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Sample records for mycobacterial virulence genes

  1. Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

    PubMed Central

    Mir, Mushtaq; Prisic, Sladjana; Kang, Choong-Min; Lun, Shichun; Guo, Haidan; Murry, Jeffrey P.; Rubin, Eric J.

    2014-01-01

    To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its environment during infection. Adaptations include changes in nutrient utilization and alterations in growth rate. M. tuberculosis Rv1422 is a conserved gene of unknown function that was found in a genetic screen to interact with the mce4 cholesterol uptake locus. The Rv1422 protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper localization of peptidoglycan synthesis. Here we show that Mycobacterium smegmatis and M. tuberculosis strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of several different carbon sources. These strains also have morphological abnormalities, including shortened and bulging cells, suggesting a cell wall defect. In both mycobacterial species, the Rv1422 protein localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis in mycobacteria. An M. tuberculosis ΔRv1422 strain is markedly attenuated for virulence in a mouse infection model, where it elicits decreased inflammation in the lungs and shows impaired bacterial persistence. These findings led us to name this gene cuvA (carbon utilization and virulence protein A) and to suggest a model in which deletion of cuvA leads to changes in nutrient uptake and/or metabolism that affect cell wall structure, morphology, and virulence. Its role in virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis during infection. PMID:25047842

  2. Mycobacterial gene cuvA is required for optimal nutrient utilization and virulence.

    PubMed

    Mir, Mushtaq; Prisic, Sladjana; Kang, Choong-Min; Lun, Shichun; Guo, Haidan; Murry, Jeffrey P; Rubin, Eric J; Husson, Robert N

    2014-10-01

    To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its environment during infection. Adaptations include changes in nutrient utilization and alterations in growth rate. M. tuberculosis Rv1422 is a conserved gene of unknown function that was found in a genetic screen to interact with the mce4 cholesterol uptake locus. The Rv1422 protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper localization of peptidoglycan synthesis. Here we show that Mycobacterium smegmatis and M. tuberculosis strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of several different carbon sources. These strains also have morphological abnormalities, including shortened and bulging cells, suggesting a cell wall defect. In both mycobacterial species, the Rv1422 protein localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis in mycobacteria. An M. tuberculosis ΔRv1422 strain is markedly attenuated for virulence in a mouse infection model, where it elicits decreased inflammation in the lungs and shows impaired bacterial persistence. These findings led us to name this gene cuvA (carbon utilization and virulence protein A) and to suggest a model in which deletion of cuvA leads to changes in nutrient uptake and/or metabolism that affect cell wall structure, morphology, and virulence. Its role in virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis during infection.

  3. Non Mycobacterial Virulence Genes in the Genome of the Emerging Pathogen Mycobacterium abscessus

    PubMed Central

    Schenowitz, Chantal; Dossat, Carole; Barbe, Valérie; Rottman, Martin; Macheras, Edouard; Heym, Beate; Herrmann, Jean-Louis; Daffé, Mamadou; Brosch, Roland; Risler, Jean-Loup; Gaillard, Jean-Louis

    2009-01-01

    Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a “mycobacterial” gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the “non mycobacterial” factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients. PMID:19543527

  4. Evaluation of mycobacterial virulence using rabbit skin liquefaction model.

    PubMed

    Zhang, Guoping; Zhu, Bingdong; Shi, Wanliang; Wang, Mingzhu; Da, Zejiao; Zhang, Ying

    2010-01-01

    Liquefaction is an important pathological process that can subsequently lead to cavitation where large numbers of bacilli can be coughed up which in turn causes spread of tuberculosis in humans. Current animal models to study the liquefaction process and to evaluate virulence of mycobacteria are tedious. In this study, we evaluated a rabbit skin model as a rapid model for liquefaction and virulence assessment using M. bovis BCG, M. tuberculosis avirulent strain H37Ra, M. smegmatis, and the H37Ra strains complemented with selected genes from virulent M. tuberculosis strain H37Rv. We found that with prime and/or boosting immunization, all of these live bacteria at enough high number could induce liquefaction, and the boosting induced stronger liquefaction and more severe lesions in shorter time compared with the prime injection. The skin lesions caused by high dose live BCG (5×10 (6) ) were the most severe followed by live M. tuberculosis H37Ra with M. smegmatis being the least pathogenic. It is of interest to note that none of the above heat-killed mycobacteria induced liquefaction. When H37Ra was complemented with certain wild type genes of H37Rv, some of the complemented H37Ra strains produced more severe skin lesions than H37Ra. These results suggest that the rabbit skin liquefaction model can be a more visual, convenient, rapid and useful model to evaluate virulence of different mycobacteria and to study the mechanisms of liquefaction.

  5. Network Analysis of Human Genes Influencing Susceptibility to Mycobacterial Infections.

    PubMed

    Lipner, Ettie M; Garcia, Benjamin J; Strong, Michael

    2016-01-01

    Tuberculosis and nontuberculous mycobacterial infections constitute a high burden of pulmonary disease in humans, resulting in over 1.5 million deaths per year. Building on the premise that genetic factors influence the instance, progression, and defense of infectious disease, we undertook a systems biology approach to investigate relationships among genetic factors that may play a role in increased susceptibility or control of mycobacterial infections. We combined literature and database mining with network analysis and pathway enrichment analysis to examine genes, pathways, and networks, involved in the human response to Mycobacterium tuberculosis and nontuberculous mycobacterial infections. This approach allowed us to examine functional relationships among reported genes, and to identify novel genes and enriched pathways that may play a role in mycobacterial susceptibility or control. Our findings suggest that the primary pathways and genes influencing mycobacterial infection control involve an interplay between innate and adaptive immune proteins and pathways. Signaling pathways involved in autoimmune disease were significantly enriched as revealed in our networks. Mycobacterial disease susceptibility networks were also examined within the context of gene-chemical relationships, in order to identify putative drugs and nutrients with potential beneficial immunomodulatory or anti-mycobacterial effects.

  6. Network Analysis of Human Genes Influencing Susceptibility to Mycobacterial Infections

    PubMed Central

    Lipner, Ettie M.; Garcia, Benjamin J.; Strong, Michael

    2016-01-01

    Tuberculosis and nontuberculous mycobacterial infections constitute a high burden of pulmonary disease in humans, resulting in over 1.5 million deaths per year. Building on the premise that genetic factors influence the instance, progression, and defense of infectious disease, we undertook a systems biology approach to investigate relationships among genetic factors that may play a role in increased susceptibility or control of mycobacterial infections. We combined literature and database mining with network analysis and pathway enrichment analysis to examine genes, pathways, and networks, involved in the human response to Mycobacterium tuberculosis and nontuberculous mycobacterial infections. This approach allowed us to examine functional relationships among reported genes, and to identify novel genes and enriched pathways that may play a role in mycobacterial susceptibility or control. Our findings suggest that the primary pathways and genes influencing mycobacterial infection control involve an interplay between innate and adaptive immune proteins and pathways. Signaling pathways involved in autoimmune disease were significantly enriched as revealed in our networks. Mycobacterial disease susceptibility networks were also examined within the context of gene-chemical relationships, in order to identify putative drugs and nutrients with potential beneficial immunomodulatory or anti-mycobacterial effects. PMID:26751573

  7. Anticytolytic screen identifies inhibitors of mycobacterial virulence protein secretion.

    PubMed

    Rybniker, Jan; Chen, Jeffrey M; Sala, Claudia; Hartkoorn, Ruben C; Vocat, Anthony; Benjak, Andrej; Boy-Röttger, Stefanie; Zhang, Ming; Székely, Rita; Greff, Zoltán; Orfi, László; Szabadkai, István; Pató, János; Kéri, György; Cole, Stewart T

    2014-10-01

    Mycobacterium tuberculosis (Mtb) requires protein secretion systems like ESX-1 for intracellular survival and virulence. The major virulence determinant and ESX-1 substrate, EsxA, arrests phagosome maturation and lyses cell membranes, resulting in tissue damage and necrosis that promotes pathogen spread. To identify inhibitors of Mtb protein secretion, we developed a fibroblast survival assay exploiting this phenotype and selected molecules that protect host cells from Mtb-induced lysis without being bactericidal in vitro. Hit compounds blocked EsxA secretion and promoted phagosome maturation in macrophages, thus reducing bacterial loads. Target identification studies led to the discovery of BTP15, a benzothiophene inhibitor of the histidine kinase MprB that indirectly regulates ESX-1, and BBH7, a benzyloxybenzylidene-hydrazine compound. BBH7 affects Mtb metal-ion homeostasis and revealed zinc stress as an activating signal for EsxA secretion. This screening approach extends the target spectrum of small molecule libraries and will help tackle the mounting problem of antibiotic-resistant mycobacteria.

  8. Agrobacterium virulence gene induction.

    PubMed

    Gelvin, Stanton B

    2006-01-01

    The ability of Agrobacterium to transform plants and other organisms is under highly regulated genetic control. Two Virulence (Vir) proteins, VirA and VirG, function as a two-component regulatory system to sense particular phenolic compounds synthesized by wounded plant tissues. Induction by these phenolic compounds, in the presence of certain neutral or acid sugars, results in activation of other vir genes, leading to the processing of T-DNA from the Ti-plasmid and transfer of T-DNA to recipient host cells. Many plant, and most nonplant, species do not provide sufficient quantities of the correct phenolic compounds to permit efficient Agrobacterium-mediated genetic transformation to occur. In order to transform these species, phenolic inducing compounds must be added to agrobacteria before and/or during cocultivation of recipient cells with the bacteria. This chapter discusses conditions for efficient induction of Agrobacterium virulence genes by phenolic compounds. PMID:16988335

  9. Identification and functional annotation of mycobacterial septum formation genes using cell division mutants of Escherichia coli.

    PubMed

    Gaiwala Sharma, Sujata S; Kishore, Vimal; Raghunand, Tirumalai R

    2016-01-01

    The major virulence trait of Mycobacterium tuberculosis is its ability to enter a latent state in the face of robust host immunity. Clues to the molecular basis of latency can emerge from understanding the mechanism of cell division, beginning with identification of proteins involved in this process. Using complementation of Escherichia coli mutants, we functionally annotated M. tuberculosis and Mycobacterium smegmatis homologs of divisome proteins FtsW and AmiC. Our results demonstrate that E. coli can be used as a surrogate model to discover mycobacterial cell division genes, and should prove invaluable in delineating the mechanisms of this fundamental process in mycobacteria.

  10. A potential target gene for the host-directed therapy of mycobacterial infection in murine macrophages.

    PubMed

    Bao, Zhang; Chen, Ran; Zhang, Pei; Lu, Shan; Chen, Xing; Yao, Yake; Jin, Xiaozheng; Sun, Yilan; Zhou, Jianying

    2016-09-01

    Mycobacterium tuberculosis (MTB), one of the major bacterial pathogens for lethal infectious diseases, is capable of surviving within the phagosomes of host alveolar macrophages; therefore, host genetic variations may alter the susceptibility to MTB. In this study, to identify host genes exploited by MTB during infection, genes were non-selectively inactivated using lentivirus-based antisense RNA methods in Raw264.7 macrophages, and the cells that survived virulent MTB infection were then screened. Following DNA sequencing of the surviving cell clones, 26 host genes affecting susceptibility to MTB were identified and their pathways were analyzed by bioinformatics analysis. In total, 9 of these genes were confirmed as positive regulators of collagen α-5(IV) chain (Col4a5) expression, a gene encoding a type IV collagen subunit present on the cell surface. The knockdown of Col4a5 consistently suppressed intracellular mycobacterial viability, promoting the survival of Raw264.7 macrophages following mycobacterial infection. Furthermore, Col4a5 deficiency lowered the pH levels of intracellular vesicles, including endosomes, lysosomes and phagosomes in the Raw264.7 cells. Finally, the knockdown of Col4a5 post-translationally increased microsomal vacuolar-type H+-ATPase activity in macrophages, leading to the acidification of intracellular vesicles. Our findings reveal a novel role for Col4a5 in the regulation of macrophage responses to mycobacterial infection and identify Col4a5 as a potential target for the host-directed anti-mycobacterial therapy. PMID:27432120

  11. A potential target gene for the host-directed therapy of mycobacterial infection in murine macrophages

    PubMed Central

    Bao, Zhang; Chen, Ran; Zhang, Pei; Lu, Shan; Chen, Xing; Yao, Yake; Jin, Xiaozheng; Sun, Yilan; Zhou, Jianying

    2016-01-01

    Mycobacterium tuberculosis (MTB), one of the major bacterial pathogens for lethal infectious diseases, is capable of surviving within the phagosomes of host alveolar macrophages; therefore, host genetic variations may alter the susceptibility to MTB. In this study, to identify host genes exploited by MTB during infection, genes were non-selectively inactivated using lentivirus-based antisense RNA methods in RAW264.7 macrophages, and the cells that survived virulent MTB infection were then screened. Following DNA sequencing of the surviving cell clones, 26 host genes affecting susceptibility to MTB were identified and their pathways were analyzed by bioinformatics analysis. In total, 9 of these genes were confirmed as positive regulators of collagen α-5(IV) chain (Col4a5) expression, a gene encoding a type IV collagen subunit present on the cell surface. The knockdown of Col4a5 consistently suppressed intracellular mycobacterial viability, promoting the survival of RAW264.7 macrophages following mycobacterial infection. Furthermore, Col4a5 deficiency lowered the pH levels of intracellular vesicles, including endosomes, lysosomes and phagosomes in the RAW264.7 cells. Finally, the knockdown of Col4a5 post-translationally increased microsomal vacuolar-type H+-ATPase activity in macrophages, leading to the acidification of intracellular vesicles. Our findings reveal a novel role for Col4a5 in the regulation of macrophage responses to mycobacterial infection and identify Col4a5 as a potential target for the host-directed anti-mycobacterial therapy. PMID:27432120

  12. Mutational and Phylogenetic Analyses of the Mycobacterial mbt Gene Cluster ▿§

    PubMed Central

    Chavadi, Sivagami Sundaram; Stirrett, Karen L.; Edupuganti, Uthamaphani R.; Vergnolle, Olivia; Sadhanandan, Gigani; Marchiano, Emily; Martin, Che; Qiu, Wei-Gang; Soll, Clifford E.; Quadri, Luis E. N.

    2011-01-01

    The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobactin based on sequence analysis. A systematic and controlled mutational analysis probing the hypothesized essential nature of each of these genes for mycobactin production has been lacking. The degree of conservation of mbt gene cluster orthologs remains to be investigated as well. In this study, we sought to conclusively establish whether each of nine mbt genes was required for mycobactin production and to examine the conservation of gene clusters orthologous to the M. tuberculosis mbt gene cluster in other bacteria. We report a systematic mutational analysis of the mbt gene cluster ortholog found in Mycobacterium smegmatis. This mutational analysis demonstrates that eight of the nine mbt genes investigated are essential for mycobactin production. Our genome mining and phylogenetic analyses reveal the presence of orthologous mbt gene clusters in several bacterial species. These gene clusters display significant organizational differences originating from an intricate evolutionary path that might have included horizontal gene transfers. Altogether, the findings reported herein advance our understanding of the genetic requirements for the biosynthesis of an important mycobacterial secondary metabolite with relevance to virulence. PMID:21873494

  13. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5.

    PubMed

    Zhang, Qi; Wang, Decheng; Jiang, Guozhong; Liu, Wei; Deng, Qing; Li, Xiujun; Qian, Wei; Ouellet, Hugues; Sun, Jianjun

    2016-01-01

    EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation. PMID:27600772

  14. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5.

    PubMed

    Zhang, Qi; Wang, Decheng; Jiang, Guozhong; Liu, Wei; Deng, Qing; Li, Xiujun; Qian, Wei; Ouellet, Hugues; Sun, Jianjun

    2016-01-01

    EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation.

  15. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5

    PubMed Central

    Zhang, Qi; Wang, Decheng; Jiang, Guozhong; Liu, Wei; Deng, Qing; Li, Xiujun; Qian, Wei; Ouellet, Hugues; Sun, Jianjun

    2016-01-01

    EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation. PMID:27600772

  16. Insights into horizontal acquisition patterns of dormancy and reactivation regulon genes in mycobacterial species using a partitioning-based framework.

    PubMed

    Mehra, Varun; Ghosh, Tarini Shankar; Mande, Sharmila S

    2016-09-01

    Horizontal Gene Transfer (HGT) events, initially thought to be rare in Mycobacterium tuberculosis, have recently been shown to be involved in the acquisition of virulence operons in M. tuberculosis. We have developed a new partitioning framework based HGT prediction algorithm, called Grid3M, and applied the same for the prediction of HGTs in Mycobacteria. Validation and testing using simulated and real microbial genomes indicated better performance of Grid3M as compared with other widely used HGT prediction methods. Specific analysis of the genes belonging to dormancy/reactivation regulons across 14 mycobacterial genomes indicated that horizontal acquisition is specifically restricted to important accessory proteins. The results also revealed Burkholderia species to be a probable source of HGT genes belonging to these regulons. The current study provides a basis for similar analyses investigating the functional/evolutionary aspects of HGT genes in other pathogens. A database of Grid3M predicted HGTs in completely sequenced genomes is available at https://metagenomics.atc.tcs.com/Grid3M/. PMID:27581938

  17. Mycobacterial virulence. Virulent strains of Mycobacteria tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity.

    PubMed

    North, R J; Izzo, A A

    1993-06-01

    The kinetics of growth of two virulent strains of mycobacteria (M. tuberculosis Erdman and M. tuberculosis H37Rv) and two attenuated strains (M. tuberculosis H37Ra and M. bovis Bacillus Calmette-Guerin [BCG]) were studied in the lungs, livers, spleens, and kidneys of severe combined immunodeficient (SCID) mice and of their coisogenic CB-17 immunocompetent counterparts. It was found, in keeping with the findings of earlier investigators (Pierce, C. H., R. J. Dubos, and W. B. Schaefer. 1953. J. Exp. Med. 97:189.), that in immunocompetent mice, virulent organisms grew progressively only in the lungs, whereas the growth of attenuated organisms was controlled in all organs. In SCID mice, in contrast, virulent mycobacteria grew rapidly and progressively in all organs, as did BCG, although at a slower rate. However, H37Ra failed to grow progressively in any organs of SCID mice, unless the mice were treated with hydrocortisone. In fact, hydrocortisone treatment enabled virulent, as well as attenuated, organisms to grow strikingly more rapidly in all organs of SCID mice and in all organs of CB-17 mice. A histological study showed that in SCID mice, multiplication of mycobacteria in the liver occurs in the cytoplasm of macrophages in granulomas and presumably in macrophages in other organs. It is suggested, therefore, that the macrophages of SCID mice possess a glucocorticoid-sensitive mycobacterial mechanism that prevents virulent and avirulent mycobacteria from expressing their true minimal doubling times. In the absence of this mechanism in the lungs of hydrocortisone-treated SCID mice, the doubling times of Erdman, H37Rv, BCG, and H37Ra were 17.7, 17.4, 44.6, and 98.6 h, respectively. The possible importance of a rapid multiplication rate for mycobacterial virulence is discussed. PMID:8496688

  18. A 1,100-year-old founder effect mutation in IL12B gene is responsible for Mendelian susceptibility to mycobacterial disease in Tunisian patients.

    PubMed

    Ben-Mustapha, Imen; Ben-Ali, Meriem; Mekki, Najla; Patin, Etienne; Harmant, Christine; Bouguila, Jihène; Elloumi-Zghal, Houda; Harbi, Abdelaziz; Béjaoui, Mohamed; Boughammoura, Lamia; Chemli, Jalel; Barbouche, Mohamed-Ridha

    2014-01-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder predisposing apparently healthy individuals to infections caused by weakly virulent mycobacteria such as bacille Calmette-Guerin (BCG), environmental mycobacteria, and poorly virulent Salmonella strains. IL-12p40 deficiency is the first reported human disease due to a cytokine gene defect and is one of the deficiencies that cause MSMD. Nine mutant alleles only have been identified in the IL12B gene, and three of them are recurrent mutations due to a founder effect in specific populations. IL-12p40 deficiency has been identified especially in countries where consanguinity is high and where BCG vaccination at birth is universal. We investigated, in such settings, the clinical, cellular, and molecular features of six IL-12p40-deficient Tunisian patients having the same mutation in IL12B gene (c.298_305del). We found that this mutation is inherited as a common founder mutation arousing ~1,100 years ago. This finding facilitates the development of a preventive approach by genetic counseling and prenatal diagnosis especially in affected families.

  19. Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence

    PubMed Central

    Kumar, Santosh; Mittal, Ekansh; Deore, Sapna; Kumar, Anil; Rahman, Aejazur; Krishnasastry, Musti V.

    2015-01-01

    The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as “hemolysin” which was re-annotated as 2′-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms. PMID:26347855

  20. Fungal virulence genes as targets for antifungal chemotherapy.

    PubMed Central

    Perfect, J R

    1996-01-01

    Fungal virulence genes have now met the age of molecular pathogenesis. The definition of virulence genes needs to be broad so that it encompasses the focus on molecular antifungal targets and vaccine epitopes. However, in the broad but simple definition of a virulence gene, there will be many complex genetic and host interactions which investigators will need to carefully define. Nevertheless, with the increasing numbers of serious fungal infections produced by old and newly reported organisms, the paucity of present antifungal drugs, and the likelihood of increasing drug resistance, the need for investigations into understanding fungal virulence at the molecular level has never been more important. PMID:8807043

  1. Massive gene acquisitions in Mycobacterium indicus pranii provide a perspective on mycobacterial evolution.

    PubMed

    Saini, Vikram; Raghuvanshi, Saurabh; Khurana, Jitendra P; Ahmed, Niyaz; Hasnain, Seyed E; Tyagi, Akhilesh K; Tyagi, Anil K

    2012-11-01

    Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria. PMID:22965120

  2. Key Hub and Bottleneck Genes Differentiate the Macrophage Response to Virulent and Attenuated Mycobacterium bovis

    PubMed Central

    Killick, Kate E.; Magee, David A.; Park, Stephen D. E.; Taraktsoglou, Maria; Browne, John A.; Conlon, Kevin M.; Nalpas, Nicolas C.; Gormley, Eamonn; Gordon, Stephen V.; MacHugh, David E.; Hokamp, Karsten

    2014-01-01

    Mycobacterium bovis is an intracellular pathogen that causes tuberculosis in cattle. Following infection, the pathogen resides and persists inside host macrophages by subverting host immune responses via a diverse range of mechanisms. Here, a high-density bovine microarray platform was used to examine the bovine monocyte-derived macrophage transcriptome response to M. bovis infection relative to infection with the attenuated vaccine strain, M. bovis Bacille Calmette–Guérin. Differentially expressed genes were identified (adjusted P-value ≤0.01) and interaction networks generated across an infection time course of 2, 6, and 24 h. The largest number of biological interactions was observed in the 24-h network, which exhibited scale-free network properties. The 24-h network featured a small number of key hub and bottleneck gene nodes, including IKBKE, MYC, NFKB1, and EGR1 that differentiated the macrophage response to virulent and attenuated M. bovis strains, possibly via the modulation of host cell death mechanisms. These hub and bottleneck genes represent possible targets for immuno-modulation of host macrophages by virulent mycobacterial species that enable their survival within a hostile environment. PMID:25324841

  3. PCR Detection of Virulence Genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and Investigation of Virulence Gene Distribution

    PubMed Central

    Thoerner, P.; Bin Kingombe, C. I.; Bögli-Stuber, K.; Bissig-Choisat, B.; Wassenaar, T. M.; Frey, J.; Jemmi, T.

    2003-01-01

    PCR-based assays were developed for the detection of plasmid- and chromosome-borne virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of Y. enterocolitica, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37°C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (ystA, ystB, and ail); however, plasmid-borne genes (yadA and virF) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only ystB was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty Y. pseudotuberculosis isolates were tested by PCR for the presence of inv, yadA, and lcrF. All isolates were inv positive, and 88% of the isolates contained the virulence plasmid genes yadA and lcrF. In conclusion, this study shows that genotyping of Yersinia spp., based on both chromosome- and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates. PMID:12620874

  4. Comparative genomics of mycobacterial proteases.

    PubMed

    Ribeiro-Guimarães, Michelle Lopes; Pessolani, Maria Cristina Vidal

    2007-01-01

    Although proteases are recognized as important virulent factors in pathogenic microorganisms, little information is available so far regarding the potential role of these enzymes in diseases caused by mycobacteria. Here we use bioinformatic tools to compare the protease-coding genes present in the genome of Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis. This analysis allowed a review of the nomenclature of the protease family present in mycobacteria. A special attention was devoted to the 'decaying genome' of M. leprae where a relatively high level of conservation of protease-coding genes was observed when compared to other genes families. A total of 39 genes out of the 49 found in M. bovis were identified in M. leprae. Of relevance, a core of well-conserved 38 protease genes shared by the four species was defined. This set of proteases is probably essential for survival in the host and disease outcome and may constitute novel targets for drug development leading to a more effective control of mycobacterial diseases.

  5. Role of iron in regulation of virulence genes.

    PubMed Central

    Litwin, C M; Calderwood, S B

    1993-01-01

    The abilities of bacterial pathogens to adapt to the environment within the host are essential to their virulence. Microorganisms have adapted to the iron limitation present in mammalian hosts by evolving diverse mechanisms for the assimilation of iron sufficient for growth. In addition, many bacterial pathogens have used the low concentration of iron present in the host as an important signal to enhance the expression of a wide variety of bacterial toxins and other virulence determinants. The molecular basis of coordinate regulation by iron has been most thoroughly studied in Escherichia coli. In this organism, coordinate regulation of gene expression by iron depends on the regulatory gene, fur. Regulation of gene expression by iron in a number of pathogenic organisms is coordinated by proteins homologous to the Fur protein of E. coli. Additional regulatory proteins may be superimposed on the Fur repressor to provide the fine-tuning necessary for the precise regulation of individual virulence genes in response to iron and other environmental signals. Studies of the mechanisms of regulation of iron acquisition systems and virulence determinants by iron should lead to a better understanding of the adaptive response of bacteria to the low-iron environment of the host and its importance in virulence. PMID:8472246

  6. Detection of putative virulence genes of Lactococcus garvieae.

    PubMed

    Ture, Mustafa; Altinok, Ilhan

    2016-04-12

    Lactococcus garvieae is the causative agent of lactococcosis and has been isolated from a wide variety of animals. In the present study, 34 strains of L. garvieae isolated from fish from different sources and locations were tested for the presence or absence of the following putative virulence genes: a capsule gene cluster (CGC), hemolysins 1, 2, and 3 (hly1, -2, -3), NADH oxidase, superoxide dismutase (sod), phosphoglucomutase (pgm), adhesin Pav (adhPav), adhesin PsaA (adhPsaA), enolase (eno), LPxTG-containing surface proteins 1, 2, 3, and 4 (LPxTG-1, LPxTG-2, LPxTG-3, LPxTG-4; where LPxTG means Leu-Pro-any-Thr-Gly), adhesin clusters 1 and 2 (adhCI, adhCII), and adhesin (adh). To determine the presence of the CGC, we developed a multiplex PCR. All strains of L. garvieae had the hly1, -2, -3, NADH oxidase, pgm, adhPav, LPxTG-2, LPxTG-3, sod, eno, adhPsaA, adhCII, and adhCII genes, while only the Lg2 strain contained the CGC. The virulent Lg2 strain contained all 17 virulent genes. All Turkish, Spanish, Italian, and French strains did not contain the CGC. The multiplex PCR assay was useful for the detection of the CGC genes. In conclusion, the CGC is not the only virulent factor in L. garvieae because strains that lack the CGC are virulent to rainbow trout. Single genes also might not be responsible for the virulence of L. garvieae.

  7. Detection of putative virulence genes of Lactococcus garvieae.

    PubMed

    Ture, Mustafa; Altinok, Ilhan

    2016-04-12

    Lactococcus garvieae is the causative agent of lactococcosis and has been isolated from a wide variety of animals. In the present study, 34 strains of L. garvieae isolated from fish from different sources and locations were tested for the presence or absence of the following putative virulence genes: a capsule gene cluster (CGC), hemolysins 1, 2, and 3 (hly1, -2, -3), NADH oxidase, superoxide dismutase (sod), phosphoglucomutase (pgm), adhesin Pav (adhPav), adhesin PsaA (adhPsaA), enolase (eno), LPxTG-containing surface proteins 1, 2, 3, and 4 (LPxTG-1, LPxTG-2, LPxTG-3, LPxTG-4; where LPxTG means Leu-Pro-any-Thr-Gly), adhesin clusters 1 and 2 (adhCI, adhCII), and adhesin (adh). To determine the presence of the CGC, we developed a multiplex PCR. All strains of L. garvieae had the hly1, -2, -3, NADH oxidase, pgm, adhPav, LPxTG-2, LPxTG-3, sod, eno, adhPsaA, adhCII, and adhCII genes, while only the Lg2 strain contained the CGC. The virulent Lg2 strain contained all 17 virulent genes. All Turkish, Spanish, Italian, and French strains did not contain the CGC. The multiplex PCR assay was useful for the detection of the CGC genes. In conclusion, the CGC is not the only virulent factor in L. garvieae because strains that lack the CGC are virulent to rainbow trout. Single genes also might not be responsible for the virulence of L. garvieae. PMID:27068503

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

    PubMed

    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.

  9. Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis

    PubMed Central

    Ferrando, M. Laura; van Baarlen, Peter; Orrù, Germano; Piga, Rosaria; Bongers, Roger S.; Wels, Michiel; De Greeff, Astrid; Smith, Hilde E.; Wells, Jerry M.

    2014-01-01

    Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition. PMID:24642967

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

    PubMed

    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.

  11. Regulation of virulence gene expression in pathogenic Listeria.

    PubMed

    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.

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

    PubMed

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

    2014-06-01

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

  13. Gene encoded antimicrobial peptides, a template for the design of novel anti-mycobacterial drugs.

    PubMed

    Carroll, James; Field, Des; O'Connor, Paula M; Cotter, Paul D; Coffey, Aidan; Hill, Colin; Ross, R Paul; O'Mahony, Jim

    2010-01-01

    Nisin A is the most widely characterized lantibiotic investigated to date. It represents one of the many antimicrobial peptides which have been the focus of much interest as potential therapeutic agents. This has resulted in the search for novel lantibiotics and more commonly, the engineering of novel variants from existing peptides with a view to increasing their activity, stability and solubility.The aim of this study was to compare the activities of nisin A and novel bioengineered hinge derivatives, nisin S, nisin T and nisin V. The microtitre alamar blue assay (MABA) was employed to identify the enhanced activity of these novel variants against M. tuberculosis (H37Ra), M. kansasii (CIT11/06), M. avium subsp. hominissuis (CIT05/03) and M. avium subsp. paratuberculosis (MAP) (ATCC 19698). All variants displayed greater anti-mycobacterial activity than nisin A. Nisin S was the most potent variant against M. tuberculosis, M. kansasii and M. avium subsp. hominissuis, retarding growth by a maximum of 29% when compared with nisin A. Sub-species variations of inhibition were also observed with nisin S reducing growth of Mycobacterium avium subsp. hominissuis by 28% and Mycobacterium avium subsp. paratuberculosis by 19% and nisin T contrastingly reducing growth of MAP by 27% and MAC by 16%.Nisin S, nisin T and nisin V are potent novel anti-mycobacterial compounds, which have the capacity to be further modified, potentially generating compounds with additional beneficial characteristics. This is the first report to demonstrate an enhancement of efficacy by any bioengineered bacteriocin against mycobacteria. PMID:21468208

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

    SciTech Connect

    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.

  15. Small Molecule Control of Virulence Gene Expression in Francisella tularensis

    PubMed Central

    Charity, James C.; Blalock, LeeAnn T.; Costante-Hamm, Michelle M.; Kasper, Dennis L.; Dove, Simon L.

    2009-01-01

    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. PMID:19876386

  16. Denitrification Genes Regulate Brucella Virulence in Mice

    PubMed Central

    Baek, Seung-Hun; Rajashekara, Gireesh; Splitter, Gary A.; Shapleigh, James P.

    2004-01-01

    Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. Genome sequencing of B. suis and B. melitensis revealed that both are complete denitrifiers. To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrification in the closely related B. neotomae was undertaken. In contrast to B. suis and B. melitensis, it was found that B. neotomae is a partial denitrifier that can reduce nitrate to nitrite but no further. Examination of the B. neotomae genome showed that a deletion in the denitrification gene cluster resulted in complete loss of nirV and the partial deletion of nirK and nnrA. Even though the nor operon is intact, a norC-lacZ promoter fusion was not expressed in B. neotomae. However, the norC-lacZ fusion was expressed in the related denitrifier Agrobacterium tumefaciens, suggesting that the lack of expression in B. neotomae is due to inactivation of NnrA. A narK-lacZ promoter fusion was found to exhibit nitrate-dependent expression consistent with the partial denitrifier phenotype. Complementation of the deleted region in B. neotomae by using nirK, nirV, and nnrA from B. melitensis restored the ability of B. neotomae to reduce nitrite. There was a significant difference in the death of IRF-1−/− mice when infected with B. neotomae containing nirK, nirV, and nnrA and those infected with wild-type B. neotomae. The wild-type strain killed all the infected mice, whereas most of the mice infected with B. neotomae containing nirK, nirV, and nnrA survived. PMID:15342571

  17. Identification and analysis of bacterial virulence genes in vivo.

    PubMed Central

    Unsworth, K E; Holden, D W

    2000-01-01

    Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies. PMID:10874734

  18. Ordered expression of virulence genes in Salmonella enterica serovar typhimurium.

    PubMed

    Papezova, K; Gregorova, D; Jonuschies, J; Rychlik, I

    2007-01-01

    Using transcriptional promoter fusions, we investigated the expression of selected SPI-1 and SPI-2 genes of Salmonella enterica serovar Typhimurium (S. Typhimurium). Promoters of genes related to the invasion of the epithelial cell (hilA, hilC, hilD, invF, sicA, sopA, sopB and sopE2) were active in Luria-Bertani (LB) medium and LB with butyrate but were suppressed by bile salts and in glucose minimal (M9) medium. Genes related to S. Typhimurium intracellular survival (phoP, ssrA, ssaB, ssaG, sifA, sifB and pipB) were characterized by their expression in stationary phase in LB and M9 medium. Activity of phoP and ssrA promoters indicated that these might be expressed inside the gut. SPI-1 genes were expressed on the transition to stationary phase while SPI-2 genes were expressed in stationary phase. Among SPI-1 genes, those with regulatory functions preceded in expression the effector genes and sop genes were expressed in the order of sopA, sopB and sopE2, showing hierarchy in the expression of S. Typhimurium virulence genes.

  19. Staphylococcus aureus CodY Negatively Regulates Virulence Gene Expression▿

    PubMed Central

    Majerczyk, Charlotte D.; Sadykov, Marat R.; Luong, Thanh T.; Lee, Chia; Somerville, Greg A.; Sonenshein, Abraham L.

    2008-01-01

    CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAII and RNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus. PMID:18156263

  20. Specific recognition of mycobacterial protein and peptide antigens by gamma-delta T cell subsets following infection with virulent Mycobacterium bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Promoting effective immunity to Mycobacterium tuberculosis complex pathogens is a challenge that is of interest to the fields of human and veterinary medicine alike. We report that gamma delta T cells from virulent Mycobacterium bovis-infected cattle respond specifically and directly to complex, pro...

  1. Malonate inhibits virulence gene expression in Vibrio cholerae.

    PubMed

    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.

  2. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

    PubMed

    Idnurm, Alexander; Reedy, Jennifer L; Nussbaum, Jesse C; Heitman, Joseph

    2004-04-01

    Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to

  3. Investigation of Specific Substitutions in Virulence Genes Characterizing Phenotypic Groups of Low-Virulence Field Strains of Listeria monocytogenes

    PubMed Central

    Roche, S. M.; Gracieux, P.; Milohanic, E.; Albert, I.; Virlogeux-Payant, I.; Témoin, S.; Grépinet, O.; Kerouanton, A.; Jacquet, C.; Cossart, P.; Velge, P.

    2005-01-01

    Several models have shown that virulence varies from one strain of Listeria monocytogenes to another, but little is known about the cause of low virulence. Twenty-six field L. monocytogenes strains were shown to be of low virulence in a plaque-forming assay and in a subcutaneous inoculation test in mice. Using the results of cell infection assays and phospholipase activities, the low-virulence strains were assigned to one of four groups by cluster analysis and then virulence-related genes were sequenced. Group I included 11 strains that did not enter cells and had no phospholipase activity. These strains exhibited a mutated PrfA; eight strains had a single amino acid substitution, PrfAK220T, and the other three had a truncated PrfA, PrfAΔ174-237. These genetic modifications could explain the low virulence of group I strains, since mutated PrfA proteins were inactive. Group II and III strains entered cells but did not form plaques. Group II strains had low phosphatidylcholine phospholipase C activity, whereas group III strains had low phosphatidylinositol phospholipase C activity. Several substitutions were observed for five out of six group III strains in the plcA gene and for one out of three group II strains in the plcB gene. Group IV strains poorly colonized spleens of mice and were practically indistinguishable from fully virulent strains on the basis of the above-mentioned in vitro criteria. These results demonstrate a relationship between the phenotypic classification and the genotypic modifications for at least group I and III strains and suggest a common evolution of these strains within a group. PMID:16204519

  4. Computational bacterial genome-wide analysis of phylogenetic profiles reveals potential virulence genes of Streptococcus agalactiae.

    PubMed

    Lin, Frank Po-Yen; Lan, Ruiting; Sintchenko, Vitali; Gilbert, Gwendolyn L; Kong, Fanrong; Coiera, Enrico

    2011-04-04

    The phylogenetic profile of a gene is a reflection of its evolutionary history and can be defined as the differential presence or absence of a gene in a set of reference genomes. It has been employed to facilitate the prediction of gene functions. However, the hypothesis that the application of this concept can also facilitate the discovery of bacterial virulence factors has not been fully examined. In this paper, we test this hypothesis and report a computational pipeline designed to identify previously unknown bacterial virulence genes using group B streptococcus (GBS) as an example. Phylogenetic profiles of all GBS genes across 467 bacterial reference genomes were determined by candidate-against-all BLAST searches,which were then used to identify candidate virulence genes by machine learning models. Evaluation experiments with known GBS virulence genes suggested good functional and model consistency in cross-validation analyses (areas under ROC curve, 0.80 and 0.98 respectively). Inspection of the top-10 genes in each of the 15 virulence functional groups revealed at least 15 (of 119) homologous genes implicated in virulence in other human pathogens but previously unrecognized as potential virulence genes in GBS. Among these highly-ranked genes, many encode hypothetical proteins with possible roles in GBS virulence. Thus, our approach has led to the identification of a set of genes potentially affecting the virulence potential of GBS, which are potential candidates for further in vitro and in vivo investigations. This computational pipeline can also be extended to in silico analysis of virulence determinants of other bacterial pathogens.

  5. Identification of novel virulence-associated genes via genome analysis of hypothetical genes.

    PubMed

    Garbom, Sara; Forsberg, Ake; Wolf-Watz, Hans; Kihlberg, Britt-Marie

    2004-03-01

    The sequencing of bacterial genomes has opened new perspectives for identification of targets for treatment of infectious diseases. We have identified a set of novel virulence-associated genes (vag genes) by comparing the genome sequences of six human pathogens that are known to cause persistent or chronic infections in humans: Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi, Streptococcus pneumoniae, and Treponema pallidum. This comparison was limited to genes annotated as hypothetical in the T. pallidum genome project. Seventeen genes with unknown functions were found to be conserved among these pathogens. Insertional inactivation of 14 of these genes generated nine mutants that were attenuated for virulence in a mouse infection model. Out of these nine genes, five were found to be specifically associated with virulence in mice as demonstrated by infection with Yersinia pseudotuberculosis in-frame deletion mutants. In addition, these five vag genes were essential only in vivo, since all the mutants were able to grow in vitro. These genes are broadly conserved among bacteria. Therefore, we propose that the corresponding vag gene products may constitute novel targets for antimicrobial therapy and that some vag mutants could serve as carrier strains for live vaccines. PMID:14977936

  6. In Silico Detection of Virulence Gene Homologues in the Human Pathogen Sphingomonas Spp.

    PubMed Central

    Saeb, Amr TM; David, Satish Kumar; Al-Brahim, Hissa

    2014-01-01

    There is an ongoing debate about the clinical significance of Sphingomonas paucimobilis as a virulent bacterial pathogen. In the present study, we investigated the presence of different virulence factors and genes in Sphingomonas bacteria. We utilized phylogenetic, comparative genomics and bioinformatics analysis to investigate the potentiality of Sphingomonas bacteria as virulent pathogenic bacteria. The 16S ribosomal RNA gene (16S rDNA) phylogenetic tree showed that the closest bacterial taxon to Sphingomonas is Brucella with a bootstrap value of 87 followed by Helicobacter, Campylobacter, Pseudomonas, and then Legionella. Sphingomonas shared no virulence factors with Helicobacter or Campylobacter, despite their close phylogenic relationship. In spite of the phylogenetic divergence between Sphingomonas and Pseudomonas, they shared many major virulence factors, such as adherence, antiphagocytosis, iron uptake, proteases, and quorum sensing. In conclusion, Sphingomonas spp. contains several major virulence factors resembling Pseudomonas sp., Legionella sp., Brucella sp., and Bordetella sp. virulence factors. Similarity of virulence factors did not match phylogenetic relationships. These findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor. Sphingomonas spp. is potential virulent bacterial pathogen. PMID:25574122

  7. The enhancin gene: One of the genetic determinants of population variation in baculoviral virulence.

    PubMed

    Martemyanov, V V; Kabilov, M R; Tupikin, A E; Baturina, O A; Belousova, I A; Podgwaite, J D; Ilynykh, A V; Vlassov, V V

    2015-01-01

    It was established that the virulence of the North American baculovirus strain LdMNPV-45 is almost two orders of magnitude higher than the virulence of the Asian strain LdMNPV-27 and does not depend on the test host population (gypsy moth). The Asian strain carries deletions in bro-p and vef-1 genes (82 and 91%, respectively). In accordance with the published data, the product of the latter can greatly increase the virulence of the virus. This result indicates that the population polymorphism of the virulence of baculoviruses can be explained by the vef-1 gene deletion. PMID:26728722

  8. Epigenetic Regulation of Virulence Gene Expression in Parasitic Protozoa.

    PubMed

    Duraisingh, Manoj T; Horn, David

    2016-05-11

    Protozoan parasites colonize numerous metazoan hosts and insect vectors through their life cycles, with the need to respond quickly and reversibly while encountering diverse and often hostile ecological niches. To succeed, parasites must also persist within individuals until transmission between hosts is achieved. Several parasitic protozoa cause a huge burden of disease in humans and livestock, and here we focus on the parasites that cause malaria and African trypanosomiasis. Efforts to understand how these pathogens adapt to survive in varied host environments, cause disease, and transmit between hosts have revealed a wealth of epigenetic phenomena. Epigenetic switching mechanisms appear to be ideally suited for the regulation of clonal antigenic variation underlying successful parasitism. We review the molecular players and complex mechanistic layers that mediate the epigenetic regulation of virulence gene expression. Understanding epigenetic processes will aid the development of antiparasitic therapeutics. PMID:27173931

  9. Ape parasite origins of human malaria virulence genes.

    PubMed

    Larremore, Daniel B; Sundararaman, Sesh A; Liu, Weimin; Proto, William R; Clauset, Aaron; Loy, Dorothy E; Speede, Sheri; Plenderleith, Lindsey J; Sharp, Paul M; Hahn, Beatrice H; Rayner, Julian C; Buckee, Caroline O

    2015-01-01

    Antigens encoded by the var gene family are major virulence factors of the human malaria parasite Plasmodium falciparum, exhibiting enormous intra- and interstrain diversity. Here we use network analysis to show that var architecture and mosaicism are conserved at multiple levels across the Laverania subgenus, based on var-like sequences from eight single-species and three multi-species Plasmodium infections of wild-living or sanctuary African apes. Using select whole-genome amplification, we also find evidence of multi-domain var structure and synteny in Plasmodium gaboni, one of the ape Laverania species most distantly related to P. falciparum, as well as a new class of Duffy-binding-like domains. These findings indicate that the modular genetic architecture and sequence diversity underlying var-mediated host-parasite interactions evolved before the radiation of the Laverania subgenus, long before the emergence of P. falciparum. PMID:26456841

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

    PubMed

    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.

  11. The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene

    PubMed Central

    Li, Yuqing; He, Zheng-Guo

    2012-01-01

    Protection against oxidative stress is one of the primary defense mechanisms contributing to the survival of Mycobacterium tuberculosis in the host. In this study, we provide evidence that OxyS, a LysR-type transcriptional regulator functions as an oxidative stress response regulator in mycobacteria. Overexpression of OxyS lowers expression of the catalase-peroxidase (KatG) gene in M. smegmatis. OxyS binds directly with the katG promoter region and a conserved, GC-rich T-N11-A motif for OxyS binding was successfully characterized in the core binding site. Interestingly, the DNA-binding activity of OxyS was inhibited by H2O2, but not by dithiothreitol. Cys25, which is situated at the DNA-binding domain of OxyS, was found to have a regulatory role for the DNA-binding ability of OxyS in response to oxidative stress. In contrast, the other three cysteine residues in OxyS do not appear to have this function. Furthermore, the mycobacterial strain over-expressing OxyS had a higher sensitivity to H2O2.Thus, OxyS responds to oxidative stress through a unique cysteine residue situated in its DNA-binding domain and negatively regulates expression of the katG gene. These findings uncover a specific regulatory mechanism for mycobacterial adaptation to oxidative stress. PMID:22272299

  12. Regulation of bacterial virulence gene expression by cell envelope stress responses

    PubMed Central

    Flores-Kim, Josué; Darwin, Andrew J

    2014-01-01

    The bacterial cytoplasm lies within a multilayered envelope that must be protected from internal and external hazards. This protection is provided by cell envelope stress responses (ESRs), which detect threats and reprogram gene expression to ensure survival. Pathogens frequently need these ESRs to survive inside the host, where their envelopes face dangerous environmental changes and attack from antimicrobial molecules. In addition, some virulence genes have become integrated into ESR regulons. This might be because these genes can protect the cell envelope from damage by host molecules, or it might help ESRs to reduce stress by moderating the assembly of virulence factors within the envelope. Alternatively, it could simply be a mechanism to coordinate the induction of virulence gene expression with entry into the host. Here, we briefly describe some of the bacterial ESRs, followed by examples where they control virulence gene expression in both Gram-negative and Gram-positive pathogens. PMID:25603429

  13. 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.

