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Sample records for bacterial virulence gene

  1. A functional gene array for detection of bacterial virulence elements.

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    Crystal Jaing

    Full Text Available Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.

  2. A functional gene array for detection of bacterial virulence elements

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    Jaing, C

    2007-11-01

    We report our development of the first of a series of microarrays designed to detect pathogens with known mechanisms of virulence and antibiotic resistance. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. To validate our approach, we developed a first generation array targeting genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for microorganism detection and discrimination, measured the required target concentration, and assessed tolerance for mismatches between probe and target sequences. Mismatch tolerance is a priority for this application, due to DNA sequence variability among members of gene families. Arrays were created using the NimbleGen Maskless Array Synthesizer at Lawrence Livermore National Laboratory. Purified genomic DNA from combinations of one or more of the four target organisms, pure cultures of four related organisms, and environmental aerosol samples with spiked-in genomic DNA were hybridized to the arrays. Based on the success of this prototype, we plan to design further arrays in this series, with the goal of detecting all known virulence and antibiotic resistance gene families in a greatly expanded set of organisms.

  3. Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

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    Coady, Alison M; Murray, Anthony L.; Elliott, Diane G.; Rhodes, Linda 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...

  4. Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

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    Li, Yuan-Yuan; Wang, Ting; Gao, Song; Xu, Guang-Mei; Niu, Hua; Huang, Rui; Wu, Shu-Yan

    2016-02-01

    Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S. typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis in vivo. We found that spvB gene could decrease the LD50 of S. typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S. typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.

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

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    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.

  6. Bacterial Human Virulence Genes across Diverse Habitats As Assessed by In silico Analysis of Environmental Metagenomes

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    Søborg, Ditte A; Hendriksen, Niels B; Kilian, Mogens;

    2016-01-01

    and glacial ice. Homologs to 16 bacterial human virulence genes, involved in urinary tract infections, gastrointestinal diseases, skin diseases, and wound and systemic infections, showed global ubiquity. A principal component analysis did not demonstrate clear trends across the metagenomes with respect...

  7. A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.

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    Samuel A Shelburne

    2010-03-01

    Full Text Available Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS suggested that the transcriptional regulator catabolite control protein A (CcpA influences many of the same genes as the control of virulence (CovRS two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both DeltaccpA and DeltacovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, DeltaccpA and DeltacovRDeltaccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the DeltaccpA and DeltacovRDeltaccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection.

  8. Bacterial proteases and virulence

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    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....

  9. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

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    Mayfield, Jeffrey A; Liang, Zhong; Agrahari, Garima; Lee, Shaun W; Donahue, Deborah L; Ploplis, Victoria A; Castellino, Francis J

    2014-01-01

    The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

  10. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

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    Jeffrey A Mayfield

    Full Text Available The cluster of virulence sensor (CovS/responder (CovR two-component operon (CovRS regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448, containing wild-type (WT CovRS (5448/CovR+S+, or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

  11. Mammalian cell entry genes in Streptomyces may provide clues to the evolution of bacterial virulence

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    Clark, Laura C.; Seipke, Ryan F.; Prieto, Pilar; Willemse, Joost; van Wezel, Gilles P.; Hutchings, Matthew I.; Hoskisson, Paul A.

    2013-01-01

    Understanding the evolution of virulence is key to appreciating the role specific loci play in pathogenicity. Streptomyces species are generally non-pathogenic soil saprophytes, yet within their genome we can find homologues of virulence loci. One example of this is the mammalian cell entry (mce) locus, which has been characterised in Mycobacterium tuberculosis. To investigate the role in Streptomyces we deleted the mce locus and studied its impact on cell survival, morphology and interaction with other soil organisms. Disruption of the mce cluster resulted in virulence towards amoebae (Acanthamoeba polyphaga) and reduced colonization of plant (Arabidopsis) models, indicating these genes may play an important role in Streptomyces survival in the environment. Our data suggest that loss of mce in Streptomyces spp. may have profound effects on survival in a competitive soil environment, and provides insight in to the evolution and selection of these genes as virulence factors in related pathogenic organisms. PMID:23346366

  12. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

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    Li, Yiping; Ingmer, Hanne; Madsen, Mogens;

    2008-01-01

    of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C......-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under...

  13. Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

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    Wilson, J. W.; Ott, C. M.; zuBentrup, K. Honer; Ramamurthy R.; Quick, L.; Porwollik, S.; Cheng, P.; McClellan, M.; Tsaprailis, G.; Radabaugh, T.; Hunt, A.; Fernandez, D.; Richter, E.; Shah, M.; Kilcoyne, M.; Joshi, L.; Nelman-Gonzalez, M.; Hing, S.; Parra, M.; Dumaras, P.; Norwood, K.; Nickerson, C. A.; Bober, R.; Devich, J.; Ruggles, A.

    2007-01-01

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth.

  14. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

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    Li, Yiping; Ingmer, Hanne; Madsen, Mogens

    2008-01-01

    of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C...... in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process....

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

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    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

  16. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

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    Bang Dang D

    2008-06-01

    Full Text Available Abstract Background Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C. jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process.

  17. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens;

    2008-01-01

    Background Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression....... jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria......-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under...

  18. 筛选细菌毒力基因方法的研究进展%Advance on methods of screening bacterial virulence genes

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    张念章; 储岳峰; 贺英; 逯忠新

    2011-01-01

    The research indicated that bacterial pathogenesis involves the interaction of several virulence genes. However, as an increasing number of microbial genome sequencing,the study of its virulence genes is lagging. Not only this affect pathogenesis and other basic research, but also restrict the development of antibiotics and vaccine. This article introduces the methods of screening bacterial virulence genes and its application in four aspects such as bioinformatics and screening genes of differential expressed, and also includes the methods to study the interaction between pathogen and host. The purpose is to develop ideas for discovering and studying new virulence factors of pathogen, and find out the relation between pathogen and host.%细菌的致病过程涉及多个毒力基因表达产物的相互作用.然而,随着越来越多的微生物基因组测序工作的相继或即将完成,对其毒力基因的研究却相对滞后.这不但影响了致病机理等基础研究的进展,还成为制约抗菌药物和疫苗研发的瓶颈.本文从生物信息学、筛选差异表达基因等4个方面介绍了筛选细茵毒力基因的方法及其应用,并综合了研究致病茵与宿主间相互作用关系的方法.为发现和研究致病茵新的毒力因子,理顺病原与宿主关系开拓了思路.

  19. Phage-mediated dispersal of biofilm and distribution of bacterial virulence genes is induced by quorum sensing.

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    Friederike S Rossmann

    2015-02-01

    Full Text Available The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 μM of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5 showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.

  20. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

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    Hensel, M; Shea, J E; Waterman, S R; Mundy, R; Nikolaus, T; Banks, G; Vazquez-Torres, A; Gleeson, C; Fang, F C; Holden, D W

    1998-10-01

    The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

  1. Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

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    Park, Duck Hwan; Mirabella, Rossana; Bronstein, Philip A; Preston, Gail M; Haring, Michel A; Lim, Chun Keun; Collmer, Alan; Schuurink, Robert C

    2010-10-01

    Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

  2. Genetic Modulation of c-di-GMP Turnover Affects Multiple Virulence Traits and Bacterial Virulence in Rice Pathogen Dickeya zeae

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    Chen, Yufan; Lv, Mingfa; Liao, Lisheng; Gu, Yanfang; Liang, Zhibin; Shi, Zurong; Liu, Shiyin; Zhou, Jianuan; Zhang, Lianhui

    2016-01-01

    The frequent outbreaks of rice foot rot disease caused by Dickeya zeae have become a significant concern in rice planting regions and countries, but the regulatory mechanisms that govern the virulence of this important pathogen remain vague. Given that the second messenger cyclic di-GMP (c-di-GMP) is associated with modulation of various virulence-related traits in various microorganisms, here we set to investigate the role of the genes encoding c-di-GMP metabolism in the regulation of the bacterial physiology and virulence by construction all in-frame deletion mutants targeting the annotated c-di-GMP turnover genes in D. zeae strain EC1. Phenotype analyses identified individual mutants showing altered production of exoenzymes and phytotoxins, biofilm formation and bacterial motilities. The results provide useful clues and a valuable toolkit for further characterization and dissection of the regulatory complex that modulates the pathogenesis and persistence of this important bacterial pathogen. PMID:27855163

  3. Citrate uptake into Pectobacterium atrosepticum is critical for bacterial virulence.

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    Urbany, Claude; Neuhaus, H Ekkehard

    2008-05-01

    To analyze whether metabolite import into Pectobacterium atrosepticum cells affects bacterial virulence, we investigated the function of a carrier which exhibits significant structural homology to characterized carboxylic-acid transport proteins. The corresponding gene, ECA3984, previously annotated as coding for a Na(+)/sulphate carrier, in fact encodes a highly specific citrate transporter (Cit1) which is energized by the proton-motive force. Expression of the cit1 gene is stimulated by the presence of citrate in the growth medium and is substantial during growth of P. atrosepticum on potato tuber tissue. Infection of tuber tissue with P. atrosepticum leads to reduced citrate levels. P. atrosepticum insertion mutants, lacking the functional Cit1 protein, did not grow in medium containing citrate as the sole carbon source, showed a substantially reduced ability to macerate potato tuber tissue, and did not provoke reduced citrate levels in the plant tissue upon infection. We propose that citrate uptake into P. atrosepticum is critical for full bacterial virulence.

  4. Regulation of bacterial virulence by Csr (Rsm) systems.

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    Vakulskas, Christopher A; Potts, Anastasia H; Babitzke, Paul; Ahmer, Brian M M; Romeo, Tony

    2015-06-01

    Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5' untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens.

  5. 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.

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

    Science.gov (United States)

    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. Impact of space flight on bacterial virulence and antibiotic susceptibility

    OpenAIRE

    Taylor, P.W.

    2015-01-01

    Manned space flight induces a reduction in immune competence among crew and is likely to cause deleterious changes to the composition of the gastrointestinal, nasal, and respiratory bacterial flora, leading to an increased risk of infection. The space flight environment may also affect the susceptibility of microorganisms within the spacecraft to antibiotics, key components of flown medical kits, and may modify the virulence characteristics of bacteria and other microorganisms that contaminat...

  8. Virulence factors genes in enterococci isolated from beavers (Castor fiber).

    Science.gov (United States)

    Lauková, Andrea; Strompfová, Viola; Kandričáková, Anna; Ščerbová, Jana; Semedo-Lemsaddek, Teresa; Miltko, Renata; Belzecki, Grzegorz

    2015-03-01

    Only limited information exists concerning the microbiota in beaver (Castor fiber). This study has been focused on the virulence factors genes detection in enterococci from beavers. In general, animals are not affected by enterococcal infections, but they can be a reservoir of, e.g. pathogenic strains. Moreover, detection of virulence factors genes in enterococci from beavers was never tested before. Free-living beavers (12), male and female (age 4-5 years) were caught in the north-east part of Poland. Sampling of lower gut and faeces was provided according to all ethical rules for animal handling. Samples were treated using a standard microbiological method. Pure bacterial colonies were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification system. Virulence factors genes-gelE (gelatinase), agg (aggregation), cylA (cytolysin A), efaAfs (adhesin Enterococcus faecalis), efaAfm (adhesin Enterococcus faecium) and esp (surface protein) were tested by PCR. Moreover, gelatinase and antibiotic phenotypes were tested. Species detected were Enterococcus thailandicus, E. faecium, E. faecalis and Enterococcus durans. In literature, enterococcal species distribution was never reported yet up to now. Strains were mostly sensitive to antibiotics. Vancomycin-resistant E. faecalis EE9Tr1 possess cylA, efaAfs, esp and gelE genes. Strains were aggregation substance genes absent. Adhesin E. faecium (efaAfm) gene was detected in two of three E. faecium strains, but it was present also in E. thailandicus. Esp gene was present in EE9Tr1 and E. durans EDTr92. The most detected were gelE, efaAfm genes; in EF 4Hc1 also gelatinase phenotype was found. Strains with virulence factors genes will be tested for their sensitivity to antimicrobial enterocins.

  9. Rapid Identification of Bacterial Virulence Factors

    Science.gov (United States)

    2014-04-15

    Arsenal, Alabama 35898 14. MARKED FOR D CODE IS. ITEW NO. 16. STOCK/PART NO. DESCRIPTION (Indicate number of shipping containers - type of...In mammals , the coatomer can only be recruited by membranes associated to ADP-ribosylation factors (ARFs), which are small GTP-binding proteins; the...different species from fungi to mammals . The high degree of conservation, in special in the nudC domain, suggests that they are genes with essential

  10. 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.

  11. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen

    KAUST Repository

    Weynberg, Karen D.

    2015-12-08

    Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.

  12. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen

    Science.gov (United States)

    Weynberg, Karen D.; Voolstra, Christian R.; Neave, Matthew J.; Buerger, Patrick; van Oppen, Madeleine J. H.

    2015-01-01

    Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements. PMID:26644037

  13. Impact of space flight on bacterial virulence and antibiotic susceptibility

    Directory of Open Access Journals (Sweden)

    Taylor PW

    2015-07-01

    Full Text Available Peter William Taylor School of Pharmacy, University College London, London, UK Abstract: Manned space flight induces a reduction in immune competence among crew and is likely to cause deleterious changes to the composition of the gastrointestinal, nasal, and respiratory bacterial flora, leading to an increased risk of infection. The space flight environment may also affect the susceptibility of microorganisms within the spacecraft to antibiotics, key components of flown medical kits, and may modify the virulence characteristics of bacteria and other microorganisms that contaminate the fabric of the International Space Station and other flight platforms. This review will consider the impact of true and simulated microgravity and other characteristics of the space flight environment on bacterial cell behavior in relation to the potential for serious infections that may appear during missions to astronomical objects beyond low Earth orbit. Keywords: Staphylococcus aureus, International Space Station, microgravity, bacterial phenotypes, low-shear modeled microgravity, spacecraft contamination

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

    Science.gov (United States)

    An, Shi-qi; Caly, Delphine L; McCarthy, Yvonne; Murdoch, Sarah L; Ward, Joseph; Febrer, Melanie; Dow, J Maxwell; Ryan, Robert P

    2014-10-01

    Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

  16. Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

    Directory of Open Access Journals (Sweden)

    Shi-qi An

    2014-10-01

    Full Text Available Bis-(3',5' cyclic di-guanylate (cyclic di-GMP is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc. This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d∼2 µM. Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

  17. Impact of space flight on bacterial virulence and antibiotic susceptibility.

    Science.gov (United States)

    Taylor, Peter William

    2015-01-01

    Manned space flight induces a reduction in immune competence among crew and is likely to cause deleterious changes to the composition of the gastrointestinal, nasal, and respiratory bacterial flora, leading to an increased risk of infection. The space flight environment may also affect the susceptibility of microorganisms within the spacecraft to antibiotics, key components of flown medical kits, and may modify the virulence characteristics of bacteria and other microorganisms that contaminate the fabric of the International Space Station and other flight platforms. This review will consider the impact of true and simulated microgravity and other characteristics of the space flight environment on bacterial cell behavior in relation to the potential for serious infections that may appear during missions to astronomical objects beyond low Earth orbit.

  18. Development of Quorum-Based Anti-Virulence Therapeutics Targeting Gram-Negative Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Wen Shan Yew

    2013-08-01

    Full Text Available Quorum sensing is a cell density-dependent signaling phenomenon used by bacteria for coordination of population-wide phenotypes, such as expression of virulence genes, antibiotic resistance and biofilm formation. Lately, disruption of bacterial communication has emerged as an anti-virulence strategy with enormous therapeutic potential given the increasing incidences of drug resistance in pathogenic bacteria. The quorum quenching therapeutic approach promises a lower risk of resistance development, since interference with virulence generally does not affect the growth and fitness of the bacteria and, hence, does not exert an associated selection pressure for drug-resistant strains. With better understanding of bacterial communication networks and mechanisms, many quorum quenching methods have been developed against various clinically significant bacterial pathogens. In particular, Gram-negative bacteria are an important group of pathogens, because, collectively, they are responsible for the majority of hospital-acquired infections. Here, we discuss the current understanding of existing quorum sensing mechanisms and present important inhibitory strategies that have been developed against this group of pathogenic bacteria.

  19. Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis

    Directory of Open Access Journals (Sweden)

    Cheng-Yen Kao

    2016-02-01

    Full Text Available Helicobacter pylori pathogenesis and disease outcomes are mediated by a complex interplay between bacterial virulence factors, host, and environmental factors. After H. pylori enters the host stomach, four steps are critical for bacteria to establish successful colonization, persistent infection, and disease pathogenesis: (1 Survival in the acidic stomach; (2 movement toward epithelium cells by flagella-mediated motility; (3 attachment to host cells by adhesins/receptors interaction; (4 causing tissue damage by toxin release. Over the past 20 years, the understanding of H. pylori pathogenesis has been improved by studies focusing on the host and bacterial factors through epidemiology researches and molecular mechanism investigations. These include studies identifying the roles of novel virulence factors and their association with different disease outcomes, especially the bacterial adhesins, cag pathogenicity island, and vacuolating cytotoxin. Recently, the development of large-scale screening methods, including proteomic, and transcriptomic tools, has been used to determine the complex gene regulatory networks in H. pylori. In addition, a more available complete genomic database of H. pylori strains isolated from patients with different gastrointestinal diseases worldwide is helpful to characterize this bacterium. This review highlights the key findings of H. pylori virulence factors reported over the past 20 years.

  20. 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...

  1. Virulent bacterial infection improves aversive learning performance in Drosophila melanogaster.

    Science.gov (United States)

    Babin, Aurélie; Kolly, Sylvain; Kawecki, Tadeusz J

    2014-10-01

    Virulent infections are expected to impair learning ability, either as a direct consequence of stressed physiological state or as an adaptive response that minimizes diversion of energy from immune defense. This prediction has been well supported for mammals and bees. Here, we report an opposite result in Drosophila melanogaster. Using an odor-mechanical shock conditioning paradigm, we found that intestinal infection with bacterial pathogens Pseudomonas entomophila or Erwinia c. carotovora improved flies' learning performance after a 1h retention interval. Infection with P. entomophila (but not E. c. carotovora) also improved learning performance after 5 min retention. No effect on learning performance was detected for intestinal infections with an avirulent GacA mutant of P. entomophila or for virulent systemic (hemocoel) infection with E. c. carotovora. Assays of unconditioned responses to odorants and shock do not support a major role for changes in general responsiveness to stimuli in explaining the changes in learning performance, although differences in their specific salience for learning cannot be excluded. Our results demonstrate that the effects of pathogens on learning performance in insects are less predictable than suggested by previous studies, and support the notion that immune stress can sometimes boost cognitive abilities.

  2. Modulation of Candida albicans virulence by bacterial biofilms on titanium surfaces.

    Science.gov (United States)

    Cavalcanti, Yuri Wanderley; Wilson, Melanie; Lewis, Michael; Del-Bel-Cury, Altair Antoninha; da Silva, Wander José; Williams, David W

    2016-01-01

    Whilst Candida albicans occurs in peri-implant biofilms, its role in peri-implantitis remains unclear. This study therefore examined the virulence of C. albicans in mixed-species biofilms on titanium surfaces. Biofilms of C. albicans (Ca), C. albicans with streptococci (Streptococcus sanguinis, S. mutans) (Ca-Ss-Sm) and those incorporating Porphyromonas gingivalis (Ca-Pg and Ca-Ss-Sm-Pg) were developed. Expression of C. albicans genes associated with adhesion (ALS1, ALS3, HWP1) and hydrolytic enzymes (SAP2, SAP4, SAP6, PLD1) was measured and hyphal production by C. albicans quantified. Compared with Ca biofilms, significant (palbicans expressed virulence factors in biofilms that could contribute to peri-implantitis, but this was dependent on associated bacterial species.

  3. 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.

  4. Pathogenicity of a Very Virulent Strain of Marek's Disease Herpesvirus Cloned as Infectious Bacterial Artificial Chromosomes

    Directory of Open Access Journals (Sweden)

    Lorraine P. Smith

    2011-01-01

    Full Text Available Bacterial artificial chromosome (BAC vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130 of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.

  5. Involvement of a 1-Cys peroxiredoxin in bacterial virulence.

    Directory of Open Access Journals (Sweden)

    Gilberto Hideo Kaihami

    2014-10-01

    Full Text Available The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H(2O(2 and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a

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

    Directory of Open Access Journals (Sweden)

    Ying Tianyi

    2010-06-01

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

  7. A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine

    DEFF Research Database (Denmark)

    Nielsen, Alex Toftgaard; Dolganov, N. A.; Rasmussen, Thomas

    2010-01-01

    A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene...... expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co...... into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients....

  8. Twin RNA polymerase-associated proteins control virulence gene expression in Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    James C Charity

    2007-06-01

    Full Text Available The MglA protein is the only known regulator of virulence gene expression in Francisella tularensis, yet it is unclear how it functions. F. tularensis also contains an MglA-like protein called SspA. Here, we show that MglA and SspA cooperate with one another to control virulence gene expression in F. tularensis. Using a directed proteomic approach, we show that both MglA and SspA associate with RNA polymerase (RNAP in F. tularensis, and that SspA is required for MglA to associate with RNAP. Furthermore, bacterial two-hybrid and biochemical assays indicate that MglA and SspA interact with one another directly. Finally, through genome-wide expression analyses, we demonstrate that MglA and SspA regulate the same set of genes. Our results suggest that a complex involving both MglA and SspA associates with RNAP to positively control virulence gene expression in F. tularensis. The F. tularensis genome is unusual in that it contains two genes encoding different alpha subunits of RNAP, and we show here that these two alpha subunits are incorporated into RNAP. Thus, as well as identifying SspA as a second critical regulator of virulence gene expression in F. tularensis, our findings provide a framework for understanding the mechanistic basis for virulence gene control in a bacterium whose transcription apparatus is unique.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Lauri Mikonranta

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

  11. Chamaecyparis obtusa Suppresses Virulence Genes in Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Eun-Hee Kim

    2016-01-01

    Full Text Available Chamaecyparis obtusa (C. obtusa is known to have antimicrobial effects and has been used as a medicinal plant and in forest bathing. This study aimed to evaluate the anticariogenic activity of essential oil of C. obtusa on Streptococcus mutans, which is one of the most important bacterial causes of dental caries and dental biofilm formation. Essential oil from C. obtusa was extracted, and its effect on bacterial growth, acid production, and biofilm formation was evaluated. C. obtusa essential oil exhibited concentration-dependent inhibition of bacterial growth over 0.025 mg/mL, with 99% inhibition at a concentration of 0.2 mg/mL. The bacterial biofilm formation and acid production were also significantly inhibited at the concentration greater than 0.025 mg/mL. The result of LIVE/DEAD® BacLight™ Bacterial Viability Kit showed a concentration-dependent bactericidal effect on S. mutans and almost all bacteria were dead over 0.8 mg/mL. Real-time PCR analysis showed that gene expression of some virulence factors such as brpA, gbpB, gtfC, and gtfD was also inhibited. In GC and GC-MS analysis, the major components were found to be α-terpinene (40.60%, bornyl acetate (12.45%, α-pinene (11.38%, β-pinene (7.22%, β-phellandrene (3.45%, and α-terpinolene (3.40%. These results show that C. obtusa essential oil has anticariogenic effect on S. mutans.

  12. Chamaecyparis obtusa Suppresses Virulence Genes in Streptococcus mutans.

    Science.gov (United States)

    Kim, Eun-Hee; Kang, Sun-Young; Park, Bog-Im; Kim, Young-Hoi; Lee, Young-Rae; Hoe, Jin-Hee; Choi, Na-Young; Ra, Ji-Young; An, So-Youn; You, Yong-Ouk

    2016-01-01

    Chamaecyparis obtusa (C. obtusa) is known to have antimicrobial effects and has been used as a medicinal plant and in forest bathing. This study aimed to evaluate the anticariogenic activity of essential oil of C. obtusa on Streptococcus mutans, which is one of the most important bacterial causes of dental caries and dental biofilm formation. Essential oil from C. obtusa was extracted, and its effect on bacterial growth, acid production, and biofilm formation was evaluated. C. obtusa essential oil exhibited concentration-dependent inhibition of bacterial growth over 0.025 mg/mL, with 99% inhibition at a concentration of 0.2 mg/mL. The bacterial biofilm formation and acid production were also significantly inhibited at the concentration greater than 0.025 mg/mL. The result of LIVE/DEAD® BacLight™ Bacterial Viability Kit showed a concentration-dependent bactericidal effect on S. mutans and almost all bacteria were dead over 0.8 mg/mL. Real-time PCR analysis showed that gene expression of some virulence factors such as brpA, gbpB, gtfC, and gtfD was also inhibited. In GC and GC-MS analysis, the major components were found to be α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%). These results show that C. obtusa essential oil has anticariogenic effect on S. mutans.

  13. Deciphering the role of coumarin as a novel quorum sensing inhibitor suppressing virulence phenotypes in bacterial pathogens.

    Science.gov (United States)

    Gutiérrez-Barranquero, José A; Reen, F Jerry; McCarthy, Ronan R; O'Gara, Fergal

    2015-04-01

    The rapid unchecked rise in antibiotic resistance over the last few decades has led to an increased focus on the need for alternative therapeutic strategies for the treatment and clinical management of microbial infections. In particular, small molecules that can suppress microbial virulence systems independent of any impact on growth are receiving increased attention. Quorum sensing (QS) is a cell-to-cell signalling communication system that controls the virulence behaviour of a broad spectrum of bacterial pathogens. QS systems have been proposed as an effective target, particularly as they control biofilm formation in pathogens, a key driver of antibiotic ineffectiveness. In this study, we identified coumarin, a natural plant phenolic compound, as a novel QS inhibitor, with potent anti-virulence activity in a broad spectrum of pathogens. Using a range of biosensor systems, coumarin was active against short, medium and long chain N-acyl-homoserine lactones, independent of any effect on growth. To determine if this suppression was linked to anti-virulence activity, key virulence systems were studied in the nosocomial pathogen Pseudomonas aeruginosa. Consistent with suppression of QS, coumarin inhibited biofilm, the production of phenazines and swarming motility in this organism potentially linked to reduced expression of the rhlI and pqsA quorum sensing genes. Furthermore, coumarin significantly inhibited biofilm formation and protease activity in other bacterial pathogens and inhibited bioluminescence in Aliivibrio fischeri. In light of these findings, coumarin would appear to have potential as a novel quorum sensing inhibitor with a broad spectrum of action.

  14. 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.

  15. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis.

    Science.gov (United States)

    Udayalaxmi, J; Bhat, G K; Kotigadde, S

    2011-01-01

    The present study was conducted to correlate the biotypes of Gardnerella vaginalis strains isolated from cases of bacterial vaginosis and their virulence factors. Thirty-two strains of G. vaginalis isolated from cases of bacterial vaginosis were biotyped. Adherence to vaginal epithelial cells, biofilm production, surface hydrophobicity, phospholipase C and protease activity were tested on these isolates. Biotype 1 was the most prevalent (8; 25%), followed by biotype 2 (7; 21.9%) and biotypes 5 and 8 (5; 15.6%). We did not find any statistical correlation between G. vaginalis biotypes and its virulence factors. Virulence factors expressed by G. vaginalis were not associated with a single biotype.

  16. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    J Udayalaxmi

    2011-01-01

    Full Text Available The present study was conducted to correlate the biotypes of Gardnerella vaginalis strains isolated from cases of bacterial vaginosis and their virulence factors. Thirty-two strains of G. vaginalis isolated from cases of bacterial vaginosis were biotyped. Adherence to vaginal epithelial cells, biofilm production, surface hydrophobicity, phospholipase C and protease activity were tested on these isolates. Biotype 1 was the most prevalent (8; 25%, followed by biotype 2 (7; 21.9% and biotypes 5 and 8 (5; 15.6%. We did not find any statistical correlation between G. vaginalis biotypes and its virulence factors. Virulence factors expressed by G. vaginalis were not associated with a single biotype.

  17. Enhanced Disease Susceptibility1 Mediates Pathogen Resistance and Virulence Function of a Bacterial Effector in Soybean.

    Science.gov (United States)

    Wang, Jialin; Shine, M B; Gao, Qing-Ming; Navarre, Duroy; Jiang, Wei; Liu, Chunyan; Chen, Qingshan; Hu, Guohua; Kachroo, Aardra

    2014-05-28

    Enhanced disease susceptibility1 (EDS1) and phytoalexin deficient4 (PAD4) are well-known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1-like (GmEDS1a/GmEDS1b) proteins and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean (Glycine max). Consistent with their significant structural conservation to Arabidopsis (Arabidopsis thaliana) counterparts, constitutive expression of GmEDS1 or GmPAD4 complemented the pathogen resistance defects of Arabidopsis eds1 and pad4 mutants, respectively. Interestingly, however, the GmEDS1 and GmPAD4 did not complement pathogen-inducible salicylic acid accumulation in the eds1/pad4 mutants. Furthermore, the GmEDS1a/GmEDS1b proteins were unable to complement the turnip crinkle virus coat protein-mediated activation of the Arabidopsis R protein Hypersensitive reaction to Turnip crinkle virus (HRT), even though both interacted with HRT. Silencing GmEDS1a/GmEDS1b or GmPAD4 reduced basal and pathogen-inducible salicylic acid accumulation and enhanced soybean susceptibility to virulent pathogens. The GmEDS1a/GmEDS1b and GmPAD4 genes were also required for Resistance to Pseudomonas syringae pv glycinea2 (Rpg2)-mediated resistance to Pseudomonas syringae. Notably, the GmEDS1a/GmEDS1b proteins interacted with the cognate bacterial effector AvrA1 and were required for its virulence function in rpg2 plants. Together, these results show that despite significant structural similarities, conserved defense signaling components from diverse plants can differ in their functionalities. In addition, we demonstrate a role for GmEDS1 in regulating the virulence function of a bacterial effector.

  18. Prevalence of clonal complexes and virulence genes among commensal and invasive Staphylococcus aureus isolates in Sweden.

    Directory of Open Access Journals (Sweden)

    Gunlög Rasmussen

    Full Text Available Staphylococcus aureus encodes a remarkable number of virulence factors which may contribute to its pathogenicity and ability to cause invasive disease. The main objective of this study was to evaluate the association between S. aureus invasiveness and bacterial genotype, in terms of the presence of virulence genes and affiliation to clonal complexes. Also, the significance of different virulence genes, mainly adhesins, for the development of infective endocarditis was investigated. DNA microarray technology was used to analyze 134 S. aureus isolates, all methicillin-susceptible, derived from three groups of clinically well-characterized patients: nasal carriers (n=46, bacteremia (n=55, and bacteremia with infective endocarditis (n=33. Invasive isolates were dominant in four of the major clonal complexes: 5, 8, 15, and 25. Of the 170 virulence genes examined, those encoding accessory gene regulator group II (agr II, capsule polysaccharide serotype 5 (cap5, and adhesins such as S. aureus surface protein G (sasG and fibronectin-binding protein B (fnbB were found to be associated with invasive disease. The same was shown for the leukocidin genes lukD/lukE, as well as the genes encoding serine protease A and B (splA/splB, staphylococcal complement inhibitor (scn and the staphylococcal exotoxin-like protein (setC or selX. In addition, there was a trend of higher prevalence of certain genes or gene clusters (sasG, agr II, cap5 among isolates causing infective endocarditis compared to other invasive isolates. In most cases, the presence of virulence genes was linked to clonal complex affiliation. In conclusion, certain S. aureus clonal lineages harboring specific sets of virulence genes seem to be more successful in causing invasive disease.

  19. Profiling of virulence associated genes of Pasteurella multocida isolated from cattle.

    Science.gov (United States)

    Verma, Subhash; Sharma, Mandeep; Katoch, Shailja; Verma, Lovit; Kumar, Sandeep; Dogra, Vishal; Chahota, Rajesh; Dhar, Prasenjit; Singh, Geetanjali

    2013-03-01

    Pasteurella multocida is a causative agent of many major diseases of which haemorrhagic septiciemia (HS) in cattle & a buffalo is responsible for significant losses to livestock sector in India and south Asia. The disease outcome is affected by various host- and pathogen-specific determinants. Several bacterial species-specific putative virulence factors including the capsular and virulence associated genes have been proposed to play a key role in this interaction. A total of 23 isolates of P. multocida were obtained from 335 cases of various clinically healthy and diseased cattle. These isolates were examined for capsule synthesis genes (capA, B, D, E and F) and eleven virulence associated genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) by PCR. A total of 19 P. multocida isolates belonging to capsular type B and 4 of capsular type A were isolated. All isolates of capsular type B harboured the virulence associated genes: tbpA, pfhA, hgbA, sodC and nanH, coding for transferrin binding protein, filamentous hemagglutinin, haemoglobin binding protein, superoxide dismutase and neuraminidases, respectively; while isolates belonging to capsular type A also carried tbpA, pfhA, hgbA and nanH genes. Only 50 % of capsular type A isolates contained sodC gene while 100 % of capsular type B isolates had sodC gene. The gene nanB and toxA were absent in all the 23 isolates. In capsular type A isolates, either sodA or sodC gene was present & these genes did not occur concurrently. The presence of virulence associated gene ptfA revealed a positive association with the disease outcome in cattle and could therefore be an important epidemiological marker gene for characterizing P. multocida isolates.

  20. Host-Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes.

    Directory of Open Access Journals (Sweden)

    Leila Masri

    2015-06-01

    Full Text Available Reciprocal coevolution between host and pathogen is widely seen as a major driver of evolution and biological innovation. Yet, to date, the underlying genetic mechanisms and associated trait functions that are unique to rapid coevolutionary change are generally unknown. We here combined experimental evolution of the bacterial biocontrol agent Bacillus thuringiensis and its nematode host Caenorhabditis elegans with large-scale phenotyping, whole genome analysis, and functional genetics to demonstrate the selective benefit of pathogen virulence and the underlying toxin genes during the adaptation process. We show that: (i high virulence was specifically favoured during pathogen-host coevolution rather than pathogen one-sided adaptation to a nonchanging host or to an environment without host; (ii the pathogen genotype BT-679 with known nematocidal toxin genes and high virulence specifically swept to fixation in all of the independent replicate populations under coevolution but only some under one-sided adaptation; (iii high virulence in the BT-679-dominated populations correlated with elevated copy numbers of the plasmid containing the nematocidal toxin genes; (iv loss of virulence in a toxin-plasmid lacking BT-679 isolate was reconstituted by genetic reintroduction or external addition of the toxins. We conclude that sustained coevolution is distinct from unidirectional selection in shaping the pathogen's genome and life history characteristics. To our knowledge, this study is the first to characterize the pathogen genes involved in coevolutionary adaptation in an animal host-pathogen interaction system.

  1. 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.

  2. Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages.

    Science.gov (United States)

    Chen, John; Carpena, Nuria; Quiles-Puchalt, Nuria; Ram, Geeta; Novick, Richard P; Penadés, José R

    2015-05-01

    Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.

  3. Construction of a multiplex promoter reporter platform to monitor Staphylococcus aureus virulence gene expression and the identification of usnic acid as a potent suppressor of psm gene expression

    Directory of Open Access Journals (Sweden)

    Peng GAO

    2016-08-01

    Full Text Available As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

  4. Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression.

    Science.gov (United States)

    Gao, Peng; Wang, Yanli; Villanueva, Iván; Ho, Pak Leung; Davies, Julian; Kao, Richard Yi Tsun

    2016-01-01

    As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

  5. Contribution of fucose-containing capsules in Klebsiella pneumoniae to bacterial virulence in mice.

    Science.gov (United States)

    Wu, June Hsieh; Wu, Albert M; Tsai, Cheng Gie; Chang, Xin-Yu; Tsai, Shih-Feng; Wu, Ting-Shu

    2008-01-01

    Bacterium Klebsiella pneumoniae (KP) contains a prominent capsule. Clinical infections usually are associated with pneumonia or urinary tract infection (UTI). Emerging evidence implicates KP in severe liver abscess especially in diabetic patients. The goal of this study was to investigate the capsular polysaccharides from KP of liver abscess (hepatic-KP) and of UTI-KP. The composition of capsular polysaccharides was analyzed by capillary high-performance liquid chromatography (HPLC, Dionex system). The terminal sugars were assayed by binding ability to lectins. The results showed that the capsule of a hepatic KP (KpL1) from a diabetic patient contained fucose, while the capsule from UTI-KP (KpU1) did not. The absence of fucose was verified by the absence of detectable polymerase chain reaction (PCR) fragment for fucose synthesis genes, gmd and wcaG in KpU1. Mice infected with the KpL1 showed high fatality, whereas those infected with the KpU1 showed high survival rate. The KpL1 capsule was reactive to lectins AAA and AAL, which detect fucose, while the KpU1 capsule was reactive to lectin GNA, which detects mannose. Phagocytosis experiment in mouse peritoneal cavity indicated that the peritoneal macrophages could interact with KpU1, while rare association of KpL1 with macrophages was observed. This study revealed that different polysaccharides were displayed on the bacterial capsules of virulent KpL1 as compared with the less virulent KpU1. Interaction of KpU1 with mice peritoneal macrophages was more prominent than that of KpL1. The possession of fucose might contribute to KpL1 virulence by avoiding phagocytosis since fucose on bacteria had been implicated in immune evasion.

  6. A comprehensive insight into bacterial virulence in drinking water using 454 pyrosequencing and Illumina high-throughput sequencing.

    Science.gov (United States)

    Huang, Kailong; Zhang, Xu-Xiang; Shi, Peng; Wu, Bing; Ren, Hongqiang

    2014-11-01

    In order to comprehensively investigate bacterial virulence in drinking water, 454 pyrosequencing and Illumina high-throughput sequencing were used to detect potential pathogenic bacteria and virulence factors (VFs) in a full-scale drinking water treatment and distribution system. 16S rRNA gene pyrosequencing revealed high bacterial diversity in the drinking water (441-586 operational taxonomic units). Bacterial diversity decreased after chlorine disinfection, but increased after pipeline distribution. α-Proteobacteria was the most dominant taxonomic class. Alignment against the established pathogen database showed that several types of putative pathogens were present in the drinking water and Pseudomonas aeruginosa had the highest abundance (over 11‰ of total sequencing reads). Many pathogens disappeared after chlorine disinfection, but P. aeruginosa and Leptospira interrogans were still detected in the tap water. High-throughput sequencing revealed prevalence of various pathogenicity islands and virulence proteins in the drinking water, and translocases, transposons, Clp proteases and flagellar motor switch proteins were the predominant VFs. Both diversity and abundance of the detectable VFs increased after the chlorination, and decreased after the pipeline distribution. This study indicates that joint use of 454 pyrosequencing and Illumina sequencing can comprehensively characterize environmental pathogenesis, and several types of putative pathogens and various VFs are prevalent in drinking water.

  7. The importance of virulence prediction and gene networks in microbial risk assessment

    DEFF Research Database (Denmark)

    Wassenaar, Gertrude Maria; Gamieldien, Junaid; Shatkin, JoAnne

    2007-01-01

    For microbial risk assessment, it is necessary to recognize and predict Virulence of bacterial pathogens, including their ability to contaminate foods. Hazard characterization requires data on strain variability regarding virulence and survival during food processing. Moreover, information on vir...

  8. Virulence Genes Profile of Multidrug Resistant Pseudomonas aeruginosa Isolated from Iranian Children with UTIs

    Directory of Open Access Journals (Sweden)

    Zohreh Heidary

    2016-04-01

    Full Text Available Virulent and resistant strains Pseudomonas aeruginosa (P. aeruginosa is one of the most important cause of UTIs in pediatrics. The present study was carried to investigate the frequency of virulence factors in the multi-drug resistant strains of P. aeruginosa isolated from pediatrics hospitalized due to the UTIs. One - hundred and forty three urine samples were collected from pediatric patients suffered from UTIs. Samples were cultured and those that were P. aeruginosa positive were analyzed for the presence of putative virulence genes. Seventy one out of 143 samples (49.65% were positive for P. aeruginosa. Monthly, sex and age-dependent prevalence were seen for P. aeruginosa. Bacterial strains had the highest levels of resistance against ampicillin (95.77%, gentamicin (92.95% and ciprofloxacin (81.69%. Of 71 P. aeruginosa isolates, 12 strains were resistant to more than 9 antibiotics (16.90%. The most commonly detected virulence factors in the cases of urethral infections were exoU and plcH while those of pyelonephritis and cystitis were were exoS and lasB. Our findings should raise awareness about antibiotic resistance in hospitalized pediatrics with UTIs in Iran. Clinicians should exercise caution in prescribing antibiotics, especially in cases of UTIs. Such information can help in identifying these virulence genes as useful diagnostic markers for clinical P. aeruginosa strains isolated from UTIs.

  9. cj0371: a novel virulence-associated gene of Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Xueqing Du

    2016-07-01

    Full Text Available Campylobacter jejuni is the major cause of human bacterial diarrhea worldwide. Its pathogenic mechanism remains poorly understood. cj0371 is a novel gene that was uncovered using immunoscreening. There have been no previous reports regarding its function. In this study, we constructed an insertion mutant and complement of this gene in C. jejuni and examined changes in virulence. We observed that the cj0371 mutant showed significantly increased invasion and colonization ability. We also investigated the role of cj0371 in motility, chemotaxis and growth kinetics to further study its function. We found that the cj0371 mutant displays hypermotility, enhanced chemotaxis and enhanced growth kinetics. In addition, we localized the Cj0371 protein at the poles of C. jejuni by fluorescence microscopy. We present data that collectively significantly proves our hypothesis that cj0371 is a new virulence-associated gene and through the influence of chemotaxis plays a negative role in C. jejuni pathogenicity.

  10. Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Jennifer M Bomberger

    2009-04-01

    Full Text Available Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane-derived vesicles (OMV secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including beta-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP-mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

  11. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis

    OpenAIRE

    Udayalaxmi, J.; Bhat, G. K.; S Kotigadde

    2011-01-01

    The present study was conducted to correlate the biotypes of Gardnerella vaginalis strains isolated from cases of bacterial vaginosis and their virulence factors. Thirty-two strains of G. vaginalis isolated from cases of bacterial vaginosis were biotyped. Adherence to vaginal epithelial cells, biofilm production, surface hydrophobicity, phospholipase C and protease activity were tested on these isolates. Biotype 1 was the most prevalent (8; 25%), followed by biotype 2 (7; 21.9%) and biotypes ...

  12. 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.

  13. Helicobacter pylori HP0231 Influences Bacterial Virulence and Is Essential for Gastric Colonization.

    Directory of Open Access Journals (Sweden)

    Yu Zhong

    Full Text Available The Dsb protein family is responsible for introducing disulfide bonds into nascent proteins in prokaryotes, stabilizing the structure of many proteins. Helicobacter pylori HP0231 is a Dsb-like protein, shown to catalyze disulfide bond formation and to participate in redox homeostasis. Notably, many H. pylori virulence factors are stabilized by the formation of disulfide bonds. By employing H. pylori HP0231 deficient strains we analyzed the effect of lack of this bacterial protein on the functionality of virulence factors containing putative disulfide bonds. The lack of H. pylori HP0231 impaired CagA translocation into gastric epithelial cells and reduced VacA-induced cellular vacuolation. Moreover, H. pylori HP0231 deficient bacteria were not able to colonize the gastric mucosa of mice, probably due to compromised motility. Together, our data demonstrate an essential function for H. pylori HP0231 in gastric colonization and proper function of bacterial virulence factors related to gastric pathology.

  14. Coincidental loss of bacterial virulence in multi-enemy microbial communities.

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    Ji Zhang

    Full Text Available The coincidental virulence evolution hypothesis suggests that outside-host selection, such as predation, parasitism and resource competition can indirectly affect the virulence of environmentally-growing bacterial pathogens. While there are some examples of coincidental environmental selection for virulence, it is also possible that the resource acquisition and enemy defence is selecting against it. To test these ideas we conducted an evolutionary experiment by exposing the opportunistic pathogen bacterium Serratia marcescens to the particle-feeding ciliate Tetrahymena thermophila, the surface-feeding amoeba Acanthamoeba castellanii, and the lytic bacteriophage Semad11, in all possible combinations in a simulated pond water environment. After 8 weeks the virulence of the 384 evolved clones were quantified with fruit fly Drosophila melanogaster oral infection model, and several other life-history traits were measured. We found that in comparison to ancestor bacteria, evolutionary treatments reduced the virulence in most of the treatments, but this reduction was not clearly related to any changes in other life-history traits. This suggests that virulence traits do not evolve in close relation with these life-history traits, or that different traits might link to virulence in different selective environments, for example via resource allocation trade-offs.

  15. Dynamics of bacterial gene regulation

    Science.gov (United States)

    Narang, Atul

    2009-03-01

    The phenomenon of diauxic growth is a classical problem of bacterial gene regulation. The most well studied example of this phenomenon is the glucose-lactose diauxie, which occurs because the expression of the lac operon is strongly repressed in the presence of glucose. This repression is often explained by appealing to molecular mechanisms such as cAMP activation and inducer exclusion. I will begin by analyzing data showing that these molecular mechanisms cannot explain the strong lac repression because they exert a relatively weak effect. I will then present a minimal model accounting only for enzyme induction and dilution, which yields strong repression despite the absence of catabolite repression and inducer exclusion. The model also explains the growth patterns observed in batch and continuous cultures of various bacterial strains and substrate mixtures. The talk will conclude with a discussion of the experimental evidence regarding positive feedback, the key component of the minimal model.

  16. Different distribution patterns of ten virulence genes in Legionella reference strains and strains isolated from environmental water and patients.

    Science.gov (United States)

    Zhan, Xiao-Yong; Hu, Chao-Hui; Zhu, Qing-Yi

    2016-04-01

    Virulence genes are distinct regions of DNA which are present in the genome of pathogenic bacteria and absent in nonpathogenic strains of the same or related species. Virulence genes are frequently associated with bacterial pathogenicity in genus Legionella. In the present study, an assay was performed to detect ten virulence genes, including iraA, iraB, lvrA, lvrB, lvhD, cpxR, cpxA, dotA, icmC and icmD in different pathogenicity islands of 47 Legionella reference strains, 235 environmental strains isolated from water, and 4 clinical strains isolated from the lung tissue of pneumonia patients. The distribution frequencies of these genes in reference or/and environmental L. pneumophila strains were much higher than those in reference non-L. pneumophila or/and environmental non-L. pneumophila strains, respectively. L. pneumophila clinical strains also maintained higher frequencies of these genes compared to four other types of Legionella strains. Distribution frequencies of these genes in reference L. pneumophila strains were similar to those in environmental L. pneumophila strains. In contrast, environmental non-L. pneumophila maintained higher frequencies of these genes compared to those found in reference non-L. pneumophila strains. This study illustrates the association of virulence genes with Legionella pathogenicity and reveals the possible virulence evolution of non-L. pneumophia strains isolated from environmental water.

  17. ``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.

  18. Stress Effects on Virulence-related Genes Expression in Enterococcus faecalis from Food Source

    Directory of Open Access Journals (Sweden)

    Ming Lei

    2014-04-01

    Full Text Available Enterococcus faecalis are ubiquitous micro-organisms which are widely used in fermented food or probiotic industry. However, they are also associated with nosocomial infections and rank among the top three nosocomial bacterial pathogens. Some virulence factors were proposed which could strongly enhance their pathogenicity. In our study, three virulence-related genes ace, gelE and efaA were found in E. faecalis SL22 which was isolated from raw milk. The expression of these genes was examined in response to different stress factors like NaCl, pH and gentamycin sulfate by using real-time PCR. Significant differences in expression level were influenced by the environment factors, which suggests that these stress factors should be taken into consideration during the selection of enterococci for use in foods or as probiotics.

