10(5) culture epimastigotes of an attenuated T. cruzi strain (TCC). The risks of TCC inoculation in terms of pathogenicity or eventual increase in virulence of TCC progeny were evaluated. No pathogenic parasites could be selected from TCC progeny by either mouse, triatome, or culture passages. Immunizing doses of live TCC did not induce in adult mice alterations res...

  14. Genomic Changes Associated with the Loss of Nocardia brasiliensis Virulence in Mice after 200 In Vitro Passages.

    Science.gov (United States)

    Gonzalez-Carrillo, Carolina; Millan-Sauceda, Cassandra; Lozano-Garza, Hector Gerardo; Ortiz-Lopez, Rocio; Elizondo-Gonzalez, Ramiro; Welsh, Oliverio; Ocampo-Candiani, Jorge; Vera-Cabrera, Lucio

    2016-09-01

    Nocardia species, particularly Nocardia brasiliensis, are etiologic agents of mycetoma, a chronic subcutaneous infection. Until now, little has been known about the pathogenic mechanisms involved in nocardial infection. Traditionally, subculture in rich media has been a simple way to induce attenuation. In this work, we report the changes in virulence toward mice and in genomic constitution of N. brasiliensis produced after 200 continuous subcultures in brain heart infusion (BHI) medium (P-200 strain). The ability of the N. brasiliensis P-200 strain to produce experimental infection was tested using BALB/c mice. P-200 was also used to immunize mice to determine whether it could induce resistance against a challenge with a nonsubcultured isolate (P-0). Comparative proteomic analysis between N. brasiliensis P-0 and P-200 was performed by two-dimensional (2-D) electrophoresis, and the genome sequence was obtained through Roche 454 sequence analysis. Virulence in BALB/c mice was completely lost, and BALB/c mice immunized with P-200 bacterial cells were resistant to mycetoma production by the nonsubcultured strain. Whole-genome sequence analysis revealed that P-200 lost a total of 262,913 bp distributed in 19 deleted regions, involving a total of 213 open reading frames (ORFs). The deleted genes included those encoding bacterial virulence factors, e.g., catalase, nitrate reductase enzymes, and a group of mammalian cell entry (MCE) family proteins, which may explain the loss of virulence of the isolate. Thus, completely attenuated N. brasiliensis was obtained after 200 passages in BHI medium, and putative Nocardia virulence genes were identified for the first time. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  15. A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

    Directory of Open Access Journals (Sweden)

    Joel Bozue

    Full Text Available Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

  16. Efflux inhibitor suppresses Streptococcus mutans virulence properties.

    Science.gov (United States)

    Zeng, Huihui; Liu, Jia; Ling, Junqi

    2017-04-01

    It is well established that efflux pumps play important roles in bacterial pathogenicity and efflux inhibitors (EIs) have been proved to be effective in suppressing bacterial virulence properties. However, little is known regarding the EI of Streptococcus mutans, a well-known caries-inducing bacterium. In this study, we identified the EI of S. mutans through ethidium bromide efflux assay and investigated how EI affected S. mutans virulence regarding the cariogenicity and stress response. Results indicated that reserpine, the identified EI, suppressed acid tolerance, mutacin production and transformation efficiency of S. mutans, and modified biofilm architecture and extracellular polysaccharide distribution. Suppressed glycosyltransferase activity was also noted after reserpine exposure. The data from quantitative real-time-PCR demonstrated that reserpine significantly altered the expression profile of quorum-sensing and virulence-associated genes. These findings suggest that reserpine represents a promising adjunct anticariogenic agent in that it suppresses virulence properties of S. mutans. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Virulence, serotype and phylogenetic groups of diarrhoeagenic ...

    African Journals Online (AJOL)

    EAEC (36%) and both EPEC and ATEC (25.6%) are the most detected pathovars (p<0.05). STEC (5.1%), NFEC (7.7) and DAEC (7.7) are less represented. Serogroups are overall diversified (89%), however, serogroups O157, O103 and O86, previously known to be associated with virulence were revealed. Most of the E.

  18. NEW VIRULENCE FACTORS OF STREPTOCOCCUS PNEUMONIAE

    NARCIS (Netherlands)

    Hermans, Peter Wilhelmus Maria; Bootsma, Jeanette Hester; Burghout, Pieter Jan; Kuipers, Oscar; Bijlsma, Johanna Jacoba Elisabeth; Kloosterman, Tomas Gerrit; Andersen, Christian O.

    2011-01-01

    The present invention provides proteins/genes, which are essential for survival, and consequently, for virulence of Streptococcus pneumoniae in vivo, and thus are ideal vaccine candidates for a vaccine preparation against pneumococcal infection. Further, also antibodies against said protein(s) are

  19. Identification of Pseudomonas aeruginosa Genes Involved in Virulence and Anaerobic Growth

    Science.gov (United States)

    Filiatrault, Melanie J.; Picardo, Kristin F.; Ngai, Helen; Passador, Luciano; Iglewski, Barbara H.

    2006-01-01

    Pseudomonas aeruginosa is a gram-negative, opportunistic pathogen and a significant cause of acute and chronic infections in patients with compromised host defenses. Evidence suggests that within infections P. aeruginosa encounters oxygen limitation and exists in microbial aggregates known as biofilms. However, there is little information that describes genes involved in anaerobic growth of P. aeruginosa and their association with virulence of this pathogen. To identify genes required for anaerobic growth, random transposon (Tn) mutagenesis was used to screen for mutants that demonstrated the inability to grow anaerobically using nitrate as a terminal electron acceptor. Of approximately 35,000 mutants screened, 57 mutants were found to exhibit no growth anaerobically using nitrate. Identification of the genes disrupted by the Tn revealed 24 distinct loci required for anaerobic growth on nitrate, including several genes not previously associated with anaerobic growth of P. aeruginosa. Several of these mutants were capable of growing anaerobically using nitrite and/or arginine, while five mutants were unable to grow anaerobically under any of the conditions tested. Three mutants were markedly attenuated in virulence in the lettuce model of P. aeruginosa infection. These studies have identified novel genes important for anaerobic growth and demonstrate that anaerobic metabolism influences virulence of P. aeruginosa. PMID:16790798

  20. Yersinia Virulence Depends on Mimicry of Host Rho-Family Nucleotide Dissociation Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Prehna,G.; Ivanov, M.; Blisha, J.; Stebbins, C.

    2006-01-01

    Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. A critical virulence determinant is the Yersinia protein kinase A, or YpkA, a multidomain protein that disrupts the eukaryotic actin cytoskeleton. Here we solve the crystal structure of a YpkA-Rac1 complex and find that YpkA possesses a Rac1 binding domain that mimics host guanidine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits nucleotide exchange in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and impair in vivo YpkA-induced cytoskeletal disruption. In cell culture experiments, the kinase and the GDI domains of YpkA act synergistically to promote cytoskeletal disruption, and a Y. pseudotuberculosis mutant lacking YpkA GDI activity shows attenuated virulence in a mouse infection assay. We conclude that virulence in Yersinia depends strongly upon mimicry of host GDI proteins by YpkA.

  1. The membrane transporter PotE is required for virulence in avian pathogenic Escherichia coli (APEC).

    Science.gov (United States)

    Guerra, Priscila Regina; Herrero-Fresno, Ana; Pors, Susanne Elisabeth; Ahmed, Shahana; Wang, Dan; Thøfner, Ida; Antenucci, Fabio; Olsen, John Elmerdahl

    2018-03-01

    Over the last few years, polyamines have been described as key-signal of virulence in pathogenic bacteria. In the current study, we investigated whether the knockout of genes related to polyamine biosynthesis and putrescine transport affected the virulence of an avian pathogenic E. coli (APEC) strain. One-week-old White Leghorn chickens were infected intratracheally with mutants in polyamine biosynthesis (ΔspeB/C and ΔspeD/E) and transport genes (ΔpotE) of a well-characterized APEC strain of ST117 (O83: H4). All polyamine mutants and the wild-type strain were able to infect chicken; however, we observed significantly fewer lesions in the lungs of the chickens infected with the polyamine mutants in comparison with chicken infected with the wild-type. Results derived from histology of infected lungs detected significantly fewer lesions in the lung of birds infected within particular the putrescine transport mutant (ΔpotE). A decrease in colonization levels was observed in the liver and spleen of birds infected with the putrescine biosynthesis mutant ΔspeB/C, and likewise, a decrease of the colonization levels of all organs from birds infected with the ΔpotE was detected. Together, our data demonstrate that the deletion of polyamine genes, and in particular the PotE membrane protein, attenuates the virulence of APEC during infection of chickens. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Influences of ORF1 on the virulence and immunogenicity of Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Yuan, Fangyan; Liu, Jinlin; Guo, Yi; Tan, Chen; Fu, Shulin; Zhao, Jin; Chen, Huanchun; Bei, Weicheng

    2011-12-01

    Actinobacillus pleuropneumoniae is a Gram-negative pathogen that causes porcine pleuropneumonia. The pathogenicity of A. pleuropneumoniae is strongly correlated with the production of active repeat-in-toxin (RTX) proteins such as ApxIVA. We evaluated the contribution of a potential ApxIVA activator, ORF1, to the virulence and immunogenicity of A. pleuropneumoniae in pigs. The orf1 gene in A. pleuropneumoniae SLW03 (serovar 1, ΔapxICΔapxIIC) was deleted, producing strain SLW05 (ΔapxICΔapxIICΔorf1). The virulence of strains SLW03 and SLW05 was compared in pigs. Clinical signs and pulmonary lesions induced by strain SLW05 were slighter than that of strain SLW03 (P pigs immunized with strain SLW03 or SLW05 developed high antibody titers against ApxIA, ApxIIA, and ApxIVA before challenge. Two weeks after a second immunization, pigs were challenged intratracheally with either a fully virulent A. pleuropneumoniae serovar 1 or serovar 3 strain. Vaccination with strains SLW03 or SLW05 provided significantly greater protection compared to the negative control (P Immunized pigs displayed significantly fewer clinical signs and lower lung lesion scores than non-immunized pigs. These results suggested that ORF1 plays an important role in the development of ApxIVA toxicity. Furthermore, strain SLW05 is a highly attenuated strain able to induce protective immunity against A. pleuropneumoniae infection.

  3. A Salmonella virulence protein that inhibits cellular trafficking.

    Science.gov (United States)

    Uchiya, K; Barbieri, M A; Funato, K; Shah, A H; Stahl, P D; Groisman, E A

    1999-07-15

    Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.

  4. Na+/H+ antiport is essential for Yersinia pestis virulence.

    Science.gov (United States)

    Minato, Yusuke; Ghosh, Amit; Faulkner, Wyatt J; Lind, Erin J; Schesser Bartra, Sara; Plano, Gregory V; Jarrett, Clayton O; Hinnebusch, B Joseph; Winogrodzki, Judith; Dibrov, Pavel; Häse, Claudia C

    2013-09-01

    Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.

  5. RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans.

    Science.gov (United States)

    Kovacs, C J; Faustoferri, R C; Quivey, R G

    2017-12-15

    Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans , the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF , the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The Δ rgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the Δ rgpF mutant strain was unable to induce activity of the F 1 F o ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF The Δ rgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the Δ rgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivo Galleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutans IMPORTANCE The cell wall of Streptococcus mutans , the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans

  6. Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hong-geller, Elizabeth [Los Alamos National Laboratory; Valdez, Yolanda E [Los Alamos National Laboratory; Shou, Yulin [Los Alamos National Laboratory; Yoshida, Thomas M [Los Alamos National Laboratory; Marrone, Babetta L [Los Alamos National Laboratory; Dunbar, John [Los Alamos National Laboratory

    2009-01-01

    Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

  7. New insights into virulence mechanisms of rice pathogen Acidovorax avenae subsp. avenae strain RS-1 following exposure to ß-lactam antibiotics.

    Science.gov (United States)

    Li, Bin; Ge, Mengyu; Zhang, Yang; Wang, Li; Ibrahim, Muhammad; Wang, Yanli; Sun, Guochang; Chen, Gongyou

    2016-02-26

    Recent research has shown that pathogen virulence can be altered by exposure to antibiotics, even when the growth rate is unaffected. Investigating this phenomenon provides new insights into understanding the virulence mechanisms of bacterial pathogens. This study investigates the phenotypic and transcriptomic responses of the rice pathogenic bacterium Acidovorax avenae subsp. avenae (Aaa) strain RS-1 to ß-lactam antibiotics especially Ampicillin (Amp). Our results indicate that exposure to Amp does not influence bacterial growth and biofilm formation, but alters the virulence, colonization capacity, composition of extracellular polymeric substances and secretion of Type VI secretion system (T6SS) effector Hcp. This attenuation in virulence is linked to unique or differential expression of known virulence-associated genes based on genome-wide transcriptomic analysis. The reliability of expression data generated by RNA-Seq was verified with quantitative real-time PCR of 21 selected T6SS genes, where significant down-regulation in expression of hcp gene, corresponding to the reduction in secretion of Hcp, was observed under exposure to Amp. Hcp is highlighted as a potential target for Amp, with similar changes observed in virulence-associated phenotypes between exposure to Amp and mutation of hcp gene. In addition, Hcp secretion is reduced in knockout mutants of 4 differentially expressed T6SS genes.

  8. Anti-biofilm, anti-hemolysis, and anti-virulence activities of black pepper, cananga, myrrh oils, and nerolidol against Staphylococcus aureus.

    Science.gov (United States)

    Lee, Kayeon; Lee, Jin-Hyung; Kim, Soon-Il; Cho, Moo Hwan; Lee, Jintae

    2014-11-01

    The long-term usage of antibiotics has resulted in the evolution of multidrug-resistant bacteria. Unlike antibiotics, anti-virulence approaches target bacterial virulence without affecting cell viability, which may be less prone to develop drug resistance. Staphylococcus aureus is a major human pathogen that produces diverse virulence factors, such as α-toxin, which is hemolytic. Also, biofilm formation of S. aureus is one of the mechanisms of its drug resistance. In this study, anti-biofilm screening of 83 essential oils showed that black pepper, cananga, and myrrh oils and their common constituent cis-nerolidol at 0.01 % markedly inhibited S. aureus biofilm formation. Furthermore, the three essential oils and cis-nerolidol at below 0.005 % almost abolished the hemolytic activity of S. aureus. Transcriptional analyses showed that black pepper oil down-regulated the expressions of the α-toxin gene (hla), the nuclease genes, and the regulatory genes. In addition, black pepper, cananga, and myrrh oils and cis-nerolidol attenuated S. aureus virulence in the nematode Caenorhabditis elegans. This study is one of the most extensive on anti-virulence screening using diverse essential oils and provides comprehensive data on the subject. This finding implies other beneficial effects of essential oils and suggests that black pepper, cananga, and myrrh oils have potential use as anti-virulence strategies against persistent S. aureus infections.

  9. Quantifying the clinical virulence of Klebsiella pneumoniae producing carbapenemase Klebsiella pneumoniae with a Galleria mellonella model and a pilot study to translate to patient outcomes.

    Science.gov (United States)

    McLaughlin, Milena M; Advincula, M Renee; Malczynski, Michael; Barajas, Grace; Qi, Chao; Scheetz, Marc H

    2014-01-15

    Previous studies may have overestimated morbidity and mortality due to Klebsiella pneumoniae producing carbapenemase (KPC) Klebsiella pneumoniae infections because of difficulties in modeling patient comorbidities. This pilot study sought to evaluate KPC virulence by combining clinical and Galleria mellonella models in patients with K. pneumoniae blood stream infections (BSIs). G. mellonella were inoculated using KPC(+) and KPC(-) isolates from these patients. Extent and rapidity of insect mortality was analyzed. Patients were stratified by KPC BSI status. Clinical outcomes of mortality and length of stay post-infection for survivors (LOS) were analyzed. Median virulence scores calculated from the insect studies were imputed in the clinical model. The in-vivo model revealed greater mortality in KPC(-) isolates (p < 0.001). Fifteen patients with KPC(+) BSI were matched with 60 patients with KPC(-) BSI. Hospital mortality was greater in the KPC(+) group versus the KPC(-) group (OR 3.79, 95% CI 1.00 - 14.34). LOS was longer in the KPC(+) group (p < 0.01). Conversely the virulence score attenuated the association between KPC(+) status and mortality and LOS in the final translational models. KPC(+) status was associated with decreased virulence in GM. Opposite findings were observed in patients. This pilot study demonstrates that measured virulence from GM may differ from human estimates of virulence.

  10. Decrease of virulence for BALB/c mice produced by continuous subculturing of Nocardia brasiliensis.

    Science.gov (United States)

    Almaguer-Chávez, Janeth A; Welsh, Oliverio; Lozano-Garza, Hector G; Said-Fernández, Salvador; Romero-Díaz, Víktor J; Ocampo-Candiani, Jorge; Vera-Cabrera, Lucio

    2011-10-26

    Subculturing has been extensively used to attenuate human pathogens. In this work we studied the effect of continuous subculturing of Nocardia brasiliensis HUJEG-1 on virulence in a murine model. Nocardia brasiliensis HUJEG-1 was subcultured up to 130 times on brain heart infusion over four years. BALB/c mice were inoculated in the right foot pad with the bacteria subcultured 0, 40, 80, 100 and 130 times (T0, T40, T80 T100 and T130). The induction of resistance was tested by using T130 to inoculate a group of mice followed by challenge with T0 12 weeks later. Biopsies were taken from the newly infected foot-pad and immunostained with antibodies against CD4, CD8 and CD14 in order to analyze the in situ immunological changes. When using T40, T80 T100 and T130 as inoculums we observed lesions in 10, 5, 0 and 0 percent of the animals, respectively, at the end of 12 weeks. In contrast, their controls produced mycetoma in 80, 80, 70 and 60% of the inoculated animals. When studying the protection of T130, we observed a partial resistance to the infection. Immunostaining revealed an intense CD4+ lymphocytic and macrophage infiltrate in healing lesions. After 130 in vitro passages of N. brasiliensis HUJEG-1 a severe decrease in its virulence was observed. Immunization of BALB/c mice, with these attenuated cells, produced a state of partial resistance to infection with the non-subcultured isolate.

  11. LPS structure and PhoQ activity are important for Salmonella Typhimurium virulence in the Galleria mellonella infection model [corrected].

    Directory of Open Access Journals (Sweden)

    Jennifer K Bender

    Full Text Available The larvae of the wax moth, Galleria mellonella, have been used experimentally to host a range of bacterial and fungal pathogens. In this study we evaluated the suitability of G. mellonella as an alternative animal model of Salmonella infection. Using a range of inoculum doses we established that the LD₅₀ of SalmonellaTyphimurium strain NCTC 12023 was 3.6 × 10³ bacteria per larva. Further, a set of isogenic mutant strains depleted of known virulence factors was tested to identify determinants essential for S. Typhimurium pathogenesis. Mutants depleted of one or both of the type III secretion systems encoded by Salmonella Pathogenicity Islands 1 and 2 showed no virulence defect. In contrast, we observed reduced pathogenic potential of a phoQ mutant indicating an important role for the PhoPQ two-component signal transduction system. Lipopolysaccharide (LPS structure was also shown to influence Salmonella virulence in G. mellonella. A waaL(rfaL mutant, which lacks the entire O-antigen (OAg, was virtually avirulent, while a wzz(ST/wzz(fepE double mutant expressing only a very short OAg was highly attenuated for virulence. Furthermore, shortly after infection both LPS mutant strains showed decreased replication when compared to the wild type in a flow cytometry-based competitive index assay. In this study we successfully established a G. mellonella model of S. Typhimurium infection. By identifying PhoQ and LPS OAg length as key determinants of virulence in the wax moth larvae we proved that there is an overlap between this and other animal model systems, thus confirming that the G. mellonella infection model is suitable for assessing aspects of Salmonella virulence function.

  12. A Novel Enterovirus 71 (EV71) Virulence Determinant: The 69th Residue of 3C Protease Modulates Pathogenicity.

    Science.gov (United States)

    Li, Bingqing; Yue, Yingying; Zhang, Yajie; Yuan, Zenglin; Li, Peng; Song, Nannan; Lin, Wei; Liu, Yan; Gu, Lichuan; Meng, Hong

    2017-01-01

    Human enterovirus type 71 (EV71), the major causative agent of hand-foot-and-mouth disease, has been known to cause fatal neurological complications. Unfortunately, the reason for neurological complications that have been seen in fatal cases of the disease and the relationship between EV71 virulence and viral genetic sequences remains largely undefined. The 3C protease (3C pro ) of EV71 plays an irreplaceable role in segmenting the precursor polyprotein during viral replication, and intervening with host life activity during viral infection. In this study, for the first time, the 69th residue of 3C protease has been identified as a novel virulence determinant of EV71. The recombinant virus with single point variation, in the 69th of 3C pro , exhibited obvious decline in replication, and virulence. We further determined the crystal structure of 3C N69D at 1.39 Ǻ resolution and found that conformation of 3C N69D demonstrated significant changes compared with a normal 3C protein, in the substrate-binding site and catalytic active site. Strikingly, one of the switch loops, essential in fixing substrates, adopts an open conformation in the 3C N69D-rupintrivir complex. Consistent with this apparent structural disruption, the catalytic activity of 3C N69D decreased sharply for host derived and viral derived substrates, detected for both in vitro and in vivo . Interestingly, in addition to EV71, Asp69 was also found in 3C proteases of other virus strains, such as CAV16, and was conserved in nearly all C type human rhinovirus. Overall, we identified a natural virulence determinant of 3C protease and revealed the mechanism of attenuated virulence is mediated by N69D substitution. Our data provides new insight into the enzymatic mechanism of a subdued 3C protease and suggests a theoretical basis for virulence determinantion of picornaviridae.

  13. Biosynthesis of Glycomonoterpenes to Attenuate Quorum Sensing Associated Virulence in Bacteria.

    Science.gov (United States)

    Patil, Amrita; Joshi-Navre, Kasturi; Mukherji, Ruchira; Prabhune, Asmita

    2017-04-01

    The acquisition of multidrug resistance in bacteria has become a bigger threat of late, mainly due to the bacterial signaling phenomenon, quorum sensing (QS). QS, among a population of bacteria, initiates the formation of biofilms and offers myriad advantages to bacteria. Burgeoning antibiotic resistance in biofilm-producing bacteria has motivated efforts toward finding new alternatives to these traditional antimicrobials. In the present study, we report the increased solubility and additional quorum quenching as well as biofilm disruption activity of glyco-derivatives of monoterpenes (citral and citronellal). Glycomonoterpenes of citral and citronellal were synthesized via conjugation of the monoterpenes with glucose by the non-pathogenic yeast Candida bombicola (ATCC 22214). Structural elucidation of newly synthesized glycomonoterpenes showed that one synthesized using citronellal contains three major lactonic forms with molecular weight 492.43, 473.47, and 330.39 Da whereas the one produced using citral has an acidic form with molecular weight 389.33 and 346.23 Da. The glycomonoterpenes were able to individually inhibit QS, mediated through various medium-chain and long-chain N-acyl homoserine lactones (AHLs). These new compounds are interesting additions to the known range of quorum sensing inhibitors (QSIs) and could be further explored for potential clinical applications.

  14. Maintenance of Paraoxonase 2 Activity as a Strategy to Attenuate P. Aeruginosa Virulence

    Science.gov (United States)

    2013-10-01

    transiently transfected into HEK 293 cells. Amino acids capable of being phosphorylated were either mutated to alanine, which would resist ...Bacterial Pathogenesis, Host Defense, Host-Pathogen Interactions, Innate Immunity, Paraoxonase, Pseudomonas aeruginosa, Quorum Sensing 16. SECURITY...should greatly accelerate the understanding of how Pseudomonas aeruginosa, as well as other gram-negative bacteria, utilize acyl- homoserine lactones

  15. Maintenance of Paraoxonase 2 Activity as a Strategy to Attenuate P. Aeruginosa Virulence

    Science.gov (United States)

    2015-12-01

    and Hoiby, N. (2005) Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis . Infect.Immun. 73, 2504-2514 6...CN) which dephosphorylated PON2, resulting in inactivation. Using inhibitory RNA techniques , we demonstrated that knockdown of calmodulin (see...GRP78 (see appendix I). This protein has a molecular mass of 78 kD. GRP78 (also known as BiP) is a molecular chaperone of the heat shock (HS) 70

  16. The role of immunity in mosquito-induced attenuation of malaria virulence.

    Science.gov (United States)

    Mackinnon, Margaret J

    2014-01-21

    A recent study found that mosquito-transmitted (MT) lines of rodent malaria parasites elicit a more effective immune response than non-transmitted lines maintained by serial blood passage (non-MT), thereby causing lower parasite densities in the blood and less pathology to the host. The authors attribute these changes to higher diversity in expression of antigen-encoding genes in MT cf. non-MT lines. Alternative explanations that are equally parsimonious with these new data, and results from previous studies, suggest that this conclusion may be premature.