    PubMed

    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.

  14. 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

    PubMed Central

    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. PMID:24968349

  15. Diverse Virulence Gene Content of Shiga Toxin-Producing Escherichia coli from Finishing Swine

    PubMed Central

    Fratamico, Pina M.; Bagi, Lori; Delannoy, Sabine; Fach, Patrick; Manning, Shannon D.; Funk, Julie A.

    2014-01-01

    Shiga toxin-producing Escherichia coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC strains. Currently, there is limited information about the virulence genes carried by swine STEC strains; therefore, this study was conducted to examine the presence and absence of 69 virulence genes in STEC strains recovered previously from finishing swine in a longitudinal study. A subset of STEC strains was analyzed by pulsed-field gel electrophoresis (PFGE) to examine their genetic relatedness. Swine STEC strains (n = 150) were analyzed by the use of a high-throughput real-time PCR array system, which included 69 virulence gene targets. Three major pathotypes consisted of 16 different combinations of virulence gene profiles, and serotypes were determined in the swine STEC strains. The majority of the swine STEC strains (n = 120) belonged to serotype O59:H21 and carried the same virulence gene profile, which consisted of 9 virulence genes: stx2e, iha, ecs1763, lpfAO113, estIa (STa), ehaA, paa, terE, and ureD. The eae, nleF, and nleH1-2 genes were detected in one swine STEC strain (O49:H21). Other genes encoding adhesins, including iha, were identified (n = 149). The PFGE results demonstrated that swine STEC strains from pigs raised in the same finishing barn were closely related. Our results revealed diverse virulence gene contents among the members of the swine STEC population and enhance understanding of the dynamics of transmission of STEC strains among pigs housed in the same barn. PMID:25107960

  16. A novel homozygous p.R1105X mutation of the AP4E1 gene in twins with hereditary spastic paraplegia and mycobacterial disease.

    PubMed

    Kong, Xiao-Fei; Bousfiha, Aziz; Rouissi, Abdelfettah; Itan, Yuval; Abhyankar, Avinash; Bryant, Vanessa; Okada, Satoshi; Ailal, Fatima; Bustamante, Jacinta; Casanova, Jean-Laurent; Hirst, Jennifer; Boisson-Dupuis, Stéphanie

    2013-01-01

    We report identical twins with intellectual disability, progressive spastic paraplegia and short stature, born to a consanguineous family. Intriguingly, both children presented with lymphadenitis caused by the live Bacillus Calmette-Guérin (BCG) vaccine. Two syndromes - hereditary spastic paraplegia (HSP) and mycobacterial disease - thus occurred simultaneously. Whole-exome sequencing (WES) revealed a homozygous nonsense mutation (p.R1105X) of the AP4E1 gene, which was confirmed by Sanger sequencing. The p.R1105X mutation has no effect on AP4E1 mRNA levels, but results in lower levels of AP-4ε protein and of the other components of the AP-4 complex, as shown by western blotting, immunoprecipitation and immunofluorescence. Thus, the C-terminal part of the AP-4ε subunit plays an important role in maintaining the integrity of the AP-4 complex. No abnormalities of the IL-12/IFN-γ axis or oxidative burst pathways were identified. In conclusion, we identified twins with autosomal recessive AP-4 deficiency associated with HSP and mycobacterial disease, suggesting that AP-4 may play important role in the neurological and immunological systems. PMID:23472171

  17. Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes

    PubMed Central

    Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Elham

    2015-01-01

    Introduction The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Material and methods Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. Results There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. Conclusions The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes. PMID:26855801

  18. Wide distribution of virulence genes among Enterococcus faecium and Enterococcus faecalis clinical isolates.

    PubMed

    Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasanthakumari; Sadeghifard, Nourkhoda; Ramli, Ramliza; Hamat, Rukman Awang

    2014-01-01

    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.

  19. Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence.

    PubMed

    Maurelli, A T; Sansonetti, P J

    1988-04-01

    Genes required for the full expression of Shigella virulence are on both the chromosome and a large virulence-associated plasmid. Expression of one or more virulence (vir) genes is temperature-regulated, wild-type strains being virulent (invasive) when grown at 37 degrees C but phenotypically avirulent (noninvasive) at 30 degrees C. A vir::lac operon fusion located on the virulence plasmid, which brings the lac genes under control of a temperature-regulated vir gene promoter, was used to select regulatory mutants constitutive for the Lac+ phenotype at the nonpermissive temperature. A transposon Tn10-induced mutant that was Lac+ at 30 degrees C and 37 degrees C was isolated, and the Tn10 insertion was transduced into a wild-type strain. The transductants all simultaneously became deregulated for virulence and invaded HeLa cells equally well at 30 degrees C and 37 degrees C. Other virulence-associated phenotypes were also deregulated and expressed at 30 degrees C. Southern hybridization with a probe for Tn10 determined the insertion to be on the chromosome. Fine mapping by transduction with phage P1L4 positioned the mutation between the galU and trp genes. A cosmid cloned fragment of Shigella chromosomal DNA containing the region around galU was used in complementation studies and showed that the closely linked regulatory gene was able to complement, in trans, the Tn10-induced mutation. We propose that this mutation defines a regulatory gene, virR, and that insertion of Tn10 into this gene inactivates a repressor that normally blocks expression of vir genes at 30 degrees C. PMID:3282241

  20. Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response.

    PubMed

    Briken, Volker; Porcelli, Steven A; Besra, Gurdyal S; Kremer, Laurent

    2004-07-01

    The cell wall component lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis is involved in the inhibition of phagosome maturation, apoptosis and interferon (IFN)-gamma signalling in macrophages and interleukin (IL)-12 cytokine secretion of dendritic cells (DC). All these processes are important for the host to mount an efficient immune response. Conversely, LAM isolated from non-pathogenic mycobacteria (PILAM) have the opposite effect, by inducing a potent proinflammatory response in macrophages and DCs. LAMs from diverse mycobacterial species differ in the modification of their terminal arabinose residues. The strong proinflammatory response induced by PILAM correlates with the presence of phospho-myo-inositol on the terminal arabinose. Interestingly, recent work indicates that the biosynthetic precursor of LAM, lipomannan (LM), which is also present in the cell wall, displays strong proinflammatory effects, independently of which mycobacterial species it is isolated from. Results from in vitro assays and knock-out mice suggest that LM, like PILAM, mediates its biological activity via Toll-like receptor 2. We hypothesize that the LAM/LM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. Recent progress in the identification of genes involved in the biosynthesis of LAM is discussed, in particular with respect to the fact that enzymes controlling the LAM/LM balance might represent targets for new antitubercular drugs. In addition, inactivation of these genes may lead to attenuated strains of M. tuberculosis for the development of new vaccine candidates.

  1. Chamaecyparis obtusa Suppresses Virulence Genes in Streptococcus mutans

    PubMed Central

    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. PMID:27293453

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

    PubMed

    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.

  3. Quantitative Trait Locus (QTL) Mapping Reveals a Role for Unstudied Genes in Aspergillus Virulence

    PubMed Central

    Christians, Julian K.; Cheema, Manjinder S.; Vergara, Ismael A.; Watt, Cortney A.; Pinto, Linda J.; Chen, Nansheng; Moore, Margo M.

    2011-01-01

    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. PMID:21559404

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

    PubMed Central

    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

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

    USGS Publications Warehouse

    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. Multilocus sequence typing and virulence gene profiles associated with Escherichia coli from human and animal sources.

    PubMed

    Manges, Amee R; Harel, Josée; Masson, Luke; Edens, Thaddeus J; Portt, Andrea; Reid-Smith, Richard J; Zhanel, George G; Kropinski, Andrew M; Boerlin, Patrick

    2015-04-01

    We investigated whether specific sequence types, and their shared virulence gene profiles, may be associated with both human and food animal reservoirs. A total of 600 Escherichia coli isolates were assembled from human (n=265) and food-animal (n=335) sources from overlapping geographic areas and time periods (2005-2010) in Canada. The entire collection was subjected to multilocus sequence typing and a subset of 286 E. coli isolates was subjected to an E. coli-specific virulence gene microarray. The most common sequence type (ST) was E. coli ST10, which was present in all human and food-animal sources, followed by ST69, ST73, ST95, ST117, and ST131. A core group of virulence genes was associated with all 10 common STs including artJ, ycfZ, csgA, csgE, fimA, fimH, gad, hlyE, ibeB, mviM, mviN, and ompA. STs 73, 92, and 95 exhibited the largest number of virulence genes, and all were exclusively identified from human infections. ST117 was found in both human and food-animal sources and shared virulence genes common in extraintestinal pathogenic E. coli lineages. Select groups of E. coli may be found in both human and food-animal reservoirs.

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

    PubMed

    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.

  8. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

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

    PubMed

    Masri, Leila; Branca, Antoine; Sheppard, Anna E; Papkou, Andrei; Laehnemann, David; Guenther, Patrick S; Prahl, Swantje; Saebelfeld, Manja; Hollensteiner, Jacqueline; Liesegang, Heiko; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Michiels, Nicolaas K; Schulte, Rebecca D; Kurtz, Joachim; Rosenstiel, Philip; Telschow, Arndt; Bornberg-Bauer, Erich; Schulenburg, Hinrich

    2015-06-01

    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. PMID:26042786

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

    PubMed Central

    de Menezes, Francisca Gleire Rodrigues; Neves, Soraya da Silva; de Sousa, Oscarina Viana; 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. PMID:25229224

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

    PubMed

    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.

  12. Temperature-dependent expression of virulence genes in fish-pathogenic bacteria

    PubMed Central

    Guijarro, José A.; Cascales, Desirée; García-Torrico, Ana I.; García-Domínguez, Mario; Méndez, Jessica

    2015-01-01

    Virulence gene expression in pathogenic bacteria is modulated by environmental parameters. A key factor in this expression is temperature. Its effect on virulence gene expression in bacteria infecting warm-blooded hosts is well documented. Transcription of virulence genes in these bacteria is induced upon a shift from low environmental to a higher host temperature (37°C). Interestingly, host temperatures usually correspond to the optimum for growth of these pathogenic bacteria. On the contrary, in ectothermic hosts such as fish, molluscs, and amphibians, infection processes generally occur at a temperature lower than that for the optimal growth of the bacteria. Therefore, regulation of virulence gene expression in response to temperature shift has to be modulated in a different way to that which is found in bacteria infecting warm-blooded hosts. The current understanding of virulence gene expression and its regulation in response to temperature in fish-pathogenic bacteria is limited, but constant extension of our knowledge base is essential to enable a rational approach to the problem of the bacterial fish diseases affecting the aquaculture industry. This is an interesting issue and progress needs to be made in order to diminish the economic losses caused by these diseases. The intention of this review is, for the first time, to compile the scattered results existing in the field in order to lay the groundwork for future research. This article is an overview of those relevant virulence genes that are expressed at temperatures lower than that for optimal bacterial growth in different fish-pathogenic bacteria as well as the principal mechanisms that could be involved in their regulation. PMID:26217329

  13. Effects of mycobacterial infection on proliferation of hematopoietic precursor cells.

    PubMed

    Choi, Hong-Hee; Kim, Kwang-Kyu; Kim, Kwang Dong; Kim, Hwa-Jung; Jo, Eun-Kyeong; Song, Chang-Hwa

    2011-12-01

    Bacterial infection can affect hematopoietic precursor cells in bone marrow, because the infected tissues produce various cytokines and chemokines. Little is known about hematopoietic precursor cells, including hematopoietic stem cells and their progenitors, during mycobacterial infection. Here, we showed that mycobacterial infections result in the expansion of not only the lin-c-kit+sca-1+ (LKS+) cell population, but also granulocyte-monocyte progenitor cells in a chronic murine tuberculosis model. Interestingly, stimulation of LKS+ cells with attenuated Mycobacterium tuberculosis H37Ra culture filtrate (RaCF) was significantly stronger than that by virulent H37Rv culture filtrate (RvCF). Lower TNF-α and IL-6 levels were observed in RvCF-stimulated bone marrow cells. Neutralization of TNF-α or IL-6 in RaCF-stimulated bone marrow cells markedly suppressed LKS+ cell clonal expansion. Additionally, numbers of LKS+ cells were lower in TLR2(-/-) and MyD88(-/-) mice after mycobacterial infection. Taken together, LKS+ cell proliferation related to mycobacterial virulence may be related to the secretion of TNF-α and IL-6 associated with TLR signaling. Expansion of hematopoietic progenitor cells may, therefore, play an important role during mycobacterial infection.

  14. 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

    PubMed Central

    Puah, Suat Moi; Chua, Kek Heng; Tan, Jin Ai Mary Anne

    2016-01-01

    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. PMID:26861367

  15. 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.

    PubMed

    Puah, Suat Moi; Chua, Kek Heng; Tan, Jin Ai Mary Anne

    2016-02-05

    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.

  16. 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.

    PubMed

    Puah, Suat Moi; Chua, Kek Heng; Tan, Jin Ai Mary Anne

    2016-02-01

    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. PMID:26861367

  17. Selection, Recombination, and Virulence Gene Diversity among Group B Streptococcal Genotypes▿ †

    PubMed Central

    Springman, A. Cody; Lacher, David W.; Wu, Guangxi; Milton, Nicole; Whittam, Thomas S.; Davies, H. Dele; Manning, Shannon D.

    2009-01-01

    Transmission of group B Streptococcus (GBS) from mothers to neonates during childbirth is a leading cause of neonatal sepsis and meningitis. Although subtyping tools have identified specific GBS phylogenetic lineages that are important in neonatal disease, little is known about the genetic diversity of these lineages or the roles that recombination and selection play in the generation of emergent genotypes. Here, we examined genetic variation, selection, and recombination in seven multilocus sequence typing (MLST) loci from 94 invasive, colonizing, and bovine strains representing 38 GBS sequence types and performed DNA sequencing and PCR-based restriction fragment length polymorphism analysis of several putative virulence genes to identify gene content differences between genotypes. Despite the low level of diversity in the MLST loci, a neighbor net analysis revealed a variable range of genetic exchange among the seven clonal complexes (CCs) identified, suggesting that recombination is partly responsible for the diversity observed between genotypes. Recombination is also important for several virulence genes, as some gene alleles had evidence for lateral gene exchange across divergent genotypes. The CC-17 lineage, which is associated with neonatal disease, is relatively homogeneous and therefore appears to have diverged independently with an exclusive set of virulence characteristics. These data suggest that different GBS genetic backgrounds have distinct virulence gene profiles that may be important for disease pathogenesis. Such profiles could be used as markers for the rapid detection of strains with an increased propensity to cause neonatal disease and may be considered useful vaccine targets. PMID:19581371

  18. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene.

    PubMed

    Proctor, R H; Hohn, T M; McCormick, S P

    1995-01-01

    The production of trichothecene mycotoxins by some plant pathogenic species of Fusarium is thought to contribute to their virulence. Gibberella zeae (F. graminearum) is an important cereal pathogen that produces the trichothecene deoxynivalenol. To determine if trichothecene production contributes to the virulence of G. zeae, we generated trichothecene-deficient mutants of the fungus by gene disruption. The disrupted gene, Tri5, encodes the enzyme trichodiene synthase, which catalyzes the first step in trichothecene biosynthesis. To disrupt Tri5, G. zeae was transformed with a plasmid carrying a doubly truncated copy of the Tri5 coding region interrupted by a hygromycin B resistance gene. Tri5- transformants were selected by screening for the inability to produce trichothecenes and by Southern blot analysis. Tri5- strains exhibited reduced virulence on seedlings of Wheaton wheat and common winter rye, but wild-type virulence on seedlings of Golden Bantam maize. On Caldwell and Marshall wheat and Porter oat seedlings, Tri5- strains were inconsistent in causing less disease than their wild-type progenitor strain. Head blight developed more slowly on Wheaton when inoculated with Tri5- mutants than when inoculated with wild-type strains. These results suggest that trichothecene production contributes to the virulence of G. zeae on some hosts. PMID:8589414

  19. The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

    PubMed Central

    Letek, Michal; González, Patricia; MacArthur, Iain; Rodríguez, Héctor; Freeman, Tom C.; Valero-Rello, Ana; Blanco, Mónica; Buckley, Tom; Cherevach, Inna; Fahey, Ruth; Hapeshi, Alexia; Holdstock, Jolyon; Leadon, Desmond; Navas, Jesús; Ocampo, Alain; Quail, Michael A.; Sanders, Mandy; Scortti, Mariela M.; Prescott, John F.; Fogarty, Ursula; Meijer, Wim G.; Parkhill, Julian; Bentley, Stephen D.; Vázquez-Boland, José A.

    2010-01-01

    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. PMID:20941392

  20. Antibiotic resistance profile and virulence genes of uropathogenic Escherichia coli isolates in relation to phylogeny.

    PubMed

    Adib, N; Ghanbarpour, R; Solatzadeh, H; Alizade, H

    2014-03-01

    Escherichia coli (E. coli) strains are the major cause of urinary tract infections (UTI) and belong to the large group of extra-intestinal pathogenic E. coli. The purposes of this study were to determine the antibiotic resistance profile, virulence genes and phylogenetic background of E. coli isolates from UTI cases. A total of 137 E. coli isolates were obtained from UTI samples. The antimicrobial susceptibility of confirmed isolates was determined by disk diffusion method against eight antibiotics. The isolates were examined to determine the presence and prevalence of selected virulence genes including iucD, sfa/focDE, papEF and hly. ECOR phylo-groups of isolates were determined by detection of yjaA and chuA genes and fragment TspE4.C2. The antibiogram results showed that 71% of the isolates were resistant to cefazolin, 60.42% to co-trimoxazole, 54.16% to nalidixic acid, 36.45% to gentamicin, 29.18% to ciprofloxacin, 14.58% to cefepime, 6.25% to nitrofurantoin and 0.00% to imipenem. Twenty-two antibiotic resistance patterns were observed among the isolates. Virulence genotyping of isolates revealed that 58.39% isolates had at least one of the four virulence genes. The iucD gene was the most prevalent gene (43.06%). The other genes including sfa/focDE, papEF and hly genes were detected in 35.76%, 18.97% and 2.18% isolates, respectively. Nine combination patterns of the virulence genes were detected in isolates. Phylotyping of 137 isolates revealed that the isolates fell into A (45.99%), B1 (13.14%), B2 (19.71%) and D (21.16%) groups. Phylotyping of multidrug resistant isolates indicated that these isolates are mostly in A (60.34%) and D (20.38%) groups. In conclusion, the isolates that possessed the iucD, sfa/focDE, papEF and hly virulence genes mostly belonged to A and B2 groups, whereas antibiotic resistant isolates were in groups A and D. Escherichia coli strains carrying virulence factors and antibiotic resistance are distributed in specific phylogenetic

  1. Distribution and pathogenic relationship of virulence associated genes among Vibrio alginolyticus from the mariculture systems.

    PubMed

    Ren, Chunhua; Hu, Chaoqun; Jiang, Xiao; Sun, Hongyan; Zhao, Zhe; Chen, Chang; Luo, Peng

    2013-01-01

    Vibrio alginolyticus has been confirmed as an important pathogen for aquatic animals. However, the pathogenic mechanism of V. alginolyticus is not completely understood. A total of 31 isolates of V. alginolyticus from sea water, fish and shrimp on the mariculture systems were fingerprinted by pulsed-field gel electrophoresis. The pathogenicity of these isolates was tested by challenge and the 21 genes associated with the virulence of Vibrio cholerae or Vibrio parahaemolyticus were examined in V. alginolyticus using PCR. The results showed that the 31 V. alginolyticus isolates belonged to 26 PFGE genotypes and the isolates from different source had different genotypes. Nine of the 31 isolates were confirmed as pathogenic strains by challenge. Moreover, 12 vibrio virulence genes were detected in this study. Of the detected genes, VCtoxR, VCtoxS, hlyA, VPtoxR and tlh were found in both pathogenic and non-pathogenic isolates. However, the other 7 virulence genes, ctxB, zot, tagA, stn, sto, tdh and trh, were only present in pathogenic isolates. Analysis of the relationship between virulence associated genes and pathogenicity of V. alginolyticus provides a possible explanation that the pathogenic mechanism of V. alginolyticus might be similar to that of V. parahaemolyticus instead of V. cholerae.

  2. Virulence characteristics of extraintestinal pathogenic Escherichia coli deletion of gene encoding the outer membrane protein X

    PubMed Central

    MENG, Xianrong; LIU, Xueling; ZHANG, Liyuan; HOU, Bo; LI, Binyou; TAN, Chen; LI, Zili; ZHOU, Rui; LI, Shaowen

    2016-01-01

    Outer membrane protein X (OmpX) and its homologues have been proposed to contribute to the virulence in various bacterial species. But, their role in virulence of extraintestinal pathogenic Escherichia coli (ExPEC) is yet to be determined. This study evaluates the role of OmpX in ExPEC virulence in vitro and in vivo using a clinical strain PPECC42 of porcine origin. The ompX deletion mutant exhibited increased swimming motility and decreased adhesion to, and invasion of pulmonary epithelial A549 cell, compared to the wild-type strain. A mild increase in LD50 and distinct decrease in bacterial load in such organs as heart, liver, spleen, lung and kidney were observed in mice infected with the ompX mutant. Complementation of the complete ompX gene in trans restored the virulence of mutant strain to the level of wild-type strain. Our results reveal that OmpX contributes to ExPEC virulence, but may be not an indispensable virulence determinant. PMID:27149893

  3. The metabolic regulator CodY links L. monocytogenes metabolism to virulence by directly activating the virulence regulatory gene, prfA

    PubMed Central

    Lobel, Lior; Sigal, Nadejda; Borovok, Ilya; Belitsky, Boris R.; Sonenshein, Abraham L.; Herskovits, Anat A.

    2015-01-01

    Summary Metabolic adaptations are critical to the ability of bacterial pathogens to grow within host cells and are normally preceded by sensing of host-specific metabolic signals, which in turn can influence the pathogen's virulence state. Previously, we reported that the intracellular bacterial pathogen Listeria monocytogenes responds to low availability of branched-chain amino acids (BCAA) within mammalian cells by up-regulating both BCAA biosynthesis and virulence genes. The induction of virulence genes required the BCAA-responsive transcription regulator, CodY, but the molecular mechanism governing this mode of regulation was unclear. In this report, we demonstrate that CodY directly binds the coding sequence of the L. monocytogenes master virulence activator gene, prfA, 15 nt downstream of its start codon, and that this binding results in up-regulation of prfA transcription specifically under low concentrations of BCAA. Mutating this site abolished CodY binding and reduced prfA transcription in macrophages, and attenuated bacterial virulence in mice. Notably, the mutated binding site did not alter prfA transcription or PrfA activity under other conditions that are known to activate PrfA, such as during growth in the presence of glucose-1-phosphate. This study highlights the tight crosstalk between L. monocytogenes metabolism and virulence' while revealing novel features of CodY-mediated regulation. PMID:25430920

  4. Co-Detection of Virulent Escherichia coli Genes in Surface Water Sources

    PubMed Central

    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. PMID:25659126

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

    PubMed Central

    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. PMID:25313356

  6. Antibiogram characterization and putative virulence genes in Aeromonas species isolated from pig fecal samples.

    PubMed

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

    2016-06-01

    Aeromonas species are broadly distributed in nature and agricultural environments and have been isolated from feces, bedding, and drinking water of healthy pigs. We assessed the incidence, virulence properties, and antimicrobial resistance profile of Aeromonas spp., isolated from pig feces. Antibiogram was done using the disc diffusion methods, and polymerase chain reaction was used for the detection of putative virulence genes. Identification of isolates revealed three phenotypic species with percentage distribution as follows: Aeromonas hydrophila 23 (45.1 %), Aeromonas caviae 16 (31.4 %), and Aeromonas sobria 12 (23.5 %). All Aeromonas isolates in the study were absolutely susceptible to cefotaxime and resistant to penicillin. A. cavaie and A. sobria demonstrated absolute susceptibility against ciprofloxacin and streptomycin. Aeromonas species showed varied susceptibility to cephalothin as follows: A. hydrophila 78.3 %, A. cavaie 93.7 %, and A. sobria 91.7 %. The percentage distribution of virulence genes among Aeromonas isolates were as follows: Aerolysin (aer) 74.5 %, flagellin gene (fla) 68.6 %, cytotoxin (hly A) 43.1 %, lipase (lip) 39.2 %, enterotoxic activities (ast) 31.3 %, and cytotonic gene (alt) 13.7 %. Reports from this study shows that Aeromonas species isolated from pig fecal samples are multi-drug resistant and possess virulence potential which may result to possible risk of human or animal infection and likely contamination of food and water from this sources.

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

    PubMed

    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.

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

    PubMed

    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.

  9. In vivo expression of Helicobacter pylori virulence genes in patients with gastritis, ulcer, and gastric cancer.

    PubMed

    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; Solnick, Jay V; Torres-López, Javier

    2012-02-01

    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.

  10. Sheeppox virus kelch-like gene SPPV-019 affects virus virulence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sheeppox virus (SPPV), a member of the Capripoxvirus genus of the Poxviridae, is the etiologic agent of a significant disease of sheep in the developing world. Genomic analysis of pathogenic and vaccine capripoxviruses identified genes with potential roles in virulence and host-range, including thr...

  11. Gene expression patterns and dynamics of the colonization of common bean (Phaseolus vulgaris L.) by highly virulent and weakly virulent strains of Fusarium oxysporum

    PubMed Central

    Niño-Sánchez, Jonathan; Tello, Vega; Casado-del Castillo, Virginia; Thon, Michael R.; Benito, Ernesto P.; Díaz-Mínguez, José María

    2015-01-01

    The dynamics of root and hypocotyl colonization, and the gene expression patterns of several fungal virulence factors and plant defense factors have been analyzed and compared in the interaction of two Fusarium oxysporum f. sp. phaseoli strains displaying clear differences in virulence, with a susceptible common bean cultivar. The growth of the two strains on the root surface and the colonization of the root was quantitatively similar although the highly virulent (HV) strain was more efficient reaching the central root cylinder. The main differences between both strains were found in the temporal and spatial dynamics of crown root and hypocotyl colonization. The increase of fungal biomass in the crown root was considerably larger for the HV strain, which, after an initial stage of global colonization of both the vascular cylinder and the parenchymal cells, restricted its growth to the newly differentiated xylem vessels. The weakly virulent (WV) strain was a much slower and less efficient colonizer of the xylem vessels, showing also growth in the intercellular spaces of the parenchyma. Most of the virulence genes analyzed showed similar expression patterns in both strains, except SIX1, SIX6 and the gene encoding the transcription factor FTF1, which were highly upregulated in root crown and hypocotyl. The response induced in the infected plant showed interesting differences for both strains. The WV strain induced an early and strong transcription of the PR1 gene, involved in SAR response, while the HV strain preferentially induced the early expression of the ethylene responsive factor ERF2. PMID:25883592

  12. Distinct Expression Levels of ALS, LIP, and SAP Genes in Candida tropicalis with Diverse Virulent Activities

    PubMed Central

    Yu, Shuanbao; Li, Wenge; Liu, Xiaoshu; Che, Jie; Wu, Yuan; Lu, Jinxing

    2016-01-01

    Candia tropicalis is an increasingly important human pathogen, causing nosocomial fungemia among patients with neutropenia or malignancy. However, limited research has been published concerning its pathogenicity. Based on the phenotypes of C. tropicalis in our previous study, we selected nine representative strains with different activities of virulence factors (adhesion, biofilm formation, secreted aspartic proteinases, and hemolysins), and one reference strain, ATCC750. The present study aimed to investigate the filamentation ability, the expression of virulence genes (ALST1-3, LIP1, LIP4, and SAPT1-4) and the cell damage of C. tropicalis strains with diverse virulences. C. tropicalis exhibited strain-dependent filamentation ability, which was positively correlated with biofilm formation. Reverse transcriptase PCR analysis showed that the ALST3 and SAPT3 genes had the highest expression in their corresponding genes for most C. tropicalis. The expressions of virulence genes, except ALST3 on polystyrene, were upregulated compared with growth in the planktonic and on human urinary bladder epithelial cell line (TCC-SUP) surface. Clustering analysis of virulence genes showed that isolates had a high biofilm forming ability on polystyrene formed a group. Lactate dehydrogenase assays showed that the cell damage induced by C. tropicalis markedly increased with longer infection time (24 and 48 h). Strain FXCT01, isolated from blood, caused the most serious cell damage; while ZRCT52, which had no filamentation ability, caused the least cell damage. Correlation analysis demonstrated significant correlation existed between adhesion on epithelial cells or the expression of ALST2-3 and cell damage. Overall, our results supported the view that adhesion and filamentation may play significant roles in the cell damage caused by C. tropicalis. PMID:27524980

  13. Expression of Agrobacterium Homolog Genes Encoding T-complex Recruiting Protein under Virulence Induction Conditions

    PubMed Central

    Yang, Jing; Wu, Meixia; Zhang, Xin; Guo, Minliang; Huang, Zhiwei

    2015-01-01

    The proteins encoded by three Agrobacterial genes, atu5117, atu4860, and atu4856, are highly homologous to each other in amino acid sequence. All three proteins can bind to VirD2 and are named VBP1, VBP2, and VBP3 (VirD2-binding protein), respectively. VBP is involved in T-DNA transfer by recruiting the T-complex from the cytosol to the polar transport apparatus T4SS (type IV secretion system) and is defined as the “T-complex recruiting protein.” However, it remains unknown how these three homologous genes co-exist in a relatively small prokaryotic genome. To understand whether these three homologous genes are expressed differentially under virulence induction conditions, we examined the effects of virulence induction conditions, including various pH values, temperatures and acetosyringone (AS, an effective virulence inducer to Agrobacterium tumefaciens) concentrations, on the expression of the three VBP-encoding genes. Our data showed that vbp1 (atu5117) and vbp3 (atu4856) maintained constant expression under the tested induction conditions, whereas the expression of vbp2 (atu4860) was affected by the conditions. Culture conditions favorable to the expression of vbp2 differed from the reported induction conditions for other virulence proteins. In particular, the pH value was a crucial factor for the expression of vbp2. In addition, the deletion of vbp1 affected the expression of vbp2. Taken together, these results suggest that the mechanisms regulating the expression of these three homologous genes are different from the virulence induction mechanism and that VBP homologs are presumably involved in other biological processes in addition to T-complex recruitment. PMID:26696988

  14. Expression of Agrobacterium Homolog Genes Encoding T-complex Recruiting Protein under Virulence Induction Conditions.

    PubMed

    Yang, Jing; Wu, Meixia; Zhang, Xin; Guo, Minliang; Huang, Zhiwei

    2015-01-01

    The proteins encoded by three Agrobacterial genes, atu5117, atu4860, and atu4856, are highly homologous to each other in amino acid sequence. All three proteins can bind to VirD2 and are named VBP1, VBP2, and VBP3 (VirD2-binding protein), respectively. VBP is involved in T-DNA transfer by recruiting the T-complex from the cytosol to the polar transport apparatus T4SS (type IV secretion system) and is defined as the "T-complex recruiting protein." However, it remains unknown how these three homologous genes co-exist in a relatively small prokaryotic genome. To understand whether these three homologous genes are expressed differentially under virulence induction conditions, we examined the effects of virulence induction conditions, including various pH values, temperatures and acetosyringone (AS, an effective virulence inducer to Agrobacterium tumefaciens) concentrations, on the expression of the three VBP-encoding genes. Our data showed that vbp1 (atu5117) and vbp3 (atu4856) maintained constant expression under the tested induction conditions, whereas the expression of vbp2 (atu4860) was affected by the conditions. Culture conditions favorable to the expression of vbp2 differed from the reported induction conditions for other virulence proteins. In particular, the pH value was a crucial factor for the expression of vbp2. In addition, the deletion of vbp1 affected the expression of vbp2. Taken together, these results suggest that the mechanisms regulating the expression of these three homologous genes are different from the virulence induction mechanism and that VBP homologs are presumably involved in other biological processes in addition to T-complex recruitment. PMID:26696988

  15. Distinct Expression Levels of ALS, LIP, and SAP Genes in Candida tropicalis with Diverse Virulent Activities.

    PubMed

    Yu, Shuanbao; Li, Wenge; Liu, Xiaoshu; Che, Jie; Wu, Yuan; Lu, Jinxing

    2016-01-01

    Candia tropicalis is an increasingly important human pathogen, causing nosocomial fungemia among patients with neutropenia or malignancy. However, limited research has been published concerning its pathogenicity. Based on the phenotypes of C. tropicalis in our previous study, we selected nine representative strains with different activities of virulence factors (adhesion, biofilm formation, secreted aspartic proteinases, and hemolysins), and one reference strain, ATCC750. The present study aimed to investigate the filamentation ability, the expression of virulence genes (ALST1-3, LIP1, LIP4, and SAPT1-4) and the cell damage of C. tropicalis strains with diverse virulences. C. tropicalis exhibited strain-dependent filamentation ability, which was positively correlated with biofilm formation. Reverse transcriptase PCR analysis showed that the ALST3 and SAPT3 genes had the highest expression in their corresponding genes for most C. tropicalis. The expressions of virulence genes, except ALST3 on polystyrene, were upregulated compared with growth in the planktonic and on human urinary bladder epithelial cell line (TCC-SUP) surface. Clustering analysis of virulence genes showed that isolates had a high biofilm forming ability on polystyrene formed a group. Lactate dehydrogenase assays showed that the cell damage induced by C. tropicalis markedly increased with longer infection time (24 and 48 h). Strain FXCT01, isolated from blood, caused the most serious cell damage; while ZRCT52, which had no filamentation ability, caused the least cell damage. Correlation analysis demonstrated significant correlation existed between adhesion on epithelial cells or the expression of ALST2-3 and cell damage. Overall, our results supported the view that adhesion and filamentation may play significant roles in the cell damage caused by C. tropicalis. PMID:27524980

  16. Genome sequencing of a virulent avian Pasteurella multocida strain GX-Pm reveals the candidate genes involved in the pathogenesis.

    PubMed

    Yu, Chengjie; Sizhu, Suolang; Luo, Qingping; Xu, Xuewen; Fu, Lei; Zhang, Anding

    2016-04-01

    Pasteurella multocida (P. multocida) was first shown to be the causative agent of fowl cholera by Louis Pasteur in 1881. First genomic study was performed on an avirulent avian strain Pm70, and until 2013, two genomes of virulent avian strains X73 and P1059 were sequenced. Comparative genome study supplied important information for further study on the pathogenesis of fowl cholera. In the previous study, a capsular serotype A strain GX-Pm was isolated from the liver of a chicken, which died during an outbreak of fowl cholera in 2011. The strain showed multiple drug resistance and was highly virulent to chickens. Therefore, the present study performed the genome sequencing and a comparative genomic analysis to reveal the candidate genes involved in virulence of P. multocida. Sequenced draft genome sequence of GX-Pm was 2,292,886 bp, contained 2941 protein-coding genes, 5 genomic islands, 4 IS elements and 2 prophage regions. Notability, all the predicted drug-resistance genes were included in predicted genomic islands. A comparative genome study on virulent avian strains P1059, X73 and GX-Pm with the avirulent avian strain Pm 70 indicated that 475 unique genes were only identified in either of virulent strains but absent in the avirulent strain. Among these genes, 20 genes were contained within genomes of all three virulent strains, including a few of putative virulence genes. Further characterization of the pathogenic functions of these genes would benefit the understanding of pathogenesis of fowl cholera.

  17. Quantitative Trait Locus Based Virulence Determinant Mapping of the HSV-1 Genome in Murine Ocular Infection: Genes Involved in Viral Regulatory and Innate Immune Networks Contribute to Virulence

    PubMed Central

    Larsen, Inna; Craven, Mark; Brandt, Curtis R.

    2016-01-01

    Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence. PMID:26962864

  18. Quantitative Trait Locus Based Virulence Determinant Mapping of the HSV-1 Genome in Murine Ocular Infection: Genes Involved in Viral Regulatory and Innate Immune Networks Contribute to Virulence.

    PubMed

    Kolb, Aaron W; Lee, Kyubin; Larsen, Inna; Craven, Mark; Brandt, Curtis R

    2016-03-01

    Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence. PMID:26962864

  19. An Ustilago maydis gene involved in H2O2 detoxification is required for virulence.

    PubMed

    Molina, Lázaro; Kahmann, Regine

    2007-07-01

    The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses. PMID:17616735

  20. Antibiotic-resistance and virulence genes in Enterococcus isolated from tropical recreational waters

    PubMed Central

    Santiago-Rodriguez, Tasha M.; Rivera, Jessica I.; Coradin, Mariel; Toranzos, Gary A.

    2014-01-01

    The prevalence of enterococci harboring tetracycline and vancomycin-resistance genes, as well as the enterococcal surface protein (esp) has mostly been determined in clinical settings, but their prevalence in tropical recreational waters remains largely unknown. The present study determined the prevalence of tetM (tetracycline-resistance), vanA and vanB (vancomycin-resistance) in the bacterial and viral fractions, enterococci and their induced phages isolated from tropical recreational marine and fresh waters, dry and wet sands. Since lysogenic phages can act as vectors for antibiotic-resistance and virulence factors, the prevalence of the mentioned genes, as well as that of an integrase-encoding gene (int) specific for Enterococcus faecalis phages was determined. Up to 60 % and 54 % of the bacterial fractions and enterococci harbored at least one of the tested genes, respectively, suggesting that bacteria in tropical environments may be reservoirs of antibiotic-resistance and virulence genes. int was detected in the viral fractions and in one Enterococcus isolate after induction. This study opens the opportunity to determine if the presence of bacteria harboring antibiotic-resistance and virulence genes in tropical recreational waters represents a threat to public health. PMID:23981868

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

    PubMed Central

    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

  2. Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation

    PubMed Central

    Ma, Yibao; Xu, Yuanxi; Yestrepsky, Bryan D.; Sorenson, Roderick J.; Chen, Meng; Larsen, Scott D.; Sun, Hongmin

    2012-01-01

    Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus. PMID:23077578

  3. Streptococcus pneumoniae arginine synthesis genes promote growth and virulence in pneumococcal meningitis.

    PubMed

    Piet, Jurgen R; Geldhoff, Madelijn; van Schaik, Barbera D C; Brouwer, Matthijs C; Valls Seron, Mercedes; Jakobs, Marja E; Schipper, Kim; Pannekoek, Yvonne; Zwinderman, Aeilko H; van der Poll, Tom; van Kampen, Antoine H C; Baas, Frank; van der Ende, Arie; van de Beek, Diederik

    2014-06-01

    Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.

  4. cj0371: A Novel Virulence-Associated Gene of Campylobacter jejuni

    PubMed Central

    Du, Xueqing; Wang, Nan; Ren, Fangzhe; Tang, Hong; Jiao, Xinan; Huang, Jinlin

    2016-01-01

    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. PMID:27471500

  5. cj0371: A Novel Virulence-Associated Gene of Campylobacter jejuni.

    PubMed

    Du, Xueqing; Wang, Nan; Ren, Fangzhe; Tang, Hong; Jiao, Xinan; Huang, Jinlin

    2016-01-01

    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.

  6. Type III secretion genes identify a putative virulence locus of Chlamydia.

    PubMed

    Hsia, R C; Pannekoek, Y; Ingerowski, E; Bavoil, P M

    1997-07-01

    Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.

  7. cj0371: A Novel Virulence-Associated Gene of Campylobacter jejuni.

    PubMed

    Du, Xueqing; Wang, Nan; Ren, Fangzhe; Tang, Hong; Jiao, Xinan; Huang, Jinlin

    2016-01-01

    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. PMID:27471500

  8. Sternal mycobacterial infections

    PubMed Central

    Yuan, Shi-Min

    2016-01-01

    Sternal mycobacterial infections are rare. Due to the rarity, its clinical characteristics, diagnoses, and regular management strategies are still scanty. A total of 76 articles on this topic were obtained by a comprehensive literature collection. The clinical features, diagnosis, management strategies and prognosis were carefully analyzed. There were totally 159 patients including 152 (95%) cases of tuberculosis (TB) and seven (5%) cases of non-TB sternal infections. Sternal mycobacterial infections can be categorized into three types: Primary, secondary, and postoperative, according to the pathogenesis; and categorized into isolated, peristernal, and multifocal, according to the extent of the lesions. Microbiological investigation is more sensitive than medical imaging and Mantoux tuberculin skin test in the diagnosis of sternal infections. Most patients show good responses to the standard four-drug regimen and a surgical intervention was necessary in 28.3% patients. The prognoses of the patients are good with a very low mortality. A delayed diagnosis of sternal mycobacterial infections may bring about recurrent sternal infections and sustained incurability. An early diagnosis and prompt antibiotic regimens may significantly improve the patients' outcomes. PMID:27168857

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

    PubMed

    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.

  10. Virulence gene profiles in Staphylococcus aureus isolated from cows with subclinical mastitis in eastern Poland.

    PubMed

    Kot, Barbara; Szweda, Piotr; Frankowska-Maciejewska, Aneta; Piechota, Małgorzata; Wolska, Katarzyna

    2016-05-01

    Staphylococcus aureus is arguably the most important pathogen involved in bovine mastitis. The aim of this study was to determine the virulence gene profiles of 124 Staph. aureus isolates from subclinical mastitis in cows in eastern Poland. The presence of 30 virulence genes encoding adhesins, proteases and superantigenic toxins was investigated by PCR. The 17 different combinations of adhesin genes were identified. Occurrence of eno (91·1%) and fib (82·3%) genes was found to be common. The frequency of other adhesion genes fnbA, fnbB, ebps were 14·5, 50, 25%, respectively, and for cna and bbp were 1·6%. The etA and etD genes, encoding exfoliative toxins, were present in genomes of 5·6 and 8·9% isolates, respectively. The splA and sspA, encoding serine protease, were detected in above 90% isolates. The most frequent enterotoxin genes were sei (21%), sem (19·4%), sen (19·4%), seg (18·5%) and seo (13·7%). The tst gene was harboured by 2·4% isolates. The 19 combinations of the superantigenic toxin genes were obtained and found in 35·5% of isolates. Three of them (seg, sei, sem, sen, seo; sec, seg, sei, sem, sen, seo and seg, sei, sem, sen) were the most frequent and found in 16·1% of the isolates. The most common virulotype, present in 17·7% of the isolates, was fib, eno, fnbB, splA, splE, sspA. The results indicate the variation in the presence of virulence genes in Staph. aureus isolates and considerable diversity of isolates that are able to cause mastitis in cows. PMID:27032339

  11. Study of the role of the htrB gene in Salmonella typhimurium virulence.

    PubMed Central

    Jones, B D; Nichols, W A; Gibson, B W; Sunshine, M G; Apicella, M A

    1997-01-01

    We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium. PMID:9353064

  12. Virulence Gene Regulation by l-Arabinose in Salmonella enterica

    PubMed Central

    López-Garrido, Javier; Puerta-Fernández, Elena; Cota, Ignacio; Casadesús, Josep

    2015-01-01

    Invasion of the intestinal epithelium is a critical step in Salmonella enterica infection and requires functions encoded in the gene cluster known as Salmonella Pathogenicity Island 1 (SPI-1). Expression of SPI-1 genes is repressed by l-arabinose, and not by other pentoses. Transport of l-arabinose is necessary to repress SPI-1; however, repression is independent of l-arabinose metabolism and of the l-arabinose-responsive regulator AraC. SPI-1 repression by l-arabinose is exerted at a single target, HilD, and the mechanism appears to be post-translational. As a consequence of SPI-1 repression, l-arabinose reduces translocation of SPI-1 effectors to epithelial cells and decreases Salmonella invasion in vitro. These observations reveal a hitherto unknown role of l-arabinose in gene expression control and raise the possibility that Salmonella may use L-arabinose as an environmental signal. PMID:25991823

  13. Virulence Gene Regulation by L-Arabinose in Salmonella enterica.

    PubMed

    López-Garrido, Javier; Puerta-Fernández, Elena; Cota, Ignacio; Casadesús, Josep

    2015-07-01

    Invasion of the intestinal epithelium is a critical step in Salmonella enterica infection and requires functions encoded in the gene cluster known as Salmonella Pathogenicity Island 1 (SPI-1). Expression of SPI-1 genes is repressed by L-arabinose, and not by other pentoses. Transport of L-arabinose is necessary to repress SPI-1; however, repression is independent of L-arabinose metabolism and of the L-arabinose-responsive regulator AraC. SPI-1 repression by L-arabinose is exerted at a single target, HilD, and the mechanism appears to be post-translational. As a consequence of SPI-1 repression, l-arabinose reduces translocation of SPI-1 effectors to epithelial cells and decreases Salmonella invasion in vitro. These observations reveal a hitherto unknown role of L-arabinose in gene expression control and raise the possibility that Salmonella may use L-arabinose as an environmental signal.