  19. Homeostatic interplay between bacterial cell-cell signaling and iron in virulence.

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    Ronen Hazan

    2010-03-01

    Full Text Available Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.

  20. Regulation of virulence gene expression in pathogenic Listeria.

    Science.gov (United States)

    Brehm, K; Kreft, J; Ripio, M T; Vázquez-Boland, J A

    1996-06-01

    Dynamic interactions between host and pathogen are characteristic of infections caused by intracellular bacteria. This has favoured the evolution of highly effective control systems by which these pathogens regulate the expression of different virulence factors during sequential steps of the infection process. In the case of the facultative intracellular bacterium Listeria monocytogenes, these steps involve internalization by eukaryotic cells, lysis of the resulting phagosome, replication as well as movement within the host cytoplasm, direct cell-to-cell spread, and subsequent lysis of a double-membrane vacuole when entering neighbouring cells. Virulence factors which are involved in each of these steps have been identified and the expression of these factors is subject to a co-ordinate and differential control exerted by the major listerial virulence regulator PrfA. This protein belongs to the Crp/Fnr-family of transcriptional activators and recognizes specific target sequences in promoter regions of several listerial virulence genes. Differential expression of these genes during sequential steps of the infection seems to be at least partially mediated by different binding affinities of PrfA to its target sequences. Activity of PrfA-dependent genes and of prfA itself is under the control of several environmental variables which are used by the pathogen to recognize its transition from the free environment into a eukaryotic host.

  1. 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.

  2. Impact of space flight on bacterial virulence and antibiotic susceptibility

    OpenAIRE

    Taylor PW

    2015-01-01

    Peter William Taylor School of Pharmacy, University College London, London, UK Abstract: Manned space flight induces a reduction in immune competence among crew and is likely to cause deleterious changes to the composition of the gastrointestinal, nasal, and respiratory bacterial flora, leading to an increased risk of infection. The space flight environment may also affect the susceptibility of microorganisms within the spacecraft to antibiotics, key components of flown medical kits, and may...

  3. Applying the ResFinder and VirulenceFinder web-services for easy identification of acquired antibiotic resistance and E. coli virulence genes in bacteriophage and prophage nucleotide sequences

    DEFF Research Database (Denmark)

    Kleinheinz, Kortine Annina; Joensen, Katrine Grimstrup; Larsen, Mette Voldby

    2014-01-01

    raised that this increased use and dissemination of phages could result in spread of deleterious genes, e.g., antibiotic resistance and virulence genes. Meanwhile, in the wake of the genomic era, several tools have been developed for characterization of bacterial genomes. Here we describe how two...... of these tools, ResFinder and VirulenceFinder, can be used to identify acquired antibiotic resistance and virulence genes in phage genomes of interest. The general applicability of the tools is demonstrated on data sets of 1,642 phage genomes and 1,442 predicted prophages.......Extensive research is currently being conducted on the use of bacteriophages for applications in human medicine, agriculture and food manufacturing. However, phages are important vehicles of horisontal gene transfer and play a significant role in bacterial evolution. As a result, concern has been...

  4. Bacteriophage-encoded shiga toxin gene in atypical bacterial host

    Directory of Open Access Journals (Sweden)

    Casas Veronica

    2011-07-01

    Full Text Available Abstract Background Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB. A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. Results Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. Conclusions The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.

  5. Microevolution of Virulence-Related Genes in Helicobacter pylori Familial Infection.

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    Yoshikazu Furuta

    Full Text Available Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene, outer membrane protein (OMP genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.

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

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    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. Bacterial ortholog of mammalian translocator protein (TSPO with virulence regulating activity.

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    Annelise Chapalain

    Full Text Available The translocator protein (TSPO, previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10(-5 M adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies.

  8. Virulence, bacterocin genes and antibacterial susceptibility in Enterococcus faecalis strains isolated from water wells for human consumption.

    Science.gov (United States)

    Padilla, Carlos; Lobos, Olga

    2013-12-01

    The objectives of this study were to detect genes for virulence and bacteriocins in addition to studying the antimicrobial susceptibility of 78 strains of E. faecalis isolated from water wells for human consumption. The virulence and bacteriocin genes of 78 E. faecalis were amplified by PCR and visualized in agarose gels. The antimicrobial susceptibility was determined through diffusion agar tests and the MIC through microdilution. It was observed that the major percentage of virulence genes in the E. faecalis strains corresponds to aggA (93.5%). The bacteriocin gene entA (64.1%) is the most frequently detected. The studied strains exhibited different virulence and bacteriocin genes, and an important antibacterial resistance. The most common resistant phenotype (n = 14) corresponds to tetracycline and chloramphenicol and the less frequent (n = 2) to ciprofloxacin and moxifloxacin. Eight different genetic profiles were observed for virulence y bacteriocin genes. It was determined a statistical association between the bacterial resistance and some of the genetic profiles detected.

  9. Helicobacter pylori virulence genes and microevolution in host and the clinical outcome: review article

    Directory of Open Access Journals (Sweden)

    Seyedeh Zahra Bakhti

    2014-12-01

    Full Text Available Helicobacter pylori (H. pylori is the causative agent in development of gastroduode-nal diseases, such as chronic atrophic gastritis, peptic ulcers, mucosa associated lym-phoid tissue (MALT lymphoma, and gastric cancer. H. pylori has been associated with inflammation in cardia, showing the fact that infection with this bacterium could also be a risk factor for gastric cardia cancer. Gastric cancer is the fourth most common cancer worldwide. This is the second leading cause of cancer-related deaths, and ap-proximately 700,000 people succumb each year to gastric adenocarcinoma. It has been estimated that 69% of the Iranian population currently harbor H. pylori infection. The prevalence of duodenal ulcer and gastric cancer is high in Iranian populations. However, this has been largely influenced by geographic and/or ethnic origin. Epidemi-ology studies have shown that host, environmental, and bacterial factors determine the outcome of H. pylori infection. The bacterium contains allelic diversity and high genet-ic variability into core- and virulence-genes and that this diversity is geographically and ethnically structured. The genetic diversity within H. pylori is greater than within most other bacteria, and its diversity is more than 50-fold higher than that of human DNA. The maintenance of high diversification makes this bacterium to cope with particular challenges in individual hosts. It has been reported that the recombination contributed to the creation of new genes and gene family. Furthermore, the microevolution in cagA and vacA genes is a common event, leading to a change in the virulence phenotype. These factors contribute to the bacterial survival in acidic conditions in stomach and protect it from host immune system, causing tissue damage and clinical disease. In this review article, we discussed the correlation between H. pylori virulence factors and clin-ical outcomes, microevolution of H. pylori virulence genes in a single host

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

    Science.gov (United States)

    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

  11. Alternative Sigma Factors and Their Roles in Bacterial Virulence

    OpenAIRE

    Kazmierczak, Mark J.; Wiedmann, Martin; Boor, Kathryn J.

    2005-01-01

    Sigma factors provide promoter recognition specificity to RNA polymerase holoenzyme, contribute to DNA strand separation, and then dissociate from the core enzyme following transcription initiation. As the regulon of a single sigma factor can be composed of hundreds of genes, sigma factors can provide effective mechanisms for simultaneously regulating expression of large numbers of prokaryotic genes. One newly emerging field is identification of the specific roles of alternative sigma factors...

  12. Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux

    Directory of Open Access Journals (Sweden)

    Hinds Jason

    2008-10-01

    Full Text Available Abstract Background Human and animal health is constantly under threat by emerging pathogens that have recently acquired genetic determinants that enhance their survival, transmissibility and virulence. We describe the construction and development of an Active Surveillance of Pathogens (ASP oligonucleotide microarray, designed to 'actively survey' the genome of a given bacterial pathogen for virulence-associated genes. Results The microarray consists of 4958 reporters from 151 bacterial species and include genes for the identification of individual bacterial species as well as mobile genetic elements (transposons, plasmid and phage, virulence genes and antibiotic resistance genes. The ASP microarray was validated with nineteen bacterial pathogens species, including Francisella tularensis, Clostridium difficile, Staphylococcus aureus, Enterococcus faecium and Stenotrophomonas maltophilia. The ASP microarray identified these bacteria, and provided information on potential antibiotic resistance (eg sufamethoxazole resistance and sulfonamide resistance and virulence determinants including genes likely to be acquired by horizontal gene transfer (e.g. an alpha-haemolysin. Conclusion The ASP microarray has potential in the clinic as a diagnostic tool, as a research tool for both known and emerging pathogens, and as an early warning system for pathogenic bacteria that have been recently modified either naturally or deliberately.

  13. A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.

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    Alex T Nielsen

    Full Text Available A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP and cholera toxin (CT were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a

  14. Detection of attaching and effacing virulence gene of E. coli

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    Maratu Soleha

    2013-07-01

    Full Text Available AbstrakLatar belakang: Bakteri Escherichia coli (E. coli ada yang telah bermutasi menjadi patogen yang menimbulkan berbagai penyakit seperti hemorrhagic colitis (HC, hemolytic uremic syndrome (HUS, sepsis, pnemonia, neonatal meningitis, dan infeksi saluran kemih. Mutasi terjadi karena bakteri ini menerima transfer gen yang virulen dari bakteri lain yang hidup di sekitarnya. E. coli yang biasanya hidup normal di dalam usus manusia telah beradaptasi sehingga bisa hidup di tanah, makanan, dan saluran kemih. Penelitian ini mendeteksi gene yang virulen pada DNA isolat E. coli. Metode: Untuk deteksi E. coli yang virulen pada penelitian ini digunakan metode Real-time PCR dengan mencocokkan hasil sekuensing dengan sekuens E. coli virulen yang telah di publikasikan sebagai rujukan. Hasil: Sekuens RT PCR menggambarkan DNA gen eae pada BLAST mempunyai kesesuaian dengan rujukan segmen E. coli yang virulen. Dari sampel yang berasal dari E. coli di sekitar perairan lingkungan didapatkan gen Eae sebagai gen yang menyebabkan E. coli menjadi virulen sebesar 7,3%. Kesimpulan: E. coli yang virulen ditemukan pada sampel E. coli yang berasal dari perairan lingkungan dengan metode realtime PCR. (Health Science Indones 2013;1:41-6 Kata kunci: gen virulen E. coli, real-time PCR, perairan lingkunganAbstractBackground: Escherichia coli(E. coli bacteria have developed into pathogenic bacteria that caused diseases such as hemorrhagic colitis (HC, hemolytic uremic syndrome (HUS, sepsis, pneumonia, neonatal meningitis, and urinary tract infections. Pathogenic E. coli have acquired pathogenic/virulence genes from other bacteria in their environment. E. coli that normally lived in the human gut had adapted to other niches such as soil, food and the urinary tract. This study investigated the presence of pathogenic E. coli from water samples by examining E. coli virulence genes present in E. coli genomes of water sourced isolates. Methods:This study used Real-time PCR to detect

  15. Growth of Yersinia pseudotuberculosis in human plasma: impacts on virulence and metabolic gene expression

    Directory of Open Access Journals (Sweden)

    Coppée Jean-Yves

    2008-12-01

    Full Text Available Abstract Background In man, infection by the Gram-negative enteropathogen Yersinia pseudotuberculosis is usually limited to the terminal ileum. However, in immunocompromised patients, the microorganism may disseminate from the digestive tract and thus cause a systemic infection with septicemia. Results To gain insight into the metabolic pathways and virulence factors expressed by the bacterium at the blood stage of pseudotuberculosis, we compared the overall gene transcription patterns (the transcriptome of bacterial cells cultured in either human plasma or Luria-Bertani medium. The most marked plasma-triggered metabolic consequence in Y. pseudotuberculosis was the switch to high glucose consumption, which is reminiscent of the acetogenic pathway (known as "glucose overflow" in Escherichia coli. However, upregulation of the glyoxylate shunt enzymes suggests that (in contrast to E. coli acetate may be further metabolized in Y. pseudotuberculosis. Our data also indicate that the bloodstream environment can regulate major virulence genes (positively or negatively; the yadA adhesin gene and most of the transcriptional units of the pYV-encoded type III secretion apparatus were found to be upregulated, whereas transcription of the pH6 antigen locus was strongly repressed. Conclusion Our results suggest that plasma growth of Y. pseudotuberculosis is responsible for major transcriptional regulatory events and prompts key metabolic reorientations within the bacterium, which may in turn have an impact on virulence.

  16. Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

    Science.gov (United States)

    Reddy, Sanjay M; Sun, Aijun; Khan, Owais A; Lee, Lucy F; Lupiani, Blanca

    2013-06-01

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

  17. Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

    Science.gov (United States)

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J; Nie, Pin

    2015-11-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.

  18. Listeria monocytogenes virulence factor Listeriolysin O favors bacterial growth in co-culture with the ciliate Tetrahymena pyriformis, causes protozoan encystment and promotes bacterial survival inside cysts

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    Ermolaeva Svetlana A

    2010-01-01

    Full Text Available Abstract Background The gram-positive pathogenic bacterium Listeria monocytogenes is widely spread in the nature. L. monocytogenes was reported to be isolated from soil, water, sewage and sludge. Listeriolysin O (LLO is a L. monocytogenes major virulence factor. In the course of infection in mammals, LLO is required for intracellular survival and apoptosis induction in lymphocytes. In this study, we explored the potential of LLO to promote interactions between L. monocytogenes and the ubiquitous inhabitant of natural ecosystems bacteriovorous free-living ciliate Tetrahymena pyriformis. Results Wild type L. monocytogenes reduced T. pyriformis trophozoite counts and stimulated encystment. The effects were observed starting from 48 h of co-incubation. On the day 14, trophozoites were eliminated from the co-culture while about 5 × 104 cells/ml remained in the axenic T. pyriformis culture. The deficient in the LLO-encoding hly gene L. monocytogenes strain failed to cause mortality among protozoa and to trigger protozoan encystment. Replenishment of the hly gene in the mutant strain restored toxicity towards protozoa and induction of protozoan encystment. The saprophytic non-haemolytic species L. innocua transformed with the LLO-expressing plasmid caused extensive mortality and encystment in ciliates. During the first week of co-incubation, LLO-producing L. monocytogenes demonstrated higher growth rates in association with T. pyriformis than the LLO-deficient isogenic strain. At latter stages of co-incubation bacterial counts were similar for both strains. T. pyriformis cysts infected with wild type L. monocytogenes caused listerial infection in guinea pigs upon ocular and oral inoculation. The infection was proved by bacterial plating from the internal organs. Conclusions The L. monocytogenes virulence factor LLO promotes bacterial survival and growth in the presence of bacteriovorous ciliate T. pyriformis. LLO is responsible for L. monocytogenes

  19. A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.

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    Hao Gong

    2011-09-01

    Full Text Available Small non-coding RNAs (sRNAs that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA and small interfering RNA (siRNA in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.

  20. Bacteraemia with Campylobacter jejuni: no association with the virulence genes iam, cdtB, capA or virB.

    Science.gov (United States)

    Nielsen, H; Persson, S; Olsen, K E P; Ejlertsen, T; Kristensen, B; Schønheyder, H C

    2010-03-01

    The role of bacterial genes in the determination of the clinical spectrum of Campylobacter jejuni infection is unclear. We compared clinical isolates from invasive blood-stream infection with stool isolates from gastroenteritis and found no association of the putative virulence genes iam, capA, virB and cdtB with clinical presentation.

  1. Distribution of putative virulence genes and antimicrobial drug resistance in Vibrio harveyi

    Digital Repository Service at National Institute of Oceanography (India)

    Parvathi, A.; Mendez, D.; Anto, C.

    environments for understanding the distribution of putative virulence genes and antimicrobial drug resistance. The putative genes targeted for PCR detection included four reversible toxin (Rtx)/hemolysin genes, a gene encoding homologue of Vibrio cholerae...

  2. Mutation in fucose synthesis gene of Klebsiella pneumoniae affects capsule composition and virulence in mice.

    Science.gov (United States)

    Pan, Po-Chang; Chen, Hui-Wen; Wu, Po-Kuan; Wu, Yu-Yang; Lin, Chun-Hung; Wu, June H

    2011-02-01

    The emerging pathogenicity of Klebsiella pneumoniae (KP) is evident by the increasing number of clinical cases of liver abscess (LA) due to KP infection. A unique property of KP is its thick mucoid capsule. The bacterial capsule has been found to contain fucose in KP strains causing LA but not in those causing urinary tract infections. The products of the gmd and wcaG genes are responsible for converting mannose to fucose in KP. A KP strain, KpL1, which is known to have a high death rate in infected mice, was mutated by inserting an apramycin-resistance gene into the gmd. The mutant expressed genes upstream and downstream of gmd, but not gmd itself, as determined by reverse transcriptase polymerase chain reaction. The DNA mapping confirmed the disruption of the gmd gene. This mutant decreased its ability to kill infected mice and showed decreased virulence in infected HepG2 cells. Compared with wild-type KpL1, the gmd mutant lost fucose in capsular polysaccharides, increased biofilm formation and interacted more readily with macrophages. The mutant displayed morphological changes with long filament forms and less uniform sizes. The mutation also converted the serotype from K1 of wild-type to K2 and weak K3. The results indicate that disruption of the fucose synthesis gene affected the pathophysiology of this bacterium and may be related to the virulence of this KpL1 strain.

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

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    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.

  4. Influence of cAMP receptor protein (CRP) on bacterial virulence and transcriptional regulation of allS by CRP in Klebsiella pneumoniae.

    Science.gov (United States)

    Xue, Jian; Tan, Bin; Yang, Shiya; Luo, Mei; Xia, Huiming; Zhang, Xian; Zhou, Xipeng; Yang, Xianxian; Yang, Ruifu; Li, Yingli; Qiu, Jingfu

    2016-11-15

    cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. The gene allS was well-known as allantoin-utilizing capability and involving in bacterial virulence in Klebsiella pneumoniae (K. pneumoniae). The specific DNA recognition motif of transcription regulator CRP was found in allS promoter region. Therefore, this study is aimed to investigate the function of CRP on virulence and its transcriptional regulation mechanism to gene allS in K. pneumoniae. The wild-type (WT) K. pneumoniae NTUH-2044, crp knockout (Kp-Δcrp) and the complemented knockout (KpC-Δcrp) strains were used to determine the function of crp gene. The lacZ fusion, qRT-PCR, electrophoretic mobility shift and DNase I footprinting assays were performed to study the transcriptional regulation of CRP on allS. The result showed a decreased virulence in crp knockout strain. Complement through supplementing crp fragment in expression plasmid partially restore virulence of knockout bacteria. The CRP could bind to the allS promoter-proximal region and the binding site was further refined to be located from 60bp to 94bp upstream of the allS promoter. Based on these results, we proposed that CRP is an essential virulence regulator and knock out of crp gene will result in reduced virulence in K. pneumoniae. In the meantime, the transcription of gene allS is positively regulated by CRP via directly binding to upstream of allS promoter.

  5. Virulence Gene Profile and Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) of Enteroinvasive Escherichia coli (EIEC) Isolates From Patients With Diarrhea in Kerman, Iran

    OpenAIRE

    Hosseini Nave, Hossein; Mansouri, Shahla; Taati Moghadam, Majid; Moradi, Mohammad

    2016-01-01

    Background Enteroinvasive Escherichia coli (EIEC) isolates cause dysentery in humans. Several virulence factors associated with EIEC pathogenesis have been characterized. Multilocus variable-number tandem-repeat analysis (MLVA) is a PCR-based method that has been used for genotyping bacterial pathogens. Objectives The aim of this study was to investigate the distribution of virulence factor genes in EIEC isolates from patients with diarrhea in Kerman, Iran, as well as the genetic relationship...

  6. The MogR Transcriptional Repressor Regulates Nonhierarchal Expression of Flagellar Motility Genes and Virulence in Listeria monocytogenes.

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    2006-04-01

    Full Text Available Flagella are surface structures critical for motility and virulence of many bacterial species. In Listeria monocytogenes, MogR tightly represses expression of flagellin (FlaA during extracellular growth at 37 degrees C and during intracellular infection. MogR is also required for full virulence in a murine model of infection. Using in vitro and in vivo infection models, we determined that the severe virulence defect of MogR-negative bacteria is due to overexpression of FlaA. Specifically, overproduction of FlaA in MogR-negative bacteria caused pleiotropic defects in bacterial division (chaining phenotype, intracellular spread, and virulence in mice. DNA binding and microarray analyses revealed that MogR represses transcription of all known flagellar motility genes by binding directly to a minimum of two TTTT-N(5-AAAA recognition sites positioned within promoter regions such that RNA polymerase binding is occluded. Analysis of MogR protein levels demonstrated that modulation of MogR repression activity confers the temperature-specificity to flagellar motility gene expression. Epistasis analysis revealed that MogR repression of transcription is antagonized in a temperature-dependent manner by the DegU response regulator and that DegU further regulates FlaA levels through a posttranscriptional mechanism. These studies provide the first known example to our knowledge of a transcriptional repressor functioning as a master regulator controlling nonhierarchal expression of flagellar motility genes.

  7. A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence

    Science.gov (United States)

    Bertolo, Lisa; Monteiro, Mario A.; Agellon, Al; Viswanathan, V. K.; Vedantam, Gayatri

    2016-01-01

    Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence. PMID:27741317

  8. Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

    Science.gov (United States)

    Smith, Roxanne P; Paxman, Jason J; Scanlon, Martin J; Heras, Begoña

    2016-07-16

    Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.

  9. 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.

  10. Brucella abortus: pathogenicity and gene regulation of virulence

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    Olga Rivas-Solano

    2015-06-01

    Full Text Available Brucella abortus is a zoonotic intracellular facultative pathogen belonging to the subdivision α2 of class Proteobacteria. It causes a worldwide distributed zoonotic disease called brucellosis. The main symptoms are abortion and sterility in cattle, as well as an undulant febrile condition in humans. In endemic regions like Central America, brucellosis has a high socioeconomic impact. A basic research project was recently conducted at the ITCR with the purpose of studying gene regulation of virulence, structure and immunogenicity in B. abortus. The present review was written as part of this project. B. abortus virulence seems to be determined by its ability to invade, survive and replicate inside professional and non-professional phagocytes. It reaches its intracellular replicative niche without the activation of host antimicrobial mechanisms of innate immunity. It also has gene regulation mechanisms for a rapid adaptation to an intracellular environment such as the two-component signal transduction system BvrR/BvrS and the quorum sensing regulator called Vjbr, as well as other transcription factors. All of them integrate a complex gene regulation network.

  11. Identification and characterization of a novel bacterial virulence factor that shares homology with mammalian Toll/interleukin-1 receptor family proteins.

    Science.gov (United States)

    Newman, Ruchi M; Salunkhe, Prabhakar; Godzik, Adam; Reed, John C

    2006-01-01

    Many important bacterial virulence factors act as mimics of mammalian proteins to subvert normal host cell processes. To identify bacterial protein mimics of components of the innate immune signaling pathway, we searched the bacterial genome database for proteins with homology to the Toll/interleukin-1 receptor (TIR) domain of the mammalian Toll-like receptors (TLRs) and their adaptor proteins. A previously uncharacterized gene, which we have named tlpA (for TIR-like protein A), was identified in the Salmonella enterica serovar Enteritidis genome that is predicted to encode a protein resembling mammalian TIR domains, We show that overexpression of TlpA in mammalian cells suppresses the ability of mammalian TIR-containing proteins TLR4, IL-1 receptor, and MyD88 to induce the transactivation and DNA-binding activities of NF-kappaB, a downstream target of the TIR signaling pathway. In addition, TlpA mimics the previously characterized Salmonella virulence factor SipB in its ability to induce activation of caspase-1 in a mammalian cell transfection model. Disruption of the chromosomal tlpA gene rendered a virulent serovar Enteritidis strain defective in intracellular survival and IL-1beta secretion in a cell culture infection model using human THP1 macrophages. Bacteria with disrupted tlpA also displayed reduced lethality in mice, further confirming an important role for this factor in pathogenesis. Taken together, our findings demonstrate that the bacterial TIR-like protein TlpA is a novel prokaryotic modulator of NF-kappaB activity and IL-1beta secretion that contributes to serovar Enteritidis virulence.

  12. Development and validation of a resistance and virulence gene microarray targeting Escherichia coli and Salmonella enterica

    Science.gov (United States)

    Davis, Margaret A.; Lim, Ji Youn; Soyer, Yesim; Harbottle, Heather; Chang, Yung-Fu; New, Daniel; Orfe, Lisa H.; Besser, Thomas E.; Call, Douglas R.

    2010-01-01

    A microarray was developed to simultaneously screen Escherichia coli and Salmonella enterica for multiple genetic traits. The final array included 203 60-mer oligonucleotide probes, including 117 for resistance genes, 16 for virulence genes, 25 for replicon markers, and 45 other markers. Validity of the array was tested by assessing interlaboratory agreement among four collaborating groups using a blinded study design. Internal validation indicated that the assay was reliable (area under the receiver-operator characteristic curve=0.97). Inter-laboratory agreement, however, was poor when estimated using the intraclass correlation coefficient, which ranged from 0.27 (95% confidence interval 0.24, 0.29) to 0.29 (0.23, 0.34). These findings suggest that extensive testing and procedure standardization will be needed before bacterial genotyping arrays can be readily shared between laboratories. PMID:20362014

  13. The type VI secretion system gene cluster of Salmonella typhimurium: required for full virulence in mice.

    Science.gov (United States)

    Liu, Ji; Guo, Ji-Tao; Li, Yong-Guo; Johnston, Randal N; Liu, Gui-Rong; Liu, Shu-Lin

    2013-07-01

    Type VI secretion system (T6SS) has increasingly been believed to participate in the infection process for many bacterial pathogens, but its role in the virulence of Salmonella typhimurium remains unclear. To look into this, we deleted the T6SS cluster from the genome of S. typhimurium 14028s and analyzed the phenotype of the resulting T6SS knockout mutant (T6SSKO mutant) in vitro and in vivo. We found that the T6SSKO mutant exhibited reduced capability in colonizing the spleen and liver in an in vivo colonization competition model in BALB/c mice infected by the oral route. Additionally, infection via intraperitoneal administration also showed that the T6SSKO mutant was less capable of colonizing the mouse spleen and liver than the wild-type strain. We did not detect significant differences between the T6SSKO and wild-type strains in epithelial cell invasion tests. However, in the macrophage RAW264.7 cell line, the T6SSKO mutant survived and proliferated significantly more poorly than the wild-type strain. These findings indicate that T6SS gene cluster is required for full virulence of S. typhimurium 14028s in BALB/c mice, possibly due to its roles in bacterial survival and proliferation in macrophages.

  14. Malonate inhibits virulence gene expression in Vibrio cholerae.

    Science.gov (United States)

    Minato, Yusuke; Fassio, Sara R; Häse, Claudia C

    2013-01-01

    We previously found that inhibition of the TCA cycle, either through mutations or chemical inhibition, increased toxT transcription in Vibrio cholerae. In this study, we found that the addition of malonate, an inhibitor of succinate dehydrogenase (SDH), decreased toxT transcription in V. cholerae, an observation inconsistent with the previous pattern observed. Unlike another SDH inhibitor, 2-thenoyltrifluoroacetone (TTFA), which increased toxT transcription and slightly inhibited V. cholerae growth, malonate inhibited toxT transcription in both the wild-type strain and TCA cycle mutants, suggesting malonate-mediated inhibition of virulence gene expression is independent to TCA cycle activity. Addition of malonate also inhibited ctxB and tcpA expressions but did not affect aphA, aphB, tcpP and toxR expressions. Malonate inhibited cholera toxin (CT) production in both V. cholerae classical biotype strains O395N1 and CA401, and El Tor biotype strain, N16961. Consistent with previous reports, we confirmed that these strains of V. cholerae did not utilize malonate as a primary carbon source. However, we found that the addition of malonate to the growth medium stimulated V. cholerae growth. All together, these results suggest that metabolizing malonate as a nutrient source negatively affects virulence gene expression in V. cholerae.

  15. Proteomics as a tool for studying bacterial virulence and antimicrobial resistance

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    Francisco José Pérez -Llarena

    2016-03-01

    Full Text Available Proteomic studies have improved our understanding of the microbial world. The most recent advances in this field have helped us to explore aspects beyond genomics. For example, by studying proteins and their regulation, researchers now understand how some pathogenic bacteria have adapted to the lethal actions of antibiotics. Proteomics has also advanced our knowledge of mechanisms of bacterial virulence and some important aspects of how bacteria interact with human cells and, thus, of the pathogenesis of infectious diseases. This review article addresses these issues in some of the most important human pathogens. It also reports some applications of MALDI-TOF mass spectrometry that may be important for the diagnosis of bacterial resistance in clinical laboratories in the future. The reported advances will enable new diagnostic and therapeutic strategies to be developed in the fight against some of the most lethal bacteria affecting humans.

  16. Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

    Science.gov (United States)

    Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-04-01

    We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

  17. Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila.

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    Mary F Fontana

    2011-02-01

    Full Text Available The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs, leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR, requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent

  18. 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

  19. Visualizing the Mechanism of Epoxide Hydrolysis by the Bacterial Virulence Enzyme Cif.

    Science.gov (United States)

    Bahl, Christopher D; Hvorecny, Kelli L; Morisseau, Christophe; Gerber, Scott A; Madden, Dean R

    2016-02-09

    The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

  20. Bacterial quorum sensing: its role in virulence and possibilities for its control.

    Science.gov (United States)

    Rutherford, Steven T; Bassler, Bonnie L

    2012-11-01

    Quorum sensing is a process of cell-cell communication that allows bacteria to share information about cell density and adjust gene expression accordingly. This process enables bacteria to express energetically expensive processes as a collective only when the impact of those processes on the environment or on a host will be maximized. Among the many traits controlled by quorum sensing is the expression of virulence factors by pathogenic bacteria. Here we review the quorum-sensing circuits of Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Vibrio cholerae. We outline these canonical quorum-sensing mechanisms and how each uniquely controls virulence factor production. Additionally, we examine recent efforts to inhibit quorum sensing in these pathogens with the goal of designing novel antimicrobial therapeutics.

  1. Influence of sublethal concentrations of common disinfectants on expression of virulence genes in Listeria monocytogenes

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Larsen, M. H.; Gram, Lone;

    2010-01-01

    , and quaternary ammonium compounds (QAC) induced the expression of the virulence genes. In general, a disinfectant had similar effects on the expression of all four virulence genes examined. Northern blot analyses confirmed the downregulation of prfA and inlA expression by Incimaxx DES (a peroxy compound...

  2. Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss

    Directory of Open Access Journals (Sweden)

    Barker Melissa

    2010-12-01

    Full Text Available Abstract Background The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains. Results To better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes. Conclusions Genome evolution in Listeria involved limited gene loss and acquisition as supported by (i a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii conserved genome size (between 2.8 and 3.2 Mb, and (iii a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus

  3. Burkholderia cepacia Complex Regulation of Virulence Gene Expression: A Review

    Science.gov (United States)

    Sousa, Sílvia A.; Feliciano, Joana R.; Pita, Tiago; Guerreiro, Soraia I.; Leitão, Jorge H.

    2017-01-01

    Burkholderia cepacia complex (Bcc) bacteria emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Their eradication is very difficult due to the high level of intrinsic resistance to clinically relevant antibiotics. Bcc bacteria have large and complex genomes, composed of two to four replicons, with variable numbers of insertion sequences. The complexity of Bcc genomes confers a high genomic plasticity to these bacteria, allowing their adaptation and survival to diverse habitats, including the human host. In this work, we review results from recent studies using omics approaches to elucidate in vivo adaptive strategies and virulence gene regulation expression of Bcc bacteria when infecting the human host or subject to conditions mimicking the stressful environment of the cystic fibrosis lung. PMID:28106859

  4. The balance of apoptotic and necrotic cell death in Mycobacterium tuberculosis infected macrophages is not dependent on bacterial virulence.

    Directory of Open Access Journals (Sweden)

    Rachel E Butler

    Full Text Available BACKGROUND: An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission. METHODS: We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237. RESULTS: We show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis--both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis. CONCLUSIONS: This reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.

  5. Host Characteristics and Bacterial Traits Predict Experimental Virulence for Escherichia coli Bloodstream Isolates From Patients With Urosepsis.

    Science.gov (United States)

    Johnson, James R; Porter, Stephen; Johnston, Brian; Kuskowski, Michael A; Spurbeck, Rachel R; Mobley, Harry L T; Williamson, Deborah A

    2015-09-01

    Background.  Extraintestinal Escherichia coli infections are common, costly, and potentially serious. A better understanding of their pathogenesis is needed. Methods.  Sixty-seven E coli bloodstream isolates from adults with urosepsis (Seattle, WA; 1980s) underwent extensive molecular characterization and virulence assessment in 2 infection models (murine subcutaneous sepsis and moth larval lethality). Statistical comparisons were made among host characteristics, bacterial traits, and experimental virulence. Results.  The 67 source patients were diverse for age, sex, and underlying medical and urological conditions. The corresponding E coli isolates exhibited diverse phylogenetic backgrounds and virulence profiles. Despite the E coli isolates' common bloodstream origin, they exhibited a broad range of experimental virulence in mice and moth larvae, in patterns that (for the murine model only) corresponded significantly with host characteristics and bacterial traits. The most highly mouse-lethal strains were enriched with classic "urovirulence" traits and typically were from younger women with anatomically and functionally normal urinary tracts. The 2 animal models corresponded poorly with one another. Conclusions.  Host compromise, including older age and urinary tract abnormalities, allows comparatively low-virulence E coli strains to cause urosepsis. Multiple E coli traits predict both experimental and epidemiological virulence. The larval lethality model cannot be a substitute for the murine sepsis model.

  6. Virulence gene content in Escherichia coli isolates from poultry flocks with clinical signs of colibacillosis in Brazil.

    Science.gov (United States)

    De Carli, Silvia; Ikuta, Nilo; Lehmann, Fernanda Kieling Moreira; da Silveira, Vinicius Proença; de Melo Predebon, Gabriela; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo

    2015-11-01

    Escherichia coli is a commensal bacterium of the bird's intestinal tract, but it can invade different tissues resulting in systemic symptoms (colibacillosis). This disease occurs only when the E. coli infecting strain presents virulence factors (encoded by specific genes) that enable the adhesion and proliferation in the host organism. Thus, it is important to differentiate pathogenic (APEC, avian pathogenic E. coli) and non-pathogenic or fecal (AFEC, avian fecal E. coli) isolates. Previous studies analyzed the occurrence of virulence factors in E. coli strains isolated from birds with colibacillosis, demonstrating a high frequency of the bacterial genes cvaC, iroN, iss, iutA, sitA, tsh, fyuA, irp-2, ompT and hlyF in pathogenic strains. The aim of the present study was to evaluate the occurrence and frequency of these virulence genes in E. coli isolated from poultry flocks in Brazil. A total of 138 isolates of E. coli was obtained from samples of different tissues and/or organs (spleen, liver, kidney, trachea, lungs, skin, ovary, oviduct, intestine, cloaca) and environmental swabs collected from chicken and turkey flocks suspected to have colibacillosis in farms from the main Brazilian producing regions. Total DNA was extracted and the 10 virulence genes were detected by traditional and/or real-time PCR. At least 11 samples of each gene were sequenced and compared to reference strains. All 10 virulence factors were detected in Brazilian E. coli isolates, with frequencies ranging from 39.9% (irp-2) to 68.8% (hlyF and sitA). Moreover, a high nucleotide similarity (over 99%) was observed between gene sequences of Brazilian isolates and reference strains. Seventy-nine isolates were defined as pathogenic (APEC) and 59 as fecal (AFEC) based on previously described criteria. In conclusion, the main virulence genes of the reference E. coli strains are also present in isolates associated with colibacillosis in Brazil. The analysis of this set of virulence factors can be

  7. Regulation of virulence gene expression resulting from Streptococcus pneumoniae and nontypeable Haemophilus influenzae interactions in chronic disease.

    Directory of Open Access Journals (Sweden)

    Emily K Cope

    Full Text Available Chronic rhinosinusitis (CRS is a common inflammatory disease of the sinonasal cavity mediated, in part, by polymicrobial communities of bacteria. Recent molecular studies have confirmed the importance of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi in CRS. Here, we hypothesize that interaction between S. pneumoniae and NTHi mixed-species communities cause a change in bacterial virulence gene expression. We examined CRS as a model human disease to validate these polymicrobial interactions. Clinical strains of S. pneumoniae and NTHi were grown in mono- and co-culture in a standard biofilm assay. Reverse transcriptase real-time PCR (RTqPCR was used to measure gene expression of key virulence factors. To validate these results, we investigated the presence of the bacterial RNA transcripts in excised human tissue from patients with CRS. Consequences of physical or chemical interactions between microbes were also investigated. Transcription of NTHi type IV pili was only expressed in co-culture in vitro, and expression could be detected ex vivo in diseased tissue. S. pneumoniae pyruvate oxidase was up-regulated in co-culture, while pneumolysin and pneumococcal adherence factor A were down-regulated. These results were confirmed in excised human CRS tissue. Gene expression was differentially regulated by physical contact and secreted factors. Overall, these data suggest that interactions between H. influenzae and S. pneumoniae involve physical and chemical mechanisms that influence virulence gene expression of mixed-species biofilm communities present in chronically diseased human tissue. These results extend previous studies of population-level virulence and provide novel insight into the importance of S. pneumoniae and NTHi in CRS.

  8. 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.

    2016-01-01

    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

  9. 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.

  10. Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease, and related strains provides insights into virulence and host specificity.

    Science.gov (United States)

    Jalan, Neha; Aritua, Valente; Kumar, Dibyendu; Yu, Fahong; Jones, Jeffrey B; Graham, James H; Setubal, João C; Wang, Nian

    2011-11-01

    Xanthomonas axonopodis pv. citrumelo is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing, and optical mapping were used to obtain a complete genome sequence of X. axonopodis pv. citrumelo strain F1, 4.9 Mb in size. The strain lacks plasmids, in contrast to other citrus Xanthomonas pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen X. campestris pv. vesicatoria 85-10, with a completely different host range. We also compared X. axonopodis pv. citrumelo to the genome of citrus canker pathogen X. axonopodis pv. citri 306. Comparative genomic analysis showed differences in several gene clusters, like those for type III effectors, the type IV secretion system, lipopolysaccharide synthesis, and others. In addition to pthA, effectors such as xopE3, xopAI, and hrpW were absent from X. axonopodis pv. citrumelo while present in X. axonopodis pv. citri. These effectors might be responsible for survival and the low virulence of this pathogen on citrus compared to that of X. axonopodis pv. citri. We also identified unique effectors in X. axonopodis pv. citrumelo that may be related to the different host range as compared to that of X. axonopodis pv. citri. X. axonopodis pv. citrumelo also lacks various genes, such as syrE1, syrE2, and RTX toxin family genes, which were present in X. axonopodis pv. citri. These may be associated with the distinct virulences of X. axonopodis pv. citrumelo and X. axonopodis pv. citri. Comparison of the complete genome sequence of X. axonopodis pv. citrumelo to those of X. axonopodis pv. citri and X. campestris pv. vesicatoria provides valuable insights into the mechanism of bacterial virulence and host specificity.

  11. A wandering pathway in plant biology: from wildflowers to phototropins to bacterial virulence.

    Science.gov (United States)

    Briggs, Winslow R

    2010-01-01

    The author describes the somewhat convoluted pathway he followed from amateur taxonomy of Minnesota wildflowers to identification of the phototropin family of blue-light receptors. He also mentions individuals who were important in moving his career first into plant taxonomy, then plant development, and finally plant photobiology (and out of music). He emphasizes the many twists and turns a research career can take, including a few that lead to blind ends. He also emphasizes the oscillatory nature of his career-back and forth between the Atlantic and Pacific oceans (with occasional forays to Freiburg, Germany) and back and forth between red-light receptors and blue-light receptors. There is a short intermission in which he describes his longtime relationship with California's Henry W. Coe State Park. Finally, he relates how he followed the unlikely pathway from plant blue-light receptors to a blue-light receptor required to maximize virulence of a bacterial animal pathogen.

  12. The bacterial alarmone (p)ppGpp is required for virulence and controls cell size and survival of Pseudomonas syringae on plants.

    Science.gov (United States)

    Chatnaparat, Tiyakhon; Li, Zhong; Korban, Schuyler S; Zhao, Youfu

    2015-11-01

    The stringent response, mediated by second messenger (p)ppGpp, results in swift and massive transcriptional reprogramming under nutrient limited conditions. In this study, the role of (p)ppGpp on virulence of Pseudomonas syringae pv. syringae B728a (PssB728a) was investigated. The virulence of the relA/spoT (ppGpp(0) ) double mutant was completely impaired on bean, and bacterial growth was significantly reduced, suggesting that (p)ppGpp is required for full virulence of P. syringae. Expression of T3SS and other virulence genes was reduced in ppGpp(0) mutants. In addition, ppGpp deficiency resulted in loss of swarming motility, reduction of pyoverdine production, increased sensitivity to oxidative stress and antibiotic tolerance, as well as reduced ability to utilize γ-amino butyric acid. Increased levels of ppGpp resulted in reduced cell size of PssB728a when grown in a minimal medium and on plant surfaces, while most ppGpp(0) mutant cells were not viable on plant surfaces 24 h after spray inoculation, suggesting that ppGpp-mediated stringent response temporarily limits cell growth, and might control cell survival on plants by limiting their growth. These results demonstrated that ppGpp-mediated stringent response plays a central role in P. syringae virulence and survival and indicated that ppGpp serves as a global signal for regulating various virulence traits in PssB728a.

  13. Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon.

    Science.gov (United States)

    Benevides-Matos, Najla; Pieri, Fabio A; Penatti, Marilene; Orlandi, Patrícia P

    2015-03-01

    The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli . Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli . Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene . EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg , aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea ( P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

  14. Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon

    Science.gov (United States)

    Benevides-Matos, Najla; Pieri, Fabio A.; Penatti, Marilene; Orlandi, Patrícia P.

    2015-01-01

    The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli . Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli . Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene . EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg , aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea ( P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC. PMID:26221098

  15. Nigribactin, a Novel Siderophore from Vibrio nigripulchritudo, Modulates Staphylococcus aureus Virulence Gene Expression

    DEFF Research Database (Denmark)

    Nielsen, Anita; Månsson, Maria; Wietz, Matthias;

    2012-01-01

    Staphylococcus aureus is a serious human pathogen that employs a number of virulence factors as part of its pathogenesis. The purpose of the present study was to explore marine bacteria as a source of compounds that modulate virulence gene expression in S. aureus. During the global marine Galathe...

  16. A study of bacterial gene regulatory mechanisms

    DEFF Research Database (Denmark)

    Hansen, Sabine

    the different regulatory mechanisms affect system dynamics. We have designed a synthetic gene regulatory network (GRN) in bacterial cells that enables us to study the dynamics of GRNs. The results presented in this PhD thesis show that model equations based on the established mechanisms of action of each...... of a particular type of regulatory mechanism. The synthetic system presented in this thesis is, to our knowledge, the first of its kind to allow a direct comparison of the dynamic behaviors of gene regulatory networks that employ different mechanisms of regulation. In addition to studying the dynamic behavior...... of GRNs this thesis also provided the first evidence of the sensor histidine kinase VC1831 being an additional player in the Vibrio cholerae quorum sensing (QS) GRN. Bacteria use a process of cell-cell communication called QS which enable the bacterial cells to collectively control their gene expression...

  17. Structural and Molecular Mechanism of CdpR Involved in Quorum-Sensing and Bacterial Virulence in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Jingru Zhao

    2016-04-01

    Full Text Available Although quorum-sensing (QS systems are important regulators of virulence gene expression in the opportunistic human pathogen Pseudomonas aeruginosa, their detailed regulatory mechanisms have not been fully characterized. Here, we show that deletion of PA2588 resulted in increased production of pyocyanin and biofilm, as well as enhanced pathogenicity in a mouse model. To gain insights into the function of PA2588, we performed a ChIP-seq assay and identified 28 targets of PA2588, including the intergenic region between PA2588 and pqsH, which encodes the key synthase of Pseudomonas quinolone signal (PQS. Though the C-terminal domain was similar to DNA-binding regions of other AraC family members, structural studies revealed that PA2588 has a novel fold at the N-terminal region (NTR, and its C-terminal HTH (helix-turn-helix domain is also unique in DNA recognition. We also demonstrated that the adaptor protein ClpS, an essential regulator of ATP-dependent protease ClpAP, directly interacted with PA2588 before delivering CdpR to ClpAP for degradation. We named PA2588 as CdpR (ClpAP-degradation and pathogenicity Regulator. Moreover, deletion of clpP or clpS/clpA promotes bacterial survival in a mouse model of acute pneumonia infection. Taken together, this study uncovered that CdpR is an important QS regulator, which can interact with the ClpAS-P system to regulate the expression of virulence factors and pathogenicity.

  18. Horizontal gene transfer and bacterial diversity

    Indian Academy of Sciences (India)

    Chitra Dutta; Archana Pan

    2002-02-01

    Bacterial genomes are extremely dynamic and mosaic in nature. A substantial amount of genetic information is inserted into or deleted from such genomes through the process of horizontal transfer. Through the introduction of novel physiological traits from distantly related organisms, horizontal gene transfer often causes drastic changes in the ecological and pathogenic character of bacterial species and thereby promotes microbial diversification and speciation. This review discusses how the recent influx of complete chromosomal sequences of various microorganisms has allowed for a quantitative assessment of the scope, rate and impact of horizontally transmitted information on microbial evolution.

  19. Adherence and virulence genes of Escherichia colifrom children diarrhoea in the Brazilian Amazon

    Directory of Open Access Journals (Sweden)

    Najla Benevides-Matos

    2015-03-01

    Full Text Available The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli. Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli. Out of 1,156 isolates obtained, 128 (11.0% were positive for eae genes corresponding to EPEC, however only 38 (29.6% of these amplified bfpAgene. EAEC were isolated from 164 (14.1% samples; of those 41(25%, 32 (19% and 16 (9.7% amplified eagg, aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea (P = 0.00006. DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

  20. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene.

    Science.gov (United States)

    Shippy, Daniel C; Eakley, Nicholas M; Bochsler, Philip N; Chopra, Ashok K; Fadl, Amin A

    2011-06-01

    Salmonella enterica serovar Typhimurium is a frequent cause of enteric disease due to the consumption of contaminated food. Identification and characterization of bacterial factors involved in Salmonella pathogenesis would help develop effective strategies for controlling salmonellosis. To investigate the role of glucose-inhibited division gene (gidA) in Salmonella virulence, we constructed a Salmonella mutant strain in which gidA was deleted. Deletion of gidA rendered Salmonella deficient in the invasion of intestinal epithelial cells, bacterial motility, intracellular survival, and induction of cytotoxicity in host cells. Deletion of gidA rendered the organism to display a filamentous morphology compared to the normal rod-shaped nature of Salmonella. Furthermore, a significant attenuation in the induction of inflammatory cytokines and chemokines, histopathological lesions, and systemic infection was observed in mice infected with the gidA mutant. Most importantly, a significant increase in LD(50) was observed in mice infected with the gidA mutant, and mice immunized with the gidA mutant were able to survive a lethal dose of wild-type Salmonella. Additionally, deletion of gidA significantly altered the expression of several bacterial factors associated with pathogenesis as indicated by global transcriptional and proteomic profiling. Taken together, our data indicate GidA as a potential regulator of Salmonella virulence genes.