  17. Substitutions at residues 300 and 389 of the VP2 capsid protein serve as the minimal determinant of attenuation for canine parvovirus vaccine strain 9985-46.

    Science.gov (United States)

    Sehata, Go; Sato, Hiroaki; Yamanaka, Morimasa; Takahashi, Takuo; Kainuma, Risa; Igarashi, Tatsuhiko; Oshima, Sho; Noro, Taichi; Oishi, Eiji

    2017-11-01

    Identifying molecular determinants of virulence attenuation in live attenuated canine parvovirus (CPV) vaccines is important for assuring their safety. To this end, we identified mutations in the attenuated CPV 9985-46 vaccine strain that arose during serial passage in Crandell-Rees feline kidney cells by comparison with the wild-type counterpart, as well as minimal determinants of the loss of virulence. Four amino acid substitutions (N93K, G300V, T389N and V562L) in VP2 of strain 9985-46 significantly restricted infection in canine A72 cells. Using an infectious molecular clone system, we constructed isogenic CPVs of the parental virulent 9985 strain carrying single or double mutations. We observed that only a single amino acid substitution in VP2, G300V or T389N, attenuated the virulent parental virus. Combinations of these mutations further attenuated CPV to a level comparable to that of 9985-46. Strains with G300V/T389N substitutions did not induce clinical symptoms in experimentally infected pups, and their ability to infect canine cells was highly restricted. We found that another G300V/V562L double mutation decreased affinity of the virus for canine cells, although its pathogenicity to dogs was maintained. These results indicate that mutation of residue 300, which plays a critical role in host tropism, is not sufficient for viral attenuation in vivo, and that attenuation of 9985-46 strain is defined by at least two mutations in residues 300 and 389 of the VP2 capsid protein. This finding is relevant for quality control of the vaccine and provides insight into the rational design of second-generation live attenuated vaccine candidates.

  18. Sigma E regulators control hemolytic activity and virulence in a shrimp pathogenic Vibrio harveyi.

    Directory of Open Access Journals (Sweden)

    Pimonsri Rattanama

    Full Text Available Members of the genus Vibrio are important marine and aquaculture pathogens. Hemolytic activity has been identified as a virulence factor in many pathogenic vibrios including V. cholerae, V. parahaemolyticus, V. alginolyticus, V. harveyi and V. vulnificus. We have used transposon mutagenesis to identify genes involved in the hemolytic activity of shrimp-pathogenic V. harveyi strain PSU3316. Out of 1,764 mutants screened, five mutants showed reduced hemolytic activity on sheep blood agar and exhibited virulence attenuation in shrimp (Litopenaeus vannamei. Mutants were identified by comparing transposon junction sequences to a draft of assembly of the PSU3316 genome. Surprisingly none of the disrupted open reading frames or gene neighborhoods contained genes annotated as hemolysins. The gene encoding RseB, a negative regulator of the sigma factor (σ(E, was interrupted in 2 out of 5 transposon mutants, in addition, the transcription factor CytR, a threonine synthetase, and an efflux-associated cytoplasmic protein were also identified. Knockout mutations introduced into the rpoE operon at the rseB gene exhibited low hemolytic activity in sheep blood agar, and were 3-to 7-fold attenuated for colonization in shrimp. Comparison of whole cell extracted proteins in the rseB mutant (PSU4030 to the wild-type by 2-D gel electrophoresis revealed 6 differentially expressed proteins, including two down-regulated porins (OmpC-like and OmpN and an upregulated protease (DegQ which have been associated with σ(E in other organisms. Our study is the first report linking hemolytic activity to the σ(E regulators in pathogenic Vibrio species and suggests expression of this virulence-linked phenotype is governed by multiple regulatory pathways within the V. harveyi.

  19. The role for TolA in enterohemorrhagic Escherichia coli pathogenesis and virulence gene transcription.

    Science.gov (United States)

    Morgan, Jason K; Ortiz, Jose A; Riordan, James T

    2014-12-01

    Loss of the periplasm spanning protein TolA in Escherichia coli leads to activation of the Rcs phosphorelay, and is required for full virulence in Gram-negative pathogens such as Salmonella enterica and Dickeya dadantii. This study explores the role for TolA in the pathogenesis of enterohemorrhagic E. coli (EHEC) and the effect of its mutation on the transcription of key EHEC virulence genes controlled by Rcs phosphorelay, including the type III secretion system (T3SS) (espA and tir), the E. coli common pilus (ecpA), and motility (fliC). Promoter activity for T3SS regulator ler was substantially higher following inactivation of tolA, and corresponded with a similar elevation in espA and tir transcription. Likewise, ecpA transcription was increased in EHECΔtolA. Conversely, and in-line with previous studies, inactivation of tolA resulted in complete loss of motility and decreased fliC transcription. For all genes examined, altered transcription observed for EHECΔtolA was dependent on the outer-membrane lipoprotein RcsF. Despite elevated virulence gene transcription, in tolA deleted strains virulence of EHEC in the Galleria mellonella wax worm model was substantially attenuated in a manner at least partly dependent on RcsF, and adherence to cultured HT-29 colonic epithelial cells was markedly reduced. The results of this study broaden the role for TolA in EHEC pathogenesis, and suggest that significant outer-membrane perturbations are able to promote transcription of important EHEC adherence factors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

    2011-06-01

    We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

  1. Characterization of the Contribution to Virulence of Three Large Plasmids of Avian Pathogenic Escherichia coli χ7122 (O78:K80:H9) ▿ †

    Science.gov (United States)

    Mellata, Melha; Ameiss, Keith; Mo, Hua; Curtiss, Roy

    2010-01-01

    Despite the fact that the presence of multiple large plasmids is a defining feature of extraintestinal pathogenic Escherichia coli (ExPEC), such as avian pathogenic E. coli (APEC), and despite the fact that these bacteria pose a considerable threat to both human and animal health, characterization of these plasmids is still limited. In this study, after successfully curing APEC of its plasmids, we were able to investigate, for the first time, the contribution to virulence of three plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb), from APEC strain χ7122 individually as well as in all combinations in the wild-type background. Characterization of the different strains revealed unique features of APEC virulence. In vivo assays showed that curing the three plasmids resulted in severe attenuation of virulence. The presence of different plasmids and combinations of plasmids resulted in strains with different pathotypes and levels of virulence, reflecting the diversity of APEC strains associated with colibacillosis in chickens. Unexpectedly, our results associated the decrease in growth of some strains in some media with the virulence of APEC, and the mechanism was associated with some combinations of plasmids that included pAPEC-1. This study provided new insights into the roles of large plasmids in the virulence, growth, and evolution of APEC by showing for the first time that both the nature of plasmids and combinations of plasmids have an effect on these phenomena. It also provided a plausible explanation for some of the conflicting results related to the virulence of ExPEC strains. This study should help us understand the virulence of other ExPEC strains and design more efficient infection control strategies. PMID:20086082

  2. Amplificação gênica alelo-específica na caracterização das hemoglobinas S, C e D e as interações entre elas e talassemias beta Allele-specific genic amplification in the characterization of hemoglobins S, C, D and interactions among them and with beta thalassemia

    Directory of Open Access Journals (Sweden)

    Luciane Cristina Bertholo

    2006-08-01

    possible interactions, based on the allele-specific genic amplification (PCR-AE with the use in parallel two primers that differ at their 3’ extremities and are complementary to the normal or mutated sequences. RESULTS AND DISCUSSION: The results make evident the validation of this methodology in the characterization of these mutations, once this procedure is easy to execute,to reproduce, as well as it is possible to be applied to a significative number of samples.

  3. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Jonnatan Pais-Morales

    Full Text Available Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.

  4. The Ribosomal Protein RplY Is Required for Pectobacterium carotovorum Virulence and Is Induced by Zantedeschia elliotiana Extract.

    Science.gov (United States)

    Jiang, Huan; Jiang, Mengyi; Yang, Liuke; Yao, Peiyan; Ma, Lin; Wang, Chunting; Wang, Huan; Qian, Gouliang; Hu, Baishi; Fan, Jiaqin

    2017-11-01

    Pectobacterium carotovorum subsp. carotovorum strain PccS1, a bacterial pathogen causing soft rot disease of Zantedeschia elliotiana (colored calla), was investigated for virulence genes induced by the host plant. Using a promoter-trap transposon (mariner), we obtained 500 transposon mutants showing kanamycin resistance dependent on extract of Z. elliotiana. One of these mutants, PM86, exhibited attenuated virulence on both Z. elliotiana and Brassica rapa subsp. pekinensis. The growth of PM86 was also reduced in minimal medium (MM), and the reduction was restored by adding plant extract to the MM. The gene containing the insertion site was identified as rplY. The deletion mutant ΔrplY, exhibited reduced virulence, motility and plant cell wall-degrading enzyme production but not biofilm formation. Analysis of gene expression and reporter fusions revealed that the rplY gene in PccS1 is up-regulated at both the transcriptional and the translational levels in the presence of plant extract. Our results suggest that rplY is induced by Z. elliotiana extract and is crucial for virulence in P. carotovorum subsp. carotovorum.

  5. Riboregulators: Fine-Tuning Virulence in Shigella.

    Science.gov (United States)

    Fris, Megan E; Murphy, Erin R

    2016-01-01

    Within the past several years, RNA-mediated regulation (ribo-regulation) has become increasingly recognized for its importance in controlling critical bacterial processes. Regulatory RNA molecules, or riboregulators, are perpetually responsive to changes within the micro-environment of a bacterium. Notably, several characterized riboregulators control virulence in pathogenic bacteria, as is the case for each riboregulator characterized to date in Shigella. The timing of virulence gene expression and the ability of the pathogen to adapt to rapidly changing environmental conditions is critical to the establishment and progression of infection by Shigella species; ribo-regulators mediate each of these important processes. This mini review will present the current state of knowledge regarding RNA-mediated regulation in Shigella by detailing the characterization and function of each identified riboregulator in these pathogens.

  6. DENGUE VIRUS VIRULENCE AND DISEASES SEVERITY.

    Science.gov (United States)

    Prommalikit, Olarn; Thisyakorn, Usa

    2015-01-01

    The dengue virus is the causative agent of a wide spectrum of clinical manifestations, ranging from mild acute febrile illness to classical dengue fever, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). DHF and DSS are the potentially fatal forms of dengue virus infection, which has become an intractable public health problem in many countries. The pathogeneses of DHF/ DSS are not clearly understood. One hypothesis concerning virus virulence and the immune enhancement hypothesis has been debated. Although dengue disease severity has been associated with evidence of genetic differences in dengue strains, virus virulence has been difficult to measure because of the lack of in vivo and in vitro models of the disease.

  7. Type IV secretion and Brucella virulence.

    Science.gov (United States)

    Boschiroli, Maria Laura; Ouahrani-Bettache, Safia; Foulongne, Vincent; Michaux-Charachon, Sylvie; Bourg, Gisele; Allardet-Servent, Annick; Cazevieille, Chantal; Lavigne, Jean-Phillipe; Liautard, Jean Pierre; Ramuz, Michel; O'Callaghan, David

    2002-12-20

    The type IV secretion system, encoded by the virB region, is a key virulence factor for Brucella. The 12 genes of the region form an operon that is specifically induced by phagosome acidification in cells after phagocytosis. We speculate that the system serves to secrete unknown effector molecules, which allow Brucella to pervert the host cell endosomal pathways and to create a novel intracellular compartment in which it can replicate. Copyright 2002 Elsevier Science B.V.

  8. aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant

    Science.gov (United States)

    Frahm, Michael; Kocijancic, Dino; Rohde, Manfred; Eckweiler, Denitsa; Bielecka, Agata; Bueno, Emilio; Cava, Felipe; Abraham, Wolf-Rainer; Curtiss, Roy; Häussler, Susanne; Erhardt, Marc; Weiss, Siegfried

    2016-01-01

    ABSTRACT Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. PMID:27601574

  9. Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model.

    Science.gov (United States)

    Arita, Minetaro; Ami, Yasushi; Wakita, Takaji; Shimizu, Hiroyuki

    2008-02-01

    Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also associated with serious neurological disorders. An attenuated EV71 strain [EV71(S1-3')] has been established in the cynomolgus monkey infection model; this strain contains the attenuation determinants derived from the type 1 poliovirus vaccine strain, Sabin 1 [PV1(Sabin)], in the 5' nontranslated region (NTR), 3D polymerase, and 3' NTR. In this study, we analyzed the effect of the attenuation determinants of PV1(Sabin) on EV71 infection in a NOD/SCID mouse infection model. We isolated a mouse-adapted EV71 strain [EV71(NOD/SCID)] that causes paralysis of the hind limbs in 3- to 4-week-old NOD/SCID mice by adaptation of the virulent EV71(Nagoya) strain in the brains of NOD/SCID mice. A single mutation at nucleotide 2876 that caused an amino acid change in capsid protein VP1 (change of the glycine at position 145 to glutamic acid) was essential for the mouse-adapted phenotype in NOD/SCID mice. Next, we introduced attenuation determinants derived from PV1(Sabin) along with the mouse adaptation mutation into the EV71(Nagoya) genome. In 4-week-old mice, the determinants in the 3D polymerase and 3' NTR, which are the major temperature-sensitive determinants, had a strong effect on attenuation. In contrast, the effect of individual determinants was weak in 3-week-old NOD/SCID mice, and all the determinants were required for substantial attenuation. These results suggest that a cooperative effect of the attenuation determinants of PV1(Sabin) is essential for attenuated neurovirulence of EV71.

  10. Generation of an infectious clone of VR-2332, a highly virulent North American type isolate of porcine reproductive and respiratory syndrome virus

    DEFF Research Database (Denmark)

    Nielsen, H.S.; Liu, G.; Nielsen, Jens

    2003-01-01

    -2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ171 restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally...... mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines....

  11. Protective efficacy of centralized and polyvalent envelope immunogens in an attenuated equine lentivirus vaccine.

    Directory of Open Access Journals (Sweden)

    Jodi K Craigo

    2015-01-01

    Full Text Available Lentiviral Envelope (Env antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated equine infectious anemia (EIAV virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully

  12. New Insights into Autoinducer-2 Signaling as a Virulence Regulator in a Mouse Model of Pneumonic Plague

    Science.gov (United States)

    Fitts, Eric C.; Andersson, Jourdan A.; Kirtley, Michelle L.; Sha, Jian; Erova, Tatiana E.; Chauhan, Sadhana; Motin, Vladimir L.

    2016-01-01

    ABSTRACT The Enterobacteriaceae family members, including the infamous Yersinia pestis, the causative agent of plague, have a highly conserved interbacterial signaling system that is mediated by the autoinducer-2 (AI-2) quorum-sensing molecule. The AI-2 system is implicated in regulating various bacterial virulence genes in diverse environmental niches. Deletion of the gene encoding the synthetic enzyme for the AI-2 substrate, luxS, leads to either no significant change or, paradoxically, an increase in in vivo bacterial virulence. We showed that deletion of the rbsA and lsrA genes, components of ABC transport systems that interact with AI-2, synergistically disrupted AI-2 signaling patterns and resulted in a more-than-50-fold decrease in Y. pestis strain CO92 virulence in a stringent pneumonic plague mouse model. Deletion of luxS or lsrK (encoding AI-2 kinase) from the ΔrbsA ΔlsrA background strain or complementation of the ΔrbsA ΔlsrA mutant with the corresponding gene(s) reverted the virulence phenotype to that of the wild-type Y. pestis CO92. Furthermore, the administration of synthetic AI-2 in mice infected with the ΔrbsA ΔlsrA ΔluxS mutant strain attenuated this triple mutant to a virulence phenotype similar to that of the ΔrbsA ΔlsrA strain in a pneumonic plague model. Conversely, the administration of AI-2 to mice infected with the ΔrbsA ΔlsrA ΔluxS ΔlsrK mutant did not rescue animals from lethality, indicating the importance of the AI-2–LsrK axis in regulating bacterial virulence. By performing high-throughput RNA sequencing, the potential role of some AI-2-signaling-regulated genes that modulated bacterial virulence was determined. We anticipate that the characterization of AI-2 signaling in Y. pestis will lead to reexamination of AI-2 systems in other pathogens and that AI-2 signaling may represent a broad-spectrum therapeutic target to combat antibiotic-resistant bacteria, which represent a global crisis of the 21st century. IMPORTANCE

  13. Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity.

    Science.gov (United States)

    Zhao, Xinxin; Liu, Qing; Xiao, Kangpeng; Hu, Yunlong; Liu, Xueyan; Li, Yanyan; Kong, Qingke

    2016-06-24

    Pasteurella multocida (P. multocida) is an important veterinary pathogen that can cause severe diseases in a wide range of mammals and birds. The global regulator crp gene has been found to regulate the virulence of some bacteria, and crp mutants have been demonstrated to be effective attenuated vaccines against Salmonella enterica and Yersinia enterocolitica. Here, we first characterized the crp gene in P. multocida, and we report the effects of a crp deletion. The P. multocida crp mutant exhibited a similar lipopolysaccharide and outer membrane protein profile but displayed defective growth and serum complement resistance in vitro compared with the parent strain. Furthermore, crp deletion decreased virulence but did not result in full attenuation. The 50 % lethal dose (LD50) of the Δcrp mutant was 85-fold higher than that of the parent strain for intranasal infection. Transcriptome sequencing analysis showed that 92 genes were up-regulated and 94 genes were down-regulated in the absence of the crp gene. Finally, we found that intranasal immunization with the Δcrp mutant triggered both systematic and mucosal antibody responses and conferred 60 % protection against virulent P. multocida challenge in ducks. The deletion of the crp gene has an inhibitory effect on bacterial growth and bacterial resistance to serum complement in vitro. The P. multocida crp mutant was attenuated and conferred moderate protection in ducks. This work affords a platform for analyzing the function of crp and aiding the formulation of a novel vaccine against P. multocida.

  14. Neutralization-resistant variants of infectious hematopoietic necrosis virus have altered virulence and tissue tropism

    Science.gov (United States)

    Kim, C.H.; Winton, J.R.; Leong, J.C.

    1994-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes an acute disease in salmon and trout. In this study, a correlation between changes in tissue tropism and specific changes in the virus genome appeared to be made by examining four IHNV neutralization-resistant variants (RB-1, RB-2, RB-3, and RB-4) that had been selected with the glycoprotein (G)-specific monoclonal antibody RB/B5. These variants were compared with the parental strain (RB-76) for their virulence and pathogenicity in rainbow trout after waterborne challenge. Variants RB-2, RB-3, and RB-4 were only slightly attenuated and showed distributions of viral antigen in the livers and hematopoietic tissues of infected fish similar to those of the parental strain. Variant RB-1, however, was highly attenuated and the tissue distribution of viral antigen in RB-1-infected fish was markedly different, with more viral antigen in brain tissue. The sequences of the G genes of all four variants and RB-76 were determined. No significant changes were found for the slightly attenuated variants, but RB-1 G had two changes at amino acids 78 and 218 that dramatically altered its predicted secondary structure. These changes are thought to be responsible for the altered tissue tropism of the virus. Thus, IHNV G, like that of rabies virus and vesicular stomatitis virus, plays an integral part in the pathogenesis of viral infection.

  15. Mutations induced by ultraviolet radiation affecting virulence in Puccinia striiformis

    International Nuclear Information System (INIS)

    Shang Hongsheng; Jing Jinxue; Li Zhenqi

    1994-01-01

    Uredospores of parent culture, cy 29-1, were treated by ultraviolet radiation and mutations to virulent were tested on resistant wheat cultivars inoculated with treated spores. 7 mutant cultures virulent to the test cultivars were developed with estimated mutation rate 10~6~10~4. The virulence of mutant cultures was different from the all known races of stripe rust. Resistance segregation to mutant cultures was detected in two test cultivars. The results suggested that mutation was important mechanism of virulence variation operative in asexual population of rust fungi

  16. Escherichia coli Type III Secretion System 2 ATPase EivC Is Involved in the Motility and Virulence of Avian Pathogenic Escherichia coli.

    Science.gov (United States)

    Wang, Shaohui; Liu, Xin; Xu, Xuan; Yang, Denghui; Wang, Dong; Han, Xiangan; Shi, Yonghong; Tian, Mingxing; Ding, Chan; Peng, Daxin; Yu, Shengqing

    2016-01-01

    Type III secretion systems (T3SSs) are crucial for bacterial infections because they deliver effector proteins into host cells. The Escherichia coli type III secretion system 2 (ETT2) is present in the majority of E. coli strains, and although it is degenerate, ETT2 regulates bacterial virulence. An ATPase is essential for T3SS secretion, but the function of the ETT2 ATPase has not been demonstrated. Here, we show that EivC is homologous to the β subunit of F0F1 ATPases and it possesses ATPase activity. To investigate the effects of ETT2 ATPase EivC on the phenotype and virulence of avian pathogenic Escherichia coli (APEC), eivC mutant and complemented strains were constructed and characterized. Inactivation of eivC led to impaired flagella production and augmented fimbriae on the bacterial surface, and, consequently, reduced bacterial motility. In addition, the eivC mutant strain exhibited attenuated virulence in ducks, diminished serum resistance, reduced survival in macrophage cells and in ducks, upregulated fimbrial gene expression, and downregulated flagellar and virulence gene expression. The expression of the inflammatory cytokines interleukin (IL)-1β and IL-8 were increased in HD-11 macrophages infected with the eivC mutant strain, compared with the wild-type strain. These virulence-related phenotypes were restored by genetic complementation. These findings demonstrate that ETT2 ATPase EivC is involved in the motility and pathogenicity of APEC.

  17. Escherichia coli Type III Secretion System 2 ATPase EivC is Involved in the Motility and Virulence of Avian Pathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Shaohui Wang

    2016-08-01

    Full Text Available Type III secretion systems (T3SSs are crucial for bacterial infections because they deliver effector proteins into host cells. The Escherichia coli type III secretion system 2 (ETT2 is present in the majority of E. coli strains, and although it is degenerate, ETT2 regulates bacterial virulence. An ATPase is essential for T3SS secretion, but the function of the ETT2 ATPase has not been demonstrated. Here we show that EivC is homologous to the β subunit of F0F1 ATPases and it possesses ATPase activity. To investigate the effects of ETT2 ATPase EivC on the phenotype and virulence of avian pathogenic Escherichia coli (APEC, eivC mutant and complemented strains were constructed and characterized. Inactivation of eivC led to impaired flagella production and augmented fimbriae on the bacterial surface, and, consequently, reduced bacterial motility. In addition, the eivC mutant strain exhibited attenuated virulence in ducks, diminished serum resistance, reduced survival in macrophage cells and in ducks, upregulated fimbrial gene expression, and downregulated flagellar and virulence gene expression. The expression of the inflammatory cytokines interleukin (IL-1β and IL-8 were increased in HD-11 macrophages infected with the eivC mutant strain, compared with the wild-type strain. These virulence-related phenotypes were restored by genetic complementation. These findings demonstrate that ETT2 ATPase EivC is involved in the motility and pathogenicity of APEC.

  18. Controlled branched-chain amino acids auxotrophy in Listeria monocytogenes allows isoleucine to serve as a host signal and virulence effector.

    Science.gov (United States)

    Brenner, Moran; Lobel, Lior; Borovok, Ilya; Sigal, Nadejda; Herskovits, Anat A

    2018-03-01

    Listeria monocytogenes (Lm) is a saprophyte and intracellular pathogen. Transition to the pathogenic state relies on sensing of host-derived metabolites, yet it remains unclear how these are recognized and how they mediate virulence gene regulation. We previously found that low availability of isoleucine signals Lm to activate the virulent state. This response is dependent on CodY, a global regulator and isoleucine sensor. Isoleucine-bound CodY represses metabolic pathways including branched-chain amino acids (BCAA) biosynthesis, however under BCAA depletion, as occurs during infection, BCAA biosynthesis is upregulated and isoleucine-unbound CodY activates virulence genes. While isoleucine was revealed as an important input signal, it was not identified how internal levels are controlled during infection. Here we show that Lm regulates BCAA biosynthesis via CodY and via a riboregulator located upstream to the BCAA biosynthesis genes, named Rli60. rli60 is transcribed when BCAA levels drop, forming a ribosome-mediated attenuator that cis-regulates the downstream genes according to BCAA supply. Notably, we found that Rli60 restricts BCAA production, essentially starving Lm, a mechanism that is directly linked to virulence, as it controls the internal isoleucine pool and thereby CodY activity. This controlled BCAA auxotrophy likely evolved to enable isoleucine to serve as a host signal and virulence effector.

