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Sample records for governs virulence expression

  1. Chromosomal “Stress-Response” Domains Govern the Spatiotemporal Expression of the Bacterial Virulence Program

    PubMed Central

    Jiang, Xuejiao; Sobetzko, Patrick; Reverchon, Sylvie; Muskhelishvili, Georgi

    2015-01-01

    ABSTRACT Recent studies strongly suggest that the gene expression sustaining both normal and pathogenic bacterial growth is governed by the structural dynamics of the chromosome. However, the mechanistic device coordinating the chromosomal configuration with selective expression of the adaptive traits remains largely unknown. We used a holistic approach exploring the inherent relationships between the physicochemical properties of the DNA and the expression of adaptive traits, including virulence factors, in the pathogen Dickeya dadantii (formerly Erwinia chrysanthemi). In the transcriptomes obtained under adverse conditions encountered during bacterial infection, we explored the patterns of chromosomal DNA sequence organization, supercoil dynamics, and gene expression densities, together with the long-range regulatory impacts of the abundant DNA architectural proteins implicated in pathogenicity control. By integrating these data, we identified transient chromosomal domains of coherent gene expression featuring distinct couplings between DNA thermodynamic stability, supercoil dynamics, and virulence traits. PMID:25922390

  2. Regulatory principles governing Salmonella and Yersinia virulence

    PubMed Central

    Erhardt, Marc; Dersch, Petra

    2015-01-01

    Enteric pathogens such as Salmonella and Yersinia evolved numerous strategies to survive and proliferate in different environmental reservoirs and mammalian hosts. Deciphering common and pathogen-specific principles for how these bacteria adjust and coordinate spatiotemporal expression of virulence determinants, stress adaptation, and metabolic functions is fundamental to understand microbial pathogenesis. In order to manage sudden environmental changes, attacks by the host immune systems and microbial competition, the pathogens employ a plethora of transcriptional and post-transcriptional control elements, including transcription factors, sensory and regulatory RNAs, RNAses, and proteases, to fine-tune and control complex gene regulatory networks. Many of the contributing global regulators and the molecular mechanisms of regulation are frequently conserved between Yersinia and Salmonella. However, the interplay, arrangement, and composition of the control elements vary between these closely related enteric pathogens, which generate phenotypic differences leading to distinct pathogenic properties. In this overview we present common and different regulatory networks used by Salmonella and Yersinia to coordinate the expression of crucial motility, cell adhesion and invasion determinants, immune defense strategies, and metabolic adaptation processes. We highlight evolutionary changes of the gene regulatory circuits that result in different properties of the regulatory elements and how this influences the overall outcome of the infection process. PMID:26441883

  3. Metabolic sensor governing bacterial virulence in Staphylococcus aureus.

    PubMed

    Ding, Yue; Liu, Xing; Chen, Feifei; Di, Hongxia; Xu, Bin; Zhou, Lu; Deng, Xin; Wu, Min; Yang, Cai-Guang; Lan, Lefu

    2014-11-18

    An effective metabolism is essential to all living organisms, including the important human pathogen Staphylococcus aureus. To establish successful infection, S. aureus must scavenge nutrients and coordinate its metabolism for proliferation. Meanwhile, it also must produce an array of virulence factors to interfere with host defenses. However, the ways in which S. aureus ties its metabolic state to its virulence regulation remain largely unknown. Here we show that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, binds to and activates the catabolite control protein E (CcpE) of S. aureus. Using structural and site-directed mutagenesis studies, we demonstrate that two arginine residues (Arg145 and Arg256) within the putative inducer-binding cavity of CcpE are important for its allosteric activation by citrate. Microarray analysis reveals that CcpE tunes the expression of 126 genes that comprise about 4.7% of the S. aureus genome. Intriguingly, although CcpE is a major positive regulator of the TCA-cycle activity, its regulon consists predominantly of genes involved in the pathogenesis of S. aureus. Moreover, inactivation of CcpE results in increased staphyloxanthin production, improved ability to acquire iron, increased resistance to whole-blood-mediated killing, and enhanced bacterial virulence in a mouse model of systemic infection. This study reveals CcpE as an important metabolic sensor that allows S. aureus to sense and adjust its metabolic state and subsequently to coordinate the expression of virulence factors and bacterial virulence.