  14. Mycobacterial Phylogenomics: An Enhanced Method for Gene Turnover Analysis Reveals Uneven Levels of Gene Gain and Loss among Species and Gene Families

    PubMed Central

    Librado, Pablo; Vieira, Filipe G.; Sánchez-Gracia, Alejandro; Kolokotronis, Sergios-Orestis; Rozas, Julio

    2014-01-01

    Species of the genus Mycobacterium differ in several features, from geographic ranges, and degree of pathogenicity, to ecological and host preferences. The recent availability of several fully sequenced genomes for a number of these species enabled the comparative study of the genetic determinants of this wide lifestyle diversity. Here, we applied two complementary phylogenetic-based approaches using information from 19 Mycobacterium genomes to obtain a more comprehensive view of the evolution of this genus. First, we inferred the phylogenetic relationships using two new approaches, one based on a Mycobacterium-specific amino acid substitution matrix and the other on a gene content dissimilarity matrix. Then, we utilized our recently developed gain-and-death stochastic models to study gene turnover dynamics in this genus in a maximum-likelihood framework. We uncovered a scenario that differs markedly from traditional 16S rRNA data and improves upon recent phylogenomic approaches. We also found that the rates of gene gain and death are high and unevenly distributed both across species and across gene families, further supporting the utility of the new models of rate heterogeneity applied in a phylogenetic context. Finally, the functional annotation of the most expanded or contracted gene families revealed that the transposable elements and the fatty acid metabolism-related gene families are the most important drivers of gene content evolution in Mycobacterium. PMID:24904011

  15. Antimicrobial resistance and virulence genes in enterococci from wild game meat in Spain.

    PubMed

    Guerrero-Ramos, Emilia; Cordero, Jorge; Molina-González, Diana; Poeta, Patrícia; Igrejas, Gilberto; Alonso-Calleja, Carlos; Capita, Rosa

    2016-02-01

    A total of 55 enterococci (45 Enterococcus faecium, 7 Enterococcus faecalis, and three Enterococcus durans) isolated from the meat of wild game animals (roe deer, boar, rabbit, pheasant, and pigeon) in North-Western Spain were tested for susceptibility to 14 antimicrobials by the disc diffusion method. All strains showed a multi-resistant phenotype (resistance to between three and 10 antimicrobials). The strains exhibited high percentages of resistance to erythromycin (89.1%), tetracycline (67.3%), ciprofloxacin (92.7%), nitrofurantoin (67.3%), and quinupristin-dalfopristin (81.8%). The lowest values (9.1%) were observed for high-level resistance to gentamicin, kanamycin, and streptomycin. The average number of resistances per strain was 5.8 for E. faecium isolates, 7.9 for E. faecalis, and 5.7 for E. durans. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. A total of 15 (57.7%) of the 26 vancomycin-resistant isolates harboured the vanA gene. Other resistance genes detected included vanB, erm(B) and/or erm(C), tet(L) and/or tet(M), acc(6')-aph(2″), and aph(3')-IIIa in strains resistant to vancomycin, erythromycin, tetracycline, gentamicin, and kanamycin, respectively. Specific genes of the Tn5397 transposon were detected in 54.8% of the tet(M)-positive enterococci. Nine virulence factors (gelE, agg, ace, cpd, frs, esp, hyl, efaAfs and efaAfm) were studied. All virulence genes, with the exception of the frs gene, were found to be present in the enterococcal isolates. At least one virulence gene was detected in 20.0% of E. faecium, 71.4% of E. faecalis and 33.3% of E. durans isolates, with ace and cpd being the most frequently detected genes (6 isolates each). This suggests that wild game meat might play a role in the spreading through the food chain of enterococci with antimicrobial resistance and virulence determinants to humans.

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

    PubMed

    Schneeberger, M; Brodard, I; Overesch, G

    2015-04-01

    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

  17. Virulence factors genes of Staphylococcus spp. isolated from caprine subclinical mastitis.

    PubMed

    Salaberry, Sandra Renata Sampaio; Saidenberg, André Becker Simões; Zuniga, Eveline; Melville, Priscilla Anne; Santos, Franklin Gerônimo Bispo; Guimarães, Ednaldo Carvalho; Gregori, Fábio; Benites, Nilson Roberti

    2015-08-01

    The aim of this study was to investigate genes involved in adhesion expression, biofilm formation, and enterotoxin production in isolates of Staphylococcus spp. from goats with subclinical mastitis and associate these results with the staphylococcal species. One hundred and twenty-four isolates were identified and polymerase chain reaction (PCR) was performed to detect the following genes: cna, ebpS, eno, fib, fnbA, fnbB, bap, sea, seb, sec, sed and see. The most commonly Staphylococcus species included S. epidermidis, S. lugdunensis, S. chromogenes, S. capitis ss capitis and S. intermedius. With the exception of fnbB, the genes were detected in different frequencies of occurrence in 86.3% of the Staphylococcus spp. isolates. Eno (73.2%) and bap (94.8%) were more frequently detected in coagulase-negative staphylococci (CNS); ebpS (76%), fib (90.9%) and fnbA (87%) were the most frequent genes in coagulase-positive staphylococci (CPS). Regarding enterotoxins, genes sed (28.2%) and see (24.2%) had a higher frequency of occurrence; sec gene was more frequently detected in CPS (58.8%). There was no association between the presence of the genes and the Staphylococcus species. Different virulence factors genes can be detected in caprine subclinical mastitis caused by CNS and CPS. The knowledge of the occurrence of these virulence factors is important for the development of effective control and prevention measures of subclinical mastitis caused by CNS and CPS in goats.

  18. Virulence factors genes of Staphylococcus spp. isolated from caprine subclinical mastitis.

    PubMed

    Salaberry, Sandra Renata Sampaio; Saidenberg, André Becker Simões; Zuniga, Eveline; Melville, Priscilla Anne; Santos, Franklin Gerônimo Bispo; Guimarães, Ednaldo Carvalho; Gregori, Fábio; Benites, Nilson Roberti

    2015-08-01

    The aim of this study was to investigate genes involved in adhesion expression, biofilm formation, and enterotoxin production in isolates of Staphylococcus spp. from goats with subclinical mastitis and associate these results with the staphylococcal species. One hundred and twenty-four isolates were identified and polymerase chain reaction (PCR) was performed to detect the following genes: cna, ebpS, eno, fib, fnbA, fnbB, bap, sea, seb, sec, sed and see. The most commonly Staphylococcus species included S. epidermidis, S. lugdunensis, S. chromogenes, S. capitis ss capitis and S. intermedius. With the exception of fnbB, the genes were detected in different frequencies of occurrence in 86.3% of the Staphylococcus spp. isolates. Eno (73.2%) and bap (94.8%) were more frequently detected in coagulase-negative staphylococci (CNS); ebpS (76%), fib (90.9%) and fnbA (87%) were the most frequent genes in coagulase-positive staphylococci (CPS). Regarding enterotoxins, genes sed (28.2%) and see (24.2%) had a higher frequency of occurrence; sec gene was more frequently detected in CPS (58.8%). There was no association between the presence of the genes and the Staphylococcus species. Different virulence factors genes can be detected in caprine subclinical mastitis caused by CNS and CPS. The knowledge of the occurrence of these virulence factors is important for the development of effective control and prevention measures of subclinical mastitis caused by CNS and CPS in goats. PMID:26026835

  19. Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains.

    PubMed

    Kim, Min-Ju; Han, Jae-Kwang; Park, Jong-Su; Lee, Jin-Sung; Lee, Soon-Ho; Cho, Joon-Il; Kim, Keun-Sung

    2015-06-01

    Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen Bacillus thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal diseases are attributed to enterotoxins. Sixteen reference strains of B. cereus and nine commercial and 12 reference strains of B. thuringiensis were screened by PCR for the presence of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR). These genes encode various enterotoxins and other virulence factors thought to play a role in infections of mammals. Amplicons were successfully generated from the strains of B. cereus and B. thuringiensis for each of these sequences, except the ces gene. Intriguingly, the majority of these B. cereus enterotoxin genes and other virulence factor genes appeared to be widespread among B. thuringiensis strains as well as B. cereus strains.

  20. Bistable expression of virulence genes in salmonella leads to the formation of an antibiotic-tolerant subpopulation.

    PubMed

    Arnoldini, Markus; Vizcarra, Ima Avalos; Peña-Miller, Rafael; Stocker, Nicolas; Diard, Médéric; Vogel, Viola; Beardmore, Robert E; Hardt, Wolf-Dietrich; Ackermann, Martin

    2014-08-01

    Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.

  1. Cytochrome c551 and the cytochrome c maturation pathway affect virulence gene expression in Bacillus cereus ATCC 14579.

    PubMed

    Han, Hesong; Sullivan, Thomas; Wilson, Adam C

    2015-02-01

    Loss of the cytochrome c maturation system in Bacillus cereus results in increased transcription of the major enterotoxin genes nhe, hbl, and cytK and the virulence regulator plcR. Increased virulence factor production occurs at 37°C under aerobic conditions, similar to previous findings in Bacillus anthracis. Unlike B. anthracis, much of the increased virulence gene expression can be attributed to loss of only c551, one of the two small c-type cytochromes. Additional virulence factor expression occurs with loss of resBC, encoding cytochrome c maturation proteins, independently of the presence of the c-type cytochrome genes. Hemolytic activity of strains missing either cccB or resBC is increased relative to that in the parental strain, while sporulation efficiency is unaffected in the mutants. Increased virulence gene expression in the ΔcccB and ΔresBC mutants occurs only in the presence of an intact plcR gene, indicating that this process is PlcR dependent. These findings suggest a new mode of regulation of B. cereus virulence and reveal intriguing similarities and differences in virulence regulation between B. cereus and B. anthracis.

  2. Expression of Streptococcus pneumoniae Virulence-Related Genes in the Nasopharynx of Healthy Children

    PubMed Central

    Sakai, Fuminori; Talekar, Sharmila J.; Klugman, Keith P.; Vidal, Jorge E.

    2013-01-01

    Colonization and persistence in the human nasopharynx are prerequisites for Streptococcus pneumoniae disease and carriage acquisition, which normally occurs during early childhood. Animal models and in vitro studies (i.e. cell adhesion and cell cytotoxicity assays) have revealed a number of colonization and virulence factors, as well as regulators, implicated in nasopharyngeal colonization and pathogenesis. Expression of genes encoding these factors has never been studied in the human nasopharynx. Therefore, this study analyzed expression of S. pneumoniae virulence-related genes in human nasopharyngeal samples. Our experiments first demonstrate that a density of ≥104 CFU/ml of S. pneumoniae cells in the nasopharynx provides enough DNA and RNA to amplify the lytA gene by conventional PCR and to detect the lytA message, respectively. A panel of 21 primers that amplified S. pneumoniae sequences was designed, and their specificity for S. pneumoniae sequences was analyzed in silico and validated against 20 related strains inhabitants of the human upper respiratory tract. These primers were utilized in molecular reactions to find out that all samples contained the genes ply, pavA, lytC, lytA, comD, codY, and mgrA, whereas nanA, nanB, pspA, and rrgB were present in ∼91–98% of the samples. Gene expression studies of these 11 targets revealed that lytC, lytA, pavA and comD were the most highly expressed pneumococcal genes in the nasopharynx whereas the rest showed a moderate to low level of expression. This is the first study to evaluate expression of virulence- and, colonization-related genes in the nasopharynx of healthy children and establishes the foundation for future gene expression studies during human pneumococcal disease. PMID:23825636

  3. Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

    PubMed Central

    Sanson, Misu; Makthal, Nishanth; Gavagan, Maire; Cantu, Concepcion; Olsen, Randall J.; Musser, James M.

    2015-01-01

    Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis. PMID:25824840

  4. Diagnostic Strategy for Identifying Avian Pathogenic Escherichia coli Based on Four Patterns of Virulence Genes

    PubMed Central

    Schaeffer, Brigitte; Brée, Annie; Mora, Azucena; Dahbi, Ghizlane; Biet, François; Oswald, Eric; Mainil, Jacques; Blanco, Jorge; Moulin-Schouleur, Maryvonne

    2012-01-01

    In order to improve the identification of avian pathogenic Escherichia coli (APEC) strains, an extensive characterization of 1,491 E. coli isolates was conducted, based on serotyping, virulence genotyping, and experimental pathogenicity for chickens. The isolates originated from lesions of avian colibacillosis (n = 1,307) or from the intestines of healthy animals (n = 184) from France, Spain, and Belgium. A subset (460 isolates) of this collection was defined according to their virulence for chicks. Six serogroups (O1, O2, O5, O8, O18, and O78) accounted for 56.5% of the APEC isolates and 22.5% of the nonpathogenic isolates. Thirteen virulence genes were more frequently present in APEC isolates than in nonpathogenic isolates but, individually, none of them could allow the identification of an isolate as an APEC strain. In order to take into account the diversity of APEC strains, a statistical analysis based on a tree-modeling method was therefore conducted on the sample of 460 pathogenic and nonpathogenic isolates. This resulted in the identification of four different associations of virulence genes that enables the identification of 70.2% of the pathogenic strains. Pathogenic strains were identified with an error margin of 4.3%. The reliability of the link between these four virulence patterns and pathogenicity for chickens was validated on a sample of 395 E. coli isolates from the collection. The genotyping method described here allowed the identification of more APEC isolates with greater reliability than the classical serotyping methods currently used in veterinary laboratories. PMID:22378905

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

    PubMed

    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.

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

    PubMed

    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. PMID:26253667

  7. Speciation and frequency of virulence genes of Enterococcus spp. isolated from rainwater tank samples in Southeast Queensland, Australia.

    PubMed

    Ahmed, W; Sidhu, J P S; Toze, S

    2012-06-19

    In this study, 212 Enterococcus isolates from 23 rainwater tank samples in Southeast Queensland (SEQ), Australia were identified to the species level. The isolates were also tested for the presence of 6 virulence genes associated with Enterococcus related infections. Among the 23 rainwater tank samples, 20 (90%), 10 (44%), 7 (30%), 5 (22%), 4 (17%), 2 (9%), and 1 (4%) samples yielded E. faecalis, E. mundtii, E. casseliflavus, E. faecium, E. hirae, E. avium, and E. durans, respectively. Among the 6 virulence genes tested, gelE and efaA were most prevalent, detected in 19 (83%) and 18 (78%) of 23 rainwater tank samples, respectively. Virulence gene ace was also detected in 14 (61%) rainwater tank samples followed by AS, esp (E. faecalis variant), and cylA genes which were detected in 3 (13%), 2 (9%), and 1 (4%) samples, respectively. In all, 120 (57%) Enterococcus isolates from 20 rainwater tank samples harbored virulence genes. Among these tank water samples, Enterococcus spp. from 5 (25%) samples harbored a single virulence gene and 15 (75%) samples were harboring two or more virulence genes. The significance of these strains in terms of health implications remains to be assessed. The potential sources of these strains need to be identified for the improved management of captured rainwater quality. Finally, it is recommended that Enterococcus spp. should be used as an additional fecal indicator bacterium in conjunction with E. coli for the microbiological assessment of rainwater tanks.

  8. Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence.

    PubMed

    Moyrand, Frédérique; Fontaine, Thierry; Janbon, Guilhem

    2007-05-01

    The polysaccharidic capsule is the main virulence factor of Cryptococcus neoformans. It primarily comprised of two polysaccharides: glucuronoxylomannan (GXM, 88% of the capsule mass) and galactoxylomannan (GalXM, 7% of the capsule mass). We constructed a large collection of mutant strains in which genes potentially involved in capsule biosynthesis were deleted. We used a new post-genomic approach to study the virulence of the strains. Primers specific for unique tags associated with the disruption cassette were used in a real-time PCR virulence assay to measure the fungal burden of each strain in different organs of mice in multi-infection experiments. With this very sensitive assay, we identified a putative UDP-glucose epimerase (Uge1p) and a putative UDP-galactose transporter (Ugt1p) essential for C. neoformans virulence. The uge1Delta and ugt1Delta strains are temperature sensitive and do not produce GalXM but synthesize a larger capsule. These mutant strains (GalXM negative, GXM positive) are not able to colonize the brain even at the first day of infection whereas GXM-negative strains (GalXM positive) can still colonize the brain, although less efficiently than the wild-type strain.

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

    PubMed

    Furian, Thales Quedi; Borges, Karen Apellanis; Laviniki, Vanessa; Rocha, Silvio Luis da Silveira; de Almeida, Camila Neves; do Nascimento, Vladimir Pinheiro; Salle, Carlos Tadeu Pippi; Moraes, Hamilton Luiz de Souza

    2016-01-01

    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. PMID:26887247

  10. Genome-Wide Identification of Pseudomonas aeruginosa Virulence-Related Genes Using a Caenorhabditis elegans Infection Model

    PubMed Central

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

    2012-01-01

    Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes. PMID:22911607

  11. Citrate synthase mutants of Agrobacterium are attenuated in virulence and display reduced vir gene induction.

    PubMed

    Suksomtip, Maneewan; Liu, Pu; Anderson, Tamara; Tungpradabkul, Sumalee; Wood, Derek W; Nester, Eugene W

    2005-07-01

    A citrate synthase (CS) deletion mutant of Agrobacterium tumefaciens C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to Escherichia coli and 77% identical to Brucella melitensis. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in Sinorhizobium. The CS mutation resulted in a 10-fold reduction in vir gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive virG [virG(Con)] locus was introduced into this mutant, the level of vir gene induction was restored to nearly wild-type level. Further, the virG(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the virG(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of A. tumefaciens C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in Sinorhizobium. The function of these loci remains unknown. PMID:15995199

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

    PubMed

    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.

  13. Reduced Set of Virulence Genes Allows High Accuracy Prediction of Bacterial Pathogenicity in Humans

    PubMed Central

    Iraola, Gregorio; Vazquez, Gustavo; Spangenberg, Lucía; Naya, Hugo

    2012-01-01

    Although there have been great advances in understanding bacterial pathogenesis, there is still a lack of integrative information about what makes a bacterium a human pathogen. The advent of high-throughput sequencing technologies has dramatically increased the amount of completed bacterial genomes, for both known human pathogenic and non-pathogenic strains; this information is now available to investigate genetic features that determine pathogenic phenotypes in bacteria. In this work we determined presence/absence patterns of different virulence-related genes among more than finished bacterial genomes from both human pathogenic and non-pathogenic strains, belonging to different taxonomic groups (i.e: Actinobacteria, Gammaproteobacteria, Firmicutes, etc.). An accuracy of 95% using a cross-fold validation scheme with in-fold feature selection is obtained when classifying human pathogens and non-pathogens. A reduced subset of highly informative genes () is presented and applied to an external validation set. The statistical model was implemented in the BacFier v1.0 software (freely available at ), that displays not only the prediction (pathogen/non-pathogen) and an associated probability for pathogenicity, but also the presence/absence vector for the analyzed genes, so it is possible to decipher the subset of virulence genes responsible for the classification on the analyzed genome. Furthermore, we discuss the biological relevance for bacterial pathogenesis of the core set of genes, corresponding to eight functional categories, all with evident and documented association with the phenotypes of interest. Also, we analyze which functional categories of virulence genes were more distinctive for pathogenicity in each taxonomic group, which seems to be a completely new kind of information and could lead to important evolutionary conclusions. PMID:22916122

  14. Orthopoxvirus Genes That Mediate Disease Virulence and Host Tropism

    PubMed Central

    Shchelkunov, Sergei N.

    2012-01-01

    In the course of evolution, viruses have developed various molecular mechanisms to evade the defense reactions of the host organism. When understanding the mechanisms used by viruses to overcome manifold defense systems of the animal organism, represented by molecular factors and cells of the immune system, we would not only comprehend better but also discover new patterns of organization and function of these most important reactions directed against infectious agents. Here, study of the orthopoxviruses pathogenic for humans, such as variola (smallpox), monkeypox, cowpox, and vaccinia viruses, may be most important. Analysis of the experimental data, presented in this paper, allows to infer that variola virus and other orthopoxviruses possess an unexampled set of genes whose protein products efficiently modulate the manifold defense mechanisms of the host organisms compared with the viruses from other families. PMID:22899927

  15. Distribution of virulence genes in clinical and environmental isolates of Aeromonas spp.

    PubMed

    Chacón, M R; Figueras, M J; Castro-Escarpulli, G; Soler, L; Guarro, J

    2003-01-01

    The distribution and phenotypic activity of the genes encoding for serine protease, glycerophospholipid-cholesterol acyltransferase, lipases, aerolysin/hemolysin and DNases were investigated in 234 isolates identified by 16S rDNA-RFLP representing all the species of Aeromonas. The former three genes were found to be highly conserved among the genus. Aerolysin/hemolysin and DNase genes and beta-hemolytic activity were significantly more frequent in clinical than in environmental isolates. Aerolysin/hemolysin and serine protease genes were present in all beta-hemolytic strains supporting serine protease as possibly important for the activation of the former gene. The high prevalence of virulence factors in clinical isolates indicates that they may play a role in the mechanisms of pathogenesis of these microorganisms.

  16. Nontuberculous mycobacterial osteomyelitis

    PubMed Central

    Bi, Sheng; Hu, Fei-Shu; Yu, Hai-Ying; Xu, Kai-Jin; Zheng, Bei-Wen; Ji, Zhong-Kang; Li, Jun-Jie; Deng, Mei; Hu, Hai-Yang; Sheng, Ji-Fang

    2015-01-01

    Abstract Osteomyelitis caused by nontuberculous mycobacteria (NTM) can have severe consequences and a poor prognosis. Physicians therefore need to be alert to this condition, especially in immunocompromised patients. Although the pathogenesis of NTM osteomyelitis is still unclear, studies in immunodeficient individuals have revealed close relationships between NTM osteomyelitis and defects associated with the interleukin-12–interferon-γ–tumor necrosis factor-α axis, as well as human immunodeficiency virus infection, various immunosuppressive conditions, and diabetes mellitus. Culture and species identification from tissue biopsies or surgical debridement tissue play crucial roles in diagnosing NTM osteomyelitis. Suitable imaging examinations are also important. Adequate surgical debridement and the choice of appropriate, combined antibiotics for long-term anti-mycobacterial chemotherapy, based on in vitro drug susceptibility tests, are the main therapies for these bone infections. Bacillus Calmette–Guerin vaccination might have limited prophylactic value. The use of multiple drugs and long duration of treatment mean that the therapeutic process needs to be monitored closely to detect potential side effects. Adequate duration of anti-mycobacterial chemotherapy together with regular monitoring with blood and imaging tests are key factors determining the recovery outcome in patients with NTM osteomyelitis. PMID:25915177

  17. Silencing a candidate nematode effector gene corresponding to the tomato resistance gene Mi-1 leads to acquisition of virulence.

    PubMed

    Gleason, Cynthia A; Liu, Qingli L; Williamson, Valerie M

    2008-05-01

    The Mi-1 gene in tomato confers effective resistance against several species of root-knot nematode, including Meloidogyne javanica. A strain of M. javanica that can reproduce on tomato with Mi-1 was obtained from a culture of an avirulent strain after greenhouse selection. DNA blots and amplified fragment length polymorphism (AFLP) analysis indicated that the two nematode strains are closely related. Expression patterns visualized as cDNA AFLPs were nearly identical except for a cDNA fragment, Cg-1, that was present in the avirulent strain but not in the virulent strain. DNA blots showed that Cg-1 corresponds to a member of a small gene family with one or more copies missing in the virulent strain compared with the avirulent strain. Except for the presence of a histone stem loop near the 3' end of the transcript, Cg-1 shows no similarity to other sequences in GenBank. The longest open reading frame is 32 amino acids and initiates at the fourth AUG in the predicted transcript. When nematode juveniles of the Mi-1-avirulent strain were soaked in dsRNA corresponding to part of the predicted Cg-1 transcript, they produced progeny that were virulent on tomato carrying the Mi-1 gene, strongly suggesting that Cg-1 is required in the nematode for Mi-1-mediated resistance.

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

    PubMed

    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.

  19. The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens.

    PubMed

    Hwang, Hau-Hsuan; Yang, Fong-Jhih; Cheng, Tun-Fang; Chen, Yi-Chun; Lee, Ying-Ling; Tsai, Yun-Long; Lai, Erh-Min

    2013-09-01

    The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a wide range of plant species. The neoplastic growth at the infection sites is caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A. tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly induced by AS under this growth condition but also constitutively expressed and moderately upregulated by AS at neutral pH 7.0. We found that the promoter activities and protein levels of several AS-induced vir genes increased in the tzs deletion mutant, a mutant with decreased tumorigenesis and transient transformation efficiencies, in Arabidopsis roots. During AS induction and infection of Arabidopsis roots, the tzs deletion mutant conferred impaired growth, which could be rescued by genetic complementation and supplementing exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities and Vir protein accumulation in both the wild-type and tzs mutant bacteria with AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in regulating AS-induced vir gene expression and, therefore, affect bacterial growth and virulence during A. tumefaciens infection. PMID:23593941

  20. The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens.

    PubMed

    Hwang, Hau-Hsuan; Yang, Fong-Jhih; Cheng, Tun-Fang; Chen, Yi-Chun; Lee, Ying-Ling; Tsai, Yun-Long; Lai, Erh-Min

    2013-09-01

    The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a wide range of plant species. The neoplastic growth at the infection sites is caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A. tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly induced by AS under this growth condition but also constitutively expressed and moderately upregulated by AS at neutral pH 7.0. We found that the promoter activities and protein levels of several AS-induced vir genes increased in the tzs deletion mutant, a mutant with decreased tumorigenesis and transient transformation efficiencies, in Arabidopsis roots. During AS induction and infection of Arabidopsis roots, the tzs deletion mutant conferred impaired growth, which could be rescued by genetic complementation and supplementing exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities and Vir protein accumulation in both the wild-type and tzs mutant bacteria with AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in regulating AS-induced vir gene expression and, therefore, affect bacterial growth and virulence during A. tumefaciens infection.

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

    PubMed

    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.

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

    PubMed

    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.

  3. Virulence Gene Profiles of Multidrug-Resistant Pseudomonas aeruginosa Isolated From Iranian Hospital Infections

    PubMed Central

    Fazeli, Nastaran; Momtaz, Hassan

    2014-01-01

    Background: The most common hospital-acquired pathogen is Pseudomonas aeruginosa. It is a multidrug resistant bacterium causing systemic infections. Objectives: The present study was carried out in order to investigate the distribution of virulence factors and antibiotic resistance properties of Pseudomonas aeruginosa isolated from various types of hospital infections in Iran. Patients and Methods: Two-hundred and seventeen human infection specimens were collected from Baqiyatallah and Payambaran hospitals in Tehran, Iran. The clinical samples were cultured immediately and samples positive for P. aeruginosa were analyzed for the presence of antibiotic resistance and bacterial virulence genes using PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed using disk diffusion methodology with Müeller–Hinton agar. Results: Fifty-eight out of 127 (45.66%) male infection specimens and 44 out of 90 (48.88%) female infection specimens harbored P. aeruginosa. Also, 65% (in male specimens) and 21% (in female specimens) of respiratory system infections were positive for P. aeruginosa, which was a high rate. The genes encoding exoenzyme S (67.64%) and phospholipases C (45.09%) were the most common virulence genes found among the strains. The incidences of various β-lactams encoding genes, including blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, and blaVEB were 94.11%, 16.66%, 15.68%, 18.62%, 21.56%, and 17.64%, respectively. The most commonly detected fluoroquinolones encoding gene was gyrA (15. 68%). High resistance levels to penicillin (100%), tetracycline (90.19%), streptomycin (64.70%), and erythromycin (43.13%) were observed too. Conclusions: Our findings should raise awareness about antibiotic resistance in hospitalized patients in Iran. Clinicians should exercise caution in prescribing antibiotics, especially in cases of human infections. PMID:25763199

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

    PubMed

    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.

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

    PubMed

    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. PMID:27166215

  6. Virulence genes of Helicobacter pylori in the Dominican Republic.

    PubMed

    Shiota, Seiji; Cruz, Modesto; Abreu, José A Jiménez; Mitsui, Takahiro; Terao, Hideo; Disla, Mildre; Iwatani, Shun; Nagashima, Hiroyuki; Matsuda, Miyuki; Uchida, Tomohisa; Tronilo, Lourdes; Rodríguez, Eduardo; Yamaoka, Yoshio

    2014-09-01

    Although the incidence of gastric cancer in the Dominican Republic is not high, the disease remains a significant health problem. We first conducted a detailed analysis of Helicobacter pylori status in the Dominican Republic. In total, 158 patients (103 females and 55 males; mean age 47.1±16.2 years) were recruited. The status of H. pylori infection was determined based on four tests: rapid urease test, culture test, histological test and immunohistochemistry. The status of cagA and vacA genotypes in H. pylori was examined using PCR and gene sequencing. The overall prevalence of H. pylori infection was 58.9 %. No relationship was found between the H. pylori infection rate and the age range of 17-91 years. Even in the youngest group (patients aged <29 years), the H. pylori infection rate was 62.5 %. Peptic ulcer was found in 23 patients and gastric cancer was found in one patient. The H. pylori infection rate in patients with peptic ulcer was significantly higher than that in patients with gastritis (82.6 versus 54.5 %, P<0.01). The cagA-positive/vacA s1m1 genotype was the most prevalent (43/64, 67.2 %). Compared with H. pylori-negative patients, H. pylori-positive patients showed more severe gastritis. Furthermore, the presence of cagA was related to the presence of more severe gastritis. All CagA-positive strains had Western-type CagA. In conclusion, we found that H. pylori infection is a risk factor for peptic ulcer in the Dominican Republic. Patients with cagA-positive H. pylori could be at higher risk for severe inflammation and atrophy.

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

    PubMed

    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.

  8. Porcine E. coli: Virulence-Associated Genes, Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

    PubMed Central

    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. PMID:23658605

  9. Enteroaggregative Escherichia coli from humans and animals differ in major phenotypical traits and virulence genes.

    PubMed

    Uber, Ana Paula; Trabulsi, Luiz R; Irino, Kinue; Beutin, Lothar; Ghilardi, Angela C R; Gomes, Tânia A T; Liberatore, Ana Maria A; de Castro, Antônio F P; Elias, Waldir P

    2006-03-01

    Enteroaggregative Escherichia coli (EAEC) is characterized by the expression of the aggregative adherence pattern to cultured epithelial cells. In this study, we determined the phenotypic and genotypic relationships among 86 EAEC strains of human and animal (calves, piglets and horses) feces. Serotypes and the presence of EAEC virulence markers were determined, and these results were associated with ribotyping. Strains harboring aggR (typical EAEC) of human origin were found carrying several of the searched markers, while atypical EAEC harbored none or a few markers. The strains of animal origin were classified as atypical EAEC (strains lacking aggR) and harbored only irp2 or shf. Strains from humans and animals belonged to several different serotypes, although none of them prevailed. Sixteen ribotypes were determined, and there was no association with virulence genes profiles or serotypes. Relationship was not found among the strains of this study, and the assessed animals may not represent a reservoir of human pathogenic typical EAEC.

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

    PubMed

    Kurushima, Jun; Kuwae, Asaomi; Abe, Akio

    2012-01-01

    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.

  11. Positive regulation of Shigella flexneri virulence genes by integration host factor.

    PubMed Central

    Porter, M E; Dorman, C J

    1997-01-01

    In Shigella flexneri, expression of the plasmid-encoded virulence genes is regulated via a complex cascade involving DNA topology, specific transactivators, and the nucleoid-associated protein H-NS, which represses transcription under inappropriate environmental conditions. We have investigated the involvement of a second nucleoid-associated protein, integration host factor (IHF), in virulence gene expression. We found that transcription of the invasion-specific genes is repressed in a strain harboring an ihfA mutation, particularly on entry into the stationary phase. Expression of the virB gene, whose product is required for the activation of these structural genes, is also enhanced by IHF in the stationary phase. In contrast, the virF gene, which encodes an activator of virB, is stimulated by IHF in both the logarithmic and early stationary phases of growth, as is another virF-regulated gene, icsA. We have identified regions of the virF, virB, and icsA promoters which form IHF-dependent protein-DNA complexes in vitro and have located sequences within these regions with similarity to the consensus IHF binding site. Moreover, results from experiments in which the virF or virB gene was expressed constitutively confirm that IHF has a direct input at the level of both virF and virB transcription. Finally, we provide evidence that at the latter promoter, the primary role of IHF may be to overcome repression by the H-NS protein. To our knowledge, this is the first report of a role for IHF in controlling gene expression in S. flexneri. PMID:9352898

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

    PubMed Central

    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. PMID:26372106

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

    PubMed

    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.

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

    USGS Publications Warehouse

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

    1999-01-01

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

  15. 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.

    PubMed

    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. PMID:27625639

  16. 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

    PubMed Central

    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.

  17. 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

    PubMed Central

    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. PMID:27625639

  18. Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli.

    PubMed

    Baranzoni, Gian Marco; Fratamico, Pina M; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-01-01

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c, the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections. PMID:27148249

  19. Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

    DOE PAGES

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-04-21

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using amore » Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. Furthermore, the present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.« less

  20. Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-01-01

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections. PMID:27148249

  1. The role of the st313-td gene in virulence of Salmonella Typhimurium ST313.

    PubMed

    Herrero-Fresno, Ana; Wallrodt, Inke; Leekitcharoenphon, Pimlapas; Olsen, John Elmerdahl; Aarestrup, Frank M; Hendriksen, Rene S

    2014-01-01

    Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02-03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.

  2. Drug Targets in Mycobacterial Sulfur Metabolism

    PubMed Central

    Bhave, Devayani P.; Muse, Wilson B.; Carroll, Kate S.

    2011-01-01

    The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress in the development of inhibitors of sulfur metabolism enzymes. PMID:17970225

  3. Rational engineering of a virulence gene from Mycobacterium tuberculosis facilitates proteomic analysis of a natural protein N-terminus

    PubMed Central

    Reyna, Cristal; Mba Medie, Felix; Champion, Matthew M.; Champion, Patricia A.

    2016-01-01

    Mass spectrometry (MS) for the detection of proteins is an indispensable tool for evaluating the biological processes of the proteome. Proteomics frequently requires proteolysis of proteins into peptide fragments. Proteins can be refractory to ideal proteolysis at the sequence level rendering them difficult to analyze by routine proteomics methods. EsxA (ESAT-6, Early Secreted Antigen, 6kDa) is a major virulence determinant of Mycobacterium tuberculosis, the cause of human tuberculosis. EsxA is routinely used to evaluate mycobacterial virulence in the laboratory and as a biomarker for tuberculosis in humans. The sequence of EsxA hinders deeper MS analysis beyond routine detection. Here we engineer the sequence of EsxA to add desirable tryptic properties aimed at improving complex MS analysis. We demonstrate that EsxA variants are amenable to MS analysis and remain functional in established in vitro and ex vivo assays of Esx-1-function. We provide the first demonstration of molecular engineering to specifically improve MS analysis of individual microbial proteins. PMID:27625110

  4. Rational engineering of a virulence gene from Mycobacterium tuberculosis facilitates proteomic analysis of a natural protein N-terminus.

    PubMed

    Reyna, Cristal; Mba Medie, Felix; Champion, Matthew M; Champion, Patricia A

    2016-01-01

    Mass spectrometry (MS) for the detection of proteins is an indispensable tool for evaluating the biological processes of the proteome. Proteomics frequently requires proteolysis of proteins into peptide fragments. Proteins can be refractory to ideal proteolysis at the sequence level rendering them difficult to analyze by routine proteomics methods. EsxA (ESAT-6, Early Secreted Antigen, 6kDa) is a major virulence determinant of Mycobacterium tuberculosis, the cause of human tuberculosis. EsxA is routinely used to evaluate mycobacterial virulence in the laboratory and as a biomarker for tuberculosis in humans. The sequence of EsxA hinders deeper MS analysis beyond routine detection. Here we engineer the sequence of EsxA to add desirable tryptic properties aimed at improving complex MS analysis. We demonstrate that EsxA variants are amenable to MS analysis and remain functional in established in vitro and ex vivo assays of Esx-1-function. We provide the first demonstration of molecular engineering to specifically improve MS analysis of individual microbial proteins. PMID:27625110

  5. Current European Labyrinthula zosterae Are Not Virulent and Modulate Seagrass (Zostera marina) Defense Gene Expression

    PubMed Central

    Brakel, Janina; Werner, Franziska Julie; Tams, Verena; Reusch, Thorsten B. H.; Bockelmann, Anna-Christina

    2014-01-01

    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. PMID:24691450

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

    PubMed

    Brakel, Janina; Werner, Franziska Julie; Tams, Verena; Reusch, Thorsten B H; Bockelmann, Anna-Christina

    2014-01-01

    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.

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

    PubMed

    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 (<1.0%) of the potential EPEC isolates were found to carry the EAF plasmid. The potential EPEC isolates mainly belonged to E. coli phylogenetic groups B1 or B2, and carried the β intimin subtype. DNA fingerprint analyses of the potential EPEC strains indicated that the isolates belonged to several genetically diverse groups, although clonal isolates were frequently detected. While the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potential EPEC strains can be found in marine beach water and their presence needs to be considered as one of the factors used in decisions concerning beach closures. PMID:20643468

  8. A mutation in the aroE gene affects pigment production, virulence, and chemotaxis in Xanthomonas oryzae pv. oryzae.

    PubMed

    Kim, Hong-Il; Noh, Tae-Hwan; Lee, Chang-Soo; Park, Young-Jin

    2015-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice. To study its function, a random insertion mutation library of Xoo was constructed using the Tn5 transposon. A mutant strain with decreased virulence against the susceptible rice cultivar IR24 was isolated from the library (aroE mutant), which also had extremely low pigment production. Thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) and sequence analysis of the mutant revealed that the transposon was inserted into the aroE gene (encoding shikimate dehydrogenase). To investigate gene expression changes in the pigment- and virulence-deficient mutant, DNA microarray analysis was performed, which showed downregulation of 20 genes involved in the chemotaxis of Xoo. Our findings reveal that mutation of the aroE gene affects virulence and pigment production, as well as expression of genes involved in Xoo chemotaxis.

  9. Polynucleotide phosphorylase negatively controls spv virulence gene expression in Salmonella enterica.

    PubMed

    Ygberg, Sofia Eriksson; Clements, Mark O; Rytkönen, Anne; Thompson, Arthur; Holden, David W; Hinton, Jay C D; Rhen, Mikael

    2006-02-01

    Mutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by the pnp gene) in Salmonella enterica serovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA 99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression of Salmonella plasmid virulence (spv) genes under in vitro growth conditions that allow induction of spv expression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which included spvABC, rtcB, entC, and STM2236. Mutational inactivation of the spvR regulator diminished the increased expression of spv observed in the pnp mutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of the pnp mutant in BALB/c mice was dependent on spvR as well. Combined, our results support the idea that in S. enterica PNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection. PMID:16428774

  10. Metabolomics: Applications and Promise in Mycobacterial Disease

    PubMed Central

    Banoei, Mohammad Mehdi; Winston, Brent W.; Schraufnagel, Dean E.

    2015-01-01

    Until recently, the study of mycobacterial diseases was trapped in culture-based technology that is more than a century old. The use of nucleic acid amplification is changing this, and powerful new technologies are on the horizon. Metabolomics, which is the study of sets of metabolites of both the bacteria and host, is being used to clarify mechanisms of disease, and can identify changes leading to better diagnosis, treatment, and prognostication of mycobacterial diseases. Metabolomic profiles are arrays of biochemical products of genes in their environment. These complex patterns are biomarkers that can allow a more complete understanding of cell function, dysfunction, and perturbation than genomics or proteomics. Metabolomics could herald sweeping advances in personalized medicine and clinical trial design, but the challenges in metabolomics are also great. Measured metabolite concentrations vary with the timing within a condition, the intrinsic biology, the instruments, and the sample preparation. Metabolism profoundly changes with age, sex, variations in gut microbial flora, and lifestyle. Validation of biomarkers is complicated by measurement accuracy, selectivity, linearity, reproducibility, robustness, and limits of detection. The statistical challenges include analysis, interpretation, and description of the vast amount of data generated. Despite these drawbacks, metabolomics provides great opportunity and the potential to understand and manage mycobacterial diseases. PMID:26196272

  11. Inactivation of thyA in Staphylococcus aureus Attenuates Virulence and Has a Strong Impact on Metabolism and Virulence Gene Expression

    PubMed Central

    Kriegeskorte, Andre; Block, Desiree; Drescher, Mike; Windmüller, Nadine; Mellmann, Alexander; Baum, Cathrin; Neumann, Claudia; Lorè, Nicola Ivan; Bragonzi, Alessandra; Liebau, Eva; Hertel, Patrick; Seggewiss, Jochen; Becker, Karsten; Proctor, Richard A.; Peters, Georg

    2014-01-01

    ABSTRACT Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. PMID:25073642

  12. Correlation of Phenotypic Profiles Using Targeted Proteomics Identifies Mycobacterial Esx-1 Substrates

    PubMed Central

    2015-01-01

    The Esx/WXG-100 (ESAT-6/Wss) exporters are multiprotein complexes that promote protein translocation across the cytoplasmic membrane in a diverse range of pathogenic and nonpathogenic bacterial species. The Esx-1 (ESAT-6 System-1) system mediates virulence factor translocation in mycobacterial pathogens, including the human pathogen Mycobacterium tuberculosis. Although several genes have been associated with Esx-1-mediated transport and virulence, the contribution of individual Esx-1 genes to export is largely undefined. A unique aspect of Esx-1 export is that several substrates require each other for export/stability. We exploited substrate “codependency” to identify Esx-1 substrates. We simultaneously quantified changes in the levels of 13 Esx-1 proteins from both secreted and cytosolic protein fractions generated from 16 Esx-1-deficient Mycobacterium marinum strains in a single experiment using MRM/SRM targeted mass spectrometry. This expansion of measurable Esx-1 proteins allowed us to define statistical rules for assigning novel substrates using phenotypic profiles of known Esx-1 substrates. Using this approach, we identified three additional Esx-1 substrates encoded by the esx-1 region. Our studies begin to address how disruption of specific genes affects several proteins in the Esx-1 complex. Overall, our findings illuminate relationships between Esx-1 proteins and create a framework for the identification of secreted substrates applicable to other protein exporters and pathways. PMID:25106450

  13. Agrobacterium tumefaciens exoR controls acid response genes and impacts exopolysaccharide synthesis, horizontal gene transfer, and virulence gene expression.

    PubMed

    Heckel, Brynn C; Tomlinson, Amelia D; Morton, Elise R; Choi, Jeong-Hyeon; Fuqua, Clay

    2014-09-01

    Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ΔexoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression. PMID:24982308

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

    SciTech Connect

    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.

  15. Comparative transcriptome profiling reveals different expression patterns in Xanthomonas oryzae pv. oryzae strains with putative virulence-relevant genes.

    PubMed

    Zhang, Fan; Du, Zhenglin; Huang, Liyu; Vera Cruz, Casiana; Zhou, Yongli; Li, Zhikang

    2013-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight, which is a major rice disease in tropical Asian countries. An attempt has been made to investigate gene expression patterns of three Xoo strains on the minimal medium XOM2, PXO99 (P6) and PXO86 (P2) from the Philippines, and GD1358 (C5) from China, which exhibited different virulence in 30 rice varieties, with putative virulence factors using deep sequencing. In total, 4,781 transcripts were identified in this study, and 1,151 and 3,076 genes were differentially expressed when P6 was compared with P2 and with C5, respectively. Our results indicated that Xoo strains from different regions exhibited distinctly different expression patterns of putative virulence-relevant genes. Interestingly, 40 and 44 genes involved in chemotaxis and motility exhibited higher transcript alterations in C5 compared with P6 and P2, respectively. Most other genes associated with virulence, including exopolysaccharide (EPS) synthesis, Hrp genes and type III effectors, including Xanthomonas outer protein (Xop) effectors and transcription activator-like (TAL) effectors, were down-regulated in C5 compared with P6 and P2. The data were confirmed by real-time quantitative RT-PCR, tests of bacterial motility, and enzyme activity analysis of EPS and xylanase. These results highlight the complexity of Xoo and offer new avenues for improving our understanding of Xoo-rice interactions and the evolution of Xoo virulence.

  16. Comparative Transcriptome Profiling Reveals Different Expression Patterns in Xanthomonas oryzae pv. oryzae Strains with Putative Virulence-Relevant Genes

    PubMed Central

    Zhang, Fan; Du, Zhenglin; Huang, Liyu; Cruz, Casiana Vera; Zhou, Yongli; Li, Zhikang

    2013-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight, which is a major rice disease in tropical Asian countries. An attempt has been made to investigate gene expression patterns of three Xoo strains on the minimal medium XOM2, PXO99 (P6) and PXO86 (P2) from the Philippines, and GD1358 (C5) from China, which exhibited different virulence in 30 rice varieties, with putative virulence factors using deep sequencing. In total, 4,781 transcripts were identified in this study, and 1,151 and 3,076 genes were differentially expressed when P6 was compared with P2 and with C5, respectively. Our results indicated that Xoo strains from different regions exhibited distinctly different expression patterns of putative virulence-relevant genes. Interestingly, 40 and 44 genes involved in chemotaxis and motility exhibited higher transcript alterations in C5 compared with P6 and P2, respectively. Most other genes associated with virulence, including exopolysaccharide (EPS) synthesis, Hrp genes and type III effectors, including Xanthomonas outer protein (Xop) effectors and transcription activator-like (TAL) effectors, were down-regulated in C5 compared with P6 and P2. The data were confirmed by real-time quantitative RT-PCR, tests of bacterial motility, and enzyme activity analysis of EPS and xylanase. These results highlight the complexity of Xoo and offer new avenues for improving our understanding of Xoo-rice interactions and the evolution of Xoo virulence. PMID:23734193

  17. Antimicrobial susceptibility, virulence gene profiles and molecular subtypes of Salmonella Newport isolated from humans and other sources.

