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Sample records for allele-specific virulence attenuation

  1. Engineering attenuated virulence of a Theileria annulata-infected macrophage.

    Directory of Open Access Journals (Sweden)

    Nadia Echebli

    Full Text Available Live attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun induced a complete failure of Theileria annulata-transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata-infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis.

  2. Physiological and Antigenic Characteristics of Virulent and Attenuated Strains of Legionella pneumophila (Philadelphia 3)

    Science.gov (United States)

    1981-07-01

    CHARACTERISTICS OF I, VIRULENT AND ATTENUATED5TRAINS OF LEGIONELLA Interim PNEUMOPHILA (PHILADELPHIA 3) 6 . PERFORMING ORG. REPORT NUMBER 70 AUTHOR(*) B...number) Several methods were used to cJvrtcterize selected virulent and attenuated strains of Legionella pneumophila serogroup 1. The cultural...Antigenic Characteristics of Virulent and Attenuated Strains of Legionella pneumophila (Philadelphia 3) JOSEPH D. RISTROPH, KENNETH W. HEDLUND, AND

  3. Variants of Sporothrix schenckii with attenuated virulence for mice.

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    de Lima, Renata Ferretti; Schäffer, Guido Vidal; Borba, Cintia de Moraes

    2003-09-01

    Strains of Sporothrix schenckii preserved under mineral oil were examined for virulence in BALB/c mice. The mice were inoculated with S. schenckii conidia and development of cutaneous lesions, signs of inactivity, weight loss, survival rates, number of viable yeast cells in lung and spleen, splenomegaly and organ lesions were evaluated. After intravenous injection of 7.5 x 10(6) conidia, two of five S. schenckii strains were unable to induce systemic disease and to kill the mice, only producing cord-like lesions on the tail that regressed with mouse maturation. Very small numbers of viable cells isolated from the spleen confirmed the lower invasive ability of these strains when compared with other strains studied here. These results suggest a relationship between the attenuation of virulence and the storage method under mineral oil after long periods of time.

  4. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. PMID:27610355

  7. A transmission-virulence evolutionary trade-off explains attenuation of HIV-1 in Uganda.

    Science.gov (United States)

    Blanquart, François; Grabowski, Mary Kate; Herbeck, Joshua; Nalugoda, Fred; Serwadda, David; Eller, Michael A; Robb, Merlin L; Gray, Ronald; Kigozi, Godfrey; Laeyendecker, Oliver; Lythgoe, Katrina A; Nakigozi, Gertrude; Quinn, Thomas C; Reynolds, Steven J; Wawer, Maria J; Fraser, Christophe

    2016-11-05

    Evolutionary theory hypothesizes that intermediate virulence maximizes pathogen fitness as a result of a trade-off between virulence and transmission, but empirical evidence remains scarce. We bridge this gap using data from a large and long-standing HIV-1 prospective cohort, in Uganda. We use an epidemiological-evolutionary model parameterised with this data to derive evolutionary predictions based on analysis and detailed individual-based simulations. We robustly predict stabilising selection towards a low level of virulence, and rapid attenuation of the virus. Accordingly, set-point viral load, the most common measure of virulence, has declined in the last 20 years. Our model also predicts that subtype A is slowly outcompeting subtype D, with both subtypes becoming less virulent, as observed in the data. Reduction of set-point viral loads should have resulted in a 20% reduction in incidence, and a three years extension of untreated asymptomatic infection, increasing opportunities for timely treatment of infected individuals.

  8. 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.

  9. Identification of Brucella melitensis Rev.1 vaccine-strain genetic markers: Towards understanding the molecular mechanism behind virulence attenuation.

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    Issa, Mohammad Nouh; Ashhab, Yaqoub

    2016-09-22

    Brucella melitensis Rev.1 is an avirulent strain that is widely used as a live vaccine to control brucellosis in small ruminants. Although an assembled draft version of Rev.1 genome has been available since 2009, this genome has not been investigated to characterize this important vaccine. In the present work, we used the draft genome of Rev.1 to perform a thorough genomic comparison and sequence analysis to identify and characterize the panel of its unique genetic markers. The draft genome of Rev.1 was compared with genome sequences of 36 different Brucella melitensis strains from the Brucella project of the Broad Institute of MIT and Harvard. The comparative analyses revealed 32 genetic alterations (30 SNPs, 1 single-bp insertion and 1 single-bp deletion) that are exclusively present in the Rev.1 genome. In silico analyses showed that 9 out of the 17 non-synonymous mutations are deleterious. Three ABC transporters are among the disrupted genes that can be linked to virulence attenuation. Out of the 32 mutations, 11 Rev.1 specific markers were selected to test their potential to discriminate Rev.1 using a bi-directional allele-specific PCR assay. Six markers were able to distinguish between Rev.1 and a set of control strains. We succeeded in identifying a panel of 32 genome-specific markers of the B. melitensis Rev.1 vaccine strain. Extensive in silico analysis showed that a considerable number of these mutations could severely affect the function of the associated genes. In addition, some of the discovered markers were able to discriminate Rev.1 strain from a group of control strains using practical PCR tests that can be applied in resource-limited settings.

  10. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

    DEFF Research Database (Denmark)

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

    2003-01-01

    Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has...... of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip((R)) microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing...... systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune...

  11. Microarray-Based Detection and Differentiation of Virulent and Attenuated Pseudorabies Virus

    Institute of Scientific and Technical Information of China (English)

    LIU Li-na; LIU Zheng-fei; RUAN Li; WANG Zhao-xiong; LI Ren-feng; ZHANG Song-lin; CHEN Huan-chun; HE Qi-gai

    2007-01-01

    DNA microchip used in this study was formed from miniature arrays of pseudorabies virus (PrV) gene-specific probes immobilized on a glass surface. Hybridization using DNA microchip (microarrays) was used for differentiation between virulent and attenuated PrV. The presence of four gene segments (gB, gD, gE+, and gE- ) encoding conservative glycoprotein B (gB), D (gD), and E (gE) of PrV was monitored using multiplex PCR. The amplicons were labeled with Cy5 or Cy3 dyes followed by hybridization to the gene-specific capture probes on the microchip. The presence of gD and gB, gE+ gene fragments was shown in virulent (gE+ genotype) and attenuated PrV (gE genotype), whereas gE- gene (deleted domain in gE gene) was demonstrated only in virulent, not in attenuated, virus. No cross-hybridization was observed when fluorescence labeled-PCR products of PrV were hybridized using capture probes of related viruses, such as porcine respiratory and reproductive syndrome virus (PRRSV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV-2). The assay was 10 times sensitive than gD gene-specific PCR. Overall, the results of this study suggested that the microarray might be very useful for detection and differentiation of virulent PrV from attenuated one.

  12. Economic game theory to model the attenuation of virulence of an obligate intracellular bacterium

    Directory of Open Access Journals (Sweden)

    Damian Tago

    2016-08-01

    Full Text Available 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.

  13. Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Sunder, Avinash Vellore; Utari, Putri Dwi; Ramasamy, Sureshkumar; van Merkerk, Ronald; Quax, Wim J.; Pundle, Archana

    2016-01-01

    Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the d

  14. Virulence Associated Genes-Deleted Salmonella Montevideo Is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens

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    Lalsiamthara, Jonathan; Lee, John H.

    2016-01-01

    To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated strains, JOL1625 (Δlon), JOL1597 (ΔcpxR), and JOL1599 (ΔlonΔcpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in EPS production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in feces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that Δlon- and/or ΔcpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections. PMID:27785128

  15. Allele-Specific DNA Methylation Detection by Pyrosequencing®

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide......-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon....

  16. 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

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

    Science.gov (United States)

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

    2016-05-31

    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.

  18. Insertional mutation of orfD of the DCW cluster of Streptococcus pneumoniae attenuates virulence.

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    Palmen, R; Ogunniyi, A D; Berroy, P; Larpin, S; Paton, J C; Trombe, M C

    1999-12-01

    Mutational analysis of a 5.5 kb fragment of the genome Streptococcus pneumoniae led to the identification of a putative new virulence gene, designated orfD. Insertion mutagenesis of flanking genes on the fragment suggested that the corresponding gene products were required for in vitro growth. In contrast, insertion mutation of orfD did not alter in vitro growth or the transformability pattern of the mutated strain. However, it did reduce bacterial growth in mice and attenuated virulence in an intraperitoneal model of infection. orfD is flanked by orfC (63 codons) and ftsL (105 codons) and all three genes are upstream of pbpx. orfC showed no similarity with other known proteins. ftsL of S. pneumoniae exhibits minimal sequence similarity with ftsL of E. coli, but shares 16% identical residues with the ftsL homologue encoded by ylld of B. subtilis. Also, ftsL of S. pneumoniae has a predicted topology similar to that described for ftsL of E. coli. Putative promoters with an extended -10 box could be identified upstream of both orfC or orfD. The four open reading frames (including pbpx) are orientated in the same direction, and polycistronic transcription could theoretically start at either promoter. Interestingly, this region shows organizational and sequence homologies with genes controlling division and cell wall biosynthesis (DCW) in other bacteria. The attenuation of virulence in the orfD insertion mutant might be due to the loss of function of the orfD gene product or to an altered level of expression of downstream genes.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Drosophila melanogaster-based screening for multihost virulence factors of Pseudomonas aeruginosa PA14 and identification of a virulence-attenuating factor, HudA.

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    Kim, Seol-Hee; Park, Shin-Young; Heo, Yun-Jeong; Cho, You-Hee

    2008-09-01

    Pseudomonas aeruginosa is an important opportunistic human pathogen that interacts with phylogenetically diverse nonmammalian hosts, including plants, nematodes, and insects. Here, we exploited the P. aeruginosa-induced killing of the fruit fly Drosophila melanogaster as an assay system to screen for virulence-attenuated mutants of P. aeruginosa PA14. Fifteen nonredundant mutants were isolated from 4,018 random transposon (TnphoA) insertion clones, and 13 out of them (86.7%) displayed significantly reduced virulence in a murine peritonitis model as well. The TnphoA insertion sites of the 15 mutants were determined; already known virulence genes (dsbA, pvdI, fhlB, pilF, and wspF) and new virulence genes such as PA0253 (hudR), PA0369, PA2077, PA0272, PA2113, PA2965 (fabF1), and PA2002 were identified; one insertion was located at the intergenic region between PA1928 and PA1929; and the other two insertions were located in the genes (PA14_35740 and PA14_36000) within a putative genomic island, indicating a potential pathogenicity island of PA14. Further characterization of hudR, a virulence gene which encodes a MarR/SlyA family transcription factor, revealed that elevated expression of PA0254 (hudA [homologous to UbiD]) was necessary and sufficient for the virulence attenuation of the hudR mutant. The HudR protein repressed the hudAR operon by directly binding to its upstream promoter region. Collectively, these results validate the relevance of the D. melanogaster model for the high-throughput identification of new virulence factors involved in the multihost pathogenesis of P. aeruginosa.

  1. Protection by attenuated and polyvalent vaccines against highly virulent strains of Marek's disease virus.

    Science.gov (United States)

    Witter, R L

    1982-01-01

    Tests confirmed that turkey herpesvirus (HVT) vaccine protected chickens poorly against challenge with the highly virulent Md5 strain of Marek's disease (MD) virus, especially in chickens with homologous HVT antibodies. The naturally avirulent SB-1 vaccine virus was likewise poorly protective against challenge with the Md5 strain. Homologous antibodies reduced the protective efficacy of both vaccines, but SB-1 was not affected by HVT antibodies. In order to provide better protection against strains of MD virus poorly protected against by HVT, such as Md5, the Md11 strain of MD virus was attenuated by 75 cell culture passages and evaluated for protective efficacy. This vaccine virus, designated Mdl 1/75C, provided good protection against challenge with Md5 and most other highly virulent MD viruses tested, but was less efficacious against challenge with the JM/102W strain, a prototype MD virus protected against well by HVT and SB-1 vaccines. Furthermore, its efficacy was consistently lower in chicks with HVT antibody. Thus, although HVT, SB-1, and Md11/75C were all efficacious against certain MD viruses, none of these vaccines protected optimally against all MD challenge viruses in all chickens. A polyvalent vaccine composed of Md11/75C, HVT and SB-1 viruses protected chickens better against a battery of five highly virulent MD challenge viruses, including three strains poorly protected against by HVT, than any single vaccine and was not influenced by HVT antibody. These data suggest that vaccinal immunity may be partially viral strain specific.

  2. Interferon-gamma responses in sheep exposed to virulent and attenuated Brucella melitensis strains.

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    Pérez-Sancho, Marta; Durán-Ferrer, Manuel; García-Seco, Teresa; Macías, Paula; García, Nerea; Martínez, Irene; Ruiz, Elena; Legaz, Emilio; Diez-Guerrier, Alberto; González, Sergio; Domínguez, Lucas; Álvarez, Julio

    2014-07-15

    Antibody detection is the basis of large-scale sheep brucellosis diagnosis because of its sensitivity and specificity. In contrast, information on the cellular mediated immune (CMI) response triggered after Brucella melitensis infection, a cornerstone in the protection against this pathogen, is more limited, particularly regarding the effect of the virulence of the infecting strain in the induced CMI reaction. Here, the interferon-gamma (IFN-γ) profiles evoked after exposure by different routes to virulent (H38) and attenuated (Rev.1) B. melitensis strains in 14 pregnant sheep and 87 ewe lambs, respectively, were characterized accounting for different host-related factors, and compared with their serological response and with the basal IFN-γ responses observed in 155 animals non exposed to Brucella. No significant differences in the IFN-γ response of Rev.1 vaccinated animals depending on the inoculation route was observed, in contrast with their serological results. Response in H38-challenged followed a similar trend although peaked later, and an effect of the abortion on the IFN-γ response was detected. This information could help to understand the interaction bacteria-host that leads to its intracellular survival and could be useful for the design of new diagnostic approaches.

  3. Deletion of virulence associated genes from attenuated African swine fever virus isolate OUR T88/3 decreases its ability to protect against challenge with virulent virus.

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    Abrams, Charles C; Goatley, Lynnette; Fishbourne, Emma; Chapman, David; Cooke, Lyndsay; Oura, Christopher A; Netherton, Christopher L; Takamatsu, Haru-Hisa; Dixon, Linda K

    2013-08-15

    African swine fever virus (ASFV) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. The attenuated ASFV strain OUR T88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including OUR T88/1. Two genes, DP71L and DP96R were deleted from the OUR T88/3 genome to create recombinant virus OUR T88/3ΔDP2. Deletion of these genes from virulent viruses has previously been shown to reduce ASFV virulence in domestic pigs. Groups of 6 pigs were immunised with deletion virus OUR T88/3ΔDP2 or parental virus OUR T88/3 and challenged with virulent OUR T88/1 virus. Four pigs (66%) were protected by inoculation with the deletion virus OUR T88/3ΔDP2 compared to 100% protection with the parental virus OUR T88/3. Thus the deletion of the two genes DP71L and DP96R from OUR T88/3 strain reduced its ability to protect pigs against challenge with virulent virus.

  4. RNA-FISH to analyze allele-specific expression.

    Science.gov (United States)

    Braidotti, G

    2001-01-01

    One of the difficulties associated with the analysis of imprinted gene expression is the need to distinguish RNA synthesis occurring at the maternal vs the paternally inherited copy of the gene. Most of the techniques used to examine allele-specific expression exploit naturally occurring polymorphisms and measure steady-state levels of RNA isolated from a pool of cells. Hence, a restriction fragment length polymorphism (RFLP) an be exploited in a heterozygote, by a reverse transcriptase polymerase chain reaction (RT-PCR)- based procedure, to analyze maternal vs paternal gene expression. The human IGF2R gene was analyzed in this way. Smrzka et al. (1) were thus able to show that the IGF2R gene possesses a hemimethylated, intronic CpG island analogous to the mouse imprinting box. However, IGF2R mRNA was detected that possessed the RFLP from both the maternal and paternal alleles in all but one of the 70 lymphoblastoid samples. (The one monoallelic sample reactivated its paternal allele with continued cell culturing.) It was concluded that monoallelic expression of the human gene is a polymorphic trait occurring in a small minority of all tested samples (reviewed in refs. 2,3). Although this is a sound conclusion, the question remains: Is the human IGF2R gene imprinted?

  5. Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

    Science.gov (United States)

    Blatti-Cardinaux, Laure; Pisoni, Giuliano; Stoffel, Michael H; Zanoni, Reto; Zahno, Marie-Luise; Bertoni, Giuseppe

    2016-01-01

    Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

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

    Science.gov (United States)

    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. PMID:27822202

  7. Evolutionary Transition from Pathogenicity to Commensalism: Global Regulator Mutations Mediate Fitness Gains through Virulence Attenuation.

    Science.gov (United States)

    Jansen, Gunther; Crummenerl, Lena L; Gilbert, Felix; Mohr, Timm; Pfefferkorn, Roxana; Thänert, Robert; Rosenstiel, Philip; Schulenburg, Hinrich

    2015-11-01

    Symbiotic interactions are indispensable for metazoan function, but their origin and evolution remain elusive. We use a controlled evolution experiment to demonstrate the emergence of novel commensal interactions between Pseudomonas aeruginosa, an initially pathogenic bacterium, and a metazoan host, Caenorhabditis elegans. We show that commensalism evolves through loss of virulence, because it provides bacteria with a double fitness advantage: Increased within-host fitness and a larger host population to infect. Commensalism arises irrespective of host immune status, as the adaptive path in immunocompromised C. elegans knockouts does not differ from that in wild type. Dissection of temporal dynamics of genomic adaptation for 125 bacterial populations reveals highly parallel evolution of incipient commensalism across independent biological replicates. Adaptation is mainly achieved through frame shift mutations in the global regulator lasR and nonsynonymous point mutations in the polymerase gene rpoB that arise early in evolution. Genetic knockouts of lasR not only corroborate its role in virulence attenuation but also show that further mutations are necessary for the fully commensal phenotype. The evolutionary transition from pathogenicity to commensalism as we observe here is facilitated by mutations in global regulators such as lasR, because few genetic changes cause pleiotropic effects across the genome with large phenotypic effects. Finally, we found that nucleotide diversity increased more quickly in bacteria adapting to immunocompromised hosts than in those adapting to immunocompetent hosts. Nevertheless, the outcome of evolution was comparable across host types. Commensalism can thus evolve independently of host immune state solely as a side-effect of bacterial adaptation to novel hosts.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. RNAi-mediated silencing of fungal acuD gene attenuates the virulence of Penicillium marneffei.

    Science.gov (United States)

    Sun, Jiufeng; Li, Xiqing; Feng, Peiying; Zhang, Junmin; Xie, Zhi; Song, Erwei; Xi, Liyan

    2014-02-01

    A number of pathogens, most of them intracellular, employ the glyoxylate cycle in order to ingest fatty acids as carbon sources as a way of coping with nutrient deprivation during the infection process. Isocitrate lyase, which is encoded by the pathogen's acuD gene, plays a pivotal role in the glyoxylate cycle, which has been implicated in fungal pathogenesis. In this study, the acuD gene of Penicillium marneffei was knocked down using siRNA expressed by a filamentous fungi expression system. The acuD siRNA reduced the acuD gene's mRNA and protein expression by 21.5 fold and 3.5 fold, respectively. When macrophages were infected with different transformants of P. marneffei, the knockdown of acuD expression with RNA interference was lethal to the pathogens. In addition, the secretion of tumor necrosis factor-alpha and interferon-gamma from the infected macrophages was reduced. Moreover, the RNAi-mediated silencing of acuD expression reduced the fungal burden in the nude mice infected with P. marneffei; inhibited the inflammatory response in the lungs, livers, and spleens during the chronic phase instead of the acute phase of infection; and thus prolonged survival of the infected animals. Collectively, our data indicate that the RNAi-mediated silencing of acuD expression could attenuate virulence of P. marneffei. The endogenous expression of the delivered siRNA vector could be used to evaluate the role of functional genes by continuous and stable expression of siRNA.

  11. Systematic annotation and analysis of "virmugens"-virulence factors whose mutants can be used as live attenuated vaccines.

    Science.gov (United States)

    Racz, Rebecca; Chung, Monica; Xiang, Zuoshuang; He, Yongqun

    2013-01-21

    Live attenuated vaccines are usually generated by mutation of genes encoding virulence factors. "Virmugen" is coined here to represent a gene that encodes for a virulent factor of a pathogen and has been proven feasible in animal models to make a live attenuated vaccine by knocking out this gene. Not all virulence factors are virmugens. VirmugenDB is a web-based virmugen database (http://www.violinet.org/virmugendb). Currently, VirmugenDB includes 225 virmugens that have been verified to be valuable for vaccine development against 57 bacterial, viral, and protozoan pathogens. Bioinformatics analysis has revealed significant patterns in virmugens. For example, 10 Gram-negative and 1 Gram-positive bacterial aroA genes are virmugens. A sequence analysis has revealed at least 50% of identities in the protein sequences of the 10 Gram-negative bacterial aroA virmugens. As a pathogen case study, Brucella virmugens were analyzed. Out of 15 verified Brucella virmugens, 6 are related to carbohydrate or nucleotide transport and metabolism, and 2 involving cell membrane biogenesis. In addition, 54 virmugens from 24 viruses and 12 virmugens from 4 parasites are also stored in VirmugenDB. Virmugens tend to involve metabolism of nutrients (e.g., amino acids, carbohydrates, and nucleotides) and cell membrane formation. Host genes whose expressions were regulated by virmugen mutation vaccines or wild type virulent pathogens have also been annotated and systematically compared. The bioinformatics annotation and analysis of virmugens helps to elucidate enriched virmugen profiles and the mechanisms of protective immunity, and further supports rational vaccine design.

  12. Mortality and kidney histopathology of Chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

    Science.gov (United States)

    O'Farrell, Caroline L.; Elliott, Diane G.; Landolt, Marsha L.

    2001-01-01

    An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.

  13. 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.

  14. 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

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  15. Mobile genetic element SCCmec-encoded psm-mec RNA suppresses translation of agrA and attenuates MRSA virulence.

    Science.gov (United States)

    Kaito, Chikara; Saito, Yuki; Ikuo, Mariko; Omae, Yosuke; Mao, Han; Nagano, Gentaro; Fujiyuki, Tomoko; Numata, Shunsuke; Han, Xiao; Obata, Kazuaki; Hasegawa, Setsuo; Yamaguchi, Hiroki; Inokuchi, Koiti; Ito, Teruyo; Hiramatsu, Keiichi; Sekimizu, Kazuhisa

    2013-01-01

    Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) is a socially problematic pathogen that infects healthy individuals, causing severe disease. CA-MRSA is more virulent than hospital associated-MRSA (HA-MRSA). The underlying mechanism for the high virulence of CA-MRSA is not known. The transcription product of the psm-mec gene, located in the mobile genetic element SCCmec of HA-MRSA, but not CA-MRSA, suppresses the expression of phenol-soluble modulin α (PSMα), a cytolytic toxin of S. aureus. Here we report that psm-mec RNA inhibits translation of the agrA gene encoding a positive transcription factor for the PSMα gene via specific binding to agrA mRNA. Furthermore, 25% of 325 clinical MRSA isolates had a mutation in the psm-mec promoter that attenuated transcription, and 9% of the strains had no psm-mec. In most of these psm-mec-mutated or psm-mec-deleted HA-MRSAs, PSMα expression was increased compared with strains carrying intact psm-mec, and some mutated strains produced high amounts of PSMα comparable with that of CA-MRSA. Deletion of psm-mec from HA-MRSA strains carrying intact psm-mec increased the expression of AgrA protein and PSMα, and virulence in mice. Thus, psm-mec RNA suppresses MRSA virulence via inhibition of agrA translation and the absence of psm-mec function in CA-MRSA causes its high virulence property.

  16. Allelic-specific expression in relation to Bombyx mori resistance to Bt toxin.

    Science.gov (United States)

    Chen, Yazhou; Li, Muwang; Islam, Iftakher; You, Lang; Wang, Yueqiang; Li, Zhiqian; Ling, Lin; Zeng, Baosheng; Xu, Jun; Huang, Yongping; Tan, Anjiang

    2014-11-01

    Understanding the mechanism of Bt resistance is one of the key elements of the effective application of Bt in pest control. The lepidopteran model insect, the silkworm, demonstrates qualities that make it an ideal species to use in achieving this understanding. We screened 45 strains of silkworm (Bombyx mori) using a Cry1Ab toxin variant. The sensitivity levels of the strains varied over a wide range. A resistant strain (P50) and a phylogenetically related susceptible strain (Dazao) were selected to profile the expressions of 12 Bt resistance-related genes. The SNPs in these genes were detected based on EST analysis and were validated by allelic-specific PCR. A comparison of allelic-specific expression between P50 and Dazao showed that the transcript levels of heterozygous genes containing two alleles rather than an imbalanced allelic expression contribute more to the resistance of P50 against Bt. The responses of the allelic-specific expression to Bt in hybrid larvae were then investigated. The results showed that the gene expression pattern of an ATP-binding cassette transporter C2 (ABCC2) and an aminopeptidase N (APN3), changed in an allelic-specific manner, with the increase of the resistant allele expression correlated with larval survival. The results suggest that a trans-regulatory mechanism in ABCC2 and APN3 allelic-specific expression is involved in the insect's response to the Bt toxin. The potential role of allelic-specific gene regulation in insect resistance to Bt toxins is discussed.

  17. A galU mutant of francisella tularensis is attenuated for virulence in a murine pulmonary model of tularemia

    Directory of Open Access Journals (Sweden)

    Re Fabio

    2011-08-01

    Full Text Available Abstract Background A number of studies have revealed that Francisella tularensis (FT suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen. Results Here we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS. We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1β following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT. Conclusions Disruption of the galU gene of FTLVS has little (if any effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in

  18. A majority of Huntington's disease patients may be treatable by individualized allele-specific RNA interference.

    Science.gov (United States)

    Lombardi, Maria Stella; Jaspers, Leonie; Spronkmans, Christine; Gellera, Cinzia; Taroni, Franco; Di Maria, Emilio; Donato, Stefano Di; Kaemmerer, William F

    2009-06-01

    Use of RNA interference to reduce huntingtin protein (htt) expression in affected brain regions may provide an effective treatment for Huntington disease (HD), but it remains uncertain whether suppression of both wild-type and mutant alleles in a heterozygous patient will provide more benefit than harm. Previous research has shown suppression of just the mutant allele is achievable using siRNA targeted to regions of HD mRNA containing single nucleotide polymorphisms (SNPs). To determine whether more than a minority of patients may be eligible for an allele-specific therapy, we genotyped DNA from 327 unrelated European Caucasian HD patients at 26 SNP sites in the HD gene. Over 86% of the patients were found to be heterozygous for at least one SNP among those tested. Because the sites are genetically linked, one cannot use the heterozygosity rates of the individual SNPs to predict how many sites (and corresponding allele-specific siRNA) would be needed to provide at least one treatment possibility for this percentage of patients. By computing all combinations, we found that a repertoire of allele-specific siRNA corresponding to seven sites can provide at least one allele-specific siRNA treatment option for 85.6% of our sample. Moreover, we provide evidence that allele-specific siRNA targeting these sites are readily identifiable using a high throughput screening method, and that allele-specific siRNA identified using this method indeed show selective suppression of endogenous mutant htt protein in fibroblast cells from HD patients. Therefore, allele-specific siRNA are not so rare as to be impractical to find and use therapeutically.

  19. Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers.

    Science.gov (United States)

    Sonneveld, T; Tobutt, K R; Robbins, T P

    2003-10-01

    PCR-based identification of all 13 known self-incompatibility (S) alleles of sweet cherry is reported. Two pairs of consensus primers were designed from our previously published cDNA sequences of S(1) to S(6) S-RNases, the stylar components of self-incompatibility, to reveal length variation of the first and the second introns. With the exception of the first intron of S(13), these also amplified S(7) to S(14) and an allele previously referred to as S(x), which we now label S(16). The genomic PCR products were cloned and sequenced. The partial sequence of S(11) matched that of S(7) and the alleles were shown to have the same functional specificity. Allele-specific primers were designed for S(7) to S(16), so that allele-specific primers are now available for all 13 S alleles of cherry (S(8), S(11) and S(15) are duplicates). These can be used to distinguish between S alleles with introns of similar size and to confirm genotypes determined with consensus primers. The reliability of the PCR with allele-specific primers was improved by the inclusion of an internal control. The use of the consensus and allele-specific primers was demonstrated by resolving conflicting genotypes that have been published recently and by determining genotypes of 18 new cherry cultivars. Two new groups are proposed, Group XXIII (S(3) S(16)), comprising 'Rodmersham Seedling' and 'Strawberry Heart', and Group XXIV (S(6) S(12)), comprising 'Aida' and 'Flamentiner'. Four new self-compatibility genotypes, S(3) S(3)', S(4)' S(6), S(4)' S(9) and S(4)' S(13), were found. The potential use of the consensus primers to reveal incompatibility alleles in other cherry species is also demonstrated.

  20. Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

    Institute of Scientific and Technical Information of China (English)

    Yi Zhong; Yuan-Yuan Li; Xiu-Gao Jiang; Guo-Ping Zhao; Shengyue Wang; Yixue Li; Rong Zeng; Xuan Li; Xiao-Kui Guo; Xiao Chang; Xing-Jun Cao; Yan Zhang; Huajun Zheng; Yongzhang Zhu; Chengsong Cai; Zelin Cui; Yunyi Zhang

    2011-01-01

    The virulence-attenuated Leptospira interrogans serovar Lai strain IPAV was derived by prolonged laboratory passage from a highly virulent ancestral strain isolated in China. We studied the genetic variations of IPAV that render it avirulent via comparative analysis against the pathogenic L. interrogans serovar Lai strain 56601. The complete genome sequence of the IPAV strain was determined and used to compare with, and then rectify and reannotate the genome sequence of strain 56601. Aside from their highly similar genomic structure and gene order, a total of 33 insertions, 53 deletions and 301 single-nucleotide variations (SNVs) were detected throughout the genome of IPAV directly affecting 101 genes, either in their 5' upstream region or within their coding region. Among them, the majority of the [44]functional genes are involved in signal transduction, stress response, transmembrane transport and nitrogen metabolism. Comparative proteomic analysis based on quantitative liquid chromatography (LC)-MS/MS data revealed that among 1 627 selected pairs of orthologs, 174 genes in the IPAV strain were upregulated, with enrichment mainly in classes of energy production and lipid metabolism. In contrast, 228 genes in strain 56601 were upregulated, with the majority enriched in the categories of protein translation and DNA replication/repair. The combination of genomic and proteomic approaches illustrated that altered expression or mutations in critical genes, such as those encoding a Ser/Thr kinase, carbon-starvation protein CstA, glutamine synthetase, GTP-binding protein BipA, ribonucleotidediphosphate reductase and phosphate transporter, and alterations in the translational profile of lipoproteins or outer membrane proteins are likely to account for the virulence attenuation in strain IPAV.

  1. Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like Attenuated Toxigenic Nonencapsulated B. anthracis Sterne in Rabbits and Mice

    Science.gov (United States)

    2011-08-01

    Microbiology. All Rights Reserved. Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like...G9241 for mice requires the presence of both plasmids. The Bacillus cereus group, of which Bacillus anthracis, Bacil- lus thuringiensis , and B... Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like Attenuated Toxigenic Nonencapsulated B

  2. Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum.

    Science.gov (United States)

    Khordadmehr, Monireh; Namavari, Mehdi; Khodakaram-Tafti, Azizollah; Mansourian, Maryam; Rahimian, Abdollah; Daneshbod, Yahya

    2013-10-01

    In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10(4), 1.0 × 10(4), 1.5 × 10(4)) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine.

  3. Genomic Changes in an Attenuated ZB Strain of Foot-and-Mouth Disease Virus Serotype Asia1 and Comparison with Its Virulent Parental Strain

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    Aiguo Xin

    2014-01-01

    Full Text Available The molecular basis of attenuation of foot-and-mouth disease virus (FMDV serotype Asia1 ZB strain remains unknown. To understand the genetic changes of attenuation, we compared the entire genomes of three different rabbit-passaged attenuated ZB strains (ZB/CHA/58(att, ZBRF168, and ZBRF188 and their virulent parental strains (ZBCF22 and YNBS/58. The results showed that attenuation may be brought about by 28 common amino acid substitutions in the coding region, with one nucleotide point mutation in the 5′-untranslated region (5′-UTR and another one in the 3′-UTR. In addition, a total of 21 nucleotides silent mutations had been found after attenuation. These substitutions, alone or in combination, may be responsible for the attenuated phenotype of the ZB strain in cattle. This will contribute to elucidation of attenuating molecular basis of the FMDV ZB strain.

  4. Epidemiological survey of Theileria parasite infection of cattle in Northeast China by allele-specific PCR.

    Science.gov (United States)

    Yu, Longzheng; Zhang, Shoufa; Liang, Wanfeng; Jin, Chunmei; Jia, Lijun; Luo, Yuzi; Li, Yan; Cao, Shinuo; Yamagishi, Junya; Nishikawa, Yoshifumi; Kawano, Suguru; Fujisaki, Kozo; Xuan, Xuenan

    2011-11-01

    An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.

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

    Science.gov (United States)

    2015-12-01

    SUPPLEMENTARY NOTES 14. ABSTRACT The P. aeruginosa signaling/virulence molecule 3OC12 mediates inactivation of the lactonase paraoxonase 2 (PON2) and induces... mediated inactivation at 3OC12 concentrations expected to be present near P. aeruginosa colonies during infection. In mouse primary cell types PON2 was not...as sensitive to 3OC12- mediated inactivation as in human cells. We also discovered that 3OC12 is rapidly hydrolyzed intracellularly by PON2 to 3OC12

  6. 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.

  7. BaalChIP: Bayesian analysis of allele-specific transcription factor binding in cancer genomes.

    Science.gov (United States)

    de Santiago, Ines; Liu, Wei; Yuan, Ke; O'Reilly, Martin; Chilamakuri, Chandra Sekhar Reddy; Ponder, Bruce A J; Meyer, Kerstin B; Markowetz, Florian

    2017-02-24

    Allele-specific measurements of transcription factor binding from ChIP-seq data are key to dissecting the allelic effects of non-coding variants and their contribution to phenotypic diversity. However, most methods of detecting an allelic imbalance assume diploid genomes. This assumption severely limits their applicability to cancer samples with frequent DNA copy-number changes. Here we present a Bayesian statistical approach called BaalChIP to correct for the effect of background allele frequency on the observed ChIP-seq read counts. BaalChIP allows the joint analysis of multiple ChIP-seq samples across a single variant and outperforms competing approaches in simulations. Using 548 ENCODE ChIP-seq and six targeted FAIRE-seq samples, we show that BaalChIP effectively corrects allele-specific analysis for copy-number variation and increases the power to detect putative cis-acting regulatory variants in cancer genomes.

  8. Allele-specific amplification and electrochemiluminescence method for single nucleotide polymorphism analysis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemiluminescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy)32+ (TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.(C) 2007 Da Xing. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  9. Determination of DQB1 alleles using PCR amplification and allele-specific primers.

    Science.gov (United States)

    Lepage, V; Ivanova, R; Loste, M N; Mallet, C; Douay, C; Naoumova, E; Charron, D

    1995-10-01

    Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

  10. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms.

    Science.gov (United States)

    Matsuda, Kazuyuki; Honda, Takayuki

    2015-01-01

    Single nucleotide alternations such as single nucleotide polymorphisms (SNPs) or single nucleotide mutations are useful genetic markers for molecular diagnosis, prognosis, drug response, and predisposition to diseases. Rapid identification of SNPs or mutations is clinically important, especially for determining drug responses and selection of molecular-targeted therapy. Here, we describe a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) by using our droplet-PCR machine (droplet-AS-PCR).

  11. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    Science.gov (United States)

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  12. 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.

  13. Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing((TM))

    NARCIS (Netherlands)

    Schaart, J.G.; Mehli, L.; Schouten, H.J.

    2005-01-01

    Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated

  14. 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.

  15. Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus.

    Science.gov (United States)

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-07-28

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes.

  16. Allele-specific amplification in cancer revealed by SNP array analysis.

    Directory of Open Access Journals (Sweden)

    Thomas LaFramboise

    2005-11-01

    Full Text Available Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site, and (b infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ.

  17. Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts.

    Science.gov (United States)

    Scholefield, Janine; Watson, Lauren; Smith, Danielle; Greenberg, Jacquie; Wood, Matthew J A

    2014-12-01

    Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.