  4. 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

  5. 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

  6. Calcineurin governs thermotolerance and virulence of Cryptococcus gattii.

    PubMed

    Chen, Ying-Lien; Lehman, Virginia N; Lewit, Yonathan; Averette, Anna F; Heitman, Joseph

    2013-03-01

    The pathogenic yeast Cryptococcus gattii, which is causing an outbreak in the Pacific Northwest region of North America, causes life-threatening pulmonary infections and meningoencephalitis in healthy individuals, unlike Cryptococcus neoformans, which commonly infects immunocompromised patients. In addition to a greater predilection for C. gattii to infect healthy hosts, the C. gattii genome sequence project revealed extensive chromosomal rearrangements compared with C. neoformans, showing genomic differences between the two Cryptococcus species. We investigated the roles of C. gattii calcineurin in three molecular types: VGIIa (R265), VGIIb (R272), and VGI (WM276). We found that calcineurin exhibits a differential requirement for growth on solid medium at 37°, as calcineurin mutants generated from R265 were more thermotolerant than mutants from R272 and WM276. We demonstrated that tolerance to calcineurin inhibitors (FK506, CsA) at 37° is linked with the VGIIa molecular type. The calcineurin mutants from the R272 background showed the most extensive growth and morphological defects (multivesicle and larger ring-like cells), as well as increased fluconazole susceptibility. Our cellular architecture examination showed that C. gattii and C. neoformans calcineurin mutants exhibit plasma membrane disruptions. Calcineurin in the C. gattii VGII molecular type plays a greater role in controlling cation homeostasis compared with that in C. gattii VGI and C. neoformans H99. Importantly, we demonstrate that C. gattii calcineurin is essential for virulence in a murine inhalation model, supporting C. gattii calcineurin as an attractive antifungal drug target.

  7. 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.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  10. Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator.

    PubMed Central

    Sheehan, B; Klarsfeld, A; Msadek, T; Cossart, P

    1995-01-01

    PrfA is a pleiotropic activator of virulence gene expression in the pathogenic bacterium Listeria monocytogenes. Several lines of evidence have suggested that a hierarchy of virulence gene activation by PrfA exists. This hypothesis was investigated by assessing the ability of PrfA to activate the expression of virulence gene fusions to lacZ in Bacillus subtilis. Expression of PrfA in this heterologous host was sufficient for activation of transcription at the hly, plcA, mpl, and actA promoters. Activation was most efficient at the divergently transcribed hly and plcA promoters. The putative PrfA binding site shared by these promoters is perfectly symmetrical and appears to represent the optimum sequence for target gene activation by PrfA. The activation of actA and mpl expression was considerably weaker and occurred more slowly than that observed at the hly and plcA promoters, suggesting that greater quantities of PrfA are required for productive interaction at these promoters. Interestingly, expression of an inlA-lacZ transcriptional fusion was very poorly activated by PrfA in B. subtilis, suggesting that other Listeria factors, in addition to PrfA, are required for PrfA-mediated activation at this promoter. Further support for the involvement of such factors was obtained by constructing and analyzing a prfA deletion mutant of L. monocytogenes. We observed that, in contrast to that of the other genes of the PrfA regulon, expression of inlA is only partially dependent on PrfA. PMID:7592422

  11. Fluoride exposure attenuates expression of Streptococcus pyogenes virulence factors.

    PubMed

    Thongboonkerd, Visith; Luengpailin, Jirapon; Cao, Junkai; Pierce, William M; Cai, Jian; Klein, Jon B; Doyle, R J