    PubMed

    Kuang, Dai; Xu, Xuebin; Meng, Jianghong; Yang, Xiaowei; Jin, Huiming; Shi, Weimin; Pan, Haijian; Liao, Ming; Su, Xudong; Shi, Xianmin; Zhang, Jianmin

    2015-12-01

    Salmonella Newport (S. Newport) is a major serotype associated with human salmonellosis. A total of 79 S. Newport recovered from humans and other sources in China were characterized for antimicrobial susceptibility, virulence gene profiles and molecular subtypes using pulsed field gel electrophoresis (PFGE). Approximately 63.3% of the isolates were susceptible to all of 16 antimicrobials tested. Nearly one third of the isolates (31.6%) were resistant to sulfisoxazole, 20.3% to tetracycline and 13.9% to nalidixic acid. Twelve isolates (15.2%) were resistant to three or more antimicrobials. Among 10 virulence genes detected, Salmonella pathogenicity island genes avrA, ssaQ, mgtC, siiD, and sopB and fimbrial gene bcfC were present in most of the isolates (93.7% to 100%). Overall, we observed nine distinct virulence gene profiles, three of which (VP1, VP2 and VP3) were most common (86.1%). A total of 56 PFGE patterns were identified and mainly grouped into seven clusters (A to G) with 80% pattern similarity. Isolates from aquatic product shared a high similarity with those from humans in several clusters, highlighting a potential risk of aquatic product as a source of S. Newport that infect humans. Furthermore, there was a strong association between certain PFGE clusters and virulence gene profiles, suggesting virulence subtyping can be a useful epidemiological tool to discriminate S. Newport isolates.

  18. Development of a DNA Microarray for Enterococcal Species, Virulence, and Antibiotic Resistance Gene Determinations among Isolates from Poultry▿

    PubMed Central

    Champagne, J.; Diarra, M. S.; Rempel, H.; Topp, E.; Greer, C. W.; Harel, J.; Masson, L.

    2011-01-01

    A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential. PMID:21335389

  19. Distribution of virulence-associated genes in a selection of Campylobacter isolates.

    PubMed

    Koolman, Leonard; Whyte, Paul; Burgess, Catherine; Bolton, Declan

    2015-05-01

    This study tested 24 Campylobacter isolates for the presence of 35 virulence genes using the polymerase chain reaction. The target genes included those involved in motility (flaA, flaB, flhA, flhB, flgB, flgE2, fliM, fliY), chemotaxis (cheA, cheB, cheR, cheW, cheY, cheZ), cell adhesion (cadF, dnaJ, jlpA, pldA, racR, virB11), invasion (iamA, ciaB, ceuE), cytotoxin production (cdtA, cdtB, cdtC, wlaN), capsule (kpsM), multidrug and bile resistance (cmeA, cmeB, cmeC), stress response/survival (katA, sodB), and the iron uptake system (cfrA, fur). The motility genes (with the exception of flaB), the CmeABC efflux system, cdtABC genes, and the sodB gene were commonly distributed among Campylobacter strains while the virB11 and wlaN genes were rarely detected. Interestingly, the findings suggest that flaB is not essential for full motility and C. coli lacking the flhA gene may be highly invasive. This study provides additional information on the distribution of Campylobacter virulence factors and the effect of their presence/absence on adhesion and invasion. It will inform future studies designed to elucidate the exact mechanisms of pathogenesis in Campylobacter.

  20. Genetic dissection of mycobacterial biofilms.

    PubMed

    Ojha, Anil K; Jacobs, William R; Hatfull, Graham F

    2015-01-01

    Our understanding of the biological principles of mycobacterial tolerance to antibiotics is crucial for developing shorter anti-tuberculosis regimens. Various in vitro approaches have been developed to identify the conditions that promote mycobacterial persistence against antibiotics. In our laboratories, we have developed a detergent-free in vitro growth model, in which mycobacteria spontaneously grow at the air-medium interface as self-organized multicellular structures, called biofilms. Mycobacterial biofilms harbor a subpopulation of drug tolerant persisters at a greater frequency than their planktonic counterpart. Importantly, development of these structures is genetically programmed, and defective biofilms of isogenic mutants harbor fewer persisters. Thus, genetic analysis of mycobacterial biofilms in vitro could potentially be a powerful tool to unravel the biology of drug tolerance in mycobacteria. In this chapter we describe a method for screening biofilm-defective mutants of mycobacteria in a 96-well format, which readily yields a clonally pure mutant for further studies. PMID:25779318

  1. Sub-lethal stress effects on virulence gene expression in Enterococcus faecalis.

    PubMed

    Lenz, Christian A; Hew Ferstl, Carrie M; Vogel, Rudi F

    2010-05-01

    Enterococci are ubiquitous lactic acid bacteria commonly associated with the human digestive tract as commensal organisms. Additionally, these organisms have a long history of use in foods improving flavor as well as providing protective mechanisms as either a probiotic or antimicrobial additive. However, Enterococcus faecalis accounts for up to 10% of all nosocomial infections of the bloodstream, wounds, urinary tract and heart. Knowledge about the regulation of virulence factors is limited and the involvement of environmental signals contributing to E. faecalis pathogenicity is poorly documented. In this study, two clinical E. faecalis isolates, TMW 2.63 and OG1RF, as well as one food isolate, TMW 2.629, were subjected to six sub-lethal food- and host-related stresses including 6.8% NaCl, 200 ppm nitrite, 51 degrees C, 80 MPa, pH 4.1 and 0.08% bile salts (cholic acid:chenodeoxycholic acid 1:1), respectively, reducing their growth rate to 10%. Relative gene expression of 15 stress and virulence-associated genes including dnaK, groEL, ctsR, clpPBCEX, gls24, efaAfs, ace, fsrB, gelE, sprE and cylB, was quantified by using real time PCR and Lightcycler((R)) technology (reference conditions: BHI broth, 37 degrees C, pH = 7.4). Apart from strain-dependent differences, sub-lethal environmental stress was capable of provoking significant alterations in the expression of virulence-associated genes in E. faecalis from clinical as well as food origins of isolation. These results help to avoid preconditioning enterococci in food production processes and to understand the complex mechanisms in E. faecalis' switch to pathogenicity. PMID:20227595

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

    PubMed

    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.

  3. Isolation of virulence genes directing surface glycosyl-phosphatidylinositol synthesis by functional complementation of Leishmania.

    PubMed Central

    Ryan, K A; Garraway, L A; Descoteaux, A; Turco, S J; Beverley, S M

    1993-01-01

    Trypanosomatid parasites of the genus Leishmania cause a spectrum of widespread tropical diseases. In the vertebrate host they reside within the macrophage phagolysosome; however, the mechanisms employed in this remarkable survival strategy are not well understood. Recent advances in the molecular genetics of these parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant lipophosphoglycan, an abundant glycosyl-phosphatidylinositol-anchored polysaccharide. LPG1, the gene product identified by complementation of the R2D2 mutant, appears to be a glycosyltransferase responsible for the addition of galactofuranosyl residues to the nascent lipophosphoglycan chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism. Images Fig. 5 PMID:8378337

  4. Contribution of CgPDR1-regulated genes in enhanced virulence of azole-resistant Candida glabrata.

    PubMed

    Ferrari, Sélène; Sanguinetti, Maurizio; Torelli, Riccardo; Posteraro, Brunella; Sanglard, Dominique

    2011-03-09

    In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1 upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance.

  5. Development and Validation of an Oligonucleotide Microarray for Detection of Multiple Virulence and Antimicrobial Resistance Genes in Escherichia coli†

    PubMed Central

    Bruant, Guillaume; Maynard, Christine; Bekal, Sadjia; Gaucher, Isabelle; Masson, Luke; Brousseau, Roland; Harel, Josée

    2006-01-01

    An oligonucleotide microarray detecting 189 Escherichia coli virulence genes or markers and 30 antimicrobial resistance genes was designed and validated using DNA from known reference strains. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging E. coli pathotypes and antimicrobial resistance, as well as for environmental, epidemiological, and phylogenetic studies including the evaluation of genome plasticity. PMID:16672535

  6. Occurrence of virulent genes among environmental isolates of Legionella pneumophila serogroup 1 strains from various parts of peninsular Malaysia.

    PubMed

    Arushothy, Revathy; Ahmad, Norazah

    2008-12-01

    Legionella pneumophila are intracellular pathogens, associated with human disease, attributed to the presence and absence of certain virulent genes. In this study, virulent gene loci (lvh and rtxA regions) associated with human disease were determined. Thirty-three cooling tower water isolates, isolated between 2004 to 2006, were analyzed for the presence of these genes by PCR method. Results showed that 19 of 33 (57.5%) of the L. pneumophila serogroup 1 isolates have both the genes. Six (18.2%) of the isolates have only the lvh gene and 2 (6.1%) of the isolates have only the rtxA gene. However, both genes were absent in 6 (18.2%) of the L. pneumophila isolates. The result of our study provides some insight into the presence of the disease causing L. pneumophila serogroup 1 in the environment. Molecular epidemiological studies will provide better understanding of the prevalence of the disease in Malaysia. PMID:19287368

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

    PubMed Central

    Behnke, Jerzy M.; Sharma, Aarti; Bearden, Rebecca; Al-Banna, Nadia

    2016-01-01

    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. PMID:27258021

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

    PubMed

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

    2011-08-01

    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.

  9. Diversity of antimicrobial resistance and virulence genes in methicillin-resistant non-Staphylococcus aureus staphylococci from veal calves.

    PubMed

    Argudín, M Angeles; Vanderhaeghen, Wannes; Butaye, Patrick

    2015-04-01

    In this study we determined whether methicillin-resistant non-Staphylococcus aureus (MRNAS) from veal calves may be a potential reservoir of antimicrobial-resistance and virulence genes. Fifty-eight MRNAS were studied by means of DNA-microarray and PCR for detection of antimicrobial resistance and virulence genes. The isolates carried a variety of antimicrobial-resistance genes [aacA-aphD, aadD, aph3, aadE, sat, spc, ampA, erm(A), erm(B), erm(C), erm(F), erm(T), lnu(A), msr(A)-msr(B), vga(A), mph(C), tet(K), tet(M), tet(L), cat, fexA, dfrA, dfrD, dfrG, dfrK, cfr, fusB, fosB, qacA, qacC, merA-merB]. Some isolates carried resistance genes without showing the corresponding resistance phenotype. Most MRNAS carried typical S. aureus virulence factors like proteases (sspP) and enterotoxins (seg) genes. Most Staphylococcus epidermidis isolates carried the arginine catabolic element, and nearly 40% of the Staphylococcus sciuri isolates carried leukocidins, and/or fibronectin-binding protein genes. MRNAS were highly multi-resistant and represent an important reservoir of antimicrobial resistance and virulence genes.

  10. Spatial Segregation of Virulence Gene Expression during Acute Enteric Infection with Salmonella enterica serovar Typhimurium

    PubMed Central

    Laughlin, Richard C.; Knodler, Leigh A.; Barhoumi, Roula; Payne, H. Ross; Wu, Jing; Gomez, Gabriel; Pugh, Roberta; Lawhon, Sara D.; Bäumler, Andreas J.; Steele-Mortimer, Olivia; Adams, L. Garry

    2014-01-01

    ABSTRACT To establish a replicative niche during its infectious cycle between the intestinal lumen and tissue, the enteric pathogen Salmonella enterica serovar Typhimurium requires numerous virulence genes, including genes for two type III secretion systems (T3SS) and their cognate effectors. To better understand the host-pathogen relationship, including early infection dynamics and induction kinetics of the bacterial virulence program in the context of a natural host, we monitored the subcellular localization and temporal expression of T3SS-1 and T3SS-2 using fluorescent single-cell reporters in a bovine, ligated ileal loop model of infection. We observed that the majority of bacteria at 2 h postinfection are flagellated, express T3SS-1 but not T3SS-2, and are associated with the epithelium or with extruding enterocytes. In epithelial cells, S. Typhimurium cells were surrounded by intact vacuolar membranes or present within membrane-compromised vacuoles that typically contained numerous vesicular structures. By 8 h postinfection, T3SS-2-expressing bacteria were detected in the lamina propria and in the underlying mucosa, while T3SS-1-expressing bacteria were in the lumen. Our work identifies for the first time the temporal and spatial regulation of T3SS-1 and -2 expression during an enteric infection in a natural host and provides further support for the concept of cytosolic S. Typhimurium in extruding epithelium as a mechanism for reseeding the lumen. PMID:24496791

  11. Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia coli.

    PubMed

    Lohrke, S M; Yang, H; Jin, S

    2001-06-01

    The ability to utilize Escherichia coli as a heterologous system in which to study the regulation of Agrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. coli containing a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization of lac promoter-driven virA and virG in combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of the virBp::lacZ fusion, and the level of virBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on vir gene expression was observed only in the presence of the chvE gene, suggesting that the glucose-binding protein of E. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the vir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for

  12. Identification of seven novel virulence genes from Xanthomonas citri subsp. citri by Tn5-based random mutagenesis.

    PubMed

    Song, Xue; Guo, Jing; Ma, Wen-xiu; Ji, Zhi-yuan; Zou, Li-fang; Chen, Gong-you; Zou, Hua-song

    2015-05-01

    To identify novel virulence genes, a mutant library of Xanthomonas citri subsp. citri 29-1 was produced using EZ-Tn5 transposon and the mutants were inoculated into susceptible grapefruit. Forty mutants with altered virulence phenotypes were identified. Nine of the mutants showed a complete loss of citrus canker induction, and the other 31 mutants resulted in attenuated canker symptoms. Southern blot analysis revealed that each of the mutants carried a single copy of Tn5. The flanking sequence was identified by plasmid rescue and 18 different ORFs were identified in the genome sequence. Of these 18 ORFs, seven had not been previously associated with the virulence of X. citri subsp. citri and were therefore confirmed by complementation analysis. Real-time PCR analysis showed that the seven genes were upregulated when the bacteria were grown in citrus plants, suggesting that the expression of these genes was essential for canker development. PMID:25935304

  13. Plasma is the main regulator of Staphylococcus epidermidis biofilms virulence genes transcription in human blood.

    PubMed

    França, Angela; Cerca, Nuno

    2016-03-01

    Staphylococcus epidermidis is frequently associated with the emergence of medical-device-associated bloodstream infections, due to its ability to form biofilms on the surface of vascular catheters. Although these biofilms may be in continuous contact with human blood, how S. epidermidis biofilm cells interact with blood and its cellular and soluble components is poorly understood. Herein, we evaluated biofilm structure, biofilm cells culturability and viability, and the transcription of a panel of genes associated with S. epidermidis biofilms virulence, upon interaction with whole human blood or plasma. Our results showed that although whole human blood caused significant alterations in biofilm structure and in the number of culturable and viable cells, plasma was the main regulator of the transcription of genes with central role in biofilm formation, maturation and immune evasion. These findings highlight the urgent need to intensify studies aiming to evaluate the impact of host soluble factors on S. epidermidis biofilms fitness and persistence. PMID:26712650

  14. Plasma is the main regulator of Staphylococcus epidermidis biofilms virulence genes transcription in human blood.

    PubMed

    França, Angela; Cerca, Nuno

    2016-03-01

    Staphylococcus epidermidis is frequently associated with the emergence of medical-device-associated bloodstream infections, due to its ability to form biofilms on the surface of vascular catheters. Although these biofilms may be in continuous contact with human blood, how S. epidermidis biofilm cells interact with blood and its cellular and soluble components is poorly understood. Herein, we evaluated biofilm structure, biofilm cells culturability and viability, and the transcription of a panel of genes associated with S. epidermidis biofilms virulence, upon interaction with whole human blood or plasma. Our results showed that although whole human blood caused significant alterations in biofilm structure and in the number of culturable and viable cells, plasma was the main regulator of the transcription of genes with central role in biofilm formation, maturation and immune evasion. These findings highlight the urgent need to intensify studies aiming to evaluate the impact of host soluble factors on S. epidermidis biofilms fitness and persistence.

  15. Improving UV resistance and virulence of Beauveria bassiana by genetic engineering with an exogenous tyrosinase gene.

    PubMed

    Shang, Yanfang; Duan, Zhibing; Huang, Wei; Gao, Qiang; Wang, Chengshu

    2012-01-01

    Insect pathogenic fungi like Beauveria bassiana have been developed as environmentally friendly biocontrol agents against arthropod pests. However, restrictive environmental factors, including solar ultraviolet (UV) radiation frequently lead to inconsistent field performance. To improve resistance to UV damage, we used Agrobacterium-mediated transformation to engineer B. bassiana with an exogenous tyrosinase gene. The results showed that the mitotically stable transformants produced larger amounts of yellowish pigments than the wild-type strain, and these imparted significantly increased UV-resistance. The virulence of the transgenic isolate was also significantly increased against the silkworm Bombyx mori and the mealworm Tenebrio molitor. This study demonstrated that genetic engineering of B. bassiana with a tyrosinase gene is an effective way to improve fungal tolerance against UV damage. PMID:22024554

  16. Homologues of Insecticidal Toxin Complex Genes in Yersinia enterocolitica Biotype 1A and Their Contribution to Virulence

    PubMed Central

    Tennant, Sharon M.; Skinner, Narelle A.; Joe, Angela; Robins-Browne, Roy M.

    2005-01-01

    Yersinia enterocolitica is an enteric pathogen that consists of six biotypes: 1A, 1B, 2, 3, 4, and 5. Strains of the latter five biotypes can carry a virulence plasmid, known as pYV, and several well-characterized chromosomally encoded virulence determinants. Y. enterocolitica strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and were once considered to be avirulent. There is growing epidemiological, clinical, and experimental evidence, however, to suggest that some biotype 1A strains are virulent and can cause gastrointestinal disease. To identify potential virulence genes of pathogenic strains of Y. enterocolitica biotype 1A, we used genomic subtractive hybridization to determine genetic differences between two biotype 1A strains: an environmental isolate, Y. enterocolitica IP2222, and a clinical isolate, Y. enterocolitica T83. Among the Y. enterocolitica T83-specific genes we identified were three, tcbA, tcaC, and tccC, that showed homology to the insecticidal toxin complex (TC) genes first discovered in Photorhabdus luminescens. The Y. enterocolitica T83 TC gene homologues were expressed by Y. enterocolitica T83 and were significantly more prevalent among clinical biotype 1A strains than other Yersinia isolates. Inactivation of the TC genes in Y. enterocolitica T83 resulted in mutants which were attenuated in the ability to colonize the gastrointestinal tracts of perorally infected mice. These results indicate that products of the TC gene complex contribute to the virulence of some strains of Y. enterocolitica biotype 1A, possibly by facilitating their persistence in vivo. PMID:16177365

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

    PubMed

    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.

  18. Prevalence of virulence genes in strains of Campylobacter jejuni isolated from human, bovine and broiler.

    PubMed

    González-Hein, Gisela; Huaracán, Bernardo; García, Patricia; Figueroa, Guillermo

    2013-12-01

    Campylobacter jejuni isolates of different origins (bovine, broiler meat, human) were screened by polymerase chain reaction for the presence of 4 genes cdtB, cst-II, ggt, and virB11, previously linked to virulence such as adherence, invasion, colonization, molecular mimicry, and cytotoxin production. In addition, the isolates were screened for the presence of the global gene regulator csrA linked to oxidative stress responses, biofilms formation, and cell adhesion. All the C. jejuni isolates were positive for cdtB gene. The csrA gene was detected in 100% and 92% of C. jejuni isolates from human and animal origin and the virB11 gene was detected in 7.3% and 3.6% isolates from chicken and human respectively. All isolates from bovine were negative for the virB11 gene. The isolates showed a wide variation for the presence of the remaining genes. Of the C. jejuni recovered from human 83.6%, and 32.7% were positive for cst-II, and ggt respectively. Out of the isolates from chicken 40% and 5.5% isolates revealed the presence of cst-II, and ggt, respectively. Finally of the C. jejuni isolates from bovine, 97.7% and 22.7% were positive for cst-II, and ggt respectively. We conclude that the genes of this study circulate among humans and animals. These results led us to hypothesize that the isolates associated with enteritis (cdtB positives) are not selected by environmental or host-specific factors. On the other hand, the high frequencies of csrA gene in C. jejuni show that this gene is important for the survival of C. jejuni in animals and humans.

  19. Mycobacterial disease, immunosuppression, and acquired immunodeficiency syndrome.

    PubMed Central

    Collins, F M

    1989-01-01

    The mycobacteria are an important group of acid-fast pathogens ranging from obligate intracellular parasites such as Mycobacterium leprae to environmental species such as M. gordonae and M. fortuitum. The latter may behave as opportunistic human pathogens if the host defenses have been depleted in some manner. The number and severity of such infections have increased markedly with the emergence of the acquired immunodeficiency syndrome (AIDS) epidemic. These nontuberculous mycobacteria tend to be less virulent for humans than M. tuberculosis, usually giving rise to self-limiting infections involving the cervical and mesenteric lymph nodes of young children. However, the more virulent serovars of M. avium complex can colonize the bronchial and intestinal mucosal surfaces of healthy individuals, becoming virtual members of the commensal gut microflora and thus giving rise to low levels of skin hypersensitivity to tuberculins prepared from M. avium and M. intracellulare. Systemic disease develops when the normal T-cell-mediated defenses become depleted as a result of old age, cancer chemotherapy, or infection with human immunodeficiency virus. As many as 50% of human immunodeficiency virus antibody-positive individuals develop mycobacterial infections at some time during their disease. Most isolates of M. avium complex from AIDS patients fall into serotypes 4 and 8. The presence of these drug-resistant mycobacteria in the lungs of the AIDS patient makes their effective clinical treatment virtually impossible. More effective chemotherapeutic, prophylactic, and immunotherapeutic reagents are urgently needed to treat this rapidly increasing patient population. PMID:2680057

  20. Clinical Escherichia coli strains carrying stx genes: stx variants and stx-positive virulence profiles.

    PubMed

    Eklund, Marjut; Leino, Kirsikka; Siitonen, Anja

    2002-12-01

    Altogether, 173 Shiga toxin-producing Escherichia coli (STEC) serotype O157 (n = 111) and non-O157 (n = 62) isolates from 170 subjects were screened by PCR-restriction fragment length polymorphism for eight different stx genes. The results were compiled according to serotypes, phage types of O157, production of Stx toxin and enterohemolysin, and the presence of eae. The stx genes occurred in 11 combinations; the most common were stx(2) with stx(2c) (42%), stx(2) alone (21%), and stx(1) alone (16%). Of the O157 strains, 64% carried stx(2) with stx(2c) versus 2% of the non-O157 strains (P < 0.001). In the non-O157 strains, the prevailing gene was stx(1) (99% versus 1% in O157 strains; P < 0.001). In addition, one strain (O Rough:H4:stx(2c)) which has not previously been described as associated with hemolytic-uremic syndrome (HUS) was found. Ten stx-positive virulence profiles were responsible for 71% of all STEC infections. Of these profiles, five accounted for 71% of the 21 strains isolated from 20 patients with HUS or thrombotic thrombocytopenic purpura (TTP). The strains having the virulence profile that caused mainly HUS or TTP or bloody diarrhea produced Stx with titers of >/=1:128 (90%) more commonly than did other strains (51%; P < 0.001). These strains were also more commonly enterohemolytic (98% versus 68% for other strains; P < 0.001) and possessed the eae gene (100%) more commonly than did other strains (74%; P < 0.001). A particular virulence profile, O157:H7:PT2:stx(2):stx(2c):eae:Ehly, was significantly more frequently associated with HUS and bloody diarrhea than were other profiles (P = 0.02) and also caused the deaths of two children. In this study, the risk factors for severe symptoms were an age of <5 years and infection by the strain of O157:H7:PT2 mentioned above.

  1. A naturally occurring single amino acid replacement in multiple gene regulator of group A Streptococcus significantly increases virulence.

    PubMed

    Sanson, Misu; O'Neill, Brian E; Kachroo, Priyanka; Anderson, Jeff R; Flores, Anthony R; Valson, Chandni; Cantu, Concepcion C; Makthal, Nishanth; Karmonik, Christof; Fittipaldi, Nahuel; Kumaraswami, Muthiah; Musser, James M; Olsen, Randall J

    2015-02-01

    Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype-patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes.

  2. A naturally occurring single amino acid replacement in multiple gene regulator of group A Streptococcus significantly increases virulence.

    PubMed

    Sanson, Misu; O'Neill, Brian E; Kachroo, Priyanka; Anderson, Jeff R; Flores, Anthony R; Valson, Chandni; Cantu, Concepcion C; Makthal, Nishanth; Karmonik, Christof; Fittipaldi, Nahuel; Kumaraswami, Muthiah; Musser, James M; Olsen, Randall J

    2015-02-01

    Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype-patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes. PMID:25476528

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

    PubMed

    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.

  4. Virulence genes and cytokine profile in systemic murine Campylobacter coli infection

    PubMed Central

    Klančnik, Anja; Pogačar, Maja Šikić; Raspor, Peter; Abram, Maja; Možina, Sonja Smole; Vučković, Darinka

    2015-01-01

    Campylobacter coli are one of the most common bacteria in bacterial gastroenteritis and acute enterocolitis in humans. However, relatively little is known regarding the mechanisms of pathogenesis and host response to C. coli infections. To investigate the influence of genetic changes, we first used PCR to demonstrate the presence of the known virulence genes cadF, virB11, cdtB, cdtC and ceuE in the clinical isolate C. coli 26536, which was isolated from the liver of infected BALB/c mice. Sequence analyses of the cadF, virB11, cdtB and ceuE genes in C. coli 26536 confirmed the stability in these virulence genes during their transmission through the host. We further investigated C. coli infection for the bacterial clearance from the liver and spleen of infected mice, and for their immune response. C. coli persisted well in both organs, with better survival in the liver. We also determined the levels of several pro-inflammatory cytokines (i.e., interleukin [IL]-6, IL-12, interferon-γ, tumor necrosis factor-α) and the anti-inflammatory cytokine IL-10 in plasma and in liver homogenates from the infected mice, using enzyme-linked immunosorbent assays. The lowest levels among these cytokines were for tumor necrosis factor-α in the plasma and IL-6 in the liver on days 1, 3 and 8 post-infection. The most pronounced production was for IL-10, in both plasma (days 1 and 8 post-infection) and liver (day 8 post-infection), which suggests that it has a role in healing of the organ inflammation. Our findings showed dynamic relationships between pro- and anti-inflammatory cytokines and thus contribute toward clarification of the healing processes involved in the resolution of C. coli infections. PMID:26039573

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

    PubMed Central

    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. PMID:27093436

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

    PubMed

    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.

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

    PubMed

    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. PMID:27093436

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

    PubMed Central

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

    2013-01-01

    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. PMID:23440111

  9. Virulence of Meloidogyne incognita to expression of N gene in pepper.

    PubMed

    Thies, Judy A

    2011-06-01

    Four pepper genotypes classified as resistant and four pepper genotypes classified as susceptible to several avirulent populations of M. incognita were compared for their reactions against a population of Meloidogyne incognita (Chitwood) Kofoid and White which had been shown to be virulent to resistant bell pepper (Capsicum annuum) in preliminary tests. The virulent population of M. incognita originated from a commercial bell pepper field in California. The resistant pepper genotypes used in all experiments were the Capsicum annuum cultivars Charleston Belle, Carolina Wonder, and Carolina Cayenne, and the C. chinense cultigen PA-426. The susceptible pepper genotypes used in the experiments were the C. annuum cultivars Keystone Resistant Giant, Yolo Wonder B, California Wonder, and the C. chinense cultigen PA-350. Root gall indices (GI) were ≥ 3.0 for all genotypes in both tests except for PA-426 (GI=2.57) in test 1 and 'Carolina Cayenne' (GI=2.83) in test 2. Numbers of eggs per gram fresh root weight ranged from 20,635 to 141,319 and reproductive indices ranged from 1.20 to 27.2 for the pepper genotypes in both tests, indicating that all eight pepper genotypes tested were susceptible to the M. incognita population used in these tests. The M. incognita population used in these studies overcame resistance conferred by the N gene in all resistant genotypes of both C. annuum and C. chinense.

  10. Virulence of Meloidogyne incognita to expression of N gene in pepper.

    PubMed

    Thies, Judy A

    2011-06-01

    Four pepper genotypes classified as resistant and four pepper genotypes classified as susceptible to several avirulent populations of M. incognita were compared for their reactions against a population of Meloidogyne incognita (Chitwood) Kofoid and White which had been shown to be virulent to resistant bell pepper (Capsicum annuum) in preliminary tests. The virulent population of M. incognita originated from a commercial bell pepper field in California. The resistant pepper genotypes used in all experiments were the Capsicum annuum cultivars Charleston Belle, Carolina Wonder, and Carolina Cayenne, and the C. chinense cultigen PA-426. The susceptible pepper genotypes used in the experiments were the C. annuum cultivars Keystone Resistant Giant, Yolo Wonder B, California Wonder, and the C. chinense cultigen PA-350. Root gall indices (GI) were ≥ 3.0 for all genotypes in both tests except for PA-426 (GI=2.57) in test 1 and 'Carolina Cayenne' (GI=2.83) in test 2. Numbers of eggs per gram fresh root weight ranged from 20,635 to 141,319 and reproductive indices ranged from 1.20 to 27.2 for the pepper genotypes in both tests, indicating that all eight pepper genotypes tested were susceptible to the M. incognita population used in these tests. The M. incognita population used in these studies overcame resistance conferred by the N gene in all resistant genotypes of both C. annuum and C. chinense. PMID:22791917

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

    PubMed

    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.

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

    PubMed

    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

  13. Virulence gene profiling and antibiotic resistance pattern of Indian isolates of Pasteurella multocida of small ruminant origin.

    PubMed

    Sarangi, Laxmi N; Thomas, P; Gupta, S K; Priyadarshini, A; Kumar, S; Nagaleekar, Viswas Konasagara; Kumar, A; Singh, Vijendra P

    2015-02-01

    Pasteurellosis in small ruminants affects the livelihood of small and marginal farmers of India. The present study was undertaken to understand the trends in gene carriage and antibiotic resistance pattern of Pasteurella multocida isolates recovered from small ruminants over a period of 10 years in India. A total of 88 P. multocida isolates of small ruminant origin were subjected to virulence gene profiling for 19 genes by PCR and antibiogram study employing 17 different antibiotics. Virulence genes like exbB, exbD, tonB, oma87, sodA, sodC, nanB and plpB (100% prevalence) and ptfA and hsf-2 (>90% prevalence) were found to be uniformly distributed among isolates. Unexpectedly, a very high prevalence (95.45%) of pfhA gene was observed in the present study. Dermonecrotoxin gene (toxA) was observed in 48.9% of isolates with highest occurrence among serotype A isolates and interestingly, one of each isolate of serotype B and F were found to carry this gene. Antimicrobial susceptibility testing revealed 17.04% isolates to be multidrug resistant. Amongst all the antibiotics tested, most of the P. multocida isolates were found to be susceptible to enrofloxacin and chloramphenicol. This study highlights novel epidemiological information on frequency and occurrence of virulence genes among Indian isolates from small ruminants.

  14. A vir-repressed gene of Bordetella pertussis is required for virulence.

    PubMed Central

    Beattie, D T; Shahin, R; Mekalanos, J J

    1992-01-01

    Coordinate regulation of gene expression in Bordetella pertussis is controlled by the products of the vir locus, BvgA and BvgS. In the presence of modulating signals such as MgSO4 and nicotinic acid, expression of vir-activated genes (vag) is reduced, while expression of vir-repressed genes (vrg) is maximal. We have cloned one of these vir-repressed genes, vrg-6, in Escherichia coli. DNA sequencing has shown that vrg-6 is contained on a single EcoRI restriction endonuclease fragment and is predicted to code for a protein of 105 amino acids with a molecular weight of 11,441. The predicted protein product appears to have two domains, one consisting of seven hydrophobic proline-rich pentameric repeats and the other consisting of five alkaline trimeric repeats. Southern blot analysis has revealed vrg-6-homologous sequences in the chromosomes of Bordetella bronchiseptica and Bordetella parapertussis, but, unlike Bordetella pertussis, these species do not express vrg-6-homologous RNA when grown under modulating conditions. In order to assess the role of vrg gene products in B. pertussis pathogenesis, two 18323 derivatives which harbor TnphoA insertions in vrg genes were analyzed in a mouse model of respiratory infection. Strain SK6, which carries a vrg-6::TnphoA mutation, failed to induce lymphocytosis and was significantly less able to colonize lungs and trachea than its parent strain 18323 or than SK18, which harbors a TnphoA fusion in the vrg-18 locus. This is the first evidence that a vir-repressed gene may play an important role in the virulence of B. pertussis and the pathogenesis of whooping cough. Images PMID:1730491

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

    PubMed

    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%).

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

    PubMed

    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.

  17. Frequency of virulence genes of Escherichia coli among newborn piglets from an intensive pig farm in Argentina.

    PubMed

    Alustiza, Fabrisio E; Picco, Natalia Y; Bellingeri, Romina V; Terzolo, Horacio R; Vivas, Adriana B

    2012-01-01

    The enterotoxigenic and porcine enteropathogenic Escherichia coli (EtEc and PEPEc) strains are agents associated with swine neonatal diarrhea, causing economic losses in swine production. The main goal of this study was to identify virulence genes of EtEc, verotoxigenic (VtEc) and PEPEc in intestinal strains responsible for swine diseases, by molecular typing using Pcr in newborn piglets from an intensive farm system. Two hundred and sixty seven rectal swabbings from 7-15 days- old landrace x large White crossbred piglets were taken, and 123 randomly selected samples, biochemically compatible with E. coli, were tested for E. coli virulence genes by Pcr. A frequency (%) compatible with: 68 EtEc, 24 VtEc, and 8 EPEc were found. of all E. coli strains studied, 19.51 % carried at least one virulence gene. These data showed conclusively that, in spite of the application of strict sanitary measures in the intensive farm, genes encoding virulence factors of intestinal pathogens compatible with EtEc are still detected; therefore these strains will probably keep circulating among animals. PMID:23267620

  18. Differential Virulence Gene Expression of Group A Streptococcus Serotype M3 in Response to Co-Culture with Moraxella catarrhalis

    PubMed Central

    Verhaegh, Suzanne J. C.; Flores, Anthony R.; van Belkum, Alex; Musser, James M.; Hays, John P.

    2013-01-01

    Streptococcus pyogenes (group A Streptococcus, GAS) and Moraxella catarrhalis are important colonizers and (opportunistic) pathogens of the human respiratory tract. However, current knowledge regarding colonization and pathogenic potential of these two pathogens is based on work involving single bacterial species, even though the interplay between respiratory bacterial species is increasingly important in niche occupation and the development of disease. Therefore, to further define and understand polymicrobial species interactions, we investigated whether gene expression (and hence virulence potential) of GAS would be affected upon co-culture with M. catarrhalis. For co-culture experiments, GAS and M. catarrhalis were cultured in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) at 37°C with 5% CO2 aeration. Each strain was grown in triplicate so that triplicate experiments could be performed. Bacterial RNA was isolated, cDNA synthesized, and microarray transcriptome expression analysis performed. We observed significantly increased (≥4-fold) expression for genes playing a role in GAS virulence such as hyaluronan synthase (hasA), streptococcal mitogenic exotoxin Z (smeZ) and IgG endopeptidase (ideS). In contrast, significantly decreased (≥4-fold) expression was observed in genes involved in energy metabolism and in 12 conserved GAS two-component regulatory systems. This study provides the first evidence that M. catarrhalis increases GAS virulence gene expression during co-culture, and again shows the importance of polymicrobial infections in directing bacterial virulence. PMID:23626831

  19. A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression

    PubMed Central

    Millet, Yves A.; Chao, Michael C.; Sasabe, Jumpei; Davis, Brigid M.

    2015-01-01

    Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression of Vibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression of tcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulate tcpA expression, the screen yielded ptsI and ptsH, which encode the enzyme I (EI) and Hpr components of the V. cholerae phosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIAGlc protein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression of tcpPH and aphAB, which themselves control expression of toxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that the V. cholerae PTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks. PMID:26056384

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Rückert, Christian; Kalinowski, Jörn

    2015-01-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  3. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation.

    PubMed

    Müller, Christine; Birmes, Franziska S; Rückert, Christian; Kalinowski, Jörn; Fetzner, Susanne

    2015-11-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    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. PMID:26039674

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

    PubMed

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

    2016-09-01

    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.

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

    PubMed

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

    2016-09-01

    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. PMID:27257743

  8. Gain of Virulence Caused by Loss of a Gene in Murine Cytomegalovirus‡

    PubMed Central

    Bubić, Ivan; Wagner, Markus; Krmpotić, Astrid; Saulig, Tanja; Kim, Sungjin; Yokoyama, Wayne M.; Jonjić, Stipan; Koszinowski, Ulrich H.

    2004-01-01

    Mouse strains are either resistant or susceptible to murine cytomegalovirus (MCMV). Resistance is determined by the Cmv1r (Ly49h) gene, which encodes the Ly49H NK cell activation receptor. The protein encoded by the m157 gene of MCMV has been defined as a ligand for Ly49H. To find out whether the m157 protein is the only Ly49H ligand encoded by MCMV, we constructed the m157 deletion mutant and a revertant virus. Viruses were tested for susceptibility to NK cell control in Ly49H+ and Ly49H− mouse strains. Deletion of the m157 gene abolished the viral activation of Ly49H+ NK cells, resulting in higher virus virulence in vivo. Thus, in the absence of m157, Ly49H+ mice react like susceptible strains. 129/SvJ mice lack the Ly49H activation NK cell receptor but express the inhibitory Ly49I NK cell receptor that binds to the m157 protein. The Δm157 inhibitory phenotype was weak because MCMV encodes a number of proteins that mediate NK inhibition, whose contribution could be shown by another mutant. PMID:15220428

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

    PubMed

    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.

  10. The agr locus regulates virulence and colonization genes in Clostridium difficile 027.

    PubMed

    Martin, Melissa J; Clare, Simon; Goulding, David; Faulds-Pain, Alexandra; Barquist, Lars; Browne, Hilary P; Pettit, Laura; Dougan, Gordon; Lawley, Trevor D; Wren, Brendan W

    2013-08-01

    The transcriptional regulator AgrA, a member of the LytTR family of proteins, plays a key role in controlling gene expression in some Gram-positive pathogens, including Staphylococcus aureus and Enterococcus faecalis. AgrA is encoded by the agrACDB global regulatory locus, and orthologues are found within the genome of most Clostridium difficile isolates, including the epidemic lineage 027/BI/NAP1. Comparative RNA sequencing of the wild type and otherwise isogenic agrA null mutant derivatives of C. difficile R20291 revealed a network of approximately 75 differentially regulated transcripts at late exponential growth phase, including many genes associated with flagellar assembly and function, such as the major structural subunit, FliC. Other differentially regulated genes include several involved in bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) synthesis and toxin A expression. C. difficile 027 R20291 agrA mutant derivatives were poorly flagellated and exhibited reduced levels of colonization and relapses in the murine infection model. Thus, the agr locus likely plays a contributory role in the fitness and virulence potential of C. difficile strains in the 027/BI/NAP1 lineage. PMID:23772065

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

    PubMed

    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. PMID:26221117

  12. Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence.

    PubMed

    Juhas, Mario; Crook, Derrick W; Hood, Derek W

    2008-12-01

    Type IV secretion systems (T4SSs) are multisubunit cell-envelope-spanning structures, ancestrally related to bacterial conjugation machines, which transfer proteins and nucleoprotein complexes across membranes. T4SSs mediate horizontal gene transfer, thus contributing to genome plasticity and the evolution of pathogens through dissemination of antibiotic resistance and virulence genes. Moreover, T4SSs are also used for the delivery of bacterial effector proteins across the bacterial membrane and the plasmatic membrane of eukaryotic host cell, thus contributing directly to pathogenicity. T4SSs are usually encoded by multiple genes organized into a single functional unit. Based on a number of features, the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several groups. Type F and P (type IVA) T4SSs resembling the archetypal VirB/VirD4 system of Agrobacterium tumefaciens are considered to be the paradigm of type IV secretion, while type I (type IVB) T4SSs are found in intracellular bacterial pathogens, Legionella pneumophila and Coxiella burnetii. Several novel T4SSs have been identified recently and their functions await investigation. The most recently described GI type T4SSs play a key role in the horizontal transfer of a wide variety of genomic islands derived from a broad spectrum of bacterial strains. PMID:18549454

  13. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.

    PubMed Central

    Pearson, J P; Gray, K M; Passador, L; Tucker, K D; Eberhard, A; Iglewski, B H; Greenberg, E P

    1994-01-01

    In Pseudomonas aeruginosa the LasR protein is required for activation of lasB and several other virulence genes. A diffusible signal molecule, the P. aeruginosa autoinducer (PAI), produced by the bacterial cell and released into the growth medium, is required for activity of LasR. By cloning a lasB::lacZ fusion and a lasR gene under control of the lac promoter in Escherichia coli, we have developed a quantitative bioassay for PAI. We have used this assay to follow the purification of PAI from cell-free culture supernatant fluids in which P. aeruginosa or E. coli containing the P. aeruginosa gene required for autoinducer synthesis, lasI, had been grown. Chemical analyses indicated the purified material was 3-oxo-N-(tetrahydro-2-oxo-3-furanyl)dodecanamide. To confirm this assignment, the compound was synthesized and the synthetic compound was shown to have chemical and biological properties identical to those of PAI purified from culture supernatant fluids. The elucidation of the PAI structure suggests therapeutic approaches toward control of P. aeruginosa infections. PMID:8278364

  14. Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence.

    PubMed

    Juhas, Mario; Crook, Derrick W; Hood, Derek W

    2008-12-01

    Type IV secretion systems (T4SSs) are multisubunit cell-envelope-spanning structures, ancestrally related to bacterial conjugation machines, which transfer proteins and nucleoprotein complexes across membranes. T4SSs mediate horizontal gene transfer, thus contributing to genome plasticity and the evolution of pathogens through dissemination of antibiotic resistance and virulence genes. Moreover, T4SSs are also used for the delivery of bacterial effector proteins across the bacterial membrane and the plasmatic membrane of eukaryotic host cell, thus contributing directly to pathogenicity. T4SSs are usually encoded by multiple genes organized into a single functional unit. Based on a number of features, the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several groups. Type F and P (type IVA) T4SSs resembling the archetypal VirB/VirD4 system of Agrobacterium tumefaciens are considered to be the paradigm of type IV secretion, while type I (type IVB) T4SSs are found in intracellular bacterial pathogens, Legionella pneumophila and Coxiella burnetii. Several novel T4SSs have been identified recently and their functions await investigation. The most recently described GI type T4SSs play a key role in the horizontal transfer of a wide variety of genomic islands derived from a broad spectrum of bacterial strains.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26221117

  16. Transcriptional modulation of enterotoxigenic Escherichia coli virulence genes in response to epithelial cell interactions.

    PubMed

    Kansal, Rita; Rasko, David A; Sahl, Jason W; Munson, George P; Roy, Koushik; Luo, Qingwei; Sheikh, Alaullah; Kuhne, Kurt J; Fleckenstein, James M

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens. PMID:23115039

  17. The cytopathology of mycobacterial infection.

    PubMed

    Michelow, Pamela; Omar, Tanvier; Field, Andrew; Wright, Colleen

    2016-03-01

    Mycobacterial infection, tuberculosis (TB) in particular, remains one of the world's deadliest communicable diseases in adults and particularly in children, in low and middle income countries. The combination of human immunodeficiency virus (HIV) and TB is often lethal with TB accounting for 25% of deaths in the HIV population. One of the cornerstones for reducing the TB epidemic is early case detection using high quality diagnostic techniques. Cytology, especially fine needle aspiration biopsy (FNAB) is able to diagnose mycobacterial infection in a rapid and cost-effective manner without requiring surgery, thus allowing appropriate management to be quickly instituted. Confirmatory ancillary tests can effectively be performed on cytologic material. In this review, the pertinent cytomorphology of mycobacterial infection in various exfoliative and FNAB specimens is presented, in both immunocompetent and immunosuppressed patients. In the immunosuppressed, the typical cytomorphology of caseating granulomatous inflammation may not be seen but suppurative necrotic inflammation, mycobacterial spindle pseudotumour or a specimen comprised entirely of necrosis may be seen instead. This review includes discussion of currently available ancillary tests that can be performed on cytologic specimens. PMID:26800030

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

    PubMed

    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

  19. An Ustilago maydis Gene Involved in H2O2 Detoxification Is Required for Virulence[W

    PubMed Central

    Molina, Lázaro; Kahmann, Regine

    2007-01-01

    The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H2O2 than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H2O2 sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H2O2 treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Δyap1 mutants. Diamino benzidine staining revealed an accumulation of H2O2 around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H2O2 detoxification system for coping with early plant defense responses. PMID:17616735

  20. Effects of partial deletion of the wzm and wzt genes on lipopolysaccharide synthesis and virulence of Brucella abortus S19.

    PubMed

    Wang, Xiuran; Wang, Lin; Lu, Tiancheng; Yang, Yanling; Chen, Si; Zhang, Rui; Lang, Xulong; Yan, Guangmou; Qian, Jing; Wang, Xiaoxu; Meng, Lingyi; Wang, Xinglong

    2014-06-01

    Brucellosis is a worldwide human and animal infectious disease, and the effective methods of its control are immunisation of animals by vaccination and elimination. Brucella abortus S19 is one of the popular vaccines with virulence in the control of cattle Brucellosis. In the present study, allelic exchange plasmids of wzm and wzt genes and partial knockout mutants of wzm and wzt were constructed to evaluate the resulting difference in virulence of B. abortus S19. PCR analysis revealed that the target genes were knocked out. The mutants were rough mutants and they could be differentiated from natural infection by the Rose Bengal plate and standard agglutination tests. The molecular weights of lipopolysaccharides of the Δwzm and Δwzt mutants were clustered between 25 and 40 kDa, and 30 and 35 kDa separately, and were markedly different from those in B. abortus S19. The virulence of B. abortus Δwzm and Δwzt was decreased compared with that of B. abortus S19 in mice. All these results identified that there were several differences between the wzm and wzt genes on lipopolysaccharide synthesis and on the virulence of B. abortus.