  18. Genome-wide survey of allele-specific splicing in humans

    Directory of Open Access Journals (Sweden)

    Scheffler Konrad

    2008-06-01

    Full Text Available Abstract Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array

  19. 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 < 0.05 and 0.01, respectively). Furthermore, in vivo studies showed that the bacterial loads of DE17ΔluxSΔaroA were reduced by 8400- and 11,333-fold in the spleen and blood of infected birds, respectively, while those of DE17ΔaroA were reduced by 743- and 1000-fold, respectively, as compared to the wild-type strain DE17. Histopathological analysis showed both that the mutants were associated with reduced pathological changes in the liver, spleen, and kidney of ducklings, and changes in DE17ΔluxSΔaroA-infected ducklings were reduced to a greater degree than those infected with DE17ΔaroA. Real-time polymerase chain reaction analysis further demonstrated that the mRNA levels of virulence-related genes (i.e., tsh, ompA, vat, iucD, pfs, fyuA, and fimC) were significantly decreased in DE17ΔaroA, especially in DE17ΔluxSΔaroA, as compared to DE17 (p < 0.05). In addition, the deletion of aroA or the double deletion of aroA and luxS reduced bacterial motility. To evaluate the potential use of DE17ΔluxSΔaroA as a vaccine candidate, 50 7-day-old ducklings were divided randomly into five groups of ten each for the experiment. The results showed that the ducklings immunized with inactivated DE17, DE17ΔluxS, DE17ΔaroA, and DE17ΔluxSΔaroA were 70.0%, 70.0%, 70.0, and 80.0% protected, respectively, after challenge with strain APEC DE17. The results of this study suggest that the double deletion of

  20. Co-expression of DevR and DevR(N-Aph proteins is associated with hypoxic adaptation defect and virulence attenuation of Mycobacterium tuberculosis.

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    Shyamasree De Majumdar

    Full Text Available BACKGROUND: The DevR response regulator is implicated in both hypoxic adaptation and virulence of Mycobacterium tuberculosis (M. tb. DevR regulon genes are powerfully induced in vivo implicating them in bacterial adaptation to host control strategies. A better understanding of DevR function will illumine the way for new strategies to control and treat tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Towards this objective, we used a combination of genetic, microbiological, biochemical, cell biological tools and a guinea pig virulence assay to compare the hypoxic adaptation and virulence properties of two novel M. tb strains, namely, a devR disruption mutant, Mut1, that expresses C-terminal truncated N-terminal domain of DevR (DevR(NTD as a fusion protein with AphI (DevR(N-Kan, and its complemented strain, Comp1, that expresses intact DevR along with DevR(N-Kan. Comp1 bacteria exhibit a defect in DevR-mediated phosphosignalling, hypoxic induction of HspX and also hypoxic survival. In addition, we find that Comp1 is attenuated in virulence in guinea pigs and shows decreased infectivity of THP-1 cells. While Mut1 bacilli are also defective in hypoxic adaptation and early growth in spleen, they exhibit an overall virulence comparable to that of wild-type bacteria. CONCLUSIONS/SIGNIFICANCE: The hypoxic defect of Comp1 is associated to a defect in DevR expression level. The demonstrated repression of DevR function by DevR(N-Kan suggests that such a knockdown approach could be useful for evaluating the activity of DevRS and other two-component signaling pathways. Further investigation is necessary to elucidate the mechanism underlying Comp1 attenuation.

  1. Therapy for dominant inherited diseases by allele-specific RNA interference: successes and pitfalls.

    Science.gov (United States)

    Trochet, Delphine; Prudhon, Bernard; Vassilopoulos, Stéphane; Bitoun, Marc

    2015-01-01

    RNA interference (RNAi) is a conserved mechanism for post-transcriptional gene silencing mediated by messenger RNA (mRNA) degradation. RNAi is commonly induced by synthetic siRNA or shRNA which recognizes the targeted mRNA by base pairing and leads to target-mRNA degradation. RNAi may discriminate between two sequences only differing by one nucleotide conferring a high specificity of RNAi for its target mRNA. This property was used to develop a particular therapeutic strategy called "allele-specific-RNA interference" devoted to silence the mutated allele of genes causing dominant inherited diseases without affecting the normal allele. Therapeutic benefit was now demonstrated in cells from patients and animal models, and promising results of the first phase Ib clinical trial using siRNA-based allele-specific therapy were reported in Pachyonychia Congenita, an inherited skin disorder due to dominant mutations in the Keratin 6 gene. Our purpose is to review the successes of this strategy aiming to treat dominant inherited diseases and to highlight the pitfalls to avoid.

  2. Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

    Science.gov (United States)

    Orue, Andrea; Rieber, Manuel

    2016-01-01

    Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting. PMID:27632281

  3. Germline allele-specific expression of DAPK1 in chronic lymphocytic leukemia.

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    Quan-Xiang Wei

    Full Text Available We previously reported a rare germline variant (c.1-6531 that resulted in allele-specific expression (ASE of death-associated protein kinase 1 (DAPK1 and predisposition to chronic lymphocytic leukemia (CLL. We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2% CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

  4. A molecular method for S-allele identification in apple based on allele-specific PCR.

    Science.gov (United States)

    Janssens, G A; Goderis, I J; Broekaert, W F; Broothaerts, W

    1995-09-01

    cDNA sequences corresponding to two self-incompatibility alleles (S-alleles) of the apple cv 'Golden Delicious' have previously been described, and now we report the identification of three additional S-allele cDNAs of apple, one of which was isolated from a pistil cDNA library of cv 'Idared' and two of which were obtained by reverse transcription-PCR (RT-PCR) on pistil RNA of cv 'Queen's Cox'. A comparison of the deduced amino acid sequences of these five S-allele cDNAs revealed an average homology of 69%. Based on the nucleotide sequences of these S-allele cDNAs, we developed a molecular technique for the diagnostic identification of the five different S-alleles in apple cultivars. The method used consists of allele-specific PCR amplification of genomic DNA followed by digestion of the amplification product with an allele-specific restriction endonuclease. Analysis of a number of apple cultivars with known S-phenotype consistently showed coincidence of phenotypic and direct molecular data of the S-allele constitution of the cultivars. It is concluded that the S-allele identification approach reported here provides a rapid and useful method to determine the S-genotype of apple cultivars.

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

    OpenAIRE

    1989-01-01

    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 co...

  6. Identification of differentially expressed proteins in porcine alveolar macrophages infected with virulent/attenuated strains of porcine reproductive and respiratory syndrome virus.

    Directory of Open Access Journals (Sweden)

    Yan-Jun Zhou

    Full Text Available The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV is still a serious threat to the swine industry. However, the pathogenic mechanism of HP-PRRSV remains unclear. We infected host porcine alveolar macrophages (PAMs with the virulent HuN4 strain and the attenuated HuN4-F112 strain and then utilized fluorescent two-dimensional difference gel electrophoresis (2D-DIGE to screen for intracellular proteins that were differentially expressed in host cells infected with the two strains. There were 153 proteins with significant different expression (P<0.01 observed, 42 of which were subjected to mass spectrometry, and 24 proteins were identified. PAM cells infected with the virulent strain showed upregulated expression of pyruvate kinase M2 (PKM2, heat shock protein beta-1 (HSPB1, and proteasome subunit alpha type 6 (PSMA6, which were downregulated in cells infected with the attenuated strain. The upregulation of PKM2 provides sufficient energy for viral replication, and the upregulation of HSPB1 inhibits host cell apoptosis and therefore facilitates mass replication of the virulent strain, while the upregulation of PSMA6 facilitates the evasion of immune surveillance by the virus. Studying on those molecules mentioned above may be able to help us to understand some unrevealed details of HP-PRRSV infection, and then help us to decrease its threat to the swine industry in the future.

  7. Attenuation of a virulent Aeromonas hydrophila with novobiocin and pathogenic characterization of the novobiocin-resistant strain

    Science.gov (United States)

    A novobiocin-resistant strain AH11NOVO was obtained from a virulent A. hydrophila strain AH11P through selection of resistance to novobiocin. AH11NOVO was found to be avirulent to channel catfish (Ictalurus punctatus) whereas AH11P was virulent. When AH11NOVO vaccinated channel catfish were challeng...

  8. Allele-specific interactions between CAST AWAY and NEVERSHED control abscission in Arabidopsis flowers

    Directory of Open Access Journals (Sweden)

    William D. Groner

    2016-10-01

    Full Text Available An advantage of analyzing abscission in genetically tractable model plants is the ability to make use of classic genetic tools such as suppression analysis. We have investigated the regulation of organ abscission by carrying out suppression analysis in Arabidopsis flowers. Plants carrying mutations in the NEVERSHED (NEV gene, which encodes an ADP-ribosylation factor GTPase-activating protein, retain their outer floral organs after fertilization. Mutant alleles of CAST AWAY (CST, which encodes a receptor-like cytoplasmic kinase, were found to restore organ abscission in nev flowers in an allele-specific manner. To further explore the basis of the interactions between CST and NEV, we tested whether the site of a nev mutation is predictive of its ability to be suppressed. Our results suggest instead that the strength of a nev allele influences whether organ abscission can be rescued by a specific allele of CST.

  9. Dideoxy single allele-specific PCR - DSASP new method to discrimination allelic

    Directory of Open Access Journals (Sweden)

    Eleonidas Moura Lima

    2015-06-01

    Full Text Available Gastric cancer (GC is a multifactorial disease with a high mortality rate in Brazil and worldwide. This work aimed to evaluate single nucleotide polymorphisms (SNP rs1695, in the Glutathione S-Transferase Pi (GSTP1 gene in GC samples by comparative analysis Specific PCR - ASP and Dideoxy Single Allele-Specific PCR - DSASP methods. The DSASP is the proposed new method for allelic discrimination. This work analyzed 60 GC samples, 26 diffuse and 34 intestinal types. The SNP rs1695 of the GSTP1 gene was significantly associated with GC analyzed by DSASP method (χ2 = 9.7, P 0.05. These results suggest that the SNP rs1695 of the GSTP1 gene was a risk factor associated with gastric carcinogens is and the DSASP method was a new successfully low-cost strategy to study allelic discrimination.

  10. Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

    Science.gov (United States)

    Peters, S E; Paterson, G K; Bandularatne, E S D; Northen, H C; Pleasance, S; Willers, C; Wang, J; Foote, A K; Constantino-Casas, F; Scase, T J; Blacklaws, B A; Bryant, C E; Mastroeni, P; Charles, I G; Maskell, D J

    2010-01-01

    In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

  11. 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.

  12. 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.

  13. Knockout of the dhfr-ts gene in Trypanosoma cruzi generates attenuated parasites able to confer protection against a virulent challenge.

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    Cecilia Perez Brandan

    2011-12-01

    Full Text Available BACKGROUND: Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes. METHODS AND FINDINGS: By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts(+/- mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8(+ T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection. CONCLUSION: This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections.

  14. Allele-specific methylation occurs at genetic variants associated with complex disease.

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    John N Hutchinson

    Full Text Available We hypothesize that the phenomenon of allele-specific methylation (ASM may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS. We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81% are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434, Celiac disease (rs2762051, Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875 and height (rs6569648. Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results.

  15. Allele-specific deposition of macroH2A1 in Imprinting Control Regions

    Energy Technology Data Exchange (ETDEWEB)

    Choo, J H; Kim, J D; Chung, J H; Stubbs, L; Kim, J

    2006-01-13

    In the current study, we analyzed the deposition patterns of macroH2A1 at a number of different genomic loci located in X chromosome and autosomes. MacroH2A1 is preferentially deposited at methylated CpG CpG-rich regions located close to promoters. The macroH2A1 deposition patterns at the methylated CpG islands of several imprinted domains, including the Imprinting Control Regions (ICRs) of Xist, Peg3, H19/Igf2 Igf2, Gtl2/Dlk1, and Gnas domains, show consistent allele-specificity towards inactive, methylated alleles. The macroH2A1 deposition levels at the ICRs and other Differentially Methylated Regions (DMRs) of these domains are also either higher or comparable to those observed at the inactive X chromosome of female mammals. Overall, our results indicate that besides DNA methylation macroH2A1 is another epigenetic component in the chromatin of ICRs displaying differential association with two parental alleles.

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

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    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.

  17. Assessing allele-specific expression across multiple tissues from RNA-seq read data

    Science.gov (United States)

    Pirinen, Matti; Lappalainen, Tuuli; Zaitlen, Noah A.; Dermitzakis, Emmanouil T.; Donnelly, Peter; McCarthy, Mark I.; Rivas, Manuel A.

    2015-01-01

    Motivation: RNA sequencing enables allele-specific expression (ASE) studies that complement standard genotype expression studies for common variants and, importantly, also allow measuring the regulatory impact of rare variants. The Genotype-Tissue Expression (GTEx) project is collecting RNA-seq data on multiple tissues of a same set of individuals and novel methods are required for the analysis of these data. Results: We present a statistical method to compare different patterns of ASE across tissues and to classify genetic variants according to their impact on the tissue-wide expression profile. We focus on strong ASE effects that we are expecting to see for protein-truncating variants, but our method can also be adjusted for other types of ASE effects. We illustrate the method with a real data example on a tissue-wide expression profile of a variant causal for lipoid proteinosis, and with a simulation study to assess our method more generally. Availability and implementation: http://www.well.ox.ac.uk/~rivas/mamba/. R-sources and data examples http://www.iki.fi/mpirinen/ Contact: matti.pirinen@helsinki.fi or rivas@well.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25819081

  18. Eugenol in combination with lactic acid bacteria attenuates Listeria monocytogenes virulence in vitro and in invertebrate model Galleria mellonella.

    Science.gov (United States)

    Upadhyay, Abhinav; Upadhyaya, Indu; Mooyottu, Shankumar; Venkitanarayanan, Kumar

    2016-06-01

    Listeria monocytogenes is a human enteric pathogen that causes severe foodborne illness in high-risk populations. Crossing the intestinal barrier is the first critical step for Listeria monocytogenes infection. Therefore, reducing L. monocytogenes colonization and invasion of intestinal epithelium and production of virulence factors could potentially control listeriosis in humans. This study investigated the efficacy of sub-inhibitory concentration (SIC) of the plant-derived antimicrobial eugenol, either alone, or in combination with five lactic acid bacteria (LAB), namely Bifidobacterium bifidum (NRRL-B41410), Lactobacillus reuteri (B-14172), Lactobacillus fermentum (B-1840), Lactobacillus plantarum (B-4496) and Lactococcus lactis subspecies lactis (B-633) in reducing Listeria monocytogenes adhesion to and invasion of human intestinal epithelial cells (Caco-2). Additionally, the effect of the aforementioned treatments on Listeria monocytogenes listeriolysin production, epithelial E-cadherin binding and expression of virulence genes was investigated. Moreover, the in vivo efficacy of eugenol-LAB treatments in reducing Listeria monocytogenes virulence in the invertebrate model Galleria mellonella was studied. Eugenol and LAB, either alone or in combination, significantly reduced Listeria monocytogenes adhesion to and invasion of intestinal cells (P eugenol-LAB treatments decreased Listeria monocytogenes haemolysin production, E-cadherin binding and virulence gene expression (P eugenol-LAB treatments significantly enhanced the survival rates of G. mellonella infected with lethal doses of Listeria monocytogenes (P eugenol either alone or in combination with LAB, and justify further investigations in a mammalian model.

  19. Self-(in)compatibility inheritance and allele-specific marker development in yellow mustard (Sinapis alba).

    Science.gov (United States)

    Zeng, Fangqin; Cheng, Bifang

    2014-01-01

    Yellow mustard (Sinapis alba) has a sporophytic self-incompatibility reproduction system. Genetically stable self-incompatible (SI) and self-compatible (SC) inbred lines have recently been developed in this crop. Understanding the S haplotype of different inbred lines and the inheritance of the self-(in)compatibility (SI/SC) trait is very important for breeding purposes. In this study, we used the S-locus gene-specific primers in Brassica rapa and Brassica oleracea to clone yellow mustard S-locus genes of SI lines Y514 and Y1130 and SC lines Y1499 and Y1501. The PCR amplification results and DNA sequences of the S-locus genes revealed that Y514 carried the class I S haplotype, while Y1130, Y1499, and Y1501 had the class II S haplotype. The results of our genetic studies indicated that self-incompatibility was dominant over self-compatibility and controlled by a one-gene locus in the two crosses of Y514 × Y1499 and Y1130 × Y1501. Of the five S-locus gene polymorphic primer pairs, Sal-SLGI and Sal-SRKI each generated one dominant marker for the SI phenotype of Y514; Sal-SLGII and Sal-SRKII produced dominant marker(s) for the SC phenotype of Y1501 and Y1499; Sal-SP11II generated one dominant marker for Y1130. These markers co-segregated with the SI/SC phenotype in the F2 populations of the two crosses. In addition, co-dominant markers were developed by mixing the two polymorphic primer pairs specific for each parent in the multiplex PCR, which allowed zygosity to be determined in the F2 populations. The SI/SC allele-specific markers have proven to be very useful for the selection of the desirable SC genotypes in our yellow mustard breeding program.

  20. Efficient and allele-specific genome editing of disease loci in human iPSCs.

    Science.gov (United States)

    Smith, Cory; Abalde-Atristain, Leire; He, Chaoxia; Brodsky, Brett R; Braunstein, Evan M; Chaudhari, Pooja; Jang, Yoon-Young; Cheng, Linzhao; Ye, Zhaohui

    2015-03-01

    Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

  1. 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.

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

    Science.gov (United States)

    Veríssimo, Carolina De Marco; Maschio, Vinícius José; Correa, Ana Paula Folmer; Brandelli, Adriano; Rott, Marilise Brittes

    2013-01-01

    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. PMID:24271042

  3. IS6110 in oriC affects the morphology and growth of Mycobacterium tuberculosis and attenuates virulence in mice.

    Science.gov (United States)

    Casart, Yveth; Turcios, Lilia; Florez, Ingrid; Jaspe, Rossana; Guerrero, Elba; de Waard, Jacobus; Aguilar, Diana; Hérnandez-Pando, Rogelio; Salazar, Leiria

    2008-11-01

    The IS6110 element is widely used in studies of molecular epidemiology of tuberculosis and it is considered the gold standard for genotyping Mycobacterium tuberculosis strains. Because of its high frequency of transposition, IS6110 is probably a major contributor to the evolution of M. tuberculosis. Nevertheless, very few studies of the effect of IS6110 insertions on the virulence of M. tuberculosis have been reported. We analysed two isogenic groups of M. tuberculosis strains isolated from the sputa of two patients. Strains belonging to the same isogenic group differed from one another by one IS6110-oriC hybridising band, but they showed identical spoligo and MIRU-VNTR profiles. Isogenic strains containing the IS6110 element in oriC exhibited a diminished growth rate and average dimensions of the bacilli were modified; moreover, they were less virulent in a mouse model.

  4. Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy.

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    Haihui Liao

    Full Text Available BACKGROUND: Meesmann epithelial corneal dystrophy (MECD is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD. METHODS AND FINDINGS: We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits. The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits. CONCLUSIONS: This study demonstrates that it is feasible to design highly potent si

  5. 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

  6. 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

  7. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

    Science.gov (United States)

    Kaulich, Manuel; Lee, Yeon J; Lönn, Peter; Springer, Aaron D; Meade, Bryan R; Dowdy, Steven F

    2015-04-20

    Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.

  8. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes [version 1; referees: 2 approved

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    Felix Krueger

    2016-06-01

    Full Text Available Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base ’N’ and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data.

  9. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes [version 2; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Felix Krueger

    2016-07-01

    Full Text Available Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data.

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

    Science.gov (United States)

    Wu, D Y; Ugozzoli, L; Pal, B K; Wallace, R B

    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' 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.

  11. Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberculosis in mice.

    Science.gov (United States)

    Bannantine, John P; Everman, Jamie L; Rose, Sasha J; Babrak, Lmar; Katani, Robab; Barletta, Raúl G; Talaat, Adel M; Gröhn, Yrjö T; Chang, Yung-Fu; Kapur, Vivek; Bermudez, Luiz E

    2014-01-01

    Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep, and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne's disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.

  12. Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberculosis in mice

    Directory of Open Access Journals (Sweden)

    John P Bannantine

    2014-07-01

    Full Text Available Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP, which results in serious economic losses worldwide in farmed livestock such as cattle, sheep and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne’s disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321 and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370 was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.

  13. 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. PMID:27911925

  14. A single tube modified allele-specific-PCR for rapid detection of erythromycin-resistant Mycoplasma pneumoniae in Beijing

    Institute of Scientific and Technical Information of China (English)

    LI Shao-li; SUN Hong-mei; ZHAO Han-qing; CAO Ling; YUAN Yi; FENG Yan-ling; XUE Guan-hua

    2012-01-01

    Background Mycoplasma pneumoniae (M.pneumoniae) is one of the common pathogens causing atypical pneumonia.In recent years,resistance to macrolides has become more common,especially in China.Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent,followed by the mutation at position 2064.Reported molecular detection methods for the identification of these mutations include direct sequencing,restriction fragment length polymorphism analysis,real-time polymerase chain reaction (PCR) with high-resolution melt analysis,and nested PCR-linked with capillary electrophoresis,etc.The most commonly used method for monitoring resistance-conferring mutations in M.pneumoniae is direct DNA sequencing of PCR or nested PCR products.However,these methods are time-consuming,labor-intensive or need expensive equipments.Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally.Methods In this study,we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis,and all results were compared with the sequencing data.We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results.Results Among 97 M.pneumoniae specimens,88 were found to possess mutations by this method,and all modified allele-specific PCR analysis results were consistent with the sequencing data.The data of the clinical courses of these 97cases showed that they suffered from severe pneumonia.Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens.However,in some cases from which mutations were detected,erythromycin monotherapy had poor efficacy,and on these patients severe symptoms improved only when azithromycin was added to the treatment.Conclusions The drug-resistant M.pneumoniae is very common in

  15. Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

    Science.gov (United States)

    Liu, Ai Hong; Guan, Gui Quan; Liu, Jun Long; Liu, Zhi Jie; Leblanc, Neil; Li, You Quan; Gao, Jin Liang; Ma, Mi Ling; Niu, Qing Li; Ren, Qiao Yun; Bai, Qi; Yin, Hong; Luo, Jian Xun

    2011-02-01

    Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

  16. Use of allele-specific FAIRE to determine functional regulatory polymorphism using large-scale genotyping arrays

    DEFF Research Database (Denmark)

    Smith, Frank Andrew; Howard, Philip; Shah, Sonia;

    2012-01-01

    identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α), rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic...... variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen) to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique...... discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006), and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly...

  17. 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.

  18. An allele-specific polymerase chain reaction assay for the differentiation of members of the Anopheles culicifacies complex

    Indian Academy of Sciences (India)

    O P Singh; Geeta Goswami; N Nanda; K Raghavendra; D Chandra; S K Subbarao

    2004-09-01

    Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminates An. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally-identified specimens of An. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.

  19. A Novel Real-time Fluorescence Mutant-allele-specific Amplification Method for Rapid Single Nucleotide Polymorphism Analysis

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Current methods for single nucleotide polymorphism (SNP) analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification (MASA) method for rapid SNP analysis. The method is a marriage of two technologies: MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.

  20. Powerful identification of cis-regulatory SNPs in human primary monocytes using allele-specific gene expression.

    Directory of Open Access Journals (Sweden)

    Jonas Carlsson Almlöf

    Full Text Available A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs by genome-wide allele-specific gene expression (ASE analysis with that of traditional expression quantitative trait locus (eQTL mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.

  1. Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny

    Science.gov (United States)

    Cuenca, José; Aleza, Pablo; Navarro, Luis; Ollitrault, Patrick

    2013-01-01

    Background Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids. Scope This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers. Conclusions Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan−1 (y/x) and y′ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis

  2. High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

    Directory of Open Access Journals (Sweden)

    Clark Taane G

    2010-04-01

    Full Text Available Abstract Background Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Results Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%. Conclusions Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Niels H Skotte

    Full Text Available 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.

  5. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    Science.gov (United States)

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish.

  6. Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.

    Science.gov (United States)

    Ginart, Paul; Kalish, Jennifer M; Jiang, Connie L; Yu, Alice C; Bartolomei, Marisa S; Raj, Arjun

    2016-03-01

    Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.

  7. Integrative analysis of hereditary nonpolyposis colorectal cancer: the contribution of allele-specific expression and other assays to diagnostic algorithms.

    Directory of Open Access Journals (Sweden)

    Laura De Lellis

    Full Text Available The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS. Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE, nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI and immunohistochemical (IHC MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5% probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

  8. 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.

  9. Oncogene mutations, copy number gains and mutant allele specific imbalance (MASI frequently occur together in tumor cells.

    Directory of Open Access Journals (Sweden)

    Junichi Soh

    Full Text Available BACKGROUND: Activating mutations in one allele of an oncogene (heterozygous mutations are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI has been observed in tumors and cell lines harboring mutations of oncogenes. METHODOLOGY/PRINCIPAL FINDINGS: We determined 1 mutational status, 2 copy number gains (CNGs and 3 relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20% in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1 MASI with CNG, either complete or partial; and 2 MASI without CNG (uniparental disomy; UPD, due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75% and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%, was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. CONCLUSIONS: MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

  10. 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.

  11. 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.

  12. Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F1 interspecies hybrids☆

    Science.gov (United States)

    Shen, Yingjia; Catchen, Julian; Garcia, Tzintzuni; Amores, Angel; Beldroth, Ion; Wagner, Jonathon R; Zhang, Ziping; Postlethwait, John; Warren, Wes; Schartl, Manfred; Walter, Ronald B.

    2011-01-01

    Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, X. maculatus and X. couchianus, and their F1 interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈ 70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈ 5–10 million years ago, were identified about every 700 bp. Using 6,524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F1 interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the insilico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F1 interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression. PMID:21466860

  13. Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F₁ interspecies hybrids.

    Science.gov (United States)

    Shen, Yingjia; Catchen, Julian; Garcia, Tzintzuni; Amores, Angel; Beldorth, Ion; Wagner, Jonathan; Zhang, Ziping; Postlethwait, John; Warren, Wes; Schartl, Manfred; Walter, Ronald B

    2012-01-01

    Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, Xiphophorus maculatus and Xiphophorus couchianus, and their F(1) interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈5-10 million years ago, were identified about every 700 bp. Using 6524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F(1) interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the in silico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F(1) interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression.

  14. Detection of Fusarium oxysporum f. sp. vasinfectum race 3 by single-base extension method and allele-specific polymerase chain reaction

    Science.gov (United States)

    We developed allele specific (AS) SNP primers for rapid detection of Fusarium oxysporum f.sp vasinfectum (FOV) race 3. FOV_BT_SNP_R3 and FOV_BT_AS_R3 primers were designed based on single nucleotide polymorphisms of partial sequence alignment of the ß-tubulin (BT) gene from several FOV races. These ...

  15. 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

  16. 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

  17. 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

  18. Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.

    Science.gov (United States)

    Ding, Yu; Xia, Bo-Hou; Liu, Qi; Li, Mei-Ya; Huang, Shui-Xian; Zhuo, Guang-Chao

    2016-10-10

    Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1

  19. 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.

  20. Attenuated virulence of pigment-producing mutant of Aeromonas veronii bv. sobria in HeLa cells and Nile tilapia (Oreochromis niloticus

    Directory of Open Access Journals (Sweden)

    Said K. Abolghait

    2013-06-01

    Full Text Available Aeromonas species are potential water/foodborne pathogens, whereas Aeromonas veronii bv. sobria is one of the most virulent species to human and fish. Most current experimental evidence has publicized that suicide plasmid dependent IS1-element untargeted integration into A. veronii bv. sobria ATCC 9071T strain was recently used to generate brown pigment-producing and spontaneous pelleting (BP+SP+ mutant. Current study was conducted to compare virulence of wild-type ATCC 9071T strain and its BP+SP+ mutant with respect to cytotoxicity in HeLa cells and lethality in Nile tilapia. It was found that the cytotoxicity of wild-type ATCC 9071T strain to HeLa cells has reached 75% versus 50% for the cytotoxicity of BP+SP+ mutant. Further, the median lethal dose (LD50 of wild-type ATCC 9071T strain in Nile tilapia was 8.25 Log10 colony-forming units (CFU/ml, compared to 9.16 Log10 CFU/ml for the LD50 of BP+SP+ mutant. Thus, current study supports the notion that non pigment-producing Aeromonas strains are more virulent than pigment-producing ones.

  1. 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.

  2. Baicalein attenuates the quorum sensing-controlled virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response in P. aeruginosa-infected macrophages by downregulating the MAPK and NFκB signal-transduction pathways

    Directory of Open Access Journals (Sweden)

    Luo J

    2016-01-01

    Full Text Available Jing Luo,* Jin-liang Kong,* Bi-ying Dong, Hong Huang, Ke Wang, Li-hong Wu, Chang-chun Hou, Yue Liang, Bing Li, Yi-qiang Chen Department of Respiratory Disease, First Affiliated Hospital of Guangxi Medical University, Nanning, People’s Republic of China *These authors contributed equally to this work Abstract: Burgeoning antibiotic resistance and unfavorable outcomes of inflammatory injury after Pseudomonas aeruginosa infection have necessitated the development of novel agents that not only target quorum sensing (QS but also combat inflammatory injury with the least risk of resistance. This study aimed to assess the anti-QS and anti-inflammatory activities of baicalein, a traditional herbal medicine that is widely used in the People’s Republic of China, against P. aeruginosa infection. We found that subminimum inhibitory concentrations of baicalein efficiently interfered with the QS-signaling pathway of P. aeruginosa via downregulation of the transcription of QS-regulated genes and the translation of QS-signaling molecules. This interference resulted in the global attenuation of QS-controlled virulence factors, such as motility and biofilm formation, and the secretion into the culture supernatant of extracellular virulence factors, including pyocyanin, LasA protease, LasB elastase, and rhamnolipids. Moreover, we examined the anti-inflammatory activity of baicalein and its mode of action via a P. aeruginosa-infected macrophage model to address its therapeutic effect. Baicalein reduced the P. aeruginosa-induced secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, and TNFα. In addition, baicalein suppressed P. aeruginosa-induced activation of the MAPK and NFκB signal-transduction pathways in cocultured macrophages; this may be the mechanism by which baicalein inhibits the production of proinflammatory cytokines. Therefore, our study demonstrates that baicalein represents a potential treatment for P. aeruginosa infection because it

  3. 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...

  4. An rfaH mutant of Salmonella enterica serovar typhimurium is attenuated in swine and reduces intestinal colonization, fecal shedding, and disease severity due to virulent Salmonella Typhimurium

    Science.gov (United States)

    Swine are often asymptomatic carriers of Salmonella spp., and interventions are needed to limit colonization of swine to enhance food safety and reduce environmental contamination. We evaluated the attenuation and potential vaccine use in pigs of a Salmonella enterica serovar Typhimurium mutant of r...

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

    Science.gov (United States)

    Manczinger, Máté; Bocsik, Alexandra; Kocsis, Gabriella F; Vörös, Andrea; Hegedűs, Zoltán; Ördögh, Lilla; Kondorosi, Éva; Marton, Annamária; Vízler, Csaba; Tubak, Vilmos; Deli, Mária; Kemény, Lajos; Nagy, István; Lakatos, Lóránt

    2015-01-01

    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.

  6. 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.

  7. Allele-specific chromatin remodeling in the ZPBP2/GSDMB/ORMDL3 locus associated with the risk of asthma and autoimmune disease.

    Science.gov (United States)

    Verlaan, Dominique J; Berlivet, Soizik; Hunninghake, Gary M; Madore, Anne-Marie; Larivière, Mathieu; Moussette, Sanny; Grundberg, Elin; Kwan, Tony; Ouimet, Manon; Ge, Bing; Hoberman, Rose; Swiatek, Marcin; Dias, Joana; Lam, Kevin C L; Koka, Vonda; Harmsen, Eef; Soto-Quiros, Manuel; Avila, Lydiana; Celedón, Juan C; Weiss, Scott T; Dewar, Ken; Sinnett, Daniel; Laprise, Catherine; Raby, Benjamin A; Pastinen, Tomi; Naumova, Anna K

    2009-09-01

    Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes.

  8. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    Science.gov (United States)

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  9. Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

    Science.gov (United States)

    Shirasu, Naoto; Kuroki, Masahide

    2014-01-01

    We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

  10. Insights into Entamoeba histolytica virulence modulation.

    Science.gov (United States)

    Padilla-Vaca, F; Anaya-Velázquez, F

    2010-08-01

    Entamoeba histolytica is able to invade human tissues by means of several molecules and biological properties related to the virulence. Pathogenic amebas use three major virulence factors, Gal/GalNAc lectin, amebapore and proteases, for lyse, phagocytose, kill and destroy a variety of cells and tissues in the host. Responses of the parasite to host components such as mucins and bacterial flora influence the behavior of pathogenic amebas altering their expression of virulence factors. The relative virulence of different strains of E. histolytica has been shown to vary as a consequence of changes in conditions of in vitro cultivation which implies substantial changes in basic metabolic aspects and factors directly and indirectly related to amebic virulence. Comparison of E. histolytica strains with different virulence phenotypes and under different conditions of growth will help to identify new virulence factor candidates and define the interplay between virulence factors and invasive phenotype. Virulence attenuate mutants of E. histolytica are useful also to uncover novel virulence determinants. The comparison of biological properties and virulence factors between E. histolytica and E. dispar, a non-pathogenic species, has been a useful approach to investigate the key factors involved in the experimental presentation of amebiasis and its complex regulation. The molecular mechanisms that regulate these variations in virulence are not yet known. Their elucidation will help us to better understand the gene expression plasticity that enables the effective adaptation of the ameba to changes in growth culture conditions and host factors.

  11. Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

    Directory of Open Access Journals (Sweden)

    Guilhem Janbon

    2014-04-01

    Full Text Available Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D representing two varieties (i.e. grubii and neoformans, respectively. Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS, over 2,000 introns in the untranslated regions (UTRs were also identified. Poly(A-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A-site-associated motif (AUGHAH. In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

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

    Science.gov (United States)

    Rothe, Jessica; Watkins, Norman E; Nagy, Marion

    2012-01-01

    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%.

  13. Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

    Science.gov (United States)

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.

  14. 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

  15. Virulence reduction in Bacteriophage resistant bacteria

    Directory of Open Access Journals (Sweden)

    Marcela eLeón

    2015-04-01

    Full Text Available Bacteriophages can influence the abundance, diversity and evolution of bacterial communities. Several bacteriophages have been reported to add virulence factors to their host and to increase bacterial virulence. However, lytic bacteriophages can also exert a selective pressure allowing the proliferation of strains with reduced virulence. This reduction can be explained because bacteriophages use structures present on the bacterial surface as receptors, which can be virulence factors in different bacterial species. Therefore, strains with modifications in these receptors will be resistant to bacteriophage infection and may also exhibit reduced virulence. This mini-review summarizes the reports on bacteriophage-resistant strains with reductions in virulence, and it discusses the potential consequences in phage therapy and in the use of bacteriophages to select attenuated strains for vaccines.

  16. The Plasmodium falciparum merozoite surface protein-1 19 KD antibody response in the Peruvian Amazon predominantly targets the non-allele specific, shared sites of this antigen

    Directory of Open Access Journals (Sweden)

    Silva Claudia

    2010-01-01

    Full Text Available Abstract Background Plasmodium falciparum re-emerged in Iquitos, Peru in 1994 and is now hypoendemic (P. falciparum infections can be followed using this population dynamic. Previous work demonstrated a strong association between this population's antibody response to PfMSP1-19KD and protection against febrile illness and parasitaemia. Therefore, some selection for PfMSP1-19KD allelic diversity would be expected if the protection is to allele-specific sites of PfMSP1-19KD. Here, the potential for allele-specific polymorphisms in this population is investigated, and the allele-specificity of antibody responses to PfMSP1-19KD are determined. Methods The 42KD region in PfMSP1 was genotyped from 160 individual infections collected between 2003 and 2007. Additionally, the polymorphic block 2 region of Pfmsp1 (Pfmsp1-B2 was genotyped in 781 infection-months to provide a baseline for population-level diversity. To test whether PfMSP1-19KD genetic diversity had any impact on antibody responses, ELISAs testing IgG antibody response were performed on individuals using all four allele-types of PfMSP1-19KD. An antibody depletion ELISA was used to test the ability of antibodies to cross-react between allele-types. Results Despite increased diversity in Pfmsp1-B2, limited diversity within Pfmsp1-42KD was observed. All 160 infections genotyped were Mad20-like at the Pfmsp1-33KD locus. In the Pfmsp1-19KD locus, 159 (99.4% were the Q-KSNG-F haplotype and 1 (0.6% was the E-KSNG-L haplotype. Antibody responses in 105 individuals showed that Q-KNG and Q-TSR alleles generated the strongest immune responses, while Q-KNG and E-KNG responses were more concordant with each other than with those from Q-TSR and E-TSR, and vice versa. The immuno-depletion ELISAs showed all samples responded to the antigenic sites shared amongst all allelic forms of PfMSP1-19KD. Conclusions A non-allele specific antibody response in PfMSP1-19KD may explain why other allelic forms have not

  17. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik

    2014-01-01

    BACKGROUND: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma...... samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence...... identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue...

  18. Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo

    Institute of Scientific and Technical Information of China (English)

    GUO Dong-xing; LI Jing-yun; LI Han-ping; LI Lin; ZHUANG Dao-min; JIAO Li-yan; WANG Zheng; BAO Zuo-yi; LIU Si-yang; LIU Yong-jian

    2010-01-01

    Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.