  1. A Bacterial Pathogen Displaying Temperature-Enhanced Virulence of the Microalga Emiliania huxleyi.

    Science.gov (United States)

    Mayers, Teaghan J; Bramucci, Anna R; Yakimovich, Kurt M; Case, Rebecca J

    2016-01-01

    shown to have acquired resistance against EhVs at elevated temperature, bacterial pathogens with temperature-dependent virulence, such as R11, may become much more important in the ecology of E. huxleyi in a warming climate.

  2. Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears.

    Science.gov (United States)

    Ramos, Laura S; Lehman, Brian L; Peter, Kari A; McNellis, Timothy W

    2014-11-01

    Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.

  3. In vitro and in vivo expression of virulence genes in Vibrio isolates belonging to the Harveyi clade in relation to their virulence towards gnotobiotic brine shrimp (Artemia franciscana).

    Science.gov (United States)

    Ruwandeepika, H A Darshanee; Defoirdt, Tom; Bhowmick, Patit Paban; Karunasagar, Indrani; Karunasagar, Iddya; Bossier, Peter

    2011-02-01

    Vibrios belonging to the Harveyi clade are pathogenic marine bacteria affecting both vertebrates and invertebrates, thereby causing a severe threat to the aquaculture industry. In this study, the expression of haemolysin, metalloprotease, serine protease, the quorum sensing master regulator LuxR and the virulence regulator ToxR in different Harveyi clade isolates was measured with reverse transcriptase real-time PCR with specific primers. There was relatively low variation in the in vitro expression levels of the quorum sensing master regulator luxR (sevenfold), whereas for the other genes, the difference in expression between the isolates showing lowest and highest expression levels was over 25-fold. Furthermore, there was a significant correlation between expression levels of toxR and luxR and between the expression levels of these regulators and the protease genes. The expression levels of luxR, toxR and haemolysin were negatively correlated with the survival of brine shrimp larvae challenged with the isolates. Finally, a non-virulent, a moderately virulent and a strongly virulent isolate were selected to study in vivo expression of the virulence genes during infection of gnotobiotic brine shrimp larvae. The in vivo gene expression study showed a clear difference in virulence gene expression between both virulent isolates and the non-virulent isolate.

  4. DNA Microarray and Gene Ontology Enrichment Analysis Reveals That a Mutation in opsX Affects Virulence and Chemotaxis in Xanthomonas oryzae pv. oryzae

    Directory of Open Access Journals (Sweden)

    Hong-Il Kim

    2016-06-01

    Full Text Available Xanthomonas oryzae pv. oryzae (Xoo causes bacterial leaf blight (BLB in rice (Oryza sativa L.. In this study, we investigated the effect of a mutation in opsX (XOO1056, which encodes a saccharide biosynthesis regulatory protein, on the virulence and bacterial chemotaxis of Xoo. We performed DNA microarray analysis, which showed that 63 of 2,678 genes, including genes related to bacterial motility (flagellar and chemotaxis proteins were significantly downregulated (<−2 log₂ fold changes by the mutation in opsX. Indeed, motility assays showed that the mutant strain was nonmotile on semisolid agar swarm plates. In addition, a mutant strain (opsX::Tn5 showed decreased virulence against the susceptible rice cultivar, IR24. Quantitative real-time RT-PCR reaction was performed to confirm the expression levels of these genes, including those related to flagella and chemotaxis, in the opsX mutant. Our findings revealed that mutation of opsX affects both virulence and bacterial motility. These results will help to improve our understanding of Xoo and provide insight into Xoo-rice interactions.

  5. Quorum sensing in Aeromonas salmonicida subsp. achromogenes and the effect of the autoinducer synthase AsaI on bacterial virulence

    DEFF Research Database (Denmark)

    Schwenteit, Johanna; Gram, Lone; Nielsen, Kristian Fog;

    2011-01-01

    The Gram-negative fish pathogenic bacterium Aeromonas salmonicida possesses the LuxIRtype quorum sensing (QS) system, termed AsaIR. In this study the role of QS in A. salmonicida subsp. achromogenes virulence and pigment production was investigated. Five wild-type Asa strains induced the N......-acyl-homoserinelactone (AHL) monitor bacteria. HPLC–HR-MS analysis identified only one type of AHL, N-butanoyl-L-homoserine lactone (C4-HSL). A knock out mutant of AsaI, constructed by allelic exchange, did not produce a detectable QS signal and its virulence in fish was significantly impaired, as LD50 of the Asa...... an important virulence factor, AsaP1, without affecting bacterial growth, makes A. salmonicida subsp. achromogenes an interesting target organism to study the effects of QS in disease development and QSI in disease control....

  6. CRISPR/Cas systems: new players in gene regulation and bacterial physiology

    Directory of Open Access Journals (Sweden)

    David eWeiss

    2014-04-01

    Full Text Available CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP. Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2, CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.

  7. A functional tonB gene is required for both virulence and competitive fitness in a chinchilla model of Haemophilus influenzae otitis media

    Directory of Open Access Journals (Sweden)

    Morton Daniel J

    2012-06-01

    Full Text Available Abstract Background Haemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. Methods An insertional mutation in tonB was constructed and the impact of the mutation on virulence and fitness in a chinchilla model of otitis media was determined. The tonB insertion mutant strain was significantly impacted in both virulence and fitness as compared to the wildtype strain in this model. Conclusions The tonB gene of H. influenzae is required for the establishment and maintenance of middle ear infection in this chinchilla model of bacterial disease.

  8. Identification of virulence genes carried by bacteriophages obtained from clinically isolated methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Karasartova, Djursun; Cavusoglu, Zeynep Burcin; Turegun, Buse; Ozsan, Murat T; Şahin, Fikret

    2016-12-01

    Bacteriophages play an important role in the pathogenicity of Staphylococcus aureus (S. aureus) either by carrying accessory virulence factors or several superantigens. Despite their importance, there are not many studies showing the actual distribution of the virulence genes carried by the prophages obtained from the clinically isolated Staphylococcus. In this study, we investigated prophages obtained from methicillin-resistant S. aureus (MRSA) strains isolated from hospital- and community-associated (HA-CA) infections for the virulence factors. In the study, 43 phages isolated from 48 MRSA were investigated for carrying toxin genes including the sak, eta, lukF-PV, sea, selp, sek, seg, seq chp, and scn virulence genes using polymerase chain reaction (PCR) and Southern blot. Restriction fragment length polymorphism was used to analyze phage genomes to investigate the relationship between the phage profiles and the toxin genes' presence. MRSA strains isolated from HA infections tended to have higher prophage presence than the MRSA strains obtained from the CA infections (97% and 67%, respectively). The study showed that all the phages with the exception of one phage contained one or more virulence genes in their genomes with different combinations. The most common toxin genes found were sea (83%) followed by sek (77%) and seq (64%). The study indicates that prophages encode a significant proportion of MRSA virulence factors.

  9. Pathways Leading from BarA/SirA to Motility and Virulence Gene Expression in Salmonella

    OpenAIRE

    Teplitski, Max; Goodier, Robert I.; Ahmer, Brian M. M.

    2003-01-01

    The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that h...

  10. A Nonsynonymous SNP Catalog of Mycobacterium tuberculosis Virulence Genes and Its Use for Detecting New Potentially Virulent Sublineages

    Science.gov (United States)

    Mikheecheva, Natalya E.; Zaychikova, Marina V.; Melerzanov, Alexander V.

    2017-01-01

    Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin–antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed. PMID:28338924

  11. Variation in virulence of Beauveria bassiana and B. pseudobassiana to the pine weevil Pissodes nemorensis in relation to mycelium characteristics and virulence genes.

    Science.gov (United States)

    Romón, Pedro; Hatting, Hardus; Goldarazena, Arturo; Iturrondobeitia, Juan Carlos

    2017-02-01

    Entomopathogenic fungi such as Beauveria spp. have potential applications in the biocontrol of insect pests but little is known regarding their infectivity to the pine weevil Pissodes nemorensis. In this study, five isolates of Beauveria pseudobassiana and five isolates of Beauveria bassiana were tested for characteristics correlating with virulence on P. nemorensis. Isolate UAMH301 had the lowest mean lethal concentration value whereas the highest value was obtained with isolate LRC137. Growth rate was negatively correlated with virulence in B. bassiana, because isolate LRC137, the least virulent isolate, grew much more rapidly than the other B. bassiana isolates on SDYA. In contrast, its growth on a hyperosmotic medium was the slowest. Sporulation rate and conidial area were not correlated with virulence. Mycelial cell density was positively correlated with virulence in both species, and the four tested genes appear to be one-copy genes. Bbchit1 and Bbhog1, genes respectively encoding a chitinase and a protein kinase, induced relative expression levels were positively correlated with virulence in B. pseudobassiana. We discuss in terms of previous morphological, physiological and genetic parameters related to virulence in Beauveria and the importance of testing the expression of putative virulence genes in comparison with their basal transcript levels.

  12. Beyond the chromosome: the prevalence of unique extra-chromosomal bacteriophages with integrated virulence genes in pathogenic Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Bryan Utter

    Full Text Available In Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01 from a vancomycin-intermediate S. aureus (VISA strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC. Our identification of several potential ExPΦs and mobile genetic elements (MGEs also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT.

  13. Nutrition and Helicobacter pylori: Host Diet and Nutritional Immunity Influence Bacterial Virulence and Disease Outcome

    Directory of Open Access Journals (Sweden)

    Kathryn P. Haley

    2016-01-01

    Full Text Available Helicobacter pylori colonizes the stomachs of greater than 50% of the world’s human population making it arguably one of the most successful bacterial pathogens. Chronic H. pylori colonization results in gastritis in nearly all patients; however in a subset of people, persistent infection with H. pylori is associated with an increased risk for more severe disease outcomes including B-cell lymphoma of mucosal-associated lymphoid tissue (MALT lymphoma and invasive adenocarcinoma. Research aimed at elucidating determinants that mediate disease progression has revealed genetic differences in both humans and H. pylori which increase the risk for developing gastric cancer. Furthermore, host diet and nutrition status have been shown to influence H. pylori-associated disease outcomes. In this review we will discuss how H. pylori is able to create a replicative niche within the hostile host environment by subverting and modifying the host-generated immune response as well as successfully competing for limited nutrients such as transition metals by deploying an arsenal of metal acquisition proteins and virulence factors. Lastly, we will discuss how micronutrient availability or alterations in the gastric microbiome may exacerbate negative disease outcomes associated with H. pylori colonization.

  14. Expression of Vibrio salmonicida virulence genes and immune response parameters in experimentally challenged Atlantic salmon (Salmo salar L.

    Directory of Open Access Journals (Sweden)

    Ane Mohn Bjelland

    2013-12-01

    Full Text Available The Gram-negative bacterium Vibrio salmonicida is the causative agent of cold-water vibriosis (CV, a hemorrhagic septicemia that primarily affects farmed Atlantic salmon (Salmo salar L.. The mechanisms of disease development, host specificity and adaptation, as well as the immunogenic properties of V. salmonicida are largely unknown. Therefore, to gain more knowledge on the pathogenesis of CV, 90 Atlantic salmon parr were injected intraperitonellay with 6 x 106 CFU of V. salmonicida LFI1238. Samples from blood and spleen tissue were taken at different time points throughout the challenge for gene expression analysis by two-step reverse transcription quantitative real-time polymerase chain reaction. Out of a panel of six housekeeping genes, accD, gapA and 16S rDNA were found to be the most suitable references for expression analysis in Vibrio salmonicida. The bacterial proliferation during challenge was monitored based on the expression of the 16S rRNA encoding gene. Before day 4, the concentrations of V. salmonicida in blood and spleen tissue demonstrated a lag phase. From day 4, the bacterial proliferation was exponential. The expression profiles of eight genes encoding potential virulence factors of V. salmonicida were studied. Surprisingly, all tested virulence genes were generally highest expressed in broth cultures compared to the in vivo samples. We hypothesize that this general muting of gene expression in vivo may be a strategy for V. salmonicida to hide from the host immune system. To further investigate this hypothesis, the expression profiles of eight genes encoding innate immune factors were analyzed. The results demonstrated a strong and rapid, but short-lasting innate immune response against V. salmonicida. These results suggest that the bacterium possesses mechanisms that inhibit and/or resist the salmon innate immune system until the host becomes exhausted of fighting the on-going and eventually overwhelming infection.

  15. Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

    Institute of Scientific and Technical Information of China (English)

    张继瑜; 刘红; 张笑兵; 杨剑; 杨帆; 杨国威; 沈岩; 侯云德; 金奇

    2003-01-01

    The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.

  16. Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species.

    Science.gov (United States)

    Whiteduck-Léveillée, Jenni; Cloutier, Michel; Topp, Edward; Lapen, David R; Talbot, Guylaine; Villemur, Richard; Khan, Izhar U H

    2016-02-01

    As the pathogenicity of Arcobacter species might be associated with various virulence factors, this study was aimed to develop and optimize three single-tube multiplex PCR (mPCR) assays that can efficiently detect multiple virulence-associated genes (VAGs) in Arcobacter spp. including the Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively. The recognized target virulence factors used in the study were fibronectin binding protein (cj1349), filamentous hemagglutinin (hecA), hemolysin activation protein (hecB), hemolysin (tlyA), integral membrane protein virulence factor (mviN), invasin (ciaB), outer membrane protein (irgA) and phospholipase (pldA). Identical results were obtained between singleplex PCR and mPCR assays and no cross- and/or non-specific amplification products were obtained when tested against other closely related bacterial species. The sensitivities of these three mPCR assays were ranging from 1ngμL(-1) to 100ngμL(-1) DNA. The developed assays with combinations of duplex or triplex PCR primer pairs of VAGs were further evaluated and validated by applying them to isolates of the A. butzleri, A. cryaerophilus and A. skirrowii recovered from fecal samples of human and animal origins. The findings revealed that the distribution of the ciaB (90%), mviN (70%), tlyA (50%) and pldA (45%) genes among these target species was significantly higher than the hecA (16%), hecB (10%) and each of irgA and cj1349 (6%) genes, respectively. The newly developed mPCR assays can be used as rapid technique and useful markers for the detection, prevalence and profiling of VAGs in the Arcobacter spp. Moreover, these assays can easily be performed with a high throughput to give a presumptive identification of the causal pathogen in epidemiological investigation of human infections.

  17. Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

    Institute of Scientific and Technical Information of China (English)

    WANG Yao; DAI Yue; ZHANG Yong; HU YangBo; YANG BaoYu; CHEN ShiYun

    2007-01-01

    The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs):N-oxododecanoyI-L-homoserine lactone (OdDHL) and N-butyryI-L-homoserine lactone (BHL). The reduced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P.aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope.

  18. Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The re- duced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope.

  19. Virulence gene profiles of avian pathogenic Escherichia coli isolated from chickens with colibacillosis in Bulawayo, Zimbabwe

    Directory of Open Access Journals (Sweden)

    Joshua Mbanga

    2015-02-01

    Full Text Available Colibacillosis, a disease caused by avian pathogenic Escherichia coli (APEC, is one of the main causes of economic losses in the poultry industry worldwide. This study was carried out in order to determine the APEC-associated virulence genes contained by E. coli isolates causing colibacillosis in chickens. A total of 45 E. coli isolates were obtained from the diagnostics and research branch of the Central Veterinary Laboratories, Bulawayo, Zimbabwe. These isolates were obtained from chickens with confirmed cases of colibacillosis after postmortem examination. The presence of the iutA, hlyF, ompT, frz, sitD, fimH, kpsM, sitA, sopB, uvrY, pstB and vat genes were investigated by multiplex polymerase chain reaction (PCR assay. Of the 45 isolates, 93% were positive for the presence of at least one virulence gene. The three most prevalent virulence genes were iutA (80%, fimH (33.3% and hlyF (24.4%. The kpsM, pstB and ompT genes had the lowest prevalence, having been detected in only 2.2% of the isolates. All 12 virulence genes studied were detected in the 45 APEC isolates. Virulence gene profiles were constructed for each APEC isolate from the multiplex data. The APEC isolates were profiled as 62.2% fitting profile A, 31.1% profile B and 6.7% profile C. None of the isolates had more than seven virulence genes. Virulence profiles of Zimbabwean APEC isolates are different from those previously reported. Zimbabwean APEC isolates appear to be less pathogenic and may rely on environmental factors and stress in hosts to establish infection.

  20. Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

    Science.gov (United States)

    Thomson, Joshua J; Withey, Jeffrey H

    2014-11-01

    The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.

  1. Quantitative trait locus (QTL mapping reveals a role for unstudied genes in Aspergillus virulence.

    Directory of Open Access Journals (Sweden)

    Julian K Christians

    Full Text Available Infections caused by the fungus Aspergillus are a major cause of morbidity and mortality in immunocompromised populations. To identify genes required for virulence that could be used as targets for novel treatments, we mapped quantitative trait loci (QTL affecting virulence in the progeny of a cross between two strains of A. nidulans (FGSC strains A4 and A91. We genotyped 61 progeny at 739 single nucleotide polymorphisms (SNP spread throughout the genome, and constructed a linkage map that was largely consistent with the genomic sequence, with the exception of one potential inversion of ∼527 kb on Chromosome V. The estimated genome size was 3705 cM and the average intermarker spacing was 5.0 cM. The average ratio of physical distance to genetic distance was 8.1 kb/cM, which is similar to previous estimates, and variation in recombination rate was significantly positively correlated with GC content, a pattern seen in other taxa. To map QTL affecting virulence, we measured the ability of each progeny strain to kill model hosts, larvae of the wax moth Galleria mellonella. We detected three QTL affecting in vivo virulence that were distinct from QTL affecting in vitro growth, and mapped the virulence QTL to regions containing 7-24 genes, excluding genes with no sequence variation between the parental strains and genes with only synonymous SNPs. None of the genes in our QTL target regions have been previously associated with virulence in Aspergillus, and almost half of these genes are currently annotated as "hypothetical". This study is the first to map QTL affecting the virulence of a fungal pathogen in an animal host, and our results illustrate the power of this approach to identify a short list of unknown genes for further investigation.

  2. Distribution of Virulence-Associated Genes of Avian Pathogenic Escherichia coli Isolates in China

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    216 avian pathogenic Escherichia coli (APEC) isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as O78, O88, and O93. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD (70.8%), iss (58.8%), and tsh (51.4%) in APEC isolates. The detection rate of high pathogenicity islands (HPI)-associatedfyuA and irp2 genes were both 44.9%, with no LEE (the locus of enterocyte effacement) island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained only fimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% O78 isolates had a gene distribution patterns of fimC+iucD+irp2+fyuA+iss+colV+tsh+.

  3. Helicobacter pylori virulence genes and host genetic polymorphisms as risk factors for peptic ulcer disease.

    Science.gov (United States)

    Miftahussurur, Muhammad; Yamaoka, Yoshio

    2015-01-01

    Helicobacter pylori infection plays an important role in the pathogenesis of peptic ulcer disease (PUD). Several factors have been proposed as possible H. pylori virulence determinants; for example, bacterial adhesins and gastric inflammation factors are associated with an increased risk of PUD. However, differences in bacterial virulence factors alone cannot explain the opposite ends of the PUD disease spectrum, that is duodenal and gastric ulcers; presumably, both bacterial and host factors contribute to the differential response. Carriers of the high-producer alleles of the pro-inflammatory cytokines IL-1B, IL-6, IL-8, IL-10, and TNF-α who also carry low-producer allele of anti-inflammatory cytokines have severe gastric mucosal inflammation, whereas carriers of the alternative alleles have mild inflammation. Recent reports have suggested that the PSCA and CYP2C19 ultra-rapid metabolizer genotypes are also associated with PUD.

  4. A nonluminescent and highly virulent Vibrio harveyi strain is associated with "bacterial white tail disease" of Litopenaeus vannamei shrimp.

    Science.gov (United States)

    Zhou, Junfang; Fang, Wenhong; Yang, Xianle; Zhou, Shuai; Hu, Linlin; Li, Xincang; Qi, Xinyong; Su, Hang; Xie, Layue

    2012-01-01

    Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by "white tail" and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of "white tail" but of non-bacterial origin, the present disease was named as "bacterial white tail disease (BWTD)". Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system.

  5. A nonluminescent and highly virulent Vibrio harveyi strain is associated with "bacterial white tail disease" of Litopenaeus vannamei shrimp.

    Directory of Open Access Journals (Sweden)

    Junfang Zhou

    Full Text Available Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by "white tail" and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905 was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN, white tail disease (WTD or penaeid white tail disease (PWTD. To differentiate from such diseases as with a sign of "white tail" but of non-bacterial origin, the present disease was named as "bacterial white tail disease (BWTD". Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system.

  6. Distribution of 13 virulence genes among clinical and environmental Aeromonas spp. in Western Australia.

    Science.gov (United States)

    Aravena-Román, M; Inglis, T J J; Riley, T V; Chang, B J

    2014-11-01

    We evaluated the pathogenic potential of 98 clinical and 31 environmental Aeromonas isolates by detecting the presence of 13 virulence genes using a polymerase chain reaction (PCR)-based method. The majority (96 %) of the strains contained at least one of the virulence genes. The overall distribution was aerA/haem (77 %), alt (53 %), lafA (51 %), ast (39 %), flaA (32 %), aspA (29 %), vasH (26 %), ascV (16 %) and aexT (13 %). No amplification products were detected for the genes encoding a bundle-forming pilus (BfpA and BfpG) or a Shiga-like toxin (stx-1 and stx-2). Five or more virulence genes were detected in 42 % of environmental and 24 % of clinical isolates. Among the major species, 48 % of A. hydrophila and 42 % of A. dhakensis isolates harboured five or more virulence genes compared with 19 % in A. veronii bv. sobria and none in A. caviae isolates. Our results suggest that, in Western Australia, strains of A. dhakensis and A. hydrophila are potentially more virulent than those of A. veronii bv. sobria and A. caviae, although the pathogenic potential of Aeromonas spp. is probably strain- rather than species-dependent.

  7. Association between antimicrobial resistance and virulence genes in Escherichia coli obtained from blood and faeces

    DEFF Research Database (Denmark)

    Bagger-Skjøt, Line; Sandvang, Dorthe; Frimodt-Møller, Niels;

    2007-01-01

    Escherichia coli isolates obtained from faeces (n = 85) and blood (n = 123) were susceptibility tested against 17 antimicrobial agents and the presence of 9 virulence genes was determined by PCR. Positive associations between several antimicrobial resistances and 2 VF genes (iutA and traT) were...

  8. Intestinal short-chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA.

    Science.gov (United States)

    Lawhon, Sara D; Maurer, Russell; Suyemoto, Mitsu; Altier, Craig

    2002-12-01

    Salmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI-1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI-1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta, the two genes required to produce acetyl-phosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl-phosphate in the bacterial cytoplasm, a pathway that bypasses barA. We also found that a Delta(ackA-pta) mutation alone had no effect on virulence but, in combination with Delta(barA), it increased the oral LD50 24-fold. Thus, the combined loss of the BarA- and acetate-dependent pathways is required to reduce virulence. Two other short-chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild-type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild-type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive

  9. Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

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    Silvia Y Bando

    2010-09-01

    Full Text Available Enteroinvasive Escherichia coli (EIEC and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i bacterial escape from macrophages after phagocytosis, (ii macrophage death induced by EIEC and S. flexneri, (iii macrophage cytokine expression in response to infection and (iv expression of plasmidial (pINV virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

  10. Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra.

    Science.gov (United States)

    Maluta, Renato P; Borges, Clarissa A; Beraldo, Lívia G; Cardozo, Marita V; Voorwald, Fabiana A; Santana, André M; Rigobelo, Everlon C; Toniollo, Gilson H; Avila, Fernando A

    2014-11-01

    Escherichia coli is the most common bacterial agent isolated from canine pyometra. The frequencies of 24 virulence genes and pulsed field gel electrophoresis (PFGE) profiles were determined for 23 E. coli isolates from cases of canine pyometra in Brazil. The frequencies of virulence genes were 91.3% fimH, 91.3% irp-2, 82.6% fyuA, 56.5% iroN, 47.8% traT, 39.1% usp, 34.8% sfaD/E, 34.8% tsh, 30.4% papC, 30.4% hlyA, 26.1% papGIII, 26.1% cnf-1, 21.7% papE/F, 21.7% iss, 17.4% iutA, 17.4% ompT, 17.4% cvaC, 17.4% hlyF, 17.4% iucD, 13.0% iucC, 13.0% astA, 4.3% papGII, 0% afaB/C and 0% papGI. The high frequency of yersiniabactin (fyuA and irp2) and salmochelin (iroN) genes suggests that iron uptake systems might be important in the pathogenesis of canine pyometra. PFGE profiles of 19 isolates were heterogeneous, confirming that E. coli isolates from canine pyometra are unlikely to be epidemic clones.

  11. Phase variation leads to the misidentification of a Neisseria gonorrhoeae virulence gene.

    Science.gov (United States)

    Anderson, Mark T; Seifert, H Steven

    2013-01-01

    Neisseria gonorrhoeae is the causative agent of gonorrhea and an obligate pathogen of humans. The Opa proteins of these bacteria are known to mediate attachment and internalization by host cells, including neutrophils. The Opa protein repertoire of a typical N. gonorrhoeae isolate is encoded on ~11 genes distributed throughout the chromosome and is subject to stochastic changes in expression through phase variation. Together, these characteristics make Opa proteins a critical yet unpredictable aspect of any experimental investigation into the interaction of N. gonorrhoeae with host cells. The goal of this study was to identify novel virulence factors of N. gonorrhoeae by assessing the contribution of a set of uncharacterized hydrogen peroxide-induced genes to bacterial survival against neutrophil-mediated killing. To this end, a strain harboring an engineered mutation in the NGO0322 gene was identified that exhibited increased sensitivity to neutrophil-mediated killing, enhanced internalization by neutrophils, and the ability to induce high levels of neutrophil-generated reactive oxygen species. Each of these phenotypes reverted to near wild-type levels following genetic complementation of the NGO0322 mutation. However, after immunoblot analysis of Opa proteins expressed by the isogenic parent, mutant, and genetically complemented strains, it was determined that phase variation had resulted in a disparity between the Opa profiles of these strains. To determine whether Opa phase variation, rather than NGO0322 mutation, was the cause of the observed neutrophil-related phenotypes, NGO0322 function was investigated in N. gonorrhoeae strains lacking all Opa proteins or constitutively expressing the OpaD variant. In both cases, mutation of NGO0322 did not alter survival of gonococci in the presence of neutrophils. These results demonstrate the importance of controlling for the frequent and random variation in Opa protein production by N. gonorrhoeae when investigating

  12. Phase variation leads to the misidentification of a Neisseria gonorrhoeae virulence gene.

    Directory of Open Access Journals (Sweden)

    Mark T Anderson

    Full Text Available Neisseria gonorrhoeae is the causative agent of gonorrhea and an obligate pathogen of humans. The Opa proteins of these bacteria are known to mediate attachment and internalization by host cells, including neutrophils. The Opa protein repertoire of a typical N. gonorrhoeae isolate is encoded on ~11 genes distributed throughout the chromosome and is subject to stochastic changes in expression through phase variation. Together, these characteristics make Opa proteins a critical yet unpredictable aspect of any experimental investigation into the interaction of N. gonorrhoeae with host cells. The goal of this study was to identify novel virulence factors of N. gonorrhoeae by assessing the contribution of a set of uncharacterized hydrogen peroxide-induced genes to bacterial survival against neutrophil-mediated killing. To this end, a strain harboring an engineered mutation in the NGO0322 gene was identified that exhibited increased sensitivity to neutrophil-mediated killing, enhanced internalization by neutrophils, and the ability to induce high levels of neutrophil-generated reactive oxygen species. Each of these phenotypes reverted to near wild-type levels following genetic complementation of the NGO0322 mutation. However, after immunoblot analysis of Opa proteins expressed by the isogenic parent, mutant, and genetically complemented strains, it was determined that phase variation had resulted in a disparity between the Opa profiles of these strains. To determine whether Opa phase variation, rather than NGO0322 mutation, was the cause of the observed neutrophil-related phenotypes, NGO0322 function was investigated in N. gonorrhoeae strains lacking all Opa proteins or constitutively expressing the OpaD variant. In both cases, mutation of NGO0322 did not alter survival of gonococci in the presence of neutrophils. These results demonstrate the importance of controlling for the frequent and random variation in Opa protein production by N. gonorrhoeae

  13. Use of Metarhizium anisopliae Chitinase Genes for Genotyping and Virulence Characterization

    Directory of Open Access Journals (Sweden)

    Saliou Niassy

    2013-01-01

    Full Text Available Virulence is the primary factor used for selection of entomopathogenic fungi (EPF for development as biopesticides. To understand the genetic mechanisms underlying differences in virulence of fungal isolates on various arthropod pests, we compared the chitinase genes, chi2 and chi4, of 8 isolates of Metarhizium anisopliae. The clustering of the isolates showed various groups depending on their virulence. However, the analysis of their chitinase DNA sequences chi2 and chi4 did not reveal major divergences. Although their protein translates have been implicated in fungal virulence, the predicted protein structure of chi2 was identical for all isolates. Despite the critical role of chitin digestion in fungal infection, we conclude that chi2 and chi4 genes cannot serve as molecular markers to characterize observed variations in virulence among M. anisopliae isolates as previously suggested. Nevertheless, processes controlling the efficient upregulation of chitinase expression might be responsible for different virulence characteristics. Further studies using comparative “in vitro” chitin digestion techniques would be more appropriate to compare the quality and the quantity of chitinase production between fungal isolates.

  14. Virulence of Klebsiella pneumoniae isolates harboring bla KPC-2 carbapenemase gene in a Caenorhabditis elegans model.

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    Jean-Philippe Lavigne

    Full Text Available Klebsiella pneumoniae carbapenemase (KPC is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla KPC-2 gene into reference strains K. pneumoniae ATCC10031/CIP53153; and (iii five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days than K. pneumoniae reference strain (LT50: 4.3 days (p<0.01. However, the worldwide spread KPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, p<0.01. The increased virulence observed in cured strains confirmed this trend. The bla KPC-2 gene itself was not associated to increased virulence.

  15. Use of Metarhizium anisopliae chitinase genes for genotyping and virulence characterization.

    Science.gov (United States)

    Niassy, Saliou; Subramanian, Sevgan; Ekesi, Sunday; Bargul, Joel L; Villinger, Jandouwe; Maniania, Nguya K

    2013-01-01

    Virulence is the primary factor used for selection of entomopathogenic fungi (EPF) for development as biopesticides. To understand the genetic mechanisms underlying differences in virulence of fungal isolates on various arthropod pests, we compared the chitinase genes, chi2 and chi4, of 8 isolates of Metarhizium anisopliae. The clustering of the isolates showed various groups depending on their virulence. However, the analysis of their chitinase DNA sequences chi2 and chi4 did not reveal major divergences. Although their protein translates have been implicated in fungal virulence, the predicted protein structure of chi2 was identical for all isolates. Despite the critical role of chitin digestion in fungal infection, we conclude that chi2 and chi4 genes cannot serve as molecular markers to characterize observed variations in virulence among M. anisopliae isolates as previously suggested. Nevertheless, processes controlling the efficient upregulation of chitinase expression might be responsible for different virulence characteristics. Further studies using comparative "in vitro" chitin digestion techniques would be more appropriate to compare the quality and the quantity of chitinase production between fungal isolates.

  16. DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

    Directory of Open Access Journals (Sweden)

    Francisca Gleire Rodrigues de Menezes

    2014-09-01

    Full Text Available The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS, and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

  17. Detection of virulence genes in environmental strains of Vibrio cholerae from estuaries in northeastern Brazil.

    Science.gov (United States)

    Menezes, Francisca Gleire Rodrigues de; Neves, Soraya da Silva; Sousa, Oscarina Viana de; Vila-Nova, Candida Machado Vieira Maia; Maggioni, Rodrigo; Theophilo, Grace Nazareth Diogo; Hofer, Ernesto; Vieira, Regine Helena Silva dos Fernandes

    2014-01-01

    The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

  18. Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes

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    Suat Moi Puah

    2016-02-01

    Full Text Available Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52 of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5% and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines.

  19. Role of Staphylococcus aureus Virulence Factors in Inducing Inflammation and Vascular Permeability in a Mouse Model of Bacterial Endophthalmitis.

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    Ajay Kumar

    Full Text Available Staphylococcus (S. aureus is a common causative agent of bacterial endophthalmitis, a vision threatening complication of eye surgeries. The relative contribution of S. aureus virulence factors in the pathogenesis of endophthalmitis remains unclear. Here, we comprehensively analyzed the development of intraocular inflammation, vascular permeability, and the loss of retinal function in C57BL/6 mouse eyes, challenged with live S. aureus, heat-killed S. aureus (HKSA, peptidoglycan (PGN, lipoteichoic acid (LTA, staphylococcal protein A (SPA, α-toxin, and Toxic-shock syndrome toxin 1 (TSST1. Our data showed a dose-dependent (range 0.01 μg/eye to 1.0 μg/eye increase in the levels of inflammatory mediators by all virulence factors. The cell wall components, particularly PGN and LTA, seem to induce higher levels of TNF-α, IL-6, KC, and MIP2, whereas the toxins induced IL-1β. Similarly, among the virulence factors, PGN induced higher PMN infiltration. The vascular permeability assay revealed significant leakage in eyes challenged with live SA (12-fold and HKSA (7.3-fold, in comparison to other virulence factors (~2-fold and controls. These changes coincided with retinal tissue damage, as evidenced by histological analysis. The electroretinogram (ERG analysis revealed a significant decline in retinal function in eyes inoculated with live SA, followed by HKSA, SPA, and α-toxin. Together, these findings demonstrate the differential innate responses of the retina to S. aureus virulence factors, which contribute to intraocular inflammation and retinal function loss in endophthalmitis.

  20. 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.

  1. The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility

    Science.gov (United States)

    Patot, Sabine; RC Imbert, Paul; Baude, Jessica; Martins Simões, Patricia; Campergue, Jean-Baptiste; Louche, Arthur; Bès, Michèle; Tristan, Anne; Laurent, Frédéric; Fischer, Adrien; Schrenzel, Jacques; François, Patrice; Lina, Gérard

    2017-01-01

    Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors are essential for initiating and propagating the eukaryotic innate immune signaling cascade. Here, we investigate TirS, a Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S. aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC) containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked. PMID:28060920

  2. Salmonella proteomics under oxidative stress reveals coordinated regulation of antioxidant defense with iron metabolism and bacterial virulence.

    Science.gov (United States)

    Fu, Jiaqi; Qi, Linlu; Hu, Mo; Liu, Yanhua; Yu, Kaiwen; Liu, Qian; Liu, Xiaoyun

    2017-02-11

    Salmonella Typhimurium is a bacterial pathogen that can cause widespread gastroenteritis. Salmonella encounters reactive oxygen species both under free-living conditions and within their mammalian host during infection. To study its response to oxidative stress, we performed the first large-scale proteomic profiling of Salmonella upon exposure to H2O2. Among 1600 detected proteins, 83 proteins showed significantly altered abundance. Interestingly, only a subset of known antioxidants was induced, likely due to distinct regulatory mechanisms. In addition, we found elevation of several Salmonella acquired phage products with potential contribution to DNA repair under oxidative stress. Furthermore, we observed robust induction of iron-uptake systems and disruption of these pathways led to bacterial survival defects under H2O2 challenge. Importantly, this work is the first to report that oxidative stress severely repressed the Salmonella type III secretion system (T3SS), reducing its virulence. Biological significance Salmonella, a Gram-negative bacterial pathogen, encounters reactive oxygen species (ROS) both endogenously and exogenously. To better understand its response to oxidative stress, we performed the first large-scale profiling of Salmonella protein expression upon H2O2 treatment. Among 1600 quantified proteins, the abundance of 116 proteins was altered significantly. Notably, iron acquisition systems were induced to promote bacterial survival under oxidative stress. Furthermore, we are the first to report that oxidative stress severely repressed Salmonella type III secretion system and hence reduced its virulence. We believe that these findings will not only help us better understand the molecular mechanisms that Salmonella has evolved to counteract ROS but also the global impact of oxidative stress on bacterial physiology.

  3. The HopQ1 effector's nucleoside hydrolase-like domain is required for bacterial virulence in arabidopsis and tomato, but not host recognition in tobacco.

    Science.gov (United States)

    Li, Wei; Chiang, Yi-Hsuan; Coaker, Gitta

    2013-01-01

    Bacterial pathogens deliver multiple effector proteins into host cells to facilitate bacterial growth. HopQ1 is an effector from Pseudomonas syringae pv. tomato DC3000 that is conserved across multiple bacterial pathogens which infect plants. HopQ1's central region possesses some homology to nucleoside hydrolases, but possesses an alternative aspartate motif not found in characterized enzymes. A structural model was generated for HopQ1 based on the E. coli RihB nucleoside hydrolase and the role of HopQ1's potential catalytic residues for promoting bacterial virulence and recognition in Nicotiana tabacum was investigated. Transgenic Arabidopsis plants expressing HopQ1 exhibit enhanced disease susceptibility to DC3000. HopQ1 can also promote bacterial virulence on tomato when naturally delivered from DC3000. HopQ1's nucleoside hydrolase-like domain alone is sufficient to promote bacterial virulence, and putative catalytic residues are required for virulence promotion during bacterial infection of tomato and in transgenic Arabidopsis lines. HopQ1 is recognized and elicits cell death when transiently expressed in N. tabacum. Residues required to promote bacterial virulence were dispensable for HopQ1's cell death promoting activities in N. tabacum. Although HopQ1 has some homology to nucleoside hydrolases, we were unable to detect HopQ1 enzymatic activity or nucleoside binding capability using standard substrates. Thus, it is likely that HopQ1 promotes pathogen virulence by hydrolyzing alternative ribose-containing substrates in planta.

  4. The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions.

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    Michal Letek

    2010-09-01

    Full Text Available We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT pathogenicity loci, including a cytoadhesive pilus determinant (rpl and the virulence plasmid vap pathogenicity island (PAI required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

  5. Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

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    Cataldi Angel A

    2009-01-01

    Full Text Available Abstract Background The exported repetitive protein (erp gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice. Results In order to elucidate the function of Erp we searched for Erp-binding proteins from M. tuberculosis by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several Actinomycetales genera, orthologues of Rv2617c are exclusively present in members of the M. tuberculosis complex (MTC. The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from Mycobacterium smegmatis and Mycobacterium leprae were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism. Conclusion We identified interactive partners of Erp, an M. tuberculosis protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.

  6. Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment

    OpenAIRE

    Peterson, Janine H.; Tian, Pu; Ieva, Raffaele; Dautin, Nathalie; Bernstein, Harris D.

    2010-01-01

    Autotransporters are bacterial virulence factors consisting of an N-terminal “passenger domain” that is secreted in a C- to-N-terminal direction and a C-terminal “β domain” that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa pas...

  7. Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil

    Directory of Open Access Journals (Sweden)

    Karen A. Borges

    2013-12-01

    Full Text Available Salmonella spp. are considered the main agents of foodborne disease and Salmonella Enteritidis is one of the most frequently isolated serovars worldwide. The virulence of Salmonella spp. and their interaction with the host are complex processes involving virulence factors to overcome host defenses. The purpose of this study was to detect virulence genes in S. Enteritidis isolates from poultry in the South of Brazil. PCR-based assays were developed in order to detect nine genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH and spvC associated with the virulence in eighty-four isolates of S. Enteritidis isolated from poultry. The invA, hilA, sivH, sefA and avrA genes were present in 100% of the isolates; lpfA and sopE were present in 99%; agfA was present in 96%; and the spvC gene was present in 92%. It was possible to characterize the isolates with four different genetic profiles (P1, P2, P3 and P4, as it follows: P1, positive for all genes; P2, negative only for spvC; P3, negative for agfA; and P4, negative for lpfA, spvC and sopE. The most prevalent profile was P1, which was present in 88% of the isolates. Although all isolates belong to the same serovar, it was possible to observe variations in the presence of these virulence-associated genes between different isolates. The characterization of the mechanisms of virulence circulating in the population of Salmonella Enteritidis is important for a better understanding of its biology and pathogenicity. The frequency of these genes and the establishment of genetic profiles can be used to determine patterns of virulence. These patterns, associated with in vivo studies, may help develop tools to predict the ability of virulence of different strains.

  8. Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes.

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    Buse, Helen Y; Lu, Jingrang; Ashbolt, Nicholas J

    2015-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems.

  9. Protective potency of clove oil and its transcriptional down-regulation of Aeromonas sobria virulence genes in African catfish (Clarias gariepinus L.).

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    Abd El-Hamid, M I; Abd El-Aziz, N K; Ali, H A

    2016-08-31

    Disease episodes of fish caused by Aeromonas species are moved to the top list of limiting problems worldwide. The present study was planned to verify the in vitro antibacterial activities as well as the in vivo potential values of clove oil and ciprofloxacin against Aeromonas sobria in African catfish (Clarias gariepinus). The in vitro phenotypic virulence activities and the successful amplification of aerolysin and hemolysin genes in the precisely identified A. sobria strain were predictive for its virulence. In the in vivo assay, virulence of A. sobria strain was fully demonstrated based on constituent mRNA expression profile of tested virulence genes and typical septicemia associated with high mortalities of infected fish. Apparent lower mortality rates were correlated well with both decrescent bacterial burden and significant down-regulated transcripts of representative genes in the treated groups with clove oil, followed by ciprofloxacin as a prophylactic use for 15 days (P < 0.0001); however, the essential oil apart from ciprofloxacin significantly enhanced different hematological parameters (P < 0.05). In addition, administration of antibiotic may be considered as a pronounced stress factor in the fish even when it used in the prophylactic dose. In conclusion, medicinal plants-derived essential oils provide a virtually safer alternative to chemotherapeutics on fish, consumers and ecosystems.

  10. Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro

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    Guo-Qi Wang

    2016-01-01

    Full Text Available Objective. To investigate the effect of negative pressure conditions induced by NPWT on P. aeruginosa. Methods. P. aeruginosa was cultured in a Luria–Bertani medium at negative pressure of −125 mmHg for 24 h in the experimental group and at atmospheric pressure in the control group. The diameters of the colonies of P. aeruginosa were measured after 24 h. ELISA kit, orcinol method, and elastin-Congo red assay were used to quantify the virulence factors. Biofilm formation was observed by staining with Alexa Fluor® 647 conjugate of concanavalin A (Con A. Virulence-regulated genes were determined by quantitative RT-PCR. Results. As compared with the control group, growth of P. aeruginosa was inhibited by negative pressure. The colony size under negative pressure was significantly smaller in the experimental group than that in the controls (p<0.01. Besides, reductions in the total amount of virulence factors were observed in the negative pressure group, including exotoxin A, rhamnolipid, and elastase. RT-PCR results revealed a significant inhibition in the expression level of virulence-regulated genes. Conclusion. Negative pressure could significantly inhibit the growth of P. aeruginosa. It led to a decrease in the virulence factor secretion, biofilm formation, and a reduction in the expression level of virulence-regulated genes.

  11. Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

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    Lone Gram

    2011-12-01

    Full Text Available During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA. To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.

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

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    Silva, Luciana M G; Baums, Christoph G; Rehm, Thomas; Wisselink, Henk J; Goethe, Ralph; Valentin-Weigand, Peter

    2006-06-15

    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 factor (EF, epf), the muramidase-released protein (MRP, mrp) and the hemolysin suilysin (SLY, sly). In this work we present a novel multiplex PCR (MP-PCR) and an mrp variant PCR for identification and characterization of virulent S. suis strains. These new methods were used to identify association of disease with particular profiles of virulence-associated genes. The MP-PCR allowed identification of S. suis through detection of the housekeeping gene gdh, differentiation of four cps types (1, 2, 7 and 9), and detection of epf, mrp, sly and arcA (arginine deiminase from S. suis). Furthermore, this study describes the first PCR assay for differentiation of at least six mrp variants. Expression of the corresponding size variants of MRP was shown for four of the six mrp variants, but was undetectable for the two larger mrp variants in the particular strains investigated. The results of this study suggest that cps7 strains are associated with pneumonia and that variation of mrp is very pronounced among these strains. Gene profiles of invasive, pneumonia and carrier S. suis isolates by combination of PCR assays allowed differentiation of 24 different genotypes among cps1, 2, 7 and 9 strains. Forty-five percent of the invasive S. suis diseases investigated in this study were caused by only two of these genotypes, namely cps2/mrp+/epf+/sly+ and cps9/mrp(*)/epf-/sly+. Thus, this study demonstrates for the first time a uniform profile of the particular virulence-associated genes for the vast majority of the investigated invasive cps9 strains.

  13. Erwinia amylovora expresses fast and simultaneously hrp/dsp virulence genes during flower infection on apple trees.

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    Doris Pester

    Full Text Available BACKGROUND: Pathogen entry through host blossoms is the predominant infection pathway of the gram-negative bacterium Erwinia amylovora leading to manifestation of the disease fire blight. Like in other economically important plant pathogens, E. amylovora pathogenicity depends on a type III secretion system encoded by hrp genes. However, timing and transcriptional order of hrp gene expression during flower infections are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative real-time PCR analyses, we addressed the questions of how fast, strong and uniform key hrp virulence genes and the effector dspA/E are expressed when bacteria enter flowers provided with the full defense mechanism of the apple plant. In non-invasive bacterial inoculations of apple flowers still attached to the tree, E. amylovora activated expression of key type III secretion genes in a narrow time window, mounting in a single expression peak of all investigated hrp/dspA/E genes around 24-48 h post inoculation (hpi. This single expression peak coincided with a single depression in the plant PR-1 expression at 24 hpi indicating transient manipulation of the salicylic acid pathway as one target of E. amylovora type III effectors. Expression of hrp/dspA/E genes was highly correlated to expression of the regulator hrpL and relative transcript abundances followed the ratio: hrpA>hrpN>hrpL>dspA/E. Acidic conditions (pH 4 in flower infections led to reduced virulence/effector gene expression without the typical expression peak observed under natural conditions (pH 7. CONCLUSION/SIGNIFICANCE: The simultaneous expression of hrpL, hrpA, hrpN, and the effector dspA/E during early floral infection indicates that speed and immediate effector transmission is important for successful plant invasion. When this delicate balance is disturbed, e.g., by acidic pH during infection, virulence gene expression is reduced, thus partly explaining the efficacy of acidification in fire blight

  14. Pseudomonas aeruginosa serA Gene Is Required for Bacterial Translocation through Caco-2 Cell Monolayers

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    Yasuda, Masashi; Nagata, Syouya; Yamane, Satoshi; Kunikata, Chinami; Kida, Yutaka; Kuwano, Koichi; Suezawa, Chigusa; Okuda, Jun

    2017-01-01

    To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function. PMID:28046014

  15. Comparative pathogenomics reveals horizontally acquired novel virulence genes in fungi infecting cereal hosts.

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    Donald M Gardiner

    2012-09-01

    Full Text Available Comparative analyses of pathogen genomes provide new insights into how pathogens have evolved common and divergent virulence strategies to invade related plant species. Fusarium crown and root rots are important diseases of wheat and barley world-wide. In Australia, these diseases are primarily caused by the fungal pathogen Fusarium pseudograminearum. Comparative genomic analyses showed that the F. pseudograminearum genome encodes proteins that are present in other fungal pathogens of cereals but absent in non-cereal pathogens. In some cases, these cereal pathogen specific genes were also found in bacteria associated with plants. Phylogenetic analysis of selected F. pseudograminearum genes supported the hypothesis of horizontal gene transfer into diverse cereal pathogens. Two horizontally acquired genes with no previously known role in fungal pathogenesis were studied functionally via gene knockout methods and shown to significantly affect virulence of F. pseudograminearum on the cereal hosts wheat and barley. Our results indicate using comparative genomics to identify genes specific to pathogens of related hosts reveals novel virulence genes and illustrates the importance of horizontal gene transfer in the evolution of plant infecting fungal pathogens.