    2002-05-10

    Fluoridation causes an obvious reduction of dental caries by interference with cariogenic streptococci. However, the effect of fluoride on group A streptococci that causes rheumatic fever and acute poststreptococcal glomerulonephritis is not known. We have used proteomic analysis to create a reference proteome map for Streptococcus pyogenes and to determine fluoride-induced protein changes in the streptococci. Cellular and extracellular proteins were resolved by two-dimensional polyacrylamide gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. 183 protein spots were visualized, and 74 spots representing 60 unique proteins were identified. A 16-h exposure to sodium fluoride caused decreased expression of proteins required to respond to cellular stress, including anti-oxidants, glycolytic enzymes, transcriptional and translational regulators, and protein folding. Fluoride caused decreased cellular expression of two well-characterized S. pyogenes virulence factors. Fluoride decreased expression of glyceraldehyde-3-phosphate dehydrogenase, which acts to bind fibronectin and promote bacterial adherence. We also performed proteomic analysis of protein released by S. pyogenes into the culture supernatant and observed decreased expression of M proteins following fluoride exposure. These data provide evidence that fluoride causes decreased expression by S. pyogenes proteins used to respond to stress, virulence factors, and implicated in non-suppurative complications of S. pyogenes, including glomerulonephritis and rheumatic fever.

  12. 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.

  13. The Cpx System Regulates Virulence Gene Expression in Vibrio cholerae

    PubMed Central

    Acosta, Nicole; Pukatzki, Stefan

    2015-01-01

    Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway in Vibrio cholerae El Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of the crp gene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production. PMID:25824837

  14. Role of feedback and network architecture in controlling virulence gene expression in Bordetella.

    PubMed

    Prajapat, Mahendra Kumar; Saini, Supreet

    2013-11-01

    Bordetella is a Gram-negative bacterium responsible for causing whooping cough in a broad range of host organisms. For successful infection, Bordetella controls expression of four distinct classes of genes (referred to as class 1, 2, 3, and 4 genes) at distinct times in the infection cycle. This control is executed by a single two-component system, BvgAS. Interestingly, the transmembrane component of the two-component system, BvgS, consists of three phospho-transfer domains leading to phosphorylation of the response regulator, BvgA. Phosphorylated BvgA then controls expression of virulence genes and also controls bvgAS transcription. In this work, we perform simulations to characterize the role of the network architecture in governing gene expression in Bordetella. Our results show that the wild-type network is locally optimal for controlling the timing of expression of the different classes of genes involved in infection. In addition, the interplay between environmental signals and positive feedback aids the bacterium identify precise conditions for and control expression of virulence genes.

  15. Erythritol triggers expression of virulence traits in Brucella melitensis.

    PubMed

    Petersen, Erik; Rajashekara, Gireesh; Sanakkayala, Neelima; Eskra, Linda; Harms, Jerome; Splitter, Gary

    2013-06-01

    Erythritol is a four-carbon sugar preferentially utilized by Brucella spp. The presence of erythritol in the placentas of goats, cows, and pigs has been used to explain the localization of Brucella to these sites and the subsequent accumulation of large amounts of bacteria, eventually leading to abortion. Here we show that Brucella melitensis will also localize to an artificial site of erythritol within a mouse, providing a potential model system to study the pathogenesis of Brucella abortion. Immunohistological staining of the sites of erythritol within infected mice indicated a higher than expected proportion of extracellular bacteria. Ensuing experiments suggested intracellular B. melitensis was unable to replicate within macrophages in the presence of erythritol and that erythritol was able to reach the site of intracellular bacteria. The intracellular inhibition of growth was found to encourage the bacteria to replicate extracellularly rather than intracellularly, a particularly interesting development in Brucella pathogenesis. To determine the effect of erythritol on expression of B. melitensis genes, bacteria grown either with or without erythritol were analyzed by microarray. Two major virulence pathways were up-regulated in response to exposure to erythritol (the type IV secretion system VirB and flagellar proteins), suggesting a role for erythritol in virulence. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. Erythritol triggers expression of virulence traits in Brucella melitensis

    PubMed Central

    Petersen, Erik; Rajashekara, Gireesh; Sanakkayala, Neelima; Eskra, Linda; Harms, Jerome; Splitter, Gary