  19. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    Science.gov (United States)

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

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

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    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

  1. Lichtheimia species exhibit differences in virulence potential.

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    Volker U Schwartze

    Full Text Available Although the number of mucormycosis cases has increased during the last decades, little is known about the pathogenic potential of most mucoralean fungi. Lichtheimia species represent the second and third most common cause of mucormycosis in Europe and worldwide, respectively. To date only three of the five species of the genus have been found to be involved in mucormycosis, namely L. corymbifera, L. ramosa and L. ornata. However, it is not clear whether the clinical situation reflects differences in virulence between the species of Lichtheimia or whether other factors are responsible. In this study the virulence of 46 strains of all five species of Lichtheimia was investigated in chicken embryos. Additionally, strains of the closest-related genus Dichotomocladium were tested. Full virulence was restricted to the clinically relevant species while all strains of L. hyalospora, L. sphaerocystis and Dichotomocladium species were attenuated. Although virulence differences were present in the clinically relevant species, no connection between origin (environmental vs clinical or phylogenetic position within the species was observed. Physiological studies revealed no clear connection of stress resistance and carbon source utilization with the virulence of the strains. Slower growth at 37°C might explain low virulence of L. hyalospora, L. spaherocystis and Dichotomocladium; however, similarly slow growing strains of L. ornata were fully virulent. Thus, additional factors or a complex interplay of factors determines the virulence of strains. Our data suggest that the clinical situation in fact reflects different virulence potentials in the Lichtheimiaceae.

  2. A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

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    Auewarakul Chirayu U

    2011-02-01

    Full Text Available Abstract Background BCR-ABL kinase domain (KD mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI in BCR-ABL-positive chronic myeloid leukemia (CML patients. T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure. A simple and sensitive method is thus required to detect T315I mutation at the earliest stage. Methods A single-tube allele specific-polymerase chain reaction (AS-PCR method was developed to detect T315I mutation in a mixture of normal and mutant alleles of varying dilutions. Denaturing high performance liquid chromatography (DHPLC and direct sequencing were performed as a comparison to AS-PCR. Results T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR. The detection sensitivity of DHPLC was around 1.5-3% dilution whereas sequencing analysis was unable to detect below 6.25% dilution. Conclusion A single-tube AS-PCR is a rapid and sensitive screening method for T315I mutation. Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.

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

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

    2014-01-01

    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.

  4. Concordance between allele-specific PCR and ultra-deep pyrosequencing for the detection of HIV-1 non-nucleoside reverse transcriptase inhibitor resistance mutations

    Science.gov (United States)

    Hunt, Gillian M; Morris, Lynn; Moorthy, Anitha; Coovadia, Ashraf; Abrams, Elaine J; Strehlau, Renate; Kuhn, Louise; Persaud, Deborah

    2014-01-01

    Recent advances in genotyping technologies have allowed for detection of HIV-1 drug resistance mutations present at low levels. The presence and percentage of Y181C and K103N drug-resistant variants in the blood of 105 subtype C HIV-infected infants who failed single-dose nevirapine prophylaxis for HIV transmission were compared using two highly sensitive genotyping methods, allele-specific PCR (AS-PCR) and ultra-deep pyrosequencing. Significant correlations in detection between both methods were found for both Y181C (correlation coefficients of 0.94 [95% CI 0.91-0.96]) and K103N (0.89 [95% CI 0.84 – 0.92]) mutations. The majority of discordant specimens (3/5 Y181C and 8/11 K103N) had wild-type variants when population sequencing was used, but mutant variants were detectable at very low levels (≤5%) with either assay. This difference is most likely due to stochastic variations in the appearance of mutant variants. Overall, both AS-PCR and ultra-deep pyrosequencing methods have proven to be sensitive and accurate, and may confidently be used where feasible. PMID:25034127

  5. Electromobility Shift Assay Reveals Evidence in Favor of Allele-Specific Binding of RUNX1 to the 5' Hypersensitive Site 4-Locus Control Region.

    Science.gov (United States)

    Dehghani, Hossein; Ghobakhloo, Sepideh; Neishabury, Maryam

    2016-08-01

    In our previous studies on the Iranian β-thalassemia (β-thal) patients, we identified an association between the severity of the β-thal phenotype and the polymorphic palindromic site at the 5' hypersensitive site 4-locus control region (5'HS4-LCR) of the β-globin gene cluster. Furthermore, a linkage disequilibrium was observed between this region and XmnI-HBG2 in the patient population. Based on this data, it was suggested that the well-recognized phenotype-ameliorating role assigned to positive XmnI could be associated with its linked elements in the LCR. To investigate the functional significance of polymorphisms at the 5'HS4-LCR, we studied its influence on binding of transcription factors. Web-based predictions of transcription factor binding revealed a binding site for runt-related transcription factor 1 (RUNX1), when the allele at the center of the palindrome (TGGGG(A/G)CCCCA) was A but not when it was G. Furthermore, electromobility shift assay (EMSA) presented evidence in support of allele-specific binding of RUNX1 to 5'HS4. Considering that RUNX1 is a well-known regulator of hematopoiesis, these preliminary data suggest the importance of further studies to confirm this interaction and consequently investigate its functional and phenotypical relevance. These studies could help us to understand the molecular mechanism behind the phenotype modifying role of the 5'HS4-LCR polymorphic palindromic region (rs16912979), which has been observed in previous studies.

  6. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

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    Zhongyang Zhang

    2015-11-01

    Full Text Available Cancer genomes exhibit profound somatic copy number alterations (SCNAs. Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1 extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2 performing joint segmentation on the two signal dimensions; 3 correcting the copy number baseline from which the SCNA state is determined; 4 calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

  7. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

    Science.gov (United States)

    Zhang, Zhongyang; Hao, Ke

    2015-11-01

    Cancer genomes exhibit profound somatic copy number alterations (SCNAs). Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1) extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2) performing joint segmentation on the two signal dimensions; 3) correcting the copy number baseline from which the SCNA state is determined; 4) calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS) as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

  8. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    Science.gov (United States)

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

  9. Genome-wide detection of allele specific copy number variation associated with insulin resistance in African Americans from the HyperGEN study.

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    Marguerite R Irvin

    Full Text Available African Americans have been understudied in genome wide association studies of diabetes and related traits. In the current study, we examined the joint association of single nucleotide polymorphisms (SNPs and copy number variants (CNVs with fasting insulin and an index of insulin resistance (HOMA-IR in the HyperGEN study, a family based study with proband ascertainment for hypertension. This analysis is restricted to 1,040 African Americans without diabetes. We generated allele specific CNV genotypes at 872,243 autosomal loci using Birdsuite, a freely available multi-stage program. Joint tests of association for SNPs and CNVs were performed using linear mixed models adjusting for covariates and familial relationships. Our results highlight SNPs associated with fasting insulin and HOMA-IR (rs6576507 and rs8026527, 3.7*10(-7≤P≤1.1*10(-5 near ATPase, class V, type 10A (ATP10A, and the L Type voltage dependent calcium channel (CACNA1D, rs1401492, P≤5.2*10(-6. ATP10A belongs to a family of aminophospholipid-transporting ATPases and has been associated with type 2 diabetes in mice. CACNA1D has been linked to pancreatic beta cell generation in mice. The two most significant copy variable markers (rs10277702 and rs361367; P<2.0*10(-4 were in the beta variable region of the T-cell receptor gene (TCRVB. Human and mouse TCR has been shown to mimic insulin and its receptor and could contribute to insulin resistance. Our findings differ from genome wide association studies of fasting insulin and other diabetes related traits in European populations, highlighting the continued need to investigate unique genetic influences for understudied populations such as African Americans.

  10. Allelic diversity of a beer haze active protein gene in cultivated and Tibetan wild barley and development of allelic specific markers.

    Science.gov (United States)

    Ye, Lingzhen; Dai, Fei; Qiu, Long; Sun, Dongfa; Zhang, Guoping

    2011-07-13

    The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ∼14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW∼14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population.

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

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    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.

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

    OpenAIRE

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

    2011-01-01

    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. Pha...

  13. Allele-specific oligonucleotide polymerase chain reaction for the determination of Rh C/c and Rh E/e antigens in thalassaemic patients

    Science.gov (United States)

    Hojjati, Mohammad Taher; Einollahi, Nahid; Nabatchian, Fariba; Pourfathollah, Ali Akbar; Mahdavi, Mohammad Reza

    2011-01-01

    Background Thalassaemia is a genetic disease in which there is a relative or complete lack of alpha or beta globin chains. Patients with moderate to severe forms of thalassaemia need transfusions from the early years of life. Antibody production against blood group antigens may cause many problems in preparing compatible blood units for transfusion. The identification of definite blood group phenotypes by the haemagglutination method can be difficult because of the mixed population of red blood cells from the donor and recipient. Materials and methods Forty multiply transfused thalassaemic patients and ten healthy controls with no history of blood transfusion were enrolled in this study. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and haemagglutination methods were used to determine the presence of Rhesus (Rh) C, c, E and e antigens. Results In this study four primer sets were used for ASO-PCR amplification of RhC/c and RhE/e. Although PCR assays for RhC/c and RHE/e genotyping have been described previously, in this study we used a new condition for PCR by decreasing the annealing temperature from 63 °C to 58 °C in order to amplify all four genes in the same condition. In order to evaluate this single run molecular method, we used the haemagglutination test as the standard method and compared the results from the two methods. We found discrepancies between phenotype and genotype results among patients with beta thalassaemia, but complete agreement between phenotype and genotype in the control group. Conclusions The advantage of this new ASO-PCR method compared to a restriction fragment length polymorphism (RFLP) PCR method is that with the former all four genes can be amplified at the same time by PCR, and electrophoresis can be performed immediately to determine individual antigen profiles. The simplicity of the ASO-PCR method makes it suitable for routine use in medical centres and it is also cheaper than RFLP-PCR. Furthermore, as shown

  14. [Factors of Salmonella typhi virulence in relation to the development of new vaccines].

    Science.gov (United States)

    García, J A; Paniagua, J; Pelayo, R; Isibasi, A; Kumate, J

    1992-01-01

    Although many vaccines against typhoid fever have been developed, none have been adapted for their further application on developing countries. In order to get better vaccines, the virulence factors of both S. typhi and S. typhimurium have been studied. Thus, some protection assays have been made using surface antigens involved on virulence or using live attenuated vaccines of bacteria mutated on virulence genes. Here we present a brief review about virulence factors studied so far for the development of new vaccines.

  15. Deciphering the Molecular Variations of Pine Wood Nematode Bursaphelenchus xylophilus with Different Virulence.

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    Xiaolei Ding

    Full Text Available Bursaphelenchus xylophilus is the causative agent of pine wilt disease which has caused huge economic losses in many countries. It has been reported that two forms of pine wood nematodes existed in its native region, i.e., with strong virulence and weak virulence. However, little is known about the molecular differences between the two forms. To better understand their molecular variations, transcriptome and genome sequences of three strongly virulent and one weakly virulent strains were analyzed. We found 238 transcripts and 84 exons which showed notable changes between the two virulent forms. Functional analyses of both differentially expressed transcripts and exons indicated that different virulence strains showed dissimilar nematode growth, reproduction, and oxidoreductase activities. In addition, we also detected a small number of exon-skipping events in B. xylophilus. Meanwhile, 117 SNPs were identified as potential genetic markers in distinguishing the two forms. Four of them were further proved to have undergone allele specific expressions and possibly interrupted the target site of evolutionary conserved B. xylophilus miR-47. These particular SNPs were experimentally verified by including eight additional strains to ensure the validity of our sequencing results. These results could help researchers to better diagnose nematode species with different virulence and facilitate the control of pine wilt disease.

  16. Bacterial virulence analysis using brine shrimp as an infection model in relation to the importance of quorum sensing and proteases.

    Science.gov (United States)

    Lee, Mi-Nan; Kim, Soo-Kyoung; Li, Xi-Hui; Lee, Joon-Hee

    2014-01-01

    Brine shrimp are aquatic crustaceans belonging to a genus of Artemia. This organism is widely used for testing the toxicity of chemicals. In this study, brine shrimp were evaluated as an infection model organism to study bacterial virulence. Artemia nauplii were infected with various pathogenic bacteria, such as Vibrio vulnificus, Pseudomonas aeruginosa, Burkholderia vietnamiensis, Staphylococcus aureus, and Escherichia coli, and the susceptibility to these bacteria was investigated by counting the survival of the infected nauplii. While all of the tested bacteria have significant virulence to brine shrimp, killing the nauplii in a few days, V. vulnificus showed the strongest virulence. P. aeruginosa also showed a dose-dependent virulence to brine shrimp, but the virulence was weaker than that of V. vulnificus. The virulence tests using the virulence-attenuated mutants of V. vulnificus and P. aeruginosa, such as quorum sensing (QS) mutants or protease-deficient mutants showed a significant attenuation of virulence, demonstrating that the QS mechanism is important in the virulence of these bacteria to brine shrimp. B. vietnamiensis, S. aureus, and E. coli were also virulent to brine shrimp and the virulence was correlated with dosage within 24 h under our conditions. Salmonella enterica Typhimurium and Bacillus subtilis were also virulent to brine shrimp, but the virulence was weak and slowly exerted compared with that of other bacteria. Taken together, we suggest that brine shrimp are a good infection model to assay bacterial virulence, especially for V. vulnificus and P. aeruginosa, and QS is important in the bacterial virulence to brine shrimp.

  17. DETEKSI MUTASI V1016G PADA GEN VOLTAGE-GATED SODIUM CHANNEL PADA POPULASI Aedes aegypti (DIPTERA: CULICIDAE DI KABUPATEN KLATEN, JAWA TENGAH DENGAN METODE ALLELE-SPECIFIC PCR

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    Dyah Widiastuti

    2015-10-01

    Full Text Available AbstrakMeluasnya kejadian resistensi pada vektor virus Dengue di Jawa Tengah memerlukan strategi pengelolaan resistensi insektisida secara efektif. Oleh karena itu, informasi mengenai mutasi gen pada posisi 1016 di domain II segmen ke­6 gen VGSC pada nyamuk Aedes aegypti yang menyebabkan perubahan asam amino valin (V menjadi glisin (G akan dapat memperkuat penelitian operasional mengenai strategi pemilihan insektisida dalam program­pengendalian­vektor­Dengue.­Penelitian­ini­menggunakan­uji­Allele-Specific­Polymerase­Chain­Reaction(AS­PCR yang dapat mendeteksi mutasi V1016G. Sampel penelitian ini adalah 22 ekor nyamuk Aedes aegypti dari Kabupaten Klaten yang berumur 2­5 hari. Hasil penelitian menunjukkan bahwa 22,7% nyamuk belum mengalami mutasi (V/V, 59,1% nyamuk mengalami mutasi heterozigot (V/G dan 18,2% nyamuk mengalami mutasi homozigot (G/G. Hal ini menunjukkan indikasi terjadinya resistensi populasi nyamuk Ae.aegypti terhadap insektisida sintetik piretroid yang disebabkan oleh mekanisme knockdown resistance.Kata Kunci:­Aedes­aegypti,­mutasi­V1016G,­Allele-Specific­PCR,­VGSCAbstractInsecticides resistance has spread rapidly among dengue vectors from Central Java, and require an effective insecticide resistance management strategies.one of the resistance mechanism in Aedes aegypti may arise through knockdown resistance or kdr which consists of single point mutation within the genes that are targeted by insecticide compounds. Mutation at position 1016 in domain II, segment 6 of the Voltage Gated Sodium Channel gene in Ae. aegypti leads to a valine to glycine substitution (V1016G is associated with resistance to the type II pyrethroid. The result of this study will help us to strengthen basic and operational research on the­development­of­strategies­for­Dengue­vector­control­in­Indonesia.­This­study­utilized­an­allele-specificPolymerase Chain Reaction (AS­PCR assay that could be used to detect the V1016G

  18. ClpP deletion causes attenuation of Salmonella Typhimurium virulence through mis-regulation of RpoS and indirect control of CsrA and the SPI genes

    DEFF Research Database (Denmark)

    Knudsen, Gitte Maegaard; Olsen, John E.; Aabo, Søren;

    2013-01-01

    Salmonella enterica serovar Typhimurium requires the type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) and controlled by the master regulator, HilA, to penetrate the intestinal epithelium. Numerous regulators affect virulence through influence on this system, including...... the proteolytic component ClpP, the stationary phase regulator RpoS and the carbon-storage regulator CsrA. However, the mechanism behind the ClpP regulation is not fully understood. To elucidate this we examined differentially expressed genes in a ΔclpP mutant compared with WT using global transcriptomic analysis......, suggesting the repression of invasion was directed through RpoS. The expression of the csrA virulence regulator was increased in the ΔclpP mutant and decreased in the rpoS : : amp and ΔclpP/rpoS : : amp mutants, indicating that ClpP affects the csrA expression level as well. Thus, this study suggests...

  19. Burkholderia pseudomallei virulence: definition, stability and association with clonality.

    Science.gov (United States)

    Ulett, G C; Currie, B J; Clair, T W; Mayo, M; Ketheesan, N; Labrooy, J; Gal, D; Norton, R; Smith, C A; Barnes, J; Warner, J; Hirst, R G

    2001-07-01

    Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.

  20. 狂犬病病毒CTN-181减毒株在豚鼠颌下腺的繁殖动态及其低毒力克隆株的筛选研究%Study on the attenuated rabies virus CTN-181 in the submandibular gland of guinea pigs and the low virulence clones from the submandibular gland

    Institute of Scientific and Technical Information of China (English)

    石磊泰; 邹剑; 俞永新; 王玲; 曹守春; 董关木; 李玉华

    2015-01-01

    目的 了解狂犬病病毒减毒株CTN-181在豚鼠颌下腺的繁殖动态并获得毒力更低的病毒纯化克隆株.方法 将CTN-181经刚离乳豚鼠颈部皮下注射,分别于1、2、3和4d取颌下腺分离狂犬病病毒,测定各天次颌下腺内的病毒滴度.用滴度高的病毒液进行下一次豚鼠颌下腺传代,连续传3代.将第3代颌下腺传代的病毒液在BHK21细胞上进行空斑试验,挑选单个病毒克隆,以3周龄小鼠脑内接种测病毒毒力.筛选对小鼠不致病的克隆株进一步测定其对乳鼠的脑内毒力,并以乳鼠脑内传代和豚鼠颌下腺传代检测其遗传稳定性.结果 CTN-181在豚鼠颌下腺能短暂轻度繁殖,通过颌下腺传代后病毒毒力明显有回升.获得19个病毒克隆,不同克隆病毒的毒力不同,为0~3.16 lg LD50;3株脑内无致病力的克隆株对乳鼠的毒力亦有减弱;乳鼠脑内和颌下腺回传后毒力无回升返祖,即传代后的病毒对小鼠均无致病力.结论 CTN-181母株对3周龄小鼠仍存在低水平毒力,经豚鼠颌下腺传代后毒力十分不稳定.筛选到的3株克隆病毒较其母株的毒力更低,遗传稳定性更高,更适合作为狂犬病兽用口服疫苗候选株.%Objective To understand the stability of an attenuated rabies virus strain CTN-181 and to obtain more attenuated and stable virus strains from the CTN-181 strain.Methods The CTN-181 virus was inoculated into the submandibular glands of guinea pigs subcutaneously.The glands were harvested on day 1,2,3 and 4 after inoculation,then the virus contents and virulence in 3-week mice were evaluated.Three generations of the submandibular gland passage were made and the gland virus sample from the third passage of the guinea pigs was used for plaque cloning.Plaque clone viruses were tested for their neurovirulence in 3-week mice or suckling mice,and the attenuation stability was performed by inoculation passage in suckling mice and submandibular gland passages

  1. 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

    Burkholderia cenocepacia H111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematode Caenorhabditis elegans. We used the C. elegans model of infection to screen a mini-Tn5 mutant library of B. cenocepacia H111 for attenuated virulence....... Of the approximately 5,500 B. cenocepacia H111 random mini-Tn5 insertion mutants that were screened, 22 showed attenuated virulence in C. elegans. Except for the quorum-sensing regulator cepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases...... 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...

  2. Preliminary virulence evaluation on mutation of pgm attenuates Brucella melitensis 16 strain%布鲁杆菌M16株pgm基因突变活菌苗株毒力初步评价

    Institute of Scientific and Technical Information of China (English)

    李鹏; 潘玉焕; 罗德炎; 王希良

    2011-01-01

    目的 通过减毒性实验评价布鲁杆菌M16株pgm基因突变活菌苗株毒力减弱情况,为新型减毒活疫苗的研制和布鲁杆菌感染免疫防御的研究奠定基础.方法 进行布鲁杆菌侵袭实验等动物和细胞实验评价突变菌株的毒力减弱情况.结果 突变菌株在小鼠体内能生存,但较强毒株存活数量有很大减少(P<0.001);突变菌株较强毒株对多粘菌素B更为敏感,存活率与强毒株相比有明显下降(P<0.001);突变菌株LPS较强毒株LPS明显缺失;突变菌株在体外HeLa细胞中能存活但复制减弱.结论 布鲁杆菌M16株pgm基因突变菌株毒力减弱.可作为未来布鲁杆菌新型减毒活疫苗的候选者,有进一步研究的价值.%Objective Evaluate the effect of attenuation and lay the foundation for the research of immunological mechanism of brucella infection and the development of novel live attenuated vaccines. Methods The effect of attenuation is evaluated by brucella invasion experiment, and other animal and cell experiments. Results Compared with wild - type strains, the mutation strains have several aspects different from it: ( 1 ) though the mutation strains of Brucella melitensis 16. can survive in the mouse model, the survival rates in the spleens of the inoculated mice were significantly lower than the wild - type strain; ( 2 ) the mutation strains were more sensitive to PmB than the wild - type parental strain; ( 3 ) the amount of LPS on the mutation strain of Brucella melitensis 16. were seen less than on the wild - type parental strain; (4) the replication rate of mutatation stains in HeLa cell line decreased dramatically although it can survive in it in vitro. Conclusions We have confirmed the attenuated effect of the mutation strain of Brucella melitensis 16, it can be used as a candidate vaccine for the control of brucellosis.

  3. spd1672基因敲除显著影响肺炎链球菌的毒力%Spd1672 gene knockout significantly attenuates the virulence of Streptococcus pneumoniae

    Institute of Scientific and Technical Information of China (English)

    黄健; 黄美容; 吴凯峰; 朱杰华; 黎兵; 闵迅

    2015-01-01

    目的 研究spd1672基因在肺炎链球菌感染过程中的作用.方法 通过腹腔攻毒实验和细菌入血载量分析、细菌的粘附侵袭实验、全血杀菌实验以及相关细胞因子检测等观察spd1672基因敲除(D39△1672菌株)后对肺炎链球菌毒力的影响.结果D39△1672菌株感染组小鼠中位生存时间和生存率显著高于野生菌株组(P0.05), the spd1672 knockout strain showed significantly lower cell invasion ability than the wild-type strain (P<0.05). The spd1672 knockout strain also had a reduced resistance to whole blood cells, and thw mice infected with spd1672 knockout strain exhibit lower levels of serum inflammatory cytokines than those infected with the wild-type strain. Conclusion Spd1672 gene is importantly related to the virulence of S. pneumoniae and plays important roles in modulating bacterial invasion, resistance to whole blood cells and proinflammatory responses.

  4. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    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....... These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host....

  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. Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP to detect the L1014F kdr-w mutation in Anopheles gambiae s. l.

    Directory of Open Access Journals (Sweden)

    Badolo Athanase

    2012-07-01

    Full Text Available Abstract Background Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides’ efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP method to detect the West African-type kdr mutation (kdr-w; L1014F in field-collected mosquitoes. Methods DNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5’ end of the backward inner primer (BIP. The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes. Results The AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75 min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method. Conclusions The AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories.

  7. Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

    NARCIS (Netherlands)

    Layre, Emilie; Lee, Ho Jun; Young, David C.; Martinot, Amanda Jezek; Buter, Jeffrey; Minnaard, Adriaan J.; Annand, John W.; Fortune, Sarah M.; Snider, Barry B.; Matsunaga, Isamu; Rubin, Eric J.; Alber, Tom; Moody, D. Branch

    2014-01-01

    To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guerin. Comparative lipidomics analysis identified more than 1,000 molecular differences

  8. 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...

  9. Hantavirus interferon regulation and virulence determinants.

    Science.gov (United States)

    Mackow, Erich R; Dalrymple, Nadine A; Cimica, Velasco; Matthys, Valery; Gorbunova, Elena; Gavrilovskaya, Irina

    2014-07-17

    Hantaviruses predominantly replicate in primary human endothelial cells and cause 2 diseases characterized by altered barrier functions of vascular endothelium. Most hantaviruses restrict the early induction of interferon-β (IFNβ) and interferon stimulated genes (ISGs) within human endothelial cells to permit their successful replication. PHV fails to regulate IFN induction within human endothelial cells which self-limits PHV replication and its potential as a human pathogen. These findings, and the altered regulation of endothelial cell barrier functions by pathogenic hantaviruses, suggest that virulence is determined by the ability of hantaviruses to alter key signaling pathways within human endothelial cells. Our findings indicate that the Gn protein from ANDV, but not PHV, inhibits TBK1 directed ISRE, kB and IFNβ induction through virulence determinants in the Gn cytoplasmic tail (GnT) that inhibit TBK1 directed IRF3 phosphorylation. Further studies indicate that in response to hypoxia induced VEGF, ANDV infection enhances the permeability and adherens junction internalization of microvascular and lymphatic endothelial cells. These hypoxia/VEGF directed responses are rapamycin sensitive and directed by mTOR signaling pathways. These results demonstrate the presence of at least two hantavirus virulence determinants that act on endothelial cell signaling pathways: one that regulates antiviral IFN signaling responses, and a second that enhances normal hypoxia-VEGF-mTOR signaling pathways to facilitate endothelial cell permeability. These findings suggest signaling pathways as potential targets for therapeutic regulation of vascular deficits that contribute to hantavirus diseases and viral protein targets for attenuating pathogenic hantaviruses.

  10. Proteomic Analysis of Pathogenic and Attenuated Alcelaphine Herpesvirus 1▿

    Science.gov (United States)

    Dry, Inga; Haig, David M.; Inglis, Neil F.; Imrie, Lisa; Stewart, James P.; Russell, George C.

    2008-01-01

    The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell. PMID:18353942

  11. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma.

    Directory of Open Access Journals (Sweden)

    Alexandre Harlé

    Full Text Available Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies.Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM PCR, Real-time allele-specific amplification (RT-ASA PCR, Next generation sequencing (NGS, immunohistochemistry (IHC and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays.BRAF mutations were found in 28(47.5%, 29(49.2%, 31(52.5%, 29(49.2% and 27(45.8% samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2% samples were found bearing a c.1799T>A (p.Val600Glu mutation, three (9.4% with a c.1798_1799delinsAA (p.Val600Lys mutation and one with c.1789_1790delinsTC (p.Leu597Ser mutation. Two samples were found bearing complex mutations.HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.

  12. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Sirirat Seekhuntod

    Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

  13. High frequency of SLC22A12 variants causing renal hypouricemia 1 in the Czech and Slovak Roma population; simple and rapid detection method by allele-specific polymerase chain reaction.

    Science.gov (United States)

    Gabrikova, Dana; Bernasovska, Jarmila; Sokolova, Jitka; Stiburkova, Blanka

    2015-10-01

    Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such as acute kidney injury. Type 1 and 2 are caused by loss-of-function mutations in the SLC22A12 and SLC2A9 gene, respectively. A cohort of 881 randomly chosen ethnic Roma from two regions in Eastern Slovakia and two regions in the Czech Republic participated. Genomic DNA was isolated from buccal swabs and/or from blood samples. The c.1245_1253del and c.1400C>T genotypes were determined using polymerase chain reaction with allele-specific primers in a multiplex arrangement and/or direct sequencing of exon 7 and 9. Allele frequencies and genotypes were tested for Hardy-Weinberg equilibrium using the Chi-square test. 25 subjects were heterozygous and three were homozygous for the c.1245_1253del, while 92 subjects were heterozygous and two were homozygous for the c.1400C>T. Moreover, two participants were compound heterozygotes. Frequencies of the c.1245_1253del and c.1400C>T variants were 1.87 and 5.56 %, respectively. Our finding confirms an uneven geographical and ethnic distribution of SLC22A12 mutant variants. We found that the c.1245_1253del and c.1400C>T variants were present in the Czech and Slovak Roma population at unexpectedly high frequencies. Renal hypouricemia should be kept in mind during differential diagnostic on Roma patients with low serum uric acid concentrations.

  14. Correlation between virulence of various strains of mycobacteria and their susceptibility to ethanolic extract of propolis (EEP).

    Science.gov (United States)

    Scheller, S; Kawalski, H; Oklek, K; Dworniczak, S; Matsuno, T; Waldemar-Klimmek, K; Rajca, M; Shani, J

    1998-01-01

    Ethanol extract of propolis (EEP) has antibacterial, antiviral, antiprotozoal and antifungal properties, in addition to many biological effects. Our laboratory has demonstrated a synergistic effect of EEP and antibiotics on the growth of Staphylococcus aureus, and suggested that the bactericidal effect of EEP was expressed mainly on virulent mycobacteria rather than on non-virulent (attenuated) ones. The present study was designed to reconfirm the latter finding, by subjecting 17 different mycobacteria strains to EEP, and evaluating whether there is a correlation between the virulence of the mycobacteria strains studied and their susceptibility to EEP. Our findings demonstrate that while the four non-virulent strains studied are not susceptible to EEP, out of the 13 virulent strains studied seven are susceptible and six are resistant to it. These results suggest that while there is no full correlation between virulence of the mycobacteria tested and their susceptibility to EEP, the few strains that were resistant to EEP were non-virulent.

  15. Transient virulence of emerging pathogens.

    Science.gov (United States)

    Bolker, Benjamin M; Nanda, Arjun; Shah, Dharmini

    2010-05-06

    Should emerging pathogens be unusually virulent? If so, why? Existing theories of virulence evolution based on a tradeoff between high transmission rates and long infectious periods imply that epidemic growth conditions will select for higher virulence, possibly leading to a transient peak in virulence near the beginning of an epidemic. This transient selection could lead to high virulence in emerging pathogens. Using a simple model of the epidemiological and evolutionary dynamics of emerging pathogens, along with rough estimates of parameters for pathogens such as severe acute respiratory syndrome, West Nile virus and myxomatosis, we estimated the potential magnitude and timing of such transient virulence peaks. Pathogens that are moderately evolvable, highly transmissible, and highly virulent at equilibrium could briefly double their virulence during an epidemic; thus, epidemic-phase selection could contribute significantly to the virulence of emerging pathogens. In order to further assess the potential significance of this mechanism, we bring together data from the literature for the shapes of tradeoff curves for several pathogens (myxomatosis, HIV, and a parasite of Daphnia) and the level of genetic variation for virulence for one (myxomatosis). We discuss the need for better data on tradeoff curves and genetic variance in order to evaluate the plausibility of various scenarios of virulence evolution.

  16. Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

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    Jason S Lehmann

    Full Text Available Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.

  17. 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.

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    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.

  18. 全血AS-PCR方法检测Wilson病ATP7B基因四个突变%Whole blood allele-specific PCR, a simple method to detect four ATP7B gene mutations in Wilson disease

    Institute of Scientific and Technical Information of China (English)

    孙玮; 管俊杰; 王进; 秦正红

    2014-01-01

    Objective To establish a simple method to detect four ATP7B gene mutations in Wilson disease using allele-specific PCR (AS-PCR) with whole blood polymerase chain reaction.Methods Four allele-specific PCR primers specific for the mutations(G2333T,C2850T,G2855A,G2975T) were designed,and PCR was optimized to screen the whole blood samples.The amplified gene products with mutation were separated with agarose gel electrophoresis to detect the pattern of point mutation and allele types.Exons 8,12 and 13 of the ATP7B gene were amplified with PCR,and the amplification products were sequenced to confirm the mutation.Results The detection of four ATP7B gene mutations by AS-PCR with whole blood was accomplished with 100% accuracy.In the 27 healthy subjects,the mutation rate of G2855A was 51.8%.No mutation was detected for G2333T,C2850T and G2975T.Among the 22 patients,11 were mutated for G2333T,C2850T or G2975T.The mutation rate was therefore 50%.Conclusion Our experiment has established an AS-PCR based method for detecting four ATP7B gene mutations using whole blood samples,which has provided a simple and effective means for the early diagnosis of Wilson disease.This method is rapid,convenient,accurate and economical for detecting point mutations of the ATP7B gene.%目的 研究肝豆状核变性ATP7B基因高频突变位点,探索全血等位基因特异性-PCR(allelespecific PCR,AS-PCR)技术在该基因4个常见突变检测中的应用.方法 针对ATP7B基因4个突变位点(G2333T、C2850T、G2855A、G2975T)设计等位基因特异性引物,应用高保真酶对人抗凝全血样本进行聚合酶链反应,扩增产物经琼脂糖凝胶电泳以判断待检样本有无基因突变及其等位基因型.PCR扩增人基因组A TP7B基因第8、12、13外显子,扩增产物直接进行基因序列测定.结果 全血AS-PCR法检测ATP7B基因4个基因突变,各位点检测结果与基因序列测定完全相符.27份健康对照血样分型结果G2333T、C2850T

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

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    Fei Sun

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  20. Brucella, nitrogen and virulence.

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    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.

  1. Evolutionary history and attenuation of myxoma virus on two continents.

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    Kerr, Peter J; Ghedin, Elodie; DePasse, Jay V; Fitch, Adam; Cattadori, Isabella M; Hudson, Peter J; Tscharke, David C; Read, Andrew F; Holmes, Edward C

    2012-01-01

    The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.

  2. Evolutionary history and attenuation of myxoma virus on two continents.

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    Peter J Kerr

    Full Text Available The attenuation of myxoma virus (MYXV following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.

  3. Uncovering the genetic basis of attenuation in Marek’s disease virus

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    While in vitro serial passage of Marek’s disease virus (MDV) is a proven method to attenuate MDV strains, the underlying genetic changes responsible for attenuation remains unknown. To identify candidate genes and mutations, a virulent MDV generated from an Md5-containing BAC clone was serially pass...

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

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    Mikonranta, Lauri; Friman, Ville-Petri; Laakso, Jouni

    2012-01-01

    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.

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

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    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.

  6. L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes

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    Lobel, Lior; Burg-Golani, Tamar; Sigal, Nadejda; Rose, Jessica; Livnat-Levanon, Nurit; Lewinson, Oded; Herskovits, Anat A.

    2017-01-01

    The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism. PMID:28114430

  7. Regulation of Francisella tularensis Virulence

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    Shipan eDai

    2011-01-01

    Full Text Available Francisella tularensis is one of the most virulent bacteria known and a Centers for Disease Control and Prevention Category A select agent. It is able to infect a variety of animals and insects and can persist in the environment, thus Francisella spp. must be able to survive in diverse environmental niches. However, F. tularensis has a surprising dearth of sensory and regulatory factors. Recent advancements in the field have identified new functions of encoded transcription factors and greatly expanded our understanding of virulence gene regulation. Here we review the current knowledge of environmental adaptation by F. tularensis, its transcriptional regulators and their relationship to animal virulence.

  8. Main functions and taxonomic distribution of virulence genes in Brucella melitensis 16 M.

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    Aniel Jessica Leticia Brambila-Tapia

    Full Text Available Many virulence genes have been detected in attenuated mutants of Brucella melitensis 16 M; nevertheless, a complete report of these genes, including the main Cluster of Orthologous Groups (COG represented as well as the taxonomical distribution among all complete bacterial and archaeal genomes, has not been analyzed. In this work a total of 160 virulence genes that have been reported in attenuated mutants in B. melitensis were included and analyzed. Additionally, we obtained 250 B. melitensis randomly selected genes as a reference group for the taxonomical comparisons. The COGs and the taxonomical distribution profile for 789 nonredundant bacterial and archaeal genomes were obtained and compared with the whole-genome COG distribution and with the 250 randomly selected genes, respectively. The main COGs associated with virulence genes corresponded to the following: intracellular trafficking, secretion and vesicular transport (U; cell motility (N; nucleotide transport and metabolism (F; transcription (K; and cell wall/membrane/envelope biogenesis (M. In addition, we found that virulence genes presented a higher proportion of orthologs in the Euryarchaeota and Proteobacteria phyla, with a significant decrease in Chlamydiae, Bacteroidetes, Tenericutes, Firmicutes and Thermotogae. In conclusion, we found that genes related to specific functions are more relevant to B. melitensis virulence, with the COG U the most significant. Additionally, the taxonomical distribution of virulence genes highlights the importance of these genes in the related Proteobacteria, being less relevant in distant groups of organisms with the exception of Euryarchaeota.

  9. [Studies on virulence of HIV and development of non-virulent live AIDS vaccine using monkeys].