  16. Co-detection of virulent Escherichia coli genes in surface water sources.

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    Ndlovu, Thando; Le Roux, Marcellous; Khan, Wesaal; Khan, Sehaam

    2015-01-01

    McNemar's test and the Pearson Chi-square were used to assess the co-detection and observed frequency, respectively, for potentially virulent E. coli genes in river water. Conventional multiplex Polymerase Chain Reaction (PCR) assays confirmed the presence of the aggR gene (69%), ipaH gene (23%) and the stx gene (15%) carried by Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Enterohermorrhagic E. coli (EHEC), respectively, in river water samples collected from the Berg River (Paarl, South Africa). Only the aggR gene was present in 23% of samples collected from the Plankenburg River system (Stellenbosch, South Africa). In a comparative study, real-time multiplex PCR assays confirmed the presence of aggR (EAEC) in 69%, stx (EHEC) in 15%, ipaH (EIEC) in 31% and eae (EPEC) in 8% of the river water samples collected from the Berg River. In the Plankenburg River, aggR (EAEC) was detected in 46% of the samples, while eae (EPEC) was present in 15% of the water samples analyzed using real-time multiplex PCR in the Plankenburg River. Pearson Chi-square showed that there was no statistical difference (p > 0.05) between the conventional and real-time multiplex PCRs for the detection of virulent E. coli genes in water samples. However, the McNemar's test showed some variation in the co-detection of virulent E. coli genes, for example, there was no statistical difference in the misclassification of the discordant results for stx versus ipaH, which implies that the ipaH gene was frequently detected with the stx gene. This study thus highlights the presence of virulent E. coli genes in river water and while early detection is crucial, quantitative microbial risk analysis has to be performed to identify and estimate the risk to human health.

  17. Co-detection of virulent Escherichia coli genes in surface water sources.

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    Thando Ndlovu

    Full Text Available McNemar's test and the Pearson Chi-square were used to assess the co-detection and observed frequency, respectively, for potentially virulent E. coli genes in river water. Conventional multiplex Polymerase Chain Reaction (PCR assays confirmed the presence of the aggR gene (69%, ipaH gene (23% and the stx gene (15% carried by Enteroaggregative E. coli (EAEC, Enteroinvasive E. coli (EIEC and Enterohermorrhagic E. coli (EHEC, respectively, in river water samples collected from the Berg River (Paarl, South Africa. Only the aggR gene was present in 23% of samples collected from the Plankenburg River system (Stellenbosch, South Africa. In a comparative study, real-time multiplex PCR assays confirmed the presence of aggR (EAEC in 69%, stx (EHEC in 15%, ipaH (EIEC in 31% and eae (EPEC in 8% of the river water samples collected from the Berg River. In the Plankenburg River, aggR (EAEC was detected in 46% of the samples, while eae (EPEC was present in 15% of the water samples analyzed using real-time multiplex PCR in the Plankenburg River. Pearson Chi-square showed that there was no statistical difference (p > 0.05 between the conventional and real-time multiplex PCRs for the detection of virulent E. coli genes in water samples. However, the McNemar's test showed some variation in the co-detection of virulent E. coli genes, for example, there was no statistical difference in the misclassification of the discordant results for stx versus ipaH, which implies that the ipaH gene was frequently detected with the stx gene. This study thus highlights the presence of virulent E. coli genes in river water and while early detection is crucial, quantitative microbial risk analysis has to be performed to identify and estimate the risk to human health.

  18. Virulence of Klebsiella pneumoniae isolates harboring bla KPC-2 carbapenemase gene in a Caenorhabditis elegans model.

    Science.gov (United States)

    Lavigne, Jean-Philippe; Cuzon, Gaelle; Combescure, Christophe; Bourg, Gisèle; Sotto, Albert; Nordmann, Patrice

    2013-01-01

    Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla KPC-2 gene into reference strains K. pneumoniae ATCC10031/CIP53153; and (iii) five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days) than K. pneumoniae reference strain (LT50: 4.3 days) (pKPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, pKPC-2 gene itself was not associated to increased virulence.

  19. 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.

  20. Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.

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    Carlos F Solis

    Full Text Available BACKGROUND: Modern RNA interference (RNAi methodologies using small interfering RNA (siRNA oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS: Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS: Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

  1. Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.

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    López-Pérez, Mario; Ballester, Ana-Rosa; González-Candelas, Luis

    2015-04-01

    The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.

  2. Differentially Expressed Genes of Virulent and Nonvirulent Entamoeba histolytica Strains Identified by Suppression Subtractive Hybridization

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    Michelle A. R. Freitas

    2014-01-01

    Full Text Available Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH technique, differential gene expressions of two E. histolytica strains, one virulent (EGG and one nonvirulent (452, have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

  3. Differentially expressed genes of virulent and nonvirulent Entamoeba histolytica strains identified by suppression subtractive hybridization.

    Science.gov (United States)

    Freitas, Michelle A R; Alvarenga, Ângela C; Fernandes, Helen C; Gil, Frederico F; Melo, Maria N; Pesquero, Jorge L; Gomes, Maria A

    2014-01-01

    Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

  4. The two CcdA proteins of Bacillus anthracis differentially affect virulence gene expression and sporulation.

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    Han, Hesong; Wilson, Adam C

    2013-12-01

    The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.

  5. Wide Distribution of Virulence Genes among Enterococcus faecium and Enterococcus faecalis Clinical Isolates

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    Sara Soheili

    2014-01-01

    Full Text Available Enterococcus, a Gram-positive facultative anaerobic cocci belonging to the lactic acid bacteria of the phylum Firmicutes, is known to be able to resist a wide range of hostile conditions such as different pH levels, high concentration of NaCl (6.5%, and the extended temperatures between 5°C and 65°C. Despite being the third most common nosocomial pathogen, our understanding on its virulence factors is still poorly understood. The current study was aimed to determine the prevalence of different virulence genes in Enterococcus faecalis and Enterococcus faecium. For this purpose, 79 clinical isolates of Malaysian enterococci were evaluated for the presence of virulence genes. pilB, fms8, efaAfm, and sgrA genes are prevalent in all clinical isolates. In conclusion, the pathogenicity of E. faecalis and E. faecium could be associated with different virulence factors and these genes are widely distributed among the enterococcal species.

  6. In Vivo Expression of Helicobacter pylori Virulence Genes in Patients with Gastritis, Ulcer, and Gastric Cancer

    Science.gov (United States)

    Avilés-Jiménez, Francisco; Reyes-Leon, Adriana; Nieto-Patlán, Erik; Hansen, Lori M.; Burgueño, Juan; Ramos, Irma P.; Camorlinga-Ponce, Margarita; Bermúdez, Hector; Blancas, Juan M.; Cabrera, Lourdes; Ribas-Aparicio, Rosa María

    2012-01-01

    The best-studied Helicobacter pylori virulence factor associated with development of peptic ulcer disease or gastric cancer (GC) rather than asymptomatic nonatrophic gastritis (NAG) is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host epithelial cells. Here we used real-time reverse transcription-PCR (RT-PCR) to measure the in vivo expression of genes on the cagPAI and of other virulence genes in patients with NAG, duodenal ulcer (DU), or GC. In vivo expression of H. pylori virulence genes was greater overall in gastric biopsy specimens of patients with GC than in those of patients with NAG or DU. However, since in vitro expression of cagA was not greater in H. pylori strains from patients with GC than in those from patients with NAG or DU, increased expression in GC in vivo is likely a result of environmental conditions in the gastric mucosa, though it may in turn cause more severe pathology. Increased expression of virulence genes in GC may represent a stress response to elevated pH or other environmental conditions in the stomach of patients with GC, which may be less hospitable to H. pylori colonization than the acidic environment in patients with NAG or DU. PMID:22124657

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

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    Mirelman D.

    2008-09-01

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

  8. Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Hayashi

    Full Text Available Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS. Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs. Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

  9. H-NS is a repressor of major virulence gene loci in Vibrio parahaemolyticus

    Directory of Open Access Journals (Sweden)

    Dongsheng eZhou

    2014-12-01

    Full Text Available Vibrio parahaemolyticus, a leading cause of seafood-associated diarrhea and gastroenteritis, harbors three major virulence gene loci T3SS1, Vp-PAI (T3SS1+tdh2 and T6SS2. As showing is this study, the nucleoid-associated DNA-binding regulator H-NS binds to multiple promoter-proximal regions in each of the above three loci to repress their transcription, and moreover H-NS inhibits the cytotoxicitiy, enterotoxicity, hemolytic activity, and mouse lethality of V. parahaemolyticus. H-NS appears to act as a major repressor of the virulence of this pathogen.

  10. Virulence Genes, Genetic Diversity, Antimicrobial Susceptibility and Phylogenetic Background of Escherichia coli Isolates

    Directory of Open Access Journals (Sweden)

    Abdi

    2015-08-01

    Full Text Available Background The epidemiology of Uropathogenic Escherichia coli (UPEC in urban and rural communities in Iran was never investigated prior to this study. Objectives The aims of this study were to detect the frequency of virulence genes and determine the antimicrobial susceptibility and phylogenetic background of Escherichia coli isolates collected from urban and rural communities. Materials and Methods A total of 100 E. coli isolates were collected from urine samples of patients with urinary tract infections (UTIs residing in two different locations, and confirmed by current biochemical tests. The phylogenetic groups were determined by the triplex-polymerase chain reaction (PCR method, and multiplex PCRs were used to detect eight Vf genes (fimH, iucD, irp2, hlyA, ompT, iha, iroN, and cnf1. The susceptibility profile of E. coli isolates was determined by the disk diffusion method. Results Ninety-five percent of UPEC showed at least one of the virulence genes, the most prevalent being fimH (95%, followed by irp2 (89%, iucD (69%, ompT (67%, iroN (29%, and iha (29%. The various combinations of detected genes were designated as virulence patterns. Phylogenetic groups, B2 (55% and D (22%, comprised the majority of isolated strains. Phenotypic tests showed that 92%, 74% and 71% of the isolates were resistant to ampicillin, ceftizoxime and cefixime, respectively. Conclusions These findings indicate that the UPEC isolates had eight virulence factors with high frequencies. Moreover, these results suggest a direct connection between virulence factors, gene diversity, phylogenetic background, and antimicrobial resistance in UPEC isolates.

  11. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

    NARCIS (Netherlands)

    Michielse, C.B.; Ram, A.F.J.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den

    2004-01-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins

  12. Bacterial virulence phenotypes of Escherichia coli and host susceptibility determine risk for urinary tract infections.

    Science.gov (United States)

    Schreiber, Henry L; Conover, Matt S; Chou, Wen-Chi; Hibbing, Michael E; Manson, Abigail L; Dodson, Karen W; Hannan, Thomas J; Roberts, Pacita L; Stapleton, Ann E; Hooton, Thomas M; Livny, Jonathan; Earl, Ashlee M; Hultgren, Scott J

    2017-03-22

    Urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC) strains. In contrast to many enteric E. coli pathogroups, no genetic signature has been identified for UPEC strains. We conducted a high-resolution comparative genomic study using E. coli isolates collected from the urine of women suffering from frequent recurrent UTIs. These isolates were genetically diverse and varied in their urovirulence, that is, their ability to infect the bladder in a mouse model of cystitis. We found no set of genes, including previously defined putative urovirulence factors (PUFs), that were predictive of urovirulence. In addition, in some patients, the E. coli strain causing a recurrent UTI had fewer PUFs than the supplanted strain. In competitive experimental infections in mice, the supplanting strain was more efficient at colonizing the mouse bladder than the supplanted strain. Despite the lack of a clear genomic signature for urovirulence, comparative transcriptomic and phenotypic analyses revealed that the expression of key conserved functions during culture, such as motility and metabolism, could be used to predict subsequent colonization of the mouse bladder. Together, our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.

  13. Differential expression of pathogenicity- and virulence-related genes of Xanthomonas axonopodis pv. citri under copper stress

    Directory of Open Access Journals (Sweden)

    Ana Carolina Basílio Palmieri

    2010-01-01

    Full Text Available In this study, we used real-time quantitative PCR (RT-qPCR to evaluate the expression of 32 genes of Xanthomonas axonopodis pv. citri related to pathogenicity and virulence that are also involved in copper detoxification. Nearly all of the genes were up-regulated, including copA and copB. Two genes homologous to members of the type II secretion system (xcsH and xcsC and two involved in the degradation of plant cell wall components (pglA and pel were the most expressed in response to an elevated copper concentration. The type II secretion system (xcs operon and a few homologues of proteins putatively secreted by this system showed enhanced expression when the bacteria were exposed to a high concentration of copper sulfate. The enhanced expression of the genes of secretion II system during copper stress suggests that this pathway may have an important role in the adaptative response of X. axonopodis pv. citri to toxic compounds. These findings highlight the potential role of these genes in attenuating the toxicity of certain metals and could represent an important means of bacterial resistance against chemicals used to control diseases.

  14. Virulence genes and antimicrobial susceptibility in Pasteurella multocida isolates from calves.

    Science.gov (United States)

    Katsuda, K; Hoshinoo, K; Ueno, Y; Kohmoto, M; Mikami, O

    2013-12-27

    A total of 378 isolates of Pasteurella multocida from clinically healthy and diseased calves were characterised for their susceptibility to 9 antimicrobial agents and screened by PCR for the presence of antimicrobial resistance genes and 22 genes virulence-associated, including capsule biosynthesis genes. Of the 378 isolates, 102 (27.0%) were resistant to at least one of the 9 tested antimicrobial agents. Resistance to oxytetracycline (21.7%) was the most frequently observed phenotype among the isolates. The tet(H) gene were the primary determinant detected. The resistance rates for thiamphenicol, ampicillin, kanamycin and florfenicol were 13.2%, 5.8%, 9.0% and 0.5%, respectively. Cefazolin, ceftiofur, cefquinome and enrofloxacin were effective antimicrobial agents, with no resistant isolates emerging over the course of the investigation. Most isolates were identified as capsular type A, only 6.3% belonged to capsular type D and no other capsular type was identified. Four of the virulence-associated genes (pfhA, tadD, tbpA and HAS) exhibited associations to the capsular type, and three (pfhA, tbpA and hgbB) were associated with the disease status of the animals. These virulence genes have been considered as epidemiological markers and are hypothesised to have a strong positive association with the outcome of disease in cattle.

  15. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells.

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    Kamalakannan Velmurugan

    2007-07-01

    Full Text Available The survival and persistence of Mycobacterium tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not been demonstrated or elucidated. Using a novel gain-of-function genetic screen, we demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Expression of M. tuberculosis nuoG in nonpathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected human or mouse macrophages, and increased their virulence in a SCID mouse model. Conversely, deletion of nuoG in M. tuberculosis ablated its ability to inhibit macrophage apoptosis and significantly reduced its virulence in mice. These results identify a key component of the genetic basis for an important virulence trait of M. tuberculosis and support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis.

  16. 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.

    Science.gov (United States)

    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.

  17. LATERAL GENE TRANSFER AND THE HISTORY OF BACTERIAL GENOMES

    Energy Technology Data Exchange (ETDEWEB)

    Howard Ochman

    2006-02-22

    The aims of this research were to elucidate the role and extent of lateral transfer in the differentiation of bacterial strains and species, and to assess the impact of gene transfer on the evolution of bacterial genomes. The ultimate goal of the project is to examine the dynamics of a core set of protein-coding genes (i.e., those that are distributed universally among Bacteria) by developing conserved primers that would allow their amplification and sequencing in any bacterial taxa. In addition, we adopted a bioinformatic approach to elucidate the extent of lateral gene transfer in sequenced genome.

  18. 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.

  19. In silico clustering of Salmonella global gene expression data reveals novel genes co-regulated with the SPI-1 virulence genes through HilD

    Science.gov (United States)

    Martínez-Flores, Irma; Pérez-Morales, Deyanira; Sánchez-Pérez, Mishael; Paredes, Claudia C.; Collado-Vides, Julio; Salgado, Heladia; Bustamante, Víctor H.

    2016-01-01

    A wide variety of Salmonella enterica serovars cause intestinal and systemic infections to humans and animals. Salmonella Patogenicity Island 1 (SPI-1) is a chromosomal region containing 39 genes that have crucial virulence roles. The AraC-like transcriptional regulator HilD, encoded in SPI-1, positively controls the expression of the SPI-1 genes, as well as of several other virulence genes located outside SPI-1. In this study, we applied a clustering method to the global gene expression data of S. enterica serovar Typhimurium from the COLOMBOS database; thus genes that show an expression pattern similar to that of SPI-1 genes were selected. This analysis revealed nine novel genes that are co-expressed with SPI-1, which are located in different chromosomal regions. Expression analyses and protein-DNA interaction assays showed regulation by HilD for six of these genes: gtgE, phoH, sinR, SL1263 (lpxR) and SL4247 were regulated directly, whereas SL1896 was regulated indirectly. Interestingly, phoH is an ancestral gene conserved in most of bacteria, whereas the other genes show characteristics of genes acquired by Salmonella. A role in virulence has been previously demonstrated for gtgE, lpxR and sinR. Our results further expand the regulon of HilD and thus identify novel possible Salmonella virulence genes. PMID:27886269

  20. Prevalence of Campylobacter species in milk and milk products, their virulence gene profile and antibiogram

    Directory of Open Access Journals (Sweden)

    Shivani Modi

    2015-01-01

    Full Text Available Aim: During the last decades, number of food poisoning cases due to Campylobacter occurred, immensely. After poultry, raw milk acts as a second main source of Campylobacter. Therefore, the present study was undertaken to detect the prevalence of Campylobacters in milk and milk products and to know the antibiotic sensitivity and virulence gene profile of Campylobacter spp. in Anand city, Gujarat, India. Materials and Methods: A total of 240 samples (85 buffalo milk, 65 cow milk, 30 cheese, 30 ice-cream and 30 paneer were collected from the different collection points in Anand city. The samples were processed by microbiological culture method, and presumptive isolates were further confirmed by genus and species-specific polymerase chain reaction using previously reported primer. The isolates were further subjected to antibiotic susceptibility assay and virulence gene detection. Result: Campylobacter species were detected in 7 (2.91% raw milk samples whereas none of the milk product was positive. All the isolate identified were Campylobacter jejuni. Most of the isolates showed resistance against nalidixic acid, ciprofloxacin, and tetracyclin. All the isolates have three virulence genes cadF, cdtB and flgR whereas only one isolate was positive for iamA gene and 6 isolates were positive for fla gene. Conclusion: The presence of Campylobacter in raw milk indicates that raw milk consumption is hazardous for human being and proper pasteurization of milk and adaptation of hygienic condition will be necessary to protect the consumer from this zoonotic pathogen.

  1. Candidate Targets for New Anti-Virulence Drugs: Selected Cases of Bacterial Adhesion and Biofilm Formation

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Kvist, Malin;

    2007-01-01

    formation are highly attractive targets for new drugs. Specific adhesion provides bacteria with target selection and prevents removal by hydrodynamic flow forces. Bacterial adhesion is of paramount importance for bacterial pathogenesis. Adhesion is also the first step in biofilm formation. Biofilm formation...

  2. Characterization of aminoglycoside resistance and virulence genes among Enterococcus spp. isolated from a hospital in China.

    Science.gov (United States)

    Li, Wanxiang; Li, Jing; Wei, Quhao; Hu, Qingfeng; Lin, Xiaowei; Chen, Mengquan; Ye, Renji; Lv, Huoyang

    2015-03-11

    This study investigated the aminoglycoside resistance phenotypes and genotypes, as well as the prevalence of virulence genes, in Enterococcus species isolated from clinical patients in China. A total of 160 enterococcal isolates from various clinical samples collected from September 2013 to July 2014 were identified to the species level using the VITEK-2 COMPACT system. The antimicrobial susceptibilities of the identified Enterococcus strains were determined by the Kirby-Bauer (K-B) disc diffusion method. PCR-based assays were used to detect the aminoglycoside resistance and virulence genes in all enterococcal isolates. Of 160 Enterococcus isolates, 105 were identified as E. faecium, 35 as E. faecalis, and 20 isolates were classified as "other" Enterococcus species. High-level aminoglycoside resistance (HLAR) for gentamicin, streptomycin, and both antibiotics was identified in 58.8, 50, and 34.4% of strains, respectively. The most common virulence gene (50.6% of isolates) was efaA, followed by asa1 (28.8%). The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(2')-Id, aph(3')-IIIa, and ant(6')-Ia, present in 49.4%, 1.3%, 48.8% and 31.3% of strains, respectively. Overall, E. faecium and E. faecalis were most frequently associated with hospital-acquired enterococcal infections in Zhejiang Province. All aminoglycoside resistance genes, except aph(2'')-Id, were significantly more prevalent in HLAR strains than amongst high level aminoglycoside susceptible (HLAS) strains, while there was no significant difference between HLAR and HLAS strains in regard to the prevalence of virulence genes, apart from esp, therefore, measures should be taken to manage infections caused by multi-drug resistant Enterococcus species.

  3. Thymidine kinase gene mutation leads to reduced virulence of pseudorabies virus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK― mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK― mutant were sequenced. Analysis of the tk gene sequence of TK― mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All other nucleotides of TK―mutant were identical to that of wt virus. Deletion and insertion of the nucleotide sequence resulted in a frameshift and a premature chain termination, and the resultant TK protein was not active. Analysis of the amino acid sequence revealed that TK protein of PRV HB contained the conserved consensus sequence of herpesviral TKs and an additional conserved-DHR-motif. The results of this work also indicated that TK― mutant was genetically stable. Compared to PRV HB, virulence of TK― mutant was greatly decreased. Mice vaccinated with TK― mutant were completely protected against a lethal challenge with virulent PRV (HB).

  4. Presence of T3SS2 and other virulence-related genes in tdh-negative Vibrio parahaemolyticus environmental strains isolated from marine samples in the area of the Venetian Lagoon, Italy.

    Science.gov (United States)

    Caburlotto, Greta; Gennari, Micol; Ghidini, Valentina; Tafi, Mariacarla; Lleo, Maria M

    2009-12-01

    Vibrio parahaemolyticus-mediated disease has traditionally been associated with two virulence factors, thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH), which are present in most clinical isolates. Recently, it has been suggested that other virulence-related factors, such as some type III secretion system (T3SS) proteins, urease and DNA-methyltransferase, among others, might also play a role in disease caused by this bacterial species and have been shown to be carried by clinical, but not by environmental strains. Screening for a number of virulence and virulence-related genes in a collection of V. parahaemolyticus strains isolated from the Italian Adriatic coast indicates that in addition to the trh-positive strains isolated (6%), a significant percentage (18%) of these strains contain one or more genes with a possible role in pathogenicity. Specifically, some of the V. parahaemolyticus strains described in this study are the first environmental strains ever detected carrying T3SS2 genes. Data obtained by reverse transcription-PCR on environmental strain RNA indicate that at least some of these genes are functional. On the basis of the results obtained, it is suggested that such strains might constitute an environmental reservoir of genes possibly contributing to V. parahaemolyticus pathogenicity and to the spread, in the marine environment, of virulence-related genes usually found in clinical strains.

  5. Detection and distribution of putative virulence associated genes in Aeromonas species from freshwater and wastewater treatment plant.

    Science.gov (United States)

    Igbinosa, Isoken H; Okoh, Anthony I

    2013-11-01

    The detection of genes responsible for Aeromonas virulence is a vital tool in establishing the potential pathogenicity of the bacteria, as these virulence genes may act alone or in synergy in the establishment of infections. Freshwater and wastewater mixed liquor samples were collected from Kat river and Fort Beaufort wastewater treatment plant in the Eastern Cape Province of South Africa. Polymerase chain reaction was utilized for the amplification of the different genes coding for virulence. All virulence associated genes screened (alt, lip, fla, aer, ast, hlyA) were detected in at least one Aeromonas isolates. In fresh water sample, virulence genes were distributed as follows: lip (67%), aer (43%), alt (33%), fla (62%), ast (10%), and hlyA (86%), while in wastewater samples the occurrence were as follows: lip (92%), aer (21%), alt (54%), fla (83%), ast (29%), and hlyA (88%). The presence of these virulence genes in environmental Aeromonas isolates is of concern to public health as these organisms are potential pathogens in the environment and the virulence determinants could be transferred to aquatic organisms and humans by one mechanism or the other.

  6. Microbial taxa and functional genes shift in degraded soil with bacterial wilt

    Science.gov (United States)

    Zhang, Hongchun; Wang, Rui; Chen, Shu; Qi, Gaofu; He, Zhili; Zhao, Xiuyun

    2017-01-01

    Soil degradation is a serious global problem, but little is known about how soil microbial communities respond to soil degradation as well as their feedback to ecosystem functioning. In this study, we found the microbial community composition, structure and functional potential significantly altered in the degraded soils with bacterial wilt (termed as degraded soils). Compared with healthy soils, OTU richness of beneficial microorganisms were significantly decreased, but OTU richness of pathogenic microorganisms were significantly increased in the degraded soils. Functional gene array (GeoChip 5.0) analysis showed the functional metabolic potential of genes involved in stress, virulence, sulfur cycle, metal resistance, degradation of plant cell wall was significantly increased in the degraded soils. Increased functional metabolic potential of these genes may be related to the acidification and severe plant disease of degraded soils. Biological activity of degraded soils was obviously decreased with weakened soil enzyme activities when compared to the healthy soils. Soil pH and enzyme activities were negatively correlated with the abundance of genes involved in sulfur cycle, virulence, and stress responses. This study provides new insights into our understanding of soil microbial community responses to soil degradation. PMID:28051173

  7. Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells.

    Science.gov (United States)

    Hayward, Richard D; Cain, Robert J; McGhie, Emma J; Phillips, Neil; Garner, Matthew J; Koronakis, Vassilis

    2005-05-01

    A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.

  8. Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

    Science.gov (United States)

    Laprade, Natacha; Cloutier, Michel; Lapen, David R; Topp, Edward; Wilkes, Graham; Villemur, Richard; Khan, Izhar U H

    2016-05-01

    Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological

  9. In silico phylogenetic and virulence gene profile analyses of avian pathogenic Escherichia coli genome sequences

    Directory of Open Access Journals (Sweden)

    Thaís C.G. Rojas

    2014-02-01

    Full Text Available Avian pathogenic Escherichia coli (APEC infections are responsible for significant losses in the poultry industry worldwide. A zoonotic risk has been attributed to APEC strains because they present similarities to extraintestinal pathogenic E. coli (ExPEC associated with illness in humans, mainly urinary tract infections and neonatal meningitis. Here, we present in silico analyses with pathogenic E. coli genome sequences, including recently available APEC genomes. The phylogenetic tree, based on multi-locus sequence typing (MLST of seven housekeeping genes, revealed high diversity in the allelic composition. Nevertheless, despite this diversity, the phylogenetic tree was able to cluster the different pathotypes together. An in silico virulence gene profile was also determined for each of these strains, through the presence or absence of 83 well-known virulence genes/traits described in pathogenic E. coli strains. The MLST phylogeny and the virulence gene profiles demonstrated a certain genetic similarity between Brazilian APEC strains, APEC isolated in the United States, UPEC (uropathogenic E. coli and diarrheagenic strains isolated from humans. This correlation corroborates and reinforces the zoonotic potential hypothesis proposed to APEC.

  10. Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

    Science.gov (United States)

    Schneeberger, M; Brodard, I; Overesch, G

    2015-04-02

    Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4

  11. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes

    DEFF Research Database (Denmark)

    Ventre, I.; Goodman, A.L.; Vallet-Gely, I.

    2006-01-01

    S, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharicles. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge...... by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection...

  12. Pyruvate kinase is necessary for Brucella abortus full virulence in BALB/c mouse

    OpenAIRE

    2016-01-01

    International audience; AbstractBrucellosis, caused by a facultative intracellular pathogen Brucella, is one of the most prevalent zoonosis worldwide. Host infection relies on several uncanonical virulence factors. A recent research hotpot is the links between carbon metabolism and bacterial virulence. In this study, we found that a carbon metabolism-related pyruvate kinase (Pyk) encoded by pyk gene (locus tag BAB_RS24320) was associated with Brucella virulence. Determination of bacterial gro...

  13. Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit

    Science.gov (United States)

    Yang, Ying; Yang, Feng; Li, Yan-Hong; Liu, He-Ping; Chen, Xiao-Yan

    2017-01-01

    Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit. PMID:28257463

  14. Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella.

    Science.gov (United States)

    Teplitski, Max; Goodier, Robert I; Ahmer, Brian M M

    2003-12-01

    The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.

  15. Bacterial determinants of importance in the virulence of Gallibacterium anatis in poultry

    DEFF Research Database (Denmark)

    Persson, Gry; Bojesen, Anders Miki

    2015-01-01

    Gallibacterium anatis, a member of the Pasteurellaceae family, constitute a part of the normal micro-flora of the upper respiratory tract and the lower genital tract in chickens. However, increasing evidence indicate that G. anatis is also associated with a wide range of pathological changes...... studied virulence determinants is a large RTX-like toxin (GtxA), which has been demonstrated to induce a strong leukotoxic effect on avian macrophages. A number of fimbria of different sizes and shapes has been described. Particularly fimbriae belonging to the F17-like family appears to be common...

  16. Detection of virulence genes in Salmonella Enteritidis isolated from different sources Detecção de genes de virulência in Salmonella Enteritidis isoladas de diferentes fontes

    Directory of Open Access Journals (Sweden)

    Sílvia Dias de Oliveira

    2003-11-01

    Full Text Available The presence of three virulence genes, invA, spvR, and spvC, was determined in Salmonella Enteritidis isolated from poultry, pigs, humans and food. All isolates were positive for the invA gene, with 91.2% being positive for spvR and 90.2% for spvC. There was no significant difference in the prevalence of the virulence genes between isolates from different sources. The results indicate that there is a putative high virulence potential for the S. Enteritidis isolates characterized.A presença de três genes de virulência (invA, spvR e spvC foi determinada em Salmonella Enteritidis isoladas de aves, suínos, humanos e alimentos. Todos os isolados foram positivos para o gene invA, 91,2% também foram positivos para o spvR e 90,2% para o spvC. Não existiu diferença significativa na prevalência dos genes de virulência entre isolados de diferentes origens. Os resultados indicaram que, provavelmente, exista um alto potencial de virulência nos isolados de S. Enteritidis caracterizados.

  17. Characterization of Antimicrobial Resistance Patterns and Detection of Virulence Genes in Campylobacter Isolates in Italy

    Directory of Open Access Journals (Sweden)

    Elisabetta Di Giannatale

    2014-02-01

    Full Text Available Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis and detection of virulence genes (sequencing and DNA microarray analysis. The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%, tetracycline (55.86% and nalidixic acid (55.17%. Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations.

  18. Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

    Directory of Open Access Journals (Sweden)

    Thales Quedi Furian

    2016-03-01

    Full Text Available Abstract Pasteurella multocida causes atrophic rhinitis in swine and fowl cholera in birds, and is a secondary agent in respiratory syndromes. Pathogenesis and virulence factors involved are still poorly understood. The aim of this study was to detect 22 virulence-associated genes by PCR, including capsular serogroups A, B and D genes and to evaluate the antimicrobial susceptibility of P. multocida strains from poultry and swine. ompH, oma87, plpB, psl, exbD-tonB, fur, hgbA, nanB, sodA, sodC, ptfA were detected in more than 90% of the strains of both hosts. 91% and 92% of avian and swine strains, respectively, were classified in serogroup A. toxA and hsf-1 showed a significant association to serogroup D; pmHAS and pfhA to serogroup A. Gentamicin and amoxicillin were the most effective drugs with susceptibility higher than 97%; however, 76.79% of poultry strains and 85% of swine strains were resistant to sulphonamides. Furthermore, 19.64% and 36.58% of avian and swine strains, respectively, were multi-resistant. Virulence genes studied were not specific to a host and may be the result of horizontal transmission throughout evolution. High multidrug resistance demonstrates the need for responsible use of antimicrobials in animals intended for human consumption, in addition to antimicrobial susceptibility testing to P. multocida.

  19. Relationship of biofilm formation and different virulence genes in uropathogenic Escherichia coli isolates from Northwest Iran

    Directory of Open Access Journals (Sweden)

    Fattahi, Sargol

    2015-07-01

    Full Text Available Background and objectives: The ( bacterium is one of the main causative agents of urinary tract infections (UTI worldwide. The ability of this bacterium to form biofilms on medical devices such as catheters plays an important role in the development of UTI. The aim of the present study was to investigate the possible relationship between virulence factors and biofilm formation of isolates responsible for urinary tract infection.Materials and methods: A total of 100 isolates isolated from patients with UTI were collected and characterized by routine bacteriological methods. In vitro biofilm formation by these isolates was determined using the 96-well microtiter-plate test, and the presence of , , and virulence genes was examined by PCR assay. Data analysis was performed using SPSS 16.0 software.Results: From 100 isolates isolated from UTIs, 92% were shown to be biofilm positive. The genes , , and were detected in 43%, 94% and 26% of isolates, respectively. Biofilm formation in isolates that expressed , , and genes was 100%, 93%, and 100%, respectively. A significant relationship was found between presence of the gene and biofilm formation in isolates isolated from UTI (<0.01, but there was no statistically significant correlation between presence of and genes with biofilm formation (<0.072, <0.104. Conclusion: Results showed that and genes do not seem to be necessary or sufficient for the production of biofilm in , but the presence of correlates with increased biofilm formation of urinary tract isolates. Overall, the presence of , , and virulence genes coincides with in vitro biofilm formation in uropathogenic

  20. The highly conserved bacterial RNase YbeY is essential in Vibrio cholerae, playing a critical role in virulence, stress regulation, and RNA processing.

    Science.gov (United States)

    Vercruysse, Maarten; Köhrer, Caroline; Davies, Bryan W; Arnold, Markus F F; Mekalanos, John J; RajBhandary, Uttam L; Walker, Graham C

    2014-06-01

    YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing of the critical 3' end of 16 S rRNA and in 70 S ribosome quality control under stress. These central roles account for YbeY's inclusion in the postulated minimal bacterial genome. However, YbeY is not essential in E. coli although loss of ybeY severely sensitizes it to multiple physiological stresses. Here, we show that YbeY is an essential endoribonuclease in Vibrio cholerae and is crucial for virulence, stress regulation, RNA processing and ribosome quality control, and is part of a core set of RNases essential in most representative pathogens. To understand its function, we analyzed the rRNA and ribosome profiles of a V. cholerae strain partially depleted for YbeY and other RNase mutants associated with 16 S rRNA processing; our results demonstrate that YbeY is also crucial for 16 S rRNA 3' end maturation in V. cholerae and that its depletion impedes subunit assembly into 70 S ribosomes. YbeY's importance to V. cholerae pathogenesis was demonstrated by the complete loss of mice colonization and biofilm formation, reduced cholera toxin production, and altered expression levels of virulence-associated small RNAs of a V. cholerae strain partially depleted for YbeY. Notably, the ybeY genes of several distantly related pathogens can fully complement an E. coli ΔybeY strain under various stress conditions, demonstrating the high conservation of YbeY's activity in stress regulation. Taken together, this work provides the first comprehensive exploration of YbeY's physiological role in a human pathogen, showing its conserved function across species in essential cellular processes.

  1. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum.

    Science.gov (United States)

    Raza, Waseem; Wang, Jichen; Wu, Yuncheng; Ling, Ning; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-09-01

    The production of volatile organic compounds (VOCs) by microbes is an important characteristic for their selection as biocontrol agents against plant pathogens. In this study, we identified the VOCs produced by the biocontrol strain Bacillus amyloliquefaciens T-5 and evaluated their impact on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum. The results showed that the VOCs of strain T-5 significantly inhibited the growth of R. solanacearum in agar medium and in soil. In addition, VOCs significantly inhibited the motility traits, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides by R. solanacearum. However, no effect of VOCs on the production of hydrolytic enzymes by R. solanacearum was observed. The strain T-5 produced VOCs, including benzenes, ketones, aldehydes, alkanes, acids, and one furan and naphthalene compound; among those, 13 VOCs showed 1-10 % antibacterial activity against R. solanacearum in their produced amounts by T-5; however, the consortium of all VOCs produced on agar medium, in sterilized soil, and in natural soil showed 75, 62, and 85 % growth inhibition of R. solanacearum, respectively. The real-time PCR analysis further confirmed the results when the expression of different virulence- and metabolism-related genes in R. solanacearum cells was decreased after exposure to the VOCs of strain T-5. The results of this study clearly revealed the significance of VOCs in the control of plant pathogens. This information would help to better comprehend the microbial interactions mediated by VOCs in nature and to develop safer strategies to control plant disease.

  2. Variant surface antigens, virulence genes and the pathogenesis of malaria

    DEFF Research Database (Denmark)

    Deitsch, Kirk W; Hviid, Lars

    2004-01-01

    The first Molecular Approaches to Malaria meeting was held 2-5 February 2000 in Lorne, Australia. Following the meeting, Brian Cooke, Mats Wahlgren and Ross Coppel predicted that research into the molecular details of the mechanisms behind sequestration of parasitized erythrocytes would "become...... increasingly more complicated, with further interactions, receptors, ligands and functional domains". Furthermore, they cautioned that "the challenge will be not to lose ourselves in the molecular detail, but remain focused on the role of [the var genes and other multigene families] in pathogenesis of malaria......". We contemplate on these statements, following the recent second Molecular Approaches to Malaria meeting, which was held at the same venue on 2-5 February 2004....

  3. Virulence of Xanthomonas oryzae pv.oryzae on Rice Near-lsogenic Lines with Single Resistance Gene and Pyramiding Lines in China

    Institute of Scientific and Technical Information of China (English)

    LIU Hong-xia; LIU Feng-quan; HU Bai-shi; YANG Wan-feng; CHEN Zhi-yi; XU Zhi-gang

    2004-01-01

    Ninety one isolates of Xanthomonas oryzae pv. Oryzae were collected from different rice growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes:IRBB1 (Xa1),IRBB2 (Xa2),IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11),IRBB13 (xa13),IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21),IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21),IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10,less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR,RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48% respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the evelopment of rice resistance breeding to bacterial blight in China.

  4. Identification of genes preferentially expressed by highly virulent piscine Streptococcus agalactiae upon interaction with macrophages.

    Directory of Open Access Journals (Sweden)

    Chang-Ming Guo

    Full Text Available Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood-brain barrier (BBB. The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB.

  5. Prevalence of ten putative virulence genes in the emerging foodborne pathogen Arcobacter isolated from food products.

    Science.gov (United States)

    Girbau, Cecilia; Guerra, Cristian; Martínez-Malaxetxebarria, Irati; Alonso, Rodrigo; Fernández-Astorga, Aurora

    2015-12-01

    Arcobacter spp. are considered to be emerging food- and waterborne pathogens for both humans and animals. However, their virulence mechanisms are still poorly understood. In this study the presence of ten virulence genes (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, tlyA and iroE) was assessed in a set of 47 strains of Arcobacter butzleri, 10 of Arcobacter cryaerophilus and 1 Arcobacter skirrowii strain recovered from different food products (pork, chicken, beef, milk, clams and mussels). Overall, the genes cadF, ciaB, cj1349, mviN, pldA and tlyA were detected in all A. butzleri and A. skirrowii strains. Lower detection rates were observed for irgA, iroE, hecA and hecB. The genes hecB and iroE were detected neither in A. cryaerophilus nor in A. skirrowii. The genes hecA and irgA were not detected in A. skirrowii. It was noteworthy that the genes hecA and hecB were significantly (P < 0.05) highly detected in A. butzleri strains isolated from clams compared with strains isolated from milk and chicken. Therefore, our findings underline clams as a source of A. butzleri strains with high prevalence of putative virulence genes. This could be hazardous to human health, especially because these bivalves are usually consumed raw or undercooked.

  6. 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.

  7. Resistance to antibiotics and inorganic ions in virulent bacterial strains from a hospital.

    Science.gov (United States)

    Vazquez, F; Fidalgo, S; Mendez, F J; Mendoza, M C

    1989-08-01

    Virulent strains of Staphylococcus aureus, S. epidermidis, Escherichia coli and Pseudomonas aeruginosa were studied for their resistance to antibiotics and inorganic ions, the correlation with their clinical use and the usefulness as an epidemioliogical tool. Multiresistance was common, the antibiotypes were similar to those previously reported, but characteristic resistotypes endemic of our county were found. A correlation between resistance and metal ion consumption was not detected. Staphylococci strains were susceptible to vancomycin, cephalothin and mercury chloride; S. epidermidis showed higher rates of resistance to antibiotics and lower to cadmium chloride and potassium iodine than S. aureus. E. coli strains were susceptible to new beta-lactamans; resistance to cephalothin, gentamicin, tobramycin and amikacin was less than 10%. P, aeruginosa was the species with the most multiresistance and antibiotype diversity, only ceftazidime, amikacin and imipenem had a resistance rate less than 11%. In both E. coli and P. aeruginosa resistance to all tested metals (except silver nitrate) was found although with different percentages.

  8. Dual-site phosphorylation of the control of virulence regulator impacts group a streptococcal global gene expression and pathogenesis.

    Directory of Open Access Journals (Sweden)

    Nicola Horstmann

    2014-05-01

    Full Text Available Phosphorylation relays are a major mechanism by which bacteria alter transcription in response to environmental signals, but understanding of the functional consequences of bacterial response regulator phosphorylation is limited. We sought to characterize how phosphorylation of the control of virulence regulator (CovR protein from the major human pathogen group A Streptococcus (GAS influences GAS global gene expression and pathogenesis. CovR mainly serves to repress GAS virulence factor-encoding genes and has been shown to homodimerize following phosphorylation on aspartate-53 (D53 in vitro. We discovered that CovR is phosphorylated in vivo and that such phosphorylation is partially heat-stable, suggesting additional phosphorylation at non-aspartate residues. Using mass spectroscopy along with targeted mutagenesis, we identified threonine-65 (T65 as an additional CovR phosphorylation site under control of the serine/threonine kinase (Stk. Phosphorylation on T65, as mimicked by the recombinant CovR T65E variant, abolished in vitro CovR D53 phosphorylation. Similarly, isoallelic GAS strains that were either unable to be phosphorylated at D53 (CovR-D53A or had functional constitutive phosphorylation at T65 (CovR-T65E had essentially an identical gene repression profile to each other and to a CovR-inactivated strain. However, the CovR-D53A and CovR-T65E isoallelic strains retained the ability to positively influence gene expression that was abolished in the CovR-inactivated strain. Consistent with these observations, the CovR-D53A and CovR-T65E strains were hypervirulent compared to the CovR-inactivated strain in a mouse model of invasive GAS disease. Surprisingly, an isoalleic strain unable to be phosphorylated at CovR T65 (CovR-T65A was hypervirulent compared to the wild-type strain, as auto-regulation of covR gene expression resulted in lower covR gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data

  9. Bacterial fitness shapes the population dynamics of antibiotic-resistant and -susceptible bacteria in a model of combined antibiotic and anti-virulence treatment

    Science.gov (United States)

    Ternent, Lucy; Dyson, Rosemary J.; Krachler, Anne-Marie; Jabbari, Sara

    2015-01-01

    Bacterial resistance to antibiotic treatment is a huge concern: introduction of any new antibiotic is shortly followed by the emergence of resistant bacterial isolates in the clinic. This issue is compounded by a severe lack of new antibiotics reaching the market. The significant rise in clinical resistance to antibiotics is especially problematic in nosocomial infections, where already vulnerable patients may fail to respond to treatment, causing even greater health concern. A recent focus has been on the development of anti-virulence drugs as a second line of defence in the treatment of antibiotic-resistant infections. This treatment, which weakens bacteria by reducing their virulence rather than killing them, should allow infections to be cleared through the body׳s natural defence mechanisms. In this way there should be little to no selective pressure exerted on the organism and, as such, a predominantly resistant population should be less likely to emerge. However, before the likelihood of resistance to these novel drugs emerging can be predicted, we must first establish whether such drugs can actually be effective. Many believe that anti-virulence drugs would not be powerful enough to clear existing infections, restricting their potential application to prophylaxis. We have developed a mathematical model that provides a theoretical framework to reveal the circumstances under which anti-virulence drugs may or may not be successful. We demonstrate that by harnessing and combining the advantages of antibiotics with those provided by anti-virulence drugs, given infection-specific parameters, it is possible to identify treatment strategies that would efficiently clear bacterial infections, while preventing the emergence of antibiotic-resistant subpopulations. Our findings strongly support the continuation of research into anti-virulence drugs and demonstrate that their applicability may reach beyond infection prevention. PMID:25701634

  10. Bacterial fitness shapes the population dynamics of antibiotic-resistant and -susceptible bacteria in a model of combined antibiotic and anti-virulence treatment.

    Science.gov (United States)

    Ternent, Lucy; Dyson, Rosemary J; Krachler, Anne-Marie; Jabbari, Sara

    2015-05-07

    Bacterial resistance to antibiotic treatment is a huge concern: introduction of any new antibiotic is shortly followed by the emergence of resistant bacterial isolates in the clinic. This issue is compounded by a severe lack of new antibiotics reaching the market. The significant rise in clinical resistance to antibiotics is especially problematic in nosocomial infections, where already vulnerable patients may fail to respond to treatment, causing even greater health concern. A recent focus has been on the development of anti-virulence drugs as a second line of defence in the treatment of antibiotic-resistant infections. This treatment, which weakens bacteria by reducing their virulence rather than killing them, should allow infections to be cleared through the body׳s natural defence mechanisms. In this way there should be little to no selective pressure exerted on the organism and, as such, a predominantly resistant population should be less likely to emerge. However, before the likelihood of resistance to these novel drugs emerging can be predicted, we must first establish whether such drugs can actually be effective. Many believe that anti-virulence drugs would not be powerful enough to clear existing infections, restricting their potential application to prophylaxis. We have developed a mathematical model that provides a theoretical framework to reveal the circumstances under which anti-virulence drugs may or may not be successful. We demonstrate that by harnessing and combining the advantages of antibiotics with those provided by anti-virulence drugs, given infection-specific parameters, it is possible to identify treatment strategies that would efficiently clear bacterial infections, while preventing the emergence of antibiotic-resistant subpopulations. Our findings strongly support the continuation of research into anti-virulence drugs and demonstrate that their applicability may reach beyond infection prevention.