  19. Drosophila melanogaster as a model to explore the effects of methicillin-resistant Staphylococcus aureus strain type on virulence and response to linezolid treatment.

    Science.gov (United States)

    Ben-Ami, Ronen; Watson, Clay C; Lewis, Russell E; Albert, Nathaniel D; Arias, Cesar A; Raad, Issam I; Kontoyiannis, Dimitrios P

    2013-02-01

    USA300 is a uniquely successful methicillin-resistant Staphylococcus aureus (MRSA) clone that has been associated with Panton-Valentine leukocidin (PVL) production and severe infections. However, conflicting experimental and epidemiological data exist regarding the virulence of USA300 relative to other MRSA clones. We aimed to address this issue using Drosophila melanogaster as a model host to study strain and PVL-dependent variations in virulence among MRSA clinical isolates. We studied the relative virulence of 39 MRSA isolates: 17 (43%) were PFGE type USA300. Lethal MRSA infection was reproducibly induced both in wild-type (WT) and Toll-deficient D. melanogaster. USA300 strains had significantly lower lethality than non-USA300 strains in a WT background but not in Toll-deficient flies. PFGE type (USA300 versus non-USA300) and PVL status did not affect the response to treatment with linezolid. Virulence was similar in strains with high vancomycin MIC (≥2 μg/mL) versus those with vancomycin MIC<2 μg/mL. D. melanogaster is a potentially useful model host to study pathogenicity and response to antibiotic treatment in S. aureus. Our results imply that the attenuated virulence of PVL(+)/USA300 requires intact host innate immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Complementation of Brucella abortus RB51 with a Functional wboA Gene Results in O-Antigen Synthesis and Enhanced Vaccine Efficacy but No Change in Rough Phenotype and Attenuation

    OpenAIRE

    Vemulapalli, Ramesh; He, Yongqun; Buccolo, Larissa S.; Boyle, Stephen M.; Sriranganathan, Nammalwar; Schurig, Gerhardt G.

    2000-01-01

    Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760–764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to pr...

  1. The NlpD lipoprotein is a novel Yersinia pestis virulence factor essential for the development of plague.

    Directory of Open Access Journals (Sweden)

    Avital Tidhar

    Full Text Available Yersinia pestis is the causative agent of plague. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the pcm gene was disrupted. In the present study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis using a set of isogenic surE, pcm, nlpD and rpoS mutants of the fully virulent Kimberley53 strain. We show that in Y. pestis, nlpD expression is controlled from elements residing within the upstream genes surE and pcm. The NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD(50 of at least 10(7 cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The filamented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrated that subcutaneous administration of the nlpD mutant could protect animals against bubonic and primary pneumonic plague. Taken together, these results demonstrate that Y. pestis NlpD is a novel virulence factor essential for the development of bubonic and pneumonic plague. Further, the nlpD mutant is superior to the EV76 prototype live vaccine strain in immunogenicity and in conferring effective protective immunity. Thus it could serve as a basis for a very potent live vaccine against bubonic and pneumonic plague.

  2. The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2.

    Science.gov (United States)

    Li, Quan; Fu, Yang; Ma, Caifeng; He, Yanan; Yu, Yanfei; Du, Dechao; Yao, Huochun; Lu, Chengping; Zhang, Wei

    2017-10-03

    Muramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Δmrp, Δmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242-596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1* (MRP-D1*, the non-conserved region of MRP from a low virulent strain, a.a. 239-598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848-1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection.

  3. Anaplasma marginale attenuated by irradiation: Kinetics of immunity and protection

    International Nuclear Information System (INIS)

    Gil, L.A.; Higuera, B.O.; Castro, J.; Chavez, M.; Ruiz, O.

    1988-01-01

    Attempts were made to attenuate Anaplasma marginale by gamma irradiation using a dose of 900 Gy. Parasites were either irradiated once and after infection of an animal isolated and re-irradiated, or irradiated and cultured in vitro for four days and then re-irradiated. In the first two treatments the animals reacted as severely as a control group that received non-irradiated organisms; upon subsequent challenge with a virulent heterologous A. marginale strain, all animals had reasonable levels of protective immunity. The group that received irradiated and cultured organisms twice was not protected upon subsequent challenge and the animals reacted as severely as controls. Antibody responses were monitored. In all instances, after patent parasitaemia, an initial IgM response was followed by an IgG 1 and IgG 2 response. It is concluded that a virulent parasite population suitable for use as a vaccine was not obtained through irradiation and further use of this technique is discouraged. (author). 17 refs, 3 figs

  4. Microbial virulence and interactions with metals

    DEFF Research Database (Denmark)

    German, N.; Lüthje, Freja Lea; Hao, X.

    2016-01-01

    Transition metals, such as iron, copper, zinc, and manganese play an important role in many bacterial biological processes that add to an overall evolutional fitness of bacteria. They are often involved in regulation of bacterial virulence as a mechanism of host invasion. However, the same transi...... reconstruction of Fe-S clusters and the use of Mn as a protectant against reactive oxygen species. Therefore, tight regulation of transition metal distribution in bacteria and hosts is a vital part of host-pathogen interactions....

  5. Metal acquisition and virulence in Brucella

    Science.gov (United States)

    Roop, R. Martin

    2013-01-01

    Similar to other bacteria, Brucella strains require several biologically essential metals for their survival in vitro and in vivo. Acquiring sufficient levels of some of these metals, particularly iron, manganese and zinc, is especially challenging in the mammalian host, where sequestration of these micronutrients is a well-documented component of both the innate and acquired immune responses. This review describes the Brucella metal transporters that have been shown to play critical roles in the virulence of these bacteria in experimental and natural hosts. PMID:22632611

  6. Omics strategies for revealing Yersinia pestis virulence

    Science.gov (United States)

    Yang, Ruifu; Du, Zongmin; Han, Yanping; Zhou, Lei; Song, Yajun; Zhou, Dongsheng; Cui, Yujun

    2012-01-01

    Omics has remarkably changed the way we investigate and understand life. Omics differs from traditional hypothesis-driven research because it is a discovery-driven approach. Mass datasets produced from omics-based studies require experts from different fields to reveal the salient features behind these data. In this review, we summarize omics-driven studies to reveal the virulence features of Yersinia pestis through genomics, trascriptomics, proteomics, interactomics, etc. These studies serve as foundations for further hypothesis-driven research and help us gain insight into Y. pestis pathogenesis. PMID:23248778

  7. Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains

    NARCIS (Netherlands)

    Bonar, Emilia; Wojcik, Iwona; Jankowska, Urszula; Kedracka-Krok, Sylwia; Bukowski, Michal; Polakowska, Klaudia; Lis, Marcin W; Kosecka-Strojek, Maja; Sabat, Artur J; Dubin, Grzegorz; Friedrich, Alexander W; Miedzobrodzki, Jacek; Dubin, Adam; Wladyka, Benedykt

    2016-01-01

    Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is

  8. Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Michael S Behnke

    2015-08-01

    Full Text Available Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5

  9. Virulence determinants of equine infectious anemia virus.

    Science.gov (United States)

    Payne, Susan L; Fuller, Frederick J

    2010-01-01

    Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that rapidly Induces disease in experimentally infected horses. Because EIAV infection and replication is centered on the monocyte/macrophage and has a pronounced acute disease stage, it is a useful model system for understanding the contribution of monocyte/macrophages to other lentivirus-induced diseases. Genetic mapping studies utilizing chimeric proviruses in which parental viruses are acutely virulent or avirulent have allowed the identification of important regions that influence acute virulence. U3 regions in the viral LTR, surface envelope (SU) protein and the accessory S2 gene strongly influence acute disease expression. While the chimeric proviruses provide insight into genes or genome regions that affect viral pathogenesis, it is then necessary to further dissect those regions to focus on specific virus-host mechanisms that lead to disease expression. The V6 region of the viral env protein is an example of one identified region that may interact with the ELR-1 receptor in an important way and we are currently identifying S2 protein motifs required for disease expression.

  10. [Virulence of Sporothrix globosa in murine models].

    Science.gov (United States)

    Cruz Choappa, Rodrigo; Pérez Gaete, Salomón; Rodríguez Badilla, Valentina; Vieille Oyarzo, Peggy; Opazo Sanchez, Héctor

    The sporothricosis disease is an infection caused by species included in Sporothrix schenkii complex. Verify the virulence of a strain of S. globosa using two different concentrations of inoculum by intraperitoneally and subcutaneously, into a mouse model. Nonrandomized pilot study, in murine inoculated with a strain of S. globosa (CBS 14.076M) by intraperitoneally and subcutaneously with inoculum concentrations of 0.5 and 4 McFarland. For this purpose 18 rodents CF-1 (ISP, Santiago, Chile) were used. The studied strain did not induce illness or injury on animals, they all survived and neither the tissue culture nor the histopathological analysis showed fungal growth or suggestive infection by organ abnormalities. The S. globosa strain did not present any virulence enough to cause disease at 0.5 and 4.0 McFarland concentration inoculum when inoculated in both intraperitoneally and subcutaneously, in murine models. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  11. Metabolism and virulence in Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    Christoph eSchoen

    2014-08-01

    Full Text Available A longstanding question in infection biology addresses the genetic basis for invasive behaviour in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions.

  12. Investigation of possible virulence factors in Candida strains isolated ...

    African Journals Online (AJOL)

    The rate of proteinase, phospholipase, and esterase positivity was higher in the C. albicans isolates. Biofilm formation was the highest in the C. parapsilosis strains. Conclusions: Higher rate of virulence factors in the most commonly isolated Candida species than other species indicates that these virulence factors play a ...

  13. Limiting opportunities for cheating stabilizes virulence in insect parasitic nematodes

    Science.gov (United States)

    Cooperative secretion of virulence factors by pathogens can often lead to social conflict as cheating mutants that benefit from collective action, but do not contribute to it, can arise and locally outcompete cooperators within hosts, leading to loss of virulence. There is a wide range of in vivo st...

  14. Production Of Some Virulence Factors Under Different Growth ...

    African Journals Online (AJOL)

    The production of some virulence factors under different growth conditions and antibiotic susceptbility pattern of Aeromonas hydrophila were investigated in this sudy. The virulence actors tested on the isolates included haemolytic activity, exopolysaccharide (capsule) and toxin production. Other cell property evaluated was ...

  15. Detection of virulence-associated genes in Brucella melitensis ...

    African Journals Online (AJOL)

    Ibrahim Eldaghayes

    2018-03-20

    Mar 20, 2018 ... isolated from goats. This discrepancies may indicate that B. melitensis field strains prevailing in Egypt are more virulent than the strains of B. melitensis isolated from caprines in Iran. As, it was emphasized that the. T4SS of Brucella encoded by the virB operon is a major virulence factor (Delrue et al., 2005).

  16. Virulence Genotype and Phylogenetic Groups in relation to Chinese ...

    African Journals Online (AJOL)

    Conclusion: PapA and papC are significant VFs with an essential role in contributing to Chinese herb-resistance. Chinese herb-resistance is associated with a shift towards more virulent strains and B2 phylogenetic group. Key words: Escherichia coli; Virulence factors; Phylogenetic group; Chinese herb-resistance.

  17. Association between antimicrobial resistance and virulence in Escherichia coli.

    Science.gov (United States)

    Da Silva, Gabriela Jorge; Mendonça, Nuno

    2012-01-01

    Escherichia coli represents a major cause of morbidity and mortality worldwide. The treatment of E. coli infections is now threatened by the emergence of antimicrobial resistance. The dissemination of resistance is associated with genetic mobile elements, such as plasmids, that may also carry virulence determinants. A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. This review aims to critically discuss the association and linked transmission of both resistance and virulence traits in strains from extraintestinal infections in E. coli, and intestinal pathotypes. Despite the numerous controversies on this topic, findings from research published to date indicate that there is a link between resistance and virulence, as illustrated by the successful E. coli ST131 epidemic clone. Perhaps the most commonly accepted view is that resistance to quinolones is linked to a loss of virulence factors. However, the low virulent phylogenetic groups might be more prone to acquire resistance to quinolones. Specific characteristics of the E. coli genome that have yet to be identified may contribute to such genetic linkages. Research based on bacterial populations is sorely needed to help understand the molecular mechanisms underlying the association between resistance and virulence, that, in turn, may help manage the future disseminations of infectious diseases in their entirety.

  18. Antibiotic Resistance and Virulence Properties in Escherichia coli ...

    African Journals Online (AJOL)

    This study determined E. coli resistance to commonly used antibiotics together with their virulence properties in Ile-Ife, Nigeria. A total of 137 E. coli isolates from cases of urinary tract infection were tested for their sensitivity to commonly used antibiotics and possession of virulence factors using standard methods.

  19. Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis.

    Science.gov (United States)

    Ganapathy, Uday; Marrero, Joeli; Calhoun, Susannah; Eoh, Hyungjin; de Carvalho, Luiz Pedro Sorio; Rhee, Kyu; Ehrt, Sabine

    2015-08-10

    The human pathogen Mycobacterium tuberculosis (Mtb) likely utilizes host fatty acids as a carbon source during infection. Gluconeogenesis is essential for the conversion of fatty acids into biomass. A rate-limiting step in gluconeogenesis is the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate by a fructose bisphosphatase (FBPase). The Mtb genome contains only one annotated FBPase gene, glpX. Here we show that, unexpectedly, an Mtb mutant lacking GLPX grows on gluconeogenic carbon sources and has detectable FBPase activity. We demonstrate that the Mtb genome encodes an alternative FBPase (GPM2, Rv3214) that can maintain gluconeogenesis in the absence of GLPX. Consequently, deletion of both GLPX and GPM2 is required for disruption of gluconeogenesis and attenuation of Mtb in a mouse model of infection. Our work affirms a role for gluconeogenesis in Mtb virulence and reveals previously unidentified metabolic redundancy at the FBPase-catalysed reaction step of the pathway.

  20. Iron – a key nexus in the virulence of Aspergillus fumigatus

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    Hubertus eHaas

    2012-02-01

    Full Text Available Iron is an essential but in excess toxic nutrient. Therefore, fungi evolved fine-tuned mechanisms for uptake and storage of iron, such as the production of siderophores (low-molecular mass iron-specific chelators. In Aspergillus fumigatus, iron starvation causes extensive transcriptional remodeling involving two central transcription factors, which are interconnected in a negative transcriptional feed-back loop: the GATA-factor SreA and the bZip-factor HapX. During iron sufficiency SreA represses iron uptake, including reductive iron assimilation and siderophore-mediated iron uptake, to avoid toxic effects. During iron starvation HapX represses iron-consuming pathways, including heme biosynthesis and respiration, to spare iron and activates synthesis of ribotoxin AspF1 and siderophores, the latter partly by ensuring supply of the precursor ornithine. In agreement with the expression pattern and mode of action, detrimental effects of inactivation of SreA and HapX are confined to growth during iron sufficiency and iron starvation, respectively. Deficiency in HapX, but not SreA, attenuates virulence of A. fumigatus in a murine model of aspergillosis, which underlines the crucial role of adaptation to iron limitation in virulence. Consistently, production of both extra- and intracellular siderophores is crucial for virulence of A. fumigatus. Recently, the sterol-regulatory element-binding protein SrbA was found to be essential for adaptation to iron starvation, thereby linking regulation of iron metabolism, ergosterol biosynthesis, azole drug resistance and hypoxia adaptation.

  1. Proteomic profiles of mouse neuro N2a cells infected with variant virulence of rabies viruses.

    Science.gov (United States)

    Wang, Xiaohu; Zhang, Shoufeng; Sun, Chenglong; Yuan, Zi-Guo; Wu, Xianfu; Wang, Dongxia; Ding, Zhuang; Hu, Rongliang

    2011-04-01

    We characterized the proteomes of murine N2a cells following infection with three rabies virus (RV) strains, characterized by distinct virulence phenotypes (i.e., virulent BD06, fixed CVS-11, and attenuated SRV9 strains), and identified 35 changes to protein expression using two-dimensional gel electrophoresis in whole-cell lysates. The annotated functions of these proteins are involved in various cytoskeletal, signal transduction, stress response, and metabolic processes. Specifically, a-enolase, prx-4, vimentin, cytokine-induced apoptosis inhibitor 1 (CIAPIN1) and prx-6 were significantly up-regulated, whereas Trx like-1 and galectin-1 were down-regulated following infection of N2a cells with all three rabies virus strains. However, comparing expressions of all 35 proteins affected between BD06-, CVS-11-, and SRV9-infected cells, specific changes in expression were also observed. The up-regulation of vimentin, CIAPIN1, prx-4, and 14-3-3 theta/delta, and downregulation of NDPK-B and HSP-1 with CVS and SRV9 infection were ≥ 2 times greater than with BD06. Meanwhile, Zfp12 protein, splicing factor, and arginine/serine-rich 1 were unaltered in the cells infected with BD06 and CVS- 11, but were up-regulated in the group infected with SRV9. The proteomic alterations described here may suggest that these changes to protein expression correlate with the rabies virus' adaptability and virulence in N2a cells, and hence provides new clues as to the response of N2a host cells to rabies virus infections, and may also aid in uncovering new pathways in these cells that are involved in rabies infections. Further characterization of the functions of the affected proteins may contribute to our understanding of the mechanisms of RV infection and pathogenesis.

  2. A Japanese encephalitis virus vaccine candidate strain is attenuated by decreasing its interferon antagonistic ability.

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    Liang, Jian-Jong; Liao, Ching-Len; Liao, Jia-Teh; Lee, Yi-Ling; Lin, Yi-Ling

    2009-05-11

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute encephalitis with high mortality in humans. To understand the virus-host interactions that influence JEV virulence, we determined the lethality of a neurovirulent (RP-9) and an attenuated (RP-2ms) variant of JEV in several immunodeficient mice strains. The attenuated phenotype of RP-2ms was completely lost in Stat-1-deficient mice, but its virulence was only slightly increased in mice lacking the components of adaptive immunity, suggesting an important role of the interferon (IFN) system in controlling JEV infection. Cell-based assays demonstrated that RP-2ms is more sensitive to IFN-alpha treatment; however, the NS5 protein of RP-2ms was still a potent antagonist of IFN, like RP-9 NS5. Using a recombinant infectious clone of RP-9, we found that a single Glu-->Lys mutation at residue 138 of the envelope protein (E-E138K) rendered the mutated RP-9 sensitive to the antiviral effect of IFN-alpha. Furthermore, IFN signaling was blocked earlier in the RP-9-infected cells relative to that in cells infected with RP-2ms or recombinant RP-9 bearing the E-E138K mutation. Thus, the E-E138K mutation of JEV appears to affect the viral growth properties, leading to a reduced efficiency in blocking IFN signaling, which then results in an attenuated phenotype in inoculated animals.

  3. Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae

    Science.gov (United States)

    Johnson, Michael D. L.; Echlin, Haley; Dao, Tina H.

    2015-01-01

    NAD is a necessary cofactor present in all living cells. Some bacteria cannot de novo synthesize NAD and must use the salvage pathway to import niacin or nicotinamide riboside via substrate importers NiaX and PnuC, respectively. Although homologues of these two importers and their substrates have been identified in other organisms, limited data exist in Streptococcus pneumoniae, specifically, on its effect on overall virulence. Here, we sought to characterize the substrate specificity of NiaX and PnuC in Str. pneumoniae TIGR4 and the contribution of these proteins to virulence of the pathogen. Although binding affinity of each importer for nicotinamide mononucleotide may overlap, we found NiaX to specifically import nicotinamide and nicotinic acid, and PnuC to be primarily responsible for nicotinamide riboside import. Furthermore, a pnuC mutant is completely attenuated during both intranasal and intratracheal infections in mice. Taken together, these findings underscore the importance of substrate salvage in pneumococcal pathogenesis and indicate that PnuC could potentially be a viable small-molecule therapeutic target to alleviate disease progression in the host. PMID:26311256

  4. Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis.

    Science.gov (United States)

    Chen, Ying-Lien; Brand, Alexandra; Morrison, Emma L; Silao, Fitz Gerald S; Bigol, Ursela G; Malbas, Fedelino F; Nett, Jeniel E; Andes, David R; Solis, Norma V; Filler, Scott G; Averette, Anna; Heitman, Joseph

    2011-06-01

    Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.

  5. Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis▿†

    Science.gov (United States)

    Chen, Ying-Lien; Brand, Alexandra; Morrison, Emma L.; Silao, Fitz Gerald S.; Bigol, Ursela G.; Malbas, Fedelino F.; Nett, Jeniel E.; Andes, David R.; Solis, Norma V.; Filler, Scott G.; Averette, Anna; Heitman, Joseph

    2011-01-01

    Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections. PMID:21531874

  6. A novel multidomain polyketide synthase is essential for zeamine production and the virulence of Dickeya zeae.

    Science.gov (United States)

    Zhou, Jianuan; Zhang, Haibao; Wu, Jien; Liu, Qiongguang; Xi, Pinggen; Lee, Jasmine; Liao, Jinling; Jiang, Zide; Zhang, Lian-Hui

    2011-10-01

    Dickeya zeae is the causal agent of the rice foot rot disease, but its mechanism of infection remains largely unknown. In this study, we identified and characterized a novel gene designated as zmsA. The gene encodes a large protein of 2,346 amino acids in length, which consists of multidomains arranged in the order of N-terminus, β-ketoacyl synthase, acyl transferase, acyl carrier protein, β-ketoacyl reductase, dehydratase. This multidomain structure and sequence alignment analysis suggest that ZmsA is a member of the polyketide synthase family. Mutation of zmsA abolished antimicrobial activity and attenuated the virulence of D. zeae. To determine the relationship between antimicrobial activity and virulence, active compounds were purified from D. zeae EC1 and were structurally characterized. This led to identification of two polyamino compounds, i.e., zeamine and zeamine II, that were phytotoxins and potent antibiotics. These results have established the essential role of ZmsA in zeamine biosynthesis and presented a new insight on the molecular mechanisms of D. zeae pathogenicity.

  7. PA-X is a virulence factor in avian H9N2 influenza virus.

    Science.gov (United States)

    Gao, Huijie; Xu, Guanlong; Sun, Yipeng; Qi, Lu; Wang, Jinliang; Kong, Weili; Sun, Honglei; Pu, Juan; Chang, Kin-Chow; Liu, Jinhua

    2015-09-01

    H9N2 influenza viruses have been circulating worldwide in multiple avian species, and regularly infect pigs and humans. Recently, a novel protein, PA-X, produced from the PA gene by ribosomal frameshifting, was demonstrated to be an antivirulence factor in pandemic 2009 H1N1, highly pathogenic avian H5N1 and 1918 H1N1 viruses. However, a similar role of PA-X in the prevalent H9N2 avian influenza viruses has not been established. In this study, we compared the virulence and cytopathogenicity of H9N2 WT virus and H9N2 PA-X-deficient virus. Loss of PA-X in H9N2 virus reduced apoptosis and had a marginal effect on progeny virus output in human pulmonary adenocarcinoma (A549) cells. Without PA-X, PA was less able to suppress co-expressed GFP in human embryonic kidney 293T cells. Furthermore, absence of PA-X in H9N2 virus attenuated viral pathogenicity in mice, which showed no mortality, reduced progeny virus production, mild-to-normal lung histopathology, and dampened proinflammatory cytokine and chemokine response. Therefore, unlike previously reported H1N1 and H5N1 viruses, we show that PA-X protein in H9N2 virus is a pro-virulence factor in facilitating viral pathogenicity and that the pro- or antivirulence role of PA-X in influenza viruses is virus strain-dependent.

  8. Vimentin binding is critical for infection by the virulent strain of Japanese encephalitis virus.

    Science.gov (United States)

    Liang, Jian-Jong; Yu, Chia-Yi; Liao, Ching-Len; Lin, Yi-Ling

    2011-09-01

    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis with high mortality in humans. We used a pair of virulent (RP-9) and attenuated (RP-2ms) variants of JEV to pull down the cell surface molecules bound with JEV particle; their identities were revealed by LC-MS/MS analysis. One major protein bound with RP-9 and weakly with RP-2ms was identified as the intermediate filament protein vimentin. Infection of RP-9 but not that of RP-2ms was blocked by anti-vimentin antibodies and by recombinant-expressed vimentin proteins. Knockdown of vimentin expression reduced the levels of viral binding and viral production of RP-9, but not that of RP-2ms. The different vimentin dependency for JEV infection could be attributed to the major structural envelope protein, as the recombinant RP-9 with an E-E138K mutation became resistant to anti-vimentin blockage. Furthermore, RP-2ms mainly depended on cell surface glycosaminoglycans for viral binding and it became vimentin-dependent only when binding to glycosaminoglycans was blocked. Thus, we suggest that vimentin contributes to virulent JEV infection and might be a new target to intervene in this deadly infection. © 2011 Blackwell Publishing Ltd.