    Science.gov (United States)

    Hayami, Masanori; Horiuchi, Reii

    2004-06-01

    A great effort for developing AIDS vaccine has been carried out in the world, designed by various new ideas based on basic research information obtained in recent virology and immunology. Withall it, to obtain effective AIDS vaccine is considered skeptical. One of the reasons of its difficulty is a lack of experimental animals susceptible to HIV-1. In our laboratory, we have succeeded in developing chimeric SIV having 3' half of HIV-1 genome including env (SHIV), which is infectious to macaque monkeys. One of SHIVs has been proved nonpathogenic in monkeys from various aspects and it afforded protective immunity to monkeys against pathogenic SHIV challenge infection. Now, we are trying to develop anti-HIV live attenuated vaccines using the nonpathogenic SHIV as a starting material. In the history of virus vaccine, live attenuated vaccines have been proved most effective in measles and polio-myelitis. However, it is not clear whether nonpathogenic HIV exists or not. Futhermore, even if nonpathogenic HIV could be obtained, there is possibility that it will easily mutate to pathogenic one. Therefore, to develop live attenuated AIDS vaccine is considered dangerous. In this article, We will introduce our research on SHIV pathogenicity using monkeys and hypothesize possibility to obtain nonpathogenic HIV which is speculated from the origin and evolution of HIV/SIV. To clarify virulence and nonvirulence of HIV and to obtain nonpathogenic virus are not only applied research but also basic science to dissolve the fundemental question why HIV can induce the disease.

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

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    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.

  11. Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    ZHANG TingTing; LI WanJie; LI Di; WANG Yue; SANG JianLi

    2008-01-01

    Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25△/△ mutants and investigated the role of the gene In morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25△/△ mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.

  12. Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25?/? mutants and investigated the role of the gene in morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25?/? mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.

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

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    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.

  14. The genomes of Marek’s disease virus exist as quasispecies at defined intervals during serial passage-induced attenuation

    Science.gov (United States)

    Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by gallid herpesvirus type 2 (GaHV-2). The disease is controlled through mass vaccination with live-attenuated strains. Attenuation of oncogenic GaHV-2 involves the serial passage of a virulent field isolate in avian embryo fib...

  15. Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

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    Anna C Llewellyn

    Full Text Available Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI, validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and

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

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    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.

  17. 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.

  18. First Streptococcus pyogenes signature-tagged mutagenesis screen identifies novel virulence determinants.

    Science.gov (United States)

    Kizy, Anne E; Neely, Melody N

    2009-05-01

    The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.

  19. Data in support of quantitative proteomics to identify potential virulence regulators in Paracoccidioides brasiliensis isolates

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    Alexandre Keiji Tashima

    2015-12-01

    Full Text Available Paracoccidioides genus are the etiologic agents of paracoccidioidomycosis (PCM, a systemic mycosis endemic in Latin America. Few virulence factors have been identified in these fungi. This paper describes support data from the quantitative proteomics of Paracoccidioides brasiliensis attenuated and virulent isolates [1]. The protein compositions of two isolates of the Pb18 strain showing distinct infection profiles were quantitatively assessed by stable isotopic dimethyl labeling and proteomic analysis. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD000804.

  20. Both msa genes in Renibacterium salmoninarum are needed for full virulence in bacterial kidney disease

    Science.gov (United States)

    Coady, A.M.; Murray, A.L.; Elliott, D.G.; Rhodes, L.D.

    2006-01-01

    Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile Chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression. Copyright ?? 2006, American Society for Microbiology. All Rights Reserved.

  1. Photonic Crystal Fiber Attenuator

    Institute of Scientific and Technical Information of China (English)

    Joo; Beom; Eom; Hokyung; Kim; Jinchae; Kim; Un-Chul; Paek; Byeong; Ha; Lee

    2003-01-01

    We propose a novel fiber attenuator based on photonic crystal fibers. The difference in the modal field diameters of a conventional single mode fiber and a photonic crystal fiber was used. A variable optical attenuator was also achieved by applying macro-bending on the PCF part of the proposed attenuator

  2. Tracer attenuation in groundwater

    Science.gov (United States)

    Cvetkovic, Vladimir

    2011-12-01

    The self-purifying capacity of aquifers strongly depends on the attenuation of waterborne contaminants, i.e., irreversible loss of contaminant mass on a given scale as a result of coupled transport and transformation processes. A general formulation of tracer attenuation in groundwater is presented. Basic sensitivities of attenuation to macrodispersion and retention are illustrated for a few typical retention mechanisms. Tracer recovery is suggested as an experimental proxy for attenuation. Unique experimental data of tracer recovery in crystalline rock compare favorably with the theoretical model that is based on diffusion-controlled retention. Non-Fickian hydrodynamic transport has potentially a large impact on field-scale attenuation of dissolved contaminants.

  3. 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.

  4. Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans.

    Science.gov (United States)

    Shah, Abdul Haseeb; Singh, Ashutosh; Dhamgaye, Sanjiveeni; Chauhan, Neeraj; Vandeputte, Patrick; Suneetha, Korivi Jyothiraj; Kaur, Rupinder; Mukherjee, Pranab K; Chandra, Jyotsna; Ghannoum, Mahmoud A; Sanglard, Dominique; Goswami, Shyamal K; Prasad, Rajendra

    2014-06-01

    The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.

  5. Vibrio vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide biosynthesis and virulence.

    Science.gov (United States)

    Valiente, Esmeralda; Jiménez, Natalia; Merino, Susana; Tomás, Juan M; Amaro, Carmen

    2008-04-01

    This work aimed to establish the role of gne (encoding UDP-GalNAc 4-epimerase activity) and galE (encoding UDP-Gal-4-epimerase activity) in the biosynthesis of surface polysaccharides, as well as in the virulence for eels and humans of the zoonotic serovar of Vibrio vulnificus biotype 2, serovar E. DNA sequence data revealed that gne and galE are quite homologous within this species (> or =90% homology). Mutation in gne of strain CECT4999 increased the surface hydrophobicity, produced deep alterations in the outer membrane architecture, and resulted in noticeable increases in the sensitivity to microcidal peptides (MP), to eel and human sera, and to phagocytosis/opsonophagocytosis. Furthermore, significant attenuation of virulence for eels and mice was observed. By contrast, mutation in galE did not alter the cellular surface, did not increase the sensitivity to MP, serum, or phagocytosis, and did not affect the virulence for fish and mice. The change in the attenuated-virulence phenotype produced by a mutation in gne was correlated with the loss of the O-antigen lipopolysaccharide (LPS), while the capsule was maintained. Complementation of a gne-deficient mutant restored the LPS structure together with the whole virulence phenotype. In conclusion, gne, but not galE, is essential for LPS biosynthesis and virulence in the zoonotic serovar of V. vulnificus biotype 2.

  6. Small molecule control of virulence gene expression in Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    James C Charity

    2009-10-01

    Full Text Available In Francisella tularensis, the SspA protein family members MglA and SspA form a complex that associates with RNA polymerase (RNAP to positively control the expression of virulence genes critical for the intramacrophage growth and survival of the organism. Although the association of the MglA-SspA complex with RNAP is evidently central to its role in controlling gene expression, the molecular details of how MglA and SspA exert their effects are not known. Here we show that in the live vaccine strain of F. tularensis (LVS, the MglA-SspA complex works in concert with a putative DNA-binding protein we have called PigR, together with the alarmone guanosine tetraphosphate (ppGpp, to regulate the expression of target genes. In particular, we present evidence that MglA, SspA, PigR and ppGpp regulate expression of the same set of genes, and show that mglA, sspA, pigR and ppGpp null mutants exhibit similar intramacrophage growth defects and are strongly attenuated for virulence in mice. We show further that PigR interacts directly with the MglA-SspA complex, suggesting that the central role of the MglA and SspA proteins in the control of virulence gene expression is to serve as a target for a transcription activator. Finally, we present evidence that ppGpp exerts its effects by promoting the interaction between PigR and the RNAP-associated MglA-SspA complex. Through its responsiveness to ppGpp, the contact between PigR and the MglA-SspA complex allows the integration of nutritional cues into the regulatory network governing virulence gene expression.

  7. Antagonistic competition moderates virulence in Bacillus thuringiensis.

    Science.gov (United States)

    Garbutt, Jennie; Bonsall, Michael B; Wright, Denis J; Raymond, Ben

    2011-08-01

    Classical models of the evolution of virulence predict that multiple infections should select for elevated virulence, if increased competitiveness arises from faster growth. However, diverse modes of parasite competition (resource-based, antagonism, immunity manipulation) can lead to adaptations with different implications for virulence. Using an experimental evolution approach we investigated the hypothesis that selection in mixed-strain infections will lead to increased antagonism that trades off against investment in virulence. Selection in mixed infections led to improved suppression of competitors in the bacterial insect pathogen Bacillus thuringiensis. Increased antagonism was associated with decreased virulence in three out of four selected lines. Moreover, mixed infections were less virulent than single-strain infections, and between-strain competition tended to decrease pathogen growth in vivo and in vitro. Spiteful interactions among these bacteria may be favoured because of the high metabolic costs of virulence factors and the high risk of mixed infections.

  8. Subinhibitory concentrations of phloretin repress the virulence of Salmonella typhimurium and protect against Salmonella typhimurium infection.

    Science.gov (United States)

    Shuai-Cheng, Wu; Ben-Dong, Fu; Xiu-Ling, Chu; Jian-Qing, Su; Yun-Xing, Fu; Zhen-Qiang, Cui; Dao-Xiu, Xu; Zong-Mei, Wu

    2016-11-01

    Phloretin, a natural component of many fruits, exhibits anti-virulence effects and provides a new alternative to counter bacterial infection. The aim of this study was to determine the effect of subinhibitory concentrations of phloretin on the virulence of Salmonella typhimurium. At concentrations where growth of Salmonella was not inhibited, phloretin significantly inhibited bacteria biofilm formation and motility. Subinhibitory concentrations of phloretin repressed eight genes involved in the Salmonella pathogenicity island 1 and 3 genes involved in flagella production. Furthermore, subinhibitory concentrations of phloretin inhibited the adhesion and invasion of Salmonella in IEC-6 cells and reduced the LDH levels of S. typhimurium-infected IEC-6 cells. Additionally, phloretin significantly decreased the cecum bacterial loads of the mice infected with live S. typhimurium containing subinhibitory concentrations of phloretin by gavage. These results suggested that subinhibitory concentrations of phloretin attenuate the virulence of S. typhimurium and protect against S. typhimurium infection.

  9. The evolution of tuberculosis virulence.

    Science.gov (United States)

    Basu, Sanjay; Galvani, Alison P

    2009-07-01

    The evolution of Mycobacterium tuberculosis presents several challenges for public health. HIV and resistance to antimycobacterial medications have evolutionary implications for how Mycobacterium tuberculosis will evolve, as these factors influence the host environment and transmission dynamics of tuberculosis strains. We present an evolutionary invasion analysis of tuberculosis that characterizes the direction of tuberculosis evolution in the context of different natural and human-driven selective pressures, including changes in tuberculosis treatment and HIV prevalence. We find that the evolution of tuberculosis virulence can be affected by treatment success rates, the relative transmissibility of emerging strains, the rate of reactivation from latency among hosts, and the life expectancy of hosts. We find that the virulence of tuberculosis strains may also increase as a consequence of rising HIV prevalence, requiring faster case detection strategies in areas where the epidemics of HIV and tuberculosis collide.

  10. Virulence Mechanisms of Enteroinvasive Pathogens

    Science.gov (United States)

    1988-01-01

    enteroinvasive strain to initiate infection, it does abortion and death can also result from inges- not address other determinants of virulence. For tion of... abortions in domestic imal models have been used in the study of animals. Humans are the natural reservoirs of enteroinvasive pathogens. The ability of...clinical manifestation rier encountered by shigellae which are invad- is dysentery (36). These data suggest that an ing the colonic mucosa. The effectiveness

  11. 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.

  12. Bacterial virulence, proinflammatory cytokines and host immunity: how to choose the appropriate Salmonella vaccine strain?

    Science.gov (United States)

    Raupach, B; Kaufmann, S H

    2001-01-01

    Salmonella infection in its mammalian host can be dissected into two main components. The co-ordinate expression of bacterial virulence genes which are designed to evade, subvert or circumvent the host response on the one hand, and the host defence mechanisms which are designed to restrict bacterial survival and replication on the other hand. The outcome of infection is determined by the one which succeeds in disturbing this equilibrium more efficiently. This delicate balance between Salmonella virulence and host immunity/inflammation has important implications for vaccine development or therapeutic intervention. Novel Salmonella vaccine candidates and live carriers for heterologous antigens are attenuated strains with defined genetic modifications of metabolic or virulence functions. Although genetic defects of different gene loci can lead to similar degrees of attenuation, effects on the course of infection may vary, thereby altering the quality of the elicited immune response. Studies with gene-deficient animals indicate that Salmonella typhimurium strains with mutations in aroA, phoP/phoQ or ssrA/ssrB invoke different immune responses and that a differential repertoire of pro-inflammatory cytokines is required for clearance. Consequently, Salmonella mutants defective in distinct virulence functions offer the potential to specifically modulate the immune response for defined medical applications.

  13. Helicobacter pylori virulence and cancer pathogenesis.

    Science.gov (United States)

    Yamaoka, Yoshio; Graham, David Y

    2014-06-01

    Helicobacter pylori is human gastric pathogen that causes chronic and progressive gastric mucosal inflammation and is responsible for the gastric inflammation-associated diseases, gastric cancer and peptic ulcer disease. Specific outcomes reflect the interplay between host-, environmental- and bacterial-specific factors. Progress in understanding putative virulence factors in disease pathogenesis has been limited and many false leads have consumed scarce resources. Few in vitro-in vivo correlations or translational applications have proved clinically relevant. Reported virulence factor-related outcomes reflect differences in relative risk of disease rather than specificity for any specific outcome. Studies of individual virulence factor associations have provided conflicting results. Since virulence factors are linked, studies of groups of putative virulence factors are needed to provide clinically useful information. Here, the authors discuss the progress made in understanding the role of H. pylori virulence factors CagA, vacuolating cytotoxin, OipA and DupA in disease pathogenesis and provide suggestions for future studies.

  14. Roles of RseB, sigmaE, and DegP in virulence and phase variation of colony morphotype of Vibrio vulnificus.

    Science.gov (United States)

    Brown, Roslyn N; Gulig, Paul A

    2009-09-01

    Vibrio vulnificus is an estuarine bacterium capable of causing serious and often fatal wound infections and primary septicemia. We used alkaline phosphatase insertion mutagenesis to identify genes necessary for the virulence of this pathogen. One mutant had an in-frame fusion of 'phoA to the gene encoding RseB, a periplasmic negative regulator of the alternative sigma factor sigma(E). sigma(E) controls an extensive regulon involved in responding to cell envelope stresses. Colonies of the rseB mutant were less opaque than wild-type colonies and underwent phase variation between translucent and opaque morphologies. rseB mutants were attenuated for virulence in subcutaneously inoculated iron-dextran-treated mice. To obtain insight into the role of rseB and the extracytoplasmic stress response in V. vulnificus, mutants with defined mutations in rseB and two important members of the extracytoplasmic stress regulon, rpoE and degP, were constructed for analysis of virulence, colony morphology, and stress-associated phenotypes. Deletion of rseB caused reversible phase variation in the colony morphotype that was associated with extracellular polysaccharides. Translucent and transparent morphotype strains were attenuated for virulence. rpoE and degP deletion mutants were sensitive to membrane-perturbing agents and heat but were not significantly attenuated for V. vulnificus virulence in mice. These results reveal complex relationships between regulation of the extracytoplasmic stress response, exopolysaccharides, and the virulence of V. vulnificus.

  15. Development of live attenuated sparfloxacin-resistant Streptococcus agalactiae polyvalent vaccines to protect Nile tilapia

    Science.gov (United States)

    To develop attenuated bacteria as potential live vaccines, sparfloxacin was used in this study to modify 40 isolates of Streptococcus agalactiae. Majority of S. agalactiae used in this study were able to develop at least 80-fold resistance to sparfloxacin. When the virulence of the sparfloxacin-resi...

  16. Complete genome sequence of an attenuated Sparfloxacin resistant Streptococcus agalactiae strain 138spar

    Science.gov (United States)

    Through selection of resistance to sparfloxacin, an attenuated Streptococcus agalactiae strain 138spar was obtained from its virulent parent strain S. agalactiae 138P. The full genome of S. agalactiae 138spar is 1,838,126 bp. The availability of this genome will allow comparative genomics to identi...

  17. Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin

    Science.gov (United States)

    To develop attenuated bacteria as potential live vaccines, sparfloxacin was used in this study to modify 40 isolates of Streptococcus agalactiae. Majority of S. agalactiae used in this study were able to develop at least 80-fold resistance to sparfloxacin. When the virulence of the sparfloxacin-resi...

  18. MspA-Mycobacterium tuberculosis-transformant with reduced virulence: the "unbirthday paradigm".

    Science.gov (United States)

    Lamrabet, Otmane; Ghigo, Eric; Mège, Jean-Louis; Lepidi, Hubert; Nappez, Claude; Raoult, Didier; Drancourt, Michel

    2014-11-01

    Expressing mspA porin gene from Mycobacterium smegmatis in Mycobacterium tuberculosis attenuated this pathogen. Intracellular growth of the transformants into free-living amoeba and murine and human macrophages decreased. Furthermore, transformants decreased the microbicidal program of human monocyte-derived macrophages. BALB/c mice inoculated with transformants exhibited higher weights, lower histological lesions and lower M. tuberculosis inoculum in the liver, spleen and lungs than control mice challenged with wild-type M. tuberculosis. Preliminary evaluation indicated that mice inoculated with this transformant showed higher weights and lower numbers of lung nodules and tissular mycobacteria than control mice when challenged with wild-type M. tuberculosis. Similar to the paradoxical "unbirthday" gift coined by Lewis Carroll in Alice's Adventures in Wonderland, adding mspA gene reduced the virulence of M. tuberculosis and yielded a protective effect. Lost of non-virulence genes is a mechanism for virulence in mycobacteria. Engineering non-virulence genes in M. tuberculosis may yield strains with decreased virulence and increased immunogenicity.

  19. Virulence of the fungal pathogen Candida albicans requires the five isoforms of protein mannosyltransferases.

    Science.gov (United States)

    Rouabhia, Mahmoud; Schaller, Martin; Corbucci, Cristina; Vecchiarelli, Anna; Prill, Stephan K-H; Giasson, Luc; Ernst, Joachim F

    2005-08-01

    The PMT gene family in Candida albicans encodes five isoforms of protein mannosyltransferases (Pmt proteins Pmt1p, Pmt2p, Pmt4p, Pmt5p, and Pmt6p) that initiate O mannosylation of secretory proteins. We compared virulence characteristics of pmt mutants in two complex, three-dimensional models of localized candidiasis, using reconstituted human epithelium (RHE) and engineered human oral mucosa (EHOM); in addition, mutants were tested in a mouse model of hematogenously disseminated candidiasis (HDC). All pmt mutants showed attenuated virulence in the HDC model and at least one model of localized candidiasis. The pmt5 mutant, which lacks in vitro growth phenotypes, was less virulent in the EHOM and HDC assays but had no consistent phenotype in the RHE assay. In contrast, the pmt4 and pmt6 mutants were less virulent in the RHE and HDC assays but not in the EHOM assay. The results stress the contribution of all Pmt isoforms to the virulence of C. albicans and suggest that the importance of individual Pmt isoforms may differ in specific host niches. We propose that Pmt proteins may be suitable targets for future novel classes of antifungal agents.

  20. A Haemophilus ducreyi CpxR deletion mutant is virulent in human volunteers.

    Science.gov (United States)

    Labandeira-Rey, Maria; Dodd, Dana; Fortney, Kate R; Zwickl, Beth; Katz, Barry P; Janowicz, Diane M; Spinola, Stanley M; Hansen, Eric J

    2011-06-15

    Haemophilus ducreyi 35000HP contains a homolog of the CpxRA 2-component signal transduction system, which controls the cell envelope stress response system in other gram-negative bacteria and regulates some important H. ducreyi virulence factors. A H. ducreyi cpxR mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule formation rate in 5 volunteers was 33% (95% confidence interval [CI], 1.3%-65.3%) at 15 parent sites and 40% (95% CI, 18.1%-61.9%) at 15 mutant sites (P = .35). Thus, the cpxR mutant was not attenuated for virulence. Inactivation of the H. ducreyi cpxR gene did not reduce the ability of this mutant to express certain proven virulence factors, including the DsrA serum resistance protein and the LspA2 protein, which inhibits phagocytosis. These results expand our understanding of the involvement of the CpxRA system in regulating virulence expression in H. ducreyi.

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

    Directory of Open Access Journals (Sweden)

    Eduardo Lopez-Medina

    2015-08-01

    Full Text Available 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.

  2. Molecular basis of live-attenuated influenza virus.

    Directory of Open Access Journals (Sweden)

    Wen He

    Full Text Available Human influenza is a seasonal disease associated with significant morbidity and mortality. The most effective means for controlling infection and thereby reducing morbidity and mortality is vaccination with a three inactivated influenza virus strains mixture, or by intranasal administration of a group of three different live attenuated influenza vaccine strains. Comparing to the inactivated vaccine, the attenuated live viruses allow better elicitation of a long-lasting and broader immune (humoral and cellular response that represents a naturally occurring transient infection. The cold-adapted (ca influenza A/AA/6/60 (H2N2 (AA ca virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. Similarly, the influenza A components of live-attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17 and A/Leningrad/134/47/57-ca (Len/47 along with virulent epidemic strains. However, the mechanism of temperature-sensitive attenuation is largely elusive. To understand how modification at genetic level of influenza virus would result in attenuation of human influenza virus A/PR/8/34 (H1N1,A/PR8, we investigated the involvement of key mutations in the PB1 and/or PB2 genes in attenuation of influenza virus in vitro and in vivo. We have demonstrated that a few of residues in PB1 and PB2 are critical for the phenotypes of live attenuated, temperature sensitive influenza viruses by minigenome assay and real-time PCR. The information of these mutation loci could be used for elucidation of mechanism of temperature-sensitive attenuation and as a new strategy for influenza vaccine development.

  3. 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.

  4. Development of allele-specific PCR for detection of herbicide-resistance in Monochoria korsakowii Regel et Maack%采用ASPCR技术检测抗药性雨久花ALS突变基因碱基种类的探讨

    Institute of Scientific and Technical Information of China (English)

    吴明根; 金万赫; 许勇男

    2011-01-01

    等位基因特异性扩增(Allele-specific PCR简称:ASPCR)技术是一项快速、简便、低成本的检测等位基因单核苷酸多态性的新方法.作者针对雨久花感抗磺酰脲类生态型ALS基因编码区第592位碱基密码(ALS氨基酸第197位)特征,采用在引物3'末端设有胸腺嘧啶核苷酸(感性)或鸟嘌呤(抗性)核苷酸的2种引物,对延边稻田发生的雨久花感抗磺酰脲类生态型ALS基因编码区第592位进行了ASPCR.检测结果显示,当以引物3'末端设有胸腺嘧啶引物时,感性类型出现ASPCR扩增带,而抗性类型无带出现,反之抗性类型有带而感性类型无带,检测到延边稻田发生的抗磺酰脲类雨久花生态型的ALS 197位点的脯氨酸被组氨酸替代,证明了该方法的有效性和可靠性.%Allele-specific PCR is a rapid, simple, and low-cost method for single-nucleotide polymorphism identification. Using this technique, a single-nucleotide polymorphism primer marker of thymine(susceptible) or guanine(resistant) at the 3'end on 592 point of ALS(acetolactate synthetase) gene encoding site (197 point of ALS amino acid) was designed to detect the herbicide-resistant type of Monochoria korsakowii Regel et Maack. in Yanbian region. The result shows that susceptibility primer marker can be used to detect the susceptibility biotype;otherwise can be detected resistant biotype; the cause of resistance to sulfonylurea herbicide type can be induced by substitution of Pro197 to histidine. It proved that the technique was efficient and feasible to detect ALS gene of herbicide-resistant type.

  5. The genome of a very virulent Marek's disease virus.

    Science.gov (United States)

    Tulman, E R; Afonso, C L; Lu, Z; Zsak, L; Rock, D L; Kutish, G F

    2000-09-01

    Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.

  6. 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.

  7. 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...

  8. 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.

  9. Effect of deletion of the lpxM gene on virulence and vaccine potential of Yersinia pestis in mice.

    Science.gov (United States)

    Anisimov, Andrey P; Shaikhutdinova, Rima Z; Pan'kina, Lyudmila N; Feodorova, Valentina A; Savostina, Elena P; Bystrova, Ol'ga V; Lindner, Buko; Mokrievich, Aleksandr N; Bakhteeva, Irina V; Titareva, Galina M; Dentovskaya, Svetlana V; Kocharova, Nina A; Senchenkova, Sof'ya N; Holst, Otto; Devdariani, Zurab L; Popov, Yuriy A; Pier, Gerald B; Knirel, Yuriy A

    2007-04-01

    Yersinia pestis undergoes an obligate flea-rodent-flea enzootic life cycle. The rapidly fatal properties of Y. pestis are responsible for the organism's sustained survival in natural plague foci. Lipopolysaccharide (LPS) plays several roles in Y. pestis pathogenesis, prominent among them being resistance to host immune effectors and induction of a septic-shock state during the terminal phases of infection. LPS is acylated with 4-6 fatty acids, the number varying with growth temperature and affecting the molecule's toxic properties. Y. pestis mutants were constructed with a deletion insertion in the lpxM gene in both virulent and attenuated strains, preventing the organisms from synthesizing the most toxic hexa-acylated lipid A molecule when grown at 25 degrees C. The virulence and/or protective potency of pathogenic and attenuated Y. pestis DeltalpxM mutants were then examined in a mouse model. The DeltalpxM mutation in a virulent strain led to no change in the LD(50) value compared to that of the parental strain, while the DeltalpxM mutation in attenuated strains led to a modest 2.5-16-fold reduction in virulence. LPS preparations containing fully hexa-acylated lipid A were ten times more toxic in actinomycin D-treated mice then preparations lacking this lipid A isoform, although this was not significant (P>0.05). The DeltalpxM mutation in vaccine strain EV caused a significant increase in its protective potency. These studies suggest there is little impact from lipid A modifications on the virulence of Y. pestis strains but there are potential improvements in the protective properties in attenuated vaccine strains.

  10. 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.

  11. 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. 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.

  12. Complete genome sequences of Brucella melitensis strains M28 and M5-90, with different virulence backgrounds.

    Science.gov (United States)

    Wang, Fangkun; Hu, Sen; Gao, Yuzhe; Qiao, Zujian; Liu, Wenxing; Bu, Zhigao

    2011-06-01

    Brucella melitensis is a Gram-negative coccobacillus bacteria belonging to the Alphaproteobacteria subclass. It is an important zoonotic pathogen that causes brucellosis, a disease affecting sheep, cattle, and sometimes humans. The B. melitensis strain M5-90, a live attenuated vaccine cultured from the B. melitensis virulent strain M28, has been an effective tool to control brucellosis in goats and sheep in China. Here we report the complete genome sequences of B. melitensis M28 and M5-90, strains with different virulence backgrounds, which will serve as a valuable reference for future studies.

  13. 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

    Directory of Open Access Journals (Sweden)

    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

  14. Proteomic Characterization of Yersinia pestis Virulence

    Energy Technology Data Exchange (ETDEWEB)

    Chromy, B; Murphy, G; Gonzales, A; Fitch, J P; McCutchen-Maloney, S L

    2005-01-05

    Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditions were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.

  15. 等位基因特异性引物延伸法在婴儿型和幼儿型神经元蜡样质脂褐质沉积病产前诊断中的应用%Prenatal diagnostic testing for infantile and late-infantile neuronal ceroid lipofusinoses (NCL) using allele specific primer extension (ASPE)

    Institute of Scientific and Technical Information of China (English)

    Nanbert ZHONG; Weina JU; Dorota MOROZIEWICZ; Anetta WRONSKA; Marilyn LI; Krystyna WISNIEWSKI; Susan Sklower BROOKS; Edmund JENKINS; W. Ted BROWN

    2005-01-01

    SUMMARY Infantile (INCL, NCL1) and late-infantile (LINCL, NCL2) neuronal ceroid lipofuscinoses have been found to result from genetic deficiency of genes CLN1 and CLN2, respectively. The application of molecular analyses can facilitate prenatal diagnosis for families affected by NCL1 or NCL2, in which the familial mutation(s) have been identified. Molecular testing with allele-specific primer extension and DNA sequencing was performed in nine pregnancies, four from two NCL1 families and five from five NCL2 families. Lysosomal enzyme activity assays were carried out as well.Four fetuses from three pregnancies in NCL1 families were found to be carriers for a mutation 451C-T in the CLN1 gene and one was normal. Prenatal testing of three NCL2 families who carried mutation R208X in the CLN2 gene showed that all fetuses were carriers. In NCL2 families who carried either mutation IVS5-1C or/and IVS5-1A two normal pregnancies were detected. Our studies indicate that DNA testing, which may provide definitive prenatal diagnosis for NCL, may be used in combination with lysosomal enzyme activity analyses.

  16. Genetically attenuated Trypanosoma cruzi parasites as a potential vaccination tool.

    Science.gov (United States)

    Pérez Brandan, Cecilia; Basombrío, Miguel Ángel

    2012-01-01

    Chagas disease is the clinical manifestation of the infection produced by the parasite Trypanosoma cruzi. Currently there is no vaccine to prevent this disease and the protection attained with vaccines containing non-replicating parasites is limited. Genetically attenuated trypanosomatid parasites can be obtained by deletion of selected genes. Gene deletion takes advantage of the fact that this parasite can undergo homologous recombination between endogenous and foreign DNA sequences artificially introduced in the cells. This approach facilitated the discovery of several unknown gene functions, as well as allowing us to speculate about the potential for genetically attenuated live organisms as experimental immunogens. Vaccination with live attenuated parasites has been used effectively in mice to reduce parasitemia and histological damage, and in dogs, to prevent vector-delivered infection in the field. However, the use of live parasites as immunogens is controversial due to the risk of reversion to a virulent phenotype. Herein, we present our results from experiments on genetic manipulation of two T. cruzi strains to produce parasites with impaired replication and infectivity, and using the mutation of the dhfr-ts gene as a safety device against reversion to virulence.

  17. Coordinated regulation of virulence during systemic infection of Salmonella enterica serovar Typhimurium.

    Directory of Open Access Journals (Sweden)

    Hyunjin Yoon

    2009-02-01

    Full Text Available To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice. Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2. These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded

  18. Virulent Salmonella enterica serovar typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells.

    Science.gov (United States)

    Tobar, Jaime A; Carreño, Leandro J; Bueno, Susan M; González, Pablo A; Mora, Jorge E; Quezada, Sergio A; Kalergis, Alexis M

    2006-11-01

    Dendritic cells (DCs) constitute the link between innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) in bacteria and by presenting bacterial antigens to T cells. Recognition of PAMPs renders DCs as professional antigen-presenting cells able to prime naïve T cells and initiate adaptive immunity against bacteria. Therefore, interfering with DC function would promote bacterial survival and dissemination. Understanding the molecular mechanisms that have evolved in virulent bacteria to evade activation of adaptive immunity requires the characterization of virulence factors that interfere with DC function. Salmonella enterica serovar Typhimurium, the causative agent of typhoid-like disease in the mouse, can prevent antigen presentation to T cells by avoiding lysosomal degradation in DCs. Here, we show that this feature of virulent Salmonella applies in vivo to prevent activation of adaptive immunity. In addition, this attribute of virulent Salmonella requires functional expression of a type three secretion system (TTSS) and effector proteins encoded within the Salmonella pathogenicity island 2 (SPI-2). In contrast to wild-type virulent Salmonella, mutant strains carrying specific deletions of SPI-2 genes encoding TTSS components or effectors proteins are targeted to lysosomes and are no longer able to prevent DCs from activating T cells in vitro or in vivo. SPI-2 mutant strains are attenuated in vivo, showing reduced tissue colonization and enhanced T-cell activation, which confers protection against a challenge with wild-type virulent Salmonella. Our data suggest that impairment of DC function by the activity of SPI-2 gene products is crucial for Salmonella pathogenesis.

  19. Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages.

    Science.gov (United States)

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

    2011-06-01

    Salmonella enterica serovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors. Salmonella virulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ∼70 amino acids and have high sequence homology to each other (>85% identity). Salmonella lacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all three Salmonella gene-encoded type III secretion systems (T3SSs)-Salmonella pathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novel Salmonella virulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.

  20. Kin selection and the evolution of virulence.

    Science.gov (United States)

    Buckling, A; Brockhurst, M A

    2008-05-01

    Social interactions between conspecific parasites are partly dependent on the relatedness of interacting parasites (kin selection), which, in turn, is predicted to affect the extent of damage they cause their hosts (virulence). High relatedness is generally assumed to favour less competitive interactions, but the relationship between relatedness and virulence is crucially dependent on the social behaviour in question. Here, we discuss the rather limited body of experimental work that addresses how kin-selected social behaviours affect virulence. First, if prudent use of host resources (a form of cooperation) maximizes the transmission success of the parasite population, decreased relatedness is predicted to result in increased host exploitation and virulence. Experimental support for this well-established theoretical result is surprisingly limited. Second, if parasite within-host growth rate is a positive function of cooperation (that is, when individuals need to donate public goods, such as extracellular enzymes), virulence is predicted to increase with increasing relatedness. The limited studies testing this hypothesis are broadly consistent with this prediction. Finally, there is some empirical evidence supporting theory that suggests that spiteful behaviours are maximized at intermediate degrees of relatedness, which, in turn, leads to minimal virulence because of the reduced growth rate of the infecting population. We highlight the need for further thorough experimentation on the role of kin selection in the evolution of virulence and identify additional biological complexities to these simple frameworks.

  1. Planetary Ices Attenuation Properties

    Science.gov (United States)

    McCarthy, Christine; Castillo-Rogez, Julie C.

    In this chapter, we review the topic of energy dissipation in the context of icy satellites experiencing tidal forcing. We describe the physics of mechanical dissipation, also known as attenuation, in polycrystalline ice and discuss the history of laboratory methods used to measure and understand it. Because many factors - such as microstructure, composition and defect state - can influence rheological behavior, we review what is known about the mechanisms responsible for attenuation in ice and what can be inferred from the properties of rocks, metals and ceramics. Since attenuation measured in the laboratory must be carefully scaled to geologic time and to planetary conditions in order to provide realistic extrapolation, we discuss various mechanical models that have been used, with varying degrees of success, to describe attenuation as a function of forcing frequency and temperature. We review the literature in which these models have been used to describe dissipation in the moons of Jupiter and Saturn. Finally, we address gaps in our present knowledge of planetary ice attenuation and provide suggestions for future inquiry.

  2. 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.

  3. Research progress in live attenuated Brucella vaccine development.

    Science.gov (United States)

    Wang, Zhen; Wu, Qingmin

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause brucellosis, which is a globally occurring zoonotic disease that is characterized by abortion in domestic animals and undulant fever, arthritis, endocarditis, and meningitis in humans. There are currently no licensed vaccines against brucellosis for human use, and only a few licensed live Brucella vaccines are available for use in animals. However, the available animal vaccines may cause abortion and are associated with lower protection rates in animals and higher virulence in humans. Much research has been performed recently to develop novel Brucella vaccines for the prevention and control of animal brucellosis. This article discusses the approaches and strategies for novel live attenuated vaccine development.

  4. Frequency Dependent Attenuation Revisited

    CERN Document Server

    Richard, Kowar; Xavier, Bonnefond

    2009-01-01

    The work is inspired by thermo-and photoacoustic imaging, where recent efforts are devoted to take into account attenuation and varying wave speed parameters. In this paper we study causal equations describing propagation of attenuated pressure waves. We review standard models like frequency power laws and and the thermo-viscous equation. The lack of causality of standard models in the parameter range relevant for photoacoustic imaging requires to derive novel equations. The main ingredients for deriving causal equations are the Kramers-Kronig relation and the mathematical concept of linear system theory. The theoretical results of this work are underpined by numerical experiments.

  5. Importance of two Enterococcus faecium loci encoding Gls-like proteins for in vitro bile salts stress response and virulence.

    Science.gov (United States)

    Choudhury, Tina; Singh, Kavindra V; Sillanpää, Jouko; Nallapareddy, Sreedhar R; Murray, Barbara E

    2011-04-15

    General stress proteins, Gls24 and GlsB, were previously shown to be involved in bile salts resistance of Enterococcus faecalis and in virulence. Here, we identified 2 gene clusters in Enterococcus faecium each encoding a homolog of Gls24 (Gls33 and Gls20; designated on the basis of their predicted sizes) and of GlsB (GlsB and GlsB1). The sequences of the gls33 and gls20 gene clusters from available genomes indicate distinct lineages, with those of hospital-associated CC17 isolates differing from non-CC17 by ∼7% and ∼3.5%, respectively. Deletion of an individual locus did not have a significant effect on virulence in a mouse peritonitis model, whereas a double-deletion mutant was highly attenuated (Pimportant for adaptation to the intestinal environment, in addition to being important for virulence functions.