  11. Enhanced Virulence Gene Activity of Agrobacterium in Muskmelon (Cucumis melo L. cv. ‘Birdie’

    Directory of Open Access Journals (Sweden)

    Abul K.M. MOHIUDDIN

    2011-05-01

    Full Text Available Muskmelon (Cucumis melo L. cultivar ‘Birdie’, was evaluated for its response to the tumorigenic Agrobacterium tumefaciens and the oncogenic A. rhizogenes strains. Stem and petiole of three week-old in vitro-grown muskmelon plants were inoculated with five strains of A. tumefaciens and A. rhizogenes each and observed phenotypic expressions i.e. induction of crown galls and hairy roots. This phenotypic expression was efficaciously increased when virulence gene activity of different strains of two Agrobacterium species was enhanced. Intensive studies on enhancement of virulence gene activity of Agrobacterium found to be correlated to the appropriate light intensity (39.3 μmol m-2 s-1 with a specific concentration of monocyclic phenolic compound, acetosyringone (20 μM. The gene activity was also influenced by several other physical factors e.g. plant tissue type, Agrobacterium species and their strains, and plant tissue-Agrobacterium interaction. Among the different A. tumefaciens strains, LBA4404 showed the best virulence gene activity in both stem and petiole through the formation of higher rate of crown galls. On the other hand, strain 15834 of A. rhizogenes showed better gene activity in stem and 8196 in petiole through the formation of higher rate of hairy roots as well as higher average number of hairy roots. Among the two different types of explants, petiole was more susceptible to both Agrobacterium species. Thus it was concluded that future muskmelon transformation study can efficiently be carried out with LBA4404, 15834 and 8196 strains using petiole explants by adding 20 μM of acetosyringone in the medium.

  12. Macrolide, glycopeptide resistance and virulence genes in Enterococcus species isolates from dairy cattle.

    Science.gov (United States)

    Iweriebor, Benson C; Obi, Larry C; Okoh, Anthony I

    2016-07-01

    The genus Enterococcus is known to possess the capacity to acquire and disseminate antimicrobial resistant determinants alongside the ability to produce various virulence genes that enables it to establish infections. We assessed the prevalence and antibiogram profiles of Enterococcus spp. in faecal samples of dairy cattle. Faecal swab samples were collected from 400 dairy cattle from two commercial cattle farms in two rural communities in the Eastern Cape, South Africa. Confirmation of enterococci isolates was carried out by PCR targeting of the tuf gene. Species delineation was by species-specific primers targeting the superoxide dismutase (sod A) gene in a multiplex PCR assay. Isolates were screened for the presence of the following virulence genes (ace, gel E, esp, efa A, cyl A and hyl E) and antimicrobial resistance determinants to erythromycin, vancomycin and streptomycin were evaluated molecularly. A total of 340 isolates were confirmed as belonging to the genus Enterococcus . Species distribution among the isolates consisted of Enterococcus faecium (52.94 %) and Enterococcus durans (23.53 %) in preponderance compared to the three other species, namely Enterococcus faecalis (8.8 %), Enterococcus hirae (8.6 %) and Enterococcus casseliflavus (5.9 %). All were resistant to vancomycin, while 99 % showed resistance to aminoglycoside and 94 % to macrolide. Three virulence genes (ace, gel E and esp) were detected in almost all the confirmed isolates. The resistance determinants van B (19.7 %), van C1 (25 %), van C2/3 (26.3 %) erm B (40.29 %) and str A (50.88 %) were detected among the isolates. A high prevalence of multidrug-resistant enterococci isolates was detected in this study and the genetic repertoire to survive in the presence of antimicrobial agents was present in these organisms.

  13. Correlation between Pr1 and Pr2 gene content and virulence in Metarhizium anisopliae strains.

    Science.gov (United States)

    Rosas-García, Ninfa M; Avalos-de-León, Osvaldo; Villegas-Mendoza, Jesús M; Mireles-Martínez, Maribel; Barboza-Corona, J E; Castañeda-Ramírez, J C

    2014-11-28

    Metarhizium anisopliae is a widely studied model to understand the virulence factors that participate in pathogenicity. Proteases such as subtilisin-like enzymes (Pr1) and trypsin-like enzymes (Pr2) are considered important factors for insect cuticle degradation. In four M. anisopliae strains (798, 6342, 6345, and 6347), the presence of pr1 and pr2 genes, as well as the enzymatic activity of these genes, was correlated with their virulence against two different insect pests. The 11 pr1 genes (A, B, C, D, E, F, G, H, I, J, and K) and pr2 gene were found in all strains. The activity of individual Pr1 and Pr2 proteases exhibited variation in time (24, 48, 72, and 96 h) and in the presence or absence of chitin as the inductor. The highest Pr1 enzymatic activity was shown by strain 798 at 48 h with chitin. The highest Pr2 enzymatic activity was exhibited by the 6342 and 6347 strains, both grown with chitin at 24 and 48 h, respectively. Highest mortality on S. exigua was caused by strain 6342 at 48 h, and strains 6342, 6345, and 6347 caused the highest mortality 7 days later. Mortality on Prosapia reached 30% without variation. The presence of subtilisin and trypsin genes and the activity of these proteases in M. anisopliae strains cannot be associated with the virulence against the two insect pests. Probably, subtilisin and trypsin enzyme production is not a vital factor for pathogenicity, but its contribution is important to the pathogenicity process.

  14. Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene.

    Science.gov (United States)

    Anyanful, Akwasi; Dolan-Livengood, Jennifer M; Lewis, Taiesha; Sheth, Seema; Dezalia, Mark N; Sherman, Melanie A; Kalman, Lisa V; Benian, Guy M; Kalman, Daniel

    2005-08-01

    Pathogenic Escherichia coli, including enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) are major causes of food and water-borne disease. We have developed a genetically tractable model of pathogenic E. coli virulence based on our observation that these bacteria paralyse and kill the nematode Caenorhabditis elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting that a secreted toxin mediates the effect. Virulence against C. elegans required tryptophan and bacterial tryptophanase, the enzyme catalysing the production of indole and other molecules from tryptophan. Thus, lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyse or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a cocultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal non-pathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyse and kill nematodes. Furthermore, tryptophanase activity was required for full activation of the LEE1 promoter, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes including sek-1, mek-1, mev-1, pgp-1,3 and vhl-1, rendered C. elegans more

  15. Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method.

    Science.gov (United States)

    Schierack, Peter; Rödiger, Stefan; Kuhl, Christoph; Hiemann, Rico; Roggenbuck, Dirk; Li, Ganwu; Weinreich, Jörg; Berger, Enrico; Nolan, Lisa K; Nicholson, Bryon; Römer, Antje; Frömmel, Ulrike; Wieler, Lothar H; Schröder, Christian

    2013-01-01

    We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.

  16. Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method.

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    Peter Schierack

    Full Text Available We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2 and their probiotic activity against infection by enteropathogenic E. coli (EPEC. 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.

  17. Diarrheagenic Escherichia coli carrying supplementary virulence genes are an important cause of moderate to severe diarrhoeal disease in Mexico.

    Science.gov (United States)

    Patzi-Vargas, Sandra; Zaidi, Mussaret Bano; Perez-Martinez, Iza; León-Cen, Magda; Michel-Ayala, Alba; Chaussabel, Damien; Estrada-Garcia, Teresa

    2015-03-01

    Diarrheagenic Escherichia coli (DEC) cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG), mainly associated with enteroaggregative E. coli (EAEC), were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%), surpassing Salmonella (12%) and Shigella (9%). Predominant DEC groups were diffusely adherent E. coli (DAEC) (35%), EAEC (24%), and enteropathogenic E. coli (EPEC) (19%). Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26%) and DAEC (18%); 30% had moderate diarrhoea mainly caused by DAEC (36%), mixed DEC infections (33%) and EAEC (32%). DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6-24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3-13.9). The presence of SVG dispersin, (aatA), dispersin-translocator (aatA), enteroaggregative heat-stable toxin 1 (astA), plasmid encoded toxin (pet), cytolethal distending toxin (cdt) was higher in DEC than non-DEC strains, (36% vs 26%, P DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2%) and non-DEC strains (21% and 13%). DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children.

  18. Evaluation of the gene encoding the enzyme βHPMEH for the bacterial wilt inhibition caused by Ralstonia solanacearum

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    Elizabeth Fernandez

    2015-10-01

    Full Text Available Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease that attacks important agricultural crops such as potato, tomato, banana, among others, causing serious yield losses. Control of R. solanacearum is difficult because of its wide range of alternate hosts, its long survival in soil, its biological and genetic variation, the lack of natural resistance sources and the insufficiency of the appropriate chemical control measures. Quorum sensing is the term that describes the phenomenon whereby the accumulation of molecules allows bacteria to know the number of bacteria found in the environment (population density. R. solanacearum has a quorum sensing system for the regulation of the expression of virulence genes; the molecule 3-OH-PAME is the self-regulatory signal. The molecule ΒHPMEH hydrolyzes 3-OH-PAME nullifying the signal of virulence, and thus, the quorum sensing communication in R. solanacearum. In order to evaluate the βhpmeh gene we designed two vectors that express this gene under the control of two different promoters. Both vectors were verified by restriction analysis and sequencing. Agroinfiltration assays were used to analyze gene expression and the effect against R. solanacearum in potato (Solanum tuberosum leaves. The results of the transient expression experiments showed that the expression of gene βhpmeh caused a delay in the appearance of symptoms of bacterial wilt and thus is a good candidate for whole genetic plant transformation.

  19. Reduced Fitness of Virulent Aphis glycines (Hemiptera: Aphididae) Biotypes May Influence the Longevity of Resistance Genes in Soybean.

    Science.gov (United States)

    Varenhorst, Adam J; McCarville, Michael T; O'Neal, Matthew E

    2015-01-01

    Sustainable use of insect resistance in crops require insect resistance management plans that may include a refuge to limit the spread of virulence to this resistance. However, without a loss of fitness associated with virulence, a refuge may not prevent virulence from becoming fixed within a population of parthenogenetically reproducing insects like aphids. Aphid-resistance in soybeans (i.e., Rag genes) prevent outbreaks of soybean aphid (Aphis glycines), yet four biotypes defined by their capacity to survive on aphid-resistant soybeans (e.g., biotype-2 survives on Rag1 soybean) are found in North America. Although fitness costs are reported for biotype-3 on aphid susceptible and Rag1 soybean, it is not clear if virulence to aphid resistance in general is associated with a decrease in fitness on aphid susceptible soybeans. In laboratory assays, we measured fitness costs for biotype 2, 3 and 4 on an aphid-susceptible soybean cultivar. In addition, we also observed negative cross-resistance for biotype-2 on Rag3, and biotype-3 on Rag1 soybean. We utilized a simple deterministic, single-locus, four compartment genetic model to account for the impact of these findings on the frequency of virulence alleles. When a refuge of aphid susceptible was included within this model, fitness costs and negative cross-resistance delayed the increase of virulence alleles when virulence was inherited recessively or additively. If virulence were inherited additively, fitness costs decreased the frequency of virulence. Combined, these results suggest that a refuge may prevent virulent A. glycines biotypes from overcoming Rag genes if this aphid-resistance were used commercially in North America.

  20. Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P.

    Science.gov (United States)

    Ko, Jae-hyeong; Izadjoo, Mina; Altman, Sidney

    2008-08-01

    External guide sequences (EGSs) targeting virulence genes from Yersinia pestis were designed and tested in vitro and in vivo in Escherichia coli. Linear EGSs and M1 RNA-linked EGSs were designed for the yscN and yscS genes that are involved in type III secretion in Y. pestis. RNase P from E. coli cleaves the messages of yscN and yscS in vitro with the cognate EGSs, and the expression of the EGSs resulted in the reduction of the levels of these messages of the virulence genes when those genes were expressed in E. coli.

  1. 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

  2. Current European Labyrinthula zosterae are not virulent and modulate seagrass (Zostera marina defense gene expression.

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    Janina Brakel

    Full Text Available Pro- and eukaryotic microbes associated with multi-cellular organisms are receiving increasing attention as a driving factor in ecosystems. Endophytes in plants can change host performance by altering nutrient uptake, secondary metabolite production or defense mechanisms. Recent studies detected widespread prevalence of Labyrinthula zosterae in European Zostera marina meadows, a protist that allegedly caused a massive amphi-Atlantic seagrass die-off event in the 1930's, while showing only limited virulence today. As a limiting factor for pathogenicity, we investigated genotype × genotype interactions of host and pathogen from different regions (10-100 km-scale through reciprocal infection. Although the endophyte rapidly infected Z. marina, we found little evidence that Z. marina was negatively impacted by L. zosterae. Instead Z. marina showed enhanced leaf growth and kept endophyte abundance low. Moreover, we found almost no interaction of protist × eelgrass-origin on different parameters of L. zosterae virulence/Z. marina performance, and also no increase in mortality after experimental infection. In a target gene approach, we identified a significant down-regulation in the expression of 6/11 genes from the defense cascade of Z. marina after real-time quantitative PCR, revealing strong immune modulation of the host's defense by a potential parasite for the first time in a marine plant. Nevertheless, one gene involved in phenol synthesis was strongly up-regulated, indicating that Z. marina plants were probably able to control the level of infection. There was no change in expression in a general stress indicator gene (HSP70. Mean L. zosterae abundances decreased below 10% after 16 days of experimental runtime. We conclude that under non-stress conditions L. zosterae infection in the study region is not associated with substantial virulence.

  3. The RpfB-Dependent Quorum Sensing Signal Turnover System Is Required for Adaptation and Virulence in Rice Bacterial Blight Pathogen Xanthomonas oryzae pv. oryzae.

    Science.gov (United States)

    Wang, Xing-Yu; Zhou, Lian; Yang, Jun; Ji, Guang-Hai; He, Ya-Wen

    2016-03-01

    Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, produces diffusible signal factor (DSF) family quorum sensing signals to regulate virulence. The biosynthesis and perception of DSF family signals require components of the rpf (regulation of pathogenicity factors) cluster. In this study, we report that RpfB plays an essential role in DSF family signal turnover in X. oryzae pv. oryzae PXO99A. The production of DSF family signals was boosted by deletion of the rpfB gene and was abolished by its overexpression. The RpfC/RpfG-mediated DSF signaling system negatively regulates rpfB expression via the global transcription regulator Clp, whose activity is reversible in the presence of cyclic diguanylate monophosphate. These findings indicate that the DSF family signal turnover system in PXO99A is generally consistent with that in Xanthomonas campestris pv. campestris. Moreover, this study has revealed several specific roles of RpfB in PXO99A. First, the rpfB deletion mutant produced high levels of DSF family signals but reduced extracellular polysaccharide production, extracellular amylase activity, and attenuated pathogenicity. Second, the rpfB/rpfC double-deletion mutant was partially deficient in xanthomonadin production. Taken together, the RpfB-dependent DSF family signal turnover system is a conserved and naturally presenting signal turnover system in Xanthomonas spp., which plays unique roles in X. oryzae pv. oryzae adaptation and pathogenesis.

  4. Detection of antibiotic resistance, virulence gene determinants and biofilm formation in Aeromonas species isolated from cattle.

    Science.gov (United States)

    Igbinosa, Isoken H; Igbinosa, Etinosa O; Okoh, Anthony I

    2015-11-01

    This study aimed to assess the antibiogram of Aeromonas strains recovered from cattle faeces and the potential pathogenic status of the isolates. The antibiogram of the Aeromonas isolates demonstrated total resistance to clindamycin oxacillin, trimethoprim, novobiocin and ticarcillin. However, Aeromonas strains were sensitive to cefotaxime, oxytetracycline and tobramycin. The Aeromonas strains from Lovedale and Fort Cox farms were found to possess some virulence genes. The percentage distribution was aer 71.4%, ast 35.7%, fla 60.7%, lip 35.7% and hlyA 25% for Lovedale farm and aer 63.1%, alt 10.5%, ast 55.2%, fla 78.9%, lip 21% and hlyA 35.9% for Fort Cox farm. Class 1 integron was present in 27% of Aeromonas isolates; the bla TEM gene was present in 34.8%, while the blaP1 class A β-lactamase gene was detected in 12.1% of the isolates. Approximately 86% of the isolates formed a biofilm on microtitre plates. The presence of multiple antibiotic resistance and virulence genes in Aeromonas isolates from cattle faeces reveals the pathogenic and infectious importance of these isolates and is of great significance to public health. The possession of a biofilm-forming capability by such isolates may lead to difficulty during the management of infection related to Aeromonas species.

  5. Genome engineering and gene expression control for bacterial strain development.

    Science.gov (United States)

    Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

    2015-01-01

    In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen.

  6. Lower Circulating Levels of Chemokine CXCL10 In Helicobacter Pylori-Infected Patients with Peptic Ulcer: Influence of the Bacterial Virulence Factor CagA

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    Abdollah Jafarzadeh

    2013-03-01

    Full Text Available Background and objectives: Alterations in CXCL10 (a Th1 chemokine expression have been associated with various diseases. The aim of this study was to evaluate the serum CXCL10 levels in H. pylori-infected patients with peptic ulcer (PU and to determine its association with bacterial virulence factor cytotoxin-associated gene A (CagA. Materials and Methods: Serum samples from 90 H. pylori infected patients (70 were anti-CagA+, 20 were anti-CagA-, 65 asymptomatic (AS carriers (40 were anti-CagA+, 25 were anti-CagA- and 30 healthy H. pylori-negative subjects (as a control were tested for the concentrations of CXCL10 by using ELISA method. Results: The mean serum levels of CXCL10 in PU patients (96.64 ± 20.85 Pg/mL was significantly lower than those observed in AS subjects (162.16 ± 53.31 Pg/mL, P < 0.01 and control group (193.93 ± 42.14 Pg/mL, P < 0.02. In the PU group, the levels of CXCL10 in anti-CagA+ subjects was significantly higher in comparison to anti-CagA- patients (P<0.04. Conclusion: These results showed that the mean concentrations of CXCL10 in H. pylori-infected-PU patients was lower than AS carriers and control group. In the PU group, the serum levels of CXCL10 were affected by bacterial factor CagA.

  7. Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung.

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    Sharna Naughton

    Full Text Available Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF, adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1, have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R and constructed a non-redundant micro-array (PANarray comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5 genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.

  8. Salmonella Modulates Metabolism During Growth under Conditions that Induce Expression of Virulence Genes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young-Mo; Schmidt, Brian; Kidwai, Afshan S.; Jones, Marcus B.; Deatherage, Brooke L.; Brewer, Heather M.; Mitchell, Hugh D.; Palsson, Bernhard O.; McDermott, Jason E.; Heffron, Fred; Smith, Richard D.; Peterson, Scott N.; Ansong, Charles; Hyduke, Daniel R.; Metz, Thomas O.; Adkins, Joshua N.

    2013-04-05

    Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative pathogen that uses complex mechanisms to invade and proliferate within mammalian host cells. To investigate possible contributions of metabolic processes in S. Typhimurium grown under conditions known to induce expression of virulence genes, we used a metabolomics-driven systems biology approach coupled with genome scale modeling. First, we identified distinct metabolite profiles associated with bacteria grown in either rich or virulence-inducing media and report the most comprehensive coverage of the S. Typhimurium metabolome to date. Second, we applied an omics-informed genome scale modeling analysis of the functional consequences of adaptive alterations in S. Typhimurium metabolism during growth under our conditions. Excitingly, we observed possible sequestration of metabolites recently suggested to have immune modulating roles. Modeling efforts highlighted a decreased cellular capability to both produce and utilize intracellular amino acids during stationary phase culture in virulence conditions, despite significant abundance increases for these molecules as observed by our metabolomics measurements. Model-guided analysis suggested that alterations in metabolism prioritized other activities necessary for pathogenesis instead, such as lipopolysaccharide biosynthesis.

  9. Identification of alkA gene related to virulence of Shigella flexneri 2a by mutational analysis

    Institute of Scientific and Technical Information of China (English)

    Zhao-Xing Shi; Heng-Liang Wang; Kun Hu; Er-Ling Feng; Xiao Yao; Guo-Fu Su; Pei-Tang Huang; Liu-Yu Huang

    2003-01-01

    AIM: In vivo induced genes are thought to play an important role during infection of host. ,AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET),but its virulence in Shigella flexneri was not reported. The purpose of this study was to identify the role of alkA gene in the pathogenesis of S. flexneriMETHODS: PCR was used to amplify alkA gene of S. flexneri 2a and fragment 028pKm. The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46. By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing. Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively.RESULTS: Deletion mutant of S. flexneri 2a alkA was successfully constructed by λ Red recombination system.The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay.CONCLUSION: AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.

  10. Characterization of the wzc gene from Pantoea sp. strain PPE7 and its influence on extracellular polysaccharide production and virulence on Pleurotus eryngii.

    Science.gov (United States)

    Kim, Min Keun; Lee, Young Han; Kim, Hyeran; Lee, Jeongyeo; Ryu, Jae San

    2015-01-01

    To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test. The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane α-helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii.

  11. Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment.

    Directory of Open Access Journals (Sweden)

    Teruaki Nakatsuji

    Full Text Available BACKGROUND: In the progression of acne vulgaris, the disruption of follicular epithelia by an over-growth of Propionibacterium acnes (P. acnes permits the bacteria to spread and become in contact with various skin and immune cells. METHODOLOGY/PRINCIPAL FINDINGS: We have demonstrated in the present study that the Christie, Atkins, Munch-Peterson (CAMP factor of P. acnes is a secretory protein with co-hemolytic activity with sphingomyelinase that can confer cytotoxicity to HaCaT keratinocytes and RAW264.7 macrophages. The CAMP factor from bacteria and acid sphingomyelinase (ASMase from the host cells were simultaneously present in the culture supernatant only when the cells were co-cultured with P. acnes. Either anti-CAMP factor serum or desipramine, a selective ASMase inhibitor, significantly abrogated the P. acnes-induced cell death of HaCaT and RAW264.7 cells. Intradermal injection of ICR mouse ears with live P. acnes induced considerable ear inflammation, macrophage infiltration, and an increase in cellular soluble ASMase. Suppression of ASMase by systemic treatment with desipramine significantly reduced inflammatory reaction induced by intradermal injection with P. acnes, suggesting the contribution of host ASMase in P. acnes-induced inflammatory reaction in vivo. Vaccination of mice with CAMP factor elicited a protective immunity against P. acnes-induced ear inflammation, indicating the involvement of CAMP factor in P. acnes-induced inflammation. Most notably, suppression of both bacterial CAMP factor and host ASMase using vaccination and specific antibody injection, respectively, cooperatively alleviated P. acnes-induced inflammation. CONCLUSIONS/SIGNIFICANCE: These findings envision a novel infectious mechanism by which P. acnes CAMP factor may hijack host ASMase to amplify bacterial virulence to degrade and invade host cells. This work has identified both CAMP factor and ASMase as potential molecular targets for the development of drugs

  12. Irregular virulence genes expression of Vibrio parahaemolyticus in shrimp or seawater matrix

    OpenAIRE

    Aijing Zhao; Haiquan Liu; Wenshuo Sun; Qin Li; Yingjie Pan; Yong Zhao

    2015-01-01

    The three virulence genes (tlh, tdh and trh) expression of five Vibrio parahaemolyticus strains (ATCC33847, ATCC17802, VP1, VP2 and VP3) in shrimp or seawater matrix at 9°C and 25°C were detected by the quantitative reverse transcription-PCR (qRT-PCR). The results showed that the transcription levels of tlh in ATCC17802 and VP3, tdh in VP1 were up-regulated, but the transcription levels of tlh and tdh in ATCC33847 were significantly (p < 0.05) down-regulated in shrimp samples compared with th...

  13. Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA.

    Science.gov (United States)

    Hamilton, Matthew J; Hadi, Asbah Z; Griffith, John F; Ishii, Satoshi; Sadowsky, Michael J

    2010-10-01

    Contamination of recreational waters with Escherichia coli and Enterococcus sp. is a widespread problem resulting in beach closures and loss of recreational activity. While E. coli is frequently used as an indicator of fecal contamination, and has been extensively measured in waterways, few studies have examined the presence of potentially pathogenic E. coli strains in beach waters. In this study, a combination of high-throughput, robot-assisted colony hybridization and PCR-based analyses were used to determine the genomic composition and frequency of virulence genes present in E. coli isolated from beach water in Avalon Bay, Santa Catalina Island, CA. A total of 24,493 E. coli isolates were collected from two sites at a popular swimming beach between August through September 2007 and from July through August 2008. All isolates were examined for the presence of shiga-like toxins (stx1/stx2), intimin (eaeA), and enterotoxins (ST/LT). Of the 24,493 isolates examined, 3.6% contained the eaeA gene, indicating that these isolates were potential EPEC strains. On five dates, however, greater than 10% of the strains were potential EPEC, suggesting that incidence of virulence genes at this beach has a strong temporal component. No STEC or ETEC isolates were detected, and only eight (water and their presence needs to be considered as one of the factors used in decisions concerning beach closures.

  14. Occurrence of virulence genes among Vibrio cholerae and Vibrio parahaemolyticus strains from treated wastewaters.

    Science.gov (United States)

    Khouadja, Sadok; Suffredini, Elisabetta; Baccouche, Besma; Croci, Luciana; Bakhrouf, Amina

    2014-10-01

    Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.

  15. Molecular screening of virulence genes in extraintestinal pathogenic Escherichia coli isolated from human blood culture in Brazil.

    Science.gov (United States)

    Koga, Vanessa L; Tomazetto, Geizecler; Cyoia, Paula S; Neves, Meiriele S; Vidotto, Marilda C; Nakazato, Gerson; Kobayashi, Renata K T

    2014-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC.

  16. 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.

  17. Defence against methylglyoxal in Group A Streptococcus: a role for Glyoxylase I in bacterial virulence and survival in neutrophils?

    Science.gov (United States)

    Zhang, May M; Ong, Cheryl-lynn Y; Walker, Mark J; McEwan, Alastair G

    2016-03-01

    Methylglyoxal is a dicarbonyl compound that acts as a toxic electrophile in biological systems. Methylglyoxal is produced in certain bacteria as a byproduct of glycolysis through methylglyoxal synthase. Like many bacteria, Group A Streptococcus (GAS), a Gram-positive human pathogen responsible for a wide spectrum of diseases, uses a two-step glyoxalase system to remove methylglyoxal. However, bioinformatic analysis revealed that no homologue of methylglyoxal synthase is present in GAS, suggesting that the role of the glyoxalase system is to detoxify methylglyoxal produced by the host. In this study, we investigated the role of methylglyoxal detoxification in the pathogenesis of GAS. A mutant (5448ΔgloA), deficient in glyoxylase I (S-lactoylglutathione lyase), was constructed and tested for susceptibility to methylglyoxal, human neutrophil survival and virulence in a murine model of infection. 5448ΔgloA was more sensitive to methylglyoxal and was also more susceptible to human neutrophil killing. Inhibition of neutrophil myeloperoxidase rescued the gloA-deficient mutant indicating that this enzyme was required for methylglyoxal production. Furthermore, the 5448ΔgloA mutant was slower at disseminating into the blood in the murine model. These data suggest that neutrophils produce methylglyoxal as an antimicrobial agent during bacterial infection, and the glyoxalase system is part of the GAS defence against the innate immune system during pathogenesis.

  18. Pseudomonas fluorescens filamentous hemagglutinin, an iron-regulated protein, is an important virulence factor that modulates bacterial pathogenicity

    Directory of Open Access Journals (Sweden)

    Yuan-yuan Sun

    2016-08-01

    Full Text Available Pseudomonas fluorescens is a common bacterial pathogen to a wide range of aquaculture animals including various species of fish. In this study, we employed proteomic analysis and identified filamentous hemagglutinin (FHA as an iron-responsive protein secreted by TSS, a pathogenic P. fluorescens isolate. In vitro study showed that compared to the wild type, the fha mutant TSSfha (i exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix, (ii displayed no apparent flagella and motility, (iii was defective in the attachment to host cells and unable to form self-aggregation, (iv displayed markedly reduced capacity of hemagglutination and surviving in host serum. In vivo infection analysis revealed that TSSfha was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality, and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen. Furthermore, when introduced into turbot as a subunit vaccine, recombinant FHA elicited a significant protection against lethal TSS challenge. Taken together, these results indicate for the first time that P. fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity.

  19. Changes in rhizosphere bacterial gene expression following glyphosate treatment.

    Science.gov (United States)

    Newman, Molli M; Lorenz, Nicola; Hoilett, Nigel; Lee, Nathan R; Dick, Richard P; Liles, Mark R; Ramsier, Cliff; Kloepper, Joseph W

    2016-05-15

    In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria.

  20. Antimicrobial resistance and virulence factor gene profiles of Enterococcus spp. isolates from wild Arctocephalus australis (South American fur seal) and Arctocephalus tropicalis (Subantarctic fur seal).

    Science.gov (United States)

    Santestevan, Naiara Aguiar; de Angelis Zvoboda, Dejoara; Prichula, Janira; Pereira, Rebeca Inhoque; Wachholz, Guilherme Raffo; Cardoso, Leonardo Almansa; de Moura, Tiane Martin; Medeiros, Aline Weber; de Amorin, Derek Blaese; Tavares, Maurício; d'Azevedo, Pedro Alves; Franco, Ana Claudia; Frazzon, Jeverson; Frazzon, Ana Paula Guedes

    2015-12-01

    Enterococci are natural inhabitants of the gastrointestinal tracts in humans and animals. Epidemiological data suggest that enterococci are important reservoirs of antimicrobial resistant genes that may be transmitted from other bacterial species The aim of this study was to investigate the species composition, antimicrobial resistance and virulence genes in enterococci recovered from fecal samples of wild Arctocephalus australis and A. tropicalis found dead along the South Coast of Brazil. From a total of 43 wild fur seals, eleven were selected for this study. Phenotypic and genotypic characterizations were used to classify Enterococcus species. Strains were tested for susceptibility to 10 antibiotics, presence of ace, gelE, asa, cylA, tet(L), tet(M) and erm(B) genes by PCR, and genetic variability using RAPD-PCR. Among the 50 enterococci isolated, 40% were Enterococcus faecalis, 40% E. hirae, 12% E. casseliflavus and 8 % other enterococcal species. Resistance profiles were observed to erythromycin, nitrofurantoin, tetracycline, norfloxacin and ciprofloxacin. The prevalence of virulence genes was ace (68%), gelE (54%), asa (22%) and cylA (4%). In erythromycin- and tetracycline strains, erm(B) and tet(M) were detected, respectively. The RAPD-PCR demonstrated a close phylogenetic relationship between the enterococci isolated from A. australis and A. tropicalis. In conclusion, different enterococcus species showing antimicrobial resistance and virulence determinates were isolated from fecal samples of fur seals. Antibiotic resistant strains in these animals could be related within food chain and aquatic pollutants or linked to environmental resistome, and demonstrates the potential importance of these animals as reservoirs and disseminators of such determinants in marine environmental.

  1. [Detection of Yersinia Enterocolitica Bacteriophage PhiYe-F10 Lysis Spectrum and Analysis of the Relationship between Lysis Ability and Virulence Gene of Yersinia Enterocolitica].

    Science.gov (United States)

    Zha, Tao; Liang, Junrong; Xiao, Yuchun; Jing, Huaiqi

    2016-03-01

    To determine the lysis spectrum of Yersinia enterocolitica bacteriophage phiYe-F10 and to analyze the relationship between the lysis ability of phiYe-F10 and the virulence gene of Yersinia enterocolitica. To observe the lysis ability of the phage phiYe-F10 to the different Yersinia strains with the double-layer technique. The strains used in this study including 213 of Yersinia enterocolitica and 36 of Yersinia pseudotuberculosis and 1 of Yersinia pestis. The virulence genes of these Yersinia enterocolitica (attachment invasion locus (ail) and enterotoxin (ystA, ystB) and yersinia adhesin A (yadA), virulence factor (virF), specific gene for lipopolysaccharide O-side chain of serotype O : 3 (rfbc) were all detected. Among the 213 Yersinia enterocolitica, 84 strains were O : 3 serotype (78 strains with rfbc gene), 10 were serotype O : 5, 13 were serotype O : 8, 34 were serotype O : 9 and 72 were other serotypes. Of these, 77 were typical pathogenic Yersinia enterocolitica harboring with virulence plasmid (ail+, ystA+, ystB-, yadA+, virF+), and 15 were pathogenic bacterial strains deficiency virulence plasmid (ail+, ystA+, ystB-, yadA-, virF-) and the rest 121 were non pathogenic genotype strains. PhiYe-F10 lysed the 71 serotype O : 3 Yersinia enterocolitica strains which were all carried with rfbc+, including 52 pathogenic Yersinia enterocolitica, 19 nonpathogenic Y. enterocolitica. The phiYe-F10 can not lysed serotype O : 5, O : 9 and other serotype Y. enterocolitica, the lysis rate of serotype O : 3 was as high as 84.5%. The phiYe-F10 can not lysed Yersinia pseudotuberculosis and Yersinia pestis. Yersinia phage phiYe-F10 is highly specific for serotype O : 3 Yersinia enterocolitic at 25 degrees C, which showed a typical narrow lysis spectrum. Phage phiYe-F10 can lysed much more pathogenic Y. enterocolitica than nonpathogenic Y. enterocolitica.

  2. PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum

    DEFF Research Database (Denmark)

    Jiang, Lubin; Mu, Jianbing; Zhang, Qingfeng;

    2013-01-01

    The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 p...... orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine....... protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown...... genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which...

  3. Virulence genes detection of Salmonella serovars isolated from pork and slaughterhouse environment in Ahmedabad, Gujarat

    Directory of Open Access Journals (Sweden)

    J. H. Chaudhary

    2015-01-01

    Full Text Available Aim: The aim was to detect virulence gene associated with the Salmonella serovars isolated from pork and Slaughterhouse environment. Materials and Methods: Salmonella isolates (n=37 used in this study were isolated from 270 pork and slaughter house environmental samples collected from the Ahmedabad Municipal Corporation Slaughter House, Ahmedabad, Gujarat, India. Salmonella serovars were isolated and identified as per BAM USFDA method and serotyped at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (Himachal Pradesh, India. Polymerase chain reaction technique was used for detection of five genes, namely invA, spvR, spvC, fimA and stn among different serovars of Salmonella. Results: Out of a total of 270 samples, 37 (13.70% Salmonella were isolated with two serovars, namely Enteritidis and Typhimurium. All Salmonella serovars produced 284 bp invA gene, 84 bp fimA and 260 bp amplicon for enterotoxin (stn gene whereas 30 isolates possessed 310 bp spvR gene, but no isolate possessed spvC gene. Conclusion: Presence of invA, fimA and stn gene in all isolates shows that they are the specific targets for Salmonella identification and are capable of producing gastroenteric illness to humans, whereas 20 Typhimurium serovars and 10 Enteritidis serovars can able to produce systemic infection.

  4. Associations between Antimicrobial Resistance Phenotypes, Antimicrobial Resistance Genes, and Virulence Genes of Fecal Escherichia coli Isolates from Healthy Grow-Finish Pigs ▿

    OpenAIRE

    2009-01-01

    Escherichia coli often carries linked antimicrobial resistance genes on transmissible genetic elements. Through coselection, antimicrobial use may select for unrelated but linked resistance or virulence genes. This study used unconditional statistical associations to investigate the relationships between antimicrobial resistance phenotypes and antimicrobial resistance genes in 151 E. coli isolates from healthy pigs. Phenotypic resistance to each drug was significantly associated with phenotyp...

  5. Molecular detection of the Aeromonas virulence aerolysin gene in retail meats from different animal sources in Egypt.

    Science.gov (United States)

    Osman, Kamelia; Aly, Magdy; Kheader, Afaf; Mabrok, Khaled

    2012-05-01

    Meat commonly contain the same Aeromonas spp. which occur in human diarrhoeal and non-diarrhoeal faecal samples. Motile Aeromonas were isolated from 5.6% of total 302 samples. The distribution of the isolates were 5.9 and 5.2% in fresh and frozen samples, respectively. Of the 302 samples taken of the four animal meat species investigated, the genus Aeromonas were isolated in 12.3% of the fresh samples collected from buffalo meat, in 6.5% of the samples collected from sheep meat and 14.0% from the samples collected from the cattle frozen meat samples. The camel meat did not reveal any Aeromonas isolates. Aeromonas hydrophila was isolated as the most prevalent species with 6.8%, followed by Aeromonas caviae with 2.7% and Aeromonas sobria with 2.1% from the total meat samples. Aerolysin toxin gene (aerA) was detected in 3/17 isolates of A. hydrophila isolated from contaminated meat. Infection due to bacterial pathogen with such virulent factor through contact with contaminated meat while handling them, poses health hazards to humans.

  6. Anaerobes and Bacterial Vaginosis in Pregnancy: Virulence Factors Contributing to Vaginal Colonisation

    Directory of Open Access Journals (Sweden)

    Charlene W. J. Africa

    2014-07-01

    Full Text Available The aetiology and pathogenesis of bacterial vaginosis (BV is unclear but it appears to be associated with factors that disrupt the normal acidity of the vagina thus altering the equilibrium between the normal vaginal microbiota. BV has serious implications for female morbidity, including reports of pelvic inflammatory disease, adverse pregnancy outcomes, increased susceptibility to sexually transmitted infections and infertility. This paper reviewed new available information regarding possible factors contributing to the establishment of the BV vaginal biofilm, examined the proposed role of anaerobic microbial species recently detected by new culture-independent methods and discusses developments related to the effects of BV on human pregnancy. The literature search included Pubmed (NLM, LISTA (EBSCO, and Web of Science. Because of the complexity and diversity of population groups, diagnosis and methodology used, no meta-analysis was performed. Several anaerobic microbial species previously missed in the laboratory diagnosis of BV have been revealed while taking cognisance of newly proposed theories of infection, thereby improving our understanding and knowledge of the complex aetiology and pathogenesis of BV and its perceived role in adverse pregnancy outcomes.

  7. Development of a miniaturized DNA microarray for identification of 66 virulence genes of Legionella pneumophila

    Directory of Open Access Journals (Sweden)

    Mariusz Żak

    2011-12-01

    Full Text Available Introduction:For the last five years, Legionella sp. infections and legionnaire’s disease in Poland have been receiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmental and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations.Material/Methods:The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected.Results.We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain.Discussion:The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of genes increasing the risk of clinical disease and to determine the pathogenic potential of environmental strains.

  8. Occurrence of motile Aeromonas in municipal drinking water and distribution of genes encoding virulence factors.

    Science.gov (United States)

    Pablos, Manuel; Rodríguez-Calleja, Jose M; Santos, Jesús A; Otero, Andrés; García-López, María-Luisa

    2009-10-31

    Aeromonas-associated cases of gastroenteritis are generally considered waterborne. The purpose of this study was to evaluate the potential microbiological risk associated with the presence of these bacteria in public drinking water. Over a period of one year, 132 drinking-water samples were monitored in León (NW of Spain, 137,000 inhabitants) for mandatory drinking-water standards and the occurrence of Aeromonas spp. Samples were taken at the municipal water treatment plant, one storage facility, and two public artesian drinking-water fountains. Because of low numbers of coliforms or Clostridium perfringens, the non-compliance rate with microbial standards was 3.8% whereas the percentage of positive samples for motile mesophilic Aeromonas was 26.5%. For all but two samples, Aeromonas was recovered between October and early March when the temperature was below 14 degrees C and the residual chlorine ranged from 0.21 to 0.72 mg/l. An apparent relationship was observed between rainfall and the incidence of Aeromonas. The 35 selected Aeromonas isolates were identified as A. caviae and A. media. The alt and laf genes were present in all isolates, the aerA gene was present in six isolates, and the four remaining genes investigated (hlyA, ast, stx1 and stx2) were absent. The combinations of putative virulence genes were: aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)/stx1(-)/stx2(-) (82.9%) and aerA(+)/hlyA(-)/alt(+)/ast(-)/laf(+)/stx1(-)/stx2(-) (17.1%). None of the isolates bore plasmids. As Aeromonas strains harbouring two or more virulence-associated genes have the potential to cause disease by direct transmission via drinking water or by water use in food preparation, it would be advisable to control excessive numbers of these bacteria in drinking-water supplies.

  9. Antimicrobial resistance, virulence genes, and genetic lineages of Staphylococcus pseudintermedius in healthy dogs in tunisia.

    Science.gov (United States)

    Gharsa, Haythem; Ben Slama, Karim; Gómez-Sanz, Elena; Lozano, Carmen; Klibi, Naouel; Jouini, Ahlem; Messadi, Lilia; Boudabous, Abdellatif; Torres, Carmen

    2013-08-01

    Nasal swabs of 100 healthy dogs were obtained in 2011 in Tunisia and tested for Staphylococcus pseudintermedius recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST) and SmaI-pulsed-field gel electrophoresis (PFGE) were investigated. S. pseudintermedius was recovered in 55 of the 100 tested samples (55 %), and one isolate per sample was further studied. All 55 S. pseudintermedius isolates were susceptible to methicillin (MSSP) but showed resistance to the following antimicrobials (% resistant isolates/resistance gene): penicillin (56.4/blaZ), tetracycline (40/tetM), trimethoprim-sulfamethoxazole (23.7), fusidic acid (9), kanamycin (3.7/aph(3´)-Ia), erythromycin-clindamycin (1.8/erm(B)), streptomycin (1.8/ant(6)-Ia), chloramphenicol (1.8) and ciprofloxacin (1.8). The following toxin genes were identified (% of isolates): lukS/F-I (98.2), expA (5.5), se-int (98.2), sec canine (1.8), siet (100), sea (5.5), seb (3.6), sec (10.9), sed (54.5), sei (5.5), sej (29.1), sek (3.6), ser (9.1), and hlg v (38.2). Ten different sequence-types were detected among 11 representative MSSP isolates: ST20, ST44, ST69, ST70, ST78, ST100, ST108, ST160, ST161, and ST162, the last three ones revealing novel alleles or allele combinations. Eleven different PFGE-patterns were identified in these isolates. The nares of healthy dogs could be a reservoir of antimicrobial resistant and virulent MSSP, highlighting the presence of the recently described exfoliating gene expA and several enterotoxin genes.

  10. Subgingival bacterial colonization profiles correlate with gingival tissue gene expression

    Directory of Open Access Journals (Sweden)

    Handfield Martin

    2009-10-01

    Full Text Available Abstract Background Periodontitis is a chronic inflammatory disease caused by the microbiota of the periodontal pocket. We investigated the association between subgingival bacterial profiles and gene expression patterns in gingival tissues of patients with periodontitis. A total of 120 patients undergoing periodontal surgery contributed with a minimum of two interproximal gingival papillae (range 2-4 from a maxillary posterior region. Prior to tissue harvesting, subgingival plaque samples were collected from the mesial and distal aspects of each tissue sample. Gingival tissue RNA was extracted, reverse-transcribed, labeled, and hybridized with whole-genome microarrays (310 in total. Plaque samples were analyzed using checkerboard DNA-DNA hybridizations with respect to 11 bacterial species. Random effects linear regression models considered bacterial levels as exposure and expression profiles as outcome variables. Gene Ontology analyses summarized the expression patterns into biologically relevant categories. Results Wide inter-species variation was noted in the number of differentially expressed gingival tissue genes according to subgingival bacterial levels: Using a Bonferroni correction (p -7, 9,392 probe sets were differentially associated with levels of Tannerella forsythia, 8,537 with Porphyromonas gingivalis, 6,460 with Aggregatibacter actinomycetemcomitans, 506 with Eikenella corrodens and only 8 with Actinomyces naeslundii. Cluster analysis identified commonalities and differences among tissue gene expression patterns differentially regulated according to bacterial levels. Conclusion Our findings suggest that the microbial content of the periodontal pocket is a determinant of gene expression in the gingival tissues and provide new insights into the differential ability of periodontal species to elicit a local host response.

  11. Exploring new roles for the rpoS gene in the survival and virulence of the fire blight pathogen Erwinia amylovora.

    Science.gov (United States)

    Santander, Ricardo D; Monte-Serrano, Mercedes; Rodríguez-Herva, José J; López-Solanilla, Emilia; Rodríguez-Palenzuela, Pablo; Biosca, Elena G

    2014-12-01

    Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits.

  12. The shf gene of a Shigella flexneri homologue on the virulent plasmid pAA2 of enteroaggregative Escherichia coli 042 is required for firm biofilm formation.

    Science.gov (United States)

    Fujiyama, Rika; Nishi, Junichiro; Imuta, Naoko; Tokuda, Koichi; Manago, Kunihiro; Kawano, Yoshifumi

    2008-05-01

    Enteroaggregative Escherichia coli (EAEC) is an increasingly important cause of diarrhea in both developing and industrialized countries, and is characterized by strong biofilm formation on the intestinal mucosa. Sequencing of the virulent plasmid pAA2 of the prototype EAEC 042 revealed a cluster of three open reading frames (ORFs; shf, capU, and virK) ca. 93% identical to a similar cluster located in Shigella flexneri. The function of the first ORF Shf protein is not known, but the closest well-characterized homologue is the IcaB protein of Staphylococcus epidermidis, which plays a crucial role in exopolysaccharide modification in bacterial biofilm formation. To investigate the role of this cluster in the virulence of EAEC, we mutated three genes at this locus. All the mutants maintained the aggregative phenotype in the liquid phase. However, the insertional mutant of shf formed a less abundant biofilm in a microtiter plate assay than did the wild type, while the capU mutant and the virK mutant did not. The complementation of the shf mutant with this cluster restored the thick biofilm similar to that of the wild type. The shf transcriptional level decreased in the transcriptional regulator aggR mutant and was restored when the mutant was complemented with aggR. These results suggest that the shf gene is required for the firm biofilm formation of EAEC 042, and transcription of the shf gene is dependent on AggR.

  13. Bacillus cereus from blood cultures: virulence genes, antimicrobial susceptibility and risk factors for blood stream infection.

    Science.gov (United States)

    Horii, Toshinobu; Notake, Shigeyuki; Tamai, Kiyoko; Yanagisawa, Hideji

    2011-11-01

    We characterized the profiles of virulence genes and antimicrobial susceptibility of Bacillus cereus isolates from blood cultures as well as the risk factors for blood stream infections (BSIs). The diversity of virulence gene patterns was found to be wide among 15 B. cereus isolates from BSIs and also among 11 isolates from contaminated blood cultures. The MicroScan broth microdilution method yielded results corresponding with those of the agar dilution (reference) method for levofloxacin, linezolid, and vancomycin, while the Etest results were consistent with the reference results for clindamycin, gentamicin, imipenem, levofloxacin, and linezolid. Compared with the reference values, however, some isolates showed marked differences of the minimum inhibitory concentrations (MICs) for ampicillin and clindamycin when determined using the MicroScan method, or the MICs for ampicillin, meropenem, and vancomycin when determined using the Etest method. Significantly more patients were treated with antimicrobials for more than 3 days during the 3-month period before isolation in the BSI group. Prior antimicrobial therapy may be a risk factor for BSIs due to B. cereus.