  1. Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence.

    PubMed

    Sevim, Ali; Donzelli, Bruno G G; Wu, Dongliang; Demirbag, Zihni; Gibson, Donna M; Turgeon, B Gillian

    2012-04-01

    Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence. PMID:22388867

  2. Horizontal Gene Transfer of the Secretome Drives the Evolution of Bacterial Cooperation and Virulence

    PubMed Central

    Nogueira, Teresa; Rankin, Daniel J.; Touchon, Marie; Taddei, François; Brown, Sam P.; Rocha, Eduardo P.C.

    2009-01-01

    Summary Background Microbes engage in a remarkable array of cooperative behaviors, secreting shared proteins that are essential for foraging, shelter, microbial warfare, and virulence. These proteins are costly, rendering populations of cooperators vulnerable to exploitation by nonproducing cheaters arising by gene loss or migration. In such conditions, how can cooperation persist? Results Our model predicts that differential gene mobility drives intragenomic variation in investment in cooperative traits. More mobile loci generate stronger among-individual genetic correlations at these loci (higher relatedness) and thereby allow the maintenance of more cooperative traits via kin selection. By analyzing 21 Escherichia genomes, we confirm that genes coding for secreted proteins—the secretome—are very frequently lost and gained and are associated with mobile elements. We show that homologs of the secretome are overrepresented among human gut metagenomics samples, consistent with increased relatedness at secretome loci across multiple species. The biosynthetic cost of secreted proteins is shown to be under intense selective pressure, even more than for highly expressed proteins, consistent with a cost of cooperation driving social dilemmas. Finally, we demonstrate that mobile elements are in conflict with their chromosomal hosts over the chimeric ensemble's social strategy, with mobile elements enforcing cooperation on their otherwise selfish hosts via the cotransfer of secretome genes with “mafia strategy” addictive systems (toxin-antitoxin and restriction-modification). Conclusion Our analysis matches the predictions of our model suggesting that horizontal transfer promotes cooperation, as transmission increases local genetic relatedness at mobile loci and enforces cooperation on the resident genes. As a consequence, horizontal transfer promoted by agents such as plasmids, phages, or integrons drives microbial cooperation. PMID:19800234

  3. Impact of virulence genes on sepsis severity and survival in Escherichia coli bacteremia

    PubMed Central

    Mora-Rillo, Marta; Fernández-Romero, Natalia; Francisco, Carolina Navarro-San; Díez-Sebastián, Jesús; Romero-Gómez, Maria Pilar; Fernández, Francisco Arnalich; López, Jose Ramon Arribas; Mingorance, Jesús

    2015-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) are a frequent cause of bacteremia and sepsis, but the role of ExPEC genetic virulence factors (VFs) in sepsis development and outcome is ill-defined. Prospective study including 120 adult patients with E. coli bacteremia to investigate the impact of bacterial and host factors on sepsis severity and mortality. Patients' clinical and demographic data were registered. Phylogenetic background of E. coli isolates was analyzed by SNP pyrosequencing and VFs by PCR. The E. coli isolates presented an epidemic population structure with 6 dominant clones making up to half of the isolates. VF gene profiles were highly diverse. Multivariate analysis for sepsis severity showed that the presence of cnf and blaTEM genes increased the risk of severe illness by 6.75 (95% confidence interval [CI] 1.79–24.71) and 2.59 (95% CI 1.04–6.43) times respectively, while each point in the Pitt score increased the risk by 1.34 (95% CI 1.02–1.76) times. Multivariate analysis for mortality showed that active chemotherapy (OR 17.87, 95% CI 3.35–95.45), McCabe-Jackson Index (OR for rapidly fatal category 120.15, 95% CI 4.19–3446.23), Pitt index (OR 1.78, 95% CI 1.25–2.56) and presence of fyuA gene (OR 8.05, 95% CI 1.37–47.12) were associated to increased mortality while the presence of P fimbriae genes had a protective role (OR 0.094, 95%IC 0.018–0.494). Bacteremic E. coli had a high diversity of genetic backgrounds and VF gene profiles. Bacterial VFs and host determinants had an impact on disease evolution and mortality. PMID:25654604

  4. Predicted highly expressed genes in Nocardia farcinica and the implication to its primary metabolism and nocardial virulence

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-02-23

    Nocardia farcinica is a gram positive, filamentous bacterium, and is considered an opportunistic pathogen. In this study, the highly expressed genes in N. farcinica were predicted using the codon adaptation index (CAI) as a numerical estimator of gene expressivity. Using ribosomal protein (RP) genes as references, the top {approx}10% of the genes were predicted to be the predicted highly expressed (PHX) genes in N. farcinica using a CAI cutoff of greater than 0.73. Consistent with early analysis in Streptomyces genomes, most of the PHX genes in N. farcinica were involved in various ''house-keeping'' functions important for cell growth. However, fifteen genes putatively involved in no cardial virulence were predicted as PHX in N. farcinica, which included genes encoding four Mce virulence proteins, cyclopropane fatty acid synthase which is involved in the modification of cell wall important for nocardia virulence, polyketide synthase PKS13 for mycolic acid synthesis and non-ribosomal peptide synthetase involved in biosynthesis of a mycobactin-related siderophore. In addition, multiple genes involved in defense against reactive oxygen species (ROS) produced by the phagocyte were predicted with high expressivity, which included alkylhydroperoxide reductase (ahpC), catalase (katG), superoxide dismutase (sodF), thioredoxin, thioredoxin reductase, glutathione peroxidase, and peptide methionine sulfoxide reductase, suggesting that combating against ROS was essential for survival of N. farcinica in host cells. The study also showed that the distribution of PHX genes in the N. farcinica circular chromosome was uneven, with more PHX genes located in the regions close to replication initiation site. The results provided the first approximates of global gene expression patterns in N. farcinica, which will be useful in guiding experimental design for further investigation.

  5. Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors.

    PubMed

    Lakshman, Dilip K; Alkharouf, Nadim; Roberts, Daniel P; Natarajan, Savithiry S; Mitra, Amitava

    2012-01-01

    Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.

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

    PubMed

    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.

  7. Detection of Virulence Genes and Growth Potential in Listeria monocytogenes Strains Isolated from Ricotta Salata Cheese.

    PubMed

    Coroneo, Valentina; Carraro, Valentina; Aissani, Nadhem; Sanna, Adriana; Ruggeri, Alessandra; Succa, Sara; Meloni, Barbara; Pinna, Antonella; Sanna, Clara

    2016-01-01

    Ricotta Salata is a traditional ripened and salted whey cheese made in Sardinia (Italy) from sheep's milk. This product is catalogued as ready-to-eat food (RTE) since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens, such as Listeria monocytogenes, can represent a health risk for consumers. In September 2012, the FDA ordered the recall of several batches of Ricotta Salata imported from Italy linked to 22 cases of Listeriosis in the United States. This study was aimed at evaluating the presence and virulence properties of L. monocytogenes in 87 samples of Ricotta Salata produced in Sardinia. The ability of this product to support its growth under foreseen packing and storing conditions was also evaluated in 252 samples. Of the 87 samples 17.2% were positive for the presence of L. monocytogenes with an average concentration of 2.2 log10 cfu/g. All virulence-associated genes (prfA, rrn, hlyA, actA, inlA, inlB, iap, plcA, and plcB) were detected in only one isolated strain. The Ricotta Salata samples were artificially inoculated and growth potential (δ) was assessed over a period of 3 mo. The value of the growth potential was always >0.5 log10 cfu/g under foreseen packing and storing conditions. This study indicates that Ricotta Salata supports the L. monocytogenes growth to levels that may present a serious risk to public health, even while stored at refrigeration temperatures. PMID:26666835

  8. Riemerella anatipestifer M949_1360 Gene Functions on the Lipopolysaccharide Biosynthesis and Bacterial Virulence.

    PubMed

    Yu, Guijing; Wang, Xiaolan; Dou, Yafeng; Wang, Shaohui; Tian, Mingxing; Qi, Jingjing; Li, Tao; Ding, Chan; Wu, Yantao; Yu, Shengqing

    2016-01-01

    Riemerella anatipestifer causes septicemic and exudative diseases in poultry, resulting in major economic losses to the duck industry. Lipopolysaccharide (LPS), as an important virulence factor in Gram-negative bacteria, can be recognized by the immune system and plays a crucial role in many interactions between bacteria and animal hosts. In this study, we screened out one LPS defective mutant strain RAΔ604 from a random transposon mutant library of R. anatipestifer serotype 1 strain CH3, which did not react with the anti-CH3 LPS monoclonal antibody 1C1 in an indirect enzyme-linked immunosorbent assay. Southern blot analysis confirmed that the genome of RAΔ604 contained a single Tn4351 insert. Then, we found that the M949_1360 gene was inactivated by insertion of the transposon. Using silver staining and western blot analyses, we found that the LPS pattern of RAΔ604 was defective, as compared with that of the wild-type (WT) strain CH3. The mutant strain RAΔ604 showed no significant influence on bacterial growth, while bacterial counting and Live/dead BacLight Bacterial Viability staining revealed that bacterial viability was decreased, as compared with the WT strain CH3. In addition, the abilities of the mutant strain RAΔ604 to adhere and invade Vero cells were significantly decreased. Animal studies revealed that the virulence of the mutant strain RAΔ604 was decreased by more than 200-fold in a duck infection model, as compared with the WT strain CH3. Furthermore, immunization with live bacteria of the mutant strain RAΔ604 protected 87.5% ducks from challenge with R. anatipestifer serotype 1 strain WJ4, indicating that the mutant strain RAΔ604 could be used as a potential vaccine candidate in the future. PMID:27500736

  9. Detection of Virulence Genes and Growth Potential in Listeria monocytogenes Strains Isolated from Ricotta Salata Cheese.

    PubMed

    Coroneo, Valentina; Carraro, Valentina; Aissani, Nadhem; Sanna, Adriana; Ruggeri, Alessandra; Succa, Sara; Meloni, Barbara; Pinna, Antonella; Sanna, Clara

    2016-01-01

    Ricotta Salata is a traditional ripened and salted whey cheese made in Sardinia (Italy) from sheep's milk. This product is catalogued as ready-to-eat food (RTE) since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens, such as Listeria monocytogenes, can represent a health risk for consumers. In September 2012, the FDA ordered the recall of several batches of Ricotta Salata imported from Italy linked to 22 cases of Listeriosis in the United States. This study was aimed at evaluating the presence and virulence properties of L. monocytogenes in 87 samples of Ricotta Salata produced in Sardinia. The ability of this product to support its growth under foreseen packing and storing conditions was also evaluated in 252 samples. Of the 87 samples 17.2% were positive for the presence of L. monocytogenes with an average concentration of 2.2 log10 cfu/g. All virulence-associated genes (prfA, rrn, hlyA, actA, inlA, inlB, iap, plcA, and plcB) were detected in only one isolated strain. The Ricotta Salata samples were artificially inoculated and growth potential (δ) was assessed over a period of 3 mo. The value of the growth potential was always >0.5 log10 cfu/g under foreseen packing and storing conditions. This study indicates that Ricotta Salata supports the L. monocytogenes growth to levels that may present a serious risk to public health, even while stored at refrigeration temperatures.

  10. Riemerella anatipestifer M949_1360 Gene Functions on the Lipopolysaccharide Biosynthesis and Bacterial Virulence

    PubMed Central

    Yu, Guijing; Wang, Xiaolan; Dou, Yafeng; Wang, Shaohui; Tian, Mingxing; Qi, Jingjing; Li, Tao; Ding, Chan; Wu, Yantao; Yu, Shengqing

    2016-01-01

    Riemerella anatipestifer causes septicemic and exudative diseases in poultry, resulting in major economic losses to the duck industry. Lipopolysaccharide (LPS), as an important virulence factor in Gram-negative bacteria, can be recognized by the immune system and plays a crucial role in many interactions between bacteria and animal hosts. In this study, we screened out one LPS defective mutant strain RAΔ604 from a random transposon mutant library of R. anatipestifer serotype 1 strain CH3, which did not react with the anti-CH3 LPS monoclonal antibody 1C1 in an indirect enzyme-linked immunosorbent assay. Southern blot analysis confirmed that the genome of RAΔ604 contained a single Tn4351 insert. Then, we found that the M949_1360 gene was inactivated by insertion of the transposon. Using silver staining and western blot analyses, we found that the LPS pattern of RAΔ604 was defective, as compared with that of the wild-type (WT) strain CH3. The mutant strain RAΔ604 showed no significant influence on bacterial growth, while bacterial counting and Live/dead BacLight Bacterial Viability staining revealed that bacterial viability was decreased, as compared with the WT strain CH3. In addition, the abilities of the mutant strain RAΔ604 to adhere and invade Vero cells were significantly decreased. Animal studies revealed that the virulence of the mutant strain RAΔ604 was decreased by more than 200-fold in a duck infection model, as compared with the WT strain CH3. Furthermore, immunization with live bacteria of the mutant strain RAΔ604 protected 87.5% ducks from challenge with R. anatipestifer serotype 1 strain WJ4, indicating that the mutant strain RAΔ604 could be used as a potential vaccine candidate in the future. PMID:27500736

  11. Evaluating virulence of waterborne and clinical Aeromonas isolates using gene expression and mortality in neonatal mice followed by assessing cell culture’s ability to predict virulence based on transcriptional response

    SciTech Connect

    Hayes, S L; Rodgers, M R; Lye, D J; Stelma, G N; McKinstry, Craig A.; Malard, Joel M.; Vesper, Sephen J.

    2007-10-01

    Aims: To assess the virulence of Aeromonas spp. using two models, a neonatal mouse assay and a mouse intestinal cell culture. Methods and Results: After artificial infection with a variety of Aeromonas spp., mRNA extracts from the two models were processed and hydridized to murine microarrays to determine host gene response. Definition of virulence was determined based on host mRNA production in murine neonatal intestinal tissue and mortality of infected animals. Infections of mouse intestinal cell cultures were then performed to determine whether this simpler model system’s mRNA responses correlated to neonatal results and therefore be predictive of virulence of Aeromonas spp. Virulent aeromonads up-regulated transcripts in both models including multiple host defense gene products (chemokines, regulation of transcription and apoptosis and cell signalling). Avirulent species exhibited little or no host response in neonates. Mortality results correlated well with both bacterial dose and average fold change of up-regulated transcripts in the neonatal mice. Conclusions: Cell culture results were less discriminating but showed promise as potentially being able to be predictive of virulence. Jun oncogene up-regulation in murine cell culture is potentially predictive of Aeromonas virulence. Significance and Impact of the Study: Having the ability to determine virulence of waterborne pathogens quickly would potentially assist public health officials to rapidly assess exposure risks.

  12. PCR detection of Shiga toxins, enterohaemolysin and intimin virulence genes of Escherichia coli O157:H7 strains isolated from faeces of Anatolian water buffaloes in Turkey.

    PubMed

    Seker, E; Kuyucuoğlu, Y; Sareyyüpoğlu, B; Yardımcı, H

    2010-12-01

    The aim of this study was to detect Shiga toxins (stx1 and stx2), enterohaemolysin (EhlyA) and intimin (eaeA) virulence genes of 11 Escherichia coli O157:H7 strains isolated from faecal samples of 300 clinically healthy Anatolian water buffaloes by PCR. Multiplex PCR was used for the detection of stx1 and stx2, and singleplex PCRs were used for the detection of EhlyA and eaeA virulence genes respectively. A total of three (27.3%) strains were determined to harbour both of the stx1 and stx2 genes, of these, one (9.1%) only harboured these two genes alone, one (9.1%) also contained the EhlyA gene and one (9.1%) additionally contained the EhlyA and the eaeA genes. EhlyA gene was obtained from eight (72.7%) strains, six (54.5%) of these were alone. eaeA gene was positive in only one (9.1%) strain. Only one (9.1%) of the 11 E. coli O157:H7 strains harboured all the four virulence genes. Two (18.2%) of the isolates had none of the virulence genes. Enterohaemolysin was found to be the most common virulence factor. In conclusion, the virulence factors of E. coli O157:H7 strains isolated from the faeces of Anatolian water buffaloes were investigated and detected for the first time in Turkey.

  13. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish

    PubMed Central

    Vemula, Mani H.; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host–pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination.

  14. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish

    PubMed Central

    Vemula, Mani H.; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host–pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. PMID:27621726

  15. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish.

    PubMed

    Vemula, Mani H; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host-pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. PMID:27621726

  16. Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes.

    PubMed

    Andino, Francisco De Jesús; Grayfer, Leon; Chen, Guangchun; Chinchar, V Gregory; Edholm, Eva-Stina; Robert, Jacques

    2015-11-01

    To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvβHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α-FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis.

  17. Antibiotic resistance genes and virulence factors in Enterococcus faecium and Enterococcus faecalis from diseased farm animals: pigs, cattle and poultry.

    PubMed

    Seputiene, V; Bogdaite, A; Ruzauskas, M; Suziedeliene, E

    2012-01-01

    Eighty enterococcal isolates (E. faecium, n = 38, E. faecalis, n = 42) from diseased farm animals (swine, cattle, poultry) in Lithuania have been studied for the prevalence of antibiotic resistance and for resistance and virulence genetic determinants. 86% of E. faecium and 71% of E. faecalis isolates were multidrug resistant (resistant to three or more unrelated antibiotics). Resistance to aminoglycosides, tetracycline and erythromycin was found most frequently in both species (61%, 69%) and was linked to aph(3')-IIIa, aac(6')-Ie-aph(2")-Ia, ant(6)-Ia (aminoglycoside resistance), tetM, tetL (tetracycline resistance), ermA, ermB (erythromycin resistance) gene combinations, which were supplemented with chloramphenicol resistance genes catA7, catA8 (E. faecalis) and catA9 (E. faecium). All E. faecalis isolates harboured genes coding for virulence factors agg, esp, fsr gelE alone or in combinations with the high prevalence of esp gene in isolates from cattle (63%) and pigs (79%). The origin-dependent incidence of agg gene variants prgB and asp1 was observed. The results indicate the existence of a large pool of potentially virulent and multidrug resistant E. faecalis in diseased farm animals posing risk to humans.

  18. Phylogroups, virulence determinants and antimicrobial resistance in stx(2) gene-carrying Escherichia coli isolated from aquatic environments.

    PubMed

    Garcia-Aljaro, Cristina; Moreno, Eva; Andreu, Antònia; Prats, Guillem; Blanch, Anicet R

    2009-10-01

    The aim of this study was to assess the prevalence of antibiotic-resistant stx(2) gene-carrying Escherichia coli isolated from human and animal wastewater with regard to their animal/human origin, serotype, phylogenetic background and virulence factors. The isolates were characterized by PCR in relation to stx variant, phylogenetic group and other virulence genes (stx(1), ehxA and saa). Antibiotic resistance was found in 92% of the stx(2) gene-carrying E. coli strains, with 77% showing intermediate resistance or full resistance to more than one antibiotic. High levels of resistance were observed to chloramphenicol, tetracycline, sulfamethoxazole, streptomycin, trimethoprim, and trimethoprim + sulfamethoxazole, with resistance values of 79%, 69%, 63%, 58%, 47% and 42%, respectively, and a higher prevalence among those strains isolated from animal wastewater. There was no association between the E. coli serotype and/or phylogroup and the antimicrobial resistance profile displayed. However, those strains carrying the stx(2) gene variant alone or in combination with other virulence factors (stx(1), ehxA or saa gene) were susceptible to most of the tested antibiotics.

  19. Prevalence of Campylobacter species in milk and milk products, their virulence gene profile and anti-bio gram

    PubMed Central

    Modi, Shivani; Brahmbhatt, M. N.; Chatur, Y. A.; Nayak, J. B.

    2015-01-01

    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. Material 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. PMID:27046986

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

    PubMed

    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.

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

    PubMed

    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.

  2. Natural Variation in the VELVET Gene bcvel1 Affects Virulence and Light-Dependent Differentiation in Botrytis cinerea

    PubMed Central

    Schumacher, Julia; Pradier, Jean-Marc; Simon, Adeline; Traeger, Stefanie; Moraga, Javier; Collado, Isidro González; Viaud, Muriel; Tudzynski, Bettina

    2012-01-01

    Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions. PMID:23118899

  3. The regulatory gene VosA affects conidiogenesis and is involved in virulence of the fungal cereal pathogen Cochliobolus sativus.

    PubMed

    Wang, Rui; Leng, Yueqiang; Zhong, Shaobin

    2015-10-01

    VosA is one of the four components in the velvet complex shown to be involved in regulation of fungal development and secondary metabolism in filamentous fungi. However, the function of VosA has only been studied in a few plant pathogenic fungi. In this study, we identified the ortholog (CsVosA) of VosA in the cereal spot blotch pathogen Cochliobolus sativus and generated gene knockout mutants for functional characterization of the gene. Conidia of the CsVosA knockout mutants (ΔCsVosA) lacked trehalose, were significantly reduced in viability, had less pigmentation, and showed a dramatic reduction in tolerance to heat, oxidative, and ion stresses. However, ΔCsVosA produced more conidia than the wild type under both constant dark, and constant light conditions, suggesting that CsVosA is a negative-feedback regulator in conidiation. Interestingly, the ΔCsVosA mutants exhibited a hypermorphic conidiation phenotype with indeterminate growth of the conidial tip cells resulting in head-to-tail (acropetal) arrays of conidiogenesis, indicating that some genes involved in conidiation are also regulated by CsVosA. The ΔCsVosA mutants showed significant reduction in virulence on susceptible barley plants and the two genes for nonribosomal peptide synthetases (NRPSs) involved in virulence during host infection were down-regulated in ΔCsVosA, suggesting that CsVosA may affect virulence of the fungus by regulating the expression of the genes for NRPSs, as well as other genes directly or indirectly involved in virulence.

  4. The regulatory gene VosA affects conidiogenesis and is involved in virulence of the fungal cereal pathogen Cochliobolus sativus.

    PubMed

    Wang, Rui; Leng, Yueqiang; Zhong, Shaobin

    2015-10-01

    VosA is one of the four components in the velvet complex shown to be involved in regulation of fungal development and secondary metabolism in filamentous fungi. However, the function of VosA has only been studied in a few plant pathogenic fungi. In this study, we identified the ortholog (CsVosA) of VosA in the cereal spot blotch pathogen Cochliobolus sativus and generated gene knockout mutants for functional characterization of the gene. Conidia of the CsVosA knockout mutants (ΔCsVosA) lacked trehalose, were significantly reduced in viability, had less pigmentation, and showed a dramatic reduction in tolerance to heat, oxidative, and ion stresses. However, ΔCsVosA produced more conidia than the wild type under both constant dark, and constant light conditions, suggesting that CsVosA is a negative-feedback regulator in conidiation. Interestingly, the ΔCsVosA mutants exhibited a hypermorphic conidiation phenotype with indeterminate growth of the conidial tip cells resulting in head-to-tail (acropetal) arrays of conidiogenesis, indicating that some genes involved in conidiation are also regulated by CsVosA. The ΔCsVosA mutants showed significant reduction in virulence on susceptible barley plants and the two genes for nonribosomal peptide synthetases (NRPSs) involved in virulence during host infection were down-regulated in ΔCsVosA, suggesting that CsVosA may affect virulence of the fungus by regulating the expression of the genes for NRPSs, as well as other genes directly or indirectly involved in virulence. PMID:26399184

  5. Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus

    PubMed Central

    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

  6. Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus.

    PubMed

    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

  7. Mycobacterial Acid Tolerance Enables Phagolysosomal Survival and Establishment of Tuberculous Infection In Vivo.

    PubMed

    Levitte, Steven; Adams, Kristin N; Berg, Russell D; Cosma, Christine L; Urdahl, Kevin B; Ramakrishnan, Lalita

    2016-08-10

    The blockade of phagolysosomal fusion is considered a critical mycobacterial strategy to survive in macrophages. However, viable mycobacteria have been observed in phagolysosomes during infection of cultured macrophages, and mycobacteria have the virulence determinant MarP, which confers acid resistance in vitro. Here we show in mice and zebrafish that innate macrophages overcome mycobacterial lysosomal avoidance strategies to rapidly deliver a substantial proportion of infecting bacteria to phagolysosomes. Exploiting the optical transparency of the zebrafish, we tracked the fates of individual mycobacteria delivered to phagosomes versus phagolysosomes and discovered that bacteria survive and grow in phagolysosomes, though growth is slower. MarP is required specifically for phagolysosomal survival, making it an important determinant for the establishment of mycobacterial infection in their hosts. Our work suggests that if pathogenic mycobacteria fail to prevent lysosomal trafficking, they tolerate the resulting acidic environment of the phagolysosome to establish infection. PMID:27512905

  8. 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.

    PubMed

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

    2014-01-01

    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 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.

  9. Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo (Bubalus bubalis).

    PubMed

    Singh, K M; Jakhesara, S J; Koringa, P G; Rank, D N; Joshi, C G

    2012-10-10

    A major research goal in rumen microbial ecology is to understand the relationship between community composition and its function, particularly involved in fermentation process is of a potential interest. The buffalo rumen microbiota impacts human food safety as well as animal health. Although the bacteria of bovine rumen have been well characterized, techniques have been lacking to correlate total community structure with gene function. We applied 454 next generations sequencing technology to characterize general microbial diversity present in buffalo rumen metagenome and also identified the repertoire of microbial genes present, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that over six percent (6.44%) of the sequences from our buffalo rumen pool sample could be categorized as virulence genes and genes associated with resistance to antibiotic and toxic compounds (RATC), which is a higher proportion of virulence genes reported from metagenome samples of chicken cecum (5.39%), cow rumen (4.43%) and Sargasso sea (2.95%). However, it was lower than the proportion found in cow milk (11.33%) cattle faeces (8.4%), Antarctic marine derived lake (8.45%), human fecal (7.7%) and farm soil (7.79%). The dynamic nature of metagenomic data, together with the large number of RATC classes observed in samples from widely different ecologies indicates that metagenomic data can be used to track potential targets and relative amounts of antibiotic resistance genes in individual animals. In addition, these data can be also used to generate antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.

  10. Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo (Bubalus bubalis).

    PubMed

    Singh, K M; Jakhesara, S J; Koringa, P G; Rank, D N; Joshi, C G

    2012-10-10

    A major research goal in rumen microbial ecology is to understand the relationship between community composition and its function, particularly involved in fermentation process is of a potential interest. The buffalo rumen microbiota impacts human food safety as well as animal health. Although the bacteria of bovine rumen have been well characterized, techniques have been lacking to correlate total community structure with gene function. We applied 454 next generations sequencing technology to characterize general microbial diversity present in buffalo rumen metagenome and also identified the repertoire of microbial genes present, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that over six percent (6.44%) of the sequences from our buffalo rumen pool sample could be categorized as virulence genes and genes associated with resistance to antibiotic and toxic compounds (RATC), which is a higher proportion of virulence genes reported from metagenome samples of chicken cecum (5.39%), cow rumen (4.43%) and Sargasso sea (2.95%). However, it was lower than the proportion found in cow milk (11.33%) cattle faeces (8.4%), Antarctic marine derived lake (8.45%), human fecal (7.7%) and farm soil (7.79%). The dynamic nature of metagenomic data, together with the large number of RATC classes observed in samples from widely different ecologies indicates that metagenomic data can be used to track potential targets and relative amounts of antibiotic resistance genes in individual animals. In addition, these data can be also used to generate antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats. PMID:22850272

  11. The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

    PubMed

    Kemner, J M; Liang, X; Nester, E W

    1997-04-01

    The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose. PMID:9079938

  12. Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice

    PubMed Central

    Carroll, Ronan K.; Shelburne, Samuel A.; Olsen, Randall J.; Suber, Bryce; Sahasrabhojane, Pranoti; Kumaraswami, Muthiah; Beres, Stephen B.; Shea, Patrick R.; Flores, Anthony R.; Musser, James M.

    2011-01-01

    Infection with different strains of the same species of bacteria often results in vastly different clinical outcomes. Despite extensive investigation, the genetic basis of microbial strain-specific virulence remains poorly understood. Recent whole-genome sequencing has revealed that SNPs are the most prevalent form of genetic diversity among different strains of the same species of bacteria. For invasive serotype M3 group A streptococci (GAS) strains, the gene encoding regulator of proteinase B (RopB) has the highest frequency of SNPs. Here, we have determined that ropB polymorphisms alter RopB function and modulate GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically divergent effects on GAS global gene expression. Additionally, generation of isoallelic GAS strains differing only by a single amino acid in RopB confirmed that variant proteins affected transcript levels of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that ropB polymorphisms influence GAS virulence and disease manifestations. These data detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections. PMID:21490401

  13. Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence.

    PubMed

    Tischler, Anna D; Leistikow, Rachel L; Ramakrishnan, Pavithra; Voskuil, Martin I; McKinney, John D

    2016-01-01

    The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ΔpstA1ΔpstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ΔpstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ΔpstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions. PMID:27557082

  14. Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence

    PubMed Central

    Leistikow, Rachel L.; Ramakrishnan, Pavithra; Voskuil, Martin I.; McKinney, John D.

    2016-01-01

    The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ΔpstA1ΔpstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ΔpstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ΔpstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions. PMID:27557082

  15. The Chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor.

    PubMed

    Carlson, John H; Whitmire, William M; Crane, Deborah D; Wicke, Luke; Virtaneva, Kimmo; Sturdevant, Daniel E; Kupko, John J; Porcella, Stephen F; Martinez-Orengo, Neysha; Heinzen, Robert A; Kari, Laszlo; Caldwell, Harlan D

    2008-06-01

    Chlamydia trachomatis possesses a cryptic 7.5-kb plasmid of unknown function. Here, we describe a comprehensive molecular and biological characterization of the naturally occurring plasmidless human C. trachomatis strain L2(25667R). We found that despite minimal chromosomal polymorphisms, the LGV strain L2(25667R) was indistinguishable from plasmid-positive strain L2(434) with regard to its in vitro infectivity characteristics such as growth kinetics, plaquing efficiency, and plaque size. The only in vitro phenotypic differences between L2(434) and L2(25667R) were the accumulation of glycogen granules in the inclusion matrix and the lack of the typical intrainclusion Brownian-like movement characteristic of C. trachomatis strains. Conversely, we observed a marked difference between the two strains in their abilities to colonize and infect the female mouse genital tract. The 50% infective dose of plasmidless strain L2(25667R) was 400-fold greater (4 x 10(6) inclusion-forming units [IFU]) than that of plasmid-bearing strain L2(434) (1 x 10(4) IFU). Transcriptome analysis of the two strains demonstrated a decrease in the transcript levels of a subset of chromosomal genes for strain L2(25667R). Among those genes was glgA, encoding glycogen synthase, a finding consistent with the failure of L2(25667R) to accumulate glycogen granules. These findings support a primary role for the plasmid in in vivo infectivity and suggest that virulence is controlled, at least in part, by the plasmid's ability to regulate the expression of chromosomal genes. Our findings have important implications in understanding a role for the plasmid in the pathogenesis of human infection and disease.

  16. Virulence and the presence of aminoglycoside resistance genes of Staphylococcus haemolyticus strains isolated from clinical specimens.

    PubMed

    Krzymińska, Sylwia; Szczuka, Ewa; Dudzińska, Kinga; Kaznowski, Adam

    2015-04-01

    We examined thirty methicillin-resistant Staphylococcus haemolyticus isolates cultured from clinical specimens for antibiotic resistance, various important interactions of the bacteria with epithelial cells and putative virulence determinants. All strains were resistant to oxacillin and carried the mecA gene. Aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene encoding nucleotidyltransferases was detected in 43 %, aminocyclitol-6'-acetyltransferase-aminocyclitol-2″-phosphotransferase (aac(6')/aph(2″)) gene encoding bifunctional acetyltransferases/phosphotransferases in 33 %, aminocyclitol-4'-adenylyltransferase (ant(4')-Ia) gene encoding phosphotransferases in 20 %. The coexistence of resistance to methicillin and aminoglycosides was investigated in multi-resistant strains. Coexisting (aac(6')/aph(2″)) and (aph(3')-IIIa) genes were detected in 33 % of isolates, whereas 63 % of isolates had at least one of these genes. All strains revealed adherence ability and most of them (63 %) were invasive to epithelial cells. Electron microscopy revealed that the bacteria were found in vacuoles inside the cells. We observed that the contact of the bacteria with host epithelial cells is a prerequisite to their cytotoxicity at 5 h-incubation. Culture supernatant of the strains induced a low effect of cytotoxicity at the same time of incubation. Cell-free supernatant of all isolates expressed cytotoxic activity which caused destruction of HEp-2 cells at 24 h. None of the strains was cytotonic towards CHO cells. Among thirty strains, 27 % revealed lipolytic activity, 43 % produced lecithinase and 20 % were positive for proteinase activity. Analyses of cellular morphology and DNA fragmentation exhibited typical characteristic features of those undergoing apoptosis. The Pearson linear test revealed positive correlations between the apoptotic index at 24 h and percentage of cytotoxicity. Our results provided new insights into the mechanisms contributing to the

  17. Microevolution During Serial Mouse Passage Demonstrates FRE3 as a Virulence Adaptation Gene in Cryptococcus neoformans

    PubMed Central

    Hu, Guowu; Chen, Shu Hui; Qiu, Jin; Bennett, John E.; Myers, Timothy G.; Williamson, Peter R.

    2014-01-01

    ABSTRACT Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a “virulence acquisition gene” during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts. PMID:24692633

  18. Dual regulatory effects of non-coding GC-rich elements on the expression of virulence genes in malaria parasites.

    PubMed

    Wei, Guiying; Zhao, Yuemeng; Zhang, Qingfeng; Pan, Weiqing

    2015-12-01

    As the primary virulence factor of falciparum malaria, var genes harboring mutually exclusive expression pattern lead to antigenic variation and immune evasion of this pathogen in human host. Although various mechanisms contribute to silence of var genes, little is known of transcriptional activation pathways of a single var gene and maintenance of its active state with other silent var loci. Here, we report a monoallelic expression pattern of the non-coding GC-elements flanking chromosomal internal var genes, and transcript from the active one was required for activation of the var gene in the same array. Meanwhile, GFP reporter assays revealed a repressive effect on the adjacent gene induced by DNA motifs of the insulator-like GC-element, which was linked to heterochromatin subnuclear localization. Taken together, these data for the first time provide experimental evidence of the dual cis- and trans-acting regulatory functions of the GC-elements in both silence and activation of var genes, which would advance our understanding of the complex regulatory network of the virulence gene family in P. falciparum.

  19. Non-tuberculous mycobacterial lymphadenitis.

    PubMed Central

    White, M P; Bangash, H; Goel, K M; Jenkins, P A

    1986-01-01

    Most cases of mycobacterial lymphadenitis in children are caused by non-tuberculous mycobacteria, previously called the atypical mycobacteria. It is important to differentiate non-tuberculous mycobacterial lymphadenitis from tuberculous lymphadenitis as the treatment is different. We reviewed 19 children (12 girls and seven boys) with non-tuberculous mycobacterial lymphadenitis to define likely presenting features, helpful diagnostic measurements, and optimum management. Mean age at diagnosis was 5.2 years. Most had no systemic upset and clear chest x ray films. Cervical nodes were the commonest affected, and enlargement was usually unilateral. Mean duration of swelling was 6.6 weeks, and 63% of the nodes had an appearance suggestive of cold abscess. Routine haematology was unhelpful, and standard tuberculin testing performed in 47% yielded negative results in two thirds. Differential Mantoux testing with human purified protein derivative and an avium-intracellular antigen may be more useful. Antituberculous drugs were ineffective. The organism was usually highly resistant. Total excision is the treatment of choice. Antituberculous drugs are unnecessary. PMID:3707188

  20. Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach.

    PubMed

    Durso, Lisa M; Harhay, Gregory P; Bono, James L; Smith, Timothy P L

    2011-02-01

    The bovine fecal microbiota impacts human food safety as well as animal health. Although the bacteria of cattle feces have been well characterized using culture-based and culture-independent methods, techniques have been lacking to correlate total community composition with community function. We used high throughput sequencing of total DNA extracted from fecal material to characterize general community composition and examine the repertoire of microbial genes present in beef cattle feces, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that traditional 16S sequencing using "universal" primers to generate full-length sequence may under represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats indicates that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time. Consequently, these data can be used to generate sample-specific and temporal antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.

  1. Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach.

    PubMed

    Durso, Lisa M; Harhay, Gregory P; Bono, James L; Smith, Timothy P L

    2011-02-01

    The bovine fecal microbiota impacts human food safety as well as animal health. Although the bacteria of cattle feces have been well characterized using culture-based and culture-independent methods, techniques have been lacking to correlate total community composition with community function. We used high throughput sequencing of total DNA extracted from fecal material to characterize general community composition and examine the repertoire of microbial genes present in beef cattle feces, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that traditional 16S sequencing using "universal" primers to generate full-length sequence may under represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats indicates that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time. Consequently, these data can be used to generate sample-specific and temporal antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats. PMID:21167876

  2. Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

    PubMed Central

    Odo, Chioma; DuPont, Herbert L.

    2016-01-01

    ABSTRACT 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. PMID:27531912

  3. Aberrant host immune response induced by highly virulent PRRSV identified by digital gene expression tag profiling

    PubMed Central

    2010-01-01

    Background There was a large scale outbreak of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China and Vietnam during 2006 and 2007 that resulted in unusually high morbidity and mortality among pigs of all ages. The mechanisms underlying the molecular pathogenesis of the highly virulent PRRS virus (H-PRRSV) remains unknown. Therefore, the relationship between pulmonary gene expression profiles after H-PRRSV infection and infection pathology were analyzed in this study using high-throughput deep sequencing and histopathology. Results H-PRRSV infection resulted in severe lung pathology. The results indicate that aberrant host innate immune responses to H-PRRSV and induction of an anti-apoptotic state could be responsible for the aggressive replication and dissemination of H-PRRSV. Prolific rapid replication of H-PRRSV could have triggered aberrant sustained expression of pro-inflammatory cytokines and chemokines leading to a markedly robust inflammatory response compounded by significant cell death and increased oxidative damage. The end result was severe tissue damage and high pathogenicity. Conclusions The systems analysis utilized in this study provides a comprehensive basis for better understanding the pathogenesis of H-PRRSV. Furthermore, it allows the genetic components involved in H-PRRSV resistance/susceptibility in swine populations to be identified. PMID:20929578

  4. CORONAVIRUS VIRULENCE GENES WITH MAIN FOCUS ON SARS-CoV ENVELOPE GENE

    PubMed Central

    DeDiego, Marta L.; Nieto-Torres, Jose L.; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Usera, Fernando; Enjuanes, Luis

    2014-01-01

    models and in humans. The modification or deletion of different motifs within E protein, including the transmembrane domain that harbors an ion channel activity, small sequences within the middle region of the carboxy-terminus of E protein, and its most carboxy-terminal end, which contains a PDZ domain-binding motif (PBM) is sufficient to attenuate the virus. Interestingly, a comprehensive collection of SARS-CoVs in which these motifs have been modified elicited full and long-term protection even in old mice, making those deletion mutants promising vaccine candidates. These data indicate that despite its small size, E protein drastically influences the replication of CoVs and their pathogenicity. Although E protein is not essential for CoV genome replication or subgenomic mRNA synthesis, it affects virus morphogenesis, budding, assembly, intracellular trafficking, and virulence. In fact, E protein is responsible in a significant proportion of the inflammasome activation and the associated inflammation elicited by SARS-CoV in the lung parenchyma. This exacerbated inflammation causes edema accumulation leading to acute respiratory distress syndrome (ARDS) and, frequently, to the death of infected animal models or human patients. PMID:25093995

  5. Modulation of virulence genes by the two-component system PhoP-PhoQ in avian pathogenic Escherichia coli.

    PubMed

    Tu, Jian; Huang, Boyan; Zhang, Yu; Zhang, Yuxi; Xue, Ting; Li, Shaocan; Qi, Kezong

    2016-01-01

    Avian pathogenic Escherichia coli (APEC) infections are a very important problem in the poultry industry. PhoP-PhoQ is a two-component system that regulates virulence genes in APEC. In this study, we constructed strains that lacked the PhoP or PhoQ genes to assess regulation of APEC pathogenicity by the PhoP-PhoQ two-component system. The PhoP mutant strain AE18, PhoQ mutant strain AE19, and PhoP/PhoQ mutant strain AE20 were constructed by the Red homologous recombination method. Swim plates were used to evaluate the motility of the APEC strains, viable bacteria counting was used to assess adhesion and invasion of chick embryo fibroblasts, and Real-Time PCR was used to measure mRNA expression of virulence genes. We first confirmed that AE18, AE19, and AE20 were successfully constructed from the wild-type AE17 strain. AE18, AE19, and AE20 showed significant decreases in motility of 70.97%, 83.87%, and 37.1%, respectively, in comparison with AE17. Moreover, in comparison with AE17, AE18, AE19, and AE20 showed significant decreases of 63.11%, 65.42%, and 30.26%, respectively, in CEF cell adhesion, and significant decreases of 59.83%, 57.82%, and 37.90%, respectively, in CEF cell invasion. In comparison with AE17, transcript levels of sodA, polA, and iss were significantly decreased in AE18, while transcript levels of fimC and iss were significantly decreased in AE19. Our results demonstrate that deletion of PhoP or PhoQ inhibits invasion and adhesion of APEC to CEF cells and significantly reduces APEC virulence by regulating transcription of virulence genes.

  6. Modulation of virulence genes by the two-component system PhoP-PhoQ in avian pathogenic Escherichia coli.

    PubMed

    Tu, Jian; Huang, Boyan; Zhang, Yu; Zhang, Yuxi; Xue, Ting; Li, Shaocan; Qi, Kezong

    2016-01-01

    Avian pathogenic Escherichia coli (APEC) infections are a very important problem in the poultry industry. PhoP-PhoQ is a two-component system that regulates virulence genes in APEC. In this study, we constructed strains that lacked the PhoP or PhoQ genes to assess regulation of APEC pathogenicity by the PhoP-PhoQ two-component system. The PhoP mutant strain AE18, PhoQ mutant strain AE19, and PhoP/PhoQ mutant strain AE20 were constructed by the Red homologous recombination method. Swim plates were used to evaluate the motility of the APEC strains, viable bacteria counting was used to assess adhesion and invasion of chick embryo fibroblasts, and Real-Time PCR was used to measure mRNA expression of virulence genes. We first confirmed that AE18, AE19, and AE20 were successfully constructed from the wild-type AE17 strain. AE18, AE19, and AE20 showed significant decreases in motility of 70.97%, 83.87%, and 37.1%, respectively, in comparison with AE17. Moreover, in comparison with AE17, AE18, AE19, and AE20 showed significant decreases of 63.11%, 65.42%, and 30.26%, respectively, in CEF cell adhesion, and significant decreases of 59.83%, 57.82%, and 37.90%, respectively, in CEF cell invasion. In comparison with AE17, transcript levels of sodA, polA, and iss were significantly decreased in AE18, while transcript levels of fimC and iss were significantly decreased in AE19. Our results demonstrate that deletion of PhoP or PhoQ inhibits invasion and adhesion of APEC to CEF cells and significantly reduces APEC virulence by regulating transcription of virulence genes. PMID:27096785

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

    PubMed

    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.

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

    PubMed Central

    2011-01-01

    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 alveolar and bronchiolar

  9. Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes

    PubMed Central

    Lu, Jingrang; Ashbolt, Nicholas J.

    2014-01-01

    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. PMID:25381242

  10. The effect of cell growth phase on the regulatory cross-talk between flagellar and Spi1 virulence gene expression.

    PubMed

    Mouslim, Chakib; Hughes, Kelly T

    2014-03-01

    The flagellar regulon controls Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD₄C₂ transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1(flhDC) and P5(flhDC), were functional in a wild-type background, while P6(flhDC) was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1(flhDC) initiates flagellar assembly and a negative autoregulatory loop through FlhD₄C₂-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1(flhDC) also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD₄C₂ transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.

  11. Prevalence of virulence genes of biofilm producing strains of Staphylococcus epidermidis isolated from clinical samples in Iran.

    PubMed

    Solati, Seyed Mostafa; Tajbakhsh, Elahe; Khamesipour, Faham; Gugnani, Harish C

    2015-12-01

    Coagulase negative staphylococci are recognized as opportunistic pathogens and are widespread in the environment. It is possible to prevent and control infections due to these bacteria if their virulence factors are recognized. Eighty isolates of Staphylococcus epidermidis (S. epidermidis) including 42 from urine (52.5%), 23 from blood (28.75%), 15 from dialysis bags (18.75%) were studied for biofilm production on Congo red agar (CRA). The virulence genes in S. aureus were investigated using polymerase chain reaction (PCR) with primers. Out of 80 isolates studied, 40 isolated (50%) formed black colonies (biofilm-forming strains) on CRA. In 22 of these isolates (25%) reaction was strongly positive; in 12 isolates (15%) reaction was moderately positive, and in the remaining 6 isolates, reaction was weakly positive. In the 22 isolates that had strong positive reaction and produced black colonies on biofilm, all virulent genes (icaC, icaD, icaA icaB, icaR) were expressed. In the 12 isolates that had moderate positive reaction, 8 expressed all genes (icaC, icaD, icaA icaB, icaR) expressed while the remaining 4 expressed only ica A, and ica D genes. Of the 6 isolated which had weak positive reaction, only 1 isolate (2.5%) expressed all the genes, in the other 5 isolates no gene was observed. Urinary isolates more frequently form biofilms than the isolates from other clinical samples. Statistical analysis using Chi square test showed that there was a significant correlation between the type of sample and the biofilm production (P < 0.05). The results of biofilm production on CRA were largely in agreement with microtiter plate assay and PCR assay. The capacity of bacteria to produce biofilm is an important factor in infectivity and happens via expression of ica genes. Recognition of bacteria that produce biofilm is thus important to control infection due to these bacteria.