  6. BCG vaccines: their mechanisms of attenuation and impact on safety and protective efficacy.

    Science.gov (United States)

    Liu, Jun; Tran, Vanessa; Leung, Andrea S; Alexander, David C; Zhu, Baoli

    2009-02-01

    Mycobacterium bovis Bacille Calmette-Guérin (BCG) was developed as an attenuated live vaccine for tuberculosis control nearly a century ago. Despite being the most widely used vaccine in human history, the mechanisms of attenuation of BCG remain poorly understood. BCG is not a single organism, but comprises a number of substrains that differ in genotypes and phenotypes. The impacts of these differences on BCG vaccine properties are largely unknown. Nevertheless, in the past decade, the development of sophisticated genome analysis techniques, coupled with advances in knowledge of the virulence mechanisms of Mycobacterium tuberculosis, have provided greater insights into the attenuation and evolution of BCG. This review article discusses these new developments, focusing on molecular mechanisms that contribute to the attenuation of BCG substrains. It is evident that BCG strains comprise natural mutants of major virulence factors of M. tb, including ESX-1, PDIM/PGL and PhoP, and that BCG substrains differ markedly in virulence level. The impacts of these findings on vaccine properties including adverse reaction effect, tuberculin reactivity and protective efficacy are discussed. These new insights have extremely important implications for national immunization programs and the development of future vaccines.

  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. 莴苣叶斑病菌的分离鉴定及弱毒菌株致病力的衰退%Isolation of the pathogens causing leaf spot of lettuce and analysis of the reason causing the attenuated virulence

    Institute of Scientific and Technical Information of China (English)

    潘显婷; 陆训; 庞茜丹; 高必达; 周倩

    2016-01-01

    Fourstrains were isolated from lettuce with leaf spot disease by tissue isolation method and identified as Stemphyliumspp. with ITS sequences analysis. An attenuated strain with debilitated pathogenicity and slower growth rate named WS–01 was found by artifical inoculation. The cellulose CF11 method was used to extract dsRNA to determine the relation between the debilitated pathogenicity and its dsRNA mycovirus, three dsRNAs ranged from 1 kb to 5 kb were detected in the WS–01 strain. High temperature-ribavirin-hyphal tip isolation method for elimination of virus was conducted. The results showed that the pathogenicity of mycovirus-eliminated strain WS–01–D was enhanced and the phenotype returned to normal compared to the original strain, indicating the abnormal phenotype of strain WS–01 was related to its dsRNA.%利用组织分离法,从感染叶斑病的莴苣叶片上分离得到4株真菌,ITS序列分析鉴定为匍柄霉(Stemphylium spp.)。科赫氏法则验证其致病性时发现,菌株 WS–01较其他菌株致病力减弱,且生长缓慢。用纤维素吸附法提取菌株WS–01的dsRNA,发现WS–01含有3条1~5 kb的dsRNA条带,提示WS–01菌株内含有dsRNA病毒。利用高温–利巴韦林–尖端脱毒结合的方法消除WS–01内的病毒,所得菌株WS–01–D与菌株WS–01相比,致病性增强且表型恢复正常,提示弱毒菌株WS–01的致病力减弱与其所含dsRNA有关。

  9. Tissue tropism in the chicken embryo of non-virulent and virulent Newcastle diseases strains that express green fluorescence protein

    NARCIS (Netherlands)

    Al-Garib, S.O.; Gielkens, A.L.J.; Gruys, E.; Peeters, B.P.H.; Koch, G.

    2003-01-01

    The tissue tropism of non-virulent and virulent Newcastle disease virus (NDV) was investigated using 8-day-old and 14-day-old embryonating chicken eggs (ECE), inoculated with an infectious clone of the non-virulent La Sota strain (NDFL-GFP) or its virulent derivative (NDFLtag-GFP). Both strains expr

  10. Use of signature-tagged mutagenesis to identify virulence determinants in Haemophilus ducreyi responsible for ulcer formation.

    Science.gov (United States)

    Yeung, Angela; Cameron, D William; Desjardins, Marc; Lee, B Craig

    2011-02-01

    Elucidating the molecular mechanisms responsible for chancroid, a genital ulcer disease caused by Haemophilus ducreyi, has been hampered in part by the relative genetic intractability of the organism. A whole genome screen using signature-tagged mutagenesis in the temperature-dependent rabbit model (TDRM) of H. ducreyi infection uncovered 26 mutants with a presumptive attenuated phenotype. Insertions in two previously recognized virulence determinants, hgbA and lspA1, validated this genome scanning technique. Database interrogation allowed assignment of 24 mutants to several functional classes, including transport, metabolism, DNA repair, stress response and gene regulation. The attenuated virulence for a 3 strain with a mutation in hicB was confirmed by individual infection in the TDRM. The results from this preliminary study indicate that this high throughput strategy will further the understanding of the pathogenesis of H. ducreyi infection.

  11. 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.

  12. Analysis of the virulence-associated RevSR two-component signal transduction system of Clostridium perfringens.

    Science.gov (United States)

    Cheung, Jackie K; Wisniewski, Jessica A; Adams, Vicki M; Quinsey, Noelene S; Rood, Julian I

    2016-09-01

    Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes human gas gangrene (clostridial myonecrosis) and food poisoning. Early studies showed that virulence was regulated by the VirSR two-component signal transduction system. However, our identification of the RevR orphan response regulator indicated that more than one system was involved in controlling virulence. To further characterize this virulence-associated regulator, gel mobility shift experiments, coupled with DNase I footprinting, were used to identify the RevR DNA binding sequence. Bioinformatics analysis suggested that an orphan sensor histidine kinase, CPE1757 (renamed RevS), was the cognate sensor of RevR. Interaction between RevS and RevR was demonstrated by use of a bacterial two-hybrid system and validated by protein-protein interaction studies using biolayer interferometry. To assess the involvement of RevS in virulence regulation, the revS gene was inactivated by Targetron insertion. When isogenic wild-type, revS and complemented revS strains were tested in a mouse myonecrosis model, the revS mutant was found to be attenuated in virulence, which was similar to the attenuation observed previously with the revR mutant. However, transcriptional analysis of selected RevR-regulated genes in the revS mutant revealed a different pattern of expression to a revR mutant, suggesting that the RevSR system is more complex than originally thought. Taken together, the results have led to the identification and characterization of the two essential parts of a new regulatory network that is involved in the regulation of virulence in C. perfringens.

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

    Science.gov (United States)

    Kellner, Ronny; Hanschke, Christian; Begerow, Dominik

    2014-01-01

    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.

  14. 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.

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

    Science.gov (United States)

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

    2005-05-10

    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.

  16. Evaluating the virulence of a Brucella melitensis hemagglutinin gene in the caprine model.

    Science.gov (United States)

    Perry, Quinesha L; Hagius, Sue D; Walker, Joel V; Elzer, Philip H

    2010-10-01

    With the completion of the genomic sequence of Brucella melitensis 16M, a putative hemagglutinin gene was identified which is present in 16M and absent in Brucella abortus. The possibility of this hemagglutinin being a potential virulence factor was evaluated via gene replacement in B. melitensis yielding 16MΔE and expression in trans in B. abortus 2308-QAE. Utilizing the caprine brucellosis model, colonization and pathogenesis studies were performed to evaluate these strains. B. melitensis 16M hemagglutinin gene expression in trans in 2308-QAE revealed a significant (p≤0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking B. melitensis 16M virulence in pregnant goats. The B. melitensis disruption mutant's colonization and abortion rates demonstrated no attenuation in colonization but displayed a 28% reduction in abortions when compared to parental B. melitensis 16M.

  17. Effect of MarA-like proteins on antibiotic resistance and virulence in Yersinia pestis.

    Science.gov (United States)

    Lister, Ida M; Mecsas, Joan; Levy, Stuart B

    2010-01-01

    MarA, an AraC/XylS transcriptional regulator in Escherichia coli, affects drug susceptibility and virulence. Two MarA-like proteins have been found in Yersinia pestis: MarA47 and MarA48. Deletion or overexpression of these proteins in the attenuated KIM 1001 Deltapgm strain led to a change in multidrug susceptibility (including susceptibility to clinically relevant drugs). Additionally, lung colonization by the marA47 or marA48 deletion mutant was decreased about 10-fold in a pneumonic plague mouse model. Complementation of the deletions by replacing the deleted genes on the chromosome restored wild-type characteristics. These findings show that two MarA homologs in Y. pestis affect antibiotic susceptibility and virulence.

  18. 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.

  19. 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.

  20. Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains

    Science.gov (United States)

    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 still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR) and non-virulent (NVIR) phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains. Staphopain C (StpC) was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC, and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model. PMID:27242969

  1. Identification of secreted exoproteome fingerprints of highly-virulent and non-virulent Staphylococcus aureus strains.

    Directory of Open Access Journals (Sweden)

    Emilia eBonar

    2016-05-01

    Full Text Available 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 still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR and non-virulent (NVIR phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains.Staphopain C (StpC was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model.

  2. 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.

  3. CodY orchestrates the expression of virulence determinants in emetic Bacillus cereus by impacting key regulatory circuits.

    Science.gov (United States)

    Frenzel, Elrike; Doll, Viktoria; Pauthner, Matthias; Lücking, Genia; Scherer, Siegfried; Ehling-Schulz, Monika

    2012-07-01

    Bacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcR-dependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.

  4. The CtsR regulator controls the expression of clpC, clpE and clpP and is required for the virulence of Enterococcus faecalis in an invertebrate model.

    Science.gov (United States)

    Cassenego, Ana Paula Vaz; de Oliveira, Naira Elane Moreira; Laport, Marinella Silva; Abranches, Jaqueline; Lemos, José A; Giambiagi-deMarval, Marcia

    2016-09-01

    The intrinsic ruggedness of Enterococcus faecalis is responsible for its widespread distribution in nature and is often viewed as an important virulence determinant. Previously, we showed that the ClpB ATPase is negatively regulated by CtsR and is required for thermotolerance and virulence in a Galleria mellonella invertebrate model. Here, we used in silico, Northern blot and quantitative real-time PCR analyses to identify additional members of the CtsR regulon, namely the clpP peptidase and the clpC and clpE ATPases. When compared to the parent strain, virulence of the ΔctsR strain in G. mellonella was significantly attenuated.

  5. Characterization of an attenuated TE3L-deficient vaccinia virus Tian Tan strain.

    Science.gov (United States)

    Wang, Yuhang; Kan, Shifu; Du, Shouwen; Qi, Yanxin; Wang, Jinhui; Liu, Liming; Ji, Huifan; He, Dongyun; Wu, Na; Li, Chang; Chi, Baorong; Li, Xiao; Jin, Ningyi

    2012-12-01

    An attenuated vaccinia virus (VACV), TE3L(-)VTT, was evaluated for virulence and safety to determine its potential use as a vaccine or as a recombinant virus vector to express foreign genes. The virulence of TE3L(-)VTT was compared with that of the wild-type VTT both in vivo and in vitro. The humoral and cellular immune responses were detected in a mouse model to test the vaccine efficacy of the TE3L mutant. The results suggested that deletion of the TE3L gene decreased the virulence and neurovirulence significantly in mice and rabbit models, yet retained the immunogenicity. Thus, the deletion of TE3L improved the safety of the VTT vector; this approach may yield a valuable resource for studies of recombinant VACV-vectored vaccines.

  6. Increased virulence of Mycobacterium tuberculosis H37Rv overexpressing LipY in a murine model.

    Science.gov (United States)

    Singh, Vipul K; Srivastava, Mrigank; Dasgupta, Arunava; Singh, Mohan P; Srivastava, Ranjana; Srivastava, Brahm S

    2014-05-01

    We have investigated the role of Rv3097c-encoded lipase (LipY) on the virulence of Mycobacterium tuberculosis. It has been shown that the overexpression of LipY in strain H37Rv induced increase in virulence of recombinant H37Rv::LipY strain. Compared to H37Rv, infection with H37Rv::LipY caused enhanced mortality, weight loss, bacterial load in lungs, splenomegaly, worsening lung morphology and pathology. Mice immunized with recombinant LipY antigen were protected against challenge with H37Rv::LipY, which correlated with enhanced survival of challenged mice and striking decrease in pathological features observed in unimmunized mice. To probe the cause of increase in virulence of H37Rv::LipY, the immune status of the host infected with H37Rv and H37Rv::LipY was compared. It was found that overexpression of LipY compromised immune responses resulting in attenuation of Th1 and Th17 responses, significant increase in IL-10, decrease in number of macrophages and T cells, and increase in numbers of Treg, and DCs in the lungs whereas in mice immunized with LipY an increased pool of T cells and DCs was observed. This led us to conclude that the increase in the virulence of H37Rv::LipY was due to downregulation of the host's protective immunity and the Rv3097c encoded LipY lipase is a virulence factor of M. tuberculosis.

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

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    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.

  8. RovM, a novel LysR-type regulator of the virulence activator gene rovA, controls cell invasion, virulence and motility of Yersinia pseudotuberculosis.

    Science.gov (United States)

    Heroven, Ann Kathrin; Dersch, Petra

    2006-12-01

    RovA is a MarR-type transcriptional regulator that controls transcription of rovA, the expression of the primary invasive factor invasin and other virulence genes of Yersinia pseudotuberculosis in response to environmental signals. Using a genetic approach to identify regulatory components that negatively influence rovA expression, we identified a new LysR-type regulatory protein, designated RovM, which exhibits homology to the virulence regulator PecT/HexA of plant pathogenic Erwinia species. DNA-binding studies revealed that RovM interacts specifically with a short binding site between promoters P1 and P2 within the rovA regulatory region and negatively modulates rovA transcription in cooperation with the histone-like protein H-NS. The rovM gene itself is under positive autoregulatory control and is significantly induced during growth in minimal media as shown in regulation studies. Disruption of the rovM gene leads to a significant increase of RovA and invasin synthesis and enhances internalization of Y. pseudotuberculosis into host cells. Finally, we show that a Y. pseudotuberculosis rovM mutant is more virulent than wild type and higher numbers of the bacteria are detectable in gut-associated lymphatic tissues and organs in the mouse infection model system. In contrast, elevated levels of the RovM protein, which exert a positive effect on flagellar motility, severely attenuate the ability of Y. pseudotuberculosis to disseminate to deeper tissues. Together, our data show, that RovM is a key regulator implicated in the environmental control of virulence factors, which are crucial for the initiation of a Yersinia infection.

  9. Comparison of CYP2D6 genotyping by allele-specific PCR with DXT phe-notype and gene chip testing%CYP2D6 PCR基因型与DXT表型和基因芯片检测的比较

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    目的:为了评价CYP2D6的基因型和表型的联系以及基因芯片在CYP2D6多基因分析中的应用.方法:242健康志愿者,口服dextromethorphan后收集尿液测定其代谢率,收集20ml血提取DNA,并通过基因特异性PCR和/(或)基因芯片分析CYP2D6*2--*11,*17和多拷贝CYP2D6基因,其中5个基因(*3、*4、*6、*7和*9)用PCR和CYD450基因芯片同时分析.结果:CYP2D6基因型比表型更富有信息和更能反映CYP2D6酶的表达.CYP2D6*3、*4、*6、*7和*9的基因检测在CYP450基因芯片和基因特异性PCR中显示高度的一致性.结论:基因芯片在检测基因多位点的多基因中是一个有发展前途和可靠的方法.%To evaluate association of genotype and phenotype of CYP2D6 and the application of oligonucleotide microarray hybridization genetic testing in CYP2D6 multiple alleles analyses. METHODS: Two hundred forty-two healthy volunteers were recruited, and a 60 mg oral dose of dextromethorphan (DXT) was administered to each for assessment of the DXT metabolic ratio [ MR]. A 20 ml blood sample was also collected for DNA isolation and testing. CYP2D6 alleles * 2-*11; * 17 and multiple CYP2D6 gene copies were tested by allele-specific PCR and/or the affymetrix CYP450 gene chip assay. Five of the CYP2D6 alleles ( * 3, * 4, *6, * 7, and * 9) were evaluated by both PCR and the CYP450 gene chip assay. RESULTS: The CYP2D6genotype was more informative and reflective in CYP2D6 enzyme expression than a phenotype. Genetic tests for the CYP2D6 * 3, * 4, * 6, * 7 and * 9 alleles showed a high degree of concordance between the CYP450 gene chip and AS-PCR methods. CONCLUSION: Oligonucleotide microarray hybridization is a promising and reliable approach for detecting multiple alleles at gene loci.

  10. Safety of a Live Attenuated Infectious Bovine Rhinotracheitis Vaccine IBRV LNM Strain

    Institute of Scientific and Technical Information of China (English)

    Guo; Li; Wang; Wei; Zhang; Shuqin; Cheng; Shipeng; Wu; Hua

    2014-01-01

    The paper was to evaluate the vaccine safety,and to prevent public health risk due to virus spread,the approach vaccination of was adopted in this research; and neck intramuscular injection of IBRV LNM attenuated vaccine strain was carried out. Blind passage for three generations in animal has tested the reversion risk to virulence. A total of 14 healthy and weaning cows at 6- 8 month old were divided into three groups. The 1st reversion of virulence trials used 105. 0TCID50/mL neck intramuscular injection of IBRV LNM attenuated vaccine strain. Then,the nose swab samples were collected for continuous 14 days. After passed through 0. 45 μm filter membrane,nasal swabs mixture was prepared as the virulence test inoculum for next generation. The body temperature was detected and clinical observation was carried out for continuous 14 days after inoculation. The inoculation dose was 1ml / cattle. Blood was collected on the 0 and 14 thdays of animal vaccination. After serum isolation,it was used for the antibody detection of serum. Research results showed that no virus was isolated from the nasal swabs from the F2 generation; vaccinated animals did not show any clinical signs of IBR; serological testing of IBRV antibody was negative,which indicated that the strain-inoculated animals did had reversion of virulence in all three generations.

  11. Ultrasonic attenuation in pearlitic steel.

    Science.gov (United States)

    Du, Hualong; Turner, Joseph A

    2014-03-01

    Expressions for the attenuation coefficients of longitudinal and transverse ultrasonic waves are developed for steel with pearlitic microstructure. This type of lamellar duplex microstructure influences attenuation because of the lamellar spacing. In addition, longitudinal attenuation measurements were conducted using an unfocused transducer with 10 MHz central frequency on the cross section of a quenched railroad wheel sample. The dependence of longitudinal attenuation on the pearlite microstructure is observed from the changes of longitudinal attenuation from the quenched tread surface to deeper locations. The results show that the attenuation value is lowest and relatively constant within the quench depth, then increases linearly. The experimental results demonstrate a reasonable agreement with results from the theoretical model. Ultrasonic attenuation provides an important non-destructive method to evaluate duplex microstructure within grains which can be implemented for quality control in conjunction with other manufacturing processes.

  12. 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.

  13. 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.

  14. 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.

  15. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Science.gov (United States)

    Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

    2015-01-01

    Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were

  16. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  17. 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.

  18. Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models.

    Science.gov (United States)

    Gácser, Attila; Schäfer, Wilhelm; Nosanchuk, Jerome S; Salomon, Siegfried; Nosanchuk, Joshua D

    2007-12-01

    Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.

  19. HtrA Is Important for Stress Resistance and Virulence in Haemophilus parasuis.

    Science.gov (United States)

    Zhang, Luhua; Li, Ying; Wen, Yiping; Lau, Gee W; Huang, Xiaobo; Wu, Rui; Yan, Qigui; Huang, Yong; Zhao, Qin; Ma, Xiaoping; Wen, Xintian; Cao, Sanjie

    2016-08-01

    Haemophilus parasuis is an opportunistic pathogen that causes Glässer's disease in swine, with polyserositis, meningitis, and arthritis. The high-temperature requirement A (HtrA)-like protease, which is involved in protein quality control, has been reported to be a virulence factor in many pathogens. In this study, we showed that HtrA of H. parasuis (HpHtrA) exhibited both chaperone and protease activities. Finally, nickel import ATP-binding protein (NikE), periplasmic dipeptide transport protein (DppA), and outer membrane protein A (OmpA) were identified as proteolytic substrates for HpHtrA. The protease activity reached its maximum at 40°C in a time-dependent manner. Disruption of the htrA gene from strain SC1401 affected tolerance to temperature stress and resistance to complement-mediated killing. Furthermore, increased autoagglutination and biofilm formation were detected in the htrA mutant. In addition, the htrA mutant was significantly attenuated in virulence in the murine model of infection. Together, these data demonstrate that HpHtrA plays an important role in the virulence of H. parasuis.

  20. DNA methylation modulates Salmonella enterica serovar Typhimurium virulence in Caenorhabditis elegans.

    Science.gov (United States)

    Oza, Javin P; Yeh, Jimmy B; Reich, Norbert O

    2005-04-01

    Salmonella enterica serovar Typhimurium was previously shown to be virulent in Caenorhabditis elegans. Here we demonstrate that DNA adenine methyltransferase (DAM) modulates Salmonella virulence in the nematode, as it does in mice. After 5 days of continual exposure to bacteria, twice as many worms died when exposed to the wild-type than the dam-mutant strain of Salmonella. Similar trends in virulence were observed when worms were exposed to Salmonella strains for 5 h and transferred to the avirulent Escherichia coli OP50. While a 10-fold attenuation was observed in the absence of DAM, the dam-strain was still able to infect and persist in the host worm. Our results further support the use of C. elegans as an accessible and readily studied animal model of bacterial pathogenesis. However, our results suggest that crucial host responses differ between the murine and nematode models. Additionally, we carried out preliminary liquid culture based experiments with the long term goal of developing high throughput animal based screens of DAM inhibitors.

  1. 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 path......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...... our laboratory and others now suggests that EF-P, previously thought to be essential, instead plays an ancillary role in translation by regulating the synthesis of a relatively limited subset of proteins. Other observations suggest that the eukaryotic homolog of EF-P, eIF5A, may illicit similar...

  2. Construction and Virulence of Filamentous Hemagglutinin Protein B1 Mutant of Pasteurella multocida in Chickens

    Institute of Scientific and Technical Information of China (English)

    GUO Dong-chun; QU Lian-dong; SUN Yan; ZHANG Ai-qin; LIU Jia-sen; LU Yan; LIU Pei-xin; YUAN Dong-wei; JIANG Qian; SI Chang-de

    2014-01-01

    Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowl cholera, bovine hemorrhagic septicemia, enzoonotic pneumonia and swine atropic rhinitis. Two iflamentous hemagglutinin genes, fhaB1 and fhaB2, are the potential virulence factors. In this study, an inactivation fhaB1 mutant of P. multocida in avian strain C48-102 was constructed by a kanamycin-resistance cassette. The virulence of the fhaB1 mutant and the wild type strain was assessed in chickens by intranasal and intramuscular challenge. The inactivation of fhaB1 resulted in a high degree of attenuation when the chickens were challenged intranasally and a lesser degree when challenged intramuscularly. The fhaB1 mutant and the wild type strain were investigated their sensitivity to the antibody-dependent classical complement-mediated killing pathway in 90%convalescent chicken serum. The fhaB1 mutant was serum sensitive as the viability has reduced between untreated serum and heat inactivated chicken serum (P<0.007). These results conifrmed that FhaB1 played the critical roles in the bacterial pathogenesis and further studies were needed to investigate the mechanism which caused reduced virulence of the fhaB1 mutant.

  3. Evaluation of an attenuated strain of Ehrlichia canis as a vaccine for canine monocytic ehrlichiosis.

    Science.gov (United States)

    Rudoler, Nir; Baneth, Gad; Eyal, Osnat; van Straten, Michael; Harrus, Shimon

    2012-12-17

    Canine monocytic ehrlichiosis is an important tick-borne disease worldwide. No commercial vaccine for the disease is currently available and tick control is the main preventive measure against the disease. The aim of this study was to evaluate the potential of a multi-passaged attenuated strain of Ehrlichia canis to serve as a vaccine for canine monocytic ehrlichiosis, and to assess the use of azithromycin in the treatment of acute ehrlichiosis. Twelve beagle dogs were divided into 3 groups of 4 dogs. Groups 1 and 2 were inoculated (vaccinated) with an attenuated strain of E. canis (#611A) twice or once, respectively. The third group consisted of naïve dogs which served as controls. All 3 groups were challenged with a wild virulent strain of E. canis by administering infected dog-blood intravenously. Transient thrombocytopenia was the only hematological abnormality observed following inoculation of dogs with the attenuated strain. Challenge with the virulent strain resulted in severe disease in all 4 control dogs while only 3 of 8 vaccinated dogs presented mild transient fever. Furthermore, the mean blood rickettsial load was significantly higher in the control group (27-92-folds higher during days 14-19 post challenge with the wild the strain) as compared to the vaccinated dogs. The use of azithromycin was assessed as a therapeutic agent for the acute disease. Four days treatment resulted in further deterioration of the clinical condition of the dogs. Molecular comparison of 4 genes known to express immunoreactive proteins and virulence factors (p30, gp19, VirB4 and VirB9) between the attenuated strain and the challenge wild strain revealed no genetic differences between the strains. The results of this study indicate that the attenuated E. canis strain may serve as an effective and secure future vaccine for canine ehrlichiosis.

  4. Virulent Aeromonas hydrophila in channel catfish

    Science.gov (United States)

    In this study, we investigated factors that predisposed catfish to motile aeromonas septicemia (MAS) caused by virulent Aeromonas hydrophila (vAh). Our results revealed that wounding on fish body surface was a prerequisite for vAh infection and disease development. A reproducible waterborne challeng...

  5. 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 i

  6. NEW VIRULENCE FACTORS OF STREPTOCOCCUS PNEUMONIAE.

    NARCIS (Netherlands)

    Bootsma, Jeanette Hester; Burghout, Pieter Jan; Hermans, Peter Wilhelmus Maria; Bijlsma, Johanna; Kuipers, Oscar; Kloosterman, Tomas Gerrit

    2012-01-01

    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 i

  7. Entamoeba histolytica: oxygen resistance and virulence.

    Science.gov (United States)

    Ramos-Martínez, Espiridión; Olivos-García, Alfonso; Saavedra, Emma; Nequiz, Mario; Sánchez, Ernesto C; Tello, Eusebio; El-Hafidi, Mohamed; Saralegui, Andrés; Pineda, Erika; Delgado, José; Montfort, Irmgard; Pérez-Tamayo, Ruy

    2009-05-01

    Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O(2). The only difference observed was a higher in vitro susceptibility of nvEh to O(2). The molecular mechanism of that difference was analyzed in both groups of amoebae after high O(2) exposure. vEh O(2) resistance correlated with: (i) higher O(2) reduction (O(2)(-) and H(2)O(2) production); (ii) increased H(2)O(2) resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O(2) exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O(2) and H(2)O(2) as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.

  8. Molecular nature of virulence in Entamoeba histolytica.

    Science.gov (United States)

    Olivos-García, Alfonso; Saavedra, Emma; Ramos-Martínez, Espiridión; Nequiz, Mario; Pérez-Tamayo, Ruy

    2009-12-01

    For many years virulence of pathogenic Entamoeba histolytica has been attributed to the capacity of the parasite to destroy tissues through the expression and/or secretion of various molecules. Such view is supported mainly by in vitro experimentation, whereas data obtained by using animal models of the disease have clearly demonstrated that the host's inflammatory response is primarily responsible for tissue damage. This review analyzes the content and/or activity of some of the presumed toxic amebic molecules present in amebic strains with different degrees of virulence compared to various parasite in vitro functions that are supposed to correlate with in vivo virulence. The analysis suggests that amebic virulence is primarily determined by the parasite's capacity to adapt and survive the aerobic conditions present in animal tissues. This initial episode in the host-parasite relationship is an absolute requirement for the further development of tissue lesions, which result from the concerted action of many molecules derived from both, the host and the parasite.

  9. 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.

  10. Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

    Institute of Scientific and Technical Information of China (English)

    ZHU Daling; LI Aihua; WANG Jianguo; LI Ming; CAI Taozhen; HU Jing

    2007-01-01

    Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin,hemolysin,and extracellular serine protease (i.e.,aerA,hlyA,and ahpA,respectively).These genes were investigated using polymerase chain reaction (PCR)with specific primers for each gene.And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed.PCR results demonstrated that the distribution patterns of aerA,hlyA,and ahpA were different in these strains.6/9 of A.hydrophila strains were aer A positive,8/9 of strains hly A positive,7/9 of strains ahp A positive,respectively.However,the assay for pathogenesis showed that two strains (A.hydrophila XS91-4-1 and C2)were strong virulent,two strains (A.hydrophila ST78-3-3 and 58-20-9)avirulent and the rest middle virulent was to the fish.In conclusion,there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio.All strong virulent A.hydrophila strains were aerA+hlyA+ahpA+genotype,and all aerA+hlyA+ahpA+strains were virulent.Strains with the genotype of aerA-hlyA-ahpA+have middle pathogenicity.In the present study,we found for the first time that all A.hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio.Additionally,there was a positive correlation between the virulence of A.hydrophila and the presence of aerA and ahpA.

  11. Salmonella enterica serovar typhimurium strains with regulated delayed attenuation in vivo.

    Science.gov (United States)

    Curtiss, Roy; Wanda, Soo-Young; Gunn, Bronwyn M; Zhang, Xin; Tinge, Steven A; Ananthnarayan, Vidya; Mo, Hua; Wang, Shifeng; Kong, Wei

    2009-03-01

    Recombinant bacterial vaccines must be fully attenuated for animal or human hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Unfortunately, many well-studied means for attenuating Salmonella render strains more susceptible to host defense stresses encountered following oral vaccination than wild-type virulent strains and/or impair their ability to effectively colonize the gut-associated and internal lymphoid tissues. This thus impairs the ability of recombinant vaccines to serve as factories to produce recombinant antigens to induce the desired protective immunity. To address these problems, we designed strains that display features of wild-type virulent strains of Salmonella at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. We recently described one means to achieve this based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen. We report here a second means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC P(BAD) cassette for the promoters of the fur, crp, phoPQ, and rpoS genes such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur, Crp, PhoPQ, and/or RpoS proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Means for achieving regulated delayed attenuation can be combined with other mutations, which together may yield safe efficacious recombinant attenuated Salmonella vaccines.

  12. 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.

  13. 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.

  14. Disruption of the Phospholipase D Gene Attenuates the Virulence of Aspergillus fumigatus

    NARCIS (Netherlands)

    Li, Xianping; Gao, Meihua; Han, Xuelin; Tao, Sha; Zheng, Dongyu; Cheng, Ying; Yu, Rentao; Han, Gaige; Schmidt, Martina; Han, Li

    2012-01-01

    Aspergillus fumigatus is the most prevalent airborne fungal pathogen that induces serious infections in immunocompromised patients. Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, knowledge o

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

    Science.gov (United States)

    2013-10-01

    µM 3OC12 (previously shown to inactivate > 90% of PON2 activity) and PON2 immunoprecipitated (IP). The IP PON2 was run on an SDS- PAGE gel and the...protein coupled taste receptor T2R38 in sinonasal epithelial cells, mediating direct antibacterial effects(10). These diverse responses suggest that

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

    Science.gov (United States)

    2014-10-01

    anesthetized mice were filled through the trachea with 2 ml of 500 µM of 3OC12 in the micellar solution and left for 1 hour before harvesting. Control...factor-induced release of endogenous fatty acids analyzed by a highly sensitive high-performance liquid chromatography method. J. Lipid Res. 38, 1913-1922...S. (2000) A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography -mass spectrometry

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

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    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.

  18. 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 (3Cpro) 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 3Cpro, 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. PMID:28217559

  19. Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.

    Science.gov (United States)

    Korte-Berwanger, Miriam; Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sören G

    2013-12-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection.

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

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    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.

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

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    Sebastian Felgner

    2016-09-01

    Full Text Available 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.

  2. Evolution of virulence when transmission occurs before disease.

    Science.gov (United States)

    Osnas, Erik E; Dobson, Andrew P

    2010-08-23

    Most models of virulence evolution assume that transmission and virulence are constant during an infection. In many viral (HIV and influenza), bacterial (TB) and prion (BSE and CWD) systems, disease-induced mortality occurs long after the host becomes infectious. Therefore, we constructed a model with two infected classes that differ in transmission rate and virulence in order to understand how the evolutionarily stable strategy (ESS) depends on the relative difference in transmission and virulence between classes, on the transition rate between classes and on the recovery rate from the second class. We find that ESS virulence decreases when expressed early in the infection or when transmission occurs late in an infection. When virulence occurred relatively equally in each class and there was disease recovery, ESS virulence increased with increased transition rate. In contrast, ESS virulence first increased and then decreased with transition rate when there was little virulence early in the infection and a rapid recovery rate. This model predicts that ESS virulence is highly dependent on the timing of transmission and pathology after infection; thus, pathogen evolution may either increase or decrease virulence after emergence in a new host.

  3. Plasmodium yoelii: induction of attenuated mutants by irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Waki, S.; Yonome, I.; Suzuki, M.

    1986-12-01

    When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number of blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.

  4. Cold Plasma Inactivation of Bacterial Biofilms and Reduction of Quorum Sensing Regulated Virulence Factors.

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    Dana Ziuzina

    Full Text Available The main objectives of this work were to investigate the effect of atmospheric cold plasma (ACP against a range of microbial biofilms commonly implicated in foodborne and healthcare associated human infections and against P. aeruginosa quorum sensing (QS-regulated virulence factors, such as pyocyanin, elastase (Las B and biofilm formation capacity post-ACP treatment. The effect of processing factors, namely treatment time and mode of plasma exposure on antimicrobial activity of ACP were also examined. Antibiofilm activity was assessed for E. coli, L. monocytogenes and S. aureus in terms of reduction of culturability and retention of metabolic activity using colony count and XTT assays, respectively. All samples were treated 'inpack' using sealed polypropylene containers with a high voltage dielectric barrier discharge ACP generated at 80 kV for 0, 60, 120 and 300 s and a post treatment storage time of 24 h. According to colony counts, ACP treatment for 60 s reduced populations of E. coli to undetectable levels, whereas 300 s was necessary to significantly reduce populations of L. monocytogenes and S. aureus biofilms. The results obtained from XTT assay indicated possible induction of viable but non culturable state of bacteria. With respect to P. aeruginosa QS-related virulence factors, the production of pyocyanin was significantly inhibited after short treatment times, but reduction of elastase was notable only after 300 s and no reduction in actual biofilm formation was achieved post-ACP treatment. Importantly, reduction of virulence factors was associated with reduction of the cytotoxic effects of the bacterial supernatant on CHO-K1 cells, regardless of mode and duration of treatment. The results of this study point to ACP technology as an effective strategy for inactivation of established biofilms and may play an important role in attenuation of virulence of pathogenic bacteria. Further investigation is warranted to propose direct evidence

  5. Type IV pili in Francisella – A virulence trait in an intracellular pathogen

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    Emelie eNäslund Salomonsson

    2011-02-01

    Full Text Available Francisella tularensis is a highly virulent intracellular human pathogen that is capable of rapid proliferation in the infected host. Mutants affected in intracellular survival and growth are highly attenuated which highlights the importance of the intracellular phase of the infection. Genomic analysis has revealed that Francisella encodes all genes required for expression of functional type IV pili (Tfp, and in this focused review we summarise recent findings regarding this system in the pathogenesis of tularemia. Tfp are dynamic adhesive structures that have been identified as major virulence determinants in several human pathogens, but it is not obvious what role these structures could have in an intracellular pathogen like Francisella. In the human pathogenic strains, genes required for secretion and assembly of Tfp and one pilin, PilA, have shown to be required for full virulence. Importantly, specific genetic differences have been identified between the different Francisella subspecies where in the most pathogenic type A variants all genes are intact while several Tfp genes are pseudogenes in the less pathogenic type B strains. This suggests that there has been a selection for expression of Tfp with different properties in the different subspecies. There is also a possibility that the genetic differences reflect adaption to different environmental niches of the subspecies and plays a role in transmission of tularemia. This is also in line with recent findings where Tfp pilins are found to be glycosylated which could reflect a role for Tfp in the environment to promote survival and transmission. We are still far from understanding the role of Tfp in virulence and transmission of tularemia, but with the genomic information and genetic tools available we are in a good position to address these issues in the future.

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

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    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.

  7. The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica.

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    Jun Kurushima

    Full Text Available Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator. The virulence of a BspR mutant (ΔbspR in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.

  8. Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence.

    Science.gov (United States)

    Li, Wei; Su, You-Lu; Mai, Yong-Zhan; Li, Yan-Wei; Mo, Ze-Quan; Li, An-Xing

    2014-05-14

    Streptococcus agalactiae is a major piscine pathogen, which causes significant morbidity and mortality among numerous fish species, and results in huge economic losses to aquaculture. Many S. agalactiae strains showing different virulence characteristics have been isolated from infected tilapia in different geographical regions throughout South China in the recent years, including natural attenuated S. agalactiae strain TFJ0901 and virulent S. agalactiae strain THN0901. In the present study, survival of tilapia challenged with S. agalactiae strain TFJ0901 and THN0901 (10(7)CFU/fish) were 93.3% and 13.3%, respectively. Moreover, there are severe lesions of the examined tissues in tilapia infected with strain THN0901, but no significant histopathological changes were observed in tilapia infected with the strain TFJ0901. In order to elucidate the factors responsible for the invasive potential of S. agalactiae between two strains TFJ0901 and THN0901, a comparative proteome analysis was applied to identify the different protein expression profiles between the two strains. 506 and 508 cellular protein spots of S. agalactiae TFJ0901 and THN0901 were separated by two dimensional electrophoresis, respectively. And 34 strain-specific spots, corresponding to 27 proteins, were identified successfully by MALDI-TOF mass spectrometry. Among them, 23 proteins presented exclusively in S. agalactiae TFJ0901 or THN0901, and the other 4 proteins presented in different isomeric forms between TFJ0901 and THN0901. Most of the strain-specific proteins were just involved in metabolic pathways, while 7 of them were presumed to be responsible for the virulence differences of S. agalactiae strain TFJ0901 and THN0901, including molecular chaperone DnaJ, dihydrolipoamide dehydrogenase, thioredoxin, manganese-dependent inorganic pyrophosphatase, elongation factor Tu, bleomycin resistance protein and cell division protein DivIVA. These virulence-associated proteins may contribute to identify new

  9. Next-Generation Bacillus anthracis Live Attenuated Spore Vaccine Based on the htrA(-) (High Temperature Requirement A) Sterne Strain.