  14. Prevalence of Virulence/Stress Genes in Campylobacter jejuni from Chicken Meat Sold in Qatari Retail Outlets.

    Directory of Open Access Journals (Sweden)

    Marawan Abu-Madi

    Full Text Available Chicken meat from the shelves of supermarkets in Qatar was tested for the presence of Campylobacter spp. and the presence of five virulence genes (htrB, cdtB, clpP, cadF and ciaB was assessed in isolates. Forty eight percent of the chickens provided for supermarkets by Saudi (53% and Qatari (45.9% producers were found to be contaminated and the most important factor affecting the overall prevalence of contaminated chickens was the store from which chicken samples originated. Variation in prevalence of Campylobacter in chicken meat from different stores was evident even when the same producer supplied the three stores in our survey. Differences in the prevalence and in the combinations of virulence genes in isolates that can and cannot grow in a classic maintenance medium (Karmali were identified, providing a starting point for linking presence/absence of particular virulence genes with actual in vivo virulence and pathogenicity. Because of the relatively low infective doses of Campylobacter that are required to initiate infection in humans, it will be important to explore further the relationships we identified between certain Campylobacter virulence genes and their capacity for survival in poultry meat, and hence their contribution to the incidence of campylobacteriosis.

  15. Comparisons of Salmonella conjugation and virulence gene hyperexpression mediated by rumen protozoa from domestic and exotic ruminants.

    Science.gov (United States)

    Brewer, Matt T; Xiong, Nalee; Dier, Jeffery D; Anderson, Kristi L; Rasmussen, Mark A; Franklin, Sharon K; Carlson, Steve A

    2011-08-05

    Recent studies have identified a phenomenon in which ciliated protozoa engulf Salmonella and the intra-protozoal environment hyperactivates virulence gene expression and provides a venue for conjugal transfer of antibiotic resistance plasmids. The former observation is relegated to Salmonella bearing the SGI1 multiresistance integron while the latter phenomenon appears to be a more generalized event for recipient Salmonella. Our previous studies have assessed virulence gene hyperexpression only with protozoa from the bovine rumen while conjugal transfer has been demonstrated in rumen protozoa from cattle and goats. The present study examined virulence gene hyperexpression for Salmonella exposed to rumen protozoa obtained from cattle, sheep, goats, or two African ruminants (giraffe and bongo). Conjugal transfer was also assessed in these protozoa using Salmonella as the recipient. Virulence gene hyperexpression was only observed following exposure to the rumen protozoa from cattle and sheep while elevated virulence was also observed in these animals. Conjugal transfer events were, however, observed in all protozoa evaluated. It therefore appears that the protozoa-based hypervirulence is not universal to all ruminants while conjugal transfer is more ubiquitous.

  16. Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae.

    LENUS (Irish Health Repository)

    Walsh, Fiona

    2010-06-01

    A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.

  17. Gene calling and bacterial genome annotation with BG7.

    Science.gov (United States)

    Tobes, Raquel; Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Kovach, Evdokim; Alekhin, Alexey; Pareja, Eduardo

    2015-01-01

    New massive sequencing technologies are providing many bacterial genome sequences from diverse taxa but a refined annotation of these genomes is crucial for obtaining scientific findings and new knowledge. Thus, bacterial genome annotation has emerged as a key point to investigate in bacteria. Any efficient tool designed specifically to annotate bacterial genomes sequenced with massively parallel technologies has to consider the specific features of bacterial genomes (absence of introns and scarcity of nonprotein-coding sequence) and of next-generation sequencing (NGS) technologies (presence of errors and not perfectly assembled genomes). These features make it convenient to focus on coding regions and, hence, on protein sequences that are the elements directly related with biological functions. In this chapter we describe how to annotate bacterial genomes with BG7, an open-source tool based on a protein-centered gene calling/annotation paradigm. BG7 is specifically designed for the annotation of bacterial genomes sequenced with NGS. This tool is sequence error tolerant maintaining their capabilities for the annotation of highly fragmented genomes or for annotating mixed sequences coming from several genomes (as those obtained through metagenomics samples). BG7 has been designed with scalability as a requirement, with a computing infrastructure completely based on cloud computing (Amazon Web Services).

  18. Genes similar to the Vibrio parahaemolyticus virulence-related genes tdh, tlh, and vscC2 occur in other vibrionaceae species isolated from a pristine estuary.

    Science.gov (United States)

    Klein, Savannah L; Gutierrez West, Casandra K; Mejia, Diana M; Lovell, Charles R

    2014-01-01

    Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.

  19. A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity.

    Science.gov (United States)

    Leavis, Helen; Top, Janetta; Shankar, Nathan; Borgen, Katrine; Bonten, Marc; van Embden, Jan; Willems, Rob J L

    2004-02-01

    Enterococcus faecalis harbors a virulence-associated surface protein encoded by the esp gene. This gene has been shown to be part of a 150-kb putative pathogenicity island. A gene similar to esp has recently been found in Enterococcus faecium isolates recovered from hospitalized patients. In the present study we analyzed the polymorphism in the esp gene of E. faecium, and we investigated the association of esp with neighboring chromosomal genes. The esp gene showed considerable sequence heterogeneity in the regions encoding the nonrepeat N- and C-terminal domains of the Esp protein as well as differences in the number of repeats. DNA sequencing of chromosomal regions flanking the esp gene of E. faecium revealed seven open reading frames, representing putative genes implicated in virulence, regulation of transcription, and antibiotic resistance. These flanking regions were invariably associated with the presence or absence of the esp gene in E. faecium, indicating that esp in E. faecium is part of a distinct genetic element. Because of the presence of virulence genes in this gene cluster, the lower G+C content relative to that of the genome, and the presence of esp in E. faecium isolates associated with nosocomial outbreaks and clinically documented infections, we conclude that this genetic element constitutes a putative pathogenicity island, the first one described in E. faecium. Except for the presence of esp and araC, this pathogenicity island is completely different from the esp-containing pathogenicity island previously disclosed in E. faecalis.

  20. Diarrheagenic Escherichia coli carrying supplementary virulence genes are an important cause of moderate to severe diarrhoeal disease in Mexico.

    Directory of Open Access Journals (Sweden)

    Sandra Patzi-Vargas

    2015-03-01

    Full Text Available Diarrheagenic Escherichia coli (DEC cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG, mainly associated with enteroaggregative E. coli (EAEC, were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%, surpassing Salmonella (12% and Shigella (9%. Predominant DEC groups were diffusely adherent E. coli (DAEC (35%, EAEC (24%, and enteropathogenic E. coli (EPEC (19%. Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26% and DAEC (18%; 30% had moderate diarrhoea mainly caused by DAEC (36%, mixed DEC infections (33% and EAEC (32%. DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6-24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3-13.9. The presence of SVG dispersin, (aatA, dispersin-translocator (aatA, enteroaggregative heat-stable toxin 1 (astA, plasmid encoded toxin (pet, cytolethal distending toxin (cdt was higher in DEC than non-DEC strains, (36% vs 26%, P <0.0001, OR = 1.5, 95% CI, 1.3-1.8. 98% of EAEC-infected children harboured strains with SVG; 85% carried the aap-aatA gene combination, and 33% of these also carried astA. 28% of both EPEC and ETEC, and 6% of DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2% and non-DEC strains (21% and 13%. DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children.

  1. The Amazonia Variant of Vibrio cholerae: Molecular Identification and Study of Virulence Genes

    Directory of Open Access Journals (Sweden)

    Baptista MAS

    1998-01-01

    Full Text Available The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a. This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the El Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.

  2. Distribution of genes encoding virulence factors and molecular analysis of Shigella spp. isolated from patients with diarrhea in Kerman, Iran.

    Science.gov (United States)

    Hosseini Nave, Hossein; Mansouri, Shahla; Emaneini, Mohammad; Moradi, Mohammad

    2016-03-01

    Shigella is one of the important causes of diarrhea worldwide. Shigella has several virulence factors contributing in colonization and invasion of epithelial cells and eventually death of host cells. The present study was performed in order to investigate the distribution of virulence factors genes in Shigella spp. isolated from patients with acute diarrhea in Kerman, Iran as well as the genetic relationship of these isolates. A total of 56 isolates including 31 S. flexneri, 18 S. sonnei and 7 S. boydii were evaluated by polymerase chain reaction (PCR) for the presence of 11 virulence genes (ipaH, ial, set1A, set1B, sen, virF, invE, sat, sigA, pic and sepA). Then, the clonal relationship of these strains was analyzed by multilocus variable-number tandem repeat analysis (MLVA) method. All isolates were positive for ipaH gene. The other genes include ial, invE and virF were found in 80.4%, 60.7% and 67.9% of the isolates, respectively. Both set1A and set1B were detected in 32.3% of S. flexneri isolates, whereas 66.1% of the isolates belonging to different serogroup carried sen gene. The sat gene was present in all S. flexneri isolates, but not in the S. sonnei and S. boydii isolates. The result showed, 30.4% of isolates were simultaneously positive and the rest of the isolates were negative for sepA and pic genes. The Shigella isolates were divided into 29 MLVA types. This study, for the first time, investigated distribution of 11 virulence genes in Shigella spp. Our results revealed heterogeneity of virulence genes in different Shigella serogroups. Furthermore, the strains belonging to the same species had little diversity.

  3. Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes.

    Science.gov (United States)

    Abdel-Ghany, Salah E; Day, Irene; Heuberger, Adam L; Broeckling, Corey D; Reddy, Anireddy S N

    2013-11-01

    1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC-MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production.

  4. Indole-3-acetic acid in plant-pathogen interactions: a key molecule for in planta bacterial virulence and fitness.

    Science.gov (United States)

    Cerboneschi, Matteo; Decorosi, Francesca; Biancalani, Carola; Ortenzi, Maria Vittoria; Macconi, Sofia; Giovannetti, Luciana; Viti, Carlo; Campanella, Beatrice; Onor, Massimo; Bramanti, Emilia; Tegli, Stefania

    The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called "indole-3-acetamide pathway" to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis. Here, two knockout mutants, ΔiaaL and ΔiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that ΔiaaL and ΔiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of ΔiaaL and ΔiaaM, as well as growth of the non-pathogenic mutant ΔhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a

  5. Identification of genes and gene products necessary for bacterial bioluminescence.

    OpenAIRE

    1984-01-01

    Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by compl...

  6. An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence

    Science.gov (United States)

    Huynh, TuAnh Ngoc; Luo, Shukun; Pensinger, Daniel; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2015-01-01

    The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network. PMID:25583510

  7. A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

    Science.gov (United States)

    Connolly, James P R; Gabrielsen, Mads; Goldstone, Robert J; Grinter, Rhys; Wang, Dai; Cogdell, Richard J; Walker, Daniel; Smith, David G E; Roe, Andrew J

    2016-01-01

    The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

  8. A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

    Directory of Open Access Journals (Sweden)

    James P R Connolly

    2016-01-01

    Full Text Available The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

  9. Comparative Genomic Analysis of Lactococcus garvieae Strains Isolated from Different Sources Reveals Candidate Virulence Genes

    Directory of Open Access Journals (Sweden)

    Eiji Miyauchi

    2012-01-01

    Full Text Available Lactococcus garvieae is a major pathogen for fish. Two complete (ATCC 49156 and Lg2 and three draft (UNIUD074, 8831, and 21881 genome sequences of L. garvieae have recently been released. We here present the results of a comparative genomic analysis of these fish and human isolates of L. garvieae. The pangenome comprised 1,542 core and 1,378 dispensable genes. The sequenced L. garvieae strains shared most of the possible virulence genes, but the capsule gene cluster was found only in fish-pathogenic strain Lg2. The absence of the capsule gene cluster in other nonpathogenic strains isolated from mastitis and vegetable was also confirmed by PCR. The fish and human isolates of L. garvieae contained the specific two and four adhesin genes, respectively, indicating that these adhesion proteins may be involved in the host specificity differences of L. garvieae. The discoveries revealed by the pangenomic analysis may provide significant insights into the biology of L. garvieae.

  10. Clinical and Genomic Analysis of Liver Abscess-Causing Klebsiella pneumoniae Identifies New Liver Abscess-Associated Virulence Genes

    Science.gov (United States)

    Ye, Meiping; Tu, Jianfei; Jiang, Jianping; Bi, Yingmin; You, Weibo; Zhang, Yanliang; Ren, Jianmin; Zhu, Taohui; Cao, Zhuo; Yu, Zuochun; Shao, Chuxiao; Shen, Zhen; Ding, Baixing; Yuan, Jinyi; Zhao, Xu; Guo, Qinglan; Xu, Xiaogang; Huang, Jinwei; Wang, Minggui

    2016-01-01

    Hypervirulent variants of Klebsiella pneumoniae (hvKp) that cause invasive community-acquired pyogenic liver abscess (PLA) have emerged globally. Little is known about the virulence determinants associated with hvKp, except for the virulence genes rmpA/A2 and siderophores (iroBCD/iucABCD) carried by the pK2044-like large virulence plasmid. Here, we collected most recent clinical isolates of hvKp from PLA samples in China, and performed clinical, molecular, and genomic sequencing analyses. We found that 90.9% (40/44) of the pathogens causing PLA were K. pneumoniae. Among the 40 LA-Kp, K1 (62.5%), and K2 (17.5%) were the dominant serotypes, and ST23 (47.5%) was the major sequence type. S1-PFGE analyses demonstrated that although 77.5% (31/40) of the LA-Kp isolates harbored a single large virulence plasmid varied in size, 5 (12.5%) isolates had no plasmid and 4 (10%) had two or three plasmids. Whole genome sequencing and comparative analysis of 3 LA-Kp and 3 non-LA-Kp identified 133 genes present only in LA-Kp. Further, large scale screening of the 133 genes in 45 LA-Kp and 103 non-LA-Kp genome sequences from public databases identified 30 genes that were highly associated with LA-Kp, including iroBCD, iucABCD and rmpA/A2 and 21 new genes. Then, these 21 new genes were analyzed in 40 LA-Kp and 86 non-LA-Kp clinical isolates collected in this study by PCR, showing that new genes were present 80–100% among LA-Kp isolates while 2–11% in K. pneumoniae isolates from sputum and urine. Several of the 21 genes have been proposed as virulence factors in other bacteria, such as the gene encoding SAM-dependent methyltransferase and pagO which protects bacteria from phagocytosis. Taken together, these genes are likely new virulence factors contributing to the hypervirulence phenotype of hvKp, and may deepen our understanding of virulence mechanism of hvKp. PMID:27965935

  11. Clinical and Genomic Analysis of Liver Abscess-Causing Klebsiella pneumoniae Identifies New Liver Abscess-Associated Virulence Genes

    Directory of Open Access Journals (Sweden)

    Meiping Ye

    2016-11-01

    Full Text Available Hypervirulent variants of Klebsiella pneumoniae (hvKp that cause invasive community-acquired pyogenic liver abscess have emerged globally. Little is known about the virulence determinants associated with hvKp, except for the virulence genes rmpA/A2 and siderophores (iroBCD/iucABCD carried by the pK2044-like large virulence plasmid. Here, we collected most recent clinical isolates of hvKp from pyogenic liver abscess (PLA samples in China, and performed clinical, molecular, and genomic sequencing analyses. We found that 90.9% (40/44 of the pathogens causing PLA were K. pneumoniae. Among the 40 LA-Kp, K1 (62.5% and K2 (17.5% were the dominant serotypes, and ST23 (47.5% was the major sequence type. S1-PFGE analyses demonstrated that although 77.5% (31/40 of the LA-Kp isolates harbored a single large virulence plasmid varied in size, 5 (12.5% isolates had no plasmid and 4 (10% had two or three plasmids. Whole genome sequencing and comparative analysis of 3 LA-Kp and 3 non-LA-Kp identified 133 genes present only in LA-Kp. Further, large scale screening of the 133 genes in 45 LA-Kp and 103 non-LA-Kp genome sequences from public databases identified 30 genes that were highly associated with LA-Kp, including iroBCD, iucABCD and rmpA/A2 and 21 new genes. Then, these 21 new genes were analyzed in 40 LA-Kp and 86 non-LA-Kp clinical isolates collected in this study by PCR, showing that new genes were present 80-100% among LA-Kp isolates while 2-11% in K. pneumoniae isolates from sputum and urine. Several of the 21 genes have been proposed as virulence factors in other bacteria, such as the gene encoding SAM-dependent methyltransferase and pagO which protects bacteria from phagocytosis. Taken together, these genes are likely new virulence factors contributing to the hypervirulence phenotype of hvKp, and may deepen our understanding of virulence mechanism of hvKp.

  12. Cooperation and the evolutionary ecology of bacterial virulence: the Bacillus cereus group as a novel study system.

    Science.gov (United States)

    Raymond, Ben; Bonsall, Michael B

    2013-08-01

    How significant is social evolution theory for the maintenance of virulence in natural populations? We assume that secreted, distantly acting virulence factors are highly likely to be cooperative public goods. Using this assumption, we discuss and critically assess the potential importance of social interactions for understanding the evolution, diversity and distribution of virulence in the Bacillus cereus group, a novel study system for microbial social biology. We conclude that dynamic equilibria in Cry toxin production, as well as strong spatial structure and population bottlenecks in hosts are the main ecological factors maintaining the cooperative secretion of virulence factors and argue that collective action has contributed to the evolution of narrow host range. Non-linearities in the benefits associated with public goods, as well as the lack of private secretion systems in the Firmicutes may also explain the prevalence and importance of distantly acting virulence factors in B. cereus and its relatives.

  13. Reciprocal interaction between dental alloy biocorrosion and Streptococcus mutans virulent gene expression.

    Science.gov (United States)

    Zhang, Songmei; Qiu, Jing; Ren, Yanfang; Yu, Weiqiang; Zhang, Fuqiang; Liu, Xiuxin

    2016-04-01

    Corrosion of dental alloys is a major concern in dental restorations. Streptococcus mutans reduces the pH in oral cavity and induces demineralization of the enamel as well as corrosion of restorative dental materials. The rough surfaces of dental alloys induced by corrosion enhance the subsequent accumulation of plaque. In this study, the corrosion process of nickel-chromium (Ni-Cr) and cobalt-chromium (Co-Cr) alloys in a nutrient-rich medium containing S. mutans was studied using inductively coupled plasma atomic emission spectrometry (ICP-AES), X-ray photoelectron spectroscopy (XPS) and electrochemical corrosion test. Our results showed that the release of Ni and Co ions increased, particularly after incubation for 3 days. The electrochemical corrosion results showed a significant decrease in the corrosion resistance (Rp) value after the alloys were immersed in the media containing S. mutans for 3 days. Correspondingly, XPS revealed a reduction in the relative dominance of Ni, Co, and Cr in the surface oxides after the alloys were immersed in the S. mutans culture. After removal of the biofilm, the pre-corroded alloys were re-incubated in S. mutans medium, and the expressions of genes associated with the adhesion and acidogenesis of S. mutans, including gtfBCD, gbpB, fif and ldh, were evaluated by detecting the mRNA levels using real-time reverse transcription polymerase chain reaction (RT-PCR). We found that the gtfBCD, gbpB, ftf and Idh expression of S. mutans were noticeably increased after incubation with pre-corroded alloys for 24 h. This study demonstrated that S. mutans enhanced the corrosion behavior of the dental alloys, on the other hand, the presence of corroded alloy surfaces up-regulated the virulent gene expression in S. mutans. Compared with smooth surfaces, the rough corroded surfaces of dental alloys accelerated the bacteria-adhesion and corrosion process by changing the virulence gene expression of S. mutans.

  14. Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes.

    Science.gov (United States)

    Sousa, Mireille Ângela Bernardes; Mendes, Edilberto Nogueira; Collares, Guilherme Birchal; Péret-Filho, Luciano Amedée; Penna, Francisco José; Magalhães, Paula Prazeres

    2013-02-01

    Diarrhoeal disease is still considered a major cause of morbidity and mortality among children. Among diarrhoeagenic agents, Shigella should be highlighted due to its prevalence and the severity of the associated disease. Here, we assessed Shigella prevalence, drug susceptibility and virulence factors. Faeces from 157 children with diarrhoea who sought treatment at the Children's Hospital João Paulo II, a reference children´s hospital in Belo Horizonte, state of Minas Gerais, Brazil, were cultured and drug susceptibility of the Shigella isolates was determined by the disk diffusion technique. Shigella virulence markers were identified by polymerase chain reaction. The bacterium was recovered from 10.8% of the children (88.2% Shigella sonnei). The ipaH, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1A was only detected in Shigella flexneri. Additionally, patients were infected by Shigella strains of different ial, sat, sen and set1A genotypes. Compared to previous studies, we observed a marked shift in the distribution of species from S. flexneri to S. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.

  15. Occurrence of putative virulence genes in arcobacter species isolated from humans and animals.

    Science.gov (United States)

    Douidah, Laid; de Zutter, Lieven; Baré, Julie; De Vos, Paul; Vandamme, Peter; Vandenberg, Olivier; Van den Abeele, Anne-Marie; Houf, Kurt

    2012-03-01

    Interest in arcobacters in veterinary and human public health has increased since the first report of the isolation of arcobacters from food of animal origin. Since then, studies worldwide have reported the occurrence of arcobacters on food and in food production animals and have highlighted possible transmission, especially of Arcobacter butzleri, to the human population. In humans, arcobacters are associated with enteritis and septicemia. To assess their clinical relevance for humans and animals, evaluation of potential virulence factors is required. However, up to now, little has been known about the mechanisms of pathogenicity. Because of their close phylogenetic affiliation to the food-borne pathogen Campylobacter and their similar clinical manifestations, the presence of nine putative Campylobacter virulence genes (cadF, ciaB, cj1349, hecA, hecB, irgA, mviN, pldA, and tlyA) previously identified in the recent Arcobacter butzleri ATCC 49616 genome sequence was determined in a large set of human and animal Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii strains after the development of rapid and accurate PCR assays and confirmed by sequencing and dot blot hybridization.

  16. Unfolded Protein Response (UPR Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Martin Hampel

    Full Text Available The unfolded protein response (UPR, a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER, coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

  17. Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

    Science.gov (United States)

    Hampel, Martin; Jakobi, Mareike; Schmitz, Lara; Meyer, Ute; Finkernagel, Florian; Doehlemann, Gunther; Heimel, Kai

    2016-01-01

    The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

  18. Benzaldehyde Schiff bases regulation to the metabolism, hemolysis, and virulence genes expression in vitro and their structure-microbicidal activity relationship.

    Science.gov (United States)

    Xia, Lei; Xia, Yu-Fen; Huang, Li-Rong; Xiao, Xiao; Lou, Hua-Yong; Liu, Tang-Jingjun; Pan, Wei-Dong; Luo, Heng

    2015-06-05

    There is an urgent need to develop new antibacterial agents because of multidrug resistance by bacteria and fungi. Schiff bases (aldehyde or ketone-like compounds) exhibit intense antibacterial characteristics, and are therefore, promising candidates as antibacterial agents. To investigate the mechanism of action of newly designed benzaldehyde Schiff bases, a series of high-yielding benzaldehyde Schiff bases were synthesized, and their structures were determined by NMR and MS spectra data. The structure-microbicidal activity relationship of derivatives was investigated, and the antibacterial mechanisms were investigated by gene assays for the expression of functional genes in vitro using Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The active compounds were selective for certain active groups. The polar substitution of the R2 group of the amino acids in the Schiff bases, affected the antibacterial activity against E. coli and S. aureus; specific active group at the R3 or R4 groups of the acylhydrazone Schiff bases could improve their inhibitory activity against these three tested organisms. The antibacterial mechanism of the active benzaldehyde Schiff bases appeared to regulate the expression of metabolism-associated genes in E. coli, hemolysis-associated genes in B. subtilis, and key virulence genes in S. aureus. Some benzaldehyde Schiff bases were bactericidal to all the three strains and appeared to regulate gene expression associated with metabolism, hemolysis, and virulence, in vitro. The newly designed benzaldehyde Schiff bases possessed unique antibacterial activity and might be potentially useful for prophylactic or therapeutic intervention of bacterial infections.

  19. Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China.

    Science.gov (United States)

    Wang, Dong; Zhang, Limei; Zhou, Xuezhang; He, Yulong; Yong, Changfu; Shen, Mingliang; Szenci, Otto; Han, Bo

    2016-12-01

    Staphylococcus aureusis the leading pathogen involved inbovine mastitis, but knowledgeabout antimicrobial resistance, virulence factors, and genotypes of Staphylococcus aureus resulting in bovine mastitis in Ningxia, China, is limited. Therefore, antimicrobial susceptibility, virulence gene, and randomly amplified polymorphic DNA (RAPD) analyses of Staph. aureus were carried out. A total of 327 milk samples from cows with clinical and subclinical mastitis in 4 regions of Ningxia were used for the isolation and identification of pathogens according to phenotypic and molecular characteristics. Antimicrobial susceptibility against 22 antimicrobial agents was determined by disk diffusion. The presence of 8 virulence genes in Staph. aureus isolates was tested by PCR. Genotypes of isolates were investigated based on RAPD. Results showed that 35 isolates obtained from mastitis milk samples were identified as Staph. aureus. The isolates were resistant to sulfamethoxazole (100%), penicillin G (94.3%), ampicillin (94.3%), erythromycin (68.6%), azithromycin (68.6%), clindamycin (25.7%), amoxicillin (11.4%), and tetracycline (5.7%). All of the isolates contained one or more virulence genes with average (standard deviation) of 6.6±1.6. The most prevalent virulence genes were hlb (97.1%), followed by fnbpA, hla, coa (94.3% each), nuc (85.7%), fnbpB (80%), clfA (77.1%), and tsst-1 (40%). Nine different gene patterns were found and 3 of them were the dominant gene combinations (77.1%). Staphylococcus aureus isolates (n=35) were divided into 6 genotypes by RAPD tying, the genotypes III and VI were the most prevalent genotypes. There was greatvariation in genotypes of Staph. aureus isolates, not only among different farms, but also within the same herd in Ningxia province. The study showed a high incidence of Staph. aureus with genomic variation of resistance genes, which is matter of great concern in public and animal health in Ningxia province of China.

  20. Bacterial Cellular Engineering by Genome Editing and Gene Silencing

    Directory of Open Access Journals (Sweden)

    Nobutaka Nakashima

    2014-02-01

    Full Text Available Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption, knock-in (insertion, and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering.

  1. High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary.

    Science.gov (United States)

    Gutierrez West, Casandra K; Klein, Savannah L; Lovell, Charles R

    2013-04-01

    Virulence factor genes encoding the thermostable direct hemolysin (tdh) and the thermostable direct hemolysin-related hemolysin (trh) are strongly correlated with virulence of the emergent human pathogen Vibrio parahaemolyticus. The gene encoding the thermolabile hemolysin (tlh) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains. V. parahaemolyticus strains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences of tdh and trh were recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenic V. parahaemolyticus strains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of the tdh and trh genes.

  2. Molecular Analysis and Identification of Virulence Gene on pRST98 from Multi-Drug Resistant Salmonella typhi

    Institute of Scientific and Technical Information of China (English)

    RuiHuang; ShuyanWu; XueguangZhang; YanyunZhang

    2005-01-01

    pRST98 is a large and conjugative resistant plasmid (R plasmid) of 98.6 mega-dalton from multi-drug resistant Salmonella typhi (S. typhi), which was classified to incompatibility group C (Inc C). It has been found that pRST98 made its host bacteria not only antibiotic resistant but also more virulent. In this study we explored the possibility of plasmid pRsr98 in S. typhi carrying the Salmonella plasmid virulence gene - spv. The plasmid pRST98 was isolated, purified and then digested by nine restriction endonucleases to make the plasmid enzyme profile. Spv-specific PCR and Southern blot were applied to identify the virulence gene on pRsr98. The amplified spv fragments spvR and spvB were cloned into pGEM-T EASY and then the DNA sequences were analysed. The fragments of pRST98 digested by endonucleases Bgl II, Pst I and Sac II were identified, which may be useful for molecular analysis and further epidemiological surveillance of pRsT98. The results of PCR and Southern blot showed that spv homologous genetic sequence which had been found in all pathogenesis Salmonella spp. except S. typhi was also presented on pRST98. The ORF of spvR and spvB of pRsr98 were 894 bp and 1,776 bp, respectively. They have more than 99% homology with that of spvR and spvB on virulence plasmid in S. typhmurium. The genotype research on pRST98 revealed that there is a plasmid carrying genes responsible for drug resistance and virulence in S. typhi. This is the first report for such kind chimerical plasmid in S. typhi. Cellular & Molecular Immunology. 2005;2(2):136-140.Key Words: s. typhi, R plasmid, virulence gene

  3. The gpsX gene encoding a glycosyltransferase is important for polysaccharide production and required for full virulence in Xanthomonas citri subsp. citri

    Directory of Open Access Journals (Sweden)

    Li Jinyun

    2012-03-01

    Full Text Available Abstract Background The Gram-negative bacterium Xanthomonas citri subsp. citri (Xac causes citrus canker, one of the most destructive diseases of citrus worldwide. In our previous work, a transposon mutant of Xac strain 306 with an insertion in the XAC3110 locus was isolated in a screening that aimed at identifying genes related to biofilm formation. The XAC3110 locus was named as bdp24 for biofilm-defective phenotype and the mutant was observed to be affected in extracellular polysaccharide (EPS and lipopolysaccharide (LPS biosynthesis and cell motility. In this study, we further characterized the bdp24 (XAC3110 gene (designated as gpsX using genetic complementation assays and expanded the knowledge about the function of the gpsX gene in Xac pathogenesis by investigating the roles of gpsX in EPS and LPS production, cell motility, biofilm formation on host leaves, stress tolerance, growth in planta, and host virulence of the citrus canker bacterium. Results The gpsX gene encodes a putative glycosyltransferase, which is highly conserved in the sequenced strains of Xanthomonas. Mutation of gpsX resulted in a significant reduction of the amount of EPS and loss of two LPS bands visualized on sodium dodecylsulphate- polyacrylamide gels. Biofilm assays revealed that the gpsX mutation affected biofilm formation by Xac on abiotic and biotic surfaces. The gpsX mutant showed delayed bacterial growth and caused reduced development of disease symptoms in susceptible citrus leaves. The gpsX mutant was more sensitive than the wild-type strain to various stresses, including the H2O2 oxidative stress. The mutant also showed attenuated ability in cell motility but not in flagellar formation. Quantitative reverse transcription-PCR assays indicated that mutation of gpsX did not affect the expression of virulence genes such as pthA in Xac strain 306. The affected phenotypes of the gpsX mutant could be complemented to wild-type levels by the intact gpsX gene

  4. Diversity of CRISPR loci and virulence genes in pathogenic Escherichia coli isolates from various sources.

    Science.gov (United States)

    Jiang, Yun; Yin, Shuang; Dudley, Edward G; Cutter, Catherine N

    2015-07-01

    Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., O26, O45, O103, O111, O121, and O145) are food-borne pathogens that pose a serious health threat to humans. Ruminants, especially cattle, are a major reservoir for O157 and non-O157 STEC. In the present study, 115 E. coli strains isolated from small and very small beef processing plants were screened for virulence genes (stx1, stx2, eae) using a multiplex polymerase chain reaction (PCR). Thirteen (11.3%) of the 115 isolates tested positive for stx1, stx2, or eae genes, but only 4 (3.5%) tested positive for either stx1 or stx2. A multiplex PCR reaction targeting eight O-serogroups (O26, O45, O103, O111, O113, O121, O145, O157) identified 12 isolates as O26, O103, O111, or O145, with E. coli O26 being the most predominant serogroup (61.5%). The thirteen isolates were further analyzed using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) subtyping. Consistent with previous studies, CRISPR alleles from strains of the same serogroup were similar in their spacer content and order, regardless of the isolation source. A completely different CRISPR allele was observed in one isolate ("7-J") which exhibited a different O-serogroup (O78). Our results confirmed previous findings that CRISPR loci are conserved among phylogenetically-related strains. In addition, 8 E. coli O26 isolates and a collection of 42 E. coli O26 isolates were screened for 12 enterohemorrhagic E. coli-specific genes. Seven genes (ECs848-Hypothetical Protein, ECs2226-Hypothetical Protein, ECs3857-nleB, ECs3858-Hypothetical Protein, ECs4552-escF, ECs4553-Hypothetical Protein, and ECs4557-sepL) were found in all 50 isolates. An additional 5 genes (ECs1322-ureA urease subunit γ, ECs1323-ureB urease subunit β, ECs1326-ureF, ECs1561-Hypothetical Protein, and ECs1568-Hypothetical Protein) were found to be highly prevalent in isolates from human sources, while lower in

  5. Trichophyton tonsurans strains from Brazil: phenotypic heterogeneity, genetic homology, and detection of virulence genes.

    Science.gov (United States)

    Sidrim, José Julio Costa; Rocha, Marcos Fábio Gadelha; Leite, João Jaime Giffoni; Maranhão, Fernanda Cristina de Albuquerque; Lima, Rita Amanda Chaves; Castelo-Branco, Débora de Souza Collares Maia; Bandeira, Tereza de Jesus Pinheiro Gomes; Cordeiro, Rossana de Aguiar; Brilhante, Raimunda Sâmia Nogueira

    2013-11-01

    The objective of this study was to establish the phenotypical and molecular patterns of clinical isolates of Trichophyton tonsurans circulating in the state of Ceará, northeastern Brazil. For this purpose, 25 T. tonsurans strains isolated from independent cases of tinea capitis in children were phenotypically evaluated regarding their macro- and micro-morphological characteristics, vitamin requirements, urease production, and antifungal susceptibility. The molecular characterization was carried out with random amplified polymorphic DNA molecular markers and M13 fingerprinting. The presence of the genes CarbM14, Sub2, CER, URE, ASP, PBL, and LAC, which encode enzymes related to fungal virulence, was also evaluated. Finally, melanin production was assessed through specific staining. The data obtained demonstrated that these T. tonsurans strains have considerable phenotypical variation, although they showed a low degree of genetic polymorphism according to the markers used. The genes CarbM14, Sub2, CER, and URE were detected in all the analyzed strains. The gene LAC was also identified in all the strains, and melanin synthesis was phenotypically confirmed. The strains were susceptible to antifungals, especially itraconazole (GM = 0.06 μg/mL) and ketoconazole (GM = 0.24 μg/mL). Therefore, T. tonsurans strains can present great phenotypical heterogeneity, even in genetically similar isolates. Moreover, the presence of the LAC gene indicates the possible participation of melanin in the pathogenesis of these dermatophytes.

  6. Screening and identification of Shigella flexneri 2a virulence-related genes induced after invasion of epithelial cells

    Institute of Scientific and Technical Information of China (English)

    SHI; Zhaoxing; WANG; Hengliang; HU; Kun; FENG; Erling; YAO

    2004-01-01

    An in vivo expression technology (IVET) was applied to screen S. flexneri2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism;one encoded a transcriptional regulator; three were identified as insertion elements; three appeared to be antisense to genes involved in the transmethylation, biosynthesis, and phosphotransferase system; and three were predicted to encode polypeptides with unknown functions.Intracellular survival assays showed that the mutants of alkA, citC and wcaJ genes had lower capability of intracellular replication or survival than the wild-type strain. The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intracellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.

  7. Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; WU Chao; LIU Mei; LIU Xu-ri; Hu Guo-cheng; SI Hua-min; SUN Zong-xiu; LIU Wen-zhen; Fu Ya-ping

    2011-01-01

    Antimierobial peptide is a polypeptide with antimicrobial activity.Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp (Fenneropenaeus Chinensis) were integrated into Oryza sativa L.subsp.japonica cv.Aichi ashahi by Agrobacterium mediated transformation system.PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in T0 generation,respectively.RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation,and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants.Four Np3 and Np5 transgenic lines in T1 generation were inoculated with ×anthomonas oryzae pv.oryzae strain CR4,and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4.The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2,Zhe 173 and OS-225.It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.

  8. Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

    OpenAIRE

    Rhodes, Linda D.; Coady, Alison M; Deinhard, Rebecca K.

    2004-01-01

    Renibacterium salmoninarum, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2. We found evidence for a third msa gene, msa3, which appears to be a duplication of msa1. Unlike msa1 and msa2, msa3 is not present in all isolates of R. salmoninarum. The presence of the...

  9. Disruption of homocitrate synthase genes in Candida albicans affects growth but not virulence.

    Science.gov (United States)

    Kur, Krzysztof; Gabriel, Iwona; Morschhäuser, Joachim; Barchiesi, Francesco; Spreghini, Elisabetta; Milewski, Sławomir

    2010-12-01

    Two genes, LYS21 and LYS22, encoding isoforms of homocitrate synthase, an enzyme catalysing the first committed step in the lysine biosynthetic pathway, were disrupted in Candida albicans using the SAT1 flipper strategy. The double null lys21Δ/lys22Δ mutant lacked homocitrate synthase activity and exhibited lysine auxotrophy in minimal media that could be fully rescued by the addition of 0.5-0.6 mM L: -lysine. On the other hand, its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that of the mother, wild-type strain. These findings strongly question a possibility of exploitation of homocitrate synthase and possibly also other enzymes of the lysine biosynthetic pathway as targets in chemotherapy of disseminated fungal infections.

  10. Frequency, virulence genes and antimicrobial resistance of Listeria spp. isolated from bovine clinical mastitis.

    Science.gov (United States)

    Jamali, Hossein; Radmehr, Behrad

    2013-11-01

    The aims of this study were to determine the prevalence, characteristics and antimicrobial resistance of Listeria spp. isolated from bovine clinical mastitis in Iran. Listeria spp. were detected in 21/207 bovine mastitic milk samples from dairy farms in Iran, comprising L. monocytogenes (n=17), L. innocua (n=3) and L. ivanovii (n=1). L. monocytogenes isolates were grouped into serogroups '4b, 4d, 4e', '1/2a, 3a', '1/2b, 3b, 7' and '1/2c, 3c'; all harboured inlA, inlC and inlJ virulence genes. Listeria spp. were most frequently resistant to penicillin G (14/21 isolates, 66.7%) and tetracyclines (11/21 isolates, 52.4%).

  11. Direct Detection of Escherichia coli Virulence Genes by Real-Time PCR in Fecal Samples from Bats in Brazil.

    Science.gov (United States)

    Cabal, Adriana; Pereira, Maria J; Aguiar, Ludmilla M S; Domínguez, Lucas; Fonseca, Carlos; Álvarez, Julio; Drexler, Jan F; Gortázar, Christian

    2015-10-01

    Guano samples from 412 Brazilian bats were screened with real-time PCR for the virulence genes (eae, est, elt, stx1, stx2, ehxA, invA, bfpA, aggR) representing five intestinal pathotypes of Escherichia coli. From 82 pooled samples, 22% contained Escherichia coli DNA, and eae, est, bfpA, aggR were detected.

  12. The effect of γ radiation on the expression of the virulence genes of Salmonella typhimurium and Vibrio spp.

    Science.gov (United States)

    Lim, Sangyong; Jung, Jinwoo; Kim, Dongho

    2007-11-01

    The principle benefit of food irradiation is the reduction of food-borne bacteria in food products. However, the microbiological safety with respect to increased virulence of surviving pathogens after irradiation remains an important issue with regard to the effectiveness of food irradiation. In this study, the transcriptional changes of virulence genes of Salmonella and Vibrio spp. after γ radiation were investigated by real-time PCR (RT-PCR). Samonella typhimurium is dependent upon the products of a large number of genes located within Salmonella pathogenicity islands (SPI) on the chromosome. The expressions of seven genes including four SPI genes, hilD, ssrB, pipB, and sopD, were measured at 1 h after 1 kGy irradiation. Compared with non-irradiated controls, the expression of hilD encoded within SPI1 and sopD encoding SPI1-related effector proteins was reduced about 4- and 16-fold, respectively. The expressions of Vibrio toxin genes, vvhA, ctxA, and tdh, were also monitored during the course of a growth cycle after re-inoculation of irradiated Vibrio spp. (0.5 and 1.0 kGy). The expressions of Vibrio toxin genes tested did not increase compared with non-irradiated counterparts. Results from this study indicate that γ radiation is much more likely to reduce the virulence gene expression of surviving pathogens.

  13. The effect of {gamma} radiation on the expression of the virulence genes of Salmonella typhimurium and Vibrio spp

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Sangyong; Jung, Jinwoo [Radiation Food Science and Biotechnology Team, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 580-185 (Korea, Republic of); Kim, Dongho [Radiation Food Science and Biotechnology Team, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 580-185 (Korea, Republic of)], E-mail: fungikim@kaeri.re.kr

    2007-11-15

    The principle benefit of food irradiation is the reduction of food-borne bacteria in food products. However, the microbiological safety with respect to increased virulence of surviving pathogens after irradiation remains an important issue with regard to the effectiveness of food irradiation. In this study, the transcriptional changes of virulence genes of Salmonella and Vibrio spp. after {gamma} radiation were investigated by real-time PCR (RT-PCR). Samonella typhimurium is dependent upon the products of a large number of genes located within Salmonella pathogenicity islands (SPI) on the chromosome. The expressions of seven genes including four SPI genes, hilD, ssrB, pipB, and sopD, were measured at 1 h after 1 kGy irradiation. Compared with non-irradiated controls, the expression of hilD encoded within SPI1 and sopD encoding SPI1-related effector proteins was reduced about 4- and 16-fold, respectively. The expressions of Vibrio toxin genes, vvhA, ctxA, and tdh, were also monitored during the course of a growth cycle after re-inoculation of irradiated Vibrio spp. (0.5 and 1.0 kGy). The expressions of Vibrio toxin genes tested did not increase compared with non-irradiated counterparts. Results from this study indicate that {gamma} radiation is much more likely to reduce the virulence gene expression of surviving pathogens.

  14. Resistance to root-knot nematodes (Meloidogyne spp. in tomato: Mi gene and occurrence of virulent populations

    Directory of Open Access Journals (Sweden)

    Gökhan AYDINLI

    2015-12-01

    Full Text Available Root-knot nematodes (Meloidogyne spp. are one of the most important agricultural pests that cause serious yield losses in tomato. Resistant tomato cultivars have commonly been used in both conventional and organic agriculture. In tomato, resistance to three most prevalent species of root-knot nematode, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, is controlled by the Mi-1 gene. However, there are two major limiting factors for efficiency of Mi-1 gene: High soil temperature and occurrence of resistance-breaking (virulent populations. These populations can emerge either naturally or after repeated exposure on tomatoes with Mi-1 gene. This review summarizes information on some strategies to prevent the emergence of virulent populations and to preserve the durability of plant resistance to Mi-1 gene.

  15. Bacteriophage Resistance Mechanisms in the Fish Pathogen Flavobacterium psychrophilum: Linking Genomic Mutations to Changes in Bacterial Virulence Factors

    DEFF Research Database (Denmark)

    Castillo, Daniel; Christiansen, Rói Hammershaimb; Dalsgaard, Inger

    2015-01-01

    Flavobacterium psychrophilum is an important fish pathogen in salmonid aquaculture worldwide. Due to increased antibiotic resistance, pathogen control using bacteriophages has been explored as a possible alternative treatment. However, the effective use of bacteriophages in pathogen control...... requires overcoming the selection for phage resistance in the bacterial populations. Here, we analyzed resistance mechanisms in F. psychrophilum after phage exposure using whole-genome sequencing of the ancestral phage-sensitive strain 950106-1/1 and six phage-resistant isolates. The phage......-resistant strains had all obtained unique insertions and/or deletions and point mutations distributed among intergenic and genic regions. Mutations in genes related to cell surface properties, gliding motility, and biosynthesis of lipopolysaccharides and cell wall were found. The observed links between phage...

  16. Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

    Science.gov (United States)

    Douëllou, T; Delannoy, S; Ganet, S; Mariani-Kurkdjian, P; Fach, P; Loukiadis, E; Montel, Mc; Thevenot-Sergentet, D

    2016-09-02

    Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples.

  17. Virulence Genes and Antimicrobial Resistance Profiles of Pasteurella multocida Strains Isolated from Rabbits in Brazil

    Directory of Open Access Journals (Sweden)

    Thais Sebastiana Porfida Ferreira

    2012-01-01

    Full Text Available Pasteurella multocida is responsible for a wide range of diseases in domestic animals. In rabbits, the agent is related to nasal discharge, pneumonia, otitis media, pyometra, orchitis, abscess, and septicemia. One hundred and forty rabbits with respiratory diseases from four rabbitries in São Paulo State, Brazil were evaluated for the detection of P. multocida in their nasal cavities. A total of twenty-nine animals were positive to P. multocida isolation, and 46 strains were selected and characterized by means of biochemical tests and PCR. P. multocida strains were tested for capsular type, virulence genes, and resistance profile. A total of 45.6% (21/46 of isolates belonged to capsular type A, and 54.34% (25/46 of the isolates were untypeable. None of the strains harboured toxA or pfhA genes. The frequency of the other twenty genes tested was variable, and the data generated was used to build a dendrogram, showing the relatedness of strains, which were clustered according to origin. Resistance revealed to be more common against sulfonamides and cotrimoxazole, followed by erythromycin, penicillin, and amoxicillin.

  18. Antimicrobial resistance and virulence gene profiles in P. multocida strains isolated from cats.

    Science.gov (United States)

    Ferreira, Thais Sebastiana Porfida; Felizardo, Maria Roberta; de Gobbi, Debora Dirani Sena; Moreno, Marina; Moreno, Andrea Micke

    2015-03-01

    Cats are often described as carriers of Pasteurella multocida in their oral microbiota. This agent is thought to cause pneumonia, conjunctivitis, rhinitis, gingivostomatitis, abscess and osteonecrosis in cats. Human infection with P. multocida has been described in several cases affecting cat owners or after cat bites. In Brazil, the cat population is approximately 21 million animals and is increasing, but there are no studies of the presence of P. multocida in the feline population or of human cases of infection associated with cats. In this study, one hundred and ninety-one healthy cats from owners and shelters in São Paulo State, Brazil, were evaluated for the presence of P. multocida in their oral cavities. Twenty animals were positive for P. multocida , and forty-one strains were selected and characterized by means of biochemical tests and PCR. The P. multocida strains were tested for capsular type, virulence genes and resistance profile. A total of 75.6% (31/41) of isolates belonged to capsular type A, and 24.4% (10/41) of the isolates were untypeable. None of the strains harboured toxA, tbpA or pfhA genes. The frequencies of the other genes tested were variable, and the data generated were used to build a dendrogram showing the relatedness of strains, which were clustered according to origin. The most common resistance profile observed was against sulfizoxazole and trimethoprim-sulphamethoxazole.

  19. Antimicrobial resistance and virulence gene profiles in P. multocida strains isolated from cats

    Directory of Open Access Journals (Sweden)

    Thais Sebastiana Porfida Ferreira

    2015-03-01

    Full Text Available Cats are often described as carriers of Pasteurella multocida in their oral microbiota. This agent is thought to cause pneumonia, conjunctivitis, rhinitis, gingivostomatitis, abscess and osteonecrosis in cats. Human infection with P. multocida has been described in several cases affecting cat owners or after cat bites. In Brazil, the cat population is approximately 21 million animals and is increasing, but there are no studies of the presence of P. multocida in the feline population or of human cases of infection associated with cats. In this study, one hundred and ninety-one healthy cats from owners and shelters in São Paulo State, Brazil, were evaluated for the presence of P. multocida in their oral cavities. Twenty animals were positive for P. multocida, and forty-one strains were selected and characterized by means of biochemical tests and PCR. The P. multocida strains were tested for capsular type, virulence genes and resistance profile. A total of 75.6% (31/41 of isolates belonged to capsular type A, and 24.4% (10/41 of the isolates were untypeable. None of the strains harboured toxA, tbpA or pfhA genes. The frequencies of the other genes tested were variable, and the data generated were used to build a dendrogram showing the relatedness of strains, which were clustered according to origin. The most common resistance profile observed was against sulfizoxazole and trimethoprim-sulphamethoxazole.