  9. Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

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    Lee, Yoon Jae [Graduate Program in Biomaterials Science and Engineering, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Vaccine Translational Research Center, Yonsei University, Seoul (Korea, Republic of); Jang, Yo Han [Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Kim, Paul; Lee, Yun Ha; Lee, Young Jae [Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Vaccine Translational Research Center, Yonsei University, Seoul (Korea, Republic of); Byun, Young Ho; Lee, Kwang-Hee; Kim, Kyusik [Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Seong, Baik Lin, E-mail: blseong@yonsei.ac.kr [Graduate Program in Biomaterials Science and Engineering, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Vaccine Translational Research Center, Yonsei University, Seoul (Korea, Republic of)

    2016-04-15

    In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.

  10. Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

    International Nuclear Information System (INIS)

    Lee, Yoon Jae; Jang, Yo Han; Kim, Paul; Lee, Yun Ha; Lee, Young Jae; Byun, Young Ho; Lee, Kwang-Hee; Kim, Kyusik; Seong, Baik Lin

    2016-01-01

    In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.

  11. Decrease of virulence for BALB/c mice produced by continuous subculturing of Nocardia brasiliensis

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    Ocampo-Candiani Jorge

    2011-10-01

    Full Text Available Abstract Background Subculturing has been extensively used to attenuate human pathogens. In this work we studied the effect of continuous subculturing of Nocardia brasiliensis HUJEG-1 on virulence in a murine model. Methods Nocardia brasiliensis HUJEG-1 was subcultured up to 130 times on brain heart infusion over four years. BALB/c mice were inoculated in the right foot pad with the bacteria subcultured 0, 40, 80, 100 and 130 times (T0, T40, T80 T100 and T130. The induction of resistance was tested by using T130 to inoculate a group of mice followed by challenge with T0 12 weeks later. Biopsies were taken from the newly infected foot-pad and immunostained with antibodies against CD4, CD8 and CD14 in order to analyze the in situ immunological changes. Results When using T40, T80 T100 and T130 as inoculums we observed lesions in 10, 5, 0 and 0 percent of the animals, respectively, at the end of 12 weeks. In contrast, their controls produced mycetoma in 80, 80, 70 and 60% of the inoculated animals. When studying the protection of T130, we observed a partial resistance to the infection. Immunostaining revealed an intense CD4+ lymphocytic and macrophage infiltrate in healing lesions. Conclusions After 130 in vitro passages of N. brasiliensis HUJEG-1 a severe decrease in its virulence was observed. Immunization of BALB/c mice, with these attenuated cells, produced a state of partial resistance to infection with the non-subcultured isolate.

  12. Analysis of Spleen-Induced Fimbria Production in Recombinant Attenuated Salmonella enterica Serovar Typhimurium Vaccine Strains

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    Paweł Łaniewski

    2017-08-01

    Full Text Available Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo. We used an in vivo expression technology (IVET strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 105 CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 109 CFU of wild-type S. Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.

  13. Deletion of the small RNA chaperone protein Hfq down regulates genes related to virulence and confers protection against wild-type Brucella challenge in mice

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    Shuangshuang eLei

    2016-01-01

    Full Text Available Brucellosis is one of the most common zoonotic epidemics worldwide. Brucella, the etiological pathogen of brucellosis, has unique virulence characteristics, including the ability to survive within the host cell. Hfq is a bacterial chaperone protein that is involved in the survival of the pathogen under stress conditions. Moreover, hfq affects the expression of a large number of target genes. In the present study, we characterized the expression and regulatory patterns of the target genes of Hfq during brucellosis. The results revealed that hfq expression is highly induced in macrophages at the early infection stage and at the late stage of mouse infection. Several genes related to virulence, including omp25, omp31, vjbR, htrA, gntR, and dnaK, were found to be regulated by hfq during infection in BALB/c mice. Gene expression and cytokine secretion analysis revealed that an hfq-deletion mutant induced different cytokine profiles compared with that induced by 16M. Infection with the hfq-deletion mutant induced protective immune responses against 16M challenge. Together, these results suggest that hfq is induced during infection and its deletion results in significant attenuation which affects the host immune response caused by Brucella infection. By regulating genes related to virulence, hfq promotes the virulence of Brucella. The unique characteristics of the hfq-deletion mutant, including its decreased virulence and the ability to induce protective immune response upon infection, suggest that it represents an attractive candidate for the design of a live attenuated vaccine against Brucella.

  14. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.

    Science.gov (United States)

    Rajashekara, Gireesh; Glover, David A; Banai, Menachem; O'Callaghan, David; Splitter, Gary A

    2006-05-01

    In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

  15. Effects of IFN-γ coding plasmid supplementation in the immune response and protection elicited by Trypanosoma cruzi attenuated parasites.

    Science.gov (United States)

    Pérez Brandán, Cecilia; Mesías, Andrea C; Parodi, Cecilia; Cimino, Rubén O; Pérez Brandán, Carolina; Diosque, Patricio; Basombrío, Miguel Ángel

    2017-11-25

    Previous studies showed that a naturally attenuated strain from Trypanosoma cruzi triggers an immune response mainly related to a Th2-type profile. Albeit this, a strong protection against virulent challenge was obtained after priming mice with this attenuated strain. However, this protection is not enough to completely clear parasites from the host. In T. cruzi infection, early Interferon-gamma (IFN-γ) is critical to lead type 1 responses able to control intracellular parasites. Therefore we evaluated whether the co-administration of a plasmid encoding murine IFN-γ could modify the immune response induced by infection with attenuated parasites and improve protection against further infections. C57BL/6J mice were infected intraperitoneally with three doses of live attenuated parasites in combination with plasmid pVXVR-mIFN-γ. Before each infection dose, sera samples were collected for parasite specific antibodies determination and cytokine quantification. To evaluate the recall response to T. cruzi, mice were challenged with virulent parasites 30 days after the last dose and parasite load in peripheral blood and heart was evaluated. As determined by ELISA, significantly increase in T. cruzi specific antibodies response was detected in the group in which pVXVR-mIFN-γ was incorporated, with a higher predominance of IgG2a subtype in comparison to the group of mice only inoculated with attenuated parasites. At our limit of detection, serum levels of IFN-γ were not detected, however a slight decrease in IL-10 concentrations was observed in groups in which pVXVR-mIFN-γ was supplemented. To analyze if the administration of pVXVR-mIFN-γ has any beneficial effect in protection against subsequent infections, all experimental groups were submitted to a lethal challenge with virulent bloodstream trypomastigotes. Similar levels of challenge parasites were detected in peripheral blood and heart of mice primed with attenuated parasites alone or combined with plasmid DNA

  16. Envelope Protein Mutations L107F and E138K Are Important for Neurovirulence Attenuation for Japanese Encephalitis Virus SA14-14-2 Strain.

    Science.gov (United States)

    Yang, Jian; Yang, Huiqiang; Li, Zhushi; Wang, Wei; Lin, Hua; Liu, Lina; Ni, Qianzhi; Liu, Xinyu; Zeng, Xianwu; Wu, Yonglin; Li, Yuhua

    2017-01-21

    The attenuated Japanese encephalitis virus (JEV) strain SA14-14-2 has been successfully utilized to prevent JEV infection; however, the attenuation determinants have not been fully elucidated. The envelope (E) protein of the attenuated JEV SA14-14-2 strain differs from that of the virulent parental SA14 strain at eight amino acid positions (E107, E138, E176, E177, E264, E279, E315, and E439). Here, we investigated the SA14-14-2-attenuation determinants by mutating E107, E138, E176, E177, and E279 in SA14-14-2 to their status in the parental virulent strain and tested the replication capacity, neurovirulence, neuroinvasiveness, and mortality associated with the mutated viruses in mice, as compared with those of JEV SA14-14-2 and SA14. Our findings indicated that revertant mutations at the E138 or E107 position significantly increased SA14-14-2 virulence, whereas other revertant mutations exhibited significant increases in neurovirulence only when combined with E138, E107, and other mutations. Revertant mutations at all eight positions in the E protein resulted in the highest degree of SA14-14-2 virulence, although this was still lower than that observed in SA14. These results demonstrated the critical role of the viral E protein in controlling JEV virulence and identified the amino acids at the E107 and E138 positions as the key determinants of SA14-14-2 neurovirulence.

  17. Envelope Protein Mutations L107F and E138K Are Important for Neurovirulence Attenuation for Japanese Encephalitis Virus SA14-14-2 Strain

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    Jian Yang

    2017-01-01

    Full Text Available The attenuated Japanese encephalitis virus (JEV strain SA14-14-2 has been successfully utilized to prevent JEV infection; however, the attenuation determinants have not been fully elucidated. The envelope (E protein of the attenuated JEV SA14-14-2 strain differs from that of the virulent parental SA14 strain at eight amino acid positions (E107, E138, E176, E177, E264, E279, E315, and E439. Here, we investigated the SA14-14-2-attenuation determinants by mutating E107, E138, E176, E177, and E279 in SA14-14-2 to their status in the parental virulent strain and tested the replication capacity, neurovirulence, neuroinvasiveness, and mortality associated with the mutated viruses in mice, as compared with those of JEV SA14-14-2 and SA14. Our findings indicated that revertant mutations at the E138 or E107 position significantly increased SA14-14-2 virulence, whereas other revertant mutations exhibited significant increases in neurovirulence only when combined with E138, E107, and other mutations. Revertant mutations at all eight positions in the E protein resulted in the highest degree of SA14-14-2 virulence, although this was still lower than that observed in SA14. These results demonstrated the critical role of the viral E protein in controlling JEV virulence and identified the amino acids at the E107 and E138 positions as the key determinants of SA14-14-2 neurovirulence.

  18. [Virulence mechanisms of enteropathogenic Escherichia coli].

    Science.gov (United States)

    Farfán-García, Ana Elvira; Ariza-Rojas, Sandra Catherine; Vargas-Cárdenas, Fabiola Andrea; Vargas-Remolina, Lizeth Viviana

    2016-08-01

    Acute diarrheal disease (ADD) is a global public health problem, especially in developing countries and is one of the causes of mortality in children under five. ADD etiologic agents include viruses, bacteria and parasites in that order. Escherichia coli bacteria it is classified as a major diarrheagenic agent and transmitted by consuming contaminated water or undercooked foods. This review compiled updates on information virulence factors and pathogenic mechanisms involved in adhesion and colonization of seven pathotypes of E. coli called enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), shigatoxigenic E. coli (STEC), enteroaggregative E. coli (EAEC) and diffusely-adherent E. coli (DAEC). A final pathotype, adherent-invasive E. coli (AIEC) associated with Crohn's disease was also reviewed. The diarrheagenic pathotypes of E. coli affect different population groups and knowledge of the molecular mechanisms involved in the interaction with the human is important to guide research towards the development of vaccines and new tools for diagnosis and control.

  19. Attenuation in Superconducting Circular Waveguides

    Directory of Open Access Journals (Sweden)

    K. H. Yeap

    2016-09-01

    Full Text Available We present an analysis on wave propagation in superconducting circular waveguides. In order to account for the presence of quasiparticles in the intragap states of a superconductor, we employ the characteristic equation derived from the extended Mattis-Bardeen theory to compute the values of the complex conductivity. To calculate the attenuation in a circular waveguide, the tangential fields at the boundary of the wall are first matched with the electrical properties (which includes the complex conductivity of the wall material. The matching of fields with the electrical properties results in a set of transcendental equations which is able to accurately describe the propagation constant of the fields. Our results show that although the attenuation in the superconducting waveguide above cutoff (but below the gap frequency is finite, it is considerably lower than that in a normal waveguide. Above the gap frequency, however, the attenuation in the superconducting waveguide increases sharply. The attenuation eventually surpasses that in a normal waveguide. As frequency increases above the gap frequency, Cooper pairs break into quasiparticles. Hence, we attribute the sharp rise in attenuation to the increase in random collision of the quasiparticles with the lattice structure.

  20. Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

    Science.gov (United States)

    Thomashow, M F; Karlinsey, J E; Marks, J R; Hurlbert, R E

    1987-01-01

    We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent. Images PMID:3597321

  1. Development of a tailored vaccine against challenge with very virulent infectious bursal disease virus of chickens using reverse genetics.

    Science.gov (United States)

    Gao, Li; Qi, Xiaole; Li, Kai; Gao, Honglei; Gao, Yulong; Qin, Liting; Wang, Yongqiang; Wang, Xiaomei

    2011-07-26

    Due to the problems associated with traditional methods for infectious bursal disease virus (IBDV) vaccine development and the pressure of evolution and variation of very virulent strains, it is urgent to develop IBDV vaccine rapidly with novel approaches. Using reverse genetics, the aim of this study was to generate a tailored vaccine strain (rGtHLJVP2) with its VP2 gene similar to very virulent IBDV (vvIBDV) to prevent the prevalence of IBDV. Characteristics of rGtHLJVP2 were evaluated in both cell culture and SPF chickens. rGtHLJVP2 replicated well as its parental strain Gt in vitro and in vivo. Immunization of SPF chickens with rGtHLJVP2 resulted in comparable antibody titers against IBDV as that of the medium virulent live vaccine B87, which was significant higher than that of attenuated vaccine Gt. Challenge studies with 10(4)ELD(50) of prevalent homogeneous or heterogeneous vvIBDV revealed complete (100%) protection in the groups immunized with rGtHLJVP2. No significant clinical and pathological lesions were observed in chickens immunized with rGtHLJVP2. Our data demonstrated that rGtHLJVP2 could be used as a novel vaccine candidate for prevention against vvIBDV. Copyright © 2011. Published by Elsevier Ltd.

  2. Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2.

    Science.gov (United States)

    Sitkiewicz, Izabela; Nagiec, Michal J; Sumby, Paul; Butler, Stephanie D; Cywes-Bentley, Colette; Musser, James M

    2006-10-24

    The molecular basis of pathogen clone emergence is relatively poorly understood. Acquisition of a bacteriophage encoding a previously unknown secreted phospholipase A(2) (designated SlaA) has been implicated in the rapid emergence in the mid-1980s of a new hypervirulent clone of serotype M3 group A Streptococcus. Although several lines of circumstantial evidence suggest that SlaA is a virulence factor, this issue has not been addressed experimentally. We found that an isogenic DeltaslaA mutant strain was significantly impaired in ability to adhere to and kill human epithelial cells compared with the wild-type parental strain. The mutant strain was less virulent for mice than the wild-type strain, and immunization with purified SlaA significantly protected mice from invasive disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We conclude that transductional acquisition of the ability of a GAS strain to produce SlaA enhanced the spread and virulence of the serotype M3 precursor strain. Hence, these studies identified a crucial molecular event underlying the evolution, rapid emergence, and widespread dissemination of unusually severe human infections caused by a distinct bacterial clone.

  3. The Two-Component Regulatory System VicRK is Important to Virulence of Streptococcus equi Subspecies equi.

    Science.gov (United States)

    Liu, Mengyao; McClure, Michael J; Zhu, Hui; Xie, Gang; Lei, Benfang

    2008-01-01

    This study aims at evaluating the importance of the two-component regulatory system VicRK to virulence of the horse pathogen Streptococcus equi subspecies equi and the potential of a vicK mutant as a live vaccine candidate using mouse infection models. The vicK gene was deleted by gene replacement. The DeltavicK mutant is attenuated in virulence in both subcutaneous and intranasal infections in mice. DeltavicK grows less slowly than the parent strain but retains the ability of S. equi to resist to phagocytosis by polymorphoneuclear leukocytes, suggesting that the vicK deletion causes growth defect. DeltavicK infection protects mice against reinfection with a wild-type S. equi strain. Intranasal DeltavicK infection induces production of anti-SeM mucosal IgA and systemic IgG. These results indicate that VicRK is important to S. equi growth and virulence and suggest that DeltavicK has the potential to be developed as a live S. equi vaccine.

  4. Inhibitory effect of live-attenuated Listeria monocytogenes-based vaccines expressing MIA gene on malignant melanoma.

    Science.gov (United States)

    Qian, Yue; Zhang, Na; Jiang, Ping; Chen, Siyuan; Chu, Shujuan; Hamze, Firas; Wu, Yan; Luo, Qin; Feng, Aiping

    2012-08-01

    Listeria monocytogenes (LM), a Gram-positive facultative intracellular bacterium, can be used as an effective exogenous antigen expression vector in tumor-target therapy. But for successful clinical application, it is necessary to construct attenuated LM stain that is safe yet retains the potency of LM based on the full virulent pathogen. In this study, attenuated LM and recombinants of LM expressing melanoma inhibitory activity (MIA) were constructed successfully. The median lethal dose (LD(50)) and invasion efficiency of attenuated LM strains were detected. The recombinants were utilized for immunotherapy of animal model of B16F10 melanoma. The level of MIA mRNA expression in tumor tissue was detected by using real-time polymerase chain reaction (PCR) with specific sequence, meanwhile the anti-tumor immune response was assayed by flow cytometric analysis and enzyme-linked immunosorbent spot (ELISPOT) assay. The results showed the toxicity and invasiveness of attenuated LM were decreased as compared with LM, and attenuated LM expressing MIA, especially the double-genes attenuated LM recombinant, could significantly induce anti-tumor immune response and inhibit tumor growth. This study implicates attenuated LM may be a safer and more effective vector for immunotherapy of melanoma.

  5. Josephson tunnel junction microwave attenuator

    DEFF Research Database (Denmark)

    Koshelets, V. P.; Shitov, S. V.; Shchukin, A. V.

    1993-01-01

    A new element for superconducting electronic circuitry-a variable attenuator-has been proposed, designed, and successfully tested. The principle of operation is based on the change in the microwave impedance of a superconductor-insulator-superconductor (SIS) Josephson tunnel junction when dc biased...... at different points in the current-voltage characteristic. Both numerical calculations based on the Tien-Gordon theory and 70-GHz microwave experiments have confirmed the wide dynamic range (more than 15-dB attenuation for one stage) and the low insertion loss in the ''open'' state. The performance of a fully...... integrated submillimeter receiver circuit which comprises a flux-flow oscillator (FFO) as local oscillator, a superconducting variable attenuator, and a microwave SIS detector with tuned-out capacitance is also reported....

  6. Coordinated zinc homeostasis is essential for the wild-type virulence of Brucella abortus.

    Science.gov (United States)

    Sheehan, Lauren M; Budnick, James A; Roop, R Martin; Caswell, Clayton C

    2015-05-01

    Metal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence of Brucella abortus; however, the capacity of Brucella strains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region of znuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion of zur or zntR alone did not result in increased zinc toxicity in the corresponding mutants; however, deletion of zntA led to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion of zntR resulted in significant attenuation of B. abortus in a mouse model of chronic infection, and subsequent experiments revealed that overexpression of zntA in the zntR mutant is the molecular basis for its decreased virulence. The importance of zinc uptake for Brucella pathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export in Brucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation of Brucella virulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria

  7. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis.

    Science.gov (United States)

    Naglik, Julian R; Challacombe, Stephen J; Hube, Bernhard

    2003-09-01

    Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.

  8. Invasion thresholds and the evolution of nonequilibrium virulence.

    Science.gov (United States)

    Bull, James J; Ebert, Dieter

    2008-02-01

    The enterprise of virulence management attempts to predict how social practices and other factors affect the evolution of parasite virulence. These predictions are often based on parasite optima or evolutionary equilibria derived from models of host-parasite dynamics. Yet even when such models accurately capture the parasite optima, newly invading parasites will typically not be at their optima. Here we show that parasite invasion of a host population can occur despite highly nonoptimal virulence. Fitness improvements soon after invasion may proceed through many steps with wide changes in virulence, because fitness depends on transmission as well as virulence, and transmission improvements can overwhelm nonoptimal virulence. This process is highly sensitive to mutation supply and the strength of selection. Importantly, the same invasion principle applies to the evolution of established parasites, whenever mutants arise that overcome host immunity/resistance. A host population may consequently experience repeated invasions of new parasite variants and possible large shifts in virulence as it evolves in an arms race with the parasite. An experimental study of phage lysis time and examples of mammalian viruses matching some of these characteristics are reviewed.

  9. In vitro markers for virulence in Yersinia ruckeri.

    Science.gov (United States)

    Tobback, E; Decostere, A; Hermans, K; Van den Broeck, W; Haesebrouck, F; Chiers, K

    2010-03-01

    In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non-virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE-214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non-virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin-D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non-virulent strains were generally serum sensitive.

  10. Virulence evolution at the front line of spreading epidemics.

    Science.gov (United States)

    Griette, Quentin; Raoul, Gaël; Gandon, Sylvain

    2015-11-01

    Understanding and predicting the spatial spread of emerging pathogens is a major challenge for the public health management of infectious diseases. Theoretical epidemiology shows that the speed of an epidemic is governed by the life-history characteristics of the pathogen and its ability to disperse. Rapid evolution of these traits during the invasion may thus affect the speed of epidemics. Here we study the influence of virulence evolution on the spatial spread of an epidemic. At the edge of the invasion front, we show that more virulent and transmissible genotypes are expected to win the competition with other pathogens. Behind the front line, however, more prudent exploitation strategies outcompete virulent pathogens. Crucially, even when the presence of the virulent mutant is limited to the edge of the front, the invasion speed can be dramatically altered by pathogen evolution. We support our analysis with individual-based simulations and we discuss the additional effects of demographic stochasticity taking place at the front line on virulence evolution. We confirm that an increase of virulence can occur at the front, but only if the carrying capacity of the invading pathogen is large enough. These results are discussed in the light of recent empirical studies examining virulence evolution at the edge of spreading epidemics. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

  11. The LCLS Gas Attenuator Revisited

    International Nuclear Information System (INIS)

    Ryutov, D

    2005-01-01

    In the report ''X-ray attenuation cell'' [1] a preliminary analysis of the gas attenuator for the Linac Coherent Light Source (LCLS) was presented. This analysis was carried out for extremely stringent set of specifications. In particular, a very large diameter for the unobstructed beam was set (1 cm) to accommodate the spontaneous radiation; the attenuator was supposed to cover the whole range of energies of the coherent radiation, from 800 eV to 8000 eV; the maximum attenuation was set at the level of 10 4 ; the use of solid attenuators was not allowed, as well as the use of rotating shutters. The need to reach a sufficient absorption at the high-energy end of the spectrum predetermined the choice of Xe as the working gas (in order to have a reasonable absorption at a not-too-high pressure). A sophisticated differential pumping system that included a Penning-type ion pump was suggested in order to minimize the gas leak into the undulator/accelerator part of the facility. A high cost of xenon meant also that an efficient (and expensive) gas-recovery system would have to be installed. The main parameter that determined the high cost and the complexity of the system was a large radius of the orifice. The present viewpoint allows for much smaller size of the orifice, r 0 = 1.5 mm. (1) The use of solid attenuators is also allowed (R.M. Bionta, private communication). It is, therefore, worthwhile to reconsider various parameters of the gas attenuator for these much less stringent conditions. This brief study should be considered as a physics input for the engineering design. As a working gas we consider now the argon, which, on the one hand, provides a reasonable absorption lengths and, on the other hand, is inexpensive enough to be exhausted into the atmosphere (no recovery). The absorption properties of argon are illustrated by Fig.1 where the attenuation factor A is shown for various beam energies, based on Ref. [2]. The other relevant parameters for argon are

  12. The effect of milk components and storage conditions on the virulence of Listeria monocytogenes as determined by a Caco-2 cell assay.

    Science.gov (United States)

    Pricope-Ciolacu, Luminita; Nicolau, Anca Ioana; Wagner, Martin; Rychli, Kathrin

    2013-08-16

    Nearly all cases of human listeriosis have been associated with consumption of contaminated food, therefore the investigation of the virulence of Listeria (L.) monocytogenes after exposure to environmental conditions in food matrices is critical in order to understand and control its impact on public health. As milk and dairy products have been implicated in more than half of the listeriosis outbreaks, we investigated the in vitro virulence of L. monocytogenes incubated in different milk types at various storage conditions. Incubation in pasteurized milk at refrigeration conditions (4°C) revealed a higher invasion and intracellular proliferation of four different L. monocytogenes strains compared to raw milk using human intestinal epithelial Caco-2 cells. Furthermore the period of storage, which increased L. monocytogenes cell numbers, decreased in vitro virulence. However, L. monocytogenes stored for 3weeks at 4°C in milk are still able to invade and proliferate into the host cell. Interestingly abused storage temperatures (25°C and 30°C) for a short time period (2h) revealed an attenuated impact on the in vitro virulence of L. monocytogenes compared to the storage temperature of 4°C. Regarding the major milk compounds, the level of milk fat significantly affected the in vitro virulence of L. monocytogenes. Pre-incubation in milk with high fat content (3.6%) resulted in a lower invasion capability compared to milk with low fat content. In contrast casein and lactose did not influence the invasiveness of L. monocytogenes into the host cell. In conclusion our study shows that the milk environment and different storage conditions influence the in vitro virulence of L. monocytogenes, both of which have to be considered in the risk assessment of contaminated food. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Temperature and Oxidative Stress as Triggers for Virulence Gene Expression in Pathogenic Leptospira spp.