    Science.gov (United States)

    Chitlaru, Theodor; Israeli, Ma'ayan; Bar-Haim, Erez; Elia, Uri; Rotem, Shahar; Ehrlich, Sharon; Cohen, Ofer; Shafferman, Avigdor

    2016-01-06

    Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10-10(4)-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

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

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    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.

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

    Science.gov (United States)

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    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.

  12. A putative P-type ATPase required for virulence and resistance to haem toxicity in Listeria monocytogenes.

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    Heather P McLaughlin

    Full Text Available Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641], which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.

  13. 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.

  14. Copper tolerance and virulence in bacteria

    Science.gov (United States)

    Ladomersky, Erik; Petris, Michael J.

    2015-01-01

    Copper (Cu) is an essential trace element for all aerobic organisms. It functions as a cofactor in enzymes that catalyze a wide variety of redox reactions due to its ability to cycle between two oxidation states, Cu(I) and Cu(II). This same redox property of copper has the potential to cause toxicity if copper homeostasis is not maintained. Studies suggest that the toxic properties of copper are harnessed by the innate immune system of the host to kill bacteria. To counter such defenses, bacteria rely on copper tolerance genes for virulence within the host. These discoveries suggest bacterial copper intoxication is a component of host nutritional immunity, thus expanding our knowledge of the roles of copper in biology. This review summarizes our current understanding of copper tolerance in bacteria, and the extent to which these pathways contribute to bacterial virulence within the host. PMID:25652326

  15. Regulation of virulence of Entamoeba histolytica.

    Science.gov (United States)

    Marie, Chelsea; Petri, William A

    2014-01-01

    Entamoeba histolytica is the third-leading cause of parasitic mortality globally. E. histolytica infection generally does not cause symptoms, but the parasite has potent pathogenic potential. The origins, benefits, and triggers of amoebic virulence are complex. Amoebic pathogenesis entails depletion of the host mucosal barrier, adherence to the colonic lumen, cytotoxicity, and invasion of the colonic epithelium. Parasite damage results in colitis and, in some cases, disseminated disease. Both host and parasite genotypes influence the development of disease, as do the regulatory responses they govern at the host-pathogen interface. Host environmental factors determine parasite transmission and shape the colonic microenvironment E. histolytica infects. Here we highlight research that illuminates novel links between host, parasite, and environmental factors in the regulation of E. histolytica virulence.

  16. Virulence Factors of Erwinia amylovora: A Review.

    Science.gov (United States)

    Piqué, Núria; Miñana-Galbis, David; Merino, Susana; Tomás, Juan M

    2015-06-05

    Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3'-5')-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

  17. Virulence Factors of Erwinia amylovora: A Review

    Directory of Open Access Journals (Sweden)

    Núria Piqué

    2015-06-01

    Full Text Available Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS, the exopolysaccharide (EPS amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′-cyclic di-GMP (c-di-GMP and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus, have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

  18. [Genetic virulence markers of opportunistic bacteria].

    Science.gov (United States)

    Bondarenko, V M

    2011-01-01

    The analysis of opportunistic bacteria phenotypic and genetic virulence markers indicates that pathogenicity formation is based on a structural modification of bacterial DNA which is linked with migration of interbacterial pathogenicity "islands" genetic determinants. Structural organization features of these mobile genetic elements determine high expression probability, and PCR detection of pathogenicity "islands" determinants that control adhesins, invasins, cytotoxic and cytolitic toxines synthesis may indicate etiopathogenetic significance of clinical isolates.

  19. Mycobacterium tuberculosis blocks annexin-1 crosslinking and thus apoptotic envelope completion on infected cells to maintain virulence

    Science.gov (United States)

    Gan, Huixian; Lee, Jinhee; Ren, Fucheng; Chen, Minjian; Kornfeld, Hardy; Remold, Heinz G.

    2017-01-01

    Macrophages infected with attenuated Mycobacterium tuberculosis strain H37Ra become apoptotic, limiting bacterial replication and facilitating antigen presentation. Here, we demonstrate that cells infected with H37Ra became apoptotic after formation of an apoptotic envelope on their surface was complete. This process required exposure of phosphatidylserine on the cell surface followed by deposition of the phospholipid-binding protein annexin-1 and then transglutaminase-mediated crosslinking of annexin-1 via its N-terminal domain. In macrophages infected with virulent strain H37Rv, in contrast, the N-terminal domain of annexin-1 was removed by proteolysis thus preventing completion of the apoptotic envelope, which results in macrophage death by necrosis. Host defense of virulent Mycobacterium tuberculosis thus occurs by failure to form the apoptotic envelope, which leads to macrophage necrosis and dissemination of infection in the lung. PMID:18794848

  20. Mutations of ferric uptake regulator (fur) impair iron homeostasis, growth, oxidative stress survival, and virulence of Xanthomonas campestris pv. campestris.

    Science.gov (United States)

    Jittawuttipoka, Thichakorn; Sallabhan, Ratiboot; Vattanaviboon, Paiboon; Fuangthong, Mayuree; Mongkolsuk, Skorn

    2010-05-01

    Iron is essential in numerous cellular functions. Intracellular iron homeostasis must be maintained for cell survival and protection against iron's toxic effects. Here, we characterize the roles of Xanthomonas campestris pv. campestris (Xcc) fur, which encodes an iron sensor and a transcriptional regulator that acts in iron homeostasis, oxidative stress, and virulence. Herein, we isolated spontaneous Xcc fur mutants that had high intracellular iron concentrations due to constitutively high siderophore levels and increased expression of iron transport genes. These mutants also had reduced aerobic plating efficiency and resistance to peroxide killing. Moreover, one fur mutant was attenuated on a host plant, thus indicating that fur has important roles in the virulence of X. campestris pv. campestris.

  1. Contribution of proton-translocating proteins to the virulence of Salmonella enterica serovars Typhimurium, Gallinarum, and Dublin in chickens and mice.

    Science.gov (United States)

    Turner, A K; Barber, L Z; Wigley, P; Muhammad, S; Jones, M A; Lovell, M A; Hulme, S; Barrow, P A

    2003-06-01

    We investigated the attenuating effects of a range of respiratory chain mutations in three Salmonella serovars which might be used in the development of live vaccines. We tested mutations in nuoG, cydA, cyoA, atpB, and atpH in three serovars of Salmonella enterica: Typhimurium, Dublin, and Gallinarum. All three serovars were assessed for attenuation in their relevant virulence assays of typhoid-like infections. Serovar Typhimurium was assessed in 1-day-old chickens and the mouse. Serovar Gallinarum 9 was assessed in 3-week-old chickens, and serovar Dublin was assessed in 6-week-old mice. Our data show variation in attenuation for the nuoG, cydA, and cyoA mutations within the different serovar-host combinations. However, mutations in atpB and atpH were highly attenuating for all three serovars in the various virulence assays. Further investigation of the mutations in the atp operon showed that the bacteria were less invasive in vivo, showing reduced in vitro survival within phagocytic cells and reduced acid tolerance. We present data showing that this reduced acid tolerance is due to an inability to adapt to conditions rather than a general sensitivity to reduced pH. The data support the targeting of respiratory components for the production of live vaccines and suggest that mutations in the atp operon provide suitable candidates for broad-spectrum attenuation of a range of Salmonella serovars.

  2. Mutations of residues 249 and 256 in VP2 are involved in the replication and virulence of infectious Bursal disease virus.

    Directory of Open Access Journals (Sweden)

    Xiaole Qi

    Full Text Available Infectious bursal disease virus (IBDV is a pathogen of worldwide significance to the poultry industry. Although the PDE and PFG domains of the capsid protein VP2 contribute significantly to virulence and fitness, the detailed molecular basis for the pathogenicity of IBDV is still not fully understood. Because residues 253 and 284 of VP2 are not the sole determinants of virulence, we hypothesized that other residues involved in virulence and fitness might exist in the PDE and PFG domains of VP2. To test this, five amino acid changes selected by sequence comparison of the PDE and PFG domains of VP2 were introduced individually using a reverse genetics system into the virulent strain (rGx-F9VP2. Then reverse mutations of the selected residues 249 and 256 were introduced individually into the attenuated strain (rGt. Seven modified viruses were generated and evaluated in vitro (CEF cells and in vivo (SPF chicken. For residue 249, Q249R could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while R249Q could reduce in vitro and elevate in vivo the replication of rGt; meanwhile Q249R reduced the virulence of rGx-F9VP2 while R249Q increased the virulence of rGt, which indicated that residue 249 significantly contributed to the replication and virulence of IBDV. For residue 256, I256V could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while V256I could reduce in vitro but didn't change in vivo the replication of rGt; although V256I didn't increase the virulence of rGt, I256V obviously reduced the virulence of virulent IBDV. The present results demonstrate for the first time, to different extent, residues 249 and 256 of VP2 are involved in the replication efficiency and virulence of IBDV; this is not only beneficial to further understanding of pathogenic mechanism but also to the design of newly tailored vaccines against IBDV.

  3. Immune response of turkey poults exposed at 1 day of age to either attenuated or wild Salmonella strains.

    Science.gov (United States)

    Hesse, Martina; Stamm, Andreas; Weber, Rita; Glünder, Gerhard; Berndt, Angela

    2016-06-01

    Salmonellosis is a foodborne zoonosis that is most often acquired by consuming poultry products such as eggs and poultry meat. Amongst other measures the vaccination of food-producing poultry is thought to contribute to a reduction in human salmonellosis. In the European Union (EU) in 2014 the licence of a commercially available Salmonella vaccine for chickens and ducks was extended to turkeys. In the present study, we examined the course of infection with a virulent Salmonella enterica ssp. enterica serovar Enteritidis (SE) strain, a virulent S. enterica ssp. enterica serovar Typhimurium (ST) strain, and the respective live vaccine containing attenuated strains of both serovars in turkey poults. Besides collecting microbiological data and detecting invading Salmonella in the caecal mucosa via immunohistochemistry, we also assessed immune reactions in terms of antibody production, influx of CD4-, CD8α- and CD28-positive cells into the caecal mucosa and the expression of four different immune-related proteins. We found that the attenuated strains were able to invade the caecum, but to a lower degree and for a shorter duration of time compared to virulent strains. Infections with virulent Salmonellae also caused an increase in CD4-, CD8α- and CD28-positive cells in the caecal mucosa and an increased transcription of iNOS, IL-8-like chemokines, and IFN-γ. In poults treated with attenuated bacteria we could not detect any evidence of immune responses. In conclusion, the vaccine showed a lower degree of caecal invasion and induced weaker immune reactions compared to the virulent Salmonella strains in turkeys. The efficiency of the vaccine has to be verified in future studies.

  4. An Attenuated Strain of Enterovirus 71 Belonging to Genotype A Showed a Broad Spectrum of Antigenicity with Attenuated Neurovirulence in Cynomolgus Monkeys▿

    OpenAIRE

    Arita, Minetaro; Nagata, Noriyo; Iwata, Naoko; Ami, Yasushi; Suzaki, Yuriko; Mizuta, Katsumi; Iwasaki, Takuya; Sata, Tetsutaro; Wakita, Takaji; SHIMIZU, Hiroyuki

    2007-01-01

    Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3′)], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3′), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 pos...

  5. Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives.

    Science.gov (United States)

    Orndorff, Paul E

    2016-11-01

    Bacteriophages (phage) that infect pathogenic bacteria often attach to surface receptors that are coincidentally required for virulence. Receptor loss or modification through mutation renders mutants both attenuated and phage resistant. Such attenuated mutants frequently have no apparent laboratory growth defects, but in the host, they fail to exhibit properties needed to produce disease such as mucosal colonization or survival within professional phagocytic cells. The connection between attenuation and phage resistance has been exploited in experimental demonstrations of phage therapy. In such experiments, phage resistant mutants that arise naturally during therapy are inconsequential because of their attenuated status. A more contemporary approach to exploiting this connection involves identifying small effector molecules, identified in high-throughput screens, that inhibit one or more of the steps needed to produce a functioning phage receptor. Since such biosynthetic steps are unique to bacteria, inhibitors can be utilized therapeutically, in lieu of antibiotics. Also, since the inhibitor is specific to a particular bacterium or group of bacteria, no off-target resistance is generated in the host's commensal bacterial population. This brief review covers examples of how mutations that confer phage resistance produce attenuation, and how this coincidental relationship can be exploited in the search for the next generation of therapeutic agents for bacterial diseases.

  6. 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

  7. 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...

  8. Semiactive control for vibration attenuation

    Energy Technology Data Exchange (ETDEWEB)

    Leitmann, G. [Univ. of California, Berkeley, CA (United States). Coll. of Engineering

    1994-12-31

    With the advent of materials, such as electrorheological fluids, whose material properties can be altered rapidly by means of external stimuli, employing such materials as actuators for the controlled attenuation of undesirable vibrations is now possible. Such control schemes are dubbed semiactive in that they attenuate vibrations whether applied actively or passively. The author investigates various such control schemes, allowing for both separate and joint control of the stiffness and damping characteristics of the material.

  9. 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. PMID:27630634

  10. Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.

    Science.gov (United States)

    Niemann, George S; Brown, Roslyn N; Gustin, Jean K; Stufkens, Afke; Shaikh-Kidwai, Afshan S; Li, Jie; McDermott, Jason E; Brewer, Heather M; Schepmoes, Athena; Smith, Richard D; Adkins, Joshua N; Heffron, Fred

    2011-01-01

    Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA' reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.

  11. 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.

  12. Distribution and Virulence Diversity of Phytophthora sojae in China

    Institute of Scientific and Technical Information of China (English)

    ZHU Zhen-dong; WANG Hua-bo; WANG Xiao-ming; CHANG Ru-zhen; WU Xiao-fei

    2004-01-01

    By investigating occurrence of Phytophthora root rot in fields and isolating P. sojae from diseased plants and soils, the distribution of P. sojae in China was surveyed. In addition to northeast region, P. sojae existed in Huanghe-Huaihe basin and Yangtze basin too. Eighty- three isolates of P. sojae isolated from different areas were identified on virulence using 13differential soybean cultivars, abundant virulence diversity was found in P. sojae. The greater diversity in virulence of P. sojae was in isolates from soil than from plants. And the greatest virulence diversity of P.sojae was found in Yangtze basin.

  13. 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 s

  14. Two mobile Pectobacterium atrosepticum prophages modulate virulence.

    Science.gov (United States)

    Evans, Terry J; Coulthurst, Sarah J; Komitopoulou, Evangelia; Salmond, George P C

    2010-03-01

    The Pectobacterium atrosepticum strain SCRI1043 genome contains two complete prophage sequences. One, ECA41, is Mu-like and is able to integrate into, and excise from, various genomic locations. The other, ECA29, is a P2 family prophage, and is also able to excise from the genome. Excision of both prophages is rare and we were unable to induce lysis of cultures. Deletion of the entire prophages, both separately and in combination, did not affect the growth rate or the secretion of plant cell wall-degrading enzymes, but swimming motility was decreased. The virulence of prophage deletion strains in the potato host was decreased.

  15. Virulence of Renibacterium salmoninarum to salmonids

    Science.gov (United States)

    Starliper, C.E.; Smith, D.R.; Shatzer, T.

    1997-01-01

    Virulence of Renibacterium salmoninarum isolates representing five origins was evaluated in eight salmonid hosts; four origins were of Lake Michigan and the fifth was of the Pacific Northwest. The species type strain, ATCC (American Type Culture Collection) 33209, was also included. Each isolate was grown in a kidney disease medium (KDM2) supplemented with 1 % ATCC 33209 culture metabolite; serial 10-fold dilutions were prepared, and groups of fish were challenged by intraperitoneal injection with 0.1 mL of each dilution. A 70-d observation period followed, and bacterial kidney disease (BKD) was diagnosed by the fluorescent antibody technique. Virulence of isolates was quantified as a dose lethal to 50% of fish (LD50) for each host–isolate challenge. In the first set of experiments, 23 isolates were used to challenge groups of brook trout Salvelinus fontinalis. The mean LD50 was 1.087 x 106 colony-forming units per milliliter (cfu/mL; SD = 2.022 x 106), and the LD50 values ranged from 8.457 x 106 to 2.227 x 104 cfu/mL. Analysis of variance to evaluate the effect of isolate origin on virulence in brook trout revealed no significant difference (F = 1.502; P = 0.243). Susceptibilities of the other salmonid hosts were evaluated by challenge with six isolates of R. salmoninarum representing each origin and the species type strain. For many of the host–isolate challenge combinations, time to death was highly dependent on the dilution (number of bacteria) injected. In general, the isolates MCO4M, B26, and A34 (all of Lake Michigan origin) tended to be more virulent. Also, LD50 values were dispersed throughout a wider range among the more susceptible hosts. Lake trout Salvelinus namaycush, rainbow trout Oncorhynchus mykiss, and brook trout were relatively resistant to challenge with the strains, whereas coho salmon O. kisutch, domestic Atlantic salmon Saltno salar, and chinook salmon O. tshawytscha were relatively susceptible. Another challenge evaluated the effect of

  16. Brucella spp. Virulence Factors and Immunity.

    Science.gov (United States)

    Byndloss, Mariana X; Tsolis, Renee M

    2016-01-01

    Brucellosis, caused by bacteria of the genus Brucella, is an important zoonotic infection that causes reproductive disease in domestic animals and chronic debilitating disease in humans. An intriguing aspect of Brucella infection is the ability of these bacteria to evade the host immune response, leading to pathogen persistence. Conversely, in the reproductive tract of infected animals, this stealthy pathogen is able to cause an acute severe inflammatory response. In this review, we discuss the different mechanisms used by Brucella to cause disease, with emphasis on its virulence factors and the dichotomy between chronic persistence and reproductive disease.

  17. Safety and vaccine efficacy of an attenuated Vibrio vulnificus strain with deletions in major cytotoxin genes.

    Science.gov (United States)

    Kim, Young Ran; Lee, Shee Eun; Kim, Jong Ro; Rhee, Joon Haeng

    2015-12-01

    Vibrio vulnificus is a human pathogen causing a rapidly progressing fatal septicemia. We have previously reported that a V. vulnificus large toxin RtxA1 causes programmed necrotic cell death through calcium-mediated mitochondrial dysfunction. Here we developed a live attenuated vaccine strain (CMM781) having deletions in three genes encoding major virulence factors: RTX cytotoxin (rtxA1), hemolysin/cytolysin (vvhA) and metalloprotease (vvpE) of a clinical isolate strain CMCP6. The CMM781 strain showed significant attenuation in cytotoxicity and mouse lethality. The safety of CMM781 was also confirmed by measuring the transepithelial electric resistance of Caco-2 cell monolayers. Intragastric immunization of mice with the live attenuated V. vulnificus strain resulted in induction of systemic and mucosal antibodies specific to the pathogen. Moreover, the vaccinated mice were protected from challenges with high doses of the virulent strain through various injection routes. These results suggest that CMM781 appears to be a safe and effective vaccine candidate that would provide significant protection against V. vulnificus infection.

  18. 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

  19. Effects of mutated replicase and movement protein genes on attenuation of tobacco mosaic virus

    Institute of Scientific and Technical Information of China (English)

    杨恭; 邱并生; 魏军亚; 刘广超

    2001-01-01

    Our previous reports showed that one opal mutation (UGA) and one ochre mutation (UAA) respectively located in the replicase and movement protein (MP) genes of the attenuated tomato mosaic virus K(ToMV-K) contribute to the viral attenuation. To explore a wider application of this attenuation pattern to other plant viruses, we have constructed three mutants which respectively contain one opal mutation of the replicase gene and/or one ochre mutation of the MP using PCR-mediated site-directed mutagenesis from a virulent tobacco mosaic virus isolated from China (TMV-Cv). Plant infection performed by in vitro transcripts revealed that the MP truncated mutant TMV-Cvmp and the replicase-MP truncated mutant TMV-Cvrase-mp were infectious on both local lesion (Nicotiana tabacum cv. Xanthi NC) and systemic (N. tabacum cv. K326) host plants, while the replicase truncated mutant TMV-Cvrase was non-infectious. The K326 plant infected by TMV- Cvrease-mp displayed only a little mild mosaic. By electronic microscopy (EM), plant re-inoculation, RNA Dot-blot, RT-PCR and sequencing we demonstrated that the progeny viruses of TMV-Cvmp and TMV-Cvrease-mp shared similar morphological character with TMV-Cv, owned the abilities to infect, replicate and propagate in the assayed plants, and maintained the mutated sites during infection. These data showed that both the opal and the ochre mutations are able to cooperatively induce the attenuated phenotypes of TMV-Cvrase-mp on plants, indicating that the mutation pattern of ToMV-K could be used to attenuate other virulent plant viruses.

  20. Virulence-associated gene profiling of Streptococcus suis isolates by PCR

    NARCIS (Netherlands)

    Silva, L.M.G.; Baums, C.G.; Rehm, T.; Wisselink, H.J.; Goethe, R.; Valentin-Weigand, P.

    2006-01-01

    Definition of virulent Streptococcus suis strains is controversial. One successful approach for identification of virulent European strains is differentiation of capsular serotypes (or the corresponding cps types) and subsequent detection of virulence-associated factors, namely the extracellular fac

  1. Herbs, Spices and Medicinal Plants Used In Hispanic Traditional Medicine Can Decrease Quorum Sensing Dependent Virulence in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    V Huerta

    2008-06-01

    alternative to antibiotic mediated bactericidal or bacteristatic approach and reduces the risk for development of resistance. Inhibition of bacterial quorum sensing by attenuating the signals can prevent the development of bacterial virulence and successful establishment of infections. Understanding the quorum sensing inhibition activity of natural bioactive phytochemicals can lead to the discovery of novel compounds and development of more effective strategies in preventing and managing microbial infections.

  2. Contribution of Asparagine Catabolism to Salmonella Virulence.

    Science.gov (United States)

    McLaughlin, Patrick A; McClelland, Michael; Yang, Hee-Jeong; Porwollik, Steffen; Bogomolnaya, Lydia; Chen, Juei-Suei; Andrews-Polymenis, Helene; van der Velden, Adrianus W M

    2017-02-01

    Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.

  3. 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.

  4. Microbial virulence and interactions with metals

    DEFF Research Database (Denmark)

    German, N.; Lüthje, F.; 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.......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...... transition metals are known to play an important role in host-defense mechanisms against bacteria through Fenton chemistry evoked toxicity as an example. Copper and zinc are used as a mechanism to poison bacteria whereas other metals, such as, iron and manganese are withheld by the predator to prevent...

  5. Regulation of Helicobacter pylori Virulence Within the Context of Iron Deficiency.

    Science.gov (United States)

    Noto, Jennifer M; Lee, Josephine Y; Gaddy, Jennifer A; Cover, Timothy L; Amieva, Manuel R; Peek, Richard M

    2015-06-01

    Helicobacter pylori strains that harbor the oncoprotein CagA increase gastric cancer risk, and this risk is augmented under iron-deficient conditions. We demonstrate here that iron depletion induces coccoid morphology in strains lacking cagA. To evaluate the stability of augmented H. pylori virulence phenotypes stimulated by low-iron conditions, H. pylori isolated from iron-depleted conditions in vivo were serially passaged in vitro. Long-term passage decreased the ability of hypervirulent strains to translocate CagA or induce interleukin 8, indicating that hypervirulent phenotypes stimulated by low-level iron conditions are reversible. Therefore, rectifying iron deficiency may attenuate disease among H. pylori-infected persons with no response to antibiotics.

  6. Photoacoustic Imaging Taking into Account Attenuation

    CERN Document Server

    Kowar, Richard

    2010-01-01

    First, we review existing attenuation models and discuss their causality properties, which we believe to be essential for algorithms for inversion with attenuated data. Then, we survey causality properties of common attenuation models. We also derive integro-differential equations which the attenuated waves are satisfying. In addition we discuss the ill--conditionness of the inverse problem for calculating the unattenuated wave from the attenuated one.

  7. 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.

  8. 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.

  9. Bacterial Adrenergic Sensors Regulate Virulence of Enteric Pathogens in the Gut

    Directory of Open Access Journals (Sweden)

    Cristiano G. Moreira

    2016-06-01

    Full Text Available Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium. Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine β-hydroxylase knockout (Dbh−/− mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI infection by a noninvasive pathogen.

  10. Streptococcus pyogenes malate degradation pathway links pH regulation and virulence.

    Science.gov (United States)

    Paluscio, Elyse; Caparon, Michael G

    2015-03-01

    The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI(-) mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP(-), MaeK(-), and MaeR(-) mutants were attenuated for virulence, whereas a MaeE(-) mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection.

  11. 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.

  12. Microbiology: EHEC downregulates virulence in response to intestinal fucose.

    Science.gov (United States)

    Keeney, Kristie M; Finlay, B Brett

    2013-02-04

    Recent work has revealed that enterohaemorrhagic Escherichia coli encodes a two-component system, termed FusKR, which responds to fucose and represses expression of virulence genes. Furthermore, a representative member of the microbiota appears to cleave fucose from host glycans, indicating that the microbiota and EHEC may act in concert to suppress virulence gene expression.

  13. Effect of phosphatidylcholine-cholesterol liposomes on Entamoeba histolytica virulence.

    Science.gov (United States)

    Serrano-Luna, Jesús; Gutiérrez-Meza, Manuel; Mejía-Zepeda, Ricardo; Galindo-Gómez, Silvia; Tsutsumi, Víctor; Shibayama, Mineko

    2010-12-01

    Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.

  14. Reconstructing the highly virulent Classical Swine Fever Virus strain Koslov

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Nielsen, Jens

    Classical swine fever virus (CSFV) may be highly virulent in pigs with a mortality rate close to 100%. The CSFV “Koslov strain” is known to be one of the most virulent CSFV, but so far a functional cloned cDNA of this strain has not been described. We suggest that this may be due to the error...

  15. Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae.

    Science.gov (United States)

    Antunes, Camila Azevedo; Sanches dos Santos, Louisy; Hacker, Elena; Köhler, Stefanie; Bösl, Korbinian; Ott, Lisa; de Luna, Maria das Graças; Hirata, Raphael; Azevedo, Vasco Ariston de Carvalho; Mattos-Guaraldi, Ana-Luíza; Burkovski, Andreas

    2015-03-01

    Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

  16. Lipidomic analysis links mycobactin synthase K to iron uptake and virulence in M. tuberculosis.

    Directory of Open Access Journals (Sweden)

    Cressida A Madigan

    2015-03-01

    Full Text Available The prolonged survival of Mycobacterium tuberculosis (M. tb in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt. Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.

  17. MarA and ramA regulate virulence in Salmonella enterica serovar Choleraesuis.

    Science.gov (United States)

    Lee, Jen-Jie; Hsuan, Shih-Ling; Kuo, Chih-Jung; Wu, Ying-Chen; Chen, Ter-Hsin

    2015-12-31

    Salmonella enterica serovar Choleraesuis is considered as an important porcine pathogen that causes serious systemic infections and exhibits poor response to treatment because of an increase in multidrug resistance (MDR). Among the various regulators of resistance, multiple antibiotic resistance factor A (marA) and regulator of acetate metabolism A (ramA) are the most effective in conferring antibiotic tolerance by activation of multidrug efflux pumps. Here we investigated the regulation of virulence in Salmonella Choleraesuis through these two transcriptional regulators. We showed that marA andramA are important for the survival of Salmonella Choleraesuis in an environment of acid and bile salts, since marA- or ramA-deficient Salmonella Choleraesuis strains failed to increase protective responses, as observed by quantitative RT-PCR (qPCR). Further, reduced invasion and survival in host cells was observed in the marA and ramA mutant strains. The results from in vitrostudies with marA- and ramA-deficient strains showed attenuated characteristics in comparison to those in the wild-type strain of Salmonella Choleraesuis when it was used to challenge BALB/c mice. The mutant strains had higher LD50 and presented poor clearance efficiency compared to the parental strain. These findings indicate that MarA and RamA not only regulate drug resistance but also play a role in the virulence of SalmonellaCholeraesuis.

  18. Role of capsule and O antigen in the virulence of uropathogenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Sohinee Sarkar

    Full Text Available Urinary tract infection (UTI is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD and O antigen (waaL mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1, RS218 (O18:K1:H7 and 1177 (O1:K1:H7. We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.

  19. Impact of Hfq on global gene expression and virulence in Klebsiella pneumoniae.

    Directory of Open Access Journals (Sweden)

    Ming-Ko Chiang

    Full Text Available Klebsiella pneumoniae is responsible for a wide range of clinical symptoms. How this bacterium adapts itself to ever-changing host milieu is still a mystery. Recently, small non-coding RNAs (sRNAs have received considerable attention for their functions in fine-tuning gene expression at a post-transcriptional level to promote bacterial adaptation. Here we demonstrate that Hfq, an RNA-binding protein, which facilitates interactions between sRNAs and their mRNA targets, is critical for K. pneumoniae virulence. A K. pneumoniae mutant lacking hfq (Δhfq failed to disseminate into extra-intestinal organs and was attenuated on induction of a systemic infection in a mouse model. The absence of Hfq was associated with alteration in composition of envelope proteins, increased production of capsular polysaccharides, and decreased resistance to H(2O(2, heat shock, and UV irradiation. Microarray-based transcriptome analyses revealed that 897 genes involved in numerous cellular processes were deregulated in the Δhfq strain. Interestingly, Hfq appeared to govern expression of many genes indirectly by affecting sigma factor RpoS and RpoE, since 19.5% (175/897 and 17.3% (155/897 of Hfq-dependent genes belong to the RpoE- and RpoS-regulon, respectively. These results indicate that Hfq regulates global gene expression at multiple levels to modulate the physiological fitness and virulence potential of K. pneumoniae.

  20. Carbon storage regulator A contributes to the virulence of Haemophilus ducreyi in humans by multiple mechanisms.

    Science.gov (United States)

    Gangaiah, Dharanesh; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Ellinger, Sheila; Zwickl, Beth; Katz, Barry P; Spinola, Stanley M

    2013-02-01

    The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog of csrA. Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

  1. Biological and virulence characteristics of the YqhC mutant of Salmonella.

    Science.gov (United States)

    Eakley, Nicholas M; Bochsler, Philip N; Gopal Reddy, P; Chopra, Ashok K; Fadl, Amin A

    2011-12-01

    Previous work by the present authors indicated a murein lipoprotein mutant of Salmonella shows a marked down-regulation in expression of yqhC. Because YqhC is a putative DNA-binding protein, it is likely involved in modulation of Salmonella genes. Deletion of yqhC renders Salmonella defective in invasion of intestinal epithelial cells, motility, and induction of cytotoxicity. In the present study, further attenuation in induction of inflammatory cytokines/chemokines and histopathological lesions was seen in mice infected with the yqhC mutant. On the other hand, deletion of yqhC did not significantly affect the LD(50) in mice or the ability of Salmonella to survive and replicate in vivo. To better understand how YqhC affects Salmonella virulence and to identify factors potentially modulated by YqhC, comparative transcriptome and proteome analysis of the yqhC mutant and the WT Salmonella was performed. Data from these experiments indicate that deletion of yqhC significantly alters the transcription of several genes associated with the SPI-1 encoded T3SS and flagellar regulons, correlating with the yqhC mutant phenotype. Overall, this study indicates that deletion of the yqhC gene causes a number of virulence-related defects in vitro, but has a modest effect in vivo, despite affecting induction of inflammatory cytokines and histopathology.

  2. 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.

  3. A live attenuated strain of Yersinia pestis KIM as a vaccine against plague.

    Science.gov (United States)

    Sun, Wei; Six, David; Kuang, Xiaoying; Roland, Kenneth L; Raetz, Christian R H; Curtiss, Roy

    2011-04-01

    Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2'-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC P(BAD) promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::P(lpxL)lpxL ΔP(crp21)::TT araC P(BAD)crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD(50) of the mutant in mice, when administered subcutaneously or intranasally was >10(7)-times and >10(4)-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6×10(7) wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2×10(4) live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.

  4. A novel lux operon in the cryptically bioluminescent fish pathogen Vibrio salmonicida is associated with virulence.

    Science.gov (United States)

    Nelson, Eric J; Tunsjø, Hege S; Fidopiastis, Pat M; Sørum, Henning; Ruby, Edward G

    2007-03-01

    The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host.

  5. Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence.

    Science.gov (United States)

    Li, Cecilia; Lev, Sophie; Saiardi, Adolfo; Desmarini, Desmarini; Sorrell, Tania C; Djordjevic, Julianne T

    2016-04-01

    Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1Δkcs1Δ and kcs1Δ lung burdens were also lower than those of ipk1Δ. Unlike ipk1Δ, ipk1Δkcs1Δ and kcs1Δ failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. Kcs1 is therefore a dual specificity (IP5 and IP6) kinase producing PP-IP4 and PP-IP5/IP7. All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced significantly in all mutants except ipk1Δ, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1Δ grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence.

  6. X-Ray Attenuation Cell

    Energy Technology Data Exchange (ETDEWEB)

    Ryutov, D.; Toor, A.

    2000-03-03

    To minimize the pulse-to-pulse variation, the LCLS FEL must operate at saturation, i.e. 10 orders of magnitude brighter spectral brilliance than 3rd-generation light sources. At this intensity, ultra-high vacuums and windowless transport are required. Many of the experiments, however, will need to be conducted at a much lower intensity thereby requiring a reliable means to reduce the x-ray intensity by many orders of magnitude without increasing the pulse-to-pulse variation. In this report we consider a possible solution for controlled attenuation of the LCLS x-ray radiation. We suggest using for this purpose a windowless gas-filled cell with the differential pumping. Although this scheme is easily realizable in principle, it has to be demonstrated that the attenuator can be made short enough to be practical and that the gas loads delivered to the vacuum line of sight (LOS) are acceptable. We are not going to present a final, optimized design. Instead, we will provide a preliminary analysis showing that the whole concept is robust and is worth further study. The spatial structure of the LCLS x-ray pulse at the location of the attenuator is shown in Fig. 1. The central high-intensity component, due to the FEL, has a FWHM of {approx}100 {micro}m. A second component, due to the undulator's broad band spontaneous radiation is seen as a much lower intensity ''halo'' with a FWHM of 1 mm. We discuss two versions of the attenuation cell. The first is directed towards a controlled attenuation of the FEL up to the 4 orders of magnitude in the intensity, with the spontaneous radiation halo being eliminated by collimators. In the second version, the spontaneous radiation is not sacrificed but the FEL component (as well as the first harmonic of the spontaneous radiation) gets attenuated by a more modest factor up to 100. We will make all the estimates assuming that the gas used in the attenuator is Xenon and that the energy of the FEL is 8.25 keV. At

  7. Virulence of Bacillus cereus: a multivariate analysis.

    Science.gov (United States)

    Minnaard, J; Delfederico, L; Vasseur, V; Hollmann, A; Rolny, I; Semorile, L; Pérez, P F

    2007-05-10

    Biological activity and presence of DNA sequences related to virulence genes were studied in 21 strains of the Bacillus cereus group. The activity of spent culture supernatants and the effect of infection by vegetative bacterial cells were assessed on cultured human enterocytes (Caco-2 cells). The effect of extracellular factors on the detachment, necrosis and mitochondrial dehydrogenase activity of cultured human enterocytes was studied. Hemolytic activity on rabbit red blood cells was also evaluated and the effect of direct procaryotic-eucaryotic interactions was assessed in infection assays with vegetative bacterial cells. Concerning virulence genes, presence of the DNA sequences corresponding to the genes entS, entFM, nhe (A, B and C), sph, hbl (A, B, C and D), piplC and bceT was assessed by PCR. Ribopatterns were determined by an automated riboprinting analysis after digestion of the DNA with EcoRI. Principal component analysis and biplots were used to address the relationship between variables. Results showed a wide range of biological activities: decrease in mitochondrial dehydrogenase activity, necrosis, cell detachment and hemolytic activity. These effects were strain-dependent. Concerning the occurrence of the DNA sequences tested, different patterns were found. In addition, ribotyping showed that strains under study grouped into two main clusters. One of these clusters includes all the strains that were positive for all the DNA sequences tested. Positive and negative correlations between variables under study were evidenced. Interestingly, high detaching strains were positively correlated with the presence of the sequences entS, nheC and sph. Within gene complexes, high correlation was found between sequences of the hbl complex. In contrast, sequences of the nhe complex were not correlated. Some strains clustered together in the biplots. These strains were positive for all the DNA sequences tested and they were able to detach enterocytes upon infection

  8. Polymerase Mechanism-Based Method of Viral Attenuation

    Science.gov (United States)

    Lee, Cheri A.; August, Avery; Arnold, Jamie J.; Cameron, Craig E.