  20. Prevalence, virulence gene distribution and genetic diversity of Arcobacter in food samples in Germany.

    Science.gov (United States)

    Lehmann, Daniel; Alter, Thomas; Lehmann, Laura; Uherkova, Simona; Seidler, Tassilo; Gölz, Greta

    2015-01-01

    This study was carried out to determine the prevalence of Arcobacter spp. in food samples in Germany. In addition, the presence of putative virulence genes and the genetic diversity was tested for Arcobacter (A.) butzleri strains isolated during this study. The prevalence of Arcobacter spp. was 34% in fish meat, 26.8% in poultry meat and 2% in minced meat (beef and pork). All investigated A. butzleri isolates carried the genes cadF, ciaB, cj1349, mviN and pldA. The gene tlyA was detectable in 97.5% of the strains. Lower detection rates were observed for hecA (47.5%), hecB (45%), iroE (40%) and irgA (35%). Genotyping by ERIC-PCR demonstrated a high genetic diversity of A. butzleri strains from different foods. In conclusion, this study shows that about one third of fish meat and poultry meat samples contained Arcobacter spp. These data highlight the need to strengthen our effort to elucidate the importance ofArcobacter on veterinary public health.

  1. 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.

  2. Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data

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    Tintle Nathan L

    2012-08-01

    Full Text Available Abstract Background Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. Results We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Conclusions Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

  3. Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada.

    Science.gov (United States)

    Aslam, Mueen; Diarra, Moussa S; Checkley, Sylvia; Bohaychuk, Valerie; Masson, Luke

    2012-06-01

    The objective of this study was to characterize antimicrobial resistance (AMR) and virulence genotypes of Enterococcus spp. particularly Enterococcus faecalis isolated from retail meats purchased (2007-2008) in Alberta, Canada. Unconditional statistical associations between AMR pheno- and genotypes and virulence genotypes were determined. A total of 532 enterococci comprising one isolate from each positive sample were analyzed for antimicrobial susceptibility. A customized enterococcal microarray was used for species identification and the detection of AMR and virulence genes. E. faecalis was found in >94% of poultry samples and in about 73% of beef and 86% of pork samples. Enterococcus faecium was not found in turkey meat and its prevalence was 2% in beef and pork and 4% in chicken samples. None of the enterococci isolates were resistant to the clinically important drugs ciprofloxacin, daptomycin, linezolid and vancomycin. Multiresistance (≥3 antimicrobials) was more common in E. faecalis (91%) isolated from chicken and turkey (91%) than those isolated from beef (14%) or pork (45%). Resistance to aminoglycosides was also noted at varying degrees. The most common resistance genes found in E. faecalis were aminoglycosides (aac, aphA3, aadE, sat4, aadA), macrolides (ermB, ermA), tetracyclines (tetM, tetL, tetO), streptogramin (vatE), bacitracin (bcrR) and lincosamide (linB). Virulence genes expressing aggregation substances (agg) and cytolysin (cylA, cylB, cylL, cylM) were found more frequently in poultry E. faecalis and were unconditionally associated with tetM, linB and bcrR resistance genes. Other virulence genes coding for adhesion (ace, efaAfs), gelatinase (gelE) were also found in the majority of E. faecalis. Significant statistical associations were found between resistance and virulence genotypes, suggesting their possible physical link on a common genetic element. This study underscores the importance of E. faecalis as a reservoir of resistance and

  4. Genotypes, Virulence Factors and Antimicrobial Resistance Genes of Staphylococcus aureus Isolated in Bovine Subclinical Mastitis from Eastern China

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    Javed Memon§, Yongchun Yang§, Jam Kashifa, Muhammad Yaqoob, Rehana Buriroa, Jamila Soomroa, Wang Liping and Fan Hongjie*

    2013-11-01

    Full Text Available This study was carried out to determine the genotypes, virulence factors and antimicrobial resistance traits of 34 Staphylococcus aureus isolated from subclinical mastitis in Eastern China. Minimal inhibitory concentration (MIC results showed resistance to erythromycin in all isolates. A high frequency of Methicillin resistant S. aureus (MRSA; 29% was observed and these isolates were also highly resistant to penicillin, oxacillin, oxytetracycline and chloramphenicol than methicillin sensitive S. aureus (MSSA isolates. Thirteen pathogenic factors and seven resistance genes including mecA and blaZ gene were checked through PCR. The spaX gene was found in all isolates, whereas cna, spaIg, nuc, clfA, fnbpB, hlA, hlB and seA were present in 35, 79, 85, 59, 35, 85, 71 and 38% isolates, respectively. Nine isolates carried a group of 8 different virulence genes. Moreover, macrolide resistance genes ermB and ermC were present in all isolates. High resistance rate against methicillin was found but no isolate was positive for mecA gene, whereas blaZ and tetK were detected in 82 and 56% isolates, respectively. Genes; fnbpA, seB, seC, seD, dfrK and tetM were not found in any isolate. The statistical association between phenotypic resistance and virulence genes showed, clfA, fnbpB, hlB and seA, were potentially associated with penicillin G, ciprofloxacin, methicillin, chloramphenicol, trimethoprim and oxytetracycline resistance (P≤0.05. REP-PCR based genotyping showed seven distinct genotypes (A-G prevalent in this region. This study reports the presence of multidrug resistant S. aureus in sub-clinical mastitis which were also highly virulent that could be a major obstacle in the treatment of mastitis in this region of China.

  5. The co-evolved Helicobacter pylori and gastric cancer: trinity of bacterial virulence, host susceptibility and lifestyle

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    Devi S Manjulata

    2007-01-01

    Full Text Available Abstract Helicobacter pylori is an important yet unproven etiological agent of gastric cancer. H. pylori infection is more prevalent in developing Asian countries like India and it is usually acquired at an early age. It has been two decades since Marshall and Warren (1984 first described curved bacilli in the stomach of ulcer and gastritis patients. This discovery has won them the Nobel Prize recently, but the debate whether H. pylori is a pathogen or a commensal organism is still hot. Associations with disease-specific factors remain illusive years after the genome sequences were made available. Cytotoxin-associated antigen A (CagA and the so-called plasticity region cluster genes are implicated in pathogenesis of the carcinoma of stomach. Another virulence factor VacA whose role is still debatable, has recently been projected in pathology of gastric cancer. Studies of the evolution through genetic variation in H. pylori populations have provided a window into the history of human population migrations and a possible co-evolution of this pathogen with its human host. Possible symbiotic relationships were seriously debated since the discovery of this pathogen. The debate has been further intensified as some studies proposed H. pylori infection to be beneficial in some humans. In this commentary, we attempt to briefly discuss about H. pylori as a human pathogen, and some of the important issues linked to its pathophysiology in different hosts. 'We dance around in a ring and suppose, the secret sits in the middle and knows' – Robert Frost

  6. Significance of tagI and mfd genes in the virulence of non-typeable Haemophilus influenzae.

    Science.gov (United States)

    Spricigo, Denis A; Cortés, Pilar; Moranta, David; Barbé, Jordi; Bengoechea, José Antonio; Lagostera, Montserrat

    2014-09-01

    Non-typeable Haemophilus influenzae (NTHi) is an opportunist pathogen well adapted to the human upper respiratory tract and responsible for many respiratory diseases. In the human airway, NTHi is exposed to pollutants, such as alkylating agents, that damage its DNA. In this study, we examined the significance of genes involved in the repair of DNA alkylation damage in NTHi virulence. Two knockout mutants, tagI and mfd, encoding N³-methyladenine-DNA glycosylase I and the key protein involved in transcription-coupled repair, respectively, were constructed and their virulence in a BALB/c mice model was examined. This work shows that N³-methyladenine-DNA glycosylase I is constitutively expressed in NTHi and that it is relevant for its virulence.

  7. Francisella tularensis subsp. tularensis induces a unique pulmonary inflammatory response: role of bacterial gene expression in temporal regulation of host defense responses.

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    Kathie-Anne Walters

    Full Text Available Pulmonary exposure to Francisella tularensis is associated with severe lung pathology and a high mortality rate. The lack of induction of classical inflammatory mediators, including IL1-β and TNF-α, during early infection has led to the suggestion that F. tularensis evades detection by host innate immune surveillance and/or actively suppresses inflammation. To gain more insight into the host response to Francisella infection during the acute stage, transcriptomic analysis was performed on lung tissue from mice exposed to virulent (Francisella tularensis ssp tularensis SchuS4. Despite an extensive transcriptional response in the lungs of animals as early as 4 hrs post-exposure, Francisella tularensis was associated with an almost complete lack of induction of immune-related genes during the initial 24 hrs post-exposure. This broad subversion of innate immune responses was particularly evident when compared to the pulmonary inflammatory response induced by other lethal (Yersinia pestis and non-lethal (Legionella pneumophila, Pseudomonas aeruginosa pulmonary infections. However, the unique induction of a subset of inflammation-related genes suggests a role for dysregulation of lymphocyte function and anti-inflammatory pathways in the extreme virulence of Francisella. Subsequent activation of a classical inflammatory response 48 hrs post-exposure was associated with altered abundance of Francisella-specific transcripts, including those associated with bacterial surface components. In summary, virulent Francisella induces a unique pulmonary inflammatory response characterized by temporal regulation of innate immune pathways correlating with altered bacterial gene expression patterns. This study represents the first simultaneous measurement of both host and Francisella transcriptome changes that occur during in vivo infection and identifies potential bacterial virulence factors responsible for regulation of host inflammatory pathways.

  8. Population structure and distribution of virulence-related genes of Bacteroides fragilis isolates from Korea and Japan.

    Science.gov (United States)

    Ko, Kwan Soo; Kuwahara, Tomomi; Lee, Kyungwon; Kook, Yoon-Hoh

    2009-07-01

    Sequences for rpoB, gyrB, pdiA, and ompA were determined from 63 Bacteroides fragilis isolates, which were from Korea and Japan and include 4 reference strains. All 4 gene sequences supported clear separation of the cfi(+) group from the cfi(-) group. Combined sequences of the 60 division I isolates (cfi(-)) produced 45 different clones. Apparent discordance of gene trees, index of association, maximum likelihood test, and homoplasy ratio all supported a high frequency of recombination. There was no association between the presence of virulence-related genes and phylogenetic clustering in any gene tree.

  9. Isolation of UmRrm75, a gene involved in dimorphism and virulence of Ustilago maydis.

    Science.gov (United States)

    Rodríguez-Kessler, Margarita; Baeza-Montañez, Lourdes; García-Pedrajas, María D; Tapia-Moreno, Alejandro; Gold, Scott; Jiménez-Bremont, Juan F; Ruiz-Herrera, José

    2012-05-20

    Ustilago maydis displays dimorphic growth, alternating between a saprophytic haploid yeast form and a filamentous dikaryon, generated by mating of haploid cells and which is an obligate parasite. Induction of the dimorphic transition of haploid strains in vitro by change in ambient pH has been used to understand the mechanisms governing this differentiation process. In this study we used suppression subtractive hybridization to generate a cDNA library of U. maydis genes up-regulated in the filamentous form induced in vitro at acid pH. Expression analysis using quantitative RT-PCR showed that the induction of two unigenes identified in this library coincided with the establishment of filamentous growth in the acid pH medium. This expression pattern suggested that they were specifically associated to hyphal development rather than merely acid pH-induced genes. One of these genes, UmRrm75, encodes a protein containing three RNA recognition motifs and glycine-rich repeats and was selected for further study. The UmRrm75 gene contains 4 introns, and produces a splicing variant by a 3'-alternative splicing site within the third exon. Mutants deleted for UmRrm75 showed a slower growth rate than wild type strains in liquid and solid media, and their colonies showed a donut-like morphology on solid medium. Interestingly, although ΔUmRrm75 strains were not affected in filamentous growth induced by acid pH and oleic acid, they exhibited reduced mating, post-mating filamentous growth and virulence. Our data suggest that UmRrm75 is probably involved in cell growth, morphogenesis, and pathogenicity in U. maydis.

  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. Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228).

    Science.gov (United States)

    Zhang, Yue-Qing; Ren, Shuang-Xi; Li, Hua-Lin; Wang, Yong-Xiang; Fu, Gang; Yang, Jian; Qin, Zhi-Qiang; Miao, You-Gang; Wang, Wen-Yi; Chen, Run-Sheng; Shen, Yan; Chen, Zhu; Yuan, Zheng-Hong; Zhao, Guo-Ping; Qu, Di; Danchin, Antoine; Wen, Yu-Mei

    2003-09-01

    Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non-pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from delta-haemolysin and beta-haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.

  12. The majority of genes in the pathogenic Neisseria species are present in non-pathogenic Neisseria lactamica, including those designated as 'virulence genes'

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    Saunders Nigel J

    2006-05-01

    Full Text Available Abstract Background Neisseria meningitidis causes the life-threatening diseases meningococcal meningitis and meningococcal septicemia. Neisseria gonorrhoeae is closely related to the meningococcus, but is the cause of the very different infection, gonorrhea. A number of genes have been implicated in the virulence of these related yet distinct pathogens, but the genes that define and differentiate the species and their behaviours have not been established. Further, a related species, Neisseria lactamica is not associated with either type of infection in normally healthy people, and lives as a harmless commensal. We have determined which of the genes so far identified in the genome sequences of the pathogens are also present in this non-pathogenic related species. Results Thirteen unrelated strains of N. lactamica were investigated using comparative genome hybridization to the pan-Neisseria microarray-v2, which contains 2845 unique gene probes. The presence of 127 'virulence genes' was specifically addressed; of these 85 are present in N. lactamica. Of the remaining 42 'virulence genes' only 11 are present in all four of the sequenced pathogenic Neisseria. Conclusion Assessment of the complete dataset revealed that the vast majority of genes present in the pathogens are also present in N. lactamica. Of the 1,473 probes to genes shared by all four pathogenic genome sequences, 1,373 hybridize to N. lactamica. These shared genes cannot include genes that are necessary and sufficient for the virulence of the pathogens, since N. lactamica does not share this behaviour. This provides an essential context for the interpretation of gene complement studies of the pathogens.

  13. Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

    Institute of Scientific and Technical Information of China (English)

    WANG Wei-xia; LAI Feng-xiang; LI Kai-long; FU Qiang

    2014-01-01

    The brown planthopperNilaparvata lugens Stål (Homoptera: Delphacidae) can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but severalN. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels inN. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes inN. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated fromN. lugens, including two different treatments (resistant or susceptible rice) and three virulentN. lugens populations. Three software programs (BestKeeper, Normfinder and geNorm) were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes18S,β-ACT,β-TUB andα-TUB as the most stable reference genes. BestKeeper identifiedETIF1 as the optimal reference gene with the least overall variation, whereas18S andα-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes18S andα-TUB were the most suitable reference genes inN. lugens. These results will facilitate future transcript profiling studies onN. lugens populations that show variation in virulence levels on different rice varieties.

  14. Occurrence of six virulence-associated genes in Arcobacter species isolated from various sources in Shiraz, Southern Iran.

    Science.gov (United States)

    Tabatabaei, Mohammad; Shirzad Aski, Hesamaddin; Shayegh, Hossein; Khoshbakht, Rahem

    2014-01-01

    In humans, arcobacters are associated with watery diarrhea and septicemia. Although, recently, more cases of diarrhea have been caused by Arcobacter species, very little is known about its pathogenesis. Therefore, the aim of this study was to evaluate the presence of six putative Arcobacter virulence genes (cadF, ciaB, cj1349, mviN, pldA, and tlyA), in a set of 113 Arcobacter butzleri, 40 Arcobacter cryaerophilus, and 15 Arcobacter skirrowii isolates that were recovered from various origins. The isolates were confirmed on the basis of polymerase chain reaction (PCR) amplification of genus and species specific PCR for determining three species. For confirmed isolates, PCR was carried out for the presence of virulence genes using specific primers. All A. butzleri isolates carried all six genes. For A. cryaerophilus and A. skirrowii, the cadF gene was detected just in 55 and 53.3%, ciaB in 97.5 and 86.6%, cj1349 in 45 and 60%, mviN in 90 and 80%, pldA in 32.5 and 13.3%, and tlyA in 37.5 and 40%, respectively. For A. cryaerophilus and A. skirrowii, the genes ciaB and mviN were significantly more prevalent than other virulence markers (P ≤ 0.05). The findings revealed that many of the important Arcobacter strains (86%) have these putative virulence genes which can be potential pathogenic properties for humans.

  15. Association between the agr locus and the presence of virulence genes and pathogenesis in Staphylococcus aureus using a Caenorhabditis elegans model

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    Terissa A. Thompson

    2017-01-01

    Conclusions: There was a strong association between the carriage of virulence genes and the presence of the agr operon in clinical strains of S. aureus. Further, agr-positive strains were more pathogenic than agr-negative strains, suggesting a correlation between the presence of agr, carriage of virulence determinants, and pathogenicity.

  16. Performance of resistance gene pyramids to races of rice bacterial blight in Zhejiang Province

    Institute of Scientific and Technical Information of China (English)

    ZHENGKangle; ZHUANGJieyun; WANGHanrong

    1998-01-01

    The effect of gene pyramiding on resistance to bacterial blight (BB) in rice was evahlated among the IR24-based near isogenic lines conraining single resistance gene and gene pyramids containing two, three or lour resistancegenes (see table).

  17. Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus

    Science.gov (United States)

    Zhou, Lifeng; Chen, Fengmao; Pan, Hongyang; Ye, Jianren; Dong, Xuejiao; Li, Chunyan; Lin, Fengling

    2016-01-01

    Bursaphelenchus mucronatus (B. mucronatus) isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while β-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus’ pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future. PMID:27618012

  18. Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus

    Directory of Open Access Journals (Sweden)

    Lifeng Zhou

    2016-09-01

    Full Text Available Bursaphelenchus mucronatus (B. mucronatus isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while β-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus’ pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future.

  19. Molecular Analysis and Identification of Virulence Gene on pRST98 from Multi-Drug Resistant Salmonella typhi

    Institute of Scientific and Technical Information of China (English)

    Rui Huang; Shuyan Wu; Xueguang Zhang; Yanyun Zhang

    2005-01-01

    pRST98 is a large and conjugative resistant plasmid (R plasmid) of 98.6 mega-dalton from multi-drug resistant Salmonella typhi (S.typhi), which was classified to incompatibility group C (Inc C). It has been found that pRST98 made its host bacteria not only antibiotic resistant but also more virulent. In this study we explored the possibility of plasmid pRST98 in S. typhi carrying the Salmonella plasmid virulence gene -spv. The plasmid pRST98 was isolated,purified and then digested by nine restriction endonucleases to make the plasmid enzyme profile. Spv-specific PCR and Southern blot were applied to identify the virulence gene on pRST98. The amplified spv fragments spvR and spvB were cloned into pGEM-T EASY and then the DNA sequences were analysed. The fragments of pRST98 digested by endonucleases Bgl Ⅱ, Pst Ⅰ and Sac Ⅱ were identified, which may be useful for molecular analysis and further epidemiological surveillance of pRST98. The results of PCR and Southern blot showed that spv homologous genetic sequence which had been found in all pathogenesis Salmonella spp. except S. typhi was also presented on pRST98. The ORF of spvR and spvB of pRST98 were 894 bp and 1,776 bp, respectively. They have more than 99% homology with that of spvR and spvB on virulence plasmid in S. typhmurium. The genotype research on pRST98 revealed that there is a plasmid carrying genes responsible for drug resistance and virulence in S. typhi. This is the first report for such kind chimerical plasmid in S. typhi. Cellular & Molecular Immunology. 2005;2(2):136-140.

  20. Expression of virulence-related genes in Listeria monocytogenes grown on Danish hard cheese as affected by NaCl content

    DEFF Research Database (Denmark)

    Larsen, Nadja; Jespersen, Lene

    2015-01-01

    Expression of virulence-related genes in Listeria monocytogenes incubated on cheese was assessed by real-time quantitative polymerase chain reaction. The objective of the study was to investigate the impact of sodium chloride concentration in cheese on transcription of virulence genes and, thereby......, virulence potential of L. monocytogenes. The expression studies were performed with L. monocytogenes strains characterized by different tolerance to salt stress. Strains ATCC(®) 51779 and DSMZ 15675 were incubated on the Danish hard-cheese type Samsoe, with low (high (3.6% [wt....../wt]) content of NaCl. Genes differentially expressed (p

  1. Neither gastric topological distribution nor principle virulence genes of Helicobacterpylori contributes to clinical outcomes

    Institute of Scientific and Technical Information of China (English)

    Yan Wing Ho; Khek Yu Ho; Felipe Ascencio; Bow Ho

    2004-01-01

    AIM: Studies on Helicobacter pylori(Hpylori) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determine if the gastric topology and genotypic distribution of H pylori were associated with different upper gastrointestinal pathologies in a multiethnic Asian population.METHODS: Gastric biopsies from the cardia, body/corpus and antrum were endoscoped from a total of 155 patients with dyspepsia and/or reflux symptoms, with informed consent. H pylori isolates obtained were tested for the presence of 26kDa, ureC, cagA, vacA, iceA1, iceA2 and babA2 genes using PCR while DNA fingerprints were generated using random amplification polymorphic DNA(RAPD).RESULTS: H pyloriwas present in 51/155 (33%) of patients studied. Of these, 16, 15 and 20 were isolated from patients with peptic ulcer diseases, gastroesophageal reflux diseases and non-ulcer dyspepsia, respectively. Of the H pylori positive patients, 75% (38/51) had H pylori in all three gastric sites.The prevalence of various genes in the H pylori isolates was shown to be similar irrespective of their colonization sites as well as among the same site of different patients.The RAPD profiles of H pylori isolates from different gastric sites were highly similar among intra-patients but varied greatly between different patients.CONCLUSION: Topographic colonization of H pylori and the virulence genes harboured by these isolates have no direct bearing to the clinical state of the patients. In multiethnic Singapore, the stomach of each patient is colonized by a predominant strain of H pylori, irrespective of the clinical diagnosis.

  2. Replication fitness determines high virulence of influenza A virus in mice carrying functional Mx1 resistance gene.

    Science.gov (United States)

    Grimm, Daniel; Staeheli, Peter; Hufbauer, Martin; Koerner, Iris; Martínez-Sobrido, Luis; Solórzano, Alicia; García-Sastre, Adolfo; Haller, Otto; Kochs, Georg

    2007-04-17

    The IFN-induced resistance factor Mx1 is a critical component of innate immunity against influenza A viruses (FLUAV) in mice. Animals carrying a wild-type Mx1 gene (Mx1(+/+)) differ from regular laboratory mice (Mx1(-/-)) in that they are highly resistant to infection with standard FLUAV strains. We identified an extraordinary variant of the FLUAV strain, A/PR/8/34 (H1N1) (designated hvPR8), which is unusually virulent in Mx1(+/+) mice. hvPR8 was well controlled in Mx1(+/+) but not Mx1(-/-) mice provided that the animals were treated with IFN before infection, indicating that hvPR8 exhibits normal sensitivity to growth restriction by Mx1. hvPR8 multiplied much faster than standard PR8 early in infection because of highly efficient viral gene expression in infected cells. Studies with reassortant viruses containing defined genome segments of both hvPR8 and standard PR8 demonstrated that the HA, neuraminidase, and polymerase genes of hvPR8 all contributed to virulence, indicating that efficient host cell entry and early gene expression renders hvPR8 highly pathogenic. These results reveal a surprisingly simple concept of how influenza viruses may gain virulence and illustrate that high speed of virus growth can outcompete the antiviral response of the infected host.

  3. Corynebacterium diphtheriae as an emerging pathogen in nephrostomy catheter-related infection: evaluation of traits associated with bacterial virulence.

    Science.gov (United States)

    Gomes, Débora L R; Martins, Carlos A S; Faria, Lúcia M D; Santos, Louisy S; Santos, Cintia S; Sabbadini, Priscila S; Souza, Mônica C; Alves, Gabriela B; Rosa, Ana C P; Nagao, Prescilla E; Pereira, Gabriela A; Hirata, Raphael; Mattos-Guaraldi, Ana L

    2009-11-01

    Corynebacterium diphtheriae still represents a global medical challenge, particularly due to the significant number of individuals susceptible to diphtheria and the emergence of non-toxigenic strains as the causative agents of invasive infections. In this study, we characterized the clinical and microbiological features of what we believe to be the first case of C. diphtheriae infection of a percutaneous nephrostomy catheter insertion site in an elderly patient with a fatal bladder cancer. Moreover, we demonstrated the potential role of adherence, biofilm formation and fibrin deposition traits in C. diphtheriae from the catheter-related infection. Non-toxigenic C. diphtheriae isolated from the purulent discharge (named strain BR-CAT5003748) was identified by the API Coryne system (code 1 010 324) and a multiplex PCR for detection of dtxR and tox genes. Strain BR-CAT5003748 showed resistance to oxacillin, ceftazidime and ciprofloxacin. In experiments performed in vitro, the catheter isolate was classified as moderately hydrophobic and as moderately adherent to polystyrene surfaces. Glass provided a more effective surface for biofilm formation than polystyrene. Micro-organisms adhered to (>1.5 x 10(6) c.f.u.) and multiplied on surfaces of polyurethane catheters. Microcolony formation (a hallmark of biofilm formation) and amorphous accretions were observed by scanning electron microscopy on both external and luminal catheter surfaces. Micro-organisms yielded simultaneous expression of localized adherence-like and aggregative-like (LAL/AAL) adherence patterns to HEp-2 cells. Interestingly, the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain. In conclusion, C. diphtheriae should be recognized as a potential cause of catheter-related infections in at-risk populations such as elderly and cancer patients. LAL/AAL strains may be associated with virulence traits that enable C

  4. Transport of magnesium by a bacterial Nramp-related gene.

    Directory of Open Access Journals (Sweden)

    Jung-Ho Shin

    2014-06-01

    Full Text Available Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5-2.0 mM. Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.

  5. The adenylate cyclase gene MaAC is required for virulence and multi-stress tolerance of Metarhizium acridum

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    Liu Shuyang

    2012-08-01

    Full Text Available Abstract Background The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors. Results Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium, Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type. Conclusion MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever.

  6. Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

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    Charles Darkoh

    2016-08-01

    Full Text Available Clostridium difficile infection (CDI is responsible for most of the definable cases of antibiotic- and hospital-associated diarrhea worldwide and is a frequent cause of morbidity and mortality in older patients. C. difficile, a multidrug-resistant anaerobic pathogen, causes disease by producing toxins A and B, which are controlled by an accessory gene regulator (Agr quorum signaling system. Some C. difficile strains encode two Agr loci in their genomes, designated agr1 and agr2. The agr1 locus is present in all of the C. difficile strains sequenced to date, whereas the agr2 locus is present in a few strains. The functional roles of agr1 and agr2 in C. difficile toxin regulation and pathogenesis were unknown until now. Using allelic exchange, we deleted components of both agr loci and examined the mutants for toxin production and virulence. The results showed that the agr1 mutant cannot produce toxins A and B; toxin production can be restored by complementation with wild-type agr1. Furthermore, the agr1 mutant is able to colonize but unable to cause disease in a murine CDI model. These findings have profound implications for CDI treatment because we have uncovered a promising therapeutic target for the development of nonantibiotic drugs to treat this life-threatening emerging pathogen by targeting the toxins directly responsible for disease.

  7. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.

    Science.gov (United States)

    Ikebe, Tadayoshi; Ato, Manabu; Matsumura, Takayuki; Hasegawa, Hideki; Sata, Tetsutaro; Kobayashi, Kazuo; Watanabe, Haruo

    2010-04-01

    Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates) and non-invasive infections (59 isolates), 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%). The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

  8. Bacteriophages of Staphylococcus aureus efficiently package various bacterial genes and mobile genetic elements including SCCmec with different frequencies.

    Science.gov (United States)

    Mašlaňová, Ivana; Doškař, Jiří; Varga, Marian; Kuntová, Lucie; Mužík, Jan; Malúšková, Denisa; Růžičková, Vladislava; Pantůček, Roman

    2013-02-01

    Staphylococcus aureus is a serious human and veterinary pathogen in which new strains with increasing virulence and antimicrobial resistance occur due to acquiring new genes by horizontal transfer. It is generally accepted that temperate bacteriophages play a major role in gene transfer. In this study, we proved the presence of various bacterial genes of the S. aureus COL strain directly within the phage particles via qPCR and quantified their packaging frequency. Non-parametric statistical analysis showed that transducing bacteriophages φ11, φ80 and φ80α of serogroup B, in contrast to serogroup A bacteriophage φ81, efficiently package selected chromosomal genes localized in 4 various loci of the chromosome and 8 genes carried on variable elements, such as staphylococcal cassette chromosome SCCmec, staphylococcal pathogenicity island SaPI1, genomic islands vSaα and vSaβ, and plasmids with various frequency. Bacterial gene copy number per ng of DNA isolated from phage particles ranged between 1.05 × 10(2) for the tetK plasmid gene and 3.86 × 10(5) for the SaPI1 integrase gene. The new and crucial finding that serogroup B bacteriophages can package concurrently ccrA1 (1.16 × 10(4)) and mecA (1.26 × 10(4)) located at SCCmec type I into their capsids indicates that generalized transduction plays an important role in the evolution and emergence of new methicillin-resistant clones.

  9. The alternative sigma factor B modulates virulence gene expression in a murine Staphylococcus aureus infection model but does not influence kidney gene expression pattern of the host.

    Science.gov (United States)

    Depke, Maren; Burian, Marc; Schäfer, Tina; Bröker, Barbara M; Ohlsen, Knut; Völker, Uwe

    2012-01-01

    Infections caused by Staphylococcus aureus are associated with significant morbidity and mortality and are an increasing threat not only in hospital settings. The expression of the staphylococcal virulence factor repertoire is known to be affected by the alternative sigma factor B (SigB). However, its impact during infection still is a matter of debate. Kidney tissues of controls or mice infected with S. aureus HG001 or its isogenic sigB mutant were analyzed by transcriptome profiling to monitor the host response, and additionally expression of selected S. aureus genes was monitored by RT-qPCR. Direct transcript analysis by RT-qPCR revealed significant SigB activity in all mice infected with the wild-type strain, but not in its isogenic sigB mutant (p<0.0001). Despite a clear-cut difference in the SigB-dependent transcription pattern of virulence genes (clfA, aur, and hla), the host reaction to infection (either wild type or sigB mutant) was almost identical. Despite its significant activity in vivo, loss of SigB did neither have an effect on the outcome of infection nor on murine kidney gene expression pattern. Thus, these data support the role of SigB as virulence modulator rather than being a virulence determinant by itself.

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

    Directory of Open Access Journals (Sweden)

    Ping Jihui

    2011-01-01

    Full Text Available Abstract Background To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV, A/Hong Kong/1/68(H3N2 (HK-wt, was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans. Results To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells and in vivo (BALB/c mouse lungs as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung

  11. A destabilized bacterial luciferase for dynamic gene expression studies

    Science.gov (United States)

    Allen, Michael S.; Wilgus, John R.; Chewning, Christopher S.; Sayler, Gary S.

    2006-01-01

    Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression. PMID:19003433

  12. The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes.

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    Alberto Elías-Villalobos

    2015-08-01

    Full Text Available Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis.

  13. The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes.

    Science.gov (United States)

    Elías-Villalobos, Alberto; Fernández-Álvarez, Alfonso; Moreno-Sánchez, Ismael; Helmlinger, Dominique; Ibeas, José I

    2015-08-01

    Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs) play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis.

  14. Transcriptomic analysis of the GCN5 gene reveals mechanisms of the epigenetic regulation of virulence and morphogenesis in Ustilago maydis.

    Science.gov (United States)

    Martínez-Soto, Domingo; González-Prieto, Juan Manuel; Ruiz-Herrera, José

    2015-09-01

    Chromatin in the eukaryotic nucleus is highly organized in the form of nucleosomes where histones wrap DNA. This structure may be altered by some chemical modifications of histones, one of them, acetylation by histone acetyltransferases (HATs) that originates relaxation of the nucleosome structure, providing access to different transcription factors and other effectors. In this way, HATs regulate cellular processes including DNA replication, and gene transcription. Previously, we isolated Ustilago maydis mutants deficient in the GCN5 HAT that are avirulent, and grow constitutively as mycelium. In this work, we proceeded to identify the genes differentially regulated by GCN5, comparing the transcriptomes of the mutant and the wild type using microarrays, to analyse the epigenetic control of virulence and morphogenesis. We identified 1203 genes, 574 positively and 629 negatively regulated in the wild type. We found that genes belonging to different categories involved in pathogenesis were downregulated in the mutant, and that genes involved in mycelial growth were negatively regulated in the wild type, offering a working hypothesis on the epigenetic control of virulence and morphogenesis of U. maydis. Interestingly, several differentially regulated genes appeared in clusters, suggesting a common regulation. Some of these belonged to pathogenesis or secondary metabolism.

  15. Mutation of a novel virulence-related gene mltD in Vibrio anguillarum enhances lethality in zebra fish.

    Science.gov (United States)

    Xu, Zinan; Wang, Ying; Han, Yin; Chen, Jixiang; Zhang, Xiao-Hua

    2011-01-01

    Vibrio anguillarum, a halophilic Gram-negative bacterium, is the causative agent of vibriosis, which is a major problem for the aquaculture industry worldwide. Previously, a virulence-related gene fragment of V. anguillarum was obtained from a suppression subtractive hybridization (SSH) library. In this study, the complete gene sequence was obtained by long and accurate PCR (LA-PCR). After sequence analysis and homologous comparison, this new virulence-related gene was revealed to encode a putative membrane-bound lytic murein transglycosylase D (MltD), which consisted of 547 amino acids, and showed 34% identity to the MltD in Escherichia coli. An mltD mutant of pathogenic V. anguillarum CW-1 was constructed by homologous recombination. Production of extracellular gelatinase and protease of the mltD mutant decreased markedly compared with those of the wild-type strain, and the hemolytic activity was totally lost. Sodium chloride challenge and antibiotic sensitivity assay showed that the resistance of the mltD mutant to high concentrations of sodium chloride, and rocephin, fortun, cefobid, gentamicin, kanamycin and carbenicillin was enhanced. Most importantly, virulence of the mltD mutant was enhanced compared with that of the wild type when it was inoculated intraperitoneally into zebra fish; the LD₅₀ of the wild type and the mutant was 3.92 × 10³ CFU and 1.01 × 10² CFU fish⁻¹, respectively. The mltD was cloned and overexpressed in E. coli, and the recombinant MltD protein showed hemolytic, phospholipase, gelatinase and diastase activities. This is the first report that MltD possibly has a virulence-related function.

  16. cDNA Array Analysis of the Differential Expression Change in Virulence-related Genes During the Development of Resistance in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    Zheng XU; Yuan-Ying JIANG; Yong-Bing CAO; Jun-Dong ZHANG; Ying-Ying CAO; Ping-Hui GAO; De-Jun WANG; Xu-Ping FU; Kang YING; Wan-Sheng CHEN

    2005-01-01

    Candida albicans is the most frequently isolated fungus in immunocompromised patients associated with mucosal and deep-tissue infections. To investigate the correlation between virulence and resistance on a gene expression profile in C. albicans, we examined the changes in virulence-related genes during the development of resistance in C. albicans from bone marrow transplant patients using a constructed cDNA array representing 3096 unigenes. In addition to the genes known to be associated with azole resistance,16 virulence-related genes were identified, whose differential expressions were newly found to be associated with the resistant phenotype. Differential expressions for these genes were confirmed by RT-PCR independently. Furthermore, the up-regulation of EFG1, CPH2, TEC1, CDC24, SAP10, ALS9, SNF1, SPO72 and BDF1, and the down-regulation of RAD32, IPF3636 and UBI4 resulted in stronger virulence and invasiveness in the resistant isolates compared with susceptible ones. These findings provide a link between the expression of virulence genes and development of resistance during C. albicans infection in bone marrow transplant (BMT) patients, where C. albicans induces hyphal formation and expression change in multiple virulence factors.

  17. Mechanisms of post-transcriptional gene regulation in bacterial biofilms

    Directory of Open Access Journals (Sweden)

    Viveka eVadyvaloo

    2014-03-01

    Full Text Available Abstract Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria.

  18. 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.

  19. Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

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    Bruno Guedes Alcoforado Aguiar

    2014-10-01

    Full Text Available Introduction Kala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2 and N-acetylglucosamine-1-phosphotransferase (NAGT, which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar. Methods To determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR, the fragments were sequenced, followed by polymorphism identification. Results The nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival. Conclusions NAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

  20. The Acinetobacter baumannii entA gene located outside the acinetobactin cluster is critical for siderophore production, iron acquisition and virulence.

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    William F Penwell

    Full Text Available Acinetobacter baumannii causes severe infections in compromised patients, who present an iron-limited environment that controls bacterial growth. This pathogen has responded to this restriction by expressing high-affinity iron acquisition systems including that mediated by the siderophore acinetobactin. Gene cloning, functional assays and biochemical tests showed that the A. baumannii genome contains a single functional copy of an entA ortholog. This gene, which is essential for the biosynthesis of the acinetobactin precursor 2,3-dihydroxybenzoic acid (DHBA, locates outside of the acinetobactin gene cluster, which otherwise harbors all genes needed for acinetobactin biosynthesis, export and transport. In silico analyses and genetic complementation tests showed that entA locates next to an entB ortholog, which codes for a putative protein that contains the isochorismatase lyase domain, which is needed for DHBA biosynthesis from isochorismic acid, but lacks the aryl carrier protein domain, which is needed for tethering activated DHBA and completion of siderophore biosynthesis. Thus, basF, which locates within the acinetobactin gene cluster, is the only fully functional entB ortholog present in ATCC 19606(T. The differences in amino acid length and sequences between these two EntB orthologs and the differences in the genetic context within which the entA and entB genes are found in different A. baumannii isolates indicate that they were acquired from different sources by horizontal transfer. Interestingly, the AYE strain proved to be a natural entA mutant capable of acquiring iron via an uncharacterized siderophore-mediated system, an observation that underlines the ability of different A. baumannii isolates to acquire iron using different systems. Finally, experimental infections using in vivo and ex vivo models demonstrate the role of DHBA and acinetobactin intermediates in the virulence of the ATCC 19606(T cells, although to a lesser extent when

  1. The Acinetobacter baumannii entA gene located outside the acinetobactin cluster is critical for siderophore production, iron acquisition and virulence.

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    Penwell, William F; Arivett, Brock A; Actis, Luis A

    2012-01-01

    Acinetobacter baumannii causes severe infections in compromised patients, who present an iron-limited environment that controls bacterial growth. This pathogen has responded to this restriction by expressing high-affinity iron acquisition systems including that mediated by the siderophore acinetobactin. Gene cloning, functional assays and biochemical tests showed that the A. baumannii genome contains a single functional copy of an entA ortholog. This gene, which is essential for the biosynthesis of the acinetobactin precursor 2,3-dihydroxybenzoic acid (DHBA), locates outside of the acinetobactin gene cluster, which otherwise harbors all genes needed for acinetobactin biosynthesis, export and transport. In silico analyses and genetic complementation tests showed that entA locates next to an entB ortholog, which codes for a putative protein that contains the isochorismatase lyase domain, which is needed for DHBA biosynthesis from isochorismic acid, but lacks the aryl carrier protein domain, which is needed for tethering activated DHBA and completion of siderophore biosynthesis. Thus, basF, which locates within the acinetobactin gene cluster, is the only fully functional entB ortholog present in ATCC 19606(T). The differences in amino acid length and sequences between these two EntB orthologs and the differences in the genetic context within which the entA and entB genes are found in different A. baumannii isolates indicate that they were acquired from different sources by horizontal transfer. Interestingly, the AYE strain proved to be a natural entA mutant capable of acquiring iron via an uncharacterized siderophore-mediated system, an observation that underlines the ability of different A. baumannii isolates to acquire iron using different systems. Finally, experimental infections using in vivo and ex vivo models demonstrate the role of DHBA and acinetobactin intermediates in the virulence of the ATCC 19606(T) cells, although to a lesser extent when compared to the

  2. Detecting rare gene transfer events in bacterial populations

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    Kaare Magne Nielsen

    2014-01-01

    Full Text Available Horizontal gene transfer (HGT enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research.

  3. Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate

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    Carignan, Bailey M.; Brumfield, Kyle D.

    2016-01-01

    ABSTRACT Vibrio cholerae is the etiological agent of the infectious disease cholera, which is characterized by vomiting and severe watery diarrhea. Recently, V. cholerae clinical isolates have demonstrated increased virulence capabilities, causing more severe symptoms with a much higher rate of disease progression than previously observed. We have identified single nucleotide polymorphisms (SNPs) in four virulence-regulatory genes (hapR, hns, luxO, and vieA) of a hypervirulent V. cholerae clinical isolate, MQ1795. Herein, all SNPs and SNP combinations of interest were introduced into the prototypical El Tor reference strain N16961, and the effects on the production of numerous virulence-related factors, including cholera toxin (CT), the toxin-coregulated pilus (TCP), and ToxT, were analyzed. Our data show that triple-SNP (hapR hns luxO and hns luxO vieA) and quadruple-SNP combinations produced the greatest increases in CT, TCP, and ToxT production. The hns and hns luxO SNP combinations were sufficient for increased TCP and ToxT production. Notably, the hns luxO vieA triple-SNP combination strain produced TCP and ToxT levels similar to those of MQ1795. Certain SNP combinations (hapR and hapR vieA) had the opposite effect on CT, TCP, and ToxT expression. Interestingly, the hns vieA double-SNP combination strain increased TCP production while decreasing CT production. Our findings suggest that SNPs identified in the four regulatory genes, in various combinations, are associated with increased virulence capabilities observed in V. cholerae clinical isolates. These studies provide insight into the evolution of highly virulent strains. IMPORTANCE Cholera, an infectious disease of the small intestine caused by the aquatic bacterium Vibrio cholerae, often results in vomiting and acute watery diarrhea. If left untreated or if the response is too slow, the symptoms can quickly lead to extreme dehydration and ultimately death of the patient. Recent anecdotal evidence of

  4. Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate.

    Science.gov (United States)

    Carignan, Bailey M; Brumfield, Kyle D; Son, Mike S

    2016-01-01

    Vibrio cholerae is the etiological agent of the infectious disease cholera, which is characterized by vomiting and severe watery diarrhea. Recently, V. cholerae clinical isolates have demonstrated increased virulence capabilities, causing more severe symptoms with a much higher rate of disease progression than previously observed. We have identified single nucleotide polymorphisms (SNPs) in four virulence-regulatory genes (hapR, hns, luxO, and vieA) of a hypervirulent V. cholerae clinical isolate, MQ1795. Herein, all SNPs and SNP combinations of interest were introduced into the prototypical El Tor reference strain N16961, and the effects on the production of numerous virulence-related factors, including cholera toxin (CT), the toxin-coregulated pilus (TCP), and ToxT, were analyzed. Our data show that triple-SNP (hapR hns luxO and hns luxO vieA) and quadruple-SNP combinations produced the greatest increases in CT, TCP, and ToxT production. The hns and hns luxO SNP combinations were sufficient for increased TCP and ToxT production. Notably, the hns luxO vieA triple-SNP combination strain produced TCP and ToxT levels similar to those of MQ1795. Certain SNP combinations (hapR and hapR vieA) had the opposite effect on CT, TCP, and ToxT expression. Interestingly, the hns vieA double-SNP combination strain increased TCP production while decreasing CT production. Our findings suggest that SNPs identified in the four regulatory genes, in various combinations, are associated with increased virulence capabilities observed in V. cholerae clinical isolates. These studies provide insight into the evolution of highly virulent strains. IMPORTANCE Cholera, an infectious disease of the small intestine caused by the aquatic bacterium Vibrio cholerae, often results in vomiting and acute watery diarrhea. If left untreated or if the response is too slow, the symptoms can quickly lead to extreme dehydration and ultimately death of the patient. Recent anecdotal evidence of cholera

  5. Comparative genomics of the family Vibrionaceae reveals the wide distribution of genes encoding virulence-associated proteins

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    Cai Hong

    2010-06-01

    Full Text Available Abstract Background Species of the family Vibrionaceae are ubiquitous in marine environments. Several of these species are important pathogens of humans and marine species. Evidence indicates that genetic exchange plays an important role in the emergence of new pathogenic strains within this family. Data from the sequenced genomes of strains in this family could show how the genes encoded by all these strains, known as the pangenome, are distributed. Information about the core, accessory and panproteome of this family can show how, for example, genes encoding virulence-associated proteins are distributed and help us understand how virulence emerges. Results We deduced the complete set of orthologs for eleven strains from this family. The core proteome consists of 1,882 orthologous groups, which is 28% of the 6,629 orthologous groups in this family. There were 4,411 accessory orthologous groups (i.e., proteins that occurred in from 2 to 10 proteomes and 5,584 unique proteins (encoded once on only one of the eleven genomes. Proteins that have been associated with virulence in V. cholerae were widely distributed across the eleven genomes, but the majority was found only on the genomes of the two V. cholerae strains examined. Conclusions The proteomes are reflective of the differing evolutionary trajectories followed by different strains to similar phenotypes. The composition of the proteomes supports the notion that genetic exchange among species of the Vibrionaceae is widespread and that this exchange aids these species in adapting to their environments.

  6. The AS87_04050 gene is involved in bacterial lipopolysaccharide biosynthesis and pathogenicity of Riemerella anatipestifer.

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    Xiaolan Wang

    Full Text Available Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. In this study, we identified a mutant strain RA2640 by Tn4351 transposon mutagenesis, in which the AS87_04050 gene was inactivated by insertion of the transposon. Southern blot analysis indicated that only one insertion was found in the genome of the mutant strain RA2640. SDS-PAGE followed by silver staining showed that the lipopolysaccharide (LPS pattern of mutant strain RA2640 was different from its wild-type strain Yb2, suggesting the LPS was defected. In addition, the phenotype of the mutant strain RA2640 was changed to rough-type, evident by altered colony morphology, autoaggregation ability and crystal violet staining characteristics. Bacterial LPS is a key factor in virulence as well as in both innate and acquired host responses to infection. The rough-type mutant strain RA2640 showed higher sensitivity to antibiotics, disinfectants and normal duck serum, and higher capability of adherence and invasion to Vero cells, compared to its wild-type strain Yb2. Moreover, the mutant strain RA2640 lost the agglutination ability of its wild-type strain Yb2 to R. anatipestifer serotype 2 positive sera, suggesting that the O-antigen is defected. Animal experiments indicated that the virulence of the mutant strain RA2640 was attenuated by more than 100,000-fold, compared to its wild-type strain Yb2. These results suggested that the AS87_04050 gene in R. anatipestifer is associated with the LPS biosynthesis and bacterial pathogenicity.

  7. Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

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    Bode, Nadine J; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen; Pearson, Melanie M

    2015-06-01

    The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence.