  12. Selected Lactic Acid-Producing Bacterial Isolates with the Capacity to Reduce Salmonella Translocation and Virulence Gene Expression in Chickens

    PubMed Central

    Yang, Xiaojian; Brisbin, Jennifer; Yu, Hai; Wang, Qi; Yin, Fugui; Zhang, Yonggang; Sabour, Parviz; Sharif, Shayan; Gong, Joshua

    2014-01-01

    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 (106–7 CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (104 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 vivo can be one

  13. Virulence Attributes of Low-Virulence Organisms

    PubMed Central

    1994-01-01

    The vast majority of infections involving female pelvic structures arise from organisms that are members of the normal flora. In addition, exogenous organisms that invade through the lower genital tract must interact with organisms that are part of the host's flora. In contrast to the concept that the normal flora is entirely innocuous, recent research has begun to identify what appear to be virulence attributes among these ordinarily low-virulence organisms. Most of our understanding of virulence has been derived from highly virulent organisms, of which Neisseria gonorrhoeae provides an example of relevance to the female genital tract. A review of the virulence factors of the gonococcus is presented to serve as an example of the variety of virulence properties associated with pathogenic bacteria. Molecular biology has begun to clarify one of the important paradigms of pathogenic bacteriology—that bacteria change their expression of virulence properties in response to their location within a host or to the stage of infection. Thus, infection involves not only the possession of virulence factors, but also the carefully controlled use of those factors. Virulence is often controlled by the coordinate expression of many virulence-associated genes in response to one environmental signal. With regard to low- virulence organisms present in the female lower genital tract, we are beginning to identify some of their virulence attributes. Examples from the work of our laboratory include the hemolysin of Gardnerella vaginalis and an immunosuppressive mycotoxin produced by Candida albicans. Demonstrating the coordinate expression (or other control mechanisms) of virulence factors in these sometimes innocuous and sometimes inimical organisms represents the next frontier in the study of normal vaginal microbiology. PMID:18475373

  14. The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes

    PubMed Central

    Elías-Villalobos, Alberto; Fernández-Álvarez, Alfonso; Moreno-Sánchez, Ismael; Helmlinger, Dominique; Ibeas, José I.

    2015-01-01

    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. PMID:26317403

  15. The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes.

    PubMed

    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.

  16. Transcriptomic analysis of the GCN5 gene reveals mechanisms of the epigenetic regulation of virulence and morphogenesis in Ustilago maydis.

    PubMed

    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.

  17. Isolation of Escherichia coli from piglets in South Korea with diarrhea and characteristics of the virulence genes.

    PubMed

    Kim, Yeong Ju; Kim, Ji Hee; Hur, Jin; Lee, John Hwa

    2010-01-01

    Escherichia coli was isolated from the feces of 122 piglets with diarrhea on 55 farms in Korea. The virulence genes of each isolate were characterized by polymerase chain reaction (PCR). Of the 562 isolates, 191 carried 1 or more of the virulence genes tested for in this study. Of the 191 isolates, 114 (60%) carried 1 or more of the genes for enterotoxigenic E. coli (ETEC) fimbriae F4, F5, F6, F18, and F41 and ETEC toxins LT, STa, and STb, 57 (30%) carried 1 or more of the genes for the Shiga-toxin-producing E. coli (STEC) toxins Stx1, Stx2, and Stx2e, and 21% and 37% carried the gene for enteropathogenic E. coli intimin and for enteroaggregative E. coli toxin, respectively. Collectively, our results indicate that other pathotypes of E. coli as well as ETEC can be strongly associated with diarrhea in piglets. In addition, detection of the genes for Stx1 and Stx2 indicates that pigs are reservoirs of human pathogenic STEC. PMID:20357961

  18. Mutation of a novel virulence-related gene mltD in Vibrio anguillarum enhances lethality in zebra fish.

    PubMed

    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. PMID:21070855

  19. Identification of Virulence Factors Genes in Escherichia coli Isolates from Women with Urinary Tract Infection in Mexico

    PubMed Central

    López-Banda, Daniela A.; Carrillo-Casas, Erika M.; Orozco-Hoyuela, Gabriel; Manjarrez-Hernández, Ángel H.; Arroyo-Escalante, Sara; Moncada-Barrón, David; Villanueva-Recillas, Silvia; Xicohtencatl-Cortes, Juan; Hernández-Castro, Rigoberto

    2014-01-01

    E coli isolates (108) from Mexican women, clinically diagnosed with urinary tract infection, were screened to identify virulence genes, phylogenetic groups, and antibiotic resistance. Isolates were identified by MicroScan4 system; additionally, the minimum inhibitory concentration (MIC) was assessed. The phylogenetic groups and 16 virulence genes encoding adhesins, toxins, siderophores, lipopolysaccharide (LPS), and invasins were identified by PCR. Phylogenetic groups distribution was as follows: B1 9.3%, A 30.6%, B2 55.6%, and D 4.6%. Virulence genes prevalence was ecp 98.1%, fimH 86.1%, traT 77.8%, sfa/focDE 74.1%, papC 62%, iutA 48.1%, fyuA 44.4%, focG 2.8%, sfaS 1.9%, hlyA 7.4%, cnf-1 6.5%, cdt-B 0.9%, cvaC 2.8%, ibeA 2.8%, and rfc 0.9%. Regarding antimicrobial resistance it was above 50% to ampicillin/sulbactam, ampicillin, piperacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and levofloxacin. Uropathogenic E. coli clustered mainly in the pathogenic phylogenetic group B2. The isolates showed a high presence of siderophores and adhesion genes and a low presence of genes encoding toxins. The high frequency of papC gene suggests that these isolates have the ability to colonize the kidneys. High resistance to drugs considered as first choice treatment such as trimethoprim/sulfamethoxazole and fluoroquinolones was consistently observed. PMID:24895634

  20. The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals

    PubMed Central

    Nomura, Kinya; Nasser, William; Kawagishi, Hirokazu; Tsuyumu, Shinji

    1998-01-01

    The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein. PMID:9826648

  1. Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate.

    PubMed

    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

  2. Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate

    PubMed Central

    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

  3. Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate

    PubMed Central

    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. Characterization of Stripe Rust Resistance in Wheat Lines with Resistance Gene Yr17 and Implications for Evaluating Resistance and Virulence.

    PubMed

    Milus, Eugene A; Lee, Kevin D; Brown-Guedira, Gina

    2015-08-01

    Stripe rust, caused by Puccinia striiformis f. sp. tritici, has been the most important foliar wheat disease in south central United States since 2000 when a new strain of the pathogen emerged. The resistance gene Yr17 was used by many breeding programs to develop resistant cultivars. Although Yr17 was classified as a seedling (all-stage) resistance gene conferring a low infection type, seedlings with Yr17 frequently had intermediate to high infection types when inoculated with isolates that caused little or no disease on adult plants of the same wheat lines. The objectives of this study were to determine how to best evaluate Yr17 resistance in wheat lines and to determine which factors made seedling tests involving Yr17 so variable. Stripe rust reactions on wheat seedlings with Yr17 were influenced by temperature, wheat genotype, pathogen isolate, and the leaf (first or second) used to assess the seedling reaction. The most critical factors for accurately evaluating Yr17 reactions at the seedling stage were to avoid night temperatures below 12°C, to use the first leaf to assess the seedling reaction, to use multiple differentials with Yr17 and known avirulent, partially virulent and virulent isolates as controls, and to recognize that intermediate infection types likely represent a level of partial virulence in the pathogen that is insufficient to cause disease on adult plants in the field.

  5. Beyond the Chromosome: The Prevalence of Unique Extra-Chromosomal Bacteriophages with Integrated Virulence Genes in Pathogenic Staphylococcus aureus

    PubMed Central

    Utter, Bryan; Deutsch, Douglas R.; Schuch, Raymond; Winer, Benjamin Y.; Verratti, Kathleen; Bishop-Lilly, Kim; Sozhamannan, Shanmuga; Fischetti, Vincent A.

    2014-01-01

    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). PMID:24963913

  6. Molecular Characterization of Equine Isolates of Streptococcus pneumoniae: Natural Disruption of Genes Encoding the Virulence Factors Pneumolysin and Autolysin

    PubMed Central

    Whatmore, Adrian M.; King, Samantha J.; Doherty, Neil C.; Sturgeon, Daniel; Chanter, Neil; Dowson, Christopher G.

    1999-01-01

    Although often considered a strict human pathogen, Streptococcus pneumoniae has been reported to infect and cause pneumonia in horses, although the pathology appears restricted compared to that of human infections. Here we report on the molecular characterization of a group of S. pneumoniae isolates obtained from horses in England and Ireland. Despite being obtained from geographically distinct locations, the isolates were found to represent a tight clonal group, virtually identical to each other but genetically distinguishable from more than 120 divergent isolates of human S. pneumoniae. A comprehensive analysis of known pneumococcal virulence determinants was undertaken in an attempt to understand the pathogenicity of equine pneumococci. Surprisingly, equine isolates appear to lack activities associated with both the hemolytic cytotoxin pneumolysin, often considered a major virulence factor of pneumococci, and the major autolysin gene lytA, also considered an important virulence factor. In support of phenotypic data, molecular studies demonstrated a deletion of parts of the coding sequences of both lytA and ply genes in equine pneumococci. The implications of these findings for the evolution and pathogenicity of equine S. pneumoniae are discussed. PMID:10338480

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

    PubMed

    Utter, Bryan; Deutsch, Douglas R; Schuch, Raymond; Winer, Benjamin Y; Verratti, Kathleen; Bishop-Lilly, Kim; Sozhamannan, Shanmuga; Fischetti, Vincent A

    2014-01-01

    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). PMID:24963913

  8. Virulence Genes among Enterococcus faecalis and Enterococcus faecium Isolated from Coastal Beaches and Human and Nonhuman Sources in Southern California and Puerto Rico

    PubMed Central

    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

  9. Virulence Genes among Enterococcus faecalis and Enterococcus faecium Isolated from Coastal Beaches and Human and Nonhuman Sources in Southern California and Puerto Rico.

    PubMed

    Ferguson, Donna M; Talavera, Ginamary Negrón; Hernández, Luis A Ríos; Weisberg, Stephen B; 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.

  10. Virulence Genes among Enterococcus faecalis and Enterococcus faecium Isolated from Coastal Beaches and Human and Nonhuman Sources in Southern California and Puerto Rico.

    PubMed

    Ferguson, Donna M; Talavera, Ginamary Negrón; Hernández, Luis A Ríos; Weisberg, Stephen B; 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

  11. [Molecular diagnosis of mycobacterial infections].

    PubMed

    Fend, F; Langer, R; Hann von Weyhern, C W; Schulz, S; Miethke, T

    2007-01-01

    Tuberculosis remains a leading cause of morbidity and mortality worldwide. A rapid and reliable diagnosis and discrimination from infections with nontuberculous mycobacteria (NTM) is critical. Frequently, formalin-fixed, paraffin-embedded (FFPE) tissues remain the only source for detection of micro-organisms in suspected cases of mycobacterial infection. Recently, numerous methods, including PCR assays, in situ hybridization and immunohistochemistry have been developed for detection of mycobacteria in FFPE samples. PCR-based assays are directed either against M.tbc.-specific sequences, such as IS6110, or amplify regions common to many mycobacterial species, e.g. the 65 kDa antigen, and then require sequencing or restriction fragment length polymorphism for species identification. Whereas the detection of DNA of M.tbc. in the correct setting is always of clinical relevance, the presence of various NTM species has to be interpreted with great caution due to their ubiquitous nature. However, the routine application of molecular tests has demonstrated that NTM infections are more common than previously thought, even in non-immunosuppressed hosts. The introduction of real-time PCR technology allows precise quantification of mycobacterial DNA and can be used for species identification through melting point analysis or appropriate DNA probes. Application of these assays originally developed for clinical microbiology offer a great opportunity for diagnostic improvement in molecular pathology as compared to qualitative PCR, mainly due to an increased specificity and a lower risk of contamination. Given the clinical impact of a positive molecular result for M. tbc., future efforts have to be aimed at standardization and quality control. PMID:18314607

  12. [Molecular diagnosis of mycobacterial infections].

    PubMed

    Fend, F; Langer, R; Hann von Weyhern, C W; Schulz, S; Miethke, T

    2007-01-01

    Tuberculosis remains a leading cause of morbidity and mortality worldwide. A rapid and reliable diagnosis and discrimination from infections with nontuberculous mycobacteria (NTM) is critical. Frequently, formalin-fixed, paraffin-embedded (FFPE) tissues remain the only source for detection of micro-organisms in suspected cases of mycobacterial infection. Recently, numerous methods, including PCR assays, in situ hybridization and immunohistochemistry have been developed for detection of mycobacteria in FFPE samples. PCR-based assays are directed either against M.tbc.-specific sequences, such as IS6110, or amplify regions common to many mycobacterial species, e.g. the 65 kDa antigen, and then require sequencing or restriction fragment length polymorphism for species identification. Whereas the detection of DNA of M.tbc. in the correct setting is always of clinical relevance, the presence of various NTM species has to be interpreted with great caution due to their ubiquitous nature. However, the routine application of molecular tests has demonstrated that NTM infections are more common than previously thought, even in non-immunosuppressed hosts. The introduction of real-time PCR technology allows precise quantification of mycobacterial DNA and can be used for species identification through melting point analysis or appropriate DNA probes. Application of these assays originally developed for clinical microbiology offer a great opportunity for diagnostic improvement in molecular pathology as compared to qualitative PCR, mainly due to an increased specificity and a lower risk of contamination. Given the clinical impact of a positive molecular result for M. tbc., future efforts have to be aimed at standardization and quality control.

  13. Identification of resistance to new virulent races of rust in sunflowers and validation of DNA markers in the gene pool.

    PubMed

    Qi, Lili; Gulya, Tom; Seiler, Gerald J; Hulke, Brent S; Vick, Brady A

    2011-02-01

    Sunflower rust, caused by Puccinia helianthi, is a prevalent disease in many countries throughout the world. The U.S. Department of Agriculture (USDA)-Agricultural Research Service, Sunflower Research Unit has released rust resistant breeding materials for several decades. However, constantly coevolving rust populations have formed new virulent races to which current hybrids have little resistance. The objectives of this study were to identify resistance to race 336, the predominant race in North America, and to race 777, the most virulent race currently known, and to validate molecular markers known to be linked to rust resistance genes in the sunflower gene pool. A total of 104 entries, including 66 released USDA inbred lines, 14 USDA interspecific germplasm lines, and 24 foreign germplasms, all developed specifically for rust resistance, were tested for their reaction to races 336 and 777. Only 13 of the 104 entries tested were resistant to both races, whereas another six were resistant only to race 336. The interspecific germplasm line, Rf ANN-1742, was resistant to both races and was identified as a new rust resistance source. A selection of 24 lines including 19 lines resistant to races 777 and/or 336 was screened with DNA markers linked to rust resistance genes R(1), R(2), R(4u), and R(5). The results indicated that the existing resistant lines are diverse in rust resistance genes. Durable genetic resistance through gene pyramiding will be effective for the control of rust.

  14. Molecular characterization reveals similar virulence gene content in unrelated clonal groups of Escherichia coli of serogroup O174 (OX3).

    PubMed

    Tarr, Cheryl L; Nelson, Adam M; Beutin, Lothar; Olsen, Katharina E P; Whittam, Thomas S

    2008-02-01

    Most severe illnesses that are attributed to Shiga toxin-producing Escherichia coli are caused by isolates that also carry a pathogenicity island called the locus of enterocyte effacement (LEE). However, many cases of severe disease are associated with LEE-negative strains. We characterized the virulence gene content and the evolutionary relationships of Escherichia coli isolates of serogroup O174 (formerly OX3), strains of which have been implicated in cases of hemorrhagic colitis and hemolytic uremic syndrome. A total of 56 isolates from humans, farm animals, and food were subjected to multilocus virulence gene profiling (MVGP), and a subset of 16 isolates was subjected to multilocus sequence analysis (MLSA). The MLSA revealed that the O174 isolates fall into four separate evolutionary clusters within the E. coli phylogeny and are related to a diverse array of clonal groups, including enteropathogenic E. coli 2 (EPEC 2), enterohemorrhagic E. coli 2 (EHEC 2), and EHEC-O121. Of the 15 genes that we surveyed with MVGP, only 6 are common in the O174 strains. The different clonal groups within the O174 serogroup appear to have independently acquired and maintained similar sets of genes that include the Shiga toxins (stx1 and stx2) and two adhesins (saa and iha). The absence of certain O island (OI) genes, such as those found on OI-122, is consistent with the notion that certain pathogenicity islands act cooperatively with the LEE island.

  15. Characterization of virulence genes cagA and vacA in Helicobacter Pylori and their prevalence in gastrointestinal disorders

    PubMed Central

    Cogo, Laura Lúcia; Monteiro, Cristina Leise Bastos; Nogueira, Keite da Silva; Palmeiro, Jussara Kasuko; Ribeiro, Marcelo Lima; de Camargo, Eloá Ramalho; Neves, Daniel Locatelli; do Nascimento, Aguinaldo José; Costa, Libera Maria Dalla

    2011-01-01

    Prevalence of H. pylori infection was determined using cultures of gastric biopsy samples of patients attended at the academic hospital of the Federal University of Paraná, Curitiba, Paraná, Brazil. Molecular methods were used to characterize the cagA and vacA genes from bacterial isolates associated with different diseases presented by patients. Out of a total of 81, forty-two gastric biopsy samples tested were positive for H. pylori, with a prevalence of 51.9%. No significant difference was found with regard to the gender (p=0.793) and age (p=0.183) of the patients. Genotype s1m1 vacA gene was found in 67% of the cases of peptic ulcer investigated (p=1.0), despite the limited number of patients with this disease (n=3). A correlation between the presence of less virulent strains (s2m2) and reflux esophagitis was found in the majority of the cases (45%), but without statistical significance. An association between the prevalence of cagA gene, found in 92% of isolates, and peptic ulcer was not observed (p=1.0), suggesting that this gene cannot be considered a specific marker of severity in our environment. The results reinforce the importance of conducting regional studies and the need to characterize H. pylori virulence genes associated with different diseases. PMID:24031754

  16. The thiG Gene Is Required for Full Virulence of Xanthomonas oryzae pv. oryzae by Preventing Cell Aggregation

    PubMed Central

    Yu, Xiaoyue; Liang, Xiaoyu; Liu, Kexue; Dong, Wenxia; Wang, Jianxin; Zhou, Ming-guo

    2015-01-01

    Bacterial blight of rice is an important serious bacterial diseases of rice in many rice-growing regions, caused by Xanthomonas oryzae pv. oryzae (Xoo). The thiG gene from Xoo strain ZJ173, which is involved with thiazole moiety production in the thiamine biosynthesis pathway, is highly conserved among the members of Xanthomonas. The thiG deletion mutant displayed impaired virulence and growth in thiamine-free medium but maintained its normal growth rate in the rice tissues, indicating that the thiG gene is involved in Xoo virulence. Compared to the wild type strain, the formation of cell-cell aggregates was affected in thiG deletion mutants. Although biofilm formation was promoted, motility and migration in rice leaves were repressed in the thiG mutants, and therefore limited the expansion of pathogen infection in rice. Quorum sensing and extracellular substance are two key factors that contribute to the formation of cell-cell aggregates. Our study found that in the thiG mutant the expression of two genes, rpfC and rpfG, which form a two-component regulatory signal system involved in the regulation of biofilm formation by a second messenger cyclic di-GMP is down-regulated. In addition, our study showed that xanthan production was not affected but the expression of some genes associated with xanthan biosynthesis, like gumD, gumE, gumH and gumM, were up-regulated in thiG mutants. Taken together, these findings are the first to demonstrate the role of the thiazole biosynthsis gene, thiG, in virulence and the formation of aggregates in Xanthomonas oryzae pv. oryzae. PMID:26222282

  17. Virulence gene profiling and pathogenicity characterization of non-typhoidal Salmonella accounted for invasive disease in humans.

    PubMed

    Suez, Jotham; Porwollik, Steffen; Dagan, Amir; Marzel, Alex; Schorr, Yosef Ilan; Desai, Prerak T; Agmon, Vered; McClelland, Michael; Rahav, Galia; Gal-Mor, Ohad

    2013-01-01

    Human infection with non-typhoidal Salmonella serovars (NTS) infrequently causes invasive systemic disease and bacteremia. To understand better the nature of invasive NTS (iNTS), we studied the gene content and the pathogenicity of bacteremic strains from twelve serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, Virchow, Newport, Bredeney, Heidelberg, Montevideo, Schwarzengrund, 9,12:l,v:- and Hadar). Comparative genomic hybridization using a Salmonella enterica microarray revealed a core of 3233 genes present in all of the iNTS strains, which include the Salmonella pathogenicity islands 1-5, 9, 13, 14; five fimbrial operons (bcf, csg, stb, sth, sti); three colonization factors (misL, bapA, sinH); and the invasion gene, pagN. In the iNTS variable genome, we identified 16 novel genomic islets; various NTS virulence factors; and six typhoid-associated virulence genes (tcfA, cdtB, hlyE, taiA, STY1413, STY1360), displaying a wider distribution among NTS than was previously known. Characterization of the bacteremic strains in C3H/HeN mice showed clear differences in disease manifestation. Previously unreported characterization of serovars Schwarzengrund, 9,12:l,v:-, Bredeney and Virchow in the mouse model showed low ability to elicit systemic disease, but a profound and elongated shedding of serovars Schwarzengrund and 9,12:l,v:- (as well as Enteritidis and Heidelberg) due to chronic infection of the mouse. Phenotypic comparison in macrophages and epithelial cell lines demonstrated a remarkable intra-serovar variation, but also showed that S. Typhimurium bacteremic strains tend to present lower intracellular growth than gastroenteritis isolates. Collectively, our data demonstrated a common core of virulence genes, which might be required for invasive salmonellosis, but also an impressive degree of genetic and phenotypic heterogeneity, highlighting that bacteremia is a complex phenotype, which cannot be attributed merely to an enhanced invasion or intracellular

  18. Whole-Genome Sequence Analysis and Genome-Wide Virulence Gene Identification of Riemerella anatipestifer Strain Yb2.

    PubMed

    Wang, Xiaolan; Ding, Chan; Wang, Shaohui; Han, Xiangan; Yu, Shengqing

    2015-08-01

    Riemerella anatipestifer is a well-described pathogen of waterfowl and other avian species that can cause septicemic and exudative diseases. In this study, we sequenced the complete genome of R. anatipestifer strain Yb2 and analyzed it against the published genomic sequences of R. anatipestifer strains DSM15868, RA-GD, RA-CH-1, and RA-CH-2. The Yb2 genome contains one circular chromosome of 2,184,066 bp with a 35.73% GC content and no plasmid. The genome has 2,021 open reading frames that occupy 90.88% of the genome. A comparative genomic analysis revealed that genome organization is highly conserved among R. anatipestifer strains, except for four inversions of a sequence segment in Yb2. A phylogenetic analysis found that the closest neighbor of Yb2 is RA-GD. Furthermore, we constructed a library of 3,175 mutants by random transposon mutagenesis, and 100 mutants exhibiting more than 100-fold-attenuated virulence were obtained by animal screening experiments. Southern blot analysis and genetic characterization of the mutants led to the identification of 49 virulence genes. Of these, 25 encode cytoplasmic proteins, 6 encode cytoplasmic membrane proteins, 4 encode outer membrane proteins, and the subcellular localization of the remaining 14 gene products is unknown. The functional classification of orthologous-group clusters revealed that 16 genes are associated with metabolism, 6 are associated with cellular processing and signaling, and 4 are associated with information storage and processing. The functions of the other 23 genes are poorly characterized or unknown. This genome-wide study identified genes important to the virulence of R. anatipestifer. PMID:26002892

  19. Rapidly Evolving Genes Are Key Players in Host Specialization and Virulence of the Fungal Wheat Pathogen Zymoseptoria tritici (Mycosphaerella graminicola)

    PubMed Central

    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

  20. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

    SciTech Connect

    Lowden, Michael J.; Skorupski, Karen; Pellegrini, Maria; Chiorazzo, Michael G.; Taylor, Ronald K.; Kull, F. Jon

    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.

  1. Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

    PubMed Central

    Geuskens, V; Vogel, J L; Grimaud, R; Desmet, L; Higgins, N P; Toussaint, A

    1991-01-01

    Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus. Images FIG. 3 FIG. 4 FIG. 5 PMID:1833383

  2. Antimicrobial resistance, virulence genes and PFGE-profiling of Escherichia coli isolates from South Korean cattle farms.

    PubMed

    Shin, Seung Won; Byun, Jae-Won; Jung, Myounghwan; Shin, Min-Kyoung; Yoo, Han Sang

    2014-09-01

    To estimate the prevalence of Escherichia coli with potential pathogenicity in cattle farm in South Korea, a total of 290 E. coli isolates were isolated from cattle farms over a period of 2 years in South Korea. These were examined for phenotypic and genotypic characteristics including antimicrobial susceptibility, serotype, and gene profiles of virulence and antimicrobial resistance. The most dominant virulence gene was f17 (26.2%), followed by stx2 (15.9%), ehxA (11.0%), stx1 (8.3%), eae (5.2%), and sta (4.1%). Some shiga-toxin producing E. coli isolates possessed eae (15.9%). All isolates except for one showed resistance to one or more antimicrobials, with 152 isolates exhibiting multidrug-resistance. The most prevalent resistance phenotype detected was streptomycin (63.1%), followed by tetracycline (54.5%), neomycin (40.3%), cephalothin (32.8%), amoxicillin (30.0%), ampicillin (29.7%), and sulphamethoxazole/trimethoprim (16.6%). The associated resistance determinants detected were strA-strB (39.0%), tet(E) (80.0%), tet(A) (27.6%), aac(3)-IV (33.1%), aphA1 (21.4%), bla TEM (23.8%), and sul2 (22.1%). When investigated by O serotyping and PFGE molecular subtyping, the high degree of diversity was exhibited in E. coli isolates. These results suggest that E. coli isolates from South Korean cattle farms are significantly diverse in terms of virulence and antimicrobial resistance. In conclusion, the gastroinstestinal flora of cattle could be a significant reservoir of diverse virulence and antimicrobial resistance determinants, which is potentially hazardous to public health.

  3. Distribution of classical and nonclassical virulence genes in enterotoxigenic Escherichia coli isolates from Chilean children and tRNA gene screening for putative insertion sites for genomic islands.

    PubMed

    Del Canto, Felipe; Valenzuela, Patricio; Cantero, Lidia; Bronstein, Jonathan; Blanco, Jesús E; Blanco, Jorge; Prado, Valeria; Levine, Myron; Nataro, James; Sommerfelt, Halvor; Vidal, Roberto

    2011-09-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNA-associated genetic insertions might identify new ETEC virulence

  4. Detecting virulence and drug resistance mycobacterial phenotypes in vivo

    PubMed Central

    Timmins, Graham

    2015-01-01

    Bacterial phenotypes are predominantly studied in culture because detection of their specific metabolic pathways in the host is challenging. Development of stable isotope breath tests allowing in situ phenotype analyses may endow diagnostics with new modalities based upon direct monitoring of in vivo microbial metabolism and host–pathogen phenotypic interactions. PMID:25800730

  5. Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission.

    PubMed

    Moreno Switt, Andrea I; den Bakker, Henk C; Cummings, Craig A; Rodriguez-Rivera, Lorraine D; Govoni, Gregory; Raneiri, Matthew L; Degoricija, Lovorka; Brown, Stephanie; Hoelzer, Karin; Peters, Joseph E; Bolchacova, Elena; Furtado, Manohar R; Wiedmann, Martin

    2012-01-01

    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.

  6. Growth temperature alters Salmonella Enteritidis heat/acid resistance, membrane lipid composition and stress/virulence related gene expression.

    PubMed

    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.

  7. The Regulatory Repertoire of Pseudomonas aeruginosa AmpC ß-Lactamase Regulator AmpR Includes Virulence Genes

    PubMed Central

    Balasubramanian, Deepak; Schneper, Lisa; Merighi, Massimo; Smith, Roger; Narasimhan, Giri; Lory, Stephen; Mathee, Kalai

    2012-01-01

    In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. In addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, we compared the transcriptional profile generated using DNA microarrays of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought, with the deletion of ampR influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Other virulence mechanisms including biofilm formation and QS-regulated acute virulence factors are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the microarray data. Further, using a Caenorhabditis elegans model, we demonstrate that a functional AmpR is required for P. aeruginosa pathogenicity. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. Further, we show differential regulation of other transcriptional regulators and sigma factors by AmpR, accounting for the extensive AmpR regulon. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating biofilm formation, a chronic infection phenotype. Unraveling this complex regulatory circuit will provide a better understanding of the bacterial response to antibiotics and how the

  8. Prevalence of Virulence Factors and Vancomycin-resistant Genes among Enterococcus faecalis and E. faecium Isolated from Clinical Specimens

    PubMed Central

    NASAJ, Mona; MOUSAVI, Seyed Masoud; HOSSEINI, Seyed Mostafa; ARABESTANI, Mohammad Reza

    2016-01-01

    Background: The aim of this study was to determine the occurrence of virulence determinants and vancomycin-resistant genes among Enterococcus faecalis and E. faecium obtained from various clinical sources. Methods: The study was performed on the 280 enterococcal isolated from clinical specimens in Hamadan hospitals, western Iran in 2012–14. Antibiotic susceptibility testing was performed using disk diffusion and Minimal Inhibitory Concentration (MIC) methods. The presence of vancomycin-resistant genes and virulence genes was investigated using PCR. Results: Totally 280 enterococcal isolates were identified as follows: E. faecalis (62.5%), E. faecium (24%) and Enterococcus spp (13.5%). The results of antibiotic susceptibility testing showed that resistance rates to vancomycin and teicoplanin in E. faecalis and E. faecium isolates were 5% and 73%, respectively. Of Sixty vancomycin-resistant Enterococci strains, fifty-one isolates were identified as E. faecium (VREfm) and nine as E. faecalis (VREfs). Prevalence of esp, hyl, and asa1 genes were determined as 82%, 71.6%, and 100%, respectively in E. faecium strains; and 78%, 56/6%, and 97%, respectively in E. faecalis strains. Conclusion: The increased frequency of VREF, as seen with rapid rise in the number of vanA isolates should be considered in infection control practices. PMID:27648425

  9. Implication of quorum sensing in Salmonella enterica serovar typhimurium virulence: the luxS gene is necessary for expression of genes in pathogenicity island 1.

    PubMed

    Choi, Jeongjoon; Shin, Dongwoo; Ryu, Sangryeol

    2007-10-01

    Despite the fact that the regulatory system sensing density of cell population and its signaling molecule have been identified in Salmonella enterica, the biological significance of this phenomenon termed as quorum sensing remains unknown. In this report, we provide evidence that the luxS gene is necessary for Salmonella virulence phenotypes. Transcription assays showed that the cell-density-dependent induction of the invF gene was abolished in a Salmonella strain with the luxS gene deleted. The effect of the luxS deletion was also investigated in other InvF-regulated genes expressed from Salmonella pathogenicity island 1 (SPI-1). The decreased expression of SPI-1 genes in the strain with luxS deleted could be restored by either the addition of a synthetic signal molecule or the introduction of a plasmid copy of the luxS gene. Thus, the reduced expression of invF and its regulated genes in Salmonella cells lacking quorum sensing resulted in the attenuation of virulence phenotypes both in vitro and in vivo.

  10. Phylogenetic Distribution of Virulence Genes Among ESBL-producing Uropathogenic Escherichia coli Isolated from Long-term Hospitalized Patients

    PubMed Central

    Zhao, Ruike; Shi, Jinfang; Shen, Yimin; Li, Yanmeng; Han, Qingzhen; Zhang, Xianfeng; Gu, Guohao

    2015-01-01

    Objectives The present study was aimed to investigate the antibiotic resistance, virulence potential and phylogenetic grouping of ESBL-producing uropathogenic Escherichia coli (EP-UPEC) isolated from long-term hospitalized patients. Materials and Methods EP-UPEC isolates from September 2013 to June 2014 at a tertiary care hospital of China were screened for ESBL-production by the double disk diffusion test. Isolates with ESBL-phenotype were further characterized by antibiotic resistance testing, PCR of different ESBL and virulence genes, and phylogenetic grouping. Results One hundred and twenty EP-UPEC were isolated from long-term hospitalized patients. All EP-UPEC isolates were resistant to Ampicillin, Cefazolin, Cefuroxime, Cefotaxime, Cefoperazone and Ceftriaxone, and the majority of EP-UPEC isolates were resistant to Piperacillin (82.5%), Ciprofloxacin (81.2%), Trimethoprim-Sulfamethoxazole (72.5%). The isolates showed the highest sensitivity against Imipenem (98.4%), Piperacillin/tazobactam (96.7%), Cefoperazone/sulbactam (91.7%), Amikacin (90.8%) and Cefepime (75.8%). Nine different ESBL genotype patterns were observed and CTX-M type was the most prevalent ESBL genotype (42.5%, 51/120). Majority of EP-UPEC isolates possess more than one ESBL genes. EP-UPEC isolates belonged mainly to phylogenetic group B2(36.7%) and D(35.0%). The prevalence of traT, ompT, iss, PAI, afa, fimH and papC were 75.8%, 63.3%, 63.3%, 60.8%, 40.8%, 19.2% and 6.7%, respectively. The number of virulence genes (VGs) detected was significantly higher in group B2 than in group A (ANOVA, p<0.001), group B1(ANOVA, p= 0.012) and D (ANOVA, p<0.001). Conclusions EP-UPEC strains showed multidrug resistance and co-resistance to other non β-lactam antibiotics. CTX-M was the most prevalent ESBL genotype and majority of EP-UPEC strains more than one ESBL genes. EP-UPEC strains belonged mainly to phylogenetic group B2 and D, and most of the virulence genes were more prevalent in group B2. PMID

  11. Evidence for Direct Control of Virulence and Defense Gene Circuits by the Pseudomonas aeruginosa Quorum Sensing Regulator, MvfR

    PubMed Central

    Maura, Damien; Hazan, Ronen; Kitao, Tomoe; Ballok, Alicia E.; Rahme, Laurence G.

    2016-01-01

    Pseudomonas aeruginosa defies eradication by antibiotics and is responsible for acute and chronic human infections due to a wide variety of virulence factors. Currently, it is believed that MvfR (PqsR) controls the expression of many of these factors indirectly via the pqs and phnAB operons. Here we provide strong evidence that MvfR may also bind and directly regulate the expression of additional 35 loci across the P. aeruginosa genome, including major regulators and virulence factors, such as the quorum sensing (QS) regulators lasR and rhlR, and genes involved in protein secretion, translation, and response to oxidative stress. We show that these anti-oxidant systems, AhpC-F, AhpB-TrxB2 and Dps, are critical for P. aeruginosa survival to reactive oxygen species and antibiotic tolerance. Considering that MvfR regulated compounds generate reactive oxygen species, this indicates a tightly regulated QS self-defense anti-poisoning system. These findings also challenge the current hierarchical regulation model of P. aeruginosa QS systems by revealing new interconnections between them that suggest a circular model. Moreover, they uncover a novel role for MvfR in self-defense that favors antibiotic tolerance and cell survival, further demonstrating MvfR as a highly desirable anti-virulence target. PMID:27678057

  12. Skin-bacteria communication: Involvement of the neurohormone Calcitonin Gene Related Peptide (CGRP) in the regulation of Staphylococcus epidermidis virulence

    PubMed Central

    N’Diaye, Awa R.; Leclerc, Camille; Kentache, Takfarinas; Hardouin, Julie; Poc, Cecile Duclairoir; Konto-Ghiorghi, Yoan; Chevalier, Sylvie; Lesouhaitier, Olivier; Feuilloley, Marc G. J.

    2016-01-01

    Staphylococci can sense Substance P (SP) in skin, but this molecule is generally released by nerve terminals along with another neuropeptide, Calcitonin Gene Related Peptide (CGRP). In this study, we investigated the effects of αCGRP on Staphylococci. CGRP induced a strong stimulation of Staphylococcus epidermidis virulence with a low threshold (<10−12 M) whereas Staphylococcus aureus was insensitive to CGRP. We observed that CGRP-treated S. epidermidis induced interleukin 8 release by keratinocytes. This effect was associated with an increase in cathelicidin LL37 secretion. S. epidermidis displayed no change in virulence factors secretion but showed marked differences in surface properties. After exposure to CGRP, the adherence of S. epidermidis to keratinocytes increased, whereas its internalization and biofilm formation activity were reduced. These effects were correlated with an increase in surface hydrophobicity. The DnaK chaperone was identified as the S. epidermidis CGRP-binding protein. We further showed that the effects of CGRP were blocked by gadolinium chloride (GdCl3), an inhibitor of MscL mechanosensitive channels. In addition, GdCl3 inhibited the membrane translocation of EfTu, the Substance P sensor. This work reveals that through interaction with specific sensors S. epidermidis integrates different skin signals and consequently adapts its virulence. PMID:27739485

  13. Sequential expression of bacterial virulence and plant defense genes during infection of tomato with Clavibacter michiganensis subsp. michiganensis.

    PubMed

    Chalupowicz, L; Cohen-Kandli, M; Dror, O; Eichenlaub, R; Gartemann, K-H; Sessa, G; Barash, I; Manulis-Sasson, S

    2010-03-01

    The molecular interactions between Clavibacter michiganensis subsp. michiganensis and tomato plant were studied by following the expression of bacterial virulence and host-defense genes during early stages of infection. The C. michiganensis subsp. michiganensis genes included the plasmid-borne cellulase (celA) and the serine protease (pat-1), and the serine proteases chpC and ppaA, residing on the chp/tomA pathogenicity island (PAI). Gene expression was measured following tomato inoculation with Cmm382 (wild type), Cmm100 (lacking the plasmids pCM1 and pCM2), and Cmm27 (lacking the PAI). Transcriptional analysis revealed that celA and pat-1 were significantly induced in Cmm382 at initial 12 to 72 h, whereas chpC and ppaA were highly expressed only 96 h after inoculation. Interdependence between the expression of chromosomal and of plasmid-located genes was revealed: expression of celA and pat-1 was substantially reduced in the absence of the chp/tomA PAI, whereas chpC and ppaA expressions were reduced in the absence of the virulence plasmids. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA, and xysB), was also induced at early stages of infection. Expression of the host-defense genes, chitinase class II and pathogenesis-related protein-5 isoform was induced in the absence of the PAI at early stages of infection, suggesting that PAI-located genes are involved in suppression of tomato basal defenses.

  14. Erwinia amylovora Expresses Fast and Simultaneously hrp/dsp Virulence Genes during Flower Infection on Apple Trees

    PubMed Central

    Pester, Doris; Milčevičová, Renáta; Schaffer, Johann; Wilhelm, Eva; Blümel, Sylvia

    2012-01-01

    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 control on a molecular

  15. Aerobactin and other virulence factor genes among strains of Escherichia coli causing urosepsis: association with patient characteristics.

    PubMed

    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.

  16. Virulence Genes and the Antimicrobial Susceptibility of Escherichia coli, Isolated from Wild Waterbirds, in the Netherlands and Poland.

    PubMed

    Kuczkowski, Maciej; Krawiec, Marta; Voslamber, Berend; Książczyk, Marta; Płoskońska-Bugla, Gabriela; Wieliczko, Alina

    2016-08-01

    Affiliation to four phylogroups (A, B1, B2, and D) was examined, among 190 Escherichia coli strains, collected from five, wild waterbird species, including the following: the Greylag goose-Anser anser (61) and the Canada goose-Branta canadensis (33) obtained in the Netherlands, and the Mallard-Anas platyrhynchos (38), the Mute swan-Cygnus olor (37), and the Great cormorant-Phalacrocorax carbo (21) obtained in Poland. Moreover, the prevalence of 10 virulence factors: astA, iss, iucD, irp2, papC, tsh, vat, cva/cvi, stx2f, and bfp, as well as antimicrobial susceptibility to amoxicillin, enrofloxacin, and tetracycline (minimum inhibitory concentration [MIC] using E-tests) were investigated, in the examined E. coli strains. Results demonstrated that the greatest number of E. coli strains belonged to phylogenetic groups, B1 (86 strains-45.3%) and D (49 strains-25.8%), whereas 40 (21.0%) and only 15 (7.9%) isolates were classified as being of phylogenetic groups, A and B2, respectively. Among the 10 tested virulence-associated genes, 7 genes were detected in 61 examined strains (32.1%) with highly varying frequency. Virulence profiles showed that astA, iss, and irp2 genes were detected most frequently among all examined E. coli strains, isolated from every chosen bird species. Antimicrobial susceptibility, as detected by MIC for the examined antibiotics, is variable among strains isolated from different species of birds. The aim of this study was to examine the prevalence of E. coli strains, isolated from different species of wild waterbirds and determine their potential pathogenicity to the environment, other birds, and people. PMID:27348207

  17. The PA-gene-mediated lethal dissemination and excessive innate immune response contribute to the high virulence of H5N1 avian influenza virus in mice.

    PubMed

    Hu, Jiao; Hu, Zenglei; Song, Qingqing; Gu, Min; Liu, Xiaowen; Wang, Xiaoquan; Hu, Shunlin; Chen, Chaoyang; Liu, Huimou; Liu, Wenbo; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2013-03-01

    Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals.

  18. Evaluation of Virulence Gene Expression Patterns in Acinetobacter baumannii Using Quantitative Real-Time Polymerase Chain Reaction Array.

    PubMed

    Lannan, Ford M; O'conor, Daniel K; Broderick, Joseph C; Tate, Jamison F; Scoggin, Jacob T; Moran, Nicholas A; Husson, Christopher M; Hegeman, Erik M; Ogrydziak, Cole E; Singh, Sneha A; Vafides, Andrew G; Brinkley, Carl C; Goodin, Jeremy L

    2016-09-01

    According to the Centers for Disease Control's recently devised National Strategy for Combating Antibiotic-Resistant Bacteria, Acinetobacter baumannii is a "serious" threat level pathogen. A. baumannii's notoriety stems from the fact that a large number of modern strains are multidrug resistant and persist in the hospital setting, thus causing numerous deaths per year. It is imperative that research focus on a more fundamental understanding of the factors responsible for the success of A. baumannii. Toward this end, our group investigated virulence gene expression patterns in a recently characterized wound isolate, AB5075, using quantitative real-time polymerase chain reaction array. Notably, several genes showed statistically significant upregulation at 37°C compared to 25°C; MviM, Wbbj, CarO, and certain genes of the Bas, Bar, and Csu operons. Additionally, we found that in vitro biofilm formation by Csu transposon insertion mutant strains is attenuated. These findings validate previous reports that suggest a link between the Csu operon and biofilm formation. More importantly, our results demonstrate a successful method for evaluating the significance of previously identified virulence factors in a modern and clinically relevant strain of A. baumannii, thereby providing a path toward a more fundamental understanding of the pathogenicity of A. baumannii. PMID:27612361

  19. Antimicrobial Effects of Blueberry, Raspberry, and Strawberry Aqueous Extracts and their Effects on Virulence Gene Expression in Vibrio cholerae.

    PubMed

    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.

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

    PubMed

    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. PMID:25201253

  1. Evaluation of Virulence Gene Expression Patterns in Acinetobacter baumannii Using Quantitative Real-Time Polymerase Chain Reaction Array.

    PubMed

    Lannan, Ford M; O'conor, Daniel K; Broderick, Joseph C; Tate, Jamison F; Scoggin, Jacob T; Moran, Nicholas A; Husson, Christopher M; Hegeman, Erik M; Ogrydziak, Cole E; Singh, Sneha A; Vafides, Andrew G; Brinkley, Carl C; Goodin, Jeremy L

    2016-09-01

    According to the Centers for Disease Control's recently devised National Strategy for Combating Antibiotic-Resistant Bacteria, Acinetobacter baumannii is a "serious" threat level pathogen. A. baumannii's notoriety stems from the fact that a large number of modern strains are multidrug resistant and persist in the hospital setting, thus causing numerous deaths per year. It is imperative that research focus on a more fundamental understanding of the factors responsible for the success of A. baumannii. Toward this end, our group investigated virulence gene expression patterns in a recently characterized wound isolate, AB5075, using quantitative real-time polymerase chain reaction array. Notably, several genes showed statistically significant upregulation at 37°C compared to 25°C; MviM, Wbbj, CarO, and certain genes of the Bas, Bar, and Csu operons. Additionally, we found that in vitro biofilm formation by Csu transposon insertion mutant strains is attenuated. These findings validate previous reports that suggest a link between the Csu operon and biofilm formation. More importantly, our results demonstrate a successful method for evaluating the significance of previously identified virulence factors in a modern and clinically relevant strain of A. baumannii, thereby providing a path toward a more fundamental understanding of the pathogenicity of A. baumannii.

  2. The ecological importance of the Staphylococcus sciuri species group as a reservoir for resistance and virulence genes.

    PubMed

    Nemeghaire, Stéphanie; Argudín, M Angeles; Feßler, Andrea T; Hauschild, Tomasz; Schwarz, Stefan; Butaye, Patrick

    2014-07-16

    The Staphylococcus sciuri species group includes five species that are most often presented as commensal animal-associated bacteria. The species of this group are Staphylococcus sciuri (with three subspecies), Staphylococcus lentus, Staphylococcus vitulinus, Staphylococcus fleurettii and Staphylococcus stepanovicii. Members of these group are commonly found in a broad range of habitats including animals, humans and the environment. However, those species have been isolated also from infections, both in veterinary and human medicine. Members of this group have been shown to be pathogenic, though infections caused by these species are infrequent. Furthermore, members of the S. sciuri species group have also been found to carry multiple virulence and resistance genes. Indeed, genes implicated in biofilm formation or coding for toxins responsible of toxic shock syndrome and multi-resistance, similar to those carried by Staphylococcus aureus, were detected. This group may thereby represent a reservoir for other bacteria. Despite its recognized abundance as commensal bacteria and its possible role as reservoir of virulence and resistance genes for other staphylococci, the S. sciuri species group is often considered harmless and, as such, not as well documented as, for example, S. aureus. More investigation into the role of the S. sciuri species group as commensal and pathogenic bacteria is required to fully assess its medical and veterinary importance.