    Directory of Open Access Journals (Sweden)

    Tricia Fraser

    2017-05-01

    Full Text Available Leptospirosis is a zooanthroponosis aetiologically caused by pathogenic bacteria belonging to the genus, Leptospira. Environmental signals such as increases in temperatures or oxidative stress can trigger response regulatory modes of virulence genes during infection. This study sought to determine the effect of temperature and oxidative stress on virulence associated genes in highly passaged Leptospira borgpeterseneii Jules and L. interrogans Portlandvere. Bacteria were grown in EMJH at 30°C, 37°C, or at 30°C before being transferred to 37°C. A total of 14 virulence-associated genes (fliY, invA, lenA, ligB, lipL32, lipL36, lipL41, lipL45, loa22, lsa21, mce, ompL1, sph2, and tlyC were assessed using endpoint PCR. Transcriptional analyses of lenA, lipL32, lipL41, loa22, sph2 were assessed by quantitative real-time RT-PCR at the temperature conditions. To assess oxidative stress, bacteria were exposed to H2O2 for 30 and 60 min with or without the temperature stress. All genes except ligB (for Portlandvere and ligB and mce (for Jules were detectable in the strains. Quantitatively, temperature stress resulted in significant changes in gene expression within species or between species. Temperature changes were more influential in gene expression for Jules, particularly at 30°C and upshift conditions; at 37°C, expression levels were higher for Portlandvere. However, compared to Jules, where temperature was influential in two of five genes, temperature was an essential element in four of five genes in Portlandvere exposed to oxidative stress. At both low and high oxidative stress levels, the interplay between genetic predisposition (larger genome size and temperature was biased towards Portlandvere particularly at 30°C and upshift conditions. While it is clear that expression of many virulence genes in highly passaged strains of Leptospira are attenuated or lost, genetic predisposition, changes in growth temperature and/or oxidative intensity and

  14. Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

    Science.gov (United States)

    Caswell, Clayton C.; Elhassanny, Ahmed E. M.; Planchin, Emilie E.; Roux, Christelle M.; Weeks-Gorospe, Jenni N.; Ficht, Thomas A.; Dunman, Paul M.

    2013-01-01

    The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants. PMID:23319565

  15. Crossing the line: selection and evolution of virulence traits.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available The evolution of pathogens presents a paradox. Pathogenic species are often absolutely dependent on their host species for their propagation through evolutionary time, yet the pathogenic lifestyle requires that the host be damaged during this dependence. It is clear that pathogenic strategies are successful in evolutionary terms because a diverse array of pathogens exists in nature. Pathogens also evolve using a broad range of molecular mechanisms to acquire and modulate existing virulence traits in order to achieve this success. Detailing the benefit of enhanced selection derived through virulence and understanding the mechanisms through which virulence evolves are important to understanding the natural world and both have implications for human health.

  16. Development and efficacy of a novel live-attenuated QX-like nephropathogenic infectious bronchitis virus vaccine in China.

    Science.gov (United States)

    Feng, Keyu; Xue, Yu; Wang, Jinglan; Chen, Weiguo; Chen, Feng; Bi, Yingzuo; Xie, Qingmei

    2015-02-25

    In this study, we attenuated a Chinese QX-like nephropathogenic infectious bronchitis virus (IBV) strain, YX10, by passaging through fertilized chicken eggs. The 90th passage strain (YX10p90) was selected as the live-attenuated vaccine candidate strain. YX10p90 was found to be safe in 7-day-old specific pathogen free chickens without induction of morbidity or mortality. YX10p90 provided nearly complete protection against QX-like (CH I genotype) strains and partial protection against other two major Chinese genotype strains. YX10p90 also showed no reversion to virulence after five back passages in chickens. An IBV polyvalent vaccine containing YX10p90 was developed and showed that it could provide better protection against major Chinese IBV virulent strains than commercial polyvalent vaccines. In addition, the complete genome sequence of YX10p90 was sequenced. Multiple-sequence alignments identified 38 nucleotide substitutions in the whole genome which resulted in 26 amino acid substitutions and a 110-bp deletion in the 3' untranslated region. In conclusion, the attenuated YX10p90 strain exhibited a fine balance between attenuation and immunogenicity, and should be considered as a candidate vaccine to prevent infection of Chinese QX-like nephropathogenic IBV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Gain attenuation of gated framing camera

    International Nuclear Information System (INIS)

    Xiao Shali; Liu Shenye; Cao Zhurong; Li Hang; Zhang Haiying; Yuan Zheng; Wang Liwei

    2009-01-01

    The theoretic model of framing camera's gain attenuation is analyzed. The exponential attenuation curve of the gain along the pulse propagation time is simulated. An experiment to measure the coefficient of gain attenuation based on the gain attenuation theory is designed. Experiment result shows that the gain follows an exponential attenuation rule with a quotient of 0.0249 nm -1 , the attenuation coefficient of the pulse is 0.00356 mm -1 . The loss of the pulse propagation along the MCP stripline is the leading reason of gain attenuation. But in the figure of a single stripline, the gain dose not follow the rule of exponential attenuation completely, instead, there is a gain increase at the stripline bottom. That is caused by the reflection of the pulse. The reflectance is about 24.2%. Combining the experiment and theory, which design of the stripline MCP can improved the gain attenuation. (authors)

  18. Involvement of nucleoprotein, phosphoprotein, and matrix protein genes of rabies virus in virulence for adult mice.

    Science.gov (United States)

    Shimizu, Kenta; Ito, Naoto; Mita, Tetsuo; Yamada, Kentaro; Hosokawa-Muto, Junji; Sugiyama, Makoto; Minamoto, Nobuyuki

    2007-02-01

    Rabies virus Ni-CE strain causes nonlethal infection in adult mice after intracerebral inoculation, whereas the parental Nishigahara strain kills mice. In this study, to identify viral gene(s) related to the difference in pathogenicity between Ni-CE and Nishigahara strains, we generated chimeric viruses with respective genes of the virulent Nishigahara strain in the background of the avirulent Ni-CE genome. Since chimeric viruses, which had the N, P, or M genes of the Nishigahara strain, respectively, killed adult mice after intracerebral inoculation, it became evident that the N, P, and M genes are related to the difference in pathogenicity between Ni-CE and Nishigahara strains. Previously, we showed that the G gene is a major contributor to the difference in pathogenicity between another avirulent strain, RC-HL, and the parental Nishigahara strain. These results imply that the attenuation mechanism of the Ni-CE strain is different from that of the RC-HL strain, thus suggesting that rabies virus can be attenuated by diverse mechanisms. This is the first report of changes in viral genes other than the G gene of rabies virus causing the reversion of pathogenicity of an avirulent strain.

  19. Autophagy is highly targeted among host comparative proteomes during infection with different virulent RABV strains.

    Science.gov (United States)

    Li, Ling; Jin, Hongli; Wang, Hualei; Cao, Zengguo; Feng, Na; Wang, Jianzhong; Zhao, Yongkun; Zheng, Xuexing; Hou, Pengfei; Li, Nan; Chi, Hang; Huang, Pei; Jiao, Cuicui; Li, Qian; Wang, Lina; Wang, Tiecheng; Sun, Weiyang; Gao, Yuwei; Tu, Changchun; Hu, Guixue; Yang, Songtao; Xia, Xianzhu

    2017-03-28

    Rabies virus (RABV) is a neurotropic virus that causes serious disease in humans and animals worldwide. It has been reported that different RABV strains can result in divergent prognoses in animal model. To identify host factors that affect different infection processes, a kinetic analysis of host proteome alterations in mouse brains infected with different virulent RABV strains was performed using isobaric tags for a relative and absolute quantification (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach, and this analysis identified 147 differentially expressed proteins (DEPs) between the pathogenic challenge virus standard (CVS)-11 strain and the attenuated SRV9 strain. Bioinformatics analyses of these DEPs revealed that autophagy and several pathways associated with autophagy, such as mammalian target of rapamycin (mTOR) signaling, p70S6K signaling, nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress and superoxide radical degradation, were dysregulated. Validation of the proteomic data showed that attenuated SRV9 induced more autophagosome accumulation than CVS-11 in an in vitro model. Our findings provide new insights into the pathogenesis of RABV and encourage further studies on this topic.

  20. Toxoplasma MIC2 is a major determinant of invasion and virulence.

    Directory of Open Access Journals (Sweden)

    My-Hang Huynh

    2006-08-01

    Full Text Available Like its apicomplexan kin, the obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and uses a unique form of gliding motility. The recent identification of several transmembrane adhesive complexes, potentially capable of gripping external receptors and the sub-membrane actinomyosin motor, suggests that the parasite has multiple options for host-cell recognition and invasion. To test whether the transmembrane adhesin MIC2, together with its partner protein M2AP, participates in a major invasion pathway, we utilized a conditional expression system to introduce an anhydrotetracycline-responsive mic2 construct, allowing us to then knockout the endogenous mic2 gene. Conditional suppression of MIC2 provided the first opportunity to directly determine the role of this protein in infection. Reduced MIC2 expression resulted in mistrafficking of M2AP, markedly defective host-cell attachment and invasion, the loss of helical gliding motility, and the inability to support lethal infection in a murine model of acute toxoplasmosis. Survival of mice infected with MIC2-deficient parasites correlated with lower parasite burden in infected tissues, an attenuated inflammatory immune response, and induction of long-term protective immunity. Our findings demonstrate that the MIC2 protein complex is a major virulence determinant for Toxoplasma infection and that MIC2-deficient parasites constitute an effective live-attenuated vaccine for experimental toxoplasmosis.

  1. Heterologous expression of Ralp3 in Streptococcus pyogenes M2 and M6 strains affects the virulence characteristics.

    Directory of Open Access Journals (Sweden)

    Nikolai Siemens

    Full Text Available BACKGROUND: Ralp3 is a transcriptional regulator present in a serotype specific fashion on the chromosome of the human pathogen Streptococcus pyogenes (group A streptococci, GAS. In serotypes harbouring the ralp3 gene either positive or negative effects on important metabolic and virulence genes involved in colonization and immune evasion in the human host were observed. A previous study revealed that deletion of ralp3 in a GAS M49 serotype significantly attenuated many virulence traits and caused metabolic disadvantages. This leads to two questions: (i which kind of consequences could Ralp3 expression have in GAS serotypes naturally lacking this gene, and (ii is Ralp3 actively lost during evolution in these serotypes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of Ralp3 in GAS M2 and M6 pathogenesis. Both serotypes lack ralp3 on their chromosome. The heterologous expression of ralp3 in both serotypes resulted in reduced attachment to and internalization into the majority of tested epithelial cells. Both ralp3 expression strains showed a decreased ability to survive in human blood and exclusively M2::ralp3 showed decreased survival in human serum. Both mutants secreted more active SpeB in the supernatant, resulting in a higher activity compared to wild type strains. The respective M2 and M6 wild type strains outcompeted the ralp3 expression strains in direct metabolic competition assays. The phenotypic changes observed in the M2:ralp3 and M6:ralp3 were verified on the transcriptional level. Consistent with the virulence data, tested genes showed transcript level changes in the same direction. CONCLUSIONS/SIGNIFICANCE: Together these data suggest that Ralp3 can take over transcriptional control of virulence genes in serotypes lacking the ralp3 gene. Those serotypes most likely lost Ralp3 during evolution since obviously expression of this gene is disadvantageous for metabolism and pathogenesis.

  2. Variation in chromosome copy number influences the virulence of Cryptococcus neoformans and occurs in isolates from AIDS patients

    Directory of Open Access Journals (Sweden)

    Hu Guanggan

    2011-10-01

    Full Text Available Abstract Background The adaptation of pathogenic fungi to the host environment via large-scale genomic changes is a poorly characterized phenomenon. Cryptococcus neoformans is the leading cause of fungal meningoencephalitis in HIV/AIDS patients, and we recently discovered clinical strains of the fungus that are disomic for chromosome 13. Here, we examined the genome plasticity and phenotypes of monosomic and disomic strains, and compared their virulence in a mouse model of cryptococcosis Results In an initial set of strains, melanin production was correlated with monosomy at chromosome 13, and disomic variants were less melanized and attenuated for virulence in mice. After growth in culture or passage through mice, subsequent strains were identified that varied in melanin formation and exhibited copy number changes for other chromosomes. The correlation between melanin and disomy at chromosome 13 was observed for some but not all strains. A survey of environmental and clinical isolates maintained in culture revealed few occurrences of disomic chromosomes. However, an examination of isolates that were freshly collected from the cerebrospinal fluid of AIDS patients and minimally cultured provided evidence for infections with multiple strains and copy number variation. Conclusions Overall, these results suggest that the genome of C. neoformans exhibits a greater degree of plasticity than previously appreciated. Furthermore, the expression of an essential virulence factor and the severity of disease are associated with genome variation. The occurrence of chromosomal variation in isolates from AIDS patients, combined with the observed influence of disomy on virulence, indicates that genome plasticity may have clinical relevance.

  3. The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus.

    Directory of Open Access Journals (Sweden)

    Hongbo Zhou

    Full Text Available The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt, WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49(th to 68(th positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.

  4. Ultrasonic attenuation in cuprate superconductors

    Indian Academy of Sciences (India)

    Ultrasonic attenuation in cuprate superconductors. T GUPTA1,∗ and D M GAITONDE1,2. 1Harish-Chandra Research Institute, Allahabad 211 019, India. 2High Pressure Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085, India. ∗. Email: gupta@mri.ernet.in. Abstract. We calculate the longitudinal ...

  5. A Generalized Correction for Attenuation.

    Science.gov (United States)

    Petersen, Anne C.; Bock, R. Darrell

    Use of the usual bivariate correction for attenuation with more than two variables presents two statistical problems. This pairwise method may produce a covariance matrix which is not at least positive semi-definite, and the bivariate procedure does not consider the possible influences of correlated errors among the variables. The method described…

  6. Attenuation of Vrancea events revisited

    International Nuclear Information System (INIS)

    Radulian, M.; Popa, M.; Grecu, B.; Panza, G.F.

    2003-11-01

    New aspects of the frequency-dependent attenuation of the seismic waves traveling from Vrancea subcrustal sources toward NW (Transylvanian Basin) and SE (Romanian Plain) are evidenced by the recent experimental data made available by the CALIXTO'99 tomography experiment. The observations validate the previous theoretical computations performed for the assessment, by means of a deterministic approach, of the seismic hazard in Romania. They reveal an essential aspect of the seismic ground motion attenuation, that has important implications on the probabilistic assessment of seismic hazard from Vrancea intermediate-depth earthquakes. The attenuation toward NW is shown to be a much stronger frequency-dependent effect than the attenuation toward SE and the seismic hazard computed by the deterministic approach fits satisfactorily well the observed ground motion distribution in the low-frequency band (< 1 Hz). The apparent contradiction with the historically-based intensity maps arises mainly from a systematic difference in the vulnerability (buildings eigenperiod) of the buildings in the intra- and extra-Carpathians regions. (author)

  7. Compact plasmonic variable optical attenuator

    DEFF Research Database (Denmark)

    Leosson, Kristjan; Rosenzveig, Tiberiu; Hermannsson, Pétur Gordon

    2008-01-01

    We demonstrate plasmonic nanowire-based thermo-optic variable optical attenuators operating in the 1525-1625 nm wavelength range. The devices have a footprint as low as 1 mm, extinction ratio exceeding 40 dB, driving voltage below 3 V, and full modulation bandwidth of 1 kHz. The polarization...

  8. Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii.

    Science.gov (United States)

    Pérez-Varela, María; Corral, Jordi; Vallejo, Juan Andrés; Rumbo-Feal, Soraya; Bou, Germán; Aranda, Jesús; Barbé, Jordi

    2017-08-01

    Acinetobacter baumannii is a major cause of antibiotic-resistant nosocomial infections worldwide. In this study, several rifampin-resistant spontaneous mutants obtained from the A. baumannii ATCC 17978 strain that differed in their point mutations in the rpoB gene, encoding the β-subunit of the RNA polymerase, were isolated. All the mutants harboring amino acid substitutions in position 522 or 540 of the RpoB protein were impaired in surface-associated motility and had attenuated virulence in the fertility model of Caenorhabditis elegans The transcriptional profile of these mutants included six downregulated genes encoding proteins homologous to transporters and metabolic enzymes widespread among A. baumannii clinical isolates. The construction of knockout mutants in each of the six downregulated genes revealed a significant reduction in the surface-associated motility and virulence of four of them in the A. baumannii ATCC 17978 strain, as well as in the virulent clinical isolate MAR002. Taken together, our results provide strong evidence of the connection between motility and virulence in this multiresistant nosocomial pathogen. Copyright © 2017 American Society for Microbiology.

  9. Isolation, genetic diversity and identification of a virulent pathogen of ...

    African Journals Online (AJOL)

    Isolation, genetic diversity and identification of a virulent pathogen of eriophyid mite, Aceria guerreronis (Acari: Eriophyidae) by DNA marker in Karnataka, India. Basavaraj Kalmath, B Mallik, S Onkarappa, R Girish, N Srinivasa ...

  10. Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides

    NARCIS (Netherlands)

    Li, C.H.; Cervantes, M.; Springer, D.J.; Boekhout, T.; Ruiz-Vazquez, R.M.; Torres-Martinez, S.R.; Heitman, J.; Lee, S.S.

    2011-01-01

    Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a

  11. Caenorhabditis elegans reveals novel Pseudomonas aeruginosa virulence mechanism

    NARCIS (Netherlands)

    Utari, Putri Dwi; Quax, Wim J.

    The susceptibility of Caenorhabditis elegans to different virulent phenotypes of Pseudomonas aeruginosa makes the worms an excellent model for studying host-pathogen interactions. Including the recently described liquid killing, five different killing assays are now available offering superb

  12. Staphylococcus hyicus virulence in relation to exudative epidermitis in pigs

    DEFF Research Database (Denmark)

    Wegener, Henrik Caspar; Andresen, Lars Ole; Bille-Hansen, Vivi

    1993-01-01

    Staphylococcus hyicus strains with different phage types, plasmid profiles, and antibiotic resistance patterns were isolated from piglets with exudative epidermitis. The strains could be divided into virulent strains, producing exudative epidermitis, and avirulent strains, producing no dermal...... changes when injected in experimental piglets. The results showed that both virulent and avirulent strains were present simultaneously on diseased piglets. This constitutes a diagnostic problem. Concentrated culture supernatants from nine virulent strains injected in the skin of healthy piglets produced...... a crusting reaction in all piglets. Acanthosis was observed in the histopathological examination of the crustaceous skin. Concentrated culture supernatants from nine avirulent strains produced no macroscopic or microscopic skin changes. Protein profiles from all virulent strains and seven out of nine...

  13. Identification of New Virulence Factors and Vaccine Candidates for Yersinia pestis.

    Science.gov (United States)

    Andersson, Jourdan A; Sha, Jian; Erova, Tatiana E; Fitts, Eric C; Ponnusamy, Duraisamy; Kozlova, Elena V; Kirtley, Michelle L; Chopra, Ashok K

    2017-01-01

    Earlier, we reported the identification of new virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM) screening approach. From this screen, the role of rbsA , which encodes an ATP-binding protein of ribose transport system, and vasK , an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during Y. pestis infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE) , and ypo1119-1120 , identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway protein E (GspE) of the T2SS, the ypo2884 -encoded protein has homology to the βγ crystallin superfamily, cyoABCDE codes for the cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp) exist in Y. pestis CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%), in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely Δ lpp Δ ypo0815 , Δ lpp Δ ypo2884 , Δ lpp Δ cyoABCDE , Δ vasK Δ hcp6 , and Δ ypo2720-2733 Δ hcp3 . We earlier showed that deletion of the lpp gene, which encodes Braun lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with Δ lpp Δ cyoABCDE , Δ vasK Δ hcp6 , and Δ ypo2720-2733 Δ hcp3 mutant strains were 55-100% protected upon subsequent re

  14. Identification of New Virulence Factors and Vaccine Candidates for Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Jourdan A. Andersson

    2017-10-01

    Full Text Available Earlier, we reported the identification of new virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM screening approach. From this screen, the role of rbsA, which encodes an ATP-binding protein of ribose transport system, and vasK, an essential component of the type VI secretion system (T6SS, were evaluated in mouse models of plague and confirmed to be important during Y. pestis infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE, and ypo1119-1120, identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway protein E (GspE of the T2SS, the ypo2884-encoded protein has homology to the βγ crystallin superfamily, cyoABCDE codes for the cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp exist in Y. pestis CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%, in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely ΔlppΔypo0815, ΔlppΔypo2884, ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3. We earlier showed that deletion of the lpp gene, which encodes Braun lipoprotein (Lpp and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3 mutant strains were 55–100% protected upon subsequent re-challenge with wild

  15. Fatal laboratory-acquired infection with an attenuated Yersinia pestis Strain--Chicago, Illinois, 2009.

    Science.gov (United States)

    2011-02-25

    On September 18, 2009, the Chicago Department of Public Health (CDPH) was notified by a local hospital of a suspected case of fatal laboratory-acquired infection with Yersinia pestis, the causative agent of plague. The patient, a researcher in a university laboratory, had been working along with other members of the laboratory group with a pigmentation-negative (pgm-) attenuated Y. pestis strain (KIM D27). The strain had not been known to have caused laboratory-acquired infections or human fatalities. Other researchers in a separate university laboratory facility in the same building had contact with a virulent Y. pestis strain (CO92) that is considered a select biologic agent; however, the pgm- attenuated KIM D27 is excluded from the National Select Agent Registry. The university, CDPH, the Illinois Department of Public Health (IDPH), and CDC conducted an investigation to ascertain the cause of death. This report summarizes the results of that investigation, which determined that the cause of death likely was an unrecognized occupational exposure (route unknown) to Y. pestis, leading to septic shock. Y. pestis was isolated from premortem blood cultures. Polymerase chain reaction (PCR) identified the clinical isolate as a pgm- strain of Y. pestis. Postmortem examination revealed no evidence of pneumonic plague. A postmortem diagnosis of hereditary hemochromatosis was made on the basis of histopathologic, laboratory, and genetic testing. One possible explanation for the unexpected fatal outcome in this patient is that hemochromatosis-induced iron overload might have provided the infecting KIM D27 strain, which is attenuated as a result of defects in its ability to acquire iron, with sufficient iron to overcome its iron-acquisition defects and become virulent. Researchers should adhere to recommended biosafety practices when handling any live bacterial cultures, even attenuated strains, and institutional biosafety committees should implement and maintain effective

  16. Invasion thresholds and the evolution of nonequilibrium virulence

    OpenAIRE

    Bull, J. J.; Ebert, D.

    2008-01-01

    Abstract The enterprise of virulence management attempts to predict how social practices and other factors affect the evolution of parasite virulence. These predictions are often based on parasite optima or evolutionary equilibria derived from models of host-parasite dynamics. Yet even when such models accurately capture the parasite optima, newly invading parasites will typically not be at their optima. Here we show that parasite invasion of a host population can occur despite highly nonopti...

  17. Investigating the ?Trojan Horse? Mechanism of Yersinia pestis Virulence

    Energy Technology Data Exchange (ETDEWEB)

    McCutchen-Maloney, S L; Fitch, J P

    2005-02-08

    Yersinia pestis, the etiological agent of plague, is a Gram-negative, highly communicable, enteric bacterium that has been responsible for three historic plague pandemics. Currently, several thousand cases of plague are reported worldwide annually, and Y. pestis remains a considerable threat from a biodefense perspective. Y. pestis infection can manifest in three forms: bubonic, septicemic, and pneumonic plague. Of these three forms, pneumonic plague has the highest fatality rate ({approx}100% if left untreated), the shortest intervention time ({approx}24 hours), and is highly contagious. Currently, there are no rapid, widely available vaccines for plague and though plague may be treated with antibiotics, the emergence of both naturally occurring and potentially engineered antibiotic resistant strains makes the search for more effective therapies and vaccines for plague of pressing concern. The virulence mechanism of this deadly bacterium involves induction of a Type III secretion system, a syringe-like apparatus that facilitates the injection of virulence factors, termed Yersinia outer membrane proteins (Yops), into the host cell. These virulence factors inhibit phagocytosis and cytokine secretion, and trigger apoptosis of the host cell. Y. pestis virulence factors and the Type III secretion system are induced thermally, when the bacterium enters the mammalian host from the flea vector, and through host cell contact (or conditions of low Ca{sup 2+} in vitro). Apart from the temperature increase from 26 C to 37 C and host cell contact (or low Ca{sup 2+} conditions), other molecular mechanisms that influence virulence induction in Y. pestis are largely uncharacterized. This project focused on characterizing two novel mechanisms that regulate virulence factor induction in Y. pestis, immunoglobulin G (IgG) binding and quorum sensing, using a real-time reporter system to monitor induction of virulence. Incorporating a better understanding of the mechanisms of virulence

  18. Amoebapore is an important virulence factor of Entamoeba histolytica

    Indian Academy of Sciences (India)

    We have previously demonstrated that inhibition of expression of amoebapore A (AP-A) by antisense RNA caused a marked decrease in the virulence of the parasite. A four-fold over-expression of AP-A was obtained with plasmid (pA7) which has the ap-a gene under the control of gene EhgLE-3-RP-L21. The virulence of ...