  8. Virulence Genes among Enterococcus faecalis and Enterococcus faecium Isolated from Coastal Beaches and Human and Nonhuman Sources in Southern California and Puerto Rico

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    Donna M. Ferguson

    2016-01-01

    Full Text Available Most Enterococcus faecalis and E. faecium are harmless to humans; however, strains harboring virulence genes, including esp, gelE, cylA, asa1, and hyl, have been associated with human infections. E. faecalis and E. faecium are present in beach waters worldwide, yet little is known about their virulence potential. Here, multiplex PCR was used to compare the distribution of virulence genes among E. faecalis and E. faecium isolated from beaches in Southern California and Puerto Rico to isolates from potential sources including humans, animals, birds, and plants. All five virulence genes were found in E. faecalis and E. faecium from beach water, mostly among E. faecalis. gelE was the most common among isolates from all source types. There was a lower incidence of asa1, esp, cylA, and hyl genes among isolates from beach water, sewage, septage, urban runoff, sea wrack, and eelgrass as compared to human isolates, indicating that virulent strains of E. faecalis and E. faecium may not be widely disseminated at beaches. A higher frequency of asa1 and esp among E. faecalis from dogs and of asa1 among birds (mostly seagull suggests that further studies on the distribution and virulence potential of strains carrying these genes may be warranted.

  9. Virulence Genes among Enterococcus faecalis and Enterococcus faecium Isolated from Coastal Beaches and Human and Nonhuman Sources in Southern California and Puerto Rico

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    Talavera, Ginamary Negrón; Hernández, Luis A. Ríos; Ambrose, Richard F.; Jay, Jennifer A.

    2016-01-01

    Most Enterococcus faecalis and E. faecium are harmless to humans; however, strains harboring virulence genes, including esp, gelE, cylA, asa1, and hyl, have been associated with human infections. E. faecalis and E. faecium are present in beach waters worldwide, yet little is known about their virulence potential. Here, multiplex PCR was used to compare the distribution of virulence genes among E. faecalis and E. faecium isolated from beaches in Southern California and Puerto Rico to isolates from potential sources including humans, animals, birds, and plants. All five virulence genes were found in E. faecalis and E. faecium from beach water, mostly among E. faecalis. gelE was the most common among isolates from all source types. There was a lower incidence of asa1, esp, cylA, and hyl genes among isolates from beach water, sewage, septage, urban runoff, sea wrack, and eelgrass as compared to human isolates, indicating that virulent strains of E. faecalis and E. faecium may not be widely disseminated at beaches. A higher frequency of asa1 and esp among E. faecalis from dogs and of asa1 among birds (mostly seagull) suggests that further studies on the distribution and virulence potential of strains carrying these genes may be warranted. PMID:27144029

  10. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

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    Lowden, Michael J.; Skorupski, Karen; Pellegrini, Maria; Chiorazzo, Michael G.; Taylor, Ronald K.; Kull, F. Jon (Dartmouth)

    2010-03-04

    Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraC-family regulator, ToxT. We have solved the 1.9 {angstrom} resolution crystal structure of ToxT, which reveals folds in the N- and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.

  11. Rapidly Evolving Genes Are Key Players in Host Specialization and Virulence of the Fungal Wheat Pathogen Zymoseptoria tritici (Mycosphaerella graminicola.

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    Stephan Poppe

    2015-07-01

    Full Text Available The speciation of pathogens can be driven by divergent host specialization. Specialization to a new host is possible via the acquisition of advantageous mutations fixed by positive selection. Comparative genome analyses of closely related species allows for the identification of such key substitutions via inference of genome-wide signatures of positive selection. We previously used a comparative genomics framework to identify genes that have evolved under positive selection during speciation of the prominent wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola. In this study, we conducted functional analyses of four genes exhibiting strong signatures of positive selection in Z. tritici. We deleted the four genes in Z. tritici and confirm a virulence-related role of three of the four genes ΔZt80707, ΔZt89160 and ΔZt103264. The two mutants ΔZt80707 and ΔZt103264 show a significant reduction in virulence during infection of wheat; the ΔZt89160 mutant causes a hypervirulent phenotype in wheat. Mutant phenotypes of ΔZt80707, ΔZt89160 and ΔZt103264 can be restored by insertion of the wild-type genes. However, the insertion of the Zt80707 and Zt89160 orthologs from Z. pseudotritici and Z. ardabiliae do not restore wild-type levels of virulence, suggesting that positively selected substitutions in Z. tritici may relate to divergent host specialization. Interestingly, the gene Zt80707 encodes also a secretion signal that targets the protein for cell secretion. This secretion signal is however only transcribed in Z. tritici, suggesting that Z. tritici-specific substitutions relate to a new function of the protein in the extracellular space of the wheat-Z. tritici interaction. Together, the results presented here highlight that Zt80707, Zt103264 and Zt89160 represent key genes involved in virulence and host-specific disease development of Z. tritici. Our findings illustrate that evolutionary predictions provide a powerful tool

  12. Rapidly Evolving Genes Are Key Players in Host Specialization and Virulence of the Fungal Wheat Pathogen Zymoseptoria tritici (Mycosphaerella graminicola)

    Science.gov (United States)

    Poppe, Stephan; Dorsheimer, Lena; Happel, Petra; Stukenbrock, Eva Holtgrewe

    2015-01-01

    The speciation of pathogens can be driven by divergent host specialization. Specialization to a new host is possible via the acquisition of advantageous mutations fixed by positive selection. Comparative genome analyses of closely related species allows for the identification of such key substitutions via inference of genome-wide signatures of positive selection. We previously used a comparative genomics framework to identify genes that have evolved under positive selection during speciation of the prominent wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola). In this study, we conducted functional analyses of four genes exhibiting strong signatures of positive selection in Z. tritici. We deleted the four genes in Z. tritici and confirm a virulence-related role of three of the four genes ΔZt80707, ΔZt89160 and ΔZt103264. The two mutants ΔZt80707 and ΔZt103264 show a significant reduction in virulence during infection of wheat; the ΔZt89160 mutant causes a hypervirulent phenotype in wheat. Mutant phenotypes of ΔZt80707, ΔZt89160 and ΔZt103264 can be restored by insertion of the wild-type genes. However, the insertion of the Zt80707 and Zt89160 orthologs from Z. pseudotritici and Z. ardabiliae do not restore wild-type levels of virulence, suggesting that positively selected substitutions in Z. tritici may relate to divergent host specialization. Interestingly, the gene Zt80707 encodes also a secretion signal that targets the protein for cell secretion. This secretion signal is however only transcribed in Z. tritici, suggesting that Z. tritici-specific substitutions relate to a new function of the protein in the extracellular space of the wheat-Z. tritici interaction. Together, the results presented here highlight that Zt80707, Zt103264 and Zt89160 represent key genes involved in virulence and host-specific disease development of Z. tritici. Our findings illustrate that evolutionary predictions provide a powerful tool for the

  13. Genes but not genomes reveal bacterial domestication of Lactococcus lactis.

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    Delphine Passerini

    Full Text Available BACKGROUND: The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE. METHODOLOGY/PRINCIPAL FINDINGS: The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST differing by up to 230 kb in genome size. CONCLUSION/SIGNIFICANCE: The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between "environmental" strains, the main contributors to the genetic diversity within the subspecies, and "domesticated" strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the "domesticated" strains essentially arose through substantial genomic flux within the dispensable

  14. Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE.

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    Shippy, Daniel C; Eakley, Nicholas M; Lauhon, Charles T; Bochsler, Philip N; Fadl, Amin A

    2013-04-01

    Salmonella is an important foodborne pathogen causing major public health problems throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division (gidA) gene significantly altered Salmonella virulence in both in vitro and in vivo models of infection. In Escherichia coli, GidA and MnmE have been shown to modify several bacterial factors by a post-transcriptional mechanism to modify tRNA. Therefore, we hypothesize that GidA and MnmE complex together to modulate virulence genes in Salmonella using a similar mechanism. To test our hypothesis, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella virulence, we constructed gidA and mnmE single mutants as well as a gidA mnmE double mutant strain of Salmonella. Results from the in vitro data displayed a reduction in growth, motility, intracellular replication, and invasion of T84 intestinal epithelial cells in the mutant strains compared to the wild-type Salmonella strain. The in vivo data showed a significant attenuation of the mutant strains as indicated by the induction of inflammatory cytokines and chemokines, as well as in the severity of histopathological lesions in the liver and spleen, compared to mice infected with the wild-type strain. Also, a significant increase in the LD50 was observed in mice infected with the mutant strains, and mice immunized with the mutants were protected against a lethal dose of wild-type Salmonella. A pull-down assay indicated that Salmonella GidA and MnmE bind together, and HPLC analysis revealed that deletion of gidA and/or mnmE altered Salmonella tRNA modification. Overall, the data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis.

  15. Analyses of genome architecture and gene expression reveal novel candidate virulence factors in the secretome of Phytophthora infestans

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    Cano Liliana M

    2010-11-01

    Full Text Available Abstract Background Phytophthora infestans is the most devastating pathogen of potato and a model organism for the oomycetes. It exhibits high evolutionary potential and rapidly adapts to host plants. The P. infestans genome experienced a repeat-driven expansion relative to the genomes of Phytophthora sojae and Phytophthora ramorum and shows a discontinuous distribution of gene density. Effector genes, such as members of the RXLR and Crinkler (CRN families, localize to expanded, repeat-rich and gene-sparse regions of the genome. This distinct genomic environment is thought to contribute to genome plasticity and host adaptation. Results We used in silico approaches to predict and describe the repertoire of P. infestans secreted proteins (the secretome. We defined the "plastic secretome" as a subset of the genome that (i encodes predicted secreted proteins, (ii is excluded from genome segments orthologous to the P. sojae and P. ramorum genomes and (iii is encoded by genes residing in gene sparse regions of P. infestans genome. Although including only ~3% of P. infestans genes, the plastic secretome contains ~62% of known effector genes and shows >2 fold enrichment in genes induced in planta. We highlight 19 plastic secretome genes induced in planta but distinct from previously described effectors. This list includes a trypsin-like serine protease, secreted oxidoreductases, small cysteine-rich proteins and repeat containing proteins that we propose to be novel candidate virulence factors. Conclusions This work revealed a remarkably diverse plastic secretome. It illustrates the value of combining genome architecture with comparative genomics to identify novel candidate virulence factors from pathogen genomes.

  16. 兰州市志贺菌相关毒力基因%Virulent genes of Shigella from Lanzhou city

    Institute of Scientific and Technical Information of China (English)

    郗宁; 韩俭; 吴玲; 李宁荫

    2009-01-01

    目的 调查兰州市分离志贺菌株的毒力基因型.方法 常规分离志贺菌,应用聚合酶链反应检测志贺菌肠毒素1基因(set1A和set1B)、志贺菌肠毒素2基因(sen)、侵袭性质粒抗原H基因(ipaH)、侵袭相关位点基因(ial)和多重调控基因(virA).结果 兰州市志贺菌毒力基因virA、set1A阳性率为100%;ipaH阳性率为93.7%;ial、sell、set1B阳性率分别为56.2%、62.5%、25.0%,set1B仅在福氏志贺菌Ⅳ型中检出.结论 兰州市志贺菌中携带的毒力基因存在差异,virA、set1A和ipaH较稳定,可作为靶基因用于兰州市志贺菌的检验,set1B可用于筛选福氏志贺菌Ⅳ型菌株.%Objective To investigate the virulent genes of Shigella spp separated from the patients from Lanzhou city. Methods Shigella spp was separated from the patients by general methods. The virulent genes of set1A,set1B,sen,ipaH,ial and virA of Shigella spp were detected by PCR. Results The virulent genes of virA and setlA were detected in all Shigella strains. The positive rates of ipaH,ial and sen genes were 93.7%,56.2% and 62.5%,respectively. The positive rate of set1B was 25% and it was only detected in some Shigella flexneri strains. Conclusion There are differences in the virulent genes of Shigella spp separated from the patients from Lanzhou city. VirA,set1A and ipaH present stably in Shigella strains from Lanzhou city and can be used as target genes to detect Shigella. Set1B can be used to screen the type Ⅳ strain of Shigella flexneri.

  17. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    Directory of Open Access Journals (Sweden)

    Benjamin R Lundgren

    Full Text Available The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  18. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    Science.gov (United States)

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  19. Virulence gene expression, adhesion and invasion of Campylobacter jejuni exposed to oxidative stress (H2O2).

    Science.gov (United States)

    Koolman, Leonard; Whyte, Paul; Burgess, Catherine; Bolton, Declan

    2016-03-02

    Studies were undertaken to investigate the effect of oxidative stress conditions (exposure to hydrogen peroxide, H2O2) on [1] the expression of 14 Campylobacter jejuni virulence-associated genes associated with motility and/or invasion (flaA, flaB, flhA, flhB, ciaB, iamA), adhesion (cadF), cytotoxin production (cdtA, cdtB, cdtC) as well as some of the regulators of these genes (rpoN, fliA, luxS, cj1000), in 10 C. jejuni strains (5 poultry and 5 human) and [2] the ability of these cells to adhere to and invade Caco-2 cells. Using 16S rRNA as the reference gene (preliminary research demonstrated that this gene was stably expressed), the expression of the 14 virulence associated genes was investigated under normal and oxidative stress conditions using reverse transcription PCR. A Caco-2 cell tissue culture assay was used to examine adhesion and invasion. The response to oxidative stress was strain-dependent. Two strains showed significant (p<0.05) up or down regulation in 7 of the 14 genes tested, while only 1-2 genes were affected in the remaining strains. Expression of cadF was significantly (p<0.05) changed in all strains, cdt B in 4 strains and cj1000 in 3 strains. Expression of the remaining genes was either unaffected or significantly altered in 1-2 strains. NCTC 11168 completely lost the ability to adhere to and invade Caco-2 cells. One other strain also demonstrated reduced adherence while two others were unable to invade Caco-2 cells after exposure to oxidative stress conditions. In contrast strain 7, a poultry isolate, showed increased invasion. It was concluded that oxidative stress affects expression of C. jejuni virulence genes in a strain-dependent manner, CadF may have a secondary survival function and the cdtB gene may have a different promoter than cdtA and cdtC.

  20. Growth temperature alters Salmonella Enteritidis heat/acid resistance, membrane lipid composition and stress/virulence related gene expression.

    Science.gov (United States)

    Yang, Yishan; Khoo, Wei Jie; Zheng, Qianwang; Chung, Hyun-Jung; Yuk, Hyun-Gyun

    2014-02-17

    The influence of growth temperature (10, 25, 37, and 42 °C) on the survival of Salmonella Enteritidis in simulated gastric fluid (SGF; pH=2.0) and during heat treatment (54, 56, 58, and 60 °C), on the membrane fatty acid composition, as well as on stress-/virulence-related gene expression was studied. Cells incubated at temperatures lower or higher than 37 °C did not increase their acid resistance, with the maximum D-value of 3.07 min in cells grown at 37 °C; while those incubated at higher temperature increased their heat resistance, with the maximum D60 °C-values of 1.4 min in cells grown at 42 °C. A decrease in the ratio of unsaturated to saturated fatty acids was observed as the growth temperature increased. Compared to the control cells grown at 37 °C, the expression of rpoS was 16.5- and 14.4-fold higher in cells cultivated at 10 and 25 °C, respectively; while the expression of rpoH was 2.9-fold higher in those cultivated at 42 °C. The increased expression of stress response gene rpoH and the decreased ratio of unsaturated to saturated fatty acids correlated with the greater heat resistance of bacteria grown at 42 °C; while the decreased expression of stress response gene rpoS at 42 °C might contribute to the decrease in acid resistance. Virulence related genes-spvR, hilA, avrA-were induced in cells cultivated at 42 °C, except sefA which was induced in the control cells. This study indicates that environmental temperature may affect the virulence potential of S. Enteritidis, thus temperature should be well controlled during food storage.

  1. Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission.

    Directory of Open Access Journals (Sweden)

    Andrea I Moreno Switt

    Full Text Available The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB; this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3, and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.

  2. A new experimental approach for studying bacterial genomic island evolution identifies island genes with bacterial host-specific expression patterns

    Directory of Open Access Journals (Sweden)

    Nickerson Cheryl A

    2006-01-01

    Full Text Available Abstract Background Genomic islands are regions of bacterial genomes that have been acquired by horizontal transfer and often contain blocks of genes that function together for specific processes. Recently, it has become clear that the impact of genomic islands on the evolution of different bacterial species is significant and represents a major force in establishing bacterial genomic variation. However, the study of genomic island evolution has been mostly performed at the sequence level using computer software or hybridization analysis to compare different bacterial genomic sequences. We describe here a novel experimental approach to study the evolution of species-specific bacterial genomic islands that identifies island genes that have evolved in such a way that they are differentially-expressed depending on the bacterial host background into which they are transferred. Results We demonstrate this approach by using a "test" genomic island that we have cloned from the Salmonella typhimurium genome (island 4305 and transferred to a range of Gram negative bacterial hosts of differing evolutionary relationships to S. typhimurium. Systematic analysis of the expression of the island genes in the different hosts compared to proper controls allowed identification of genes with genera-specific expression patterns. The data from the analysis can be arranged in a matrix to give an expression "array" of the island genes in the different bacterial backgrounds. A conserved 19-bp DNA site was found upstream of at least two of the differentially-expressed island genes. To our knowledge, this is the first systematic analysis of horizontally-transferred genomic island gene expression in a broad range of Gram negative hosts. We also present evidence in this study that the IS200 element found in island 4305 in S. typhimurium strain LT2 was inserted after the island had already been acquired by the S. typhimurium lineage and that this element is likely not

  3. Identification and Isolation of Brucella suis Virulence Genes Involved in Resistance to the Human Innate Immune System▿

    Science.gov (United States)

    Liautard, Janny; Ouahrani-Bettache, Safia; Jubier-Maurin, Véronique; Lafont, Virginie; Köhler, Stephan; Liautard, Jean-Pierre

    2007-01-01

    Brucella strains are facultative intracellular pathogens that induce chronic diseases in humans and animals. This observation implies that Brucella subverts innate and specific immune responses of the host to develop its full virulence. Deciphering the genes involved in the subversion of the immune system is of primary importance for understanding the virulence of the bacteria, for understanding the pathogenic consequences of infection, and for designing an efficient vaccine. We have developed an in vitro system involving human macrophages infected by Brucella suis and activated syngeneic γ9δ2 T lymphocytes. Under these conditions, multiplication of B. suis inside macrophages is only slightly reduced. To identify the genes responsible for this reduced sensitivity, we screened a library of 2,000 clones of transposon-mutated B. suis. For rapid and quantitative analysis of the multiplication of the bacteria, we describe a simple method based on Alamar blue reduction, which is compatible with screening a large library. By comparing multiplication inside macrophages alone and multiplication inside macrophages with activated γ9δ2 T cells, we identified four genes of B. suis that were necessary to resist to the action of the γ9δ2 T cells. The putative functions of these genes are discussed in order to propose possible explanations for understanding their exact role in the subversion of innate immunity. PMID:17709411

  4. Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution

    Science.gov (United States)

    Erickson, Keesha E.; Otoupal, Peter B.

    2017-01-01

    ABSTRACT Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment

  5. 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

  6. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.

    Directory of Open Access Journals (Sweden)

    Tadayoshi Ikebe

    2010-04-01

    Full Text Available Streptococcal toxic shock syndrome (STSS is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates and non-invasive infections (59 isolates, 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%. The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

  7. Aerobactin and other virulence factor genes among strains of Escherichia coli causing urosepsis: association with patient characteristics.

    Science.gov (United States)

    Johnson, J R; Moseley, S L; Roberts, P L; Stamm, W E

    1988-02-01

    To assess the role of aerobactin as a virulence factor among uropathogenic Escherichia coli, we determined the prevalence, location, and phenotypic expression of aerobactin determinants among 58 E. coli strains causing bacteremic urinary tract infections. We correlated the presence of the aerobactin system with antimicrobial-agent resistance, the presence and phenotypic expression of other uropathogenic virulence factor determinants (P fimbriae, hemolysin, and type 1 fimbriae), and characteristics of patients. Colony and Southern hybridization of total and plasmid DNA with DNA probes for each virulence factor showed that aerobactin determinants were present in 78% of the strains and were plasmid associated in 21%, whereas P fimbria, hemolysin, and type 1 fimbria determinants were present in 74, 43, and 98% of the strains, respectively, and were always chromosomal. Chromosomal aerobactin, P fimbria, and hemolysin determinants occurred together on the chromosome more often in strains from patients without predisposing urological or medical conditions (P = 0.04). Strains with plasmid-encoded aerobactin lacked determinants for P fimbriae (P = 0.004) and hemolysin (P = 0.0004), were resistant to multiple antimicrobial agents (P = 0.0001), and were found only in compromised patients. Mating experiments demonstrated that some aerobactin plasmids also encoded antimicrobial-agent resistance. These findings suggest that the determinants for aerobactin, P fimbriae, and hemolysin are conserved on the chromosome of the antimicrobial-agent-susceptible uropathogenic strains of E. coli which invade noncompromised patients. In contrast, these chromosomal virulence factors are often absent from E. coli strains causing urosepsis in compromised hosts; these strains may acquire plasmid aerobactin in conjunction with antimicrobial-agent resistance genes.

  8. Primary study on the resistance to bacterial blight (X. oryzae) in Cecropin B gene transgenic rices

    Institute of Scientific and Technical Information of China (English)

    HUAZhihua; HUANGDanian; XUERui; WANGXiaoling; GAOZhenya

    1998-01-01

    Bacterial blight (BB) is one of the major diseases to rice. Antibacterial Cecropin B gene has been cloned and transformed into rice. We studied the resistance to bacterial blight in Cecropin B gene tronsgenic rices.

  9. Genetic Screening for Bacterial Mutants in Liquid Growth Media By Fluorescence-Activated Cell Sorting

    Science.gov (United States)

    Abuaita, Basel H.; Withey, Jeffrey H.

    2010-01-01

    Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and make screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction. PMID:21094189

  10. Antimicrobial Effects of Blueberry, Raspberry, and Strawberry Aqueous Extracts and their Effects on Virulence Gene Expression in Vibrio cholerae.

    Science.gov (United States)

    Khalifa, Hazim O; Kamimoto, Maki; Shimamoto, Toshi; Shimamoto, Tadashi

    2015-11-01

    The antimicrobial effects of aqueous extracts of blueberry, raspberry, and strawberry on 13 pathogenic bacteria were evaluated. The minimum inhibitory concentrations and minimum bactericidal concentrations of the extracts were determined before and after neutralization to pH 7.03 ± 0.15. Both Gram-positive and Gram-negative pathogenic bacteria were selectively inhibited by the non-neutralized berries. Blueberry was the best inhibitor, and Vibrio and Listeria were the most sensitive bacteria. After neutralization, blueberry affected only Vibrio and Listeria, whereas the antimicrobial activities of raspberry and strawberry were abolished. The total contents of phenolics, flavonoids, and proanthocyanidins in the extracts were measured with colorimetric methods and were highest in strawberry, followed by raspberry, and then blueberry. We also studied the effects of sub-bactericidal concentrations of the three berry extracts on virulence gene expression in Vibrio cholerae. Real-time quantitative reverse transcription-polymerase chain reaction revealed that the three berry extracts effectively repressed the transcription of the tcpA gene. Raspberry also repressed the transcription of the ctxA gene, whereas blueberry and strawberry did not. However, the three berry extracts did not affect the transcription of toxT. These results suggest that the three berry extracts exert potent antimicrobial effects and inhibit the expression of the virulence factors of V. cholerae.

  11. Inactivation of the Haemophilus ducreyi luxS gene affects the virulence of this pathogen in human subjects.

    Science.gov (United States)

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

    2009-08-01

    Haemophilus ducreyi 35000HP contains a homologue of the luxS gene, which encodes an enzyme that synthesizes autoinducer 2 (AI-2) in other gram-negative bacteria. H. ducreyi 35000HP produced AI-2 that functioned in a Vibrio harveyi-based reporter system. A H. ducreyi luxS mutant was constructed by insertional inactivation of the luxS gene and lost the ability to produce AI-2. Provision of the H. ducreyi luxS gene in trans partially restored AI-2 production by the mutant. The luxS mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule-formation rate in 5 volunteers was 93.3% (95% confidence interval, 81.7%-99.9%) at 15 parent sites and 60.0% (95% confidence interval, 48.3%-71.7%) at 15 mutant sites (1-tailed P < .001). Thus, the luxS mutant was partially attenuated for virulence. This is the first report of AI-2 production contributing to the pathogenesis of a genital ulcer disease.

  12. Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Baldry, Mara; Nielsen, Anita; Bojer, Martin S.;

    2016-01-01

    Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than viab...

  13. 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...

  14. Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model

    Science.gov (United States)

    Pantel, Alix; Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Mesureur, Jennifer; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène

    2016-01-01

    Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence. PMID:26926643

  15. Systems analysis of multiple regulator perturbations allows discovery of virulence factors in Salmonella

    Directory of Open Access Journals (Sweden)

    Heffron Fred

    2011-06-01

    Full Text Available Abstract Background Systemic bacterial infections are highly regulated and complex processes that are orchestrated by numerous virulence factors. Genes that are coordinately controlled by the set of regulators required for systemic infection are potentially required for pathogenicity. Results In this study we present a systems biology approach in which sample-matched multi-omic measurements of fourteen virulence-essential regulator mutants were coupled with computational network analysis to efficiently identify Salmonella virulence factors. Immunoblot experiments verified network-predicted virulence factors and a subset was determined to be secreted into the host cytoplasm, suggesting that they are virulence factors directly interacting with host cellular components. Two of these, SrfN and PagK2, were required for full mouse virulence and were shown to be translocated independent of either of the type III secretion systems in Salmonella or the type III injectisome-related flagellar mechanism. Conclusions Integrating multi-omic datasets from Salmonella mutants lacking virulence regulators not only identified novel virulence factors but also defined a new class of translocated effectors involved in pathogenesis. The success of this strategy at discovery of known and novel virulence factors suggests that the approach may have applicability for other bacterial pathogens.

  16. Systems analysis of multiple regulator perturbations allows discovery of virulence factors in Salmonella

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Hyunjin; Ansong, Charles; McDermott, Jason E.; Gritsenko, Marina A.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.

    2011-06-28

    Background: Systemic bacterial infections are highly regulated and complex processes that are orchestrated by numerous virulence factors. Genes that are coordinately controlled by the set of regulators required for systemic infection are potentially required for pathogenicity. Results: In this study we present a systems biology approach in which sample-matched multi-omic measurements of fourteen virulence-essential regulator mutants were coupled with computational network analysis to efficiently identify Salmonella virulence factors. Immunoblot experiments verified network-predicted virulence factors and a subset was determined to be secreted into the host cytoplasm, suggesting that they are virulence factors directly interacting with host cellular components. Two of these, SrfN and PagK2, were required for full mouse virulence and were shown to be translocated independent of either of the type III secretion systems in Salmonella or the type III injectisome-related flagellar mechanism. Conclusions: Integrating multi-omic datasets from Salmonella mutants lacking virulence regulators not only identified novel virulence factors but also defined a new class of translocated effectors involved in pathogenesis. The success of this strategy at discovery of known and novel virulence factors suggests that the approach may have applicability for other bacterial pathogens.

  17. Hemolysin activities as virulence factor of Enterococcus faecalis isolated from saliva and periapical abscess (gene detection by PCR

    Directory of Open Access Journals (Sweden)

    Dewa Ayu N.P.A

    2013-03-01

    Full Text Available Background: Enterococcus faecalis is a normal flora of the oral cavity, commonly detected in saliva and persistence in endodontic infections. These bacteria have diverse survival and virulence factors. Hemolysin is one of the factor and still had unclear role as a virulence factor of the Enterococcus faecalis to survive in the root canal. Purpose: The purpose of this research was to analyze the presence and activity of hemolysin gene and its activity as a virulence factor isolated from saliva and root canals with periapical abscess. Yet by understanding one of the phenotypes characters which is hemolysin, it is expected a successful endodontic treatment can be provided with the persistent of Enterococcus faecalis bacteria. Methods: Method of the research starting with the identification of Enterococcus faecalis bacteria in isolated saliva and periapical abscess was done in the first part of the study. Then the phenotypes character of Enterococcus faecalis such as gene detection and expression of hemolysin in blood agar cultures of the 60 colonies samples were performed in the later part. Results: Not all of the colonies cultured were identified as Enterococcus faecalis. All positive detection on hemolysin gene showed hemolysin expresion in both isolated samples. However, there were samples with hemolysin expression eventough no hemolysin gene detected. Hemolysin expression detection in saliva was higher due to different activation phase of hemolysin in saliva. The study with just one primer could lead to the possibility of undetected hemolysin gene, eventough there were samples that did not have hemolysin gene. The proportion of hemolysin expression in root canals were less than saliva, this could be influenced by environmental factors. However, Hemolysin was considered as important virulence factor, particularly for disease therapy. Conclusion: The conclusion of this research was hemolysin gene discovered in clinical isolated saliva and root

  18. Abundance of Pathogenic Escherichia coli Virulence-Associated Genes in Well and Borehole Water Used for Domestic Purposes in a Peri-Urban Community of South Africa

    Science.gov (United States)

    Abia, Akebe Luther King; Schaefer, Lisa; Ubomba-Jaswa, Eunice; Le Roux, Wouter

    2017-01-01

    In the absence of pipe-borne water, many people in Africa, especially in rural communities, depend on alternative water sources such as wells, boreholes and rivers for household and personal hygiene. Poor maintenance and nearby pit latrines, however, lead to microbial pollution of these sources. We evaluated the abundance of Escherichia coli and the prevalence of pathogenic E. coli virulence genes in water from wells, boreholes and a river in a South African peri-urban community. Monthly samples were collected between August 2015 and November 2016. In all, 144 water samples were analysed for E. coli using the Colilert 18 system. Virulence genes (eagg, eaeA, stx1, stx2, flichH7, ST, ipaH, ibeA) were investigated using real-time polymerase chain reaction. Mean E. coli counts ranged between 0 and 443.1 Most Probable Number (MPN)/100 mL of water sample. Overall, 99.3% of samples were positive for at least one virulence gene studied, with flicH7 being the most detected gene (81/140; 57.6%) and the stx2 gene the least detected gene (8/140; 5.7%). Both intestinal and extraintestinal pathogenic E. coli genes were detected. The detection of virulence genes in these water sources suggests the presence of potentially pathogenic E. coli strains and is a public health concern. PMID:28335539

  19. The lysine-peptoid hybrid LP5 maintain activity under physiological conditions and affects virulence gene expression in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Gottschalk, Sanne; Ingmer, Hanne; Thomsen, Line E.

    2016-01-01

    , we found that LP5 was protease resistant. However, the dltA and vraF genes, involved in reducing the net anionic charge of the bacterial cell envelope and sensing of antimicrobial peptides, respectively, played a role in the tolerance of S. aureus against LP5. In addition, the exposure of S. aureus...

  20. The balance of apoptotic and necrotic cell death in Mycobacterium tuberculosis infected macrophages is not dependent on bacterial virulence.

    OpenAIRE

    2012-01-01

    BACKGROUND: An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission. METHODS: We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and ...

  1. Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC.

    Directory of Open Access Journals (Sweden)

    Bryon A Nicholson

    Full Text Available Neonatal Meningitis Escherichia coli (NMEC is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.

  2. Impact of lux gene insertion on bacterial surface properties and transport.

    Science.gov (United States)

    Chen, Gang; Srinivasa Ranga, Vijay Penagonda; Mao, Yongjun; Chen, Kevin; Qiao, Hanzi

    2008-03-01

    Genetic markers have been in popular use for tracing microbial movement in the environment. However, the impact of genetic marker insertion on microbial surface properties and consequent transport is often ignored. For this research, we investigated the impact of luminescence-based genetic marker insertion on bacterial surface properties and transport. Typical Gram-positive bacterial strains of Lactobacillus casei, Streptococcus mitis and Micrococcus luteus were used as model bacterial strains in this research. We manipulated gene transfer to observe the impact of lux gene insertion on bacterial surface properties based on contact angle measurements, and we conducted column experiments to evaluate the impact of lux gene insertion on bacterial transport. After lux gene insertion, bacterial interactions with the porous media increased, demonstrating stronger deposition potential in the porous media. Accordingly, retention of the daughter strains increased. Lux gene insertion also resulted in an increase in bacterial dispersion and equilibrium adsorption in the porous media. The bacterial deposition coefficient was found to correlate with the free energy of interactions between bacteria and the porous media.

  3. Effect Of Spaceflight On Microbial Gene Expression And Virulence: Preliminary Results From Microbe Payload Flown On-Board STS-115

    Science.gov (United States)

    Wilson, J. W.; HonerzuBentrup, K,; Schurr, M. J.; Buchanan, K.; Morici, L.; Hammond, T.; Allen, P.; Baker, C.; Ott, C. M.; Nelman-Gonzalez M.; Schurr, J. R.; Pierson, D. L.; Stodieck, L.; Hing, S.; Hammond, T.; Allen, P.; Baker, C.; Parra, M.; Dumars, P.; Stefanyshyn-Piper, H. M.; Nickerson, C. A.

    2007-01-01

    Human presence in space, whether permanent or temporary, is accompanied by the presence of microbes. However, the extent of microbial changes in response to spaceflight conditions and the corresponding changes to infectious disease risk is unclear. Previous studies have indicated that spaceflight weakens the immune system in humans and animals. In addition, preflight and in-flight monitoring of the International Space Station (ISS) and other spacecraft indicates the presence of opportunistic pathogens and the potential of obligate pathogens. Altered antibiotic resistance of microbes in flight has also been shown. As astronauts and cosmonauts live for longer periods in a closed environment, especially one using recycled water and air, there is an increased risk to crewmembers of infectious disease events occurring in-flight. Therefore, understanding how the space environment affects microorganisms and their disease potential is critically important for spaceflight missions and requires further study. The goal of this flight experiment, operationally called MICROBE, is to utilize three model microbial pathogens, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans to examine the global effects of spaceflight on microbial gene expression and virulence attributes. Specifically, the aims are (1) to perform microarray-mediated gene expression profiling of S. typhimurium, P. aeruginosa, and C. albicans, in response to spaceflight in comparison to ground controls and (2) to determine the effect of spaceflight on the virulence potential of these microorganisms immediately following their return from spaceflight using murine models. The model microorganisms were selected as they have been isolated from preflight or in-flight monitoring, represent different degrees of pathogenic behavior, are well characterized, and have sequenced genomes with available microarrays. In particular, extensive studies of S. typhimurium by the Principal Investigator, Dr. Nickerson

  4. Cnm is a major virulence factor of invasive Streptococcus mutans and part of a conserved three-gene locus.

    Science.gov (United States)

    Avilés-Reyes, A; Miller, J H; Simpson-Haidaris, P J; Lemos, J A; Abranches, J

    2014-02-01

    Cnm, a collagen- and laminin-binding protein present in a subset of Streptococcus mutans strains, mediates binding to extracellular matrices (ECM), intracellular invasion and virulence in the Galleria mellonella model. Antibodies raised against Cnm were used to confirm expression and the cell surface localization of Cnm in the highly invasive OMZ175 strain. Sequence analysis identified two additional genes (cnaB and cbpA) encoding putative surface proteins immediately upstream of cnm. Inactivation of cnaB and cbpA in OMZ175, individually or in combination, did not decrease the ability of this highly invasive and virulent strain to bind to different ECM proteins, invade human coronary artery endothelial cells (HCAEC), or kill G. mellonella. Similarly, expression of cnaB and cbpA in the cnm(-) strain UA159 revealed that these genes did not enhance Cnm-related phenotypes. However, integration of cnm in the chromosome of UA159 significantly increased its ability to bind to collagen and laminin, invade HCAEC, and kill G. mellonella. Moreover, the presence of antibodies against Cnm nearly abolished the ability of OMZ175 to bind to collagen and laminin and invade HCAEC, and significantly protected G. mellonella against OMZ175 infection. We concluded that neither CnaB nor CbpA is necessary for the expression of Cnm-related traits. We also provided definitive evidence that Cnm is an important virulence factor and a suitable target for the development of novel preventive and therapeutic strategies to combat invasive S. mutans strains.

  5. Identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host.

    Science.gov (United States)

    Zygmunt, Michel S; Hagius, Sue D; Walker, Joel V; Elzer, Philip H

    2006-01-01

    Brucella species are gram-negative bacteria which belong to alpha-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified.

  6. 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.

  7. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  8. Relationship between eae and stx virulence genes and Escherichia coli in an agricultural watershed: implications for irrigation water standards and leafy green commodities.

    Science.gov (United States)

    Shelton, Daniel R; Karns, Jeffrey S; Coppock, Cary; Patel, Jitu; Sharma, Manan; Pachepsky, Yakov A

    2011-01-01

    The California Leafy Greens Marketing Agreement (LGMA) was adopted in an effort to minimize the risk of contamination of leafy greens with enteric pathogens from a variety of sources, including ground and surface irrigation waters. The LGMA contains standards similar to those established for recreational waters, based on Escherichia coli concentrations. However, no correlation between E. coli and any specific waterborne pathogen(s) has been reported. We conducted this monitoring study in an agricultural watershed to (i) evaluate spatial and temporal fluctuations in E. coli populations and virulence genes associated with pathogenic E. coli and (ii) investigate whether a relationship could be established between E. coli and virulence genes. The virulence genes targeted for analysis were the eae and stx genes, encoding for intimin and Shiga-like toxins, respectively; they were detected with PCR methods. E. coli concentrations and eae and stx prevalence varied both spatially and temporally. In general, both were higher in agricultural than in forested areas and were higher in the summer and fall seasons than in winter. The eae and stx genes were prevalent throughout the watershed. However, in the absence of actual isolates, no conclusions could be drawn regarding the prevalence of specific pathogenic E. coli. No correlation was observed between E. coli concentrations and virulence genes; lower E. coli concentrations were not necessarily associated with decreased prevalence of eae and stx genes. These results suggest that the LGMA standards might not adequately address the issue of waterborne contamination, and that alternative criteria might be required.

  9. Virulence of Klebsiella pneumoniae Isolates Harboring bla KPC-2 Carbapenemase Gene in a Caenorhabditis elegans Model

    OpenAIRE

    Jean-Philippe Lavigne; Gaelle Cuzon; Christophe Combescure; Gisèle Bourg; Albert Sotto; Patrice Nordmann

    2013-01-01

    Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids...

  10. Bacterial genes in the aphid genome: absence of functional gene transfer from Buchnera to its host.

    Directory of Open Access Journals (Sweden)

    Naruo Nikoh

    2010-02-01

    Full Text Available Genome reduction is typical of obligate symbionts. In cellular organelles, this reduction partly reflects transfer of ancestral bacterial genes to the host genome, but little is known about gene transfer in other obligate symbioses. Aphids harbor anciently acquired obligate mutualists, Buchnera aphidicola (Gammaproteobacteria, which have highly reduced genomes (420-650 kb, raising the possibility of gene transfer from ancestral Buchnera to the aphid genome. In addition, aphids often harbor other bacteria that also are potential sources of transferred genes. Previous limited sampling of genes expressed in bacteriocytes, the specialized cells that harbor Buchnera, revealed that aphids acquired at least two genes from bacteria. The newly sequenced genome of the pea aphid, Acyrthosiphon pisum, presents the first opportunity for a complete inventory of genes transferred from bacteria to the host genome in the context of an ancient obligate symbiosis. Computational screening of the entire A. pisum genome, followed by phylogenetic and experimental analyses, provided strong support for the transfer of 12 genes or gene fragments from bacteria to the aphid genome: three LD-carboxypeptidases (LdcA1, LdcA2,psiLdcA, five rare lipoprotein As (RlpA1-5, N-acetylmuramoyl-L-alanine amidase (AmiD, 1,4-beta-N-acetylmuramidase (bLys, DNA polymerase III alpha chain (psiDnaE, and ATP synthase delta chain (psiAtpH. Buchnera was the apparent source of two highly truncated pseudogenes (psiDnaE and psiAtpH. Most other transferred genes were closely related to genes from relatives of Wolbachia (Alphaproteobacteria. At least eight of the transferred genes (LdcA1, AmiD, RlpA1-5, bLys appear to be functional, and expression of seven (LdcA1, AmiD, RlpA1-5 are highly upregulated in bacteriocytes. The LdcAs and RlpAs appear to have been duplicated after transfer. Our results excluded the hypothesis that genome reduction in Buchnera has been accompanied by gene transfer to the

  11. Role of Staphylococcus aureus global regulators sae and sigmaB in virulence gene expression during device-related infection.

    Science.gov (United States)

    Goerke, Christiane; Fluckiger, Ursula; Steinhuber, Andrea; Bisanzio, Vittoria; Ulrich, Martina; Bischoff, Markus; Patti, Joseph M; Wolz, Christiane

    2005-06-01

    The ability of Staphylococcus aureus to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators sae and sigmaB of S. aureus and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of sae, asp23, hla, clfA, coa, and fnbA was determined in strain Newman and its isogenic saeS/R and sigB mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators. sae had a dominant effect on target gene expression during device-related infection. SigmaB seemed to be less active throughout the infection than under induced conditions in vitro.

  12. Short communication: Emergence of a new race of leaf rust with combined virulence to Lr14a and Lr72 genes on durum wheat

    Energy Technology Data Exchange (ETDEWEB)

    Soleiman, N.H; Solis, I.; Soliman, M.H.; Sillero, J.C.; Villegas, D.; Alvaro, F.; Royo, C.; Serra, J.; Ammar, K.; Martínez-Moreno, F.

    2016-11-01

    Leaf rust is a foliar disease caused by the fungus Puccinia triticina that may severely reduce durum wheat yield. Resistance to this pathogen is common in modern durum germplasm but is frequently based on Lr72 and Lr14a. After accounts of races with virulence to Lr14a gene in France in 2000, the present study reports the detection in 2013 for the first time of a new race with virulence to Lr14a and Lr72. The aim of this work was to characterize the virulence pattern of four Spanish isolates with virulence to Lr14a, and to discuss the consequences of this presence. Rusted leaves from cultivars ‘Don Jaime’ (Lr14a) and ‘Gallareta’ (Lr72) were collected in 2013 in the field at two Spanish sites, one in the south (near Cadiz) and another in the north (near Girona). Spores from single pustule for each cultivar and site were multiplied on susceptible cultivar ‘Don Rafael’. Then, the four isolates were inoculated on a set of 19 isogenic lines Thatcher to characterize their virulence spectrum. All isolates presented the same virulence pattern. They were virulent on both Lr14a and Lr72 and the race was named DBB/BS. This race was very similar to those reported in 2009-11, but with added virulence to Lr14a. The resistance based on Lr14a has therefore been overcome in Spain, by a new race that has likely emerged via stepwise mutation from the local predominating races. This information is important to guide breeders in their breeding programmes and gene deployment strategies. (Author)

  13. The UmGcn5 gene encoding histone acetyltransferase from Ustilago maydis is involved in dimorphism and virulence.

    Science.gov (United States)

    González-Prieto, Juan Manuel; Rosas-Quijano, Raymundo; Domínguez, Angel; Ruiz-Herrera, José

    2014-10-01

    We isolated a gene encoding a histone acetyltransferase from Ustilago maydis (DC.) Cda., which is orthologous to the Saccharomyces cerevisiae GCN5 gene. The gene was isolated from genomic clones identified by their specific hybridization to a gene fragment obtained by the polymerase chain reaction (PCR). This gene (Umgcn5; um05168) contains an open reading frame (ORF) of 1421bp that encodes a putative protein of 473 amino acids with a Mr. of 52.6kDa. The protein exhibits a high degree of homology with histone acetyltransferases from different organisms. Null a2b2 ΔUmgcn5 mutants were constructed by substitution of the region encoding the catalytic site with a hygromycin B resistance cassette. Null a1b1 ΔUmgcn5 mutants were isolated from genetic crosses of a2b2 ΔUmgcn5 and a1b1 wild-type strains in maize. Mutants displayed a slight reduction in growth rate under different conditions, and were more sensitive than the wild type to stress conditions, but more important, they grew as long mycelial cells, and formed fuzz-like colonies under all conditions where wild-type strains grew in the yeast-like morphology and formed smooth colonies. This phenotype was not reverted by cAMP addition. Mutants were not virulent to maize plants, and were unable to form teliospores. These phenotypic alterations of the mutants were reverted by their transformation with the wild-type gene.

  14. Broiler chickens, broiler chicken meat, pigs and pork as sources of ExPEC related virulence genes and resistance in Escherichia coli isolates from community-dwelling humans and UTI patients.

    Science.gov (United States)

    Jakobsen, Lotte; Spangholm, Daniel J; Pedersen, Karl; Jensen, Lars B; Emborg, Hanne-Dorthe; Agersø, Yvonne; Aarestrup, Frank M; Hammerum, Anette M; Frimodt-Møller, Niels

    2010-08-15

    Urinary tract infection (UTI) is one of the most common bacterial infections. UTI is primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) from the patients' own fecal flora. The ExPEC often belong to phylogroups B2 and D, the groups which include potent human ExPEC isolates causing UTI, bacteremia, and meningitis. The external sources of these ExPEC in the human intestine are unknown. The food supply may transmit ExPEC to humans. However, evidence of this hypothesis is limited. To assess this hypothesis, the objective of our study was to investigate the presence of ExPEC related virulence genes in E. coli isolates from UTI patients, community-dwelling humans, meat, and production animals. Accordingly, we included 964 geographically and temporally matched E. coli isolates from UTI patients (n=102), community-dwelling humans (n=109), fresh Danish (n=197) and imported broiler chicken meat (n=86), broiler chickens (n=138), fresh Danish (n=177) and imported pork (n=10), and pigs (n=145) in the study. All isolates were investigated for the presence of eight ExPEC related genes (kpsM II, papA, papC, iutA, sfaS, focG, afa, hlyD) using PCR. To investigate any similarities between isolates from the different origins, we performed a cluster analysis including antimicrobial resistance data previously published. We detected seven of the eight ExPEC related genes in isolates from broiler chicken meat, broiler chickens, pork and pigs. Our findings suggest that broiler chicken meat, broiler chickens, pork and pigs could be the source of strains with these ExPEC related virulence genes in community-dwelling humans and UTI patients. Especially detection of ExPEC related virulence genes in isolates belonging to phylogroups B2 and D is very concerning and may have a significant medical impact. The cluster analysis of virulence gene and antimicrobial resistance profiles showed strong similarities between UTI patient, community-dwelling human isolates, meat, and

  15. Evaluating bacterial gene-finding HMM structures as probabilistic logic programs

    DEFF Research Database (Denmark)

    Mørk, Søren; Holmes, Ian

    2012-01-01

    Motivation: Probabilistic logic programming offers a powerful way to describe and evaluate structured statistical models. To investigate the practicality of probabilistic logic programming for structure learning in bioinformatics, we undertook a simplified bacterial gene-finding benchmark in PRIS...

  16. Molecular methods for bacterial genotyping and analyzed gene regions

    Directory of Open Access Journals (Sweden)

    İbrahim Halil Yıldırım1, Seval Cing Yıldırım2, Nadir Koçak3

    2011-06-01

    Full Text Available Bacterial strain typing is an important process for diagnosis, treatment and epidemiological investigations. Current bacterial strain typing methods may be classified into two main categories: phenotyping and genotyping. Phenotypic characters are the reflection of genetic contents. Genotyping, which refers discrimination of bacterial strains based on their genetic content, has recently become widely used for bacterial strain typing. The methods already used in genotypingof bacteria are quite different from each other. In this review we tried to summarize the basic principles of DNA-based methods used in genotyping of bacteria and describe some important DNA regions that are used in genotyping of bacteria. J Microbiol Infect Dis 2011;1(1:42-46.

  17. Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

    Science.gov (United States)

    Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

    2011-11-01

    A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein intera