  3. Enhancing the Stress Tolerance and Virulence of an Entomopathogen by Metabolic Engineering of Dihydroxynaphthalene Melanin Biosynthesis Genes ▿ †

    PubMed Central

    Tseng, Min N.; Chung, Pei C.; Tzean, Shean S.

    2011-01-01

    Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed. PMID:21571888

  4. Genome of the facultative scuticociliatosis pathogen Pseudocohnilembus persalinus provides insight into its virulence through horizontal gene transfer

    PubMed Central

    Xiong, Jie; Wang, Guangying; Cheng, Jun; Tian, Miao; Pan, Xuming; Warren, Alan; Jiang, Chuanqi; Yuan, Dongxia; Miao, Wei

    2015-01-01

    Certain ciliates of the subclass Scuticociliatia (scuticociliates) are facultative parasites of fishes in which they cause a suite of diseases collectively termed scuticociliatosis. Hitherto, comparatively little was known about genetics and genomics of scuticociliates or the mechanism of scuticociliatosis. In this study, a laboratory culture of the facultatively pathogenic scuticociliate Pseudocohnilembus persalinus was established and its genome sequenced, giving the first genome of a marine ciliate. Genome-wide horizontal gene transfer (HGT) analysis showed P. persalinus has acquired many unique prokaryote-derived genes that potentially contribute to the virulence of this organism, including cell adhesion, hemolysis and heme utilization genes. These findings give new insights into our understanding of the pathology of scuticociliates. PMID:26486372

  5. Genome of the facultative scuticociliatosis pathogen Pseudocohnilembus persalinus provides insight into its virulence through horizontal gene transfer.

    PubMed

    Xiong, Jie; Wang, Guangying; Cheng, Jun; Tian, Miao; Pan, Xuming; Warren, Alan; Jiang, Chuanqi; Yuan, Dongxia; Miao, Wei

    2015-01-01

    Certain ciliates of the subclass Scuticociliatia (scuticociliates) are facultative parasites of fishes in which they cause a suite of diseases collectively termed scuticociliatosis. Hitherto, comparatively little was known about genetics and genomics of scuticociliates or the mechanism of scuticociliatosis. In this study, a laboratory culture of the facultatively pathogenic scuticociliate Pseudocohnilembus persalinus was established and its genome sequenced, giving the first genome of a marine ciliate. Genome-wide horizontal gene transfer (HGT) analysis showed P. persalinus has acquired many unique prokaryote-derived genes that potentially contribute to the virulence of this organism, including cell adhesion, hemolysis and heme utilization genes. These findings give new insights into our understanding of the pathology of scuticociliates. PMID:26486372

  6. Identification of Quantitative Proteomic Differences between Mycobacterium tuberculosis Lineages with Altered Virulence

    PubMed Central

    Peters, Julian S.; Calder, Bridget; Gonnelli, Giulia; Degroeve, Sven; Rajaonarifara, Elinambinina; Mulder, Nicola; Soares, Nelson C.; Martens, Lennart; Blackburn, Jonathan M.

    2016-01-01

    Evidence currently suggests that as a species Mycobacterium tuberculosis exhibits very little genomic sequence diversity. Despite limited genetic variability, members of the M. tuberculosis complex (MTBC) have been shown to exhibit vast discrepancies in phenotypic presentation in terms of virulence, elicited immune response and transmissibility. Here, we used qualitative and quantitative mass spectrometry tools to investigate the proteomes of seven clinically-relevant mycobacterial strains—four M. tuberculosis strains, M. bovis, M. bovis BCG, and M. avium—that show varying degrees of pathogenicity and virulence, in an effort to rationalize the observed phenotypic differences. Following protein preparation, liquid chromatography mass spectrometry (LC MS/MS) and data capture were carried out using an LTQ Orbitrap Velos. Data analysis was carried out using a novel bioinformatics strategy, which yielded high protein coverage and was based on high confidence peptides. Through this approach, we directly identified a total of 3788 unique M. tuberculosis proteins out of a theoretical proteome of 4023 proteins and identified an average of 3290 unique proteins for each of the MTBC organisms (representing 82% of the theoretical proteomes), as well as 4250 unique M. avium proteins (80% of the theoretical proteome). Data analysis showed that all major classes of proteins are represented in every strain, but that there are significant quantitative differences between strains. Targeted selected reaction monitoring (SRM) assays were used to quantify the observed differential expression of a subset of 23 proteins identified by comparison to gene expression data as being of particular relevance to virulence. This analysis revealed differences in relative protein abundance between strains for proteins which may promote bacterial fitness in the more virulent W. Beijing strain. These differences may contribute to this strain's capacity for surviving within the host and resisting

  7. New Targets and Inhibitors of Mycobacterial Sulfur Metabolism§

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2015-01-01

    The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress made in the development of inhibitors of sulfur metabolism enzymes. PMID:23808874

  8. Use of siRNA molecular beacons to detect and attenuate mycobacterial infection in macrophages

    PubMed Central

    George, Remo; Cavalcante, Renata; Jr, Celso Carvalho; Marques, Elyana; Waugh, Jonathan B; Unlap, M Tino

    2015-01-01

    Tuberculosis is one of the leading infectious diseases plaguing mankind and is mediated by the facultative pathogen, Mycobacterium tuberculosis (MTB). Once the pathogen enters the body, it subverts the host immune defenses and thrives for extended periods of time within the host macrophages in the lung granulomas, a condition called latent tuberculosis (LTB). Persons with LTB are prone to reactivation of the disease when the body’s immunity is compromised. Currently there are no reliable and effective diagnosis and treatment options for LTB, which necessitates new research in this area. The mycobacterial proteins and genes mediating the adaptive responses inside the macrophage is largely yet to be determined. Recently, it has been shown that the mce operon genes are critical for host cell invasion by the mycobacterium and for establishing a persistent infection in both in vitro and in mouse models of tuberculosis. The YrbE and Mce proteins which are encoded by the MTB mce operons display high degrees of homology to the permeases and the surface binding protein of the ABC transports, respectively. Similarities in structure and cell surface location impute a role in cell invasion at cholesterol rich regions and immunomodulation. The mce4 operon is also thought to encode a cholesterol transport system that enables the mycobacterium to derive both energy and carbon from the host membrane lipids and possibly generating virulence mediating metabolites, thus enabling the bacteria in its long term survival within the granuloma. Various deletion mutation studies involving individual or whole mce operon genes have shown to be conferring varying degrees of attenuation of infectivity or at times hypervirulence to the host MTB, with the deletion of mce4A operon gene conferring the greatest degree of attenuation of virulence. Antisense technology using synthetic siRNAs has been used in knocking down genes in bacteria and over the years this has evolved into a powerful tool for

  9. Further studies on the GS element. A novel mycobacterial insertion sequence (IS1612), inserted into an acetylase gene (mpa) in Mycobacterium avium subsp. silvaticum but not in Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Bull, T J; Sheridan, J M; Martin, H; Sumar, N; Tizard, M; Hermon-Taylor, J

    2000-12-20

    We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

  10. Effect Of Spaceflight On Microbial Gene Expression And Virulence: Preliminary Results From Microbe Payload Flown On-Board STS-115

    NASA Technical Reports Server (NTRS)

    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

  11. Presence of pathogenicity island related and plasmid encoded virulence genes in cytolethal distending toxin producing Escherichia coli isolates from diarrheal cases

    PubMed Central

    Oloomi, Mana; Javadi, Maryam; Bouzari, Saeid

    2015-01-01

    Context: Mobile genetic elements such as plasmids, bacteriophages, insertion elements, and genomic islands play a critical role in virulence of bacterial pathogens. These elements transfer horizontally and could play an important role in the evolution and virulence of many pathogens. A broad spectrum of gram-negative bacterial species has been shown to produce a cytolethal distending toxin (CDT). On the other hand, Shiga toxin producing Escherichia coli are the one carry virulence genes such as stx 1 and stx 2 (Shiga toxin) and these genes can be acquired by horizontal gene transfer. Aim: The aim of this study was to investigate the presence of other virulence associated genes among CDT producing E. coli strains. Materials and Methods: Thirty CDT positive strains isolated from patients with diarrhea were characterized. Thereafter, the association with virulent genetic elements in known pathogenicity islands (PAIs) was assessed by polymerase chain reaction. Results: In this study, it was shown that the most CDT producing E. coli isolates express Shiga toxin. Moreover, the presence of prophages framing cdt genes (like P2 phage) was also identified in each cdt-type genomic group. Flanked regions of cdt-I, cdt-IV, and cdt-V-type was similar to plasmid sequences while cdt-II and cdt-III-type regions similarity with hypothetical protein (orf3) was observed. Conclusion: The occurrence of each cdt-type groups with specific virulence genes and PAI genetic elements is indicative of horizontal gene transfer by these mobile genetic elements, which could lead to diversity among the isolates. PMID:26539367

  12. Virulence Characterization of Salmonella enterica by a New Microarray: Detection and Evaluation of the Cytolethal Distending Toxin Gene Activity in the Unusual Host S. Typhimurium

    PubMed Central

    Figueiredo, Rui; Card, Roderick; Nunes, Carla; AbuOun, Manal; Bagnall, Mary C.; Nunez, Javier; Mendonça, Nuno; Anjum, Muna F.; da Silva, Gabriela Jorge

    2015-01-01

    Salmonella enterica is a zoonotic foodborne pathogen that causes acute gastroenteritis in humans. We assessed the virulence potential of one-hundred and six Salmonella strains isolated from food animals and products. A high through-put virulence genes microarray demonstrated Salmonella Pathogenicity Islands (SPI) and adherence genes were highly conserved, while prophages and virulence plasmid genes were variably present. Isolates were grouped by serotype, and virulence plasmids separated S. Typhimurium in two clusters. Atypical microarray results lead to whole genome sequencing (WGS) of S. Infantis Sal147, which identified deletion of thirty-eight SPI-1 genes. Sal147 was unable to invade HeLa cells and showed reduced mortality in Galleria mellonella infection model, in comparison to a SPI-1 harbouring S. Infantis. Microarray and WGS of S. Typhimurium Sal199, established for the first time in S. Typhimurium presence of cdtB and other Typhi-related genes. Characterization of Sal199 showed cdtB genes were upstream of transposase IS911, and co-expressed with other Typhi-related genes. Cell cycle arrest, cytoplasmic distension, and nuclear enlargement were detected in HeLa cells infected by Sal199, but not with S. Typhimurium LT2. Increased mortality of Galleria was detected on infection with Sal199 compared to LT2. Thus, Salmonella isolates were rapidly characterized using a high through-put microarray; helping to identify unusual virulence features which were corroborated by further characterisation. This work demonstrates that the use of suitable screening methods for Salmonella virulence can help assess the potential risk associated with certain Salmonella to humans. Incorporation of such methodology into surveillance could help reduce the risk of emergence of epidemic Salmonella strains. PMID:26244504

  13. Virulence Characterization of Salmonella enterica by a New Microarray: Detection and Evaluation of the Cytolethal Distending Toxin Gene Activity in the Unusual Host S. Typhimurium.

    PubMed

    Figueiredo, Rui; Card, Roderick; Nunes, Carla; AbuOun, Manal; Bagnall, Mary C; Nunez, Javier; Mendonça, Nuno; Anjum, Muna F; da Silva, Gabriela Jorge

    2015-01-01

    Salmonella enterica is a zoonotic foodborne pathogen that causes acute gastroenteritis in humans. We assessed the virulence potential of one-hundred and six Salmonella strains isolated from food animals and products. A high through-put virulence genes microarray demonstrated Salmonella Pathogenicity Islands (SPI) and adherence genes were highly conserved, while prophages and virulence plasmid genes were variably present. Isolates were grouped by serotype, and virulence plasmids separated S. Typhimurium in two clusters. Atypical microarray results lead to whole genome sequencing (WGS) of S. Infantis Sal147, which identified deletion of thirty-eight SPI-1 genes. Sal147 was unable to invade HeLa cells and showed reduced mortality in Galleria mellonella infection model, in comparison to a SPI-1 harbouring S. Infantis. Microarray and WGS of S. Typhimurium Sal199, established for the first time in S. Typhimurium presence of cdtB and other Typhi-related genes. Characterization of Sal199 showed cdtB genes were upstream of transposase IS911, and co-expressed with other Typhi-related genes. Cell cycle arrest, cytoplasmic distension, and nuclear enlargement were detected in HeLa cells infected by Sal199, but not with S. Typhimurium LT2. Increased mortality of Galleria was detected on infection with Sal199 compared to LT2. Thus, Salmonella isolates were rapidly characterized using a high through-put microarray; helping to identify unusual virulence features which were corroborated by further characterisation. This work demonstrates that the use of suitable screening methods for Salmonella virulence can help assess the potential risk associated with certain Salmonella to humans. Incorporation of such methodology into surveillance could help reduce the risk of emergence of epidemic Salmonella strains.

  14. Mendelian susceptibility to mycobacterial disease: genetic, immunological, and clinical features of inborn errors of IFN-γ immunity.

    PubMed

    Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Abel, Laurent; Casanova, Jean-Laurent

    2014-12-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is a rare condition characterized by predisposition to clinical disease caused by weakly virulent mycobacteria, such as BCG vaccines and environmental mycobacteria, in otherwise healthy individuals with no overt abnormalities in routine hematological and immunological tests. MSMD designation does not recapitulate all the clinical features, as patients are also prone to salmonellosis, candidiasis and tuberculosis, and more rarely to infections with other intramacrophagic bacteria, fungi, or parasites, and even, perhaps, a few viruses. Since 1996, nine MSMD-causing genes, including seven autosomal (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, and IRF8) and two X-linked (NEMO, and CYBB) genes have been discovered. The high level of allelic heterogeneity has already led to the definition of 18 different disorders. The nine gene products are physiologically related, as all are involved in IFN-γ-dependent immunity. These disorders impair the production of (IL12B, IL12RB1, IRF8, ISG15, NEMO) or the response to (IFNGR1, IFNGR2, STAT1, IRF8, CYBB) IFN-γ. These defects account for only about half the known MSMD cases. Patients with MSMD-causing genetic defects may display other infectious diseases, or even remain asymptomatic. Most of these inborn errors do not show complete clinical penetrance for the case-definition phenotype of MSMD. We review here the genetic, immunological, and clinical features of patients with inborn errors of IFN-γ-dependent immunity.

  15. Mendelian susceptibility to mycobacterial disease: genetic, immunological, and clinical features of inborn errors of IFN-γ immunity

    PubMed Central

    Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Abel, Laurent; Casanova, Jean-Laurent

    2014-01-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is a rare condition characterized by predisposition to clinical disease caused by weakly virulent mycobacteria, such as BCG vaccines and environmental mycobacteria, in otherwise healthy individuals with no overt abnormalities in routine hematological and immunological tests. MSMD designation does not recapitulate all the clinical features, as patients are also prone to salmonellosis, candidiasis and tuberculosis, and more rarely to infections with other intramacrophagic bacteria, fungi, or parasites, and even, perhaps, a few viruses. Since 1996, nine MSMD-causing genes, including seven autosomal (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, and IRF8) and two X-linked (NEMO, CYBB) genes have been discovered. The high level of allelic heterogeneity has already led to the definition of 18 different disorders. The nine gene products are physiologically related, as all are involved in IFN-γ-dependent immunity. These disorders impair the production of (IL12B, IL12RB1, IRF8, ISG15, NEMO) or the response to (IFNGR1, IFNGR2, STAT1, IRF8, CYBB) IFN-γ. These defects account for only about half the known MSMD cases. Patients with MSMD-causing genetic defects may display other infectious diseases, or even remain asymptomatic. Most of these inborn errors do not show complete clinical penetrance for the case-definition phenotype of MSMD. We review here the genetic, immunological, and clinical features of patients with inborn errors of IFN-γ-dependent immunity. PMID:25453225

  16. [Hydrocarbon-Oxidizing potential and the genes for n-alkane biodegradation in a new acidophilic mycobacterial association from sulfur blocks].

    PubMed

    Ivanova, I E; Sukhacheva, M V; Kanat'eva, A Yu; Kravchenko, I K; Kurganov, A A

    2014-01-01

    Capacity of AG(S10), a new aerobic acidophilic (growing within the pH range from 1.3 to 4.5 with the optimum at 2.0-2.5) bacterial association from sulfur blocks of the Astrakhan gas-processing complex (AGC), for oxidation of hydrocarbons of various chemical structure was investigated. A broad spectrum of normal (C10-C21) and iso-alkanes, toluene, naphthalene, andphenanthrene, as well as isoprenoids resistant to microbial degradation, pristane and phytane (components of paraffin oil), and 2,2,4,4,6,8,8,-heptamethylnonane, a branched hydrocarbon, were biodegraded under acidic conditions. Microbiological investigation revealed the dominance of mycobacteria in the AGS10 association, which was confirmed by analysis of the 16S rRNA gene clone library. In the phylogenetic tree, the 16S rRNA sequences formed a branch within the cluster of slow-growing mycobacteria, with 98% homology to the closest species Mycobacterium florentinum. Genomic DNA of AG(S10) culture grown on C14-C17 n-alkanes at pH 2.5 was found to contain the genes of two hydroxylase families, alkB and Cyp 153, indicating their combined involvement in hydrocarbon biodegradation. The high hydrocarbon-oxidizing potential of the AGS10 bacterial association, indicated that further search for the genes responsible for degradation of various hydrocarbons in acidophilic mycobacteria could be promising.

  17. Relationship between eae and stx virulence genes and Escherichia coli in an agricultural watershed: implications for irrigation water standards and leafy green commodities.

    PubMed

    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. PMID:21219758

  18. Relationship between eae and stx virulence genes and Escherichia coli in an agricultural watershed: implications for irrigation water standards and leafy green commodities.

    PubMed

    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.

  19. Molecular characterization of virulence genes of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in equines

    PubMed Central

    Javed, R.; Taku, A. K.; Gangil, Rakhi; Sharma, R. K.

    2016-01-01

    Aim: The aim was to determine the occurrence of streptococci in equines in Jammu (R. S. Pura, Katra), characterization of Streptococci equi subsp. equi and Streptococcus equi subsp. zooepidemicus with respect to their virulence traits and to determine antibiotic sensitivity pattern of virulent Streptococcus isolates. Materials and Methods: A total of 96 samples were collected from both clinically affected animals (exhibiting signs of respiratory tract disease) and apparently healthy animals and were sent to laboratory. The organisms were isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and confirmed by cultural characteristics and biochemical tests. Molecular detection of Streptococcus was done directly from cultures using sodA and seM gene-based polymerase chain reaction (PCR). Antibiogram was performed against five antibiotics such as amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During this study, a total 40 streptococcal isolates were obtained out of which 2 isolates were of S. equi subsp. equi, 12 isolates were from S. equi subsp. zooepidemicus. In the PCR-based detection, we revealed amplicons of 235 bp and 679 bp for confirmation of sodA and seM gene, respectively. In antibiogram, two isolates of S. equi subsp. equi were found resistant to penicillin G, and all other isolates were found sensitive to amoxicillin and streptomycin. Conclusion: The majority of streptococcal infections was due to S. equi subsp. Zooepidemicus, and thus was recognized as a potential pathogen of diseases of equines besides S. equi subsp. equi.

  20. A recombinant rabies virus encoding two copies of the glycoprotein gene confers protection in dogs against a virulent challenge.

    PubMed

    Liu, Xiaohui; Yang, Youtian; Sun, Zhaojin; Chen, Jing; Ai, Jun; Dun, Can; Fu, Zhen F; Niu, Xuefeng; Guo, Xiaofeng

    2014-01-01

    The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines. PMID:24498294

  1. Phylogenetic grouping, epidemiological typing, analysis of virulence genes, and antimicrobial susceptibility of Escherichia coli isolated from healthy broilers in Japan

    PubMed Central

    2014-01-01

    Background The aim of our study was to investigate the possible etiology of avian colibacillosis by examining Escherichia coli isolates from fecal samples of healthy broilers. Findings Seventy-eight E. coli isolates from fecal samples of healthy broilers in Japan were subjected to analysis of phylogenetic background, virulence-associated gene profiling, multi-locus sequence typing (MLST), and antimicrobial resistance profiling. Phylogenetic analysis demonstrated that 35 of the 78 isolates belonged to group A, 28 to group B1, one to group B2, and 14 to group D. Virulence-associated genes iutA, iss, cvaC, tsh, iroN, ompT, and hlyF were found in 23 isolates (29.5%), 16 isolates (20.5%), nine isolates (11.5%), five isolates (6.4%), 19 isolates (24.4%), 23 isolates (29.5%), and 22 isolates (28.2%) respectively. Although the genetic diversity of group D isolates was revealed by MLST, the group D isolates harbored iutA (10 isolates, 71.4%), iss (6 isolates, 42.9%), cvaC (5 isolates, 35.7%), tsh (3 isolates, 21.4%), hlyF (9 isolates, 64.3%), iroN (7 isolates, 50.0%), and ompT (9 isolates, 64.3%). Conclusions Our results indicated that E. coli isolates inhabiting the intestines of healthy broilers pose a potential risk of causing avian colibacillosis. PMID:25061511

  2. Identification of ABC Transporter Genes of Fusarium graminearum with Roles in Azole Tolerance and/or Virulence

    PubMed Central

    Döll, Katharina; Karlovsky, Petr; Deising, Holger B.; Wirsel, Stefan G. R.

    2013-01-01

    Fusarium graminearum is a plant pathogen infecting several important cereals, resulting in substantial yield losses and mycotoxin contamination of the grain. Triazole fungicides are used to control diseases caused by this fungus on a worldwide scale. Our previous microarray study indicated that 15 ABC transporter genes were transcriptionally upregulated in response to tebuconazole treatment. Here, we deleted four ABC transporter genes in two genetic backgrounds of F. graminearum representing the DON (deoxynivalenol) and the NIV (nivalenol) trichothecene chemotypes. Deletion of FgABC3 and FgABC4 belonging to group I of ABC-G and to group V of ABC-C subfamilies of ABC transporters, respectively, considerably increased the sensitivity to the class I sterol biosynthesis inhibitors triazoles and fenarimol. Such effects were specific since they did not occur with any other fungicide class tested. Assessing the contribution of the four ABC transporters to virulence of F. graminearum revealed that, irrespective of their chemotypes, deletion mutants of FgABC1 (ABC-C subfamily group V) and FgABC3 were impeded in virulence on wheat, barley and maize. Phylogenetic context and analyses of mycotoxin production suggests that FgABC3 may encode a transporter protecting the fungus from host-derived antifungal molecules. In contrast, FgABC1 may encode a transporter responsible for the secretion of fungal secondary metabolites alleviating defence of the host. Our results show that ABC transporters play important and diverse roles in both fungicide resistance and pathogenesis of F. graminearum. PMID:24244413

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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. PMID:26470920

  6. Genome sequencing reveals widespread virulence gene exchange among human Neisseria species.

    PubMed

    Marri, Pradeep Reddy; Paniscus, Mary; Weyand, Nathan J; Rendón, María A; Calton, Christine M; Hernández, Diana R; Higashi, Dustin L; Sodergren, Erica; Weinstock, George M; Rounsley, Steven D; So, Magdalene

    2010-07-28

    Commensal bacteria comprise a large part of the microbial world, playing important roles in human development, health and disease. However, little is known about the genomic content of commensals or how related they are to their pathogenic counterparts. The genus Neisseria, containing both commensal and pathogenic species, provides an excellent opportunity to study these issues. We undertook a comprehensive sequencing and analysis of human commensal and pathogenic Neisseria genomes. Commensals have an extensive repertoire of virulence alleles, a large fraction of which has been exchanged among Neisseria species. Commensals also have the genetic capacity to donate DNA to, and take up DNA from, other Neisseria. Our findings strongly suggest that commensal Neisseria serve as reservoirs of virulence alleles, and that they engage extensively in genetic exchange.

  7. Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains.

    PubMed Central

    Steck, T R; Kado, C I

    1990-01-01

    Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone. PMID:2318813

  8. An antisense RNA that governs the expression kinetics of a multifunctional virulence gene

    PubMed Central

    Lee, Eun-Jin; Groisman, Eduardo A.

    2010-01-01

    Summary Genome-wide transcriptome analyses of several bacterial species have recently uncovered a hitherto unappreciated amount of antisense transcription. However, the physiological role, regulation and significance of such antisense transcripts are presently unclear. We now report the identification of a cis-encoded 1.2 kb long antisense RNA – termed AmgR – that is complementary to the mgtC portion of the mgtCBR polycistronic message from Salmonella enterica. The mgtCBR mRNA specifies the MgtC protein, which is necessary for survival within macrophages, virulence in mice and growth in low Mg2+; the Mg2+ transporter MgtB with no apparent role in virulence; and the membrane peptide MgtR mediating MgtC degradation. Expression of AmgR dimished both MgtC and MgtB protein levels in a process requiring RNase E but independent of RNase III, the RNA chaperone Hfq, and the regulatory peptide MgtR. Inactivation of the chromosomal amgR promoter increased MgtC and MgtB protein levels and enhanced Salmonella virulence. Surprisingly, AmgR transcription is governed by the regulatory protein PhoP, which also directs transcription of the sense mgtCBR mRNA. AmgR may function as a timing device that alters MgtC and MgtB levels after the onset of PhoP-inducing conditions. PMID:20398218

  9. Transcriptional regulation by iron of a Vibrio cholerae virulence gene and homology of the gene to the Escherichia coli fur system.

    PubMed

    Goldberg, M B; Boyko, S A; Calderwood, S B

    1990-12-01

    We have previously described an iron-regulated virulence determinant in Vibrio cholerae. Strain MBG40, which contains a TnphoA insertion mutation in the iron-regulated gene irgA, has reduced virulence in a newborn mouse model and has lost the major 77-kDa iron-regulated outer membrane protein. We report here the cloning of the irgA'-'phoA gene fusion, the sequencing of the 5'-proximal portion of irgA, and the definition of its promoter region by primer extension. The deduced amino acid sequence of the amino-terminal portion of IrgA is homologous to the ferrienterochelin receptor of Escherichia coli (FepA), suggesting that IrgA may be the iron-vibriobactin outer membrane receptor. Iron regulation of irgA in an E. coli background and that of the E. coli gene slt-IA in a V. cholerae background are reciprocal, suggesting a common mechanism of iron regulation. Regulation of irgA by iron in V. cholerae occurs at the transcriptional level, and there is an interrupted dyad symmetric sequence in the vicinity of the promoter that is homologous to Fur binding sites of E. coli. Unlike iron-regulated genes in E. coli, however, transcription of irgA requires an additional 900 bp of upstream DNA that contains an open reading frame in inverse orientation to irgA.

  10. The UmGcn5 gene encoding histone acetyltransferase from Ustilago maydis is involved in dimorphism and virulence.

    PubMed

    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.

  11. A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite Plasmodium falciparum.

    PubMed

    Ukaegbu, Uchechi E; Zhang, Xu; Heinberg, Adina R; Wele, Mamadou; Chen, Qijun; Deitsch, Kirk W

    2015-05-01

    Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent "true" switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent "default" switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in

  12. A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Heinberg, Adina R.; Wele, Mamadou; Chen, Qijun; Deitsch, Kirk W.

    2015-01-01

    Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent “true” switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent “default” switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation

  13. Altered virulence potential of Salmonella Enteritidis cultured in different foods: A cumulative effect of differential gene expression and immunomodulation.

    PubMed

    Jaiswal, Sangeeta; Sahoo, Prakash Kumar; Ryan, Daniel; Das, Jugal Kishore; Chakraborty, Eesha; Mohakud, Nirmal Kumar; Suar, Mrutyunjay

    2016-08-01

    Salmonella enterica serovars Enteritidis (S. Enteritidis) is one of the most common causes of food borne illness. Bacterial growth environment plays an important role in regulating gene expression thereby affecting the virulence profile of the bacteria. Different foods present diverse growth conditions which may affect the pathogenic potential of the bacteria. In the present study, the effect of food environments on the pathogenic potential of S. Enteritidis has been evaluated. S. Enteritidis was grown in different foods e.g. egg white, peanut butter and milk, and virulent phenotypes were compared to those grown in Luria Bertani broth. In-vivo experiments in C57BL/6 mice revealed S. Enteritidis grown in egg white did not induce significant (p<0.001) production of proinflammatory cytokines in mice and were unable to cause colitis despite efficient colonization in cecum, mesenteric lymph node, spleen and liver. Further studies revealed that bacteria grown in LB activated MAP Kinase and NFκB pathways efficiently, while those grown in egg white poorly activated the above pathways which can account for the decreased production of proinflammatory cytokines. qRT PCR analysis revealed SPI-1 effectors were downregulated in bacteria grown in egg white. Interestingly, bacteria grown in egg white showed reversal of phenotype upon change in growth media to LB. Additionally, bacteria grown in milk and peanut butter showed different degrees of virulence in mice as compared to those grown in LB media. Thus, the present study demonstrates that, S. Enteritidis grown in egg white colonizes systemic sites without causing colitis in a mouse model, while bacteria grown in milk and peanut butter show different pathogenicity profiles suggesting that food environments significantly affect the pathogenicity of S. Enteritidis.

  14. Altered virulence potential of Salmonella Enteritidis cultured in different foods: A cumulative effect of differential gene expression and immunomodulation.

    PubMed

    Jaiswal, Sangeeta; Sahoo, Prakash Kumar; Ryan, Daniel; Das, Jugal Kishore; Chakraborty, Eesha; Mohakud, Nirmal Kumar; Suar, Mrutyunjay

    2016-08-01

    Salmonella enterica serovars Enteritidis (S. Enteritidis) is one of the most common causes of food borne illness. Bacterial growth environment plays an important role in regulating gene expression thereby affecting the virulence profile of the bacteria. Different foods present diverse growth conditions which may affect the pathogenic potential of the bacteria. In the present study, the effect of food environments on the pathogenic potential of S. Enteritidis has been evaluated. S. Enteritidis was grown in different foods e.g. egg white, peanut butter and milk, and virulent phenotypes were compared to those grown in Luria Bertani broth. In-vivo experiments in C57BL/6 mice revealed S. Enteritidis grown in egg white did not induce significant (p<0.001) production of proinflammatory cytokines in mice and were unable to cause colitis despite efficient colonization in cecum, mesenteric lymph node, spleen and liver. Further studies revealed that bacteria grown in LB activated MAP Kinase and NFκB pathways efficiently, while those grown in egg white poorly activated the above pathways which can account for the decreased production of proinflammatory cytokines. qRT PCR analysis revealed SPI-1 effectors were downregulated in bacteria grown in egg white. Interestingly, bacteria grown in egg white showed reversal of phenotype upon change in growth media to LB. Additionally, bacteria grown in milk and peanut butter showed different degrees of virulence in mice as compared to those grown in LB media. Thus, the present study demonstrates that, S. Enteritidis grown in egg white colonizes systemic sites without causing colitis in a mouse model, while bacteria grown in milk and peanut butter show different pathogenicity profiles suggesting that food environments significantly affect the pathogenicity of S. Enteritidis. PMID:27132148

  15. Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way.

    PubMed

    Schorey, Jeffrey S; Cooper, Andrea M

    2003-03-01

    Mycobacteria activate a series of macrophage signalling pathways upon engaging host cell receptors and during the invasion process. These signals initiate a cascade of events leading to the production of immune effector molecules including cytokines, chemokines and reactive nitrogen intermediates. This response by the macrophage is critical for the control of the mycobacterial infection and, not surprisingly, pathogenic mycobacteria have evolved mechanisms to limit this macrophage activation. Recent data has suggested that macrophages infected with pathogenic compared to non-pathogenic mycobacteria are restricted in their activation of the mitogen activated protein kinase (MAPK) pathways. Mitogen activated protein kinase activation in macrophages appears to play an important role in promoting antimycobacterial activity and in the production of various effector molecules following a mycobacterial infection. Therefore, the ability of pathogenic mycobacteria to limit MAPK activity is likely an important virulence mechanism and may be a potential therapeutic target.

  16. Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii.

    PubMed

    Rommereim, Leah M; Bellini, Valeria; Fox, Barbara A; Pètre, Graciane; Rak, Camille; Touquet, Bastien; Aldebert, Delphine; Dubremetz, Jean-François; Cesbron-Delauw, Marie-France; Mercier, Corinne; Bzik, David J

    2016-01-01

    Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection. PMID:27458822

  17. Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii

    PubMed Central

    Fox, Barbara A.; Pètre, Graciane; Rak, Camille; Touquet, Bastien; Aldebert, Delphine; Dubremetz, Jean-François; Cesbron-Delauw, Marie-France; Mercier, Corinne; Bzik, David J.

    2016-01-01

    Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection. PMID:27458822

  18. The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

    PubMed

    Niu, Xiaolei; Zhou, Meiliang; Henkel, Christiaan V; van Heusden, G Paul H; Hooykaas, Paul J J

    2015-12-01

    During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process. PMID:26461850

  19. Molecular characterization and virulence gene profiling of pathogenic Streptococcus agalactiae populations from tilapia (Oreochromis sp.) farms in Thailand.

    PubMed

    Kayansamruaj, Pattanapon; Pirarat, Nopadon; Katagiri, Takayuki; Hirono, Ikuo; Rodkhum, Channarong

    2014-05-19

    Streptococcus spp. were recovered from diseased tilapia in Thailand during 2009-2010 (n = 33), and were also continually collected from environmental samples (sediment and water) from tilapia farms for 9 months in 2011 (n = 25). The relative percent recovery of streptococci from environmental samples was 13-67%. All streptococcal isolates were identified as S. agalactiae (group B streptococci [GBS]) by a species-specific polymerase chain reaction. In molecular characterization assays, 4 genotypic categories comprised of 1) molecular serotypes, 2) the infB allele, 3) virulence gene profiling patterns (cylE, hylB, scpB, lmb, cspA, dltA, fbsA, fbsB, bibA, gap, and pili backbone-encoded genes), and 4) randomly amplified polymorphic DNA (RAPD) fingerprinting patterns, were used to describe the genotypic diversity of the GBS isolates. There was only 1 isolate identified as molecular serotype III, while the others were serotype Ia. Most GBS serotype Ia isolates had an identical infB allele and virulence gene profiling patterns, but a large diversity was established by RAPD analysis with diversity tending to be geographically dependent. Experimental infection of Nile tilapia (Oreochromis niloticus) revealed that the GBS serotype III isolate was nonpathogenic in the fish, while all 5 serotype Ia isolates (3 fish and 2 environmental isolates) were pathogenic, with a median lethal dose of 6.25-7.56 log10 colony-forming units. In conclusion, GBS isolates from tilapia farms in Thailand showed a large genetic diversity, which was associated with the geographical origins of the bacteria. PMID:24842288

  20. A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

    PubMed

    Kapperud, G; Dommarsnes, K; Skurnik, M; Hornes, E

    1990-01-01

    We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.

  1. CHARACTERIZATION OF VIRULENCE GENES AND ANTIMICROBIAL RESISTANCE OF LUNG PATHOGENIC ESCHERICHIA COLI ISOLATES IN FOREST MUSK DEER (MOSCHUS BEREZOVSKII).

    PubMed

    Luo, Xi; Wang, Peng; Cheng, Jian-guo; Luo, Yan; Dai, Lei; Zhou, Xin; Zou, Li-kou; Li, Bei; Xiao, Jiu-Jin

    2016-06-01

    This study investigated genotypic diversity, 26 virulence genes, and antimicrobial susceptibility of lung pathogenic Escherichia coli (LPEC) isolated from forest musk deer. Associations between virulence factors (VFs) and phylogenetic group, between antimicrobial resistance (AMR) and phylogenetic group, and between AMR and VFs were subsequently assessed. The results showed 30 LPEC isolated were grouped into seven different clusters (A, B, C, D, E, F, and G). The detection rates of crl (90%), kpsMT II (76.67%), mat (76.67%), and ompA (80%) were over 75%. The most frequent types of resistance were to amoxicillin (100%), sulfafurazole (100%), ampicillin (96.67%), and tetracycline (96.67%), with 93.33% (n = 28) of isolates resistant to more than eight types of drugs. There were significant relationships between resistance to cefalotin and the presence of iucD(a) (P < 0.001), papC (P = 0.032), and kpsMT II (P = 0.028); between resistance to chloromycetin and the presence of irp2 (P = 0.004) and vat (P = 0.047); between resistance to nalidixic acid and the presence of crl (P = 0.002) and iucD(a) (P = 0.004); and between resistance to ampicillin/sulbactam and the presence of vat (P = 0.013). These results indicated there could be some association between resistance and VFs, and there is a great need for the prudent use of antimicrobial agents in LPEC. PMID:27468027

  2. PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets.

    PubMed

    Turton, Jane F; Perry, Claire; Elgohari, Suzanne; Hampton, Catherine V

    2010-05-01

    A multiplex PCR is described which detects capsular types K1, K2, K5, K54 and K57, which are those most associated with invasive disease or pathogenicity, a further capsular type (K20), two putative virulence factors (rmpA and wcaG) and the 16S-23S internal transcribed spacer unit of Klebsiella pneumoniae, facilitating identification of this organism. wcaG encodes capsular fucose production and was associated with capsular types K1 and K54, but was also found in strains of other capsular types; 18 of the 543 isolates screened were PCR-positive for this gene. An eight-locus variable number tandem repeat (VNTR) scheme was designed, which provided discrimination at a level similar to that afforded by PFGE among a panel of 36 isolates representing 29 PFGE types. All isolates tested of the virulent K1 clone of CC23, associated with pyogenic liver abscesses, shared the same VNTR profile, which may be helpful in identifying this clone; such isolates were also PCR-positive for allS. These methods provide a rapid means of characterizing and typing isolates of this important agent of community-acquired and nosocomial infection.

  3. CHARACTERIZATION OF VIRULENCE GENES AND ANTIMICROBIAL RESISTANCE OF LUNG PATHOGENIC ESCHERICHIA COLI ISOLATES IN FOREST MUSK DEER (MOSCHUS BEREZOVSKII).

    PubMed

    Luo, Xi; Wang, Peng; Cheng, Jian-guo; Luo, Yan; Dai, Lei; Zhou, Xin; Zou, Li-kou; Li, Bei; Xiao, Jiu-Jin

    2016-06-01

    This study investigated genotypic diversity, 26 virulence genes, and antimicrobial susceptibility of lung pathogenic Escherichia coli (LPEC) isolated from forest musk deer. Associations between virulence factors (VFs) and phylogenetic group, between antimicrobial resistance (AMR) and phylogenetic group, and between AMR and VFs were subsequently assessed. The results showed 30 LPEC isolated were grouped into seven different clusters (A, B, C, D, E, F, and G). The detection rates of crl (90%), kpsMT II (76.67%), mat (76.67%), and ompA (80%) were over 75%. The most frequent types of resistance were to amoxicillin (100%), sulfafurazole (100%), ampicillin (96.67%), and tetracycline (96.67%), with 93.33% (n = 28) of isolates resistant to more than eight types of drugs. There were significant relationships between resistance to cefalotin and the presence of iucD(a) (P < 0.001), papC (P = 0.032), and kpsMT II (P = 0.028); between resistance to chloromycetin and the presence of irp2 (P = 0.004) and vat (P = 0.047); between resistance to nalidixic acid and the presence of crl (P = 0.002) and iucD(a) (P = 0.004); and between resistance to ampicillin/sulbactam and the presence of vat (P = 0.013). These results indicated there could be some association between resistance and VFs, and there is a great need for the prudent use of antimicrobial agents in LPEC.

  4. Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Agrobacterium-mediated transformation (AMT) was used to identify potential virulence factors in Sclerotinia sclerotiorum. Screening AMT transformants identified two mutants showing significantly reduced virulence. The mutants showed similar growth rate, colony morphology, and sclerotial and oxalate ...

  5. Expression of Virulence-Related Genes in Listeria monocytogenes Grown on Danish Hard Cheese as Affected by NaCl Content.

    PubMed

    Larsen, Nadja; Jespersen, Lene

    2015-06-01

    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 (<0.15% [wt/wt]) and high (3.6% [wt/wt]) content of NaCl. Genes differentially expressed (p<0.05) through the 48-h incubation were transcriptional regulators prfA and agrA, genes of the main virulence cluster inlA, hly, actA, involved in invasion of the epithelial cells, and genes bsh, opuC, gadC, clpP, and ami, associated with osmotic stress responses in L. monocytogenes. The more sensitive strain ATCC(®) 51779 was most responsive, showing significant upregulation of prfA, actA, hly, and bsh both at low and high NaCl. Strain DSMZ 15675 was less responsive to NaCl stress, showing reduced or consistent gene transcription at all conditions. Decreased transcription of agrA, ami, gadC, and opuC in both strains was promoted by low NaCl content. The study indicated that virulence gene expression of L. monocytogenes grown in cheese was affected by NaCl content and that effect was more significant in strains sensitive to both hypo- and hyperosmotic stresses.

  6. Expression of Virulence-Related Genes in Listeria monocytogenes Grown on Danish Hard Cheese as Affected by NaCl Content.

    PubMed

    Larsen, Nadja; Jespersen, Lene

    2015-06-01

    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 (<0.15% [wt/wt]) and high (3.6% [wt/wt]) content of NaCl. Genes differentially expressed (p<0.05) through the 48-h incubation were transcriptional regulators prfA and agrA, genes of the main virulence cluster inlA, hly, actA, involved in invasion of the epithelial cells, and genes bsh, opuC, gadC, clpP, and ami, associated with osmotic stress responses in L. monocytogenes. The more sensitive strain ATCC(®) 51779 was most responsive, showing significant upregulation of prfA, actA, hly, and bsh both at low and high NaCl. Strain DSMZ 15675 was less responsive to NaCl stress, showing reduced or consistent gene transcription at all conditions. Decreased transcription of agrA, ami, gadC, and opuC in both strains was promoted by low NaCl content. The study indicated that virulence gene expression of L. monocytogenes grown in cheese was affected by NaCl content and that effect was more significant in strains sensitive to both hypo- and hyperosmotic stresses. PMID:26067229

  7. Prevalence of the Most Common Virulence-Associated Genes among Brucella Melitensis Isolates from Human Blood Cultures in Hamadan Province, West of Iran.

    PubMed

    Naseri, Zahra; Alikhani, Mohammad Yousef; Hashemi, Seyed Hamid; Kamarehei, Farideh; Arabestani, Mohammad Reza

    2016-09-01

    Brucellosis is a widespread zoonotic disease causing considerable economic and public health problems. Despite animal vaccination, brucellosis remains endemic in some areas such as Iran, especially in the western Iranian province of Hamadan. We sought to detect some of the most common virulence-associated genes in Brucella isolated from human blood cultures to determine the prevalence of some virulence genes among Brucella isolates. Fifty-seven isolates were studied from patients with a clinical diagnosis of brucellosis who referred to the Infectious Diseases Ward of Sina Hospital in Hamadan Province, Iran, between April 2013 and July 2014. Blood samples were collected for the diagnosis of brucellosis using the BACTEC blood culture system. All of these isolates were confirmed by the bcsp31 Brucella-specific gene. We detected 11 virulence-associated genes of Brucella, namely cβg, virB, znuA, ure, bvfA, omp25, omp31, wbkA, mviN, manA, and manB, which are important for the pathogenesis of this bacterium in the intracellular environment by multiplex PCR. Totally, 149 patients with a clinical diagnosis of brucellosis were enrolled in this study. Fifty-seven (38.3%) patients had positive blood cultures. On biochemical and molecular testing, all of the isolates were Brucella melitensis. Ten of the virulence genes were detected among all of the 57 isolates, but the bvf gene was detected in 53 (93%) isolates. The high prevalence of virulence-associated genes among the Brucella isolates detected in Hamadan Province, Iran, underscores the pathogenicity of this bacterium in this region. PMID:27582592

  8. Prevalence of the Most Common Virulence-Associated Genes among Brucella Melitensis Isolates from Human Blood Cultures in Hamadan Province, West of Iran

    PubMed Central

    Naseri, Zahra; Alikhani, Mohammad Yousef; Hashemi, Seyed Hamid; Kamarehei, Farideh; Arabestani, Mohammad Reza

    2016-01-01

    Brucellosis is a widespread zoonotic disease causing considerable economic and public health problems. Despite animal vaccination, brucellosis remains endemic in some areas such as Iran, especially in the western Iranian province of Hamadan. We sought to detect some of the most common virulence-associated genes in Brucella isolated from human blood cultures to determine the prevalence of some virulence genes among Brucella isolates. Fifty-seven isolates were studied from patients with a clinical diagnosis of brucellosis who referred to the Infectious Diseases Ward of Sina Hospital in Hamadan Province, Iran, between April 2013 and July 2014. Blood samples were collected for the diagnosis of brucellosis using the BACTEC blood culture system. All of these isolates were confirmed by the bcsp31 Brucella-specific gene. We detected 11 virulence-associated genes of Brucella, namely cβg, virB, znuA, ure, bvfA, omp25, omp31, wbkA, mviN, manA, and manB, which are important for the pathogenesis of this bacterium in the intracellular environment by multiplex PCR. Totally, 149 patients with a clinical diagnosis of brucellosis were enrolled in this study. Fifty-seven (38.3%) patients had positive blood cultures. On biochemical and molecular testing, all of the isolates were Brucella melitensis. Ten of the virulence genes were detected among all of the 57 isolates, but the bvf gene was detected in 53 (93%) isolates. The high prevalence of virulence-associated genes among the Brucella isolates detected in Hamadan Province, Iran, underscores the pathogenicity of this bacterium in this region. PMID:27582592

  9. Characterization of Salmonella enterica and detection of the virulence genes specific to diarrheagenic Escherichia coli from poultry carcasses in Ouagadougou, Burkina Faso.