  19. Silkworm Apolipophorin Protein Inhibits Staphylococcus aureus Virulence*

    Science.gov (United States)

    Hanada, Yuichi; Sekimizu, Kazuhisa; Kaito, Chikara

    2011-01-01

    Silkworm hemolymph inhibits hemolysin production by Staphylococcus aureus. We purified a factor in the silkworm hemolymph responsible for this inhibitory activity. The final fraction with the greatest specific activity contained 220- and 74-kDa proteins. Determination of the N-terminal amino acid sequence revealed that the 220- and 74-kDa proteins were apolipophorin I and apolipophorin II, respectively, indicating that the factor was apolipophorin (ApoLp). The purified ApoLp fraction showed decreased expression of S. aureus hla encoding α-hemolysin, hlb encoding β-hemolysin, saeRS, and RNAIII, which activate the expression of these hemolysin genes. Injection of an anti-ApoLp antibody into the hemolymph increased the sensitivity of silkworms to the lethal effect of S. aureus. Hog gastric mucin, a mammalian homologue of ApoLp, decreased the expression of S. aureus hla and hlb. These findings suggest that ApoLp in the silkworm hemolymph inhibits S. aureus virulence and contributes to defense against S. aureus infection and that its activity is conserved in mammalian mucin. PMID:21937431

  20. Regulatory principles governing Salmonella and Yersinia virulence

    Science.gov (United States)

    Erhardt, Marc; Dersch, Petra

    2015-01-01

    Enteric pathogens such as Salmonella and Yersinia evolved numerous strategies to survive and proliferate in different environmental reservoirs and mammalian hosts. Deciphering common and pathogen-specific principles for how these bacteria adjust and coordinate spatiotemporal expression of virulence determinants, stress adaptation, and metabolic functions is fundamental to understand microbial pathogenesis. In order to manage sudden environmental changes, attacks by the host immune systems and microbial competition, the pathogens employ a plethora of transcriptional and post-transcriptional control elements, including transcription factors, sensory and regulatory RNAs, RNAses, and proteases, to fine-tune and control complex gene regulatory networks. Many of the contributing global regulators and the molecular mechanisms of regulation are frequently conserved between Yersinia and Salmonella. However, the interplay, arrangement, and composition of the control elements vary between these closely related enteric pathogens, which generate phenotypic differences leading to distinct pathogenic properties. In this overview we present common and different regulatory networks used by Salmonella and Yersinia to coordinate the expression of crucial motility, cell adhesion and invasion determinants, immune defense strategies, and metabolic adaptation processes. We highlight evolutionary changes of the gene regulatory circuits that result in different properties of the regulatory elements and how this influences the overall outcome of the infection process. PMID:26441883

  1. Proteomics Analysis Reveals Previously Uncharacterized Virulence Factors in Vibrio proteolyticus

    Directory of Open Access Journals (Sweden)

    Ann Ray

    2016-07-01

    Full Text Available Members of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells.

  2. Virulence potential of Staphylococcus aureus isolates from Buruli ulcer patients.

    Science.gov (United States)

    Amissah, Nana Ama; Chlebowicz, Monika A; Ablordey, Anthony; Tetteh, Caitlin S; Prah, Isaac; van der Werf, Tjip S; Friedrich, Alex W; van Dijl, Jan Maarten; Stienstra, Ymkje; Rossen, John W

    2017-06-01

    Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like α-hemolysin, and the α- and β-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (SCC) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply that the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  3. Global regulation of virulence and the stress response by CsrA in the highly adapted human gastric pathogen Helicobacter pylori

    DEFF Research Database (Denmark)

    Barnard, F.M.; Loughlin, M.F.; Fainberg, H.P.

    2004-01-01

    Although successful and persistent colonization of the gastric mucosa depends on the ability to respond to changing environmental conditions and co-ordinate the expression of virulence factors during the course of infection, Helicobacter pylori possesses relatively few transcriptional regulators....... We therefore investigated the contribution of the regulatory protein CsrA to global gene regulation in this important human pathogen. CsrA was necessary for full motility and survival of H. pylori under conditions of oxidative stress. Loss of csrA expression deregulated the oxidant...... NapA protein was produced in the mutant strain. Finally, H. pylori strains deficient in the production of CsrA were significantly attenuated for virulence in a mouse model of infection. This work provides evidence that CsrA has a broad role in regulating the physiology of H. pylori in response...

  4. Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.

    Directory of Open Access Journals (Sweden)

    Ping Chu

    2014-10-01

    Full Text Available Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt, leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP. The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.

  5. Deletion of ssnA Attenuates the Pathogenicity of Streptococcus suis and Confers Protection against Serovar 2 Strain Challenge.

    Directory of Open Access Journals (Sweden)

    Miao Li

    Full Text Available Streptococcus suis serotype 2 (SS2 is a major porcine and human pathogen which causes arthritis, meningitis, and septicemia. Streptococcus suis nuclease A (SsnA is a recently discovered deoxyribonuclease (DNase, which has been demonstrated to contribute to escape killing in neutrophil extracellular traps (NETs. To further determine the effects of ssnA on virulence, the ssnA deletion mutant (ΔssnA and its complemented strain (C-ΔssnA were constructed. The ability of ΔssnA mutant to interact with human laryngeal epithelial cell (Hep-2 was evaluated and it exhibited dramatically decreased ability to adhere to and invade Hep-2 cells. This mutation was found to exhibit significant attenuation of virulence when evaluated in CD1 mice, suggesting ssnA plays a critical role in the pathogenesis of SS2. Finally, we found that immunization with the ΔssnA mutant triggered both antibody responses and cell-mediated immunity, and conferred 80% protection against virulent SS2 challenge in mice. Taken together, our results suggest that ΔssnA represents an attractive candidate for designing an attenuated live vaccine against SS2.

  6. Association of the Host Immune Response with Protection Using a Live Attenuated African Swine Fever Virus Model.

    Science.gov (United States)

    Carlson, Jolene; O'Donnell, Vivian; Alfano, Marialexia; Velazquez Salinas, Lauro; Holinka, Lauren G; Krug, Peter W; Gladue, Douglas P; Higgs, Stephen; Borca, Manuel V

    2016-10-22

    African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus, ASF virus (ASFV). There is no vaccine to prevent the disease and current control measures are limited to culling and restricting animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pretoriuskop/96/4 (Pret4) virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 days post infection (dpi) showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response (ASFV-specific antibody and interferon (IFN)-γ responses, or specific cytokine profiles) and protection against challenge. With the exception of ASFV-specific antibodies in survivors challenged at 21 and 28 dpi, no association between the parameters assessed and protection could be established. These results, encompassing data from 65 immunized swine, underscore the complexity of the system under study, suggesting that protection relies on the concurrence of different host immune mechanisms.

  7. Association of the Host Immune Response with Protection Using a Live Attenuated African Swine Fever Virus Model

    Directory of Open Access Journals (Sweden)

    Jolene Carlson

    2016-10-01

    Full Text Available African swine fever (ASF is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus, ASF virus (ASFV. There is no vaccine to prevent the disease and current control measures are limited to culling and restricting animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pretoriuskop/96/4 (Pret4 virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus is completely attenuated. Swine infected with Pret4Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 days post infection (dpi showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi. This animal model was used to associate the presence of host immune response (ASFV-specific antibody and interferon (IFN-γ responses, or specific cytokine profiles and protection against challenge. With the exception of ASFV-specific antibodies in survivors challenged at 21 and 28 dpi, no association between the parameters assessed and protection could be established. These results, encompassing data from 65 immunized swine, underscore the complexity of the system under study, suggesting that protection relies on the concurrence of different host immune mechanisms.

  8. Changes in proteome of the Δhfq strain derived from Francisella tularensis LVS correspond with its attenuated phenotype.

    Science.gov (United States)

    Lenco, Juraj; Tambor, Vojtech; Link, Marek; Klimentova, Jana; Dresler, Jiri; Peterek, Miroslav; Charbit, Alain; Stulik, Jiri

    2014-11-01

    The posttranscriptional regulatory protein Hfq was shown to be an important determinant of the stress resistance and full virulence in the dangerous human pathogen Francisella tularensis. Transcriptomics brought rather limited clues to the precise contribution of Hfq in virulence. To reveal the molecular basis of the attenuation caused by hfq inactivation, we employed iTRAQ in the present study and compared proteomes of the parent and isogenic Δhfq strains. We show that Hfq modulates the level of 76 proteins. Most of them show decreased abundance in the ∆hfq mutant, thereby indicating that Hfq widely acts rather as a positive regulator of Francisella gene expression. Several key Francisella virulence factors including those encoded within the Francisella pathogenicity island were found among the downregulated proteins, which is in a good agreement with the attenuated phenotype of the Δhfq strain. To further validate the iTRAQ exploratory findings, we subsequently performed targeted LC-SRM analysis of selected proteins. This accurate quantification method corroborated the trends found in the iTRAQ data. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. miR-126-5p by direct targeting of JNK-interacting protein-2 (JIP-2) plays a key role in Theileria-infected macrophage virulence

    KAUST Repository

    Haidar, Malak

    2018-03-23

    Theileria annulata is an apicomplexan parasite that infects and transforms bovine macrophages that disseminate throughout the animal causing a leukaemia-like disease called tropical theileriosis. Using deep RNAseq of T. annulata-infected B cells and macrophages we identify a set of microRNAs induced by infection, whose expression diminishes upon loss of the hyper-disseminating phenotype of virulent transformed macrophages. We describe how infection-induced upregulation of miR-126-5p ablates JIP-2 expression to release cytosolic JNK to translocate to the nucleus and trans-activate AP-1-driven transcription of mmp9 to promote tumour dissemination. In non-disseminating attenuated macrophages miR-126-5p levels drop, JIP-2 levels increase, JNK1 is retained in the cytosol leading to decreased c-Jun phosphorylation and dampened AP-1-driven mmp9 transcription. We show that variation in miR-126-5p levels depends on the tyrosine phosphorylation status of AGO2 that is regulated by Grb2-recruitment of PTP1B. In attenuated macrophages Grb2 levels drop resulting in less PTP1B recruitment, greater AGO2 phosphorylation, less miR-126-5p associated with AGO2 and a consequent rise in JIP-2 levels. Changes in miR-126-5p levels therefore, underpin both the virulent hyper-dissemination and the attenuated dissemination of T. annulata-infected macrophages.

  10. ENHANCEMENTS TO NATURAL ATTENUATION: SELECTED CASE STUDIES

    Energy Technology Data Exchange (ETDEWEB)

    Vangelas, K; W. H. Albright, W; E. S. Becvar, E; C. H. Benson, C; T. O. Early, T; E. Hood, E; P. M. Jardine, P; M. Lorah, M; E. Majche, E; D. Major, D; W. J. Waugh, W; G. Wein, G; O. R. West, O

    2007-05-15

    In 2003 the US Department of Energy (DOE) embarked on a project to explore an innovative approach to remediation of subsurface contaminant plumes that focused on introducing mechanisms for augmenting natural attenuation to achieve site closure. Termed enhanced attenuation (EA), this approach has drawn its inspiration from the concept of monitored natural attenuation (MNA).

  11. Macroseismic intensity attenuation in Iran

    Science.gov (United States)

    Yaghmaei-Sabegh, Saman

    2018-01-01

    Macroseismic intensity data plays an important role in the process of seismic hazard analysis as well in developing of reliable earthquake loss models. This paper presents a physical-based model to predict macroseismic intensity attenuation based on 560 intensity data obtained in Iran in the time period 1975-2013. The geometric spreading and energy absorption of seismic waves have been considered in the proposed model. The proposed easy to implement relation describes the intensity simply as a function of moment magnitude, source to site distance and focal depth. The prediction capability of the proposed model is assessed by means of residuals analysis. Prediction results have been compared with those of other intensity prediction models for Italy, Turkey, Iran and central Asia. The results indicate the higher attenuation rate for the study area in distances less than 70km.

  12. Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials.

    Science.gov (United States)

    Arbues, Ainhoa; Aguilo, Juan I; Gonzalo-Asensio, Jesus; Marinova, Dessislava; Uranga, Santiago; Puentes, Eugenia; Fernandez, Conchita; Parra, Alberto; Cardona, Pere Joan; Vilaplana, Cristina; Ausina, Vicente; Williams, Ann; Clark, Simon; Malaga, Wladimir; Guilhot, Christophe; Gicquel, Brigitte; Martin, Carlos

    2013-10-01

    The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Campylobacter polysaccharide capsules: virulence and vaccines

    Directory of Open Access Journals (Sweden)

    Patricia eGuerry

    2012-02-01

    Full Text Available Campylobacter jejuni remains a major cause of bacterial diarrhea worldwide and is associated with numerous sequelae, including Guillain Barre Syndrome, inflammatory bowel disease, reactive arthritis, and irritable bowel syndrome. C. jejuni is unusual for an intestinal pathogen in its ability to coat its surface with a polysaccharide capsule (CPS. These capsular polysaccharides vary in sugar composition and linkage, especially those involving heptoses of unusual configuration and O-methyl phosphoramidate linkages. This structural diversity is consistent with CPS being the major serodeterminant of the Penner scheme, of which there are 47 C. jejuni serotypes. Both CPS expression and expression of modifications are subject to phase variation by slip strand mismatch repair. Although capsules are virulence factors for other pathogens, the role of CPS in C. jejuni disease has not been well defined beyond descriptive studies demonstrating a role in serum resistance and for diarrhea in a ferret model of disease. However, perhaps the most compelling evidence for a role in pathogenesis are data that CPS conjugate vaccines protect against diarrheal disease in non-human primates. A CPS conjugate vaccine approach against this pathogen is intriguing, but several questions need to be addressed, including the valency of CPS types required for an effective vaccine. There have been numerous studies of prevalence of CPS serotypes in the developed world, but few studies from developing countries where the disease incidence is higher. The complexity and cost of Penner serotyping has limited its usefulness, and a recently developed multiplex PCR method for determination of capsule type offers the potential of a more rapid and affordable method. Comparative studies have shown a strong correlation of the two methods and studies are beginning to ascertain CPS-type distribution worldwide, as well as examination of correlation of severity of illness with specific CPS types.

  14. A comparison of computational methods for identifying virulence factors.

    Directory of Open Access Journals (Sweden)

    Lu-Lu Zheng

    Full Text Available Bacterial pathogens continue to threaten public health worldwide today. Identification of bacterial virulence factors can help to find novel drug/vaccine targets against pathogenicity. It can also help to reveal the mechanisms of the related diseases at the molecular level. With the explosive growth in protein sequences generated in the postgenomic age, it is highly desired to develop computational methods for rapidly and effectively identifying virulence factors according to their sequence information alone. In this study, based on the protein-protein interaction networks from the STRING database, a novel network-based method was proposed for identifying the virulence factors in the proteomes of UPEC 536, UPEC CFT073, P. aeruginosa PAO1, L. pneumophila Philadelphia 1, C. jejuni NCTC 11168 and M. tuberculosis H37Rv. Evaluated on the same benchmark datasets derived from the aforementioned species, the identification accuracies achieved by the network-based method were around 0.9, significantly higher than those by the sequence-based methods such as BLAST, feature selection and VirulentPred. Further analysis showed that the functional associations such as the gene neighborhood and co-occurrence were the primary associations between these virulence factors in the STRING database. The high success rates indicate that the network-based method is quite promising. The novel approach holds high potential for identifying virulence factors in many other various organisms as well because it can be easily extended to identify the virulence factors in many other bacterial species, as long as the relevant significant statistical data are available for them.

  15. Differentiation in quinolone resistance by virulence genotype in Pseudomonas aeruginosa.

    Science.gov (United States)

    Agnello, Melissa; Wong-Beringer, Annie

    2012-01-01

    Pseudomonas aeruginosa is a leading pathogen that has become increasingly resistant to the fluoroquinolone antibiotics due to widespread prescribing. Adverse outcomes have been shown for patients infected with fluoroquinolone-resistant strains. The type III secretion system (TTSS) is a major virulence determinant during acute infections through the injection of effector toxins into host cells. Most strains exhibit a unique TTSS virulence genotype defined by the presence of either exoS or exoU gene encoding two of the effector toxins, ExoS and ExoU, respectively. Specific TTSS effector genotype has been shown previously to differentially impact virulence in pneumonia. In this study, we examined the relationship between TTSS effector genotype and fluoroquinolone resistance mechanisms in a collection of 270 respiratory isolates. We found that a higher proportion of exoU+ strains were fluoroquinolone-resistant compared to exoS+ strains (63% vs 49%, p = 0.03) despite its lower overall prevalence (38% exoU+ vs 56% exoS+). Results from sequencing the quinolone resistance determining regions (QRDRs) of the 4 target genes (gyrA, gyrB, parC, parE) indicated that strains containing the exoU gene were more likely to acquire ≥ 2 mutations than exoS+ strains at MICs ≤ 8 µg/ml (13% vs none) and twice as likely to have mutations in both gyrA and parC than exoS+ strains (48% vs 24% p = 0.0439). Our findings indicate that P. aeruginosa strains differentially develop resistance-conferring mutations that correlate with TTSS effector genotype and the more virulent exoU+ subpopulation. Differences in mutational processes by virulence genotype that were observed suggest co-evolution of resistance and virulence traits favoring a more virulent genotype in the quinolone-rich clinical environment.

  16. Attenuated Bioluminescent Brucella melitensis Mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) Confer Protection in Mice

    OpenAIRE

    Rajashekara, Gireesh; Glover, David A.; Banai, Menachem; O'Callaghan, David; Splitter, Gary A.

    2006-01-01

    In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B....

  17. A medicinal herb Cassia alata attenuates quorum sensing in Chromobacterium violaceum and Pseudomonas aeruginosa.

    Science.gov (United States)

    Rekha, P D; Vasavi, H S; Vipin, C; Saptami, K; Arun, A B

    2017-03-01

    Quorum sensing (QS) has been shown to play a crucial role in the pathogenesis in many bacteria, and attenuation of QS is one of the targets of antimicrobial therapy with particular interest in combating drug resistance. This study reports the QS inhibitory activity of metabolites from Cassia alata L. (Ca. alata), an important medicinal herb widely used in the treatment of microbial infections. For investigating the QS inhibition (QSI), the potential of Ca. alata L., initially, metabolites of the leaves extracted using ethanol was tested against biosensor strain Chromobacterium violaceum CV026 and C. violaceum wild-type strains. Furthermore, a purified fraction rich in flavonoids (F-AF) was used for establishing QSI activity by studying the inhibition of violacein production in C. violaceum, and QS controlled virulence and biofilm formation in Pseudomonas aeruginosa PAO1. The study results showed 50% inhibition of violacein production in C. violaceum at 0·05 mg ml -1 concentration of F-AF. In P. aeruginosa PAO1, it inhibited the tested virulence factors and biofilm formation significantly. The F-AF contained major flavonoids namely, quercetin, quercetrin and kaempferol displaying QSI activity individually against the test organisms. Present study demonstrates the quorum sensing inhibitory activity of metabolites from Cassia alata, an important medicinal herb which is commonly used worldwide in the treatment of infections caused by microorganisms. An extract prepared from the leaves of the plant showed activity against quorum sensing in Chromobacterium violaceum and was also effective against attenuating the quorum sensing controlled virulence factors in Pseudomonas aeruginosa. Activity is attributed to the rich flavonoid composition of the plant. Results of the present investigation throw an insight into the possibility of developing drug formulations using the isolated compounds against infections caused by quorum sensing-mediated pathogenicity of bacteria

  18. Development of a human live attenuated West Nile infectious DNA vaccine: Identification of a minimal mutation set conferring the attenuation level acceptable for a human vaccine.

    Science.gov (United States)

    Yamshchikov, Vladimir; Manuvakhova, Marina; Rodriguez, Efrain; Hébert, Charles

    2017-01-01

    For the development of a human West Nile (WN) infectious DNA (iDNA) vaccine, we created highly attenuated chimeric virus W1806 with the serological identity of highly virulent WN-NY99. Earlier, we attempted to utilize mutations found in the E protein of the SA14-14-2 vaccine to bring safety of W1806 to the level acceptable for human use (Yamshchikov et al., 2016). Here, we analyzed effects of the SA14-14-2 changes on growth properties and neurovirulence of W1806. A set including the E138K, K279M, K439R and G447D changes was identified as the perspective subset for satisfying the target safety profile without compromising immunogenicity of the vaccine candidate. The genetic stability of the attenuated phenotype was found to be unsatisfactory being dependent on a subset of attenuating changes incorporated in W1806. Elucidation of underlying mechanisms influencing selection of pathways for restoration of the envelope protein functionality will facilitate resolution of the emerged genetic stability issue. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Vitamin B6 Is Required for Full Motility and Virulence in Helicobacter pylori

    Science.gov (United States)

    Grubman, Alexandra; Phillips, Alexandra; Thibonnier, Marie; Kaparakis-Liaskos, Maria; Johnson, Chad; Thiberge, Jean-Michel; Radcliff, Fiona J.; Ecobichon, Chantal; Labigne, Agnès; de Reuse, Hilde; Mendz, George L.; Ferrero, Richard L.

    2010-01-01

    Despite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. To identify new virulence factors in H. pylori, we generated low-infectivity variants of a mouse-colonizing H. pylori strain using the classical technique of in vitro attenuation. The resulting variants and their highly infectious progenitor bacteria were then analyzed by global gene expression profiling. The gene expression levels of five open reading frames (ORFs) were significantly reduced in low-infectivity variants, with the most significant changes observed for ORFs HP1583 and HP1582. These ORFs were annotated as encoding homologs of the Escherichia coli vitamin B6 biosynthesis enzymes PdxA and PdxJ. Functional complementation studies with E. coli confirmed H. pylori PdxA and PdxJ to be bona fide homologs of vitamin B6 biosynthesis enzymes. Importantly, H. pylori PdxA was required for optimal growth in vitro and was shown to be essential for chronic colonization in mice. In addition to having a well-known metabolic role, vitamin B6 is necessary for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori. Thus, for the first time, we identify vitamin B6 biosynthesis enzymes as novel virulence factors in bacteria. Interestingly, pdxA and pdxJ orthologs are present in a number of human pathogens, but not in mammalian cells. We therefore propose that PdxA/J enzymes may represent ideal candidates for therapeutic targets against bacterial pathogens. PMID:21151756

  20. A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses.

    Science.gov (United States)

    Szpara, Moriah L; Tafuri, Yolanda R; Parsons, Lance; Shamim, S Rafi; Verstrepen, Kevin J; Legendre, Matthieu; Enquist, L W

    2011-10-01

    Alphaherpesviruses are widespread in the human population, and include herpes simplex virus 1 (HSV-1) and 2, and varicella zoster virus (VZV). These viral pathogens cause epithelial lesions, and then infect the nervous system to cause lifelong latency, reactivation, and spread. A related veterinary herpesvirus, pseudorabies (PRV), causes similar disease in livestock that result in significant economic losses. Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine. We present full genome sequence comparisons of the PRV vaccine strain Bartha, and two virulent PRV isolates, Kaplan and Becker. These genome sequences were determined by high-throughput sequencing and assembly, and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain. We find many previously unknown coding differences between PRV Bartha and the virulent strains, including changes to the fusion proteins gH and gB, and over forty other viral proteins. Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome. Almost 20% of the PRV genome contains tandem short sequence repeats (SSRs), a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency, transcriptional regulation, and protein interactions. We find SSRs throughout the herpesvirus family, and provide the first global characterization of SSRs in viruses, both within and between strains. We find SSR length variation between different isolates of PRV and HSV-1, which may provide a new mechanism for phenotypic variation between strains. Finally, we detected a small number of polymorphic bases within each plaque-purified PRV strain, and we characterize the effect of passage and plaque-purification on these polymorphisms. These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit limited sequence

  1. Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

    Science.gov (United States)

    Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

    2015-07-01

    Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

  2. The copper-responsive RicR regulon contributes to Mycobacterium tuberculosis virulence.

    Science.gov (United States)

    Shi, Xiaoshan; Festa, Richard A; Ioerger, Thomas R; Butler-Wu, Susan; Sacchettini, James C; Darwin, K Heran; Samanovic, Marie I

    2014-02-18

    As with most life on Earth, the transition metal copper (Cu) is essential for the viability of the human pathogen Mycobacterium tuberculosis. However, infected hosts can also use Cu to control microbial growth. Several Cu-responsive pathways are present in M. tuberculosis, including the regulated in copper repressor (RicR) regulon, which is unique to pathogenic mycobacteria. In this work, we describe the contribution of each RicR-regulated gene to Cu resistance in vitro and to virulence in animals. We found that the deletion or disruption of individual RicR-regulated genes had no impact on virulence in mice, although several mutants had Cu hypersensitivity. In contrast, a mutant unable to activate the RicR regulon was not only highly susceptible to Cu but also attenuated in mice. Thus, these data suggest that several genes of the RicR regulon are required simultaneously to combat Cu toxicity in vivo or that this regulon is also important for resistance against Cu-independent mechanisms of host defense. Mycobacterium tuberculosis is the causative agent of tuberculosis, killing millions of people every year. Therefore, understanding the biology of M. tuberculosis is crucial for the development of new therapies to treat this devastating disease. Our studies reveal that although host-supplied Cu can suppress bacterial growth, M. tuberculosis has a unique pathway, the RicR regulon, to defend against Cu toxicity. These findings suggest that Cu homeostasis pathways in both the host and the pathogen could be exploited for the treatment of tuberculosis.