    2016-01-01

    Vaccines remain the most effective way of preventing infection and spread of infectious diseases. These prophylactics have been used for centuries but still to this day only three main design strategies exist: (1) live attenuated virus (LAV) vaccines, (2) killed or inactivated virus vaccines, (3) and subunit vaccines of the three, the most efficacious vaccines remain LAVs. LAVs replicate in relevant tissues, elicit strong cellular and humoral responses, and often confer lifelong immunity. While this vaccine strategy has produced the majority of successful vaccines in use today, there are also important safety concerns to consider with this approach. In the past, the development of LAVs has been empirical. Blind passage of viruses in various cell types results in the accumulation of multiple attenuating mutations leaving the molecular mechanisms of attenuation unknown. Also, due to the high error rate of RNA viruses and selective pressures of the host environment, these LAVs, derived from such viruses, can potentially revert back to wild-type virulence. This not only puts the vaccinee at risk, but if shed can put those that are unvaccinated at risk as well. While these vaccines have been successful there still remains a need for a rational design strategy by which to create additional LAVs. One approach for rational vaccine design involves increasing the fidelity of the viral RdRp. Increased fidelity decreases the viral mutational frequency thereby reducing the genetic variation the virus needs in order to evade the host imposed bottlenecks to infection. While polymerase mutants exist which decrease viral mutation frequency the mutations are not in conserved regions of the polymerase, which doesn’t lend itself toward using a common mutant approach toward developing a universal vaccine strategy for all RNA viruses. We have identified a conserved lysine residue in the active site of the PV RdRp that acts as a general acid during nucleotide incorporation. Mutation

  9. Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse.

    Science.gov (United States)

    Alaganan, Aditi; Fentress, Sarah J; Tang, Keliang; Wang, Qiuling; Sibley, L David

    2014-01-21

    The intracellular parasite Toxoplasma gondii enjoys a wide host range and is adept at surviving in both naive and activated macrophages. Previous studies have emphasized the importance of the active serine-threonine protein kinase rhoptry protein 18 (ROP18), which targets immunity-related GTPases (IRGs), in mediating macrophage survival and acute virulence of T. gondii in mice. Here, we demonstrate that ROP18 exists in a complex with the pseudokinases rhoptry proteins 8 and 2 (ROP8/2) and dense granule protein 7 (GRA7). Individual deletion mutant gra7 or rop18 was partially attenuated for virulence in mice, whereas the combined gra7rop18 mutant was avirulent, suggesting these proteins act together in the same pathway. The virulence defect of the double mutant was mirrored by increased recruitment of IRGs and clearance of the parasite in IFN-γ-activated macrophages in vitro. GRA7 was shown to recognize a conserved feature of IRGs, binding directly to the active dimer of immunity-related GTPase a6 in a GTP-dependent manner. Binding of GRA7 to immunity-related GTPase a6 led to enhanced polymerization, rapid turnover, and eventual disassembly. Collectively, these studies suggest that ROP18 and GRA7 act in a complex to target IRGs by distinct mechanisms that are synergistic.

  10. Attenuation in silica-based optical fibers

    DEFF Research Database (Denmark)

    Wandel, Marie Emilie

    2006-01-01

    In this thesis on attenuation in silica based optical fibers results within three main topics are reported. Spectral attenuation measurements on transmission fibers are performed in the wide wavelength range 290 nm – 1700 nm. The measured spectral attenuation is analyzed with special emphasis...... on absorption peaks in order to investigate the cause of an unusual high attenuation in a series of transmission fibers. Strong indications point to Ni2+ in octahedral coordination as being the cause of the high attenuation. The attenuation of fibers having a high core refractive index is analyzed and the cause...... of the high attenuation measured in such fibers is described as being due to scattering of light on fluctuations of the core diameter. A novel semi-empirical model for predicting the attenuation of high index fibers is presented. The model is shown to be able to predict the attenuation of high index fibers...

  11. The Central Metabolism Regulator EIIAGlc Switches Salmonella from Growth Arrest to Acute Virulence through Activation of Virulence Factor Secretion

    Directory of Open Access Journals (Sweden)

    Alain Mazé

    2014-06-01

    Full Text Available The ability of Salmonella to cause disease depends on metabolic activities and virulence factors. Here, we show that a key metabolic protein, EIIAGlc, is absolutely essential for acute infection, but not for Salmonella survival, in a mouse typhoid fever model. Surprisingly, phosphorylation-dependent EIIAGlc functions, including carbohydrate transport and activation of adenylate cyclase for global regulation, do not explain this virulence phenotype. Instead, biochemical studies, in vitro secretion and translocation assays, and in vivo genetic epistasis experiments suggest that EIIAGlc binds to the type three secretion system 2 (TTSS-2 involved in systemic virulence, stabilizes its cytoplasmic part including the crucial TTSS-2 ATPase, and activates virulence factor secretion. This unexpected role of EIIAGlc reveals a striking direct link between central Salmonella metabolism and a crucial virulence mechanism.

  12. Selected lactic acid-producing bacterial isolates with the capacity to reduce Salmonella translocation and virulence gene expression in chickens.

    Directory of Open Access Journals (Sweden)

    Xiaojian Yang

    Full Text Available BACKGROUND: Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. METHODOLOGY/PRINCIPAL FINDINGS: In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0 and high bile salt (0.3-1.5% and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10(6-7 CFU/chick or phosphate-buffered saline (PBS at 1 day of age followed by Salmonella challenge (10(4 CFU/chick next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1. These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10 in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. CONCLUSIONS/SIGNIFICANCE: The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in

  13. Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.

    Science.gov (United States)

    Thompson, C; Baravalle, M E; Valentini, B; Mangold, A; Torioni de Echaide, S; Ruybal, P; Farber, M; Echaide, I

    2014-06-01

    The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones.

  14. BCG-PPD、H37Rv-PPD诱导入巨噬细胞死亡及TNF-α、IL-1β和IL-10的表达差异%Different cell death of THP-1 induced by virulent/attenuated purfied protein derivatives tuberculin and the different expression of TNF-α,IL-1β and IL-10 in Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    史会连; 路蝉伊; 虞胜镭; 张文宏; 翁心华; 陈澍

    2010-01-01

    expression of IL-1β was higher than the latter.Conclusion It indicated that the necrosis of cells stimulated by H37Rv-PPD was asossiated with the excessive expression of TNF-α and IL-10,and the apoptosis of cells induced by BCG-PPD was IL-1β related.Perhaps the mechanism of differences in virulence exist in protein of strain,and associated with cytokines IL-1β,TNF-α and IL-10.

  15. 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 bias...

  16. 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...

  17. Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1 (DHAV-1) Isolates in Shandong Province of China

    Institute of Scientific and Technical Information of China (English)

    Jiming Gao; Junhao Chen; Xingkui Si; Zhijing Xie; Yanli Zhu; Xingxiao Zhang; Shujing Wang; Shijin Jiang

    2012-01-01

    To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD50s) and the median lethal doses(LD50s),respectively.The results showed that the ELD5s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 106/mL to 1.44 × 107/mL,while the LD50s were 2.39 × 105/mL to 6.15 × 106/mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(as 158-160,180-193 and 205-219) and other variable points in VPI protein,but which didn't cause virulence of DHAV-1 change.

  18. Stormwater Attenuation by Green Roofs

    Science.gov (United States)

    Sims, A.; O'Carroll, D. M.; Robinson, C. E.; Smart, C. C.

    2014-12-01

    Innovative municipal stormwater management technologies are urgently required in urban centers. Inadequate stormwater management can lead to excessive flooding, channel erosion, decreased stream baseflows, and degraded water quality. A major source of urban stormwater is unused roof space. Green roofs can be used as a stormwater management tool to reduce roof generated stormwater and generally improve the quality of runoff. With recent legislation in some North American cities, including Toronto, requiring the installation of green roofs on large buildings, research on the effectiveness of green roofs for stormwater management is important. This study aims to assess the hydrologic response of an extensive sedum green roof in London, Ontario, with emphasis on the response to large precipitation events that stress municipal stormwater infrastructure. A green roof rapidly reaches field capacity during large storm events and can show significantly different behavior before and after field capacity. At field capacity a green roof has no capillary storage left for retention of stormwater, but may still be an effective tool to attenuate peak runoff rates by transport through the green roof substrate. The attenuation of green roofs after field capacity is linked to gravity storage, where gravity storage is the water that is temporarily stored and can drain freely over time after field capacity has been established. Stormwater attenuation of a modular experimental green roof is determined from water balance calculations at 1-minute intervals. Data is used to evaluate green roof attenuation and the impact of field capacity on peak flow rates and gravity storage. In addition, a numerical model is used to simulate event based stormwater attenuation. This model is based off of the Richards equation and supporting theory of multiphase flow through porous media.

  19. 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.

  20. Polyamines Are Required for Virulence in Salmonella enterica Serovar Typhimurium

    DEFF Research Database (Denmark)

    Jelsbak, Lotte; Thomsen, Line Elnif; Wallrodt, Inke;

    2012-01-01

    for studying typhoid fever. Central to its virulence are two major virulence loci Salmonella Pathogenicity Island 1 and 2 (SPI1 and SPI2). SPI1 promotes invasion of epithelial cells, whereas SPI2 enables S. Typhimurium to survive and proliferate within specialized compartments inside host cells. In this study......, 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...

  1. Role of pathogenicity determinant protein C (PdpC in determining the virulence of the Francisella tularensis subspecies tularensis SCHU.

    Directory of Open Access Journals (Sweden)

    Akihiko Uda

    Full Text Available Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

  2. Expression of interferon gamma by a highly virulent strain of Newcastle disease virus decreases its pathogenicity in chickens.

    Science.gov (United States)

    Susta, Leonardo; Cornax, Ingrid; Diel, Diego G; Garcia, Stivalis Cardenas; Miller, Patti J; Liu, Xiufan; Hu, Shunlin; Brown, Corrie C; Afonso, Claudio L

    2013-01-01

    The role of interferon gamma (IFN-γ) expression during Newcastle disease virus (NDV) infection in chickens is unknown. Infection of chickens with highly virulent NDV results in rapid death, which is preceded by increased expression of IFN-γ in target tissues. IFN-γ is a cytokine that has pleiotropic biological effects including intrinsic antiviral activity and immunomodulatory effects that may increase morbidity and mortality during infections. To better understand how IFN-γ contributes to NDV pathogenesis, the coding sequence of the chicken IFN-γ gene was inserted in the genome of the virulent NDV strain ZJ1 (rZJ1-IFNγ), and the effects of high levels of IFN-γ expression during infection were determined in vivo and in vitro. IFN-γ expression did not significantly affect NDV replication in fibroblast or in macrophage cell lines. However, it affected the pathogenesis of rZJ1-IFNγ in vivo. Relative to the virus expressing the green fluorescent protein (rZJ1-GFP) or lacking the IFN-γ insert (rZJ1-rev), expression of IFN-γ by rZJ1-IFNγ produced a marked decrease of pathogenicity in 4-week-old chickens, as evidenced by lack of mortality, decreased disease severity, virus shedding, and antigen distribution. These results suggest that early expression of IFN-γ had a significant protective role against the effects of highly virulent NDV infection in chickens, and further suggests that the level and timing of expression of this cytokine may be critical for the disease outcome. This is the first description of an in vivo attenuation of a highly virulent NDV by avian cytokines, and shows the feasibility to use NDV for cytokine delivery in chicken organs. This approach may facilitate the study of the role of other avian cytokines on the pathogenesis of NDV.

  3. A pathoadaptive deletion in an enteroaggregative Escherichia coli outbreak strain enhances virulence in a Caenorhabditis elegans model.

    Science.gov (United States)

    Hwang, Jennifer; Mattei, Lisa M; VanArendonk, Laura G; Meneely, Philip M; Okeke, Iruka N

    2010-09-01

    Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase (cad) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli. To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans, and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC, which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.

  4. ``Black Holes" and Bacterial Pathogenicity: A Large Genomic Deletion that Enhances the Virulence of Shigella spp. and Enteroinvasive Escherichia coli

    Science.gov (United States)

    Maurelli, Anthony T.; Fernandez, Reinaldo E.; Bloch, Craig A.; Rode, Christopher K.; Fasano, Alessio

    1998-03-01

    Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylate (LDC) activity is present in ≈ 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these ``black holes,'' deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

  5. 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.

  6. Ferrite attenuator modulation improves antenna performance

    Science.gov (United States)

    Hooks, J. C.; Larson, S. G.; Shorkley, F. H.; Williams, B. T.

    1970-01-01

    Ferrite attenuator inserted into appropriate waveguide reduces the gain of the antenna element which is causing interference. Modulating the ferrite attenuator to change the antenna gain at the receive frequency permits ground tracking until the antenna is no longer needed.

  7. Effects of the nuclear localization of the N(pro) protein of classical swine fever virus on its virulence in pigs.

    Science.gov (United States)

    Li, Yongfeng; Shen, Liang; Sun, Yuan; Wang, Xiao; Li, Chao; Huang, Junhua; Chen, Jianing; Li, Lianfeng; Zhao, Bibo; Luo, Yuzi; Li, Su; Qiu, Hua-Ji

    2014-12-01

    The N(pro) protein of classical swine fever virus (CSFV) is localized in the cytoplasm and nucleus. However, it is unknown whether the nuclear localization of N(pro) correlates with the virulence of CSFV in the host. Previously, we showed that the N(pro) protein fused with interferon regulatory factor 3 (IRF3) was present only in the cytoplasm. Here, we generated and evaluated a recombinant CSFV vSM-IRF3 harboring the IRF3 gene inserted into the N(pro) gene of the highly virulent CSFV Shimen strain. Compared to the even nuclear and cytoplasmic distribution of the enhanced green fluorescent protein (EGFP)-N(pro) fusion expressed by the recombinant CSFV EGFP-CSFV, vSM-IRF3 expressed an IRF3-N(pro) fusion protein that only was localized in the cytoplasm. vSM-IRF3 was markedly attenuated in vitro and in vivo, and the inoculated pigs were completely protected from lethal CSFV challenge, whereas the parental virus as well as EGFP-CSFV exhibited a typical virulent phenotype. Taken together, the nuclear localization of N(pro) plays a significant role in the CSFV replication and virulence.

  8. Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model.

    Science.gov (United States)

    Sayeed, Sameera; Uzal, Francisco A; Fisher, Derek J; Saputo, Juliann; Vidal, Jorge E; Chen, Yue; Gupta, Phalguni; Rood, Julian I; McClane, Bruce A

    2008-01-01

    Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log-phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log-phase vegetative cultures of wild-type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre-incubation of wild-type CN3685 with a CPB-neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild-type CN3685 and that damage was prevented by pre-incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685-induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.

  9. 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).

  10. 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.

  11. 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.

  12. 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.

    2016-01-01

    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. PMID:27782090

  13. 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.

  14. 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...

  15. Caenorhabditis elegans reveals novel Pseudomonas aeruginosa virulence mechanism

    NARCIS (Netherlands)

    Utari, Putri Dwi; Quax, Wim J.

    2013-01-01

    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 possibi

  16. Correlation between antimicrobial resistance and virulence in Klebsiella pneumoniae.

    Science.gov (United States)

    Hennequin, C; Robin, F

    2016-03-01

    Klebsiella pneumoniae is responsible for a wide range of infections, including urinary tract infections, pneumonia, bacteremia, and liver abscesses. In addition to susceptible clinical isolates involved in nosocomial infections, multidrug-resistant (MDR) and hypervirulent (hvKP) strains have evolved separately in distinct clonal groups. The rapid geographic spread of these isolates is of particular concern. However, we still know little about the virulence of K. pneumoniae except for hvKP, whose secrets are beginning to be revealed. The treatment of K. pneumoniae infections is 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. Here, we review current knowledge on the topic.

  17. Mechanisms of geometrical seismic attenuation

    Directory of Open Access Journals (Sweden)

    Igor B. Morozov

    2011-07-01

    Full Text Available In several recent reports, we have explained the frequency dependence of the apparent seismic quality-factor (Q observed in many studies according to the effects of geometrical attenuation, which was defined as the zero-frequency limit of the temporal attenuation coefficient. In particular, geometrical attenuation was found to be positive for most waves traveling within the lithosphere. Here, we present three theoretical models that illustrate the origin of this geometrical attenuation, and we investigate the causes of its preferential positive values. In addition, we discuss the physical basis and limitations of both the conventional and new attenuation models. For waves in media with slowly varying properties, geometrical attenuation is caused by variations in the wavefront curvature, which can be both positive (for defocusing and negative (for focusing. In media with velocity/density contrasts, incoherent reflectivity leads to geometrical-attenuation coefficients which are proportional to the mean squared reflectivity and are always positive. For «coherent» reflectivity, the geometrical attenuation is approximately zero, and the attenuation process can be described according to the concept of «scattering Q». However, the true meaning of this parameter is in describing the mean reflectivity within the medium, and not that of the traditional resonator quality factor known in mechanics. The general conclusion from these models is that non-zero and often positive levels of geometrical attenuation are common in realistic, heterogeneous media, both observationally and theoretically. When transformed into the conventional Q-factor form, this positive geometrical attenuation leads to Q values that quickly increase with frequency. These predictions show that the positive frequency-dependent Q observed in many datasets might represent artifacts of the transformations of the attenuation coefficients into Q.

  1. The evolution and maintenance of virulence in microparasites.

    OpenAIRE

    Levin, B R

    1996-01-01

    In recent years, population and evolutionary biologists have questioned the traditional view that parasite-mediated morbidity and mortality¿virulence¿is a primitive character and an artifact of recent associations between parasites and their hosts. A number of hypotheses have been proposed that favor virulence and suggest that it will be maintained by natural selection. According to some of these hypotheses, the pathogenicity of HIV, Vibrio cholerae, Mycobacterium tuberculosis,theShigella,as ...

  2. 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

  3. Cross-protection against Salmonella Typhimurium infection conferred by a live attenuated Salmonella Enteritidis vaccine.

    Science.gov (United States)

    Nandre, Rahul M; Lee, Dajeong; Lee, John Hwa

    2015-01-01

    In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.

  4. Plasma membrane lipids and their role in fungal virulence.

    Science.gov (United States)

    Rella, Antonella; Farnoud, Amir M; Del Poeta, Maurizio

    2016-01-01

    There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies.

  5. Virulence parameters in the characterization of strains of Entamoeba histolytica.

    Science.gov (United States)

    Gomes, M A; Melo, M N; Pena, G P; Silva, E F

    1997-01-01

    Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic disease.

  6. Diverse virulent pneumophages infect Streptococcus mitis.

    Directory of Open Access Journals (Sweden)

    Siham Ouennane

    Full Text Available Streptococcus mitis has emerged as one of the leading causes of bacterial endocarditis and is related to Streptococcus pneumoniae. Antibiotic resistance has also increased among strains of S. mitis and S. pneumoniae. Phages are being reinvestigated as alternatives to antibiotics for managing infections. In this study, the two virulent phages Cp-1 (Podoviridae and Dp-1 (Siphoviridae, previously isolated from S. pneumoniae, were found to also infect S. mitis. Microbiological assays showed that both pneumophages could not only replicate in S. mitis but also produced more visible plaques on this host. However, the burst size and phage adsorption data were lower in S. mitis as compared to S. pneumoniae. A comparison of the genomes of each phage grown on both hosts produced identical nucleotide sequences, confirming that the same phages infect both bacterial species. We also discovered that the genomic sequence of podophage Cp-1 of the Félix d'Hérelle collection is different than the previously reported sequence and thus renamed SOCP.

  7. Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

    Science.gov (United States)

    Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

    2016-02-02

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain.

  8. Immunity to avian pneumovirus infection in turkeys following in ovo vaccination with an attenuated vaccine.

    Science.gov (United States)

    Worthington, Karen J; Sargent, Barbara A; Davelaar, F G; Jones, R C

    2003-03-28

    Fertile turkey eggs after 24 days of incubation were vaccinated in ovo with a commercial live attenuated subtype A avian pneumovirus (APV) vaccine. Hatchability was not adversely affected. When a high dose (10 times maximum commercial dose) of vaccine was tested in maternal antibody negative (MA-) eggs, mild clinical signs developed in a small proportion of the poults for 1-4 days only. Post-vaccination antibody titres at 3 weeks of age were significantly higher than those seen when the same dose was administered by eyedrop or spray at day-old. A low dose (end of shelf-life titre) of vaccine given to MA- eggs did not cause disease and vaccinated poults were 100% protected against virulent APV challenge at 3 or 5 weeks of age. Post-vaccination antibody titres reached significant levels at 3 weeks of age, whereas those from MA- poults vaccinated by spray at day-old with a similar low dose did not. In a 'worst-case' scenario, maternal antibody positive (MA+) poults vaccinated in ovo with the low dose were still 77% protected against clinical disease, despite lack of seroconversion. The recommended commercial dose of vaccine given to MA- eggs in ovo induced 100% protection against virulent APV challenge for up to 14 weeks of age, even though post-vaccination antibody titres had dropped to insignificant levels at this age. In ovo vaccination with a mixture of the recommended commercial doses of live APV and Newcastle disease (ND) vaccines had no detrimental affect on the efficacy of the APV vaccine. This is the first report of the successful use of an APV vaccine being given in ovo. The results indicate that for turkeys, in ovo vaccination with a live attenuated APV vaccine is safe and effective against virulent challenge and comparable with vaccination by conventional methods.

  9. Thogoto virus lacking interferon-antagonistic protein ML is strongly attenuated in newborn Mx1-positive but not Mx1-negative mice.

    Science.gov (United States)

    Pichlmair, Andreas; Buse, Johanna; Jennings, Stephanie; Haller, Otto; Kochs, Georg; Staeheli, Peter

    2004-10-01

    The Thogoto virus ML protein suppresses interferon synthesis in infected cells. Nevertheless, a virus mutant lacking ML remained highly pathogenic in standard laboratory mice. It was strongly attenuated, however, in mice carrying the interferon-responsive Mx1 gene found in wild mice, demonstrating that enhanced interferon synthesis is protective only if appropriate antiviral effector molecules are present. Our study shows that the virulence-enhancing effects of some viral interferon antagonists may escape detection in conventional animal models.

  10. 用等位基因特异性PCR/限制性片段长度多态性策略构建原发性青光眼致病基因CYP1B1的单倍型%Construction of CYP1B1 gene haplotypes predisposing to primary congenital glaucoma through allele-specific PCR/restriction fragment length polymorphism analysis

    Institute of Scientific and Technical Information of China (English)

    张爱平; 李圣杰; 欧阳琦; 汤荔; 王晓蕾; 吉建; 曹文俊

    2015-01-01

    目的 建立等位基因特异性PCR/限制性片段长度多态性(allele-specific PCR/restriction fragment length polymorphism,AS-PCR/RFLP)法检测原发性先天性青光眼(primary congenital glaucoma,PCG)致病基因CYP1B1常见单核苷酸多态性(single nucleotide polymorphisms,SNPs)及其单倍型的方法.方法 收集20例原发性先天性青光眼患者和20名正常对照为研究对象,首先经测序筛查SNP位点后,再分别以PCR-RFLP和AS-PCR/RFLP策略构建CYP1B1基因rs10012(S1)和rs1056827(S2)及rs1056836(S3)和rs1056837(S4)位点的单倍型,并对这两种策略进行评价.结果 测序共发现4个SNP位点,为第2外显子rs10012 G/C(S1)及rs1056827 T/G (S2)、第3外显子rs1056836C/G(S3)及rs1056837T/C(S4).这些位点在PCG患者和正常对照中的分布呈现不同特点,同时存在rs10012 (S1)和rs1056827(S2)位点的PCG患者和正常对照分别为10例(50%)和5人(25%);同时存在rs1056836 (S3)和rs1056837(S4)位点的PCG患者和正常对照分别为5例(25%)和2人(10%);均未发现上述SNP位点单独存在.确定各位点的分布特点后,首先用PCR-RFLP策略构建rs10012(S1)和rs1056827(S2)位点单倍型,显示杂合突变型除出现目的条带外还存在底物条带,虽提示同时存在rs10012(S1)和rs1056827 (S2),但仍不能证实存在位点间的连锁;AS-PCR/RFLP构建的结果显示,AS-PCR扩增rs10012(S1)位点获得阳性结果的同时,针对rs1056827 (S2)位点的特异性RFLP分析也获得阳性结果.AS-PCR/RFLP对位点rs1056836(S3)和rs1056837 (S4)的分析获得了类似的结果.应用AS-PCR/RFLP成功构建了C-G[rs10012(S1)-rs1056827(S2)]和G-C[rs1056836 (S3)-rs1056837 (S4)]两种单倍型.结论 应用AS-PCR/RFLP策略成功构建PCG致病基因CYP1B1单倍型,该方法准确高效特异可用于构建遗传性疾病基因的单倍型.%Objective To develop an allele-specific PCR (AS-PCR)/restriction fragment length polymorphism (RFLP) assay for CYP1B1 gene haplotypes

  11. Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR

    NARCIS (Netherlands)

    Wisselink, H.J.; Reek, F.H.; Vecht, U.; Stockhofe-Zurwieden, N.; Smits, M.A.; Smith, H.E.

    1999-01-01

    We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP EF ) and hi

  12. PSM-Mec-A Virulence Determinant that Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci.

    Science.gov (United States)

    Qin, Li; McCausland, Joshua W; Cheung, Gordon Y C; Otto, Michael

    2016-01-01

    PSM-mec is a secreted virulence factor that belongs to the phenol-soluble modulin (PSM) family of amphipathic, alpha-helical peptide toxins produced by Staphylococcus species. All known PSMs are core genome-encoded with the exception of PSM-mec, whose gene is found in specific sub-types of SCCmec methicillin resistance mobile genetic elements present in methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci. In addition to the cytolytic translational product, PSM-mec, the psm-mec locus encodes a regulatory RNA. In S. aureus, the psm-mec locus influences cytolytic capacity, methicillin resistance, biofilm formation, cell spreading, and the expression of other virulence factors, such as other PSMs, which results in a significant impact on immune evasion and disease. However, these effects are highly strain-dependent, which is possibly due to differences in PSM-mec peptide vs. psm-mec RNA-controlled effects. Here, we summarize the functional properties of PSM-mec and the psm-mec RNA molecule and their roles in staphylococcal pathogenesis and physiology.

  13. 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.

  14. Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides.

    Directory of Open Access Journals (Sweden)

    Charles H Li

    2011-06-01

    Full Text Available 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 heterothallic fungus: (+ sex allele encodes SexP and (- sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl (- mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+ isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc. Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types flanked by genes encoding triose phosphate transporter (TPT and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in

  15. Imaging Rayleigh wave attenuation with USArray

    Science.gov (United States)

    Bao, Xueyang; Dalton, Colleen A.; Jin, Ge; Gaherty, James B.; Shen, Yang

    2016-07-01

    The EarthScope USArray provides an opportunity to obtain detailed images of the continental upper mantle at an unprecedented scale. The majority of mantle models derived from USArray data to date contain spatial variations in seismic-wave speed; however, in many cases these data sets do not by themselves allow a non-unique interpretation. Joint interpretation of seismic attenuation and velocity models can improve upon the interpretations based only on velocity and provide important constraints on the temperature, composition, melt content, and volatile content of the mantle. The surface wave amplitudes that constrain upper-mantle attenuation are sensitive to factors in addition to attenuation, including the earthquake source excitation, focusing and defocusing by elastic structure, and local site amplification. Because of the difficulty of isolating attenuation from these other factors, little is known about the attenuation structure of the North American upper mantle. In this study, Rayleigh wave traveltime and amplitude in the period range 25-100 s are measured using an interstation cross-correlation technique, which takes advantage of waveform similarity at nearby stations. Several estimates of Rayleigh wave attenuation and site amplification are generated at each period, using different approaches to separate the effects of attenuation and local site amplification on amplitude. It is assumed that focusing and defocusing effects can be described by the Laplacian of the traveltime field. All approaches identify the same large-scale patterns in attenuation, including areas where the attenuation values are likely contaminated by unmodelled focusing and defocusing effects. Regionally averaged attenuation maps are constructed after removal of the contaminated attenuation values, and the variations in intrinsic shear attenuation that are suggested by these Rayleigh wave attenuation maps are explored.

  16. 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

  17. 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.

  18. 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.

  19. Identification of a novel virulence determinant with serum opacification activity in Streptococcus suis.

    Science.gov (United States)

    Baums, Christoph G; Kaim, Ute; Fulde, Marcus; Ramachandran, Girish; Goethe, Ralph; Valentin-Weigand, Peter

    2006-11-01

    Streptococcus suis serotype 2 is a porcine and human pathogen with adhesive and invasive properties. In other streptococci, large surface-associated proteins (>100 kDa) of the MSCRAMM family (microbial surface components recognizing adhesive matrix molecules) are key players in interactions with host tissue. In this study, we identified a novel opacity factor of S. suis (OFS) with structural homology to members of the MSCRAMM family. The N-terminal region of OFS is homologous to the respective regions of fibronectin-binding protein A (FnBA) of Streptococcus dysgalactiae and the serum opacity factor (SOF) of Streptococcus pyogenes. Similar to these two proteins, the N-terminal domain of OFS opacified horse serum. Serum opacification activity was detectable in sodium dodecyl sulfate extracts of wild-type S. suis but not in extracts of isogenic ofs knockout mutants. Heterologous expression of OFS in Lactococcus lactis demonstrated that a high level of expression of OFS is sufficient to provide surface-associated serum opacification activity. Furthermore, serum opacification could be inhibited by an antiserum against recombinant OFS. The C-terminal repetitive sequence elements of OFS differed significantly from the respective repeat regions of FnBA and SOF as well as from the consensus sequence of the fibronectin-binding repeats of MSCRAMMs. Accordingly, fibronectin binding was not detectable in recombinant OFS. To investigate the putative function of OFS in the pathogenesis of invasive S. suis diseases, piglets were experimentally infected with an isogenic mutant strain in which the ofs gene had been knocked out by an in-frame deletion. The mutant was severely attenuated in virulence but not in colonization, demonstrating that OFS represents a novel virulence determinant of S. suis.

  20. Corneal replication is an interferon response-independent bottleneck for virulence of herpes simplex virus 1 in the absence of virion host shutoff.

    Science.gov (United States)

    Pasieka, Tracy Jo; Menachery, Vineet D; Rosato, Pamela C; Leib, David A

    2012-07-01

    Herpes simplex viruses lacking the virion host shutoff function (Δvhs) are avirulent and hypersensitive to type I and type II interferon (IFN). In this study, we demonstrate that even in the absence of IFN responses in AG129 (IFN-αβγR(-/-)) mice, Δvhs remains highly attenuated via corneal infection but is fully virulent via intracranial infection. The data demonstrate that the interferon-independent inherent replication defect of Δvhs has a significant impact upon peripheral replication and neuroinvasion.

  1. 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.

  2. Yersinia pestis with regulated delayed attenuation as a vaccine candidate to induce protective immunity against plague.

    Science.gov (United States)

    Sun, Wei; Roland, Kenneth L; Kuang, Xiaoying; Branger, Christine G; Curtiss, Roy

    2010-03-01

    Two mutant strains of Yersinia pestis KIM5+, a Deltacrp mutant and a mutant with arabinose-dependent regulated delayed-shutoff crp expression (araC P(BAD) crp), were constructed, characterized in vitro, and evaluated for virulence, immunogenicity, and protective efficacy in mice. Both strains were highly attenuated by the subcutaneous (s.c.) route. The 50% lethal doses (LD(50)s) of the Deltacrp and araC P(BAD) crp mutants were approximately 1,000,000-fold and 10,000-fold higher than those of Y. pestis KIM5+, respectively, indicating that both strains were highly attenuated. Mice vaccinated s.c. with 3.8 x 10(7) CFU of the Deltacrp mutant developed high anti-Y. pestis and anti-LcrV serum IgG titers, both with a strong Th2 bias, and induced protective immunity against subcutaneous challenge with virulent Y. pestis (80% survival) but no protection against pulmonary challenge. Mice vaccinated with 3.0 x 10(4) CFU of the araC P(BAD) crp mutant also developed high anti-Y. pestis and anti-LcrV serum IgG titers but with a more balanced Th1/Th2 response. This strain induced complete protection against s.c. challenge and partial protection (70% survival) against pulmonary challenge. Our results demonstrate that arabinose-dependent regulated crp expression is an effective strategy to attenuate Y. pestis while retaining strong immunogenicity, leading to protection against the pneumonic and bubonic forms of plague.

  3. Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

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    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

  4. 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.

  5. Magnetoelectric Composite Based Microwave Attenuator

    Science.gov (United States)

    Tatarenko, A. S.; Srinivasan, G.

    2005-03-01

    Ferrite-ferroelectric composites are magnetoelectric (ME) due to their response to elastic and electromagnetic force fields. The ME composites are characterized by tensor permittivity, permeability and ME susceptibility. The unique combination of magnetic, electrical, and ME interactions, therefore, opens up the possibility of electric field tunable ferromagnetic resonance (FMR) based devices [1]. Here we discuss an ME attenuator operating at 9.3 GHz based on FMR in a layered sample consisting of lead magnesium niobate-lead titanate bonded to yttrium iron garnet (YIG) film on a gadolinium gallium garnet substrate. Electrical tuning is realized with the application of a control voltage due to ME effect; the shift is 0-15 Oe as E is increased from 0 to 3 kV/cm. If the attenuator is operated at FMR, the corresponding insertion loss will range from 25 dB to 2 dB. 1. S. Shastry and G. Srinivasan, M.I. Bichurin, V.M. Petrov, A.S. Tatarenko. Phys. Rev. B, 70 064416 (2004). - supported by grants the grants from the National Science Foundation (DMR-0302254), from Russian Ministry of Education (Å02-3.4-278) and from Universities of Russia Foundation (UNR 01.01.026).

  6. Attenuation map reconstruction from TOF PET data

    CERN Document Server

    Yang, Qingsong; Wang, Ge

    2013-01-01

    To reconstruct a radioactive tracer distribution with positron emission tomography (PET), the background attenuation correction is needed to eliminate image artifacts. Recent research shows that time-of-flight (TOF) PET data determine the attenuation sinogram up to a constant, and its gradient can be computed using an analytic algorithm. In this paper, we study a direct estimation of the sinogram only from TOF PET data. First, the gradient of the attenuation sinogram is estimated using the aforementioned algorithm. Then, a relationship is established to link the differential attenuation sinogram and the underlying attenuation background. Finally, an iterative algorithm is designed to determine the attenuation sinogram accurately and stably. A 2D numerical simulation study is conducted to verify the correctness of our proposed approach.

  7. 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

    2016-12-28

    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.

  8. 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.

  9. 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.

  10. The link between morphotype transition and virulence in Cryptococcus neoformans.

    Science.gov (United States)

    Wang, Linqi; Zhai, Bing; Lin, Xiaorong

    2012-01-01

    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. [Virulence factors and pathophysiology of extraintestinal pathogenic Escherichia coli].

    Science.gov (United States)

    Bidet, P; Bonarcorsi, S; Bingen, E

    2012-11-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) causing urinary tract infections, bacteraemia or meningitis are characterized by a particular genetic background (phylogenetic group B2 and D) and the presence, within genetic pathogenicity islands (PAI) or plasmids, of genes encoding virulence factors involved in adhesion to epithelia, crossing of the body barriers (digestive, kidney, bloodbrain), iron uptake and resistance to the immune system. Among the many virulence factors described, two are particularly linked with a pathophysiological process: type P pili PapGII adhesin is linked with acute pyelonephritis, in the absence of abnormal flow of urine, and the K1 capsule is linked with neonatal meningitis. However, if the adhesin PapGII appears as the key factor of pyelonephritis, such that its absence in strain causing the infection is predictive of malformation or a vesico-ureteral reflux, the meningeal virulence of E. coli can not be reduced to a single virulence factor, but results from a combination of factors unique to each clone, and an imbalance between the immune defenses of the host and bacterial virulence.

  12. Cyclo(valine-valine) inhibits Vibrio cholerae virulence gene expression.

    Science.gov (United States)

    Vikram, Amit; Ante, Vanessa M; Bina, X Renee; Zhu, Qin; Liu, Xinyu; Bina, James E

    2014-06-01

    Vibrio cholerae has been shown to produce a cyclic dipeptide, cyclo(phenylalanine-proline) (cFP), that functions to repress virulence factor production. The objective of this study was to determine if heterologous cyclic dipeptides could repress V. cholerae virulence factor production. To that end, three synthetic cyclic dipeptides that differed in their side chains from cFP were assayed for virulence inhibitory activity in V. cholerae. The results revealed that cyclo(valine-valine) (cVV) inhibited virulence factor production by a ToxR-dependent process that resulted in the repression of the virulence regulator aphA. cVV-dependent repression of aphA was found to be independent of known aphA regulatory genes. The results demonstrated that V. cholerae was able to respond to exogenous cyclic dipeptides and implicated the hydrophobic amino acid side chains on both arms of the cyclo dipeptide scaffold as structural requirements for inhibitory activity. The results further suggest that cyclic dipeptides have potential as therapeutics for cholera treatment.