    PubMed

    Kagambèga, Assèta; Barro, Nicolas; Traoré, Alfred S; Siitonen, Anja; Haukka, Kaisa

    2012-07-01

    One hundred chicken carcasses purchased from three markets selling poultry in Ouagadougou, Burkina Faso, between June 2010 and October 2010 were examined for their microbiological quality. The presence of Salmonella was investigated using standard bacteriological procedures, and the isolates obtained were serotyped and tested for antimicrobial susceptibility. The presence of virulence-associated genes of the five main pathogroups of diarrheagenic Escherichia coli-Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli, and enteroinvasive E. coli-was investigated using 16-plex polymerase chain reaction (PCR) on the mixed bacterial cultures from the poultry samples. Of the 100 chicken carcasses studied, 57 were contaminated by Salmonella; 16 different serotypes were identified, the most frequent being Salmonella Derby, found in 28 samples. Four Salmonella strains were resistant to tetracycline, and two were resistant to streptomycin. Based on the PCR detection of the virulence genes, in total, 45 carcasses were contaminated by three pathogroups of E. coli: STEC, EPEC, or EAEC. The STEC and EPEC virulence genes were detected on six and 39 carcasses, respectively. EAEC virulence genes were only detected in combination with those of EPEC (on 11 carcasses) or STEC (on two carcasses). The STEC-positive carcasses contained the genes stx(1), stx(2), eaeA, escV, and ent in different combinations. None of the EPEC-positive carcasses contained the bfp gene, indicating that only atypical EPEC was present. EAEC virulence genes detected were aggR and/or pic. The high proportion of chicken carcasses contaminated by Salmonella and diarrheagenic E. coli indicates a potential food safety risk for consumers and highlights the necessity of public awareness of these pathogens. PMID:22551070

  10. Natural selection on coding and noncoding DNA sequences is associated with virulence genes in a plant pathogenic fungus.

    PubMed

    Rech, Gabriel E; Sanz-Martín, José M; Anisimova, Maria; Sukno, Serenella A; Thon, Michael R

    2014-09-04

    Natural selection leaves imprints on DNA, offering the opportunity to identify functionally important regions of the genome. Identifying the genomic regions affected by natural selection within pathogens can aid in the pursuit of effective strategies to control diseases. In this study, we analyzed genome-wide patterns of selection acting on different classes of sequences in a worldwide sample of eight strains of the model plant-pathogenic fungus Colletotrichum graminicola. We found evidence of selective sweeps, balancing selection, and positive selection affecting both protein-coding and noncoding DNA of pathogenicity-related sequences. Genes encoding putative effector proteins and secondary metabolite biosynthetic enzymes show evidence of positive selection acting on the coding sequence, consistent with an Arms Race model of evolution. The 5' untranslated regions (UTRs) of genes coding for effector proteins and genes upregulated during infection show an excess of high-frequency polymorphisms likely the consequence of balancing selection and consistent with the Red Queen hypothesis of evolution acting on these putative regulatory sequences. Based on the findings of this work, we propose that even though adaptive substitutions on coding sequences are important for proteins that interact directly with the host, polymorphisms in the regulatory sequences may confer flexibility of gene expression in the virulence processes of this important plant pathogen.

  11. Prevalence of enterococcus species and their virulence genes in fresh water prior to and after storm events.

    PubMed

    Sidhu, J P S; Skelly, E; Hodgers, L; Ahmed, W; Li, Y; Toze, S

    2014-01-01

    Enterococcus spp. isolates (n = 286) collected from six surface water bodies in subtropical Brisbane, Australia, prior to and after storm events, were identified to species level and tested for the presence of seven clinically important virulence genes (VGs). Enterococcus faecalis (48%), Enterococcus faecium (14%), Enterococcus mundtii (13%), and Enterococcus casseliflavus (13%) were frequently detected at all sites. The frequency of E. faecium occurrence increased from 6% in the dry period to 18% after the wet period. The endocarditis antigen (efaA), gelatinase (gelE), collagen-binding protein (ace), and aggregation substance (asa1) were detected in 61%, 43%, 43%, and 23% of Enterococcus isolates, respectively. The chances of occurrence of ace, gelE, efaA, and asa1 genes in E. faecalis were found to be much higher compared to the other Enterococcus spp. The observed odds ratio of occurrence of ace and gelE genes in E. faecalis was much higher at 7.96 and 6.40 times, respectively. The hyl gene was 3.84 times more likely to be detected in E. casseliflavus. The presence of multiple VGs in most of the E. faecalis isolates underscores the importance of E. faecalis as a reservoir of VGs in the fresh water aquatic environment. Consequently, if contaminated surface water is to be used for production of potable and nonpotable water some degree of treatment depending upon intended use such as detention in basins prior to use or chlorination is required.

  12. Transcriptome-Based Identification of Differently Expressed Genes from Xanthomonas oryzae pv. oryzae Strains Exhibiting Different Virulence in Rice Varieties

    PubMed Central

    Noh, Tae-Hwan; Song, Eun-Sung; Kim, Hong-Il; Kang, Mi-Hyung; Park, Young-Jin

    2016-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice (Oryza sativa L.). In this study, we investigated the genome-wide transcription patterns of two Xoo strains (KACC10331 and HB1009), which showed different virulence patterns against eight rice cultivars, including IRBB21 (carrying Xa21). In total, 743 genes showed a significant change (p-value < 0.001 in t-tests) in their mRNA expression levels in the HB1009 (K3a race) strain compared with the Xoo KACC10331 strain (K1 race). Among them, four remarkably enriched GO terms, DNA binding, transposition, cellular nitrogen compound metabolic process, and cellular macromolecule metabolic process, were identified in the upregulated genes. In addition, the expression of 44 genes was considerably higher (log2 fold changes > 2) in the HB1009 (K3a race) strain than in the Xoo KACC10331 (K1 race) strain. Furthermore, 13 and 12 genes involved in hypersensitive response and pathogenicity (hrp) and two-component regulatory systems (TCSs), respectively, were upregulated in the HB1009 (K3a race) strain compared with the Xoo KACC10331 (K1 race) strain, which we determined using either quantitative real-time PCR analysis or next-generation RNA sequencing. These results will be helpful to improve our understanding of Xoo and to gain a better insight into the Xoo–rice interactions. PMID:26907259

  13. Natural Selection on Coding and Noncoding DNA Sequences Is Associated with Virulence Genes in a Plant Pathogenic Fungus

    PubMed Central

    Rech, Gabriel E.; Sanz-Martín, José M.; Anisimova, Maria; Sukno, Serenella A.; Thon, Michael R.

    2014-01-01

    Natural selection leaves imprints on DNA, offering the opportunity to identify functionally important regions of the genome. Identifying the genomic regions affected by natural selection within pathogens can aid in the pursuit of effective strategies to control diseases. In this study, we analyzed genome-wide patterns of selection acting on different classes of sequences in a worldwide sample of eight strains of the model plant-pathogenic fungus Colletotrichum graminicola. We found evidence of selective sweeps, balancing selection, and positive selection affecting both protein-coding and noncoding DNA of pathogenicity-related sequences. Genes encoding putative effector proteins and secondary metabolite biosynthetic enzymes show evidence of positive selection acting on the coding sequence, consistent with an Arms Race model of evolution. The 5′ untranslated regions (UTRs) of genes coding for effector proteins and genes upregulated during infection show an excess of high-frequency polymorphisms likely the consequence of balancing selection and consistent with the Red Queen hypothesis of evolution acting on these putative regulatory sequences. Based on the findings of this work, we propose that even though adaptive substitutions on coding sequences are important for proteins that interact directly with the host, polymorphisms in the regulatory sequences may confer flexibility of gene expression in the virulence processes of this important plant pathogen. PMID:25193312

  14. Mycobacterial species as case-study of comparative genome analysis.

    PubMed

    Zakham, F; Belayachi, L; Ussery, D; Akrim, M; Benjouad, A; El Aouad, R; Ennaji, M M

    2011-02-08

    The genus Mycobacterium represents more than 120 species including important pathogens of human and cause major public health problems and illnesses. Further, with more than 100 genome sequences from this genus, comparative genome analysis can provide new insights for better understanding the evolutionary events of these species and improving drugs, vaccines, and diagnostics tools for controlling Mycobacterial diseases. In this present study we aim to outline a comparative genome analysis of fourteen Mycobacterial genomes: M. avium subsp. paratuberculosis K—10, M. bovis AF2122/97, M. bovis BCG str. Pasteur 1173P2, M. leprae Br4923, M. marinum M, M. sp. KMS, M. sp. MCS, M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis H37Ra, M. tuberculosis H37Rv, M. tuberculosis KZN 1435 , M. ulcerans Agy99,and M. vanbaalenii PYR—1, For this purpose a comparison has been done based on their length of genomes, GC content, number of genes in different data bases (Genbank, Refseq, and Prodigal). The BLAST matrix of these genomes has been figured to give a lot of information about the similarity between species in a simple scheme. As a result of multiple genome analysis, the pan and core genome have been defined for twelve Mycobacterial species. We have also introduced the genome atlas of the reference strain M. tuberculosis H37Rv which can give a good overview of this genome. And for examining the phylogenetic relationships among these bacteria, a phylogenic tree has been constructed from 16S rRNA gene for tuberculosis and non tuberculosis Mycobacteria to understand the evolutionary events of these species.

  15. MvirDB: Microbial Database of Protein Toxins, Virulence Factors and Antibiotic Resistance Genes for Bio-Defense Applications

    DOE Data Explorer

    Zhou, C. E.; Smith, J.; Lam, M.; Zemla, M. D.; Slezak, T.

    MvirDB is a cenntralized resource (data warehouse) comprising all publicly accessible, organized sequence data for protein toxins, virulence factors, and antibiotic resistance genes. Protein entries in MvirDB are annotated using a high-throughput, fully automated computational annotation system; annotations are updated periodically to ensure that results are derived using current public database and open-source tool releases. Tools provided for using MvirDB include a web-based browser tool and BLAST interfaces. MvirDB serves researchers in the bio-defense and medical fields. (taken from page 3 of PI's paper of same title published in Nucleic Acids Research, 2007, Vol.35, Database Issue (Open Source)

  16. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  17. The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants.

    PubMed

    Harris, S J; Shih, Y L; Bentley, S D; Salmond, G P

    1998-05-01

    We have identified a gene important for the regulation of exoenzyme virulence factor synthesis in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc) and virulence and motility in Erwinia carotovora ssp. atroseptica (Eca). This gene, hexA (hyperproduction of exoenzymes), is a close relative of the Erwinia chrysanthemi (Echr) gene pecT and encodes a member of the LysR family of transcriptional regulators. hexA mutants in both Ecc and Eca produce abnormally high levels of the exoenzyme virulence factors pectate lyase, cellulase and protease. In addition, Eca hexA mutants show increased expression of the fliA and fliC genes and hypermotility. Consistent with a role as a global regulator, expression of hexA from even a low-copy plasmid can suppress exoenzyme production in Ecc and Eca and motility in Eca. Production of the quorum-sensing pheromone OHHL in Ecc hexA is higher throughout the growth curve compared with the wild-type strain. Overexpression of Ecc hexA also caused widespread effects in several strains of the opportunistic human pathogen, Serratia. Low-copy hexA expression resulted in repression of exoenzyme, pigment and antibiotic production and repression of the spreading phenotype. Finally, mutations in hexA were shown to increase Ecc or Eca virulence in planta.

  18. Molecular characterization of virulence genes of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in equines

    PubMed Central

    Javed, R.; Taku, A. K.; Gangil, Rakhi; Sharma, R. K.

    2016-01-01

    Aim: The aim was to determine the occurrence of streptococci in equines in Jammu (R. S. Pura, Katra), characterization of Streptococci equi subsp. equi and Streptococcus equi subsp. zooepidemicus with respect to their virulence traits and to determine antibiotic sensitivity pattern of virulent Streptococcus isolates. Materials and Methods: A total of 96 samples were collected from both clinically affected animals (exhibiting signs of respiratory tract disease) and apparently healthy animals and were sent to laboratory. The organisms were isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and confirmed by cultural characteristics and biochemical tests. Molecular detection of Streptococcus was done directly from cultures using sodA and seM gene-based polymerase chain reaction (PCR). Antibiogram was performed against five antibiotics such as amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During this study, a total 40 streptococcal isolates were obtained out of which 2 isolates were of S. equi subsp. equi, 12 isolates were from S. equi subsp. zooepidemicus. In the PCR-based detection, we revealed amplicons of 235 bp and 679 bp for confirmation of sodA and seM gene, respectively. In antibiogram, two isolates of S. equi subsp. equi were found resistant to penicillin G, and all other isolates were found sensitive to amoxicillin and streptomycin. Conclusion: The majority of streptococcal infections was due to S. equi subsp. Zooepidemicus, and thus was recognized as a potential pathogen of diseases of equines besides S. equi subsp. equi. PMID:27651677

  19. Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology.

    PubMed

    Strauß, Lena; Ruffing, Ulla; Abdulla, Salim; Alabi, Abraham; Akulenko, Ruslan; Garrine, Marcelino; Germann, Anja; Grobusch, Martin Peter; Helms, Volkhard; Herrmann, Mathias; Kazimoto, Theckla; Kern, Winfried; Mandomando, Inácio; Peters, Georg; Schaumburg, Frieder; von Müller, Lutz; Mellmann, Alexander

    2016-04-01

    Staphylococcus aureusis a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed anin silicotyping scheme for the software SeqSphere(+)(Ridom GmbH, Münster, Germany). The implemented target genes (n= 182) correspond to those queried by the IdentibacS. aureusGenotyping DNA microarray (Alere Technologies, Jena, Germany). Thein silicoscheme was evaluated by comparing the typing results of microarray and of WGS for 154 humanS. aureusisolates. A total of 96.8% (n= 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosomemecelement types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.

  20. Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology

    PubMed Central

    Strauß, Lena; Ruffing, Ulla; Abdulla, Salim; Alabi, Abraham; Akulenko, Ruslan; Garrine, Marcelino; Germann, Anja; Grobusch, Martin Peter; Helms, Volkhard; Herrmann, Mathias; Kazimoto, Theckla; Kern, Winfried; Mandomando, Inácio; Peters, Georg; Schaumburg, Frieder; von Müller, Lutz

    2016-01-01

    Staphylococcus aureus is a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed an in silico typing scheme for the software SeqSphere+ (Ridom GmbH, Münster, Germany). The implemented target genes (n = 182) correspond to those queried by the Identibac S. aureus Genotyping DNA microarray (Alere Technologies, Jena, Germany). The in silico scheme was evaluated by comparing the typing results of microarray and of WGS for 154 human S. aureus isolates. A total of 96.8% (n = 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosome mec element types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences. PMID:26818676

  1. Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but Nonculturable Vibrio parahaemolyticus after Recovery of Culturability▿

    PubMed Central

    Coutard, François; Lozach, Solen; Pommepuy, Monique; Hervio-Heath, Dominique

    2007-01-01

    A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4°C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37°C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state. PMID:17557845

  2. Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes.

    PubMed Central

    Cookson, B T; Berg, D E; Goldman, W E

    1990-01-01

    The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of

  3. Integrated Bioinformatic and Targeted Deletion Analyses of the SRS Gene Superfamily Identify SRS29C as a Negative Regulator of Toxoplasma Virulence

    PubMed Central

    Wasmuth, James D.; Pszenny, Viviana; Haile, Simon; Jansen, Emily M.; Gast, Alexandra T.; Sher, Alan; Boyle, Jon P.; Boulanger, Martin J.; Parkinson, John; Grigg, Michael E.

    2012-01-01

    ABSTRACT The Toxoplasma gondii SRS gene superfamily is structurally related to SRS29B (formerly SAG1), a surface adhesin that binds host cells and stimulates host immunity. Comparative genomic analyses of three Toxoplasma strains identified 182 SRS genes distributed across 14 chromosomes at 57 genomic loci. Eight distinct SRS subfamilies were resolved. A core 69 functional gene orthologs were identified, and strain-specific expansions and pseudogenization were common. Gene expression profiling demonstrated differential expression of SRS genes in a developmental-stage- and strain-specific fashion and identified nine SRS genes as priority targets for gene deletion among the tissue-encysting coccidia. A Δsag1 ∆sag2A mutant was significantly attenuated in murine acute virulence and showed upregulated SRS29C (formerly SRS2) expression. Transgenic overexpression of SRS29C in the virulent RH parent was similarly attenuated. Together, these findings reveal SRS29C to be an important regulator of acute virulence in mice and demonstrate the power of integrated genomic analysis to guide experimental investigations. PMID:23149485

  4. A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus

    PubMed Central

    Gu, Dan; Guo, Min; Yang, Minjun; Zhang, Yuanxing; Zhou, Xiaohui; Wang, Qiyao

    2016-01-01

    In vibrios, the expression of virulence factors is often controlled by LuxR, the master quorum-sensing regulator. Here, we investigate the interplay between LuxR and σE, an alternative sigma factor, during the control of virulence-related gene expression and adaptations to temperature elevations in the zoonotic pathogen Vibrio alginolyticus. An rpoE null V. alginolyticus mutant was unable to adapt to various stresses and was survival-deficient in fish. In wild type V. alginolyticus, the expression of LuxR-regulated virulence factors increased as the temperature was increased from 22°C to 37°C, but mutants lacking σE did not respond to temperature, indicating that σE is critical for the temperature-dependent upregulation of virulence genes. Further analyses revealed that σE binds directly to -10 and -35 elements in the luxR promoter that drive its transcription. ChIP assays showed that σE binds to the promoter regions of luxR, rpoH and rpoE at high temperatures (e.g., 30°C and 37°C). However, at higher temperatures (42°C) that induce thermal stress, σE binding to the luxR promoter decreased, while its binding to the rpoH and rpoE promoters was unchanged. Thus, the temperature-dependent binding of σE to distinct promoters appears to underlie a σE-controlled switch between the expression of virulence genes and adaptation to thermal stress. This study illustrates how a conserved temperature response mechanism integrates into quorum-sensing circuits to regulate both virulence and stress adaptation. PMID:27253371

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

    NASA Technical Reports Server (NTRS)

    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.

  6. Molecular epidemiologic evaluation of transmissibility and virulence of Mycobacterium tuberculosis.

    PubMed

    Rhee, J T; Piatek, A S; Small, P M; Harris, L M; Chaparro, S V; Kramer, F R; Alland, D

    1999-06-01

    Discovery of genotypic markers associated with increased transmissibility in Mycobacterium tuberculosis would represent an important step in advancing mycobacterial virulence studies. M. tuberculosis strains may be classified into one of three genotypes on the basis of the presence of specific nucleotide substitutions in codon 463 of the katG gene (katG-463) and codon 95 of the gyrA gene (gyrA-95). It has previously been reported that two of these three genotypes are associated with increased IS6110-based clustering, a potential proxy of virulence. We designed a case-control analysis of U.S.-born patients with tuberculosis in San Francisco, Calif., between 1991 and 1997 to investigate associations between katG-463 and gyrA-95 genotypes and epidemiologically determined measures of strain-specific infectivity and pathogenicity and IS6110-based clustering status. We used a new class of molecular probes called molecular beacons to genotype the isolates rapidly. Infectivity was defined as the propensity of isolates to cause tuberculin skin test conversions among named contacts, and pathogenicity was defined as their propensity to cause active disease among named contacts. The molecular beacon assay was a simple and reproducible method for the detection of known single nucleotide polymorphisms in large numbers of clinical M. tuberculosis isolates. The results showed that no genotype of the katG-463- and gyrA-95-based classification system was associated with increased infectivity and pathogenicity or with increased IS6110-based clustering in San Francisco during the study period. We speculate that molecular epidemiologic studies investigating clinically relevant outcomes may contribute to the knowledge of the significance of laboratory-derived virulence factors in the propagation of tuberculosis in human communities.

  7. CD36 deficiency attenuates experimental mycobacterial infection

    PubMed Central

    2010-01-01

    Background Members of the CD36 scavenger receptor family have been implicated as sensors of microbial products that mediate phagocytosis and inflammation in response to a broad range of pathogens. We investigated the role of CD36 in host response to mycobacterial infection. Methods Experimental Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection in Cd36+/+ and Cd36-/- mice, and in vitro co-cultivation of M. tuberculosis, BCG and M. marinum with Cd36+/+ and Cd36-/-murine macrophages. Results Using an in vivo model of BCG infection in Cd36+/+ and Cd36-/- mice, we found that mycobacterial burden in liver and spleen is reduced (83% lower peak splenic colony forming units, p < 0.001), as well as the density of granulomas, and circulating tumor necrosis factor (TNF) levels in Cd36-/- animals. Intracellular growth of all three mycobacterial species was reduced in Cd36-/- relative to wild type Cd36+/+ macrophages in vitro. This difference was not attributable to alterations in mycobacterial uptake, macrophage viability, rate of macrophage apoptosis, production of reactive oxygen and/or nitrogen species, TNF or interleukin-10. Using an in vitro model designed to recapitulate cellular events implicated in mycobacterial infection and dissemination in vivo (i.e., phagocytosis of apoptotic macrophages containing mycobacteria), we demonstrated reduced recovery of viable mycobacteria within Cd36-/- macrophages. Conclusions Together, these data indicate that CD36 deficiency confers resistance to mycobacterial infection. This observation is best explained by reduced intracellular survival of mycobacteria in the Cd36-/- macrophage and a role for CD36 in the cellular events involved in granuloma formation that promote early bacterial expansion and dissemination. PMID:20950462

  8. Immunomodulatory action of mycobacterial secretory proteins.

    PubMed

    Trajkovic, Vladimir; Natarajan, Krishnamurthy; Sharma, Pawan

    2004-04-01

    The recently discovered RD1 locus encodes proteins that are actively secreted by pathogenic mycobacteria, including Mycobacterium tuberculosis. Since they are missing in non-tuberculous mycobacteria, these proteins are promising not only as candidates for vaccination and diagnostic tests, but also in understanding mycobacterial evasion of protective immunity in susceptible individuals. Here we analyze the possible role of M. tuberculosis secretory proteins in immunity against tuberculosis, with emphasis on their immunomodulatory action and the potential involvement in mycobacterial subversion of the host immune defense.

  9. An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression.

    PubMed

    Reniere, Michelle L; Whiteley, Aaron T; Portnoy, Daniel A

    2016-07-01

    Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5' untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. PMID:27414028

  10. An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression

    PubMed Central

    Portnoy, Daniel A.

    2016-01-01

    Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5’ untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. PMID:27414028

  11. Prevalence of Virulence Genes Associated with Pathogenic Escherichia coli Strains Isolated from Domestically Harvested Rainwater during Low- and High-Rainfall Periods

    PubMed Central

    Dobrowsky, P. H.; van Deventer, A.; De Kwaadsteniet, M.; Ndlovu, T.; Khan, S.; Cloete, T. E.

    2014-01-01

    The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting. PMID:24375127

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

    PubMed

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

    2016-01-01

    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. PMID:27609474

  13. Genome-Wide Analysis of Oceanimonas sp. GK1 Isolated from Gavkhouni Wetland (Iran) Demonstrates Presence of Genes for Virulence and Pathogenicity

    PubMed Central

    Parsa Yeganeh, Laleh; Azarbaijani, Reza; Mousavi, Hossein; Shahzadeh Fazeli, Seyed Abolhassan; Amoozgar, Mohammad Ali; Salekdeh, Ghasem Hosseini

    2015-01-01

    Objective The bacterium Oceanimonas sp. (O. sp.) GK1 is a member of the Aeromonadaceae family and its genome represents several virulence genes involved in fish and human pathogenicity. In this original research study we aimed to identify and characterize the putative virulence factors and pathogenicity of this halotolerant marine bacterium using genome wide analysis. Materials and Methods The genome data of O. sp. GK1 was obtained from NCBI. Comparative genomic study was done using MetaCyc database. Results Whole genome data analysis of the O. sp. GK1 revealed that the bacterium possesses some important virulence genes (e.g. ZOT, RTX toxin, thermostable hemolysin, lateral flagella and type IV pili) which have been implicated in adhesion and biofilm formation and infection in some other pathogenic bacteria. Conclusion This is the first report of the putative pathogenicity of O. sp.GK1. The genome wide analysis of the bacterium demonstrates the presence of virulence genes causing infectious diseases in many warmand cold-blooded animals. PMID:26464816

  14. Infection of human airway epithelial cells by different subtypes of Dobrava-Belgrade virus reveals gene expression patterns corresponding to their virulence potential.

    PubMed

    Witkowski, Peter T; Bourquain, Daniel; Bankov, Katrin; Auste, Brita; Dabrowski, Piotr W; Nitsche, Andreas; Krüger, Detlev H; Schaade, Lars

    2016-06-01

    Dobrava-Belgrade virus (DOBV) is a pathogen causing hemorrhagic fever with renal syndrome in Europe. Virulence and case fatality rate are associated with virus genotype; however the reasons for these differences are not well understood. In this work we present virus-specific effects on the gene expression profiles of human lung epithelial cells (A549) infected with different genotypes of DOBV (Dobrava, Kurkino, and Sochi), as well as the low-virulent Tula virus (TULV). The data was collected by whole-genome gene expression microarrays and confirmed by quantitative real-time PCR. Despite their close genetic relationship, the expression profiles induced by infection with different hantaviruses are significantly varying. Major differences were observed in regulation of immune response genes, which were especially induced by highly virulent DOBV genotypes Dobrava and Sochi in contrast to less virulent DOBV-Kurkino and TULV. This work gives first insights into the differences of virus - host interactions of DOBV on genotype level. PMID:27058765

  15. Prevalence of virulence genes associated with pathogenic Escherichia coli strains isolated from domestically harvested rainwater during low- and high-rainfall periods.

    PubMed

    Dobrowsky, P H; van Deventer, A; De Kwaadsteniet, M; Ndlovu, T; Khan, S; Cloete, T E; Khan, W

    2014-03-01

    The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting.

  16. The impact of oregano (Origanum heracleoticum) essential oil and carvacrol on virulence gene transcription by Escherichia coli O157:H7.

    PubMed

    Mith, Hasika; Clinquart, Antoine; Zhiri, Abdesselam; Daube, Georges; Delcenserie, Véronique

    2015-01-01

    The aim of the current study was to determine, via reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the effect of oregano essential oil (Origanum heracleoticum) and carvacrol, its major component, on the expression of virulence-associated genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 ATCC strain 35150. Both oregano oil and carvacrol demonstrated their efficacy firstly, by inhibiting the transcription of the ler gene involved in upregulation of the LEE2, LEE3 and LEE4 promoters and of attaching and effacing lesions and secondly by decreasing both Shiga toxin and fliC genes expression. In addition, a decrease in luxS gene transcription involved in quorum sensing was observed. These results were dose dependent and showed a specific effect of O. heracleoticum and carvacrol in downregulating the expression of virulence genes in EHEC O157:H7. These findings suggest that oregano oil and carvacrol have the potential to mitigate the adverse health effects caused by virulence gene expression in EHEC O157:H7, through the use of these substances as natural antibacterial additives in foods or as an alternative to antibiotics. PMID:25790499

  17. The impact of oregano (Origanum heracleoticum) essential oil and carvacrol on virulence gene transcription by Escherichia coli O157:H7.

    PubMed

    Mith, Hasika; Clinquart, Antoine; Zhiri, Abdesselam; Daube, Georges; Delcenserie, Véronique

    2015-01-01

    The aim of the current study was to determine, via reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the effect of oregano essential oil (Origanum heracleoticum) and carvacrol, its major component, on the expression of virulence-associated genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 ATCC strain 35150. Both oregano oil and carvacrol demonstrated their efficacy firstly, by inhibiting the transcription of the ler gene involved in upregulation of the LEE2, LEE3 and LEE4 promoters and of attaching and effacing lesions and secondly by decreasing both Shiga toxin and fliC genes expression. In addition, a decrease in luxS gene transcription involved in quorum sensing was observed. These results were dose dependent and showed a specific effect of O. heracleoticum and carvacrol in downregulating the expression of virulence genes in EHEC O157:H7. These findings suggest that oregano oil and carvacrol have the potential to mitigate the adverse health effects caused by virulence gene expression in EHEC O157:H7, through the use of these substances as natural antibacterial additives in foods or as an alternative to antibiotics.

  18. The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd.

    PubMed

    Xu, Jia; Tan, Xiao; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2015-11-01

    Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese

  19. The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd.

    PubMed

    Xu, Jia; Tan, Xiao; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2015-11-01

    Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese

  20. Role of the rttA gene in morphogenesis, stress response, and virulence in the human pathogenic fungus Penicillium marneffei.

    PubMed

    Suwunnakorn, Sumanun; Cooper, Chester R; Kummasook, Aksarakorn; Pongpom, Monsicha; Vanittanakom, Pramote; Vanittanakom, Nongnuch

    2015-02-01

    Penicillium marneffei is a human pathogenic fungus and the only thermally dimorphic species of the genus. At 25°C, P. marneffei grows as a mycelium that produces conidia in chains. However, when incubated at 37°C or following infection of host tissue, the fungus develops as a fission yeast. Previously, a mutant (strain I133) defective in morphogenesis was generated via Agrobacterium-mediated transformation. Specifically, the rtt109 gene (subsequently designated rttA) in this mutant was interrupted by T-DNA insertion. We characterized strain I133 and the possible roles of the mutated rttA gene in altered P. marneffei phenotypes. At 25°C, the rttA mutant produces fewer conidia than the wild type and a complemented mutant strain, as well as slower rates of conidial germination; however, strain I133 continued to grow as a yeast in 37°C-incubated cultures. Furthermore, whereas the wild type exhibited increased expression of rttA at 37°C in response to the DNA-damaging agent methyl methane sulfonate, strain I133 was hypersensitive to this and other genotoxic agents. Under similar conditions, the rttA mutant exhibited decreased expression of genes associated with carbohydrate metabolism and oxidative stress. Importantly, when compared with the wild-type and the complemented strain, I133 was significantly less virulent in a Galleria infection model when the larvae were incubated at 37°C. Moreover, the mutant exhibited inappropriate phase transition in vivo. In conclusion, the rttA gene plays important roles in morphogenesis, carbohydrate metabolism, stress response, and pathogenesis in P. marneffei, suggesting that this gene may be a potential target for the development of antifungal compounds.

  1. Effect of high-fructose corn syrup on Streptococcus mutans virulence gene expression and on tooth demineralization.

    PubMed

    Sun, Minmin; Kang, Qiongyi; Li, Tingting; Huang, Lili; Jiang, Yuntao; Xia, Wenwei

    2014-06-01

    High-fructose corn syrup-55 (HFCS-55) has been widely welcomed in recent years as a substitute for sucrose on the basis of its favourable properties and price. The objective of this study was to determine the influence of HFCS-55 on the expression of Streptococcus mutans UA159 virulence genes and on tooth demineralization. Real-time reverse-transcription PCR (real-time RT-PCR) and microhardness evaluations were performed to examine gene expression and enamel demineralization, respectively, after treatment with HFCS-55 and/or sucrose. Significant up-regulation of glucosyltransferase B (gtfB) by HFCS-55 was found. A mixture of HFCS-55 and sucrose could positively enhance expression of glucan-binding protein (gbp) genes. Regarding acidogenicity, expression of the lactate dehydrogenase (ldh) gene was unaffected by HFCS-55. A notable finding in this study was that 5% HFCS-55 significantly enhanced expression of the intracellular response gene of the two-component VicRK signal transduction system (vicR). Demineralization testing showed that the microhardness of teeth decreased by a greater extent in response to HFCS-55 than in response to sucrose. The results indicate that HFCS-55 can enhance S. mutans biofilm formation indirectly in the presence of sucrose and that HFCS-55 has a more acidogenic potential than does sucrose. Summing up the real-time PCR and demineralization results, HFCS-55 appears to be no less cariogenic than sucrose in vitro - at least, not under the conditions of our experiments. PMID:24813075

  2. Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.

    PubMed

    Gouran, Hossein; Chakraborty, Sandeep; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya

    2014-01-01

    Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction. PMID:25717364

  3. Detection of genes encoding multidrug resistance and biofilm virulence factor in uterine pathogenic bacteria in postpartum dairy cows.

    PubMed

    Kasimanickam, V R; Owen, K; Kasimanickam, R K

    2016-01-15

    Reckless use of antibiotics and/or development of biofilm are the rationale for the development of multidrug resistance (MDR) of pathogenic bacteria. The objective of the present study was to detect MDR genes in Trueperella pyogenes and to detect biofilm virulence factor (VF) genes in Escherichia coli isolated from the uterus of postpartum dairy cows. Uterine secretions from different parity postpartum Holstein cows (n = 40) were collected using cytobrush technique after a sterile procedure from cows with varying degree of uterine inflammatory conditions. The cytobrush was stored in a specimen collector, placed in a cooler with ice, and transported to the laboratory within 2 hours. The pathogens were isolated and were identified initially by their colony morphology and biochemical characteristics. To further identify and classify the single species, and to determine the presence of MDR and VF genes, the genes fragments were amplified using the respective primers by either singleplex or multiplex polymerase chain reaction protocol, and amplicons were detected by electrophoresis method. T pyogenes was isolated in 17 of 40 (42.5%) cows in the study population as recognized by the 16S rRNA gene. Of the positive T pyogenes samples, 8 of 17 (42.1%) were positive for integron type 1 (intI I), and none were positive for integron type 2 (intI II). Of those 8 positive for intI I, six of eight (66.7%) were positive for amplicons aadA5 and aadA24-ORF1 at 1048 and 1608 bp, respectively, associated with specific drug resistance. Presence of addA5 indicated resistance to sulfadiazine, bacitracin, florfenicol, and ceftiofur. Presence of addA24-ORF1 indicated resistant to sulfadiazine, bacitracin, penicillin, clindamycin, and erythromycin. E coli was isolated in 18 of 40 (45.0%) cows in the study population. The genes for VF, Agn43a, and Agn43 b, associated with biofilm production, were found in 6 of 18 (33.3%) of the positive isolates. Both T pyogenes MDR gene and E coli

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

    PubMed Central

    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 relationships between these isolates. Patients and Methods A total of 620 diarrheic stool samples were collected from patients attending two hospitals in Kerman from June 2013 to August 2014. All isolates were confirmed as EIEC by PCR for the ipaH gene. The EIEC isolates were evaluated by PCR for the presence of nine virulence genes (ial, set1A, sen, virF, invE, sat, sigA, pic, and sepA). MLVA was performed for all EIEC isolates. Results A total of 11 EIEC isolates were identified, and all were positive for the ial gene. The invE and virF genes were observed in 81.8% of the isolates, while sen, sigA, and pic were detected in 72.7%, 63.6%, and 27.3% of the isolates, respectively. None of the isolates were positive for the sat, set, and sepA genes. Using MLVA, the 11 total isolates were divided into five types. Conclusions By studying the profiles of virulence genes and MLVA, it can be concluded that EIEC isolates do not have high heterogeneity and are derived from a limited number of clones. PMID:27635212

  5. Polyamines are essential for virulence in Salmonella enterica serovar Gallinarum despite evolutionary decay of polyamine biosynthesis genes.

    PubMed

    Schroll, Casper; Christensen, Jens P; Christensen, Henrik; Pors, Susanne E; Thorndahl, Lotte; Jensen, Peter R; Olsen, John E; Jelsbak, Lotte

    2014-05-14

    Serovars of Salmonella enterica exhibit different host-specificities where some have broad host-ranges and others, like S. Gallinarum and S. Typhi, are host-specific for poultry and humans, respectively. With the recent availability of whole genome sequences it has been reported that host-specificity coincides with accumulation of pseudogenes, indicating adaptation of host-restricted serovars to their narrow niches. Polyamines are small cationic amines and in Salmonella they can be synthesized through two alternative pathways directly from l-ornithine to putrescine and from l-arginine via agmatine to putrescine. The first pathway is not active in S. Gallinarum and S. Typhi, and this prompted us to investigate the importance of polyamines for virulence in S. Gallinarum. Bioinformatic analysis of all sequenced genomes of Salmonella revealed that pseudogene formation of the speC gene was exclusive for S. Typhi and S. Gallinarum and happened through independent events. The remaining polyamine biosynthesis pathway was found to be essential for oral infection with S. Gallinarum since single and double mutants in speB and speE, encoding the pathways from agmatine to putrescine and from putrescine to spermidine, were attenuated. In contrast, speB was dispensable after intraperitoneal challenge, suggesting that putrescine was less important for the systemic phase of the disease. In support of this hypothesis, a ΔspeE;ΔpotCD mutant, unable to synthesize and import spermidine, but with retained ability to import and synthesize putrescine, was attenuated after intraperitoneal infection. We therefore conclude that polyamines are essential for virulence of S. Gallinarum. Furthermore, our results point to distinct roles for putrescine and spermidine during systemic infection.

  6. ESX-1-induced apoptosis during mycobacterial infection: to be or not to be, that is the question

    PubMed Central

    Aguiló, Nacho; Marinova, Dessislava; Martín, Carlos; Pardo, Julián

    2013-01-01

    The major Mycobacterium tuberculosis virulence factor ESAT-6 exported by the ESX-1 secretion system has been described as a pro-apoptotic factor by several independent groups in recent years, sustaining a role for apoptosis in M. tuberculosis pathogenesis. This role has been supported by independent studies in which apoptosis has been shown as a hallmark feature in human and mouse lungs infected with virulent strains. Nevertheless, the role of apoptosis during mycobacterial infection is subject to an intense debate. Several works maintain that apoptosis is more evident with attenuated strains, whereas virulent mycobacteria tend to inhibit this process, suggesting that apoptosis induction may be a host mechanism to control infection. In this review, we summarize the evidences that support the involvement of ESX-1-induced apoptosis in virulence, intending to provide a rational treatise for the role of programmed cell death during M. tuberculosis infection. PMID:24364000

  7. Characterization of Virulence-Associated Genes, Antimicrobial Resistance Genes, and Class 1 Integrons in Salmonella enterica serovar Typhimurium Isolates from Chicken Meat and Humans in Egypt.

    PubMed

    Ahmed, Heba A; El-Hofy, Fatma I; Shafik, Saleh M; Abdelrahman, Mahmoud A; Elsaid, Gamilat A

    2016-06-01

    Foodborne pathogens are leading causes of illness especially in developing countries. The current study aimed to characterize virulence-associated genes and antimicrobial resistance in 30 Salmonella Typhimurium isolates of chicken and human origin at Mansoura, Egypt. The results showed that invA, avrA, mgtC, stn, and bcfC genes were identified in all the examined isolates, while 96.7% and 6.7% were positive for sopB and pef genes, respectively. The highest resistance frequencies of the isolates were to chloramphenicol and trimethoprim-sulfamethoxazole (73.3%, each), followed by streptomycin (56.7%), tetracycline and ampicillin (53.3%, each), and gentamicin (30%). However, only 2.7% of the isolates were resistant to cefotaxime and ceftriaxone each. Different resistance-associated genes, including blaTEM, aadB, aadC, aadA1, aadA2, floR, tetA(A), tetA(B), and sul1, were identified in Salmonella Typhimurium isolates with the respective frequencies of 53.3%, 6.7%, 23.3%, 46.7%, 63.3%, 73.3%, 60%, 20%, and 96.7%. None of the isolates was positive for blaSHV, blaOXA, and blaCMY genes. The results showed that the intI1 gene was detected in 24 (80%) of the examined Salmonella Typhimurium isolates. Class 1 integrons were found in 19 (79.2%) isolates that were intI1 positive. Seven integron profiles (namely: P-I to P-VII) were identified with P-V (gene cassette dfrA15, aadA2), the most prevalent profile. To the best of our knowledge, this is the first study to characterize the unusual gene cassette array dfrA12-OrfF-aadA27 from Salmonella Typhimurium isolates in Egypt. PMID:26977940

  8. Mutations in the bvgA gene of Bordetella pertussis that differentially affect regulation of virulence determinants.

    PubMed Central

    Stibitz, S

    1994-01-01

    By using chemical mutagenesis and genetic mapping, a search was undertaken for previously undescribed genes which may be involved in different regulatory mechanisms governing different virulence factors of Bordetella pertussis. Previous studies have shown that the fha locus encoding filamentous hemagglutinin is regulated directly by the bvgAS two component system, while regulation of ptx encoding pertussis toxin is less direct or occurs by a different mechanism. With a strain containing gene fusions to each of these regulated loci, screening was done for mutations which were defective for ptx expression but maintained normal or nearly normal levels of fha expression. Two mutations which had such a phenotype and were also deficient in adenylate cyclase toxin/hemolysin expression were found and characterized more fully. Both were found to affect residues in the C-terminal portion of the BvgA response regulator protein, a domain which shares sequence similarity with a family of regulatory proteins including FixJ, UhpA, MalT, RcsA, RcsB, and LuxR. The residues affected are within a region which, by extension from studies on the LuxR protein, may be involved in transcriptional activation. Images PMID:8083156

  9. Genetic diversity and virulence gene determinants of antibiotic-resistant Salmonella isolated from preharvest turkey production sources.

    PubMed

    Nayak, R; Stewart, T; Wang, R-F; Lin, J; Cerniglia, C E; Kenney, P B

    2004-02-15

    This study evaluated the molecular diversity of 29 Salmonella serotypes isolated from turkey ceca and the production environment. Isolates were resistant to bacitracin (100%), erythromycin (100%), novobiocin (100%), rifampin (100%), streptomycin (62%), gentamicin (52%), spectinomycin (48%), tetracycline (31%), sulfamethoxazole/trimethoprim (SXT) (3%) and tobramycin (3%). The minimum inhibitory concentration (MIC) values ranged from 32 to >/=1024 microg/ml. The pulsed-field gel electrophoresis (PFGE) and ribotyping patterns were identical within each of the serotypes Heidelberg, Worthington and Muenster. The plasmid profiles were identical within each of the Salmonella serotypes. Two different clones of Salmonella anatum were differentiated by PFGE typing but not by ribotyping. Heidelberg isolates from nine turkey ceca and three drinker samples had identical antibiotic resistance, PFGE, ribotype and plasmid patterns, suggesting that transmission of this particular clone may have occurred between the birds and the drinkers. Identical PFGE, ribotype and plasmid patterns were observed in one Salmonella worthington isolate from turkey ceca in one flock and two S. worthington isolates from feeder contents and drinkers from a subsequent flock, suggesting transmission of this pathogen between flocks. Individual and multiple polymerase chain reaction (PCR) analyses revealed the presence of the virulence genes invA, aceK and sopB and the absence of the h-1i gene in all isolates. A combination of genotypic and phenotypic markers can be useful in studying genetic variation among natural salmonellae populations in turkey production and delineating possible transmission pathways.

  10. The mtfA Transcription Factor Gene Controls Morphogenesis, Gliotoxin Production, and Virulence in the Opportunistic Human Pathogen Aspergillus fumigatus

    PubMed Central

    Smith, Timothy D.

    2014-01-01

    Aspergillus fumigatus is the leading causative agent of invasive aspergillosis (IA). The number of cases is on the rise, with mortality rates as high as 90% among immunocompromised patients. Molecular genetic studies in A. fumigatus could provide novel targets to potentially set the basis for antifungal therapies. In the current study, we investigated the role of the transcription factor gene mtfA in A. fumigatus. Our results revealed that mtfA plays a role in the growth and development of the fungus. Deletion or overexpression of mtfA leads to a slight reduction in colony growth, as well as a reduction in conidiation levels, in the overexpression strain compared to the wild-type strain. Furthermore, production of the secondary metabolite gliotoxin increased when mtfA was overexpressed, coinciding with an increase in the transcription levels of the gliotoxin genes gliZ and gliP with respect to the wild type. In addition, our study showed that mtfA is also necessary for normal protease activity in A. fumigatus; deletion of mtfA resulted in a reduction of protease activity compared to wild-type levels. Importantly, the absence of mtfA caused a decrease in virulence in the Galleria mellonella infection model, indicating that mtfA is necessary for A. fumigatus wild-type pathogenesis. PMID:24728192

  11. Molecular Evolution of Typical Enteropathogenic Escherichia coli: Clonal Analysis by Multilocus Sequence Typing and Virulence Gene Allelic Profiling▿ †

    PubMed Central

    Lacher, David W.; Steinsland, Hans; Blank, T. Eric; Donnenberg, Michael S.; Whittam, Thomas S.

    2007-01-01

    Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Typical EPEC isolates are differentiated from other types of pathogenic E. coli by two distinctive phenotypes, attaching effacement and localized adherence. The genes specifying these phenotypes are found on the locus of enterocyte effacement (LEE) and the EPEC adherence factor (EAF) plasmid. To describe how typical EPEC has evolved, we characterized a diverse collection of strains by multilocus sequence typing (MLST) and performed restriction fragment length polymorphism (RFLP) analysis of three virulence genes (eae, bfpA, and perA) to assess allelic variation. Among 129 strains representing 20 O-serogroups, 21 clonal genotypes were identified using MLST. RFLP analysis resolved nine eae, nine bfpA, and four perA alleles. Each bfpA allele was associated with only one perA allele class, suggesting that recombination has not played a large role in shuffling the bfpA and perA loci between separate EAF plasmids. The distribution of eae alleles among typical EPEC strains is more concordant with the clonal relationships than the distribution of the EAF plasmid types. These results provide further support for the hypothesis that the EPEC pathotype has evolved multiple times within E. coli through separate acquisitions of the LEE island and EAF plasmid. PMID:17098897