  3. Bordetella Pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance.

    Science.gov (United States)

    Dorji, Dorji; Mooi, Frits; Yantorno, Osvaldo; Deora, Rajendar; Graham, Ross M; Mukkur, Trilochan K

    2018-02-01

    Despite high vaccine coverage, whooping cough caused by Bordetella pertussis remains one of the most common vaccine-preventable diseases worldwide. Introduction of whole-cell pertussis (wP) vaccines in the 1940s and acellular pertussis (aP) vaccines in 1990s reduced the mortality due to pertussis. Despite induction of both antibody and cell-mediated immune (CMI) responses by aP and wP vaccines, there has been resurgence of pertussis in many countries in recent years. Possible reasons hypothesised for resurgence have ranged from incompliance with the recommended vaccination programmes with the currently used aP vaccine to infection with a resurged clinical isolates characterised by mutations in the virulence factors, resulting in antigenic divergence with vaccine strain, and increased production of pertussis toxin, resulting in dampening of immune responses. While use of these vaccines provide varying degrees of protection against whooping cough, protection against infection and transmission appears to be less effective, warranting continuation of efforts in the development of an improved pertussis vaccine formulations capable of achieving this objective. Major approaches currently under evaluation for the development of an improved pertussis vaccine include identification of novel biofilm-associated antigens for incorporation in current aP vaccine formulations, development of live attenuated vaccines and discovery of novel non-toxic adjuvants capable of inducing both antibody and CMI. In this review, the potential roles of different accredited virulence factors, including novel biofilm-associated antigens, of B. pertussis in the evolution, formulation and delivery of improved pertussis vaccines, with potential to block the transmission of whooping cough in the community, are discussed.

  4. The involvement of the Mid1/Cch1/Yvc1 calcium channels in Aspergillus fumigatus virulence.

    Directory of Open Access Journals (Sweden)

    Patrícia Alves de Castro

    Full Text Available Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Calcium homeostasis and signaling is essential for numerous biological processes and also influences A. fumigatus pathogenicity. The presented study characterized the function of the A. fumigatus homologues of three Saccharomyces cerevisiae calcium channels, voltage-gated Cch1, stretch-activated Mid1 and vacuolar Yvc1. The A. fumigatus calcium channels cchA, midA and yvcA were regulated at transcriptional level by increased calcium levels. The YvcA::GFP fusion protein localized to the vacuoles. Both ΔcchA and ΔmidA mutant strains showed reduced radial growth rate in nutrient-poor minimal media. Interestingly, this growth defect in the ΔcchA strain was rescued by the exogenous addition of CaCl2. The ΔcchA, ΔmidA, and ΔcchA ΔmidA strains were also sensitive to the oxidative stress inducer, paraquat. Restriction of external Ca(2+ through the addition of the Ca(2+-chelator EGTA impacted upon the growth of the ΔcchA and ΔmidA strains. All the A. fumigatus ΔcchA, ΔmidA, and ΔyvcA strains demonstrated attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Infection with the parental strain resulted in a 100% mortality rate at 15 days post-infection, while the mortality rate of the ΔcchA, ΔmidA, and ΔyvcA strains after 15 days post-infection was only 25%. Collectively, this investigation strongly indicates that CchA, MidA, and YvcA play a role in A. fumigatus calcium homeostasis and virulence.

  5. A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses.

    Directory of Open Access Journals (Sweden)

    Moriah L Szpara

    2011-10-01

    Full Text Available Alphaherpesviruses are widespread in the human population, and include herpes simplex virus 1 (HSV-1 and 2, and varicella zoster virus (VZV. These viral pathogens cause epithelial lesions, and then infect the nervous system to cause lifelong latency, reactivation, and spread. A related veterinary herpesvirus, pseudorabies (PRV, causes similar disease in livestock that result in significant economic losses. Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine. We present full genome sequence comparisons of the PRV vaccine strain Bartha, and two virulent PRV isolates, Kaplan and Becker. These genome sequences were determined by high-throughput sequencing and assembly, and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain. We find many previously unknown coding differences between PRV Bartha and the virulent strains, including changes to the fusion proteins gH and gB, and over forty other viral proteins. Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome. Almost 20% of the PRV genome contains tandem short sequence repeats (SSRs, a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency, transcriptional regulation, and protein interactions. We find SSRs throughout the herpesvirus family, and provide the first global characterization of SSRs in viruses, both within and between strains. We find SSR length variation between different isolates of PRV and HSV-1, which may provide a new mechanism for phenotypic variation between strains. Finally, we detected a small number of polymorphic bases within each plaque-purified PRV strain, and we characterize the effect of passage and plaque-purification on these polymorphisms. These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit

  6. Proteomics Analysis Reveals Previously Uncharacterized Virulence Factors in Vibrio proteolyticus.

    Science.gov (United States)

    Ray, Ann; Kinch, Lisa N; de Souza Santos, Marcela; Grishin, Nick V; Orth, Kim; Salomon, Dor

    2016-07-26

    Members of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells. The pan-genome of the genus Vibrio is a potential reservoir of unidentified toxins that can provide insight into how members of this genus have successfully risen as emerging pathogens worldwide. We focused on Vibrio proteolyticus, a marine bacterium that was previously implicated in virulence toward marine animals, and characterized its interaction with eukaryotic cells. We found that this bacterium causes actin cytoskeleton rearrangements and leads to cell death. Using a

  7. Empirical support for optimal virulence in a castrating parasite.

    Directory of Open Access Journals (Sweden)

    Knut Helge Jensen

    2006-07-01

    Full Text Available The trade-off hypothesis for the evolution of virulence predicts that parasite transmission stage production and host exploitation are balanced such that lifetime transmission success (LTS is maximised. However, the experimental evidence for this prediction is weak, mainly because LTS, which indicates parasite fitness, has been difficult to measure. For castrating parasites, this simple model has been modified to take into account that parasites convert host reproductive resources into transmission stages. Parasites that kill the host too early will hardly benefit from these resources, while postponing the killing of the host results in diminished returns. As predicted from optimality models, a parasite inducing castration should therefore castrate early, but show intermediate levels of virulence, where virulence is measured as time to host killing. We studied virulence in an experimental system where a bacterial parasite castrates its host and produces spores that are not released until after host death. This permits estimating the LTS of the parasite, which can then be related to its virulence. We exposed replicate individual Daphnia magna (Crustacea of one host clone to the same amount of bacterial spores and followed individuals until their death. We found that the parasite shows strong variation in the time to kill its host and that transmission stage production peaks at an intermediate level of virulence. A further experiment tested for the genetic basis of variation in virulence by comparing survival curves of daphniids infected with parasite spores obtained from early killing versus late killing infections. Hosts infected with early killer spores had a significantly higher death rate as compared to those infected with late killers, indicating that variation in time to death was at least in part caused by genetic differences among parasites. We speculate that the clear peak in lifetime reproductive success at intermediate killing times

  8. Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

    Directory of Open Access Journals (Sweden)

    Ping Jihui

    2011-01-01

    Full Text Available Abstract Background To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV, A/Hong Kong/1/68(H3N2 (HK-wt, was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans. Results To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells and in vivo (BALB/c mouse lungs as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung

  9. A meta-analysis of parasite virulence in nestling birds.

    Science.gov (United States)

    Møller, A P; Arriero, E; Lobato, E; Merino, S

    2009-11-01

    Parasitism is a common cause of host mortality, but little is known about the ecological factors affecting parasite virulence (the rate of mortality among infected hosts). We reviewed 117 field estimates of parasite-induced nestling mortality in birds, showing that there was significant consistency in mortality among host and parasite taxa. Virulence increased towards the tropics in analyses of both species-specific data and phylogenetic analyses. We found evidence of greater parasite prevalence being associated with reduced virulence. Furthermore, bird species breeding in open nest sites suffered from greater parasite-induced mortality than hole-nesting species. By contrast, parasite specialization and generation time of parasites relative to that of hosts explained little variation in virulence. Likewise, there were little or no significant effects of host genetic variability, host sociality, host migration, host insular distribution or host survival on parasite virulence. These findings suggest that parasite-induced nestling mortality in birds is mainly determined by geographical location and to a smaller extent nest site and prevalence.

  10. The link between morphotype transition and virulence in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Linqi Wang

    Full Text Available Cryptococcus neoformans is a ubiquitous human fungal pathogen. This pathogen can undergo morphotype transition between the yeast and the filamentous form and such morphological transition has been implicated in virulence for decades. Morphotype transition is typically observed during mating, which is governed by pheromone signaling. Paradoxically, components specific to the pheromone signaling pathways play no or minimal direct roles in virulence. Thus, the link between morphotype transition and virulence and the underlying molecular mechanism remain elusive. Here, we demonstrate that filamentation can occur independent of pheromone signaling and mating, and both mating-dependent and mating-independent morphotype transition require the transcription factor Znf2. High expression of Znf2 is necessary and sufficient to initiate and maintain sex-independent filamentous growth under host-relevant conditions in vitro and during infection. Importantly, ZNF2 overexpression abolishes fungal virulence in murine models of cryptococcosis. Thus, Znf2 bridges the sex-independent morphotype transition and fungal pathogenicity. The impacts of Znf2 on morphological switch and pathogenicity are at least partly mediated through its effects on cell adhesion property. Cfl1, a Znf2 downstream factor, regulates morphogenesis, cell adhesion, biofilm formation, and virulence. Cfl1 is the first adhesin discovered in the phylum Basidiomycota of the Kingdom Fungi. Together with previous findings in other eukaryotic pathogens, our findings support a convergent evolution of plasticity in morphology and its impact on cell adhesion as a critical adaptive trait for pathogenesis.

  11. The Complex Relationship between Virulence and Antibiotic Resistance

    Science.gov (United States)

    Schroeder, Meredith; Brooks, Benjamin D.; Brooks, Amanda E.

    2017-01-01

    Antibiotic resistance, prompted by the overuse of antimicrobial agents, may arise from a variety of mechanisms, particularly horizontal gene transfer of virulence and antibiotic resistance genes, which is often facilitated by biofilm formation. The importance of phenotypic changes seen in a biofilm, which lead to genotypic alterations, cannot be overstated. Irrespective of if the biofilm is single microbe or polymicrobial, bacteria, protected within a biofilm from the external environment, communicate through signal transduction pathways (e.g., quorum sensing or two-component systems), leading to global changes in gene expression, enhancing virulence, and expediting the acquisition of antibiotic resistance. Thus, one must examine a genetic change in virulence and resistance not only in the context of the biofilm but also as inextricably linked pathologies. Observationally, it is clear that increased virulence and the advent of antibiotic resistance often arise almost simultaneously; however, their genetic connection has been relatively ignored. Although the complexities of genetic regulation in a multispecies community may obscure a causative relationship, uncovering key genetic interactions between virulence and resistance in biofilm bacteria is essential to identifying new druggable targets, ultimately providing a drug discovery and development pathway to improve treatment options for chronic and recurring infection. PMID:28106797

  12. Genetic Regulation of Virulence and Antibiotic Resistance in Acinetobacter baumannii

    Directory of Open Access Journals (Sweden)

    Carsten Kröger

    2016-12-01

    Full Text Available Multidrug resistant microorganisms are forecast to become the single biggest challenge to medical care in the 21st century. Over the last decades, members of the genus Acinetobacter have emerged as bacterial opportunistic pathogens, in particular as challenging nosocomial pathogens because of the rapid evolution of antimicrobial resistances. Although we lack fundamental biological insight into virulence mechanisms, an increasing number of researchers are working to identify virulence factors and to study antibiotic resistance. Here, we review current knowledge regarding the regulation of virulence genes and antibiotic resistance in Acinetobacter baumannii. A survey of the two-component systems AdeRS, BaeSR, GacSA and PmrAB explains how each contributes to antibiotic resistance and virulence gene expression, while BfmRS regulates cell envelope structures important for pathogen persistence. A. baumannii uses the transcription factors Fur and Zur to sense iron or zinc depletion and upregulate genes for metal scavenging as a critical survival tool in an animal host. Quorum sensing, nucleoid-associated proteins, and non-classical transcription factors such as AtfA and small regulatory RNAs are discussed in the context of virulence and antibiotic resistance.

  13. The Complex Relationship between Virulence and Antibiotic Resistance

    Directory of Open Access Journals (Sweden)

    Meredith Schroeder

    2017-01-01

    Full Text Available Antibiotic resistance, prompted by the overuse of antimicrobial agents, may arise from a variety of mechanisms, particularly horizontal gene transfer of virulence and antibiotic resistance genes, which is often facilitated by biofilm formation. The importance of phenotypic changes seen in a biofilm, which lead to genotypic alterations, cannot be overstated. Irrespective of if the biofilm is single microbe or polymicrobial, bacteria, protected within a biofilm from the external environment, communicate through signal transduction pathways (e.g., quorum sensing or two-component systems, leading to global changes in gene expression, enhancing virulence, and expediting the acquisition of antibiotic resistance. Thus, one must examine a genetic change in virulence and resistance not only in the context of the biofilm but also as inextricably linked pathologies. Observationally, it is clear that increased virulence and the advent of antibiotic resistance often arise almost simultaneously; however, their genetic connection has been relatively ignored. Although the complexities of genetic regulation in a multispecies community may obscure a causative relationship, uncovering key genetic interactions between virulence and resistance in biofilm bacteria is essential to identifying new druggable targets, ultimately providing a drug discovery and development pathway to improve treatment options for chronic and recurring infection.

  14. Genetic Regulation of Virulence and Antibiotic Resistance in Acinetobacter baumannii

    Science.gov (United States)

    Kröger, Carsten; Kary, Stefani C.; Schauer, Kristina; Cameron, Andrew D. S.

    2016-01-01

    Multidrug resistant microorganisms are forecast to become the single biggest challenge to medical care in the 21st century. Over the last decades, members of the genus Acinetobacter have emerged as bacterial opportunistic pathogens, in particular as challenging nosocomial pathogens because of the rapid evolution of antimicrobial resistances. Although we lack fundamental biological insight into virulence mechanisms, an increasing number of researchers are working to identify virulence factors and to study antibiotic resistance. Here, we review current knowledge regarding the regulation of virulence genes and antibiotic resistance in Acinetobacter baumannii. A survey of the two-component systems AdeRS, BaeSR, GacSA and PmrAB explains how each contributes to antibiotic resistance and virulence gene expression, while BfmRS regulates cell envelope structures important for pathogen persistence. A. baumannii uses the transcription factors Fur and Zur to sense iron or zinc depletion and upregulate genes for metal scavenging as a critical survival tool in an animal host. Quorum sensing, nucleoid-associated proteins, and non-classical transcription factors such as AtfA and small regulatory RNAs are discussed in the context of virulence and antibiotic resistance. PMID:28036056

  15. OrfX, a Nucleomodulin Required for Listeria monocytogenes Virulence

    Directory of Open Access Journals (Sweden)

    Andrzej Prokop

    2017-10-01

    Full Text Available Listeria monocytogenes is a bacterial pathogen causing severe foodborne infections in humans and animals. Listeria can enter into host cells and survive and multiply therein, due to an arsenal of virulence determinants encoded in different loci on the chromosome. Several key Listeria virulence genes are clustered in Listeria pathogenicity island 1. This important locus also contains orfX (lmo0206, a gene of unknown function. Here, we found that OrfX is a small, secreted protein whose expression is positively regulated by PrfA, the major transcriptional activator of Listeria virulence genes. We provide evidence that OrfX is a virulence factor that dampens the oxidative response of infected macrophages, which contributes to intracellular survival of bacteria. OrfX is targeted to the nucleus and interacts with the regulatory protein RybP. We show that in macrophages, the expression of OrfX decreases the level of RybP, which controls cellular infection. Collectively, these data reveal that Listeria targets RybP and evades macrophage oxidative stress for efficient infection. Altogether, OrfX is after LntA, the second virulence factor acting directly in the nucleus.

  16. Bacterial Prostatitis: Bacterial Virulence, Clinical Outcomes, and New Directions.

    Science.gov (United States)

    Krieger, John N; Thumbikat, Praveen

    2016-02-01

    Four prostatitis syndromes are recognized clinically: acute bacterial prostatitis, chronic bacterial prostatitis, chronic prostatitis/chronic pelvic pain syndrome, and asymptomatic prostatitis. Because Escherichia coli represents the most common cause of bacterial prostatitis, we investigated the importance of bacterial virulence factors and antimicrobial resistance in E. coli strains causing prostatitis and the potential association of these characteristics with clinical outcomes. A structured literature review revealed that we have limited understanding of the virulence-associated characteristics of E. coli causing acute prostatitis. Therefore, we completed a comprehensive microbiological and molecular investigation of a unique strain collection isolated from healthy young men. We also considered new data from an animal model system suggesting certain E. coli might prove important in the etiology of chronic prostatitis/chronic pelvic pain syndrome. Our human data suggest that E. coli needs multiple pathogenicity-associated traits to overcome anatomic and immune responses in healthy young men without urological risk factors. The phylogenetic background and accumulation of an exceptional repertoire of extraintestinal pathogenic virulence-associated genes indicate that these E. coli strains belong to a highly virulent subset of uropathogenic variants. In contrast, antibiotic resistance confers little added advantage to E. coli strains in these healthy outpatients. Our animal model data also suggest that certain pathogenic E. coli may be important in the etiology of chronic prostatitis/chronic pelvic pain syndrome through mechanisms that are dependent on the host genetic background and the virulence of the bacterial strain.

  17. Protective efficacy of a live attenuated anti-coccidial vaccine administered to 1-day-old chickens.

    Science.gov (United States)

    Crouch, C F; Andrews, S J; Ward, R G; Francis, M J

    2003-06-01

    The efficacy of a live attenuated anti-coccidial vaccine, Paracox-5, administered to 1-day-old chicks was investigated by assessing protection against changes in weight gain following virulent challenge. Vaccinated birds were challenged independently 28 days later with each of the component species (Eimeria acervulina, Eimeria maxima, Eimeria mitis or Eimeria tenella), and protection was demonstrated against associated reduction in weight gain and lesion formation. In addition, an improvement in bird performance, in terms of feed conversion ratio, was also observed following vaccination. Furthermore, under conditions designed to more closely mimic those in the field and using hatchery spray administration, protection against a mixed virulent challenge introduced by 'seeder birds' was demonstrated evenly across a flock of broiler birds within 21 days after vaccination. These data demonstrate that Paracox-5 vaccine will protect broiler chickens against the adverse effects on performance induced by Eimeria spp.

  18. Live attenuated hepatitis A vaccines developed in China.

    Science.gov (United States)

    Xu, Zhi-Yi; Wang, Xuan-Yi

    2014-01-01

    Two live, attenuated hepatitis A vaccines, H 2 and LA-1 virus strains, were developed through serial passages of the viruses in cell cultures at 32 °C and 35 °C respectively. Both vaccines were safe and immunogenic, providing protection against clinical hepatitis A in 95% of the vaccinees, with a single dose by subcutaneous injection. The vaccine recipients were not protected from asymptomatic, subclinical hepatitis A virus (HAV) infection, which induced a similar antibody response as for unvaccinated subjects. A second dose caused anamnestic response and can be used for boosting. Oral immunization of human with H 2 vaccine or of marmoset with LA-1 vaccine failed, and no evidence was found for person-to-person transmission of the H 2 strain or for marmoset-to-marmoset transmission of LA-1 strain, by close contact. H 2 strain was genetically stable when passaged in marmosets, humans or cell cultures at 37 °C; 3 consecutive passages of the virus in marmosets did not cause virulence mutation. The live vaccines offer the benefits of low cost, single dose injection, long- term protection, and increased duration of immunity through subclinical infection. Improved sanitation and administration of 150 million doses of the live vaccines to children had led to a 90% reduction in the annual national incidence rate of hepatitis A in China during the 16-year period, from 1991 to 2006. Hepatitis A immunization with both live and inactivated HA vaccines was implemented in the national routine childhood immunization program in 2008 and around 92% of the 16 million annual births received the affordable live, attenuated vaccines at 18 months of age. Near elimination of the disease was achieved in China for 14 years following introduction of the H 2 live vaccine into the Expanded Immunization Program (EPI) in 1992.

  19. Live attenuated hepatitis A vaccines developed in China

    Science.gov (United States)

    Xu, Zhi-Yi; Wang, Xuan-Yi

    2014-01-01

    Two live, attenuated hepatitis A vaccines, H2 and LA-1 virus strains, were developed through serial passages of the viruses in cell cultures at 32 °C and 35 °C respectively. Both vaccines were safe and immunogenic, providing protection against clinical hepatitis A in 95% of the vaccinees, with a single dose by subcutaneous injection. The vaccine recipients were not protected from asymptomatic, subclinical hepatitis A virus (HAV) infection, which induced a similar antibody response as for unvaccinated subjects. A second dose caused anamnestic response and can be used for boosting. Oral immunization of human with H2 vaccine or of marmoset with LA-1 vaccine failed, and no evidence was found for person-to-person transmission of H2 strain or for marmoset-to-marmoset transmission of LA-1 strain by close contact. H2 strain was genetically stable when passaged in marmosets, humans or cell cultures at 37 °C; 3 consecutive passages of the virus in marmosets did not cause virulence mutation. The live vaccines offer the benefits of low cost, single dose injection, long- term protection, and increased duration of immunity through subclinical infection. Improved sanitation and administration of 150 million doses of the live vaccines to children had led to a 90% reduction in the annual national incidence rate of hepatitis A in China during the 16-year period, from 1991 to 2006. Hepatitis A (HA) immunization with both live and inactivated HA vaccines was implemented in the national routine childhood immunization program in 2008 and around 92% of the 16 million annual births received the affordable live, attenuated vaccines at 18 months of age. Near elimination of the disease was achieved in a county of China for 14 years following introduction of the H2 live vaccine into the Expanded Immunization Program (EPI) in 1992. PMID:24280971

  20. Frequency Dependent Attenuation in Rocks

    Science.gov (United States)

    1990-01-20

    WORK UNIT ELEMENT NO NO NO ACCESSION NO 62714E 7A10 DA DA 1𔃻 T. ,E (Incude Security Clasification ) Frequency Dependent Attenuation in Rocks Ŗ 0E;SO’.A_...P., and L. Peselnick, Investigation of internal friction in fused quartz, steel , plexiglass, and Westerly granite from 0.01 to 1.00 Hertz at 10- to 10...P., and L. Peselnick, Investigation of internal friction in fused quartz, steel , plexiglas, and Westerly granite from 0.01 to 1.00 Hertz at 10-8 to 10

  1. Low virulent oral Candida albicans strains isolated from smokers.

    Science.gov (United States)

    de Azevedo Izidoro, Ana Claudia Santos; Semprebom, Andressa Marafon; Baboni, Fernanda Brasil; Rosa, Rosimeire Takaki; Machado, Maria Angela Naval; Samaranayake, Lakshman Perera; Rosa, Edvaldo Antonio Ribeiro

    2012-02-01

    It is widely accepted that tabagism is a predisposing factor to oral candidosis and cumulate data suggest that cigarette compounds may increase candidal virulence. To verify if enhanced virulence occurs in Candida albicans from chronic smokers, a cohort of 42 non-smokers and other of 58 smokers (all with excellent oral conditions and without signs of candidosis) were swabbed on tong dorsum and jugal mucosa. Results showed that oral candidal loads do not differ between smoker and non-smokers. Activities of secreted aspartyl-protease (Sap), phospholipase, chondroitinase, esterase-lipase, and haemolysin secretions were screened for thirty-two C. albicans isolates. There were detected significant increments in phospholipasic and chondroitinasic activities in isolates from non-smokers. For other virulence factors, no differences between both cohorts were achieved. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Virulence - associated genes in Avian Pathogenic Escherichia coli of turkey

    Directory of Open Access Journals (Sweden)

    Antonio Camarda

    2010-01-01

    Full Text Available 50 Escherichia coli (APEC-Avian Pathogenic Escherichia coli strains and 15 E. coli (AFEC-Avian Faecal Escherichia coli from turkeys affected by colibacillosis and from healthy turkeys were tested for the presence of eight different virulence-associated genes. Besides, APEC were serotyped. O78 has been the most detected serotyped. The presence of the tested virulence genes was prevalently related to the APEC isolates. With reference to serogroup, all the tested O78 resulted iss and irp2 positive. Besides, tsh e cva/cvi were respectively present in 88.9 and 83.3% of O78. Nevertheless, the finding of a not typeable strains equipped with all the eight tested virulence genes among the APEC isolates suggest the importance of a careful and complete characterisation of the isolate to evaluate the real potential pathogenic attitude of the bacterium.