  13. Genes involved in virulence of the entomopathogenic fungus Beauveria bassiana.

    Science.gov (United States)

    Valero-Jiménez, Claudio A; Wiegers, Harm; Zwaan, Bas J; Koenraadt, Constantianus J M; van Kan, Jan A L

    2016-01-01

    Pest insects cause severe damage to global crop production and pose a threat to human health by transmitting diseases. Traditionally, chemical pesticides (insecticides) have been used to control such pests and have proven to be effective only for a limited amount of time because of the rapid spread of genetic insecticide resistance. The basis of this resistance is mostly caused by (co)dominant mutations in single genes, which explains why insecticide use alone is an unsustainable solution. Therefore, robust solutions for insect pest control need to be sought in alternative methods such as biological control agents for which single-gene resistance is less likely to evolve. The entomopathogenic fungus Beauveria bassiana has shown potential as a biological control agent of insects, and insight into the mechanisms of virulence is essential to show the robustness of its use. With the recent availability of the whole genome sequence of B. bassiana, progress in understanding the genetics that constitute virulence toward insects can be made more quickly. In this review we divide the infection process into distinct steps and provide an overview of what is currently known about genes and mechanisms influencing virulence in B. bassiana. We also discuss the need for novel strategies and experimental methods to better understand the infection mechanisms deployed by entomopathogenic fungi. Such knowledge can help improve biocontrol agents, not only by selecting the most virulent genotypes, but also by selecting the genotypes that use combinations of virulence mechanisms for which resistance in the insect host is least likely to develop.

  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. Non-invasive prenatal diagnosis for homozygous α0-thalassemia based on paternal different SNP with allele-specific real-time fluorescence PCR: establishment and application%基于父源差异SNP位点的等位基因特异性实时荧光PCR方法建立及其在α0-地中海贫血无创产前诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    刘彦慧; 吴亚敏; 石少权; 娄季武; 马秋林; 何怡; 徐婉芳; 林洋洋; 周万军

    2012-01-01

    目的 建立并评价1种纯合α0-地中海贫血排除性无创产前诊断的等位基因特异性实时荧光PCR技术(AS-qPCR).方法 用PCR微测序技术,检测SEA高风险夫妇基因缺失区内9个SNP以确定双亲差异位点;然后以AS-qPCR技术检测孕妇血浆中父源差异SNP位点,判断父方是否将正常单倍型遗传给胎儿.结果 167个家系中,160个家系找到1个或多个有双亲差异的SNP位点,检测率为98.16%;94例检测到父源性正常等位基因SNP而免于创伤性产前诊断,检出率57.67%;所有家系的绒毛或羊水基因检测结果与本方法的符合率为100%.结论 采用AS-qPCR检测孕妇血浆游离DNA中双亲差异SNP位点的方法,在孕早期即可进行纯合α0-地中海贫血的排除性无创产前诊断,可使约50%高风险孕产妇免于创伤性产前诊断.%Objective To establish and evaluate an allele-specific real-time fluorescence PCR ( AS-qPCR) assay for non-invasive exclusive prenatal diagnosis of homozygous a -thalassemia. Methods Among 9 SNPs within the SEA deletion range, the different SNPs between parents were determined with PCR micro-sequencing technology. The paternal SNP was detected in maternal plasma DNA with AS-qPCR to determine whether the paternal normal haplotype had been inherited to the fetus. Results Among 167 families, 160 were found to have one or more parents-different SNPs with the detection rates over 98. 16%. The paternal normal SNPs were detected in 94 maternal plasma DNA. In this way, they avoided invasive prenatal diagnosis with a detection rate of 57.67%. These results were 100% coincidence with the results of CVS or amniocentesis. Conclusion Using AS-qPCR to detect the parents-difference SNPs in maternal plasma DNA in the early stage of gestation, is a non-invasive exclusive prenatal diagnosis of homozygous a?thalassemia. This method can make about 50% high-risk pregnant women to avoid invasive prenatal diagnosis.

  16. A mycobacterium ESX-1-secreted virulence factor with unique requirements for export.

    Directory of Open Access Journals (Sweden)

    Bryant McLaughlin

    2007-08-01

    Full Text Available Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1 locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6 system 1 (ESX-1 secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10, and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates.

  17. Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

    Science.gov (United States)

    Vilchèze, Catherine; Molle, Virginie; Carrère-Kremer, Séverine; Leiba, Jade; Mourey, Lionel; Shenai, Shubhada; Baronian, Grégory; Tufariello, Joann; Hartman, Travis; Veyron-Churlet, Romain; Trivelli, Xavier; Tiwari, Sangeeta; Weinrick, Brian; Alland, David; Guérardel, Yann; Jacobs, William R; Kremer, Laurent

    2014-05-01

    Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase

  18. Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Catherine Vilchèze

    2014-05-01

    Full Text Available Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of

  19. Optimal ultrasonic array focusing in attenuative media.

    Science.gov (United States)

    Ganguli, A; Gao, R X; Liang, K; Jundt, J

    2011-12-01

    This paper presents a parametric study on the efficiency of ultrasound focusing in an attenuative medium, using phased arrays. Specifically, an analytical model of ultrasound wave focusing in a homogeneous, isotropic and attenuative fluid with point sources is presented. Calculations based on the model have shown that in an attenuative medium, an optimum frequency exists for the best focusing performance for a particular size of aperture and focal distance. The effect of different f numbers on the focusing performance in the attenuative medium is further investigated. The information obtained from the analytical model provides insights into the design and installation of a phased transducer array for energy efficient wave focusing.

  20. Virulence parameters in the characterization of strains of Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Maria A GOMES

    1997-03-01

    Full Text Available Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic diseaseParâmetros de virulência na caracterização de Cepas de Entamoeba histolytica. A caracterização quanto a virulência das diferentes cepas de E. histolytica foi avaliada através de ensaios in vivo e in vitro. Discrepâncias nesta caracterização têm surgido quando se compara os diferentes ensaios. Avaliamos alguns parâmetros de virulência na caracterização de 5 cepas axênicas de E. histolytica. Estas cepas foram caracterizadas com relação à sua capacidade para induzir abscesso em fígado de hamster

  1. Wideband, 50 dB Attenuation Range Liquid Crystal Based Variable Optical Attenuator

    Institute of Scientific and Technical Information of China (English)

    J.J.; Pan; Henry; He; Eric; Zhang

    2003-01-01

    A compact variable optical attenuator, covering C and L bands with over 50 dB attenuation range, is realized using a single liquid crystal cell with a tilted fused silica coating compensating the cell's small residual birefringence.

  2. A Neisseria meningitidis NMB1966 mutant is impaired for invasion of respiratory epithelial cells, survival in human blood and for virulence in vivo.

    Science.gov (United States)

    Li, Ming-Shi; Chow, Noel Y S; Sinha, Sunita; Halliwell, Denise; Finney, Michelle; Gorringe, Andrew R; Watson, Mark W; Kroll, J Simon; Langford, Paul R; Webb, Steven A R

    2009-02-01

    We sought to determine whether NMB1966, encoding a putative ABC transporter, has a role in pathogenesis. Compared to its isogenic wild-type parent strain Neisseria meningitidis MC58, the NMB1966 knockout mutant was less adhesive and invasive for human bronchial epithelial cells, had reduced survival in human blood and was attenuated in a systemic mouse model of infection. The transcriptome of the wild-type and the NMB1966 mutant was compared. The data are consistent with a previous functional assignment of NMB1966 being the ABC transporter component of a glutamate transporter operon. Forty-seven percent of all the differentially regulated genes encoded known outer membrane proteins or pathways generating complex surface structures such as adhesins, peptidoglycan and capsule. The data show that NMB1966 has a role in virulence and that remodelling of the outer membrane and surface/structures is associated with attenuation of the NMB1966 mutant.

  3. Viability and Virulence of Entomopathogenic Nematodes Exposed to Ultraviolet Radiation.

    Science.gov (United States)

    Shapiro-Ilan, David I; Hazir, Selcuk; Lete, Luis

    2015-09-01

    Entomopathogenic nematodes (EPNs) can be highly effective biocontrol agents, but their efficacy can be reduced due to exposure to environmental stress such as from ultraviolet (UV) radiation. Our objectives were to 1) compare UV tolerance among a broad array of EPN species, and 2) investigate the relationship between reduced nematode viability (after exposure to UV) and virulence. Nematodes exposed to a UV radiation (254 nm) for 10 or 20 min were assessed separately for viability (survival) and virulence to Galleria mellonella. We compared 9 different EPN species and 15 strains: Heterorhabditis bacteriophora (Baine, fl11, Oswego, and Vs strains), H. floridensis (332), H. georgiana (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All, Cxrd, DD136, and Sal strains), S. feltiae (SN), S. rarum (17C&E), and S. riobrave (355). In viability assessments, steinernematids, particularly strains of S. carpocapsae, generally exhibited superior UV tolerance compared with the heterorhabditids. However, some heterorhabditids tended to be more tolerant than others, e.g., H. megidis and H. bacteriophora (Baine) were most susceptible and H. bacteriophora (Vs) was the only heterorhabditid that did not exhibit a significant effect after 10 min of exposure. All heterorhabditids experienced reduced viability after 20 min exposure though several S. carpocapsae strains did not. In total, after 10 or 20 min exposure, the viability of seven nematode strains did not differ from their non-UV exposed controls. In virulence assays, steinernematids (particularly S. carpocapsae strains) also tended to exhibit higher UV tolerance. However, in contrast to the viability measurements, all nematodes experienced a reduction in virulence relative to their controls. Correlation analysis revealed that viability among nematode strains is not necessarily related to virulence. In conclusion, our results indicate that the impact of UV varies substantially among EPNs, and viability alone

  4. Ultrasonic attenuation in cuprate superconductors

    Indian Academy of Sciences (India)

    T Gupta; D M Gaitonde

    2002-05-01

    We calculate the longitudinal ultrasonic attenuation rate (UAR) in clean d-wave superconductors in the Meissner and the mixed phases. In the Meissner phase we calculate the contribution of previously ignored processes involving the excitation of a pair of quasi-holes or quasi-particles. There is a contribution ∝ in the regime B ≪ F ≪ 0 and a contribution ∝ 1/ in the regime F ≪ B ≪ 0. We find that these contributions to the UAR are large and cannot be ignored. In the mixed phase, using a semi-classical description, we calculate the electronic quasi-particle contribution to the UAR which at very low , has a independent term proportional to $\\sqrt{H}$.

  5. 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.

  6. Regulated expression of virulence gene mviN provides protective immunity and colonization control of Salmonella in poultry.

    Science.gov (United States)

    Rubinelli, Peter M; Lee, Sang In; Roto, Stephanie M; Park, Si Hong; Ricke, Steven C

    2015-10-05

    Current live attenuated vaccines for control of Salmonella in poultry persist in the ceca and may persist in the environment. In this paper we report the construction and characterization of the vaccine efficacy of a Salmonella mutant strain with inducible mviN expression and rapid clearance from the host. The mutant was effective in oral immunization of the broiler chicken host against a virulent Salmonella oral challenge strain, having a mean 7×10(6)CFU/g in the ceca of unvaccinated controls compared to a mean 2×10(3)CFU/g in the ceca of vaccinated chickens at 4 weeks post-challenge (6 weeks of age). The mutant strain also demonstrated immunogenicity, reduced organ colonization, and rapid clearance in broiler chickens within 3 weeks of inoculation.

  7. DSM-5: ATTENUATED PSYCHOSIS SYNDROME?

    Directory of Open Access Journals (Sweden)

    Eduardo Fonseca-Pedrero

    2013-09-01

    Full Text Available Psychotic syndrome includes several devastating mental disorders characterized by a rupture of higher mental functions. The signs and symptoms of psychosis begin in adolescence or early adulthood and usually begin gradually and progress over time. Attenuated psychosis syndrome is a new DSM-5 diagnostic proposal which deals with identifying people at high-risk mental state (ARMS/UHR which may be a predictor of conversion to psychosis. The potential benefit would be that if psychotic disorder is treated more effectively in its early stages, it could produce a lasting beneficial effect that probably could not be achieved with later intervention. This syndrome has generated intense discussion in specialized scientific and professional forums, crisscrossing arguments in favor and against its inclusion. HRMS is preferentially evaluated in the adolescent or young adult population. HRMS evolution is associated with a higher rate of transition toward nonaffective psychosis, although it can evolve toward other mental disorders, remain stable or remit over time. Empirical evidence shows that early intervention seems to have a certain beneficial effect, although for now the results are still insufficient and contradictory. The lack of specificity of symptoms in predicting psychosis, presence of certain limitations (e.g., stigmatization, results found in early interventions and lack of empirical evidence, have led to include the attenuated psychosis syndrome in the DSM-5 Appendix III. The main benefits and limitations of including this supposed category, possible lessons learned from this type of study and future lines of action are discussed in the light of these findings.

  8. Pilus gene pool variation and the virulence of Corynebacterium diphtheriae clinical isolates during infection of a nematode.

    Science.gov (United States)

    Broadway, Melissa M; Rogers, Elizabeth A; Chang, Chungyu; Huang, I-Hsiu; Dwivedi, Prabhat; Yildirim, Suleyman; Schmitt, Michael P; Das, Asis; Ton-That, Hung

    2013-08-01

    Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.

  9. The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection.

    Science.gov (United States)

    Zimmerman, Shawn M; Michel, Frank; Hogan, Robert J; Lafontaine, Eric R

    2015-01-01

    Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥ 300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

  10. The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection.

    Directory of Open Access Journals (Sweden)

    Shawn M Zimmerman

    Full Text Available Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥ 300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

  11. The role of two periplasmic copper- and zinc-cofactored superoxide dismutases in the virulence of Salmonella choleraesuis.

    Science.gov (United States)

    Sansone, Assunta; Watson, Patricia R; Wallis, Timothy S; Langford, Paul R; Kroll, J Simon

    2002-03-01

    Periplasmic copper- and zinc-cofactored superoxide dismutases ([Cu,Zn]-SODs, SodC) of several Gram-negative pathogens can protect against superoxide-radical-mediated host defences, and thus contribute to virulence. This role has been previously defined for one [Cu,Zn]-SOD in various Salmonella serovars. Following the recent discovery of a second periplasmic [Cu,Zn]-SOD in Salmonella, the effect of knockout mutations in one or both of the original sodC-1 and the new sodC-2 on the virulence of the porcine pathogen Salmonella choleraesuis is investigated here. In comparison to wild-type, while sodC mutants--whether single or double--showed no impairment in growth, they all showed equally enhanced sensitivity to superoxide and a dramatically increased sensitivity to the combination of superoxide and nitric oxide in vitro. This observation had its correlate in experimental infection both ex vivo and in vivo. Mutation of sodC significantly impaired survival of S. choleraesuis in interferon gamma-stimulated murine macrophages compared to wild-type organisms, and all S. choleraesuis sodC mutants persisted in significantly lower numbers than wild-type in BALB/c (Ity(s)) and C3H/HeN (Ity(r)) mice after experimental infection, but in no experimental system were sodC-1 sodC-2 double mutants more attenuated than either single mutant. These data suggest that both [Cu,Zn]-SODs are needed to protect bacterial periplasmic or membrane components. While SodC plays a role in S. choleraesuis virulence, the data presented here suggest that this is through overcoming a threshold effect, probably achieved by acquisition of sodC-1 on a bacteriophage. Loss of either sodC gene confers maximum vulnerability to superoxide on S. choleraesuis.

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

    Science.gov (United States)

    Herrero-Fresno, Ana; Leekitcharoenphon, Pimlapas; Hendriksen, Rene S; Olsen, John E; Aarestrup, Frank M

    2014-03-01

    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 regions of unknown function (sb7-sb11, sb46, sb49-sb50 and sb54) were mapped by PCR in two strain collections: (i) 310 isolates of S. Typhimurium from human blood or stool samples, and from food, animal and environmental reservoirs; and (ii) 90 isolates belonging to other serovars. The region sb49-sb50 was found to be unique to S. Typhimurium and was strongly associated with strains isolated from blood samples (100  and 28.4 % of the blood and non-blood isolates, respectively). The region was cloned into LT2 and knocked out in SL1344, and these strains were compared to wild-type isogenic strains in in vitro assays used to predict virulence association. No difference in invasion of the Int407 human cell line was observed between the wild-type and mutated strains, but the isolate carrying the whole ST64B prophage was found to have a slightly better survival in blood. The study showed a high prevalence and a strong association between the prophage ST64B and isolates of S. Typhimurium collected from blood, and may indicate that such strains constitute a selected subpopulation within this serovar. Further studies are indicated to determine whether the slight increase in blood survival observed in the strain carrying ST64B genes is of paramount importance for systemic infections.

  13. 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....

  14. Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties

    Science.gov (United States)

    Orihuela, C. J.; Janssen, R.; Robb, C. W.; Watson, D. A.; Niesel, D. W.

    2000-01-01

    We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

  15. Attenuation characteristics of a light attenuator combined by polarizers with different extinction ratios

    Institute of Scientific and Technical Information of China (English)

    Huang Chong; Deng Peng; Zhao Shuang; Chen Hai-Qing

    2011-01-01

    This paper deals with a systematical analysis and an algorithm of attenuation characteristics of a light attenuator combined by n pieces of polarizers(n-LACP)whose extinction ratios are different from each other.The attenuation ratio expression of a two-LACP is deduced. We find that the monotonic attenuation interval depends on the first polarizer and that the attenuation range depends on the second one.For the three-LACP,a method for obtaining a monotonic attenuation interval is proposed.Moreover,the attenuation ratio expression is demonstrated.Analysis and experiment show that when the initial status of the three-LACP is at the maximum output,if the second or third polarizer rotates alone,the minimum attenuation ratios can reach K2-1and K3-1,respectively,and if the first polarizer rotates,a minimum attenuation ratio of K2-1K3-1can be obtained(K1,K2 and K3 represent the extinction ratios of the three polarizers in turn).Furthermore,the attenuation ratio expression of n-LACP and the relevant attenuation characteristics are proposed.The minimum attenuation ratio of an n-LACP is(K2K3...Kn)-1.

  16. Allele-specific regulation of MTTP expression influences the risk of ischemic heart disease

    DEFF Research Database (Denmark)

    Aminoff, Anna; Ledmyr, Helena; Thulin, Petra;

    2010-01-01

    Promoter polymorphisms in microsomal triglyceride transfer protein (MTTP) have been associated with decreased plasma lipids but an increased risk for ischemic heart disease (IHD), indicating that MTTP influences the susceptibility for IHD independent of plasma lipids. The objective of this study...

  17. Rapid identification of capybara (Hydrochaeris hydrochaeris through allele-specific PCR

    Directory of Open Access Journals (Sweden)

    Flávio Henrique-Silva

    2005-07-01

    Full Text Available The capybara is the largest rodent in the world and is widely distributed throughout Central and South America.  It is an animal of economic interest due to the pleasant flavor of its meat and higher protein content in comparison  to beef and pork meat.  The hide, hair and fat also have economic advantages. Thus,  as an animal with such high economic potential, it is the target of hunters, even though  hunting capybara is prohibited by law in Brazil.   Due to their  similarities,  capybara meat  is easily confused with  pork  meat.   This  occurs  upon  the apprehension of the  meat  from hunters, as well as in some restaurants that serve capybara meat that was slaughtered clandestinely. In both cases, when the meat is confiscated, those responsible for the crimes claim it is pork meat,  hindering  the enforcement of the law. A practical  course was ministered  to undergraduate biology students enrolled in the elective course Introduction to Genetic  Engineering  at Federal  University  of Sao Carlos (UFSCar, Sao Paulo  State, Brazil.  The  objective  of the  course was to establish  and  apply  a Polymerase  Chain  Reaction  (PCR assay to identify capybara meat and discriminate it in relation  to other types of meat,  including pork. Primers  were designed based  on 12S rRNA,  transthyretin and  growth  hormone  receptor  genes.  The primers generated  capybara specific fragments  of approximately 220, 290 and 330 bp for transthyretin,12S rRNA  and  growth  hormone  receptor,  respectively.   The  duplexes  developed  in the  present work can be used effectively to discriminate capybara meat  from other  animals,  contributing to combating predatory capybara hunting. The results were extensively discussed and the students have contributed to written a paper  to be submitted to a publication.

  18. Rapid identification of capybara (Hydrochaeris hydrochaeris) through allele-specific PCR

    OpenAIRE

    2005-01-01

    The capybara is the largest rodent in the world and is widely distributed throughout Central and South America.  It is an animal of economic interest due to the pleasant flavor of its meat and higher protein content in comparison  to beef and pork meat.  The hide, hair and fat also have economic advantages. Thus,  as an animal with such high economic potential, it is the target of hunters, even though  hunting capybara is prohibited by law in Brazil.   Due to their  similarities,  capybara me...

  19. Accurate quantitation of allele-specific expression patterns by analysis of DNA melting

    OpenAIRE

    Jeong, Sangkyun; Hahn, Yoonsoo; Rong, Qi; Pfeifer, Karl

    2007-01-01

    Epigenetic and genetic mechanisms can result in large differences in expression levels of the two alleles in a diploid organism. Furthermore, these differences may be critical to phenotypic variations among individuals. In this study, we present a novel procedure and algorithm to precisely and accurately quantitate the relative expression of each allele. This method uses the differential melting properties of DNAs differing at even a single base pair. By referring to the melting characteristi...

  20. Ploidy mosaicism and allele-specific gene expression differences in the allopolyploid Squalius alburnoides

    Directory of Open Access Journals (Sweden)

    Matos Isa

    2011-12-01

    Full Text Available Abstract Background Squalius alburnoides is an Iberian cyprinid fish resulting from an interspecific hybridisation between Squalius pyrenaicus females (P genome and males of an unknown Anaecypris hispanica-like species (A genome. S. alburnoides is an allopolyploid hybridogenetic complex, which makes it a likely candidate for ploidy mosaicism occurrence, and is also an interesting model to address questions about gene expression regulation and genomic interactions. Indeed, it was previously suggested that in S. alburnoides triploids (PAA composition silencing of one of the three alleles (mainly of the P allele occurs. However, not a whole haplome is inactivated but a more or less random inactivation of alleles varying between individuals and even between organs of the same fish was seen. In this work we intended to correlate expression differences between individuals and/or between organs to the occurrence of mosaicism, evaluating if mosaics could explain previous observations and its impact on the assessment of gene expression patterns. Results To achieve our goal, we developed flow cytometry and cell sorting protocols for this system generating more homogenous cellular and transcriptional samples. With this set-up we detected 10% ploidy mosaicism within the S. alburnoides complex, and determined the allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin in cells from liver and kidney of mosaic and non-mosaic individuals coming from different rivers over a wide geographic range. Conclusions Ploidy mosaicism occurs sporadically within the S. alburnoides complex, but in a frequency significantly higher than reported for other organisms. Moreover, we could exclude the influence of this phenomenon on the detection of variable allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin in cells from liver and kidney of triploid individuals. Finally, we determined that the expression patterns previously detected only in a narrow geographic range is not a local restricted phenomenon but is pervasive in rivers where S. pyrenaicus is sympatric with S. alburnoides. We discuss mechanisms that could lead to the formation of mosaic S. alburnoides and hypothesise about a relaxation of the mechanisms that impose a tight control over mitosis and ploidy control in mixoploids.

  1. Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data

    DEFF Research Database (Denmark)

    Favero, Francesco; Joshi, Tejal; Marquard, Andrea Marion;

    2015-01-01

    Background : Exome or whole genome deep sequencing of tumor DNA along with paired normal DNA can potentially provide a detailed picture of the somatic mutations that characterize the tumor. However, analysis of such sequence data can be complicated by the presence of normal cells in the tumor spe...

  2. DQB1*06:02 allele-specific expression varies by allelic dosage, not narcolepsy status

    DEFF Research Database (Denmark)

    Weiner Lachmi, Karin; Lin, Ling; Kornum, Birgitte Rahbek;

    2012-01-01

    The association of narcolepsy-cataplexy, a sleep disorder caused by the loss of hypocretin/orexin neurons in the hypothalamus, with DQA1*01:02-DQB1*06:02 is one of the tightest known single-allele human leukocyte antigen (HLA) associations. In this study, we explored genome-wide expression...... in peripheral white blood cells of 50 narcolepsy versus 47 controls (half of whom were DQB1*06:02 positive) and observed the largest differences between the groups in the signal from HLA probes. Further studies of HLA-DQ expression (mRNA and protein in a subset) in 125 controls and 147 narcolepsy cases did...... indicate that allelic dosage is transmitted into changes in heterodimer availability, a phenomenon that may explain the increased risk for narcolepsy in DQB1*06:02 homozygotes versus heterozygotes....

  3. Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer.

    Directory of Open Access Journals (Sweden)

    Kerstin B Meyer

    2008-05-01

    Full Text Available The recent whole-genome scan for breast cancer has revealed the FGFR2 (fibroblast growth factor receptor 2 gene as a locus associated with a small, but highly significant, increase in the risk of developing breast cancer. Using fine-scale genetic mapping of the region, it has been possible to narrow the causative locus to a haplotype of eight strongly linked single nucleotide polymorphisms (SNPs spanning a region of 7.5 kilobases (kb in the second intron of the FGFR2 gene. Here we describe a functional analysis to define the causative SNP, and we propose a model for a disease mechanism. Using gene expression microarray data, we observed a trend of increased FGFR2 expression in the rare homozygotes. This trend was confirmed using real-time (RT PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028. To elucidate which SNPs might be responsible for this difference, we examined protein-DNA interactions for the eight most strongly disease-associated SNPs in different breast cell lines. We identify two cis-regulatory SNPs that alter binding affinity for transcription factors Oct-1/Runx2 and C/EBPbeta, and we demonstrate that both sites are occupied in vivo. In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression. We propose a model in which the Oct-1/Runx2 and C/EBPbeta binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation.

  4. Allele-specific down-regulation of RPTOR expression induced by retinoids contributes to climate adaptations.

    Directory of Open Access Journals (Sweden)

    Chang Sun

    2010-10-01

    Full Text Available The mechanistic target of rapamycin (MTOR pathway regulates cell growth, energy homeostasis, apoptosis, and immune response. The regulatory associated protein of MTOR encoded by the RPTOR gene is a key component of this pathway. A previous survey of candidate genes found that RPTOR contains multiple SNPs with strong correlations between allele frequencies and climate variables, consistent with the action of selective pressures that vary across environments. Using data from a recent genome scan for selection signals, we honed in on a SNP (rs11868112 26 kb upstream to the transcription start site of RPTOR that exhibits the strongest association with temperature variables. Transcription factor motif scanning and mining of recently mapped transcription factor binding sites identified a binding site for POU class 2 homeobox 1 (POU2F1 spanning the SNP and an adjacent retinoid acid receptor (RAR binding site. Using expression quantification, chromatin immunoprecipitation (ChIP, and reporter gene assays, we demonstrate that POU2F1 and RARA do bind upstream of the RPTOR gene to regulate its expression in response to retinoids; this regulation is affected by the allele status at rs11868112 with the derived allele resulting in lower expression levels. We propose a model in which the derived allele influences thermogenesis or immune response by altering MTOR pathway activity and thereby increasing fitness in colder climates. Our results show that signatures of genetic adaptations can identify variants with functional effects, consistent with the idea that selection signals may be used for SNP annotation.

  5. A four-element based transposon system for allele specific tagging in plants – Theoretical considerations

    Indian Academy of Sciences (India)

    Sanjay Phogat; Pradeep Kumar Burma; Deepak Pental

    2000-03-01

    The two-element transposon constructs, utilizing either Ac/Ds or Spm/dSpm, allow random tagging of genes in heterologous model species, but are inadequate for directed tagging of specific alleles of agronomic importance. We propose the use of Ac/Ds in conjunction with Spm/dSpm to develop a four-element system for directed tagging of crop-specific alleles. The four-element based construct would include both Ds and dSpm along with relevant marker genes and would function in two steps. In the first step dSpm(Ds) stocks (a minimum of two) would be crossed to a line containing transposases of Spm and unlinked integrations would be selected from segregating population by the use of a negative selection marker to develop stocks representing integration of dSpm(Ds) at a large number of locations in the genome. Selections would be made for a line in which dSpm(Ds) shows partial or complete linkage to the allele of interest. In the second step selected line would be crossed to a line containing Ac transposase to induce transpositions of Ds element to linked sites thereby exploiting the natural tendency of Ds element to jump to linked sites. Unlinked jumps of dSpm(Ds) and linked jumps of Ds could be monitored by appropriate marker genes. The proposed model would allow tagging of allele of interest in chromosome addition lines and also help in the efficient use of genic male sterility systems for hybrid seed production by tightly marking the fertility restorer gene with a negative selection marker.

  6. Using Allele-Specific PCR with Molecular Beams as a Means for Genotyping the Diallelic Indels

    Energy Technology Data Exchange (ETDEWEB)

    Doktycz, M.J.; Weber, J.L. (Marshfield Medical Research Foundation)

    2000-06-01

    The first Specific Aim for this grant was to identify and characterize an average of 500 human insertion/deletion polymorphisms per grant year (1500 total). This task was carried out entirely at MMRF. They substantially exceeded this goal by confirming about 2,300 diallelic indels. Complete characterization information for these polymorphisms is available from the Marshfield web site. A manuscript describing results for the first 2,000 diallelic indels was published earlier this year in the American Journal of Human Genetics. The Second Specific Aim of the grant was to investigate and develop improved methods for analysis of diallelic polymorphisms using miniaturized DNA arrays. The initial genotyping technology efforts focused on various hybridization and extension protocols with oligo arrays on flow-through channel glass. Channel glass is a porous material that permits reagents to be passed through the arrays. They devoted roughly 19 months at the beginning of the grant in pursuit of this methodology, but for various technological reasons, progress was limited.

  7. Lack of allele-specific efficacy of a bivalent AMA1 malaria vaccine

    Directory of Open Access Journals (Sweden)

    Ellis Ruth D

    2010-06-01

    Full Text Available Abstract Background Extensive genetic diversity in vaccine antigens may contribute to the lack of efficacy of blood stage malaria vaccines. Apical membrane antigen-1 (AMA1 is a leading blood stage malaria vaccine candidate with extreme diversity, potentially limiting its efficacy against infection and disease caused by Plasmodium falciparum parasites with diverse forms of AMA1. Methods Three hundred Malian children participated in a Phase 2 clinical trial of a bivalent malaria vaccine that found no protective efficacy. The vaccine consists of recombinant AMA1 based on the 3D7 and FVO strains of P. falciparum adjuvanted with aluminum hydroxide (AMA1-C1. The gene encoding AMA1 was sequenced from P. falciparum infections experienced before and after immunization with the study vaccine or a control vaccine. Sequences of ama1 from infections in the malaria vaccine and control groups were compared with regard to similarity to the vaccine antigens using several measures of genetic diversity. Time to infection with parasites carrying AMA1 haplotypes similar to the vaccine strains with respect to immunologically important polymorphisms and the risk of infection with vaccine strain haplotypes were compared. Results Based on 62 polymorphic AMA1 residues, 186 unique ama1 haplotypes were identified among 315 ama1 sequences that were included in the analysis. Eight infections had ama1 sequences identical to 3D7 while none were identical to FVO. Several measures of genetic diversity showed that ama1 sequences in the malaria vaccine and control groups were comparable both at baseline and during follow up period. Pre- and post-immunization ama1 sequences in both groups all had a similar degree of genetic distance from FVO and 3D7 ama1. No differences were found in the time of first clinical episode or risk of infection with an AMA1 haplotype similar to 3D7 or FVO with respect to a limited set of immunologically important polymorphisms found in the cluster 1 loop of domain I of AMA1. Conclusion This Phase 2 trial of a bivalent AMA1 malaria vaccine found no evidence of vaccine selection or strain-specific efficacy, suggesting that the extreme genetic diversity of AMA1 did not account for failure of the vaccine to provide protection.

  8. Allele-specific expression of the low density lipoprotein receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Minnich, A.; Lussier-Cacan, S.; Roy, M. [Clincial Research Institute of Montreal, Quebec (Canada)

    1994-09-01

    Approximately 60% of familial hypercholesterolemia (FH) in French Canadians is due to a > 10 kb deletion of the promoter region of the gene encoding the low density lipoprotein (LDL) receptor (LDL-R), allowing determination of the influence of a single LDL-R allele on phenotypic expression of FH. Normal allele haplotypes of approximately 250 heterozygotes were determined with 7 RFLPs. In vitro maximal LDL-R activity of blood lymphocytes from a subset of approximately 150 heterozygotes, measured by immunocytofluorometry, was significantly higher (20 to 30%) in subjects with LDL-R normal allele haplotype G (n=11), and O (n=7) compared to the most frequent haplotype F (n=43), while no differences were observed among F, E (n=11), and the 2 other most prevalent haplotypes (n=43). LDL-R mRNA in these lymphocytes was significantly elevated 2.3-, 1.7-, and 1.8- fold, in G, O, and E, respectively, compared to F, while no significant differences were apparent between F and the other two most frequent haplotyes. Large interindividual variability in lymphocyte LDL-R mRNA levels and activity was observed even among subjects with the same LDL-R normal allele haplotype. However, maximally induced lymphocyte LDL-R mRNA levels correlated poorly with levels measured in freshly isolated cells (n=14). Relative to haplotype F (n=47 women (W), 39 men (M)), mean plasma LDL cholesterol levels adjusted for age and apolipoprotein E genotype were 5-10% lower in men and women with haplotypes G (n=16 W, 12 M) and O (n=8 W, 6 M), and 20% lower in 7 W with haplotype E. These results suggest that (1) normal LDL-R allele haplotype G and O may contain sequence variations which confer relatively high gene expression and (2) environmental and genetic influences other than the LDL-R gene contribute substantially to variability in LDL-R expression and plasma LDL cholesterol levels in French Canadian FH heterozygotes.

  9. Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate.

    Science.gov (United States)

    Langevin, Stanley A; Bowen, Richard A; Ramey, Wanichaya N; Sanders, Todd A; Maharaj, Payal D; Fang, Ying; Cornelius, Jennine; Barker, Christopher M; Reisen, William K; Beasley, David W C; Barrett, Alan D T; Kinney, Richard M; Huang, Claire Y-H; Brault, Aaron C

    2011-12-01

    The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.

  10. Deep-sequencing analysis of the mouse transcriptome response to infection with Brucella melitensis strains of differing virulence.

    Science.gov (United States)

    Wang, Fangkun; Hu, Sen; Liu, Wenxing; Qiao, Zujian; Gao, Yuzhe; Bu, Zhigao

    2011-01-01

    Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE) analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK) pathways. Further analysis employed gene ontology (GO) analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy.

  11. Deep-sequencing analysis of the mouse transcriptome response to infection with Brucella melitensis strains of differing virulence.

    Directory of Open Access Journals (Sweden)

    Fangkun Wang

    Full Text Available Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK pathways. Further analysis employed gene ontology (GO analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy.

  12. Temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys.

    Science.gov (United States)

    Arita, Minetaro; Shimizu, Hiroyuki; Nagata, Noriyo; Ami, Yasushi; Suzaki, Yuriko; Sata, Tetsutaro; Iwasaki, Takuya; Miyamura, Tatsuo

    2005-05-01

    Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is sometimes associated with serious neurological disorders. In this study, an attempt was made to identify molecular determinants of EV71 attenuation of neurovirulence in a monkey infection model. An infectious cDNA clone of the virulent strain of EV71 prototype BrCr was constructed; temperature-sensitive (ts) mutations of an attenuated strain of EV71 or of poliovirus (PV) Sabin vaccine strains were then introduced into the infectious clone. In vitro and in vivo phenotypes of the parental and mutant viruses were analysed in cultured cells and in cynomolgus monkeys, respectively. Mutations in 3D polymerase (3D(pol)) and in the 3' non-translated region (NTR), corresponding to ts determinants of Sabin 1, conferred distinct temperature sensitivity to EV71. An EV71 mutant [EV71(S1-3')] carrying mutations in the 5' NTR, 3D(pol) and in the 3' NTR showed attenuated neurovirulence, resulting in limited spread of virus in the central nervous system of monkeys. These results indicate that EV71 and PV1 share common genetic determinants of neurovirulence in monkeys, despite the distinct properties in their original pathogenesis.

  13. Attenuated strains of Mycobacterium avium subspecies paratuberculosis as vaccine candidates against Johne's disease.

    Science.gov (United States)

    Settles, Erik W; Kink, John A; Talaat, Adel

    2014-04-11

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease in ruminants. Johne's disease has a severe economic impact on the dairy industry in the USA and worldwide. In an effort to combat this disease, we screened several transposon mutants that were attenuated in the murine model of paratuberculosis for the potential use as live attenuated vaccines. Using the murine model, two vaccine candidates (pgs1360, pgs3965 with mutations of fabG2_2 and umaA1, respectively) were at or below the limit of detection for tissue colonization suggesting their low level persistence and hence safety. Prior to challenge, both candidates induced a M. paratuberculosis-specific IFN-γ, an indication of eliciting cell-mediated immunity. Following challenge with a virulent strain of M. paratuberculosis, the two vaccine candidates significantly reduced bacterial colonization in organs with reduced histological scores compared to control animals. In addition, one of the vaccine candidates (pgs3965) also induced IL-17a, a cytokine associated with protective immunity in mycobacterial infection. Our analysis suggested that the pgs3965 vaccine candidate is a potential live-