    2013-01-01

    Erythritol is a four-carbon sugar preferentially utilized by Brucella spp. The presence of erythritol in the placentas of goats, cows, and pigs has been used to explain the localization of Brucella to these sites and the subsequent accumulation of large amounts of bacteria, eventually leading to abortion. Here we show that B. melitensis will also localize to an artificial site of erythritol within a mouse, providing a potential model system to study the pathogenesis of Brucella abortion. Immunohistological staining of the sites of erythritol within infected mice indicated a higher than expected proportion of extracellular bacteria. Ensuing experiments suggested intracellular B. melitensis was unable to replicate within macrophages in the presence of erythritol and that erythritol was able to reach the site of intracellular bacteria. The intracellular inhibition of growth was found to encourage the bacteria to replicate extracellularly rather than intracellularly, a particularly interesting development in Brucella pathogenesis. To determine the effect of erythritol on expression of B. melitensis genes, bacteria grown either with or without erythritol were analyzed by microarray. Two major virulence pathways were up-regulated in response to exposure to erythritol (the type IV secretion system VirB and flagellar proteins), suggesting a role for erythritol in virulence. PMID:23421980

  17. Expression of virulence genes in luminescent and nonluminescent isogenic vibrios and virulence towards gnotobiotic brine shrimp (Artemia franciscana).

    PubMed

    Ruwandeepika, H A D; Defoirdt, T; Bhowmick, P P; Karunasagar, I; Bossier, P

    2011-02-01

    This study aimed to evaluate the expression levels of virulence gene regulators (luxR and toxR) and virulence factors (serine protease, metalloprotease and haemolysin) in luminescent and nonluminescent isogenic Vibrio harveyi and Vibrio campbellii. Nonluminescent variants have been reported before to become dominant in cultures of luminescent vibrios when grown under static conditions in the dark. Wild-type V. harveyi BB120, V. campbellii LMG 21363, quorum sensing mutants of V. harveyi BB120 and their previously reported nonluminescent isogenic counterparts were used in this study. The expression level of the virulence genes srp serine protease, vhp metalloprotease and vhh haemolysin, the quorum sensing master regulator gene luxR and the virulence regulator gene toxR in isogenic luminescent and nonluminescent strains were quantified using reverse transcriptase real-time PCR. These experiments revealed that the nonluminescent strains produced lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene (which is known to be regulated by quorum sensing). Finally, challenge tests with gnotobiotic brine shrimp (Artemia franciscana) larvae revealed that the nonluminescent strains are less virulent than their luminescent isogenic counterparts. Nonluminescent variants of V. harveyi and V. campbellii strains produce lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene and are less virulent to brine shrimp than their isogenic luminescent counterparts. These results indicate that adaptation of luminescent vibrios to specific growth conditions that result in a dominant nonluminescent phenotype is accompanied by a decreased adaptation to a host environment because of altered virulence gene regulation. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

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

    PubMed

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

    2017-01-19

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

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

    PubMed Central

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

    2017-01-01

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

  20. Influence of Sublethal Concentrations of Common Disinfectants on Expression of Virulence Genes in Listeria monocytogenes ▿

    PubMed Central

    Kastbjerg, Vicky G.; Larsen, Marianne Halberg; Gram, Lone; Ingmer, Hanne

    2010-01-01

    Listeria monocytogenes is a food-borne human pathogen that causes listeriosis, a relatively rare infection with a high fatality rate. The regulation of virulence gene expression is influenced by several environmental factors, and the aim of the present study was to determine how disinfectants used routinely in the food industry affect the expression of different virulence genes in L. monocytogenes when added at sublethal concentrations. An agar-based assay was developed to screen the effect of disinfectants on virulence gene promoter expression and was validated at the transcriptional level by Northern blot analysis. Eleven disinfectants representing four different groups of active components were evaluated in this study. Disinfectants with the same active ingredients had a similar effect on gene expression. Peroxy and chlorine compounds reduced the expression of the virulence genes, and quaternary ammonium compounds (QAC) induced the expression of the virulence genes. In general, a disinfectant had similar effects on the expression of all four virulence genes examined. Northern blot analyses confirmed the downregulation of prfA and inlA expression by Incimaxx DES (a peroxy compound) and their upregulation by Triquart Super (a QAC) in L. monocytogenes EGD. Hence, sublethal concentrations of disinfectants routinely used in the food industry affect virulence gene expression in the human pathogen L. monocytogenes, and the effect depends on the active components of the disinfectant. From a practical perspective, the study underlines that disinfectants should be used at the lethal concentrations recommended by the manufacturers. Further studies are needed to elucidate whether the changes in virulence gene expression induced by the disinfectants have impact on virulence or other biological properties, such as antibiotic resistance. PMID:19897753

  1. Differential expression of microRNAs of Litopenaeus vannamei in response to different virulence WSSV infection.

    PubMed

    Sun, Xinying; Liu, Qing-Hui; Yang, Bing; Huang, Jie

    2016-11-01

    WSSV is one of the most harmful pathogeny in the pacific white shrimp, and genetic variations caused the strains of different virulence. MicroRNAs (miRNAs) involved in the regulation of virus defense. To understand the different virulence of WSSV on miRNA expression in Litopeneaus vannamei, the deep sequencing was performed to compare two small RNA libraries prepared from hepatopancreas of Litopeneaus vannamei infected with normal-virulence or low-virulence WSSV. Approximately 29,398,623 raw reads from normal-virulence library and 35,291,803 raw reads from low-virulence library were obtained. There were about 37 miRNAs homologs identified. Sixteen miRNAs were significantly up-regulated and twenty-one miRNAs were significantly down-regulated in normal-virulence infection library compared with low-virulence infection library. Of these, Igi-miR-1175-3p was the most significant different miRNA, followed by bmo-miR-1175-3p and ipu-miR-26b, respectively. The putative target genes for differentially expressed miRNAs were concerned with biological processes, signal meditated, cell differentiation and apoptosis, immune recognition and other more functions. The results will help to understand the miRNAs response to different virulence WSSV infection.

  2. Temperature control of molecular circuit switch responsible for virulent phenotype expression in uropathogenic Escherichia coli

    NASA Astrophysics Data System (ADS)

    Samoilov, Michael

    2010-03-01

    The behavior and fate of biological organisms are to a large extent dictated by their environment, which can be often viewed as a collection of features and constraints governed by physics laws. Since biological systems comprise networks of molecular interactions, one such key physical property is temperature, whose variations directly affect the rates of biochemical reactions involved. For instance, temperature is known to control many gene regulatory circuits responsible for pathogenicity in bacteria. One such example is type 1 fimbriae (T1F) -- the foremost virulence factor in uropathogenic E. coli (UPEC), which accounts for 80-90% of all community-acquired urinary tract infections (UTIs). The expression of T1F is randomly `phase variable', i.e. individual cells switch between virulent/fimbriate and avirulent/afimbriate phenotypes, with rates regulated by temperature. Our computational investigation of this process, which is based on FimB/FimE recombinase-mediated inversion of fimS DNA element, offers new insights into its discrete-stochastic kinetics. In particular, it elucidates the logic of T1F control optimization to the host temperature and contributes further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs.

  3. Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB

    PubMed Central

    Liu, Zhi; Yang, Menghua; Peterfreund, Gregory L.; Tsou, Amy M.; Selamoglu, Nur; Daldal, Fevzi; Zhong, Zengtao; Kan, Biao; Zhu, Jun

    2011-01-01

    Bacterial pathogens have evolved sophisticated signal transduction systems to coordinately control the expression of virulence determinants. For example, the human pathogen Vibrio cholerae is able to respond to host environmental signals by activating transcriptional regulatory cascades. The host signals that stimulate V. cholerae virulence gene expression, however, are still poorly understood. Previous proteomic studies indicated that the ambient oxygen concentration plays a role in V. cholerae virulence gene expression. In this study, we found that under oxygen-limiting conditions, an environment similar to the intestines, V. cholerae virulence genes are highly expressed. We show that anaerobiosis enhances dimerization and activity of AphB, a transcriptional activator that is required for the expression of the key virulence regulator TcpP, which leads to the activation of virulence factor production. We further show that one of the three cysteine residues in AphB, C235, is critical for oxygen responsiveness, as the AphBC235S mutant can activate virulence genes under aerobic conditions in vivo and can bind to tcpP promoters in the absence of reducing agents in vitro. Mass spectrometry analysis suggests that under aerobic conditions, AphB is modified at the C235 residue. This modification is reversible between oxygen-rich aquatic environments and oxygen-limited human hosts, suggesting that V. cholerae may use a thiol-based switch mechanism to sense intestinal signals and activate virulence. PMID:21187377

  4. 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

  5. Golden pigment production and virulence gene expression are affected by metabolisms in Staphylococcus aureus.

    PubMed

    Lan, Lefu; Cheng, Alice; Dunman, Paul M; Missiakas, Dominique; He, Chuan

    2010-06-01

    The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of approximately 400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival.

  6. Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus▿ †

    PubMed Central

    Lan, Lefu; Cheng, Alice; Dunman, Paul M.; Missiakas, Dominique; He, Chuan

    2010-01-01

    The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of ∼400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival. PMID:20400547

  7. Virulence genes in clinical and environmental Stenotrophomas maltophilia isolates: a genome sequencing and gene expression approach.

    PubMed

    Adamek, Martina; Linke, Burkhard; Schwartz, Thomas

    2014-01-01

    The rate of nosocomial infections with the opportunistic pathogen Stenotrophomonas maltophilia has remarkably increased in the last decade. To determine S. maltophilia virulence genes, the complete genome sequences of two S. maltophilia isolates were compared. The clinical strain SKK35 was proved virulent in an amoeba host-pathogen model, and wastewater strain RA8 was determined as non-virulent in the amoeba model. The genome sequences of three additional S. maltophilia strains, K279a (clinical, non-virulent against amoeba), R511-3 and SKA14 (both environmental, non-virulent against amoeba) were taken into account as reference strains. We were able to show that all clinical and environmental S. maltophilia strains presented comparable distribution of so far identified potential virulence genes, regardless to their virulence potential against amoebae. Aside from that, strain SKK35 was found harboring a putative, strain specific pathogenicity island, encoding two proteins from the RTX (repeats-in-toxin) family. The actual expression of the RTX genes was verified in growth experiments in different culture media containing blood or blood components and in co-cultures with amoeba.

  8. 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

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

    PubMed

    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.

  10. The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon

    PubMed Central

    Fernández de Marco, María del Mar; Alejo, Alí; Hudson, Paul; Damon, Inger K.; Alcami, Antonio

    2010-01-01

    Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.—Fernández de Marco, M. M., Alejo, A., Hudson, P., Damon, I. K., Alcami, A. The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon. PMID:20019241

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

    PubMed Central

    Nielsen, Alex T.; Dolganov, Nadia A.; Rasmussen, Thomas; Otto, Glen; Miller, Michael C.; Felt, Stephen A.; Torreilles, Stéphanie; Schoolnik, Gary K.

    2010-01-01

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

  12. 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.

  13. Hypoxia Reduces the Pathogenicity of Pseudomonas aeruginosa by Decreasing the Expression of Multiple Virulence Factors.

    PubMed

    Schaible, Bettina; Rodriguez, Javier; Garcia, Amaya; von Kriegsheim, Alexander; McClean, Siobhán; Hickey, Caitríona; Keogh, Ciara E; Brown, Eric; Schaffer, Kirsten; Broquet, Alexis; Taylor, Cormac T

    2017-05-01

    Our understanding of how the course of opportunistic bacterial infection is influenced by the microenvironment is limited. We demonstrate that the pathogenicity of Pseudomonas aeruginosa strains derived from acute clinical infections is higher than that of strains derived from chronic infections, where tissues are hypoxic. Exposure to hypoxia attenuated the pathogenicity of strains from acute (but not chronic) infections, implicating a role for hypoxia in regulating bacterial virulence. Mass spectrometric analysis of the secretome of P. aeruginosa derived from an acute infection revealed hypoxia-induced repression of multiple virulence factors independent of altered bacterial growth. Pseudomonas aeruginosa lacking the Pseudomonas prolyl-hydroxylase domain-containing protein, which has been implicated in bacterial oxygen sensing, displays reduced virulence factor expression. Furthermore, pharmacological hydroxylase inhibition reduces virulence factor expression and pathogenicity in a murine model of pneumonia. We hypothesize that hypoxia reduces P. aeruginosa virulence at least in part through the regulation of bacterial hydroxylases. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  14. Expression of virulence genes by Listeria monocytogenes J0161 in natural environment

    PubMed Central

    Tirumalai, Prem Saran; Prakash, Soam

    2012-01-01

    Majority of studies concerning the gene expression of Listeria monocytogenes have been done on pure culture states. Our objective was to study L.monocytogenes in a co-cultured state and to understand if microbes in their natural state of existence are different in their expression than that of the purely cultured lab grown forms. For a long period discussions have been on the expression of prfA, (which is a virulence gene regulator) in a mammalian host and its role in causing the switch from a saprophytic to pathogenic form of L.monocytogenes. We, in this paper for the first time report the expression of prfA and other virulence genes by L.monocytogenes under different extracellular conditions, and also as a pure culture biofilms, that is different from the previous reports. We also report that the expression of prfA seems to vary considerably when co-cultured with Bacillus subtilis. PMID:24031897

  15. Intracellular and Interstitial Expression of Helicobacter pylori Virulence Genes in Gastric Precancerous Intestinal Metaplasia and Adenocarcinoma

    PubMed Central

    Semino-Mora, Cristina; Doi, Sonia Q.; Marty, Aileen; Simko, Vlado; Carlstedt, Ingemar; Dubois, Andre

    2008-01-01

    Gastric intestinal metaplasia (IM) and gastric cancer are associated with Helicobacter pylori, but the bacterium often is undetectable in these lesions. To unravel this apparent paradox, IM, H. pylori presence, and the expression of H. pylori virulence genes were quantified concurrently using histologic testing, in situ hybridization, and immunohistochemistry. H. pylori was detected inside metaplastic, dysplastic, and neoplastic epithelial cells, and cagA and babA2 expression was colocalized. Importantly, expression of cagA was significantly higher in patients with IM and adenocarcinoma than in control subjects. The preneoplastic “acidic” MUC2 mucin was detected only in the presence of H. pylori, and MUC2 expression was higher in patients with IM, dysplasia, and cancer. These novel findings are compatible with the hypothesis that all stages of gastric carcinogenesis are fostered by persistent intracellular expression of H. pylori virulence genes, especially cagA inside MUC2-producing precancerous gastric cells and pleomorphic cancer cells. PMID:12695995

  16. 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.

  17. Attenuation and quantitation of virulence gene expression in quorum-quenched Dickeya chrysanthemi.

    PubMed

    Hosseinzadeh, Saeed; Shams-Bakhsh, Masoud; Sadeghizadeh, Majid

    2017-01-01

    N-Acyl-homoserine lactones (AHLs)-dependent quorum sensing (QS) system(s) is recruited by the soft rot bacterium Dickeya chrysanthemi for coordinating its social activities such as secretion of plant cell wall-degrading enzymes, while the main signal molecule and quantity dependence of virulence to QS in this bacterium have not been clarified. To do this end, the involvement of AHLs in African violet leaves and potato tuber maceration; swarming motility; pectate lyase and polygalacturonase enzymes production and in planta expression of virulence genes including pelE, pehX and pemA by electroporating two quorum-quenching vectors. The expression of two types of AHL-lactonase expressing vector caused dramatic decrease in swarming motility, production of pectinolytic enzymes and macerating of plant tissues. The maximum ability of quenching of QS in repression of D. chrysanthemi virulence was assessed quantitatively by q-RT-PCR, as expression of pelE, pehX and pemA genes were decreased 90.5-92.18 % in quenched cells. We also showed that virulence and pathogenicity of this bacterium was under the control of DHL-dependent QS system and that the existence of second DHL operating system is probable for this bacterium. Thus, this signal molecule would be the key point for future research to design DHL-specific lactonase enzymes using bioinformatics methods.

  18. Low-shear modelled microgravity alters expression of virulence determinants of Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Rosado, Helena; Doyle, Marie; Hinds, Jason; Taylor, Peter W.

    2010-02-01

    Microbiological monitoring of air and surfaces within the ISS indicate that bacteria of the genus Staphylococcus are found with high frequency. Staphylococcus aureus, an opportunistic pathogen with the capacity to cause severe debilitating infection, constitutes a significant proportion of these isolates. Experiments conducted during short-term flight suggest that growth in microgravity leads to increases in bacterial antibiotic resistance and to cell wall changes. Growth under low-shear modelled microgravity (LSMMG) indicated that a reduced gravitational field acts as an environmental signal for expression of enhanced bacterial virulence in gram-negative pathogens. We therefore examined the effect of simulated microgravity on parameters of antibiotic susceptibility and virulence in methicillin-susceptible S. aureus isolates RF1, RF6 and RF11; these strains were grown in a high aspect ratio vessel under LSMMG and compared with cells grown under normal gravity (NG). There were no significant differences in antibiotic susceptibility of staphylococci grown under LSMMG compared to NG. LSMMG-induced reductions in synthesis of the pigment staphyloxanthin and the major virulence determinant α-toxin were noted. Significant changes in global gene expression were identified by DNA microarray analysis; with isolate RF6, the expression of hla and genes of the regulatory system saeR/saeS were reduced approximately two-fold. These data provide strong evidence that growth of S. aureus under modelled microgravity leads to a reduction in expression of virulence determinants.

  19. Role of IS1 in the Conversion of Virulence (Vi) Antigen Expression in Enterobacteriaceae,

    DTIC Science & Technology

    1992-01-01

    into viaB to presumably a virulence factor for Salmonella typhi . Its occur, the host bacterium must carry IS/ or an IS1-like expression is controlled...Noon KF, Baron LS (1985) Development National Science Council Grant NSC80-0418-BI82-01 of Exec- of a DNA probe to detect Salmonella typhi . J Clin

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

    USDA-ARS?s Scientific Manuscript database

    Infection of chickens with highly virulent NDV results in rapid death, which is preceded by increased expression of interferon gamma (IFN-g) in target tissues. IFN-g is a cytokine that has pleiotropic biological effects including intrinsic antiviral activity and immunomodulatory effects. Here we a...

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

    PubMed Central

    Wang, Guo-Qi; Li, Tong-Tong; Li, Zhi-Rui; Zhang, Li-Cheng

    2016-01-01

    Objective. To investigate the effect of negative pressure conditions induced by NPWT on P. aeruginosa. Methods. P. aeruginosa was cultured in a Luria–Bertani medium at negative pressure of −125 mmHg for 24 h in the experimental group and at atmospheric pressure in the control group. The diameters of the colonies of P. aeruginosa were measured after 24 h. ELISA kit, orcinol method, and elastin-Congo red assay were used to quantify the virulence factors. Biofilm formation was observed by staining with Alexa Fluor® 647 conjugate of concanavalin A (Con A). Virulence-regulated genes were determined by quantitative RT-PCR. Results. As compared with the control group, growth of P. aeruginosa was inhibited by negative pressure. The colony size under negative pressure was significantly smaller in the experimental group than that in the controls (p < 0.01). Besides, reductions in the total amount of virulence factors were observed in the negative pressure group, including exotoxin A, rhamnolipid, and elastase. RT-PCR results revealed a significant inhibition in the expression level of virulence-regulated genes. Conclusion. Negative pressure could significantly inhibit the growth of P. aeruginosa. It led to a decrease in the virulence factor secretion, biofilm formation, and a reduction in the expression level of virulence-regulated genes. PMID:28074188

  2. Aerococcus viridans expression of Cpn60 is associated with virulence during infection of the American lobster, Homarus americanus Milne Edwards.

    PubMed

    Clark, K F; Greenwood, S J

    2011-11-01

    The Gram-positive bacterium Aerococcus viridans var. homari is a well-documented causative agent of the lethal systemic disease gaffkemia in both the American lobster, Homarus americanus, and the European lobster, Homarus gammarus. Previous phenotypic characterization has been unsuccessful at differentiating avirulent from virulent strains without performing lethal animal infection trials. Recent genetic characterization of A. viridans strains through 16S rRNA sequencing and random