  3. Polyamines Are Required for Virulence in Salmonella enterica Serovar Typhimurium

    DEFF Research Database (Denmark)

    Jelsbak, Lotte; Thomsen, Line Elnif; Wallrodt, Inke

    2012-01-01

    Sensing and responding to environmental cues is a fundamental characteristic of bacterial physiology and virulence. Here we identify polyamines as novel environmental signals essential for virulence of Salmonella enterica serovar Typhimurium, a major intracellular pathogen and a model organism......, we show that an S. Typhimurium polyamine mutant is defective for invasion, intracellular survival, killing of the nematode Caenorhabditis elegans and systemic infection of the mouse model of typhoid fever. Virulence of the mutant could be restored by genetic complementation, and invasion...... and intracellular survival could, as well, be complemented by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection. Interestingly, intracellular survival of the polyamine mutant was significantly enhanced above the wild type level by the addition of exogenous putrescine...

  4. A pilot study on an attenuated Chinese EIAV vaccine inducing broadly neutralizing antibodies.

    Science.gov (United States)

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Liang, Hua; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2011-08-01

    The attenuated Chinese equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. In this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated Chinese vaccine strain EIAVFDDV and then observed the evolution of neutralizing antibodies against different EIAV strains. During the vaccination phase, all vaccinees rapidly developed high levels of neutralizing antibodies against the homologous vaccine strain (pLGFD3V), and 3 out of 4 horses showed a gradual increase in serum neutralizing activity against two relatively heterologous virulent variants of the challenge strain (pLGFD3Mu12V and DLV34). After challenge, the three horses that had developed high levels of neutralizing antibodies against pLGFD3Mu12V and DLV34 did not show signs of infection, which was demonstrated by immune suppression, while the one horse producing serum that could only neutralize pLGFD3V developed a febrile episode during the 8-month observation period. To assess whether the broadly neutralizing activity is associated with immune protection, sera drawn on the day of challenge from these four vaccinees and an additional four EIAVFDDV-vaccinated horses were analyzed for neutralizing antibodies against pLGFD3V, pLGFD3Mu12V and DLV34. Although there was no significant correlation between protection from infection and serum neutralizing activity against any of these three viral strains, protection from infection was observed to correlate better with serum neutralizing activity against the two heterologous virulent strains than against the homologous vaccine strain. These data indicate that EIAVFDDV induced broadly neutralizing antibodies, which might confer enhanced protection of vaccinees from infection by the challenge virus.

  5. Virulence of Colletotrichum acutatum isolates to several host plants

    Directory of Open Access Journals (Sweden)

    Barbora Staňková

    2011-01-01

    Full Text Available Colletotrichum acutatum belongs to polyphagous fungal pathogens and is widespread in many countries on all continents. C. acutatum causes the most serious economic damage in strawberry (Fragaria x ananassa Duch.. Considering the wide variability of the pathogen may be assumed spread to other areas which constitutes danger not only for strawberry, but also other economically important fruit crops, vegetables and fruits.The main objective of our study was to verify the cross infection of eleven C. acutatum isolates from different host plants (strawberry, safflower, lupine, pepper and Hypericum perforatum to selected host plants (strawberry, pepper and safflower. Two varieties from each of the experimental plant species were selected and virulence of isolates C. acutatum was evaluated.Based on results of statistical evaluation, virulence of C. acutatum isolates was different on strawberry, pepper and safflower. The strawberry variety Pegasus was more susceptible to C. acutatum than the variety Elkas. Isolate 710 from H. perforatum showed the highest virulence for both varieties in terms of index of infection intensity. The pepper variety Pirouet was more susceptible than the variety Cynthia. The highest degree of virulence was found for isolate 29267 from pepper in the variety Cynthia, the highest virulence was proved for isolate 231 from strawberry in the variety Pirouet. No statistical difference was confirmed between susceptibility of the safflower varieties. Isolate 1209 from safflower showed the most important effect on tested plants of safflower. Isolates 710 from H. perforatum, isolate 1209 from safflower, isolate 29267 from pepper and isolate 231 from strawberry showed different virulence for tested host plants.

  6. Natural Selection in Virulence Genes of Francisella tularensis.

    Science.gov (United States)

    Gunnell, Mark K; Robison, Richard A; Adams, Byron J

    2016-06-01

    A fundamental tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage. Negatively selected alleles are removed from the gene pool and are eventually extinguished from the population. Conversely, alleles under positive selection do confer an evolutionary advantage and lead to an increase in the overall fitness of the organism. These alleles increase in frequency until they eventually become fixed in the population. Francisella tularensis is a zoonotic pathogen and a potential biothreat agent. The most virulent type of F. tularensis, Type A, is distributed across North America with Type A.I occurring mainly in the east and Type A.II appearing mainly in the west. F. tularensis is thought to be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. To test this hypothesis, we sequenced and compared whole genomes of Type A.I and A.II isolates. We analyzed a subset of virulence and housekeeping genes from several F. tularensis subspecies genomes to ascertain the presence and extent of positive selection. Eleven previously identified virulence genes were screened for positive selection along with 10 housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection at loci implicated in cell surface structures and membrane proteins, metabolism and biosynthesis, transcription, translation and cell separation, and substrate binding and transport. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution

  7. Virulence of the zoonotic agent of leptospirosis: still terra incognita?

    Science.gov (United States)

    Picardeau, Mathieu

    2017-05-01

    Pathogenic leptospires are the bacterial agents of leptospirosis, which is an emerging zoonotic disease that affects animals and humans worldwide. The success of leptospires as pathogens is explained by their spiral shape and endoflagellar motility (which enable these spirochetes to rapidly cross connective tissues and barriers), as well as by their ability to escape or hijack the host immune system. However, the basic biology and virulence factors of leptospires remain poorly characterized. In this Review, we discuss the recent advances in our understanding of the epidemiology, taxonomy, genomics and the molecular basis of virulence in leptospires, and how these properties contribute to the mechanism of pathogenesis of leptospirosis.

  8. Reconstructing the highly virulent Classical Swine Fever Virus strain Koslov

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Nielsen, Jens

    by reconstructing ancestral sequences. To test this hypothesis, we inferred sequences that correspond to ancestral nodes in a phylogenetic tree built from full-length nucleotide sequences of non-functional Koslov cDNAs and then proceeded to test the reconstructions. Specifically, we altered a non-functional c......, when tested in pigs, were at least as virulent as the Koslov strain. The ancestral reconstruction therefore proved to give rise to a functional cDNA of the highly virulent Koslov strain. In vivo studies confirmed our methods and enabled us to identify nucleotide positions within the viral genome...

  9. Variations in virulence between different electrophoretic types of Listeria monocytogenes

    DEFF Research Database (Denmark)

    Nørrung, Birgit; Andersen, Jens Kirk

    2000-01-01

    A total of 245 strains of Listeria monocytogenes, representing 33 different electrophoretic types (ETs), were examined quantitatively for haemolytic activity. No significant difference was observed in the mean haemolytic activity between different ETs. Eighty four out of 91 strains examined were...... compared with 3.64 among food isolates). The explanation for this may be that more virulent strains are more prone to cause human infection. It is, however, also possible that strains oft. monocytogenes may become more virulent while multiplying in a living organism compared with multiplying in foods....

  10. Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Reimann, Ilona

    2007-01-01

    A chimeric pestivirus of border disease virus Gifhorn and bovine viral diarrhea virus CP7 (Meyers et al., 1996) was constructed. Virulence, immunogenicity and vaccine properties of the chimeric virus were studied in a vaccination–challenge experiment in pigs. The chimeric virus proved to be aviru......A chimeric pestivirus of border disease virus Gifhorn and bovine viral diarrhea virus CP7 (Meyers et al., 1996) was constructed. Virulence, immunogenicity and vaccine properties of the chimeric virus were studied in a vaccination–challenge experiment in pigs. The chimeric virus proved...

  11. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    Energy Technology Data Exchange (ETDEWEB)

    Joel Walls; M.T. Taner; Naum Derzhi; Gary Mavko; Jack Dvorkin

    2003-12-01

    We have developed and tested technology for a new type of direct hydrocarbon detection. The method uses inelastic rock properties to greatly enhance the sensitivity of surface seismic methods to the presence of oil and gas saturation. These methods include use of energy absorption, dispersion, and attenuation (Q) along with traditional seismic attributes like velocity, impedance, and AVO. Our approach is to combine three elements: (1) a synthesis of the latest rock physics understanding of how rock inelasticity is related to rock type, pore fluid types, and pore microstructure, (2) synthetic seismic modeling that will help identify the relative contributions of scattering and intrinsic inelasticity to apparent Q attributes, and (3) robust algorithms that extract relative wave attenuation attributes from seismic data. This project provides: (1) Additional petrophysical insight from acquired data; (2) Increased understanding of rock and fluid properties; (3) New techniques to measure reservoir properties that are not currently available; and (4) Provide tools to more accurately describe the reservoir and predict oil location and volumes. These methodologies will improve the industry's ability to predict and quantify oil and gas saturation distribution, and to apply this information through geologic models to enhance reservoir simulation. We have applied for two separate patents relating to work that was completed as part of this project.

  12. Chlorine signal attenuation in concrete.

    Science.gov (United States)

    Naqvi, A A; Maslehuddin, M; Ur-Rehman, Khateeb; Al-Amoudi, O S B

    2015-11-01

    The intensity of prompt gamma-ray was measured at various depths from chlorine-contaminated silica fume (SF) concrete slab concrete specimens using portable neutron generator-based prompt gamma-ray setup. The intensity of 6.11MeV chloride gamma-rays was measured from the chloride contaminated slab at distance of 15.25, 20.25, 25.25, 30.25 and 35.25cm from neutron target in a SF cement concrete slab specimens. Due to attenuation of thermal neutron flux and emitted gamma-ray intensity in SF cement concrete at various depths, the measured intensity of chlorine gamma-rays decreases non-linearly with increasing depth in concrete. A good agreement was noted between the experimental results and the results of Monte Carlo simulation. This study has provided useful experimental data for evaluating the chloride contamination in the SF concrete utilizing gamma-ray attenuation method. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Assessment of Mycobacterium bovis deleted in p27-p55 virulence operon as candidate vaccine against tuberculosis in animal models.

    Science.gov (United States)

    Bianco, María V; Clark, Simon; Blanco, Federico C; Garbaccio, Sergio; García, Elizabeth; Cataldi, Angel A; Williams, Ann; Bigi, Fabiana

    2014-01-01

    A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.

  14. Assessment of Mycobacterium bovis Deleted in p27-p55 Virulence Operon as Candidate Vaccine against Tuberculosis in Animal Models

    Directory of Open Access Journals (Sweden)

    María V. Bianco

    2014-01-01

    Full Text Available A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.

  15. Genetic Modulation of c-di-GMP Turnover Affects Multiple Virulence Traits and Bacterial Virulence in Rice Pathogen Dickeya zeae.

    Directory of Open Access Journals (Sweden)

    Yufan Chen

    Full Text Available The frequent outbreaks of rice foot rot disease caused by Dickeya zeae have become a significant concern in rice planting regions and countries, but the regulatory mechanisms that govern the virulence of this important pathogen remain vague. Given that the second messenger cyclic di-GMP (c-di-GMP is associated with modulation of various virulence-related traits in various microorganisms, here we set to investigate the role of the genes encoding c-di-GMP metabolism in the regulation of the bacterial physiology and virulence by construction all in-frame deletion mutants targeting the annotated c-di-GMP turnover genes in D. zeae strain EC1. Phenotype analyses identified individual mutants showing altered production of exoenzymes and phytotoxins, biofilm formation and bacterial motilities. The results provide useful clues and a valuable toolkit for further characterization and dissection of the regulatory complex that modulates the pathogenesis and persistence of this important bacterial pathogen.

  16. Genetic Modulation of c-di-GMP Turnover Affects Multiple Virulence Traits and Bacterial Virulence in Rice Pathogen Dickeya zeae.

    Science.gov (United States)

    Chen, Yufan; Lv, Mingfa; Liao, Lisheng; Gu, Yanfang; Liang, Zhibin; Shi, Zurong; Liu, Shiyin; Zhou, Jianuan; Zhang, Lianhui

    2016-01-01

    The frequent outbreaks of rice foot rot disease caused by Dickeya zeae have become a significant concern in rice planting regions and countries, but the regulatory mechanisms that govern the virulence of this important pathogen remain vague. Given that the second messenger cyclic di-GMP (c-di-GMP) is associated with modulation of various virulence-related traits in various microorganisms, here we set to investigate the role of the genes encoding c-di-GMP metabolism in the regulation of the bacterial physiology and virulence by construction all in-frame deletion mutants targeting the annotated c-di-GMP turnover genes in D. zeae strain EC1. Phenotype analyses identified individual mutants showing altered production of exoenzymes and phytotoxins, biofilm formation and bacterial motilities. The results provide useful clues and a valuable toolkit for further characterization and dissection of the regulatory complex that modulates the pathogenesis and persistence of this important bacterial pathogen.

  17. Synergistic interactions between the NS3(hel and E proteins contribute to the virulence of dengue virus type 1.

    Directory of Open Access Journals (Sweden)

    Luana de Borba

    Full Text Available BACKGROUND: Dengue includes a broad range of symptoms, ranging from fever to hemorrhagic fever and may occasionally have alternative clinical presentations. Many possible viral genetic determinants of the intrinsic virulence of dengue virus (DENV in the host have been identified, but no conclusive evidence of a correlation between viral genotype and virus transmissibility and pathogenicity has been obtained. METHODOLOGY/PRINCIPAL FINDINGS: We used reverse genetics techniques to engineer DENV-1 viruses with subsets of mutations found in two different neuroadapted derivatives. The mutations were inserted into an infectious clone of DENV-1 not adapted to mice. The replication and viral production capacity of the recombinant viruses were assessed in vitro and in vivo. The results demonstrated that paired mutations in the envelope protein (E and in the helicase domain of the NS3 (NS3(hel protein had a synergistic effect enhancing viral fitness in human and mosquito derived cell lines. E mutations alone generated no detectable virulence in the mouse model; however, the combination of these mutations with NS3(hel mutations, which were mildly virulent on their own, resulted in a highly neurovirulent phenotype. CONCLUSIONS/SIGNIFICANCE: The generation of recombinant viruses carrying specific E and NS3(hel proteins mutations increased viral fitness both in vitro and in vivo by increasing RNA synthesis and viral load (these changes being positively correlated with central nervous system damage, the strength of the immune response and animal mortality. The introduction of only pairs of amino acid substitutions into the genome of a non-mouse adapted DENV-1 strain was sufficient to alter viral fitness substantially. Given current limitations to our understanding of the molecular basis of dengue neuropathogenesis, these results could contribute to the development of attenuated strains for use in vaccinations and provide insights into virus/host interactions

  18. Type VI Secretion is a Major Virulence Determinant in Burkholderia Mallei

    National Research Council Canada - National Science Library

    Schell, Mark A; Ulrich, Ricky L; Ribot, Wilson J; Brueggemann, Ernst E; Hines, Harry B; Chen, Dan; Lipscomb, Lyla; Kim, H. S; Mrazek, Jan; Nierman, William C; DeShazer, David

    2007-01-01

    Burkholderia mallei is a host-adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two-component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown...

  19. Estimating Rain Attenuation In Satellite Communication Links

    Science.gov (United States)

    Manning, R. M.

    1991-01-01

    Attenuation computed with help of statistical model and meteorological data. NASA Lewis Research Center Satellite Link Attenuation Model (SLAM) program QuickBASIC computer program evaluating static and dynamic statistical assessment of impact of rain attenuation on communication link established between Earth terminal and geosynchronous satellite. Application in specification, design, and assessment of satellite communication links for any terminal location in continental United States. Written in Microsoft QuickBASIC.

  20. Attenuation correction in SPECT images using attenuation map estimation with its emission data

    Science.gov (United States)

    Tavakoli, Meysam; Naji, Maryam; Abdollahi, Ali; Kalantari, Faraz

    2017-03-01

    Photon attenuation during SPECT imaging significantly degrades the diagnostic outcome and the quantitative accuracy of final reconstructed images. It is well known that attenuation correction can be done by using iterative reconstruction methods if we access to attenuation map. Two methods have been used to calculate the attenuation map: transmission-based and transmissionless techniques. In this phantom study, we evaluated the importance of attenuation correction by quantitative evaluation of errors associated with each method. For transmissionless approach, the attenuation map was estimated from the emission data only. An EM algorithm with attenuation model was developed and used for attenuation correction during image reconstruction. Finally, a comparison was done between reconstructed images using our OSEM code and analytical FBP method before and after attenuation correction. The results of measurements showed that: our programs are capable to reconstruct SPECT images and correct the attenuation effects. Moreover, to evaluate reconstructed image quality before and after attenuation correction we applied a novel approach using Image Quality Index. Attenuation correction increases the quality and quantitative accuracy in both methods. This increase is independent of activity in quantity factor and decreases with activity in quality factor. In EM algorithm, it is necessary to use regularization to obtain true distribution of attenuation coefficients.

  1. Virulence evolution in response to anti-infection resistance: toxic food plants can select for virulent parasites of monarch butterflies.

    Science.gov (United States)

    de Roode, J C; de Castillejo, C Lopez Fernandez; Faits, T; Alizon, S

    2011-04-01

    Host resistance to parasites can come in two main forms: hosts may either reduce the probability of parasite infection (anti-infection resistance) or reduce parasite growth after infection has occurred (anti-growth resistance). Both resistance mechanisms are often imperfect, meaning that they do not fully prevent or clear infections. Theoretical work has suggested that imperfect anti-growth resistance can select for higher parasite virulence by favouring faster-growing and more virulent parasites that overcome this resistance. In contrast, imperfect anti-infection resistance is thought not to select for increased parasite virulence, because it is assumed that it reduces the number of hosts that become infected, but not the fitness of parasites in successfully infected hosts. Here, we develop a theoretical model to show that anti-infection resistance can in fact select for higher virulence when such resistance reduces the effective parasite dose that enters a host. Our model is based on a monarch butterfly-parasite system in which larval food plants confer resistance to the monarch host. We carried out an experiment and showed that this environmental resistance is most likely a form of anti-infection resistance, through which toxic food plants reduce the effective dose of parasites that initiates an infection. We used these results to build a mathematical model to investigate the evolutionary consequences of food plant-induced resistance. Our model shows that when the effective infectious dose is reduced, parasites can compensate by evolving a higher per-parasite growth rate, and consequently a higher intrinsic virulence. Our results are relevant to many insect host-parasite systems, in which larval food plants often confer imperfect anti-infection resistance. Our results also suggest that - for parasites where the infectious dose affects the within-host dynamics - vaccines that reduce the effective infectious dose can select for increased parasite virulence.

  2. Type VI secretion system contributes to Enterohemorrhagic Escherichia coli virulence by secreting catalase against host reactive oxygen species (ROS).

    Science.gov (United States)

    Wan, Baoshan; Zhang, Qiufen; Ni, Jinjing; Li, Shuxian; Wen, Donghua; Li, Jun; Xiao, Haihan; He, Ping; Ou, Hong-Yu; Tao, Jing; Teng, Qihui; Lu, Jie; Wu, Wenjuan; Yao, Yu-Feng

    2017-03-01

    Enterohemorrhagic Escherichia coli (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of katN is promoted by RpoS and OxyR and repressed by H-NS, and katN contributes to bacterial growth under oxidative stress in vitro. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of katN had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells.

  3. Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

    Science.gov (United States)

    Burnside, Kellie; Lembo, Annalisa; de Los Reyes, Melissa; Iliuk, Anton; Binhtran, Nguyen-Thao; Connelly, James E; Lin, Wan-Jung; Schmidt, Byron Z; Richardson, Anthony R; Fang, Ferric C; Tao, Weiguo Andy; Rajagopal, Lakshmi

    2010-06-11

    Exotoxins, including the hemolysins known as the alpha (alpha) and beta (beta) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

  4. Type VI secretion system contributes to Enterohemorrhagic Escherichia coli virulence by secreting catalase against host reactive oxygen species (ROS.

    Directory of Open Access Journals (Sweden)

    Baoshan Wan

    2017-03-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of katN is promoted by RpoS and OxyR and repressed by H-NS, and katN contributes to bacterial growth under oxidative stress in vitro. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of katN had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells.

  5. Identification of Salmonella typhimurium genes required for colonization of the chicken alimentary tract and for virulence in newly hatched chicks.

    Science.gov (United States)

    Turner, A K; Lovell, M A; Hulme, S D; Zhang-Barber, L; Barrow, P A

    1998-05-01

    From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC. Transduction of most of the transposon mutations to a fresh S. typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S. typhimurium F98 supported the role in colonization of all but the pts locus. The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested. All but the ptsC and rfaY mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described.

  6. Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

    Directory of Open Access Journals (Sweden)

    Kellie Burnside

    2010-06-01

    Full Text Available Exotoxins, including the hemolysins known as the alpha (alpha and beta (beta toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1 were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1 increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU, serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE and a hypothetical protein (NWMN_1123 were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

  7. Contribution of eukaryotic-type serine/threonine kinase to stress response and virulence of Streptococcus suis.

    Directory of Open Access Journals (Sweden)

    Haodan Zhu

    Full Text Available Streptococcus suis serotype 2 (SS2 is an important swine and human pathogen responsible for septicemia and meningitis. The bacterial homologues of eukaryotic-type serine/threonine kinases (ESTKs have been reported to play critical roles in various cellular processes. To investigate the role of STK in SS2, an isogenic stk mutant strain (Δstk and a complemented strain (CΔstk were constructed. The Δstk showed a significant decrease in adherence to HEp-2 cells, compared with the wild-type strain, and a reduced survival ratio in whole blood. In addition, the Δstk exhibited a notable reduced tolerance of environmental stresses including high temperature, acidic pH, oxidative stress, and high osmolarity. More importantly, the Δstk was attenuated in both the CD1 mouse and piglet models of infection. The results of quantitative reverse transcription-PCR (qRT-PCR analysis indicated that the expressions of a few genes involving in adherence, stress response and virulence were clearly decreased in the Δstk mutant strain. Our data suggest that SsSTK is required for virulence and stress response in SS2.

  8. Shoot the Message, Not the Messenger—Combating Pathogenic Virulence in Plants by Inhibiting Quorum Sensing Mediated Signaling Molecules

    Directory of Open Access Journals (Sweden)

    Ganesh Alagarasan

    2017-04-01

    Full Text Available Immunity, virulence, biofilm formation, and survival in the host environment are regulated by the versatile nature of density dependent microbial cell signaling, also called quorum sensing (QS. The QS molecules can associate with host plant tissues and, at times, cause a change in its gene expression at the downstream level through inter-kingdom cross talking. Progress in controlling QS through fungicide/bactericide in pathogenic microscopic organisms has lead to a rise of antibiotic resistance pathogens. Here, we review the application of selective quorum quenching (QQ endophytes to control phytopathogens that are shared by most, if not all, terrestrial plant species as well as aquatic plants. Allowing the plants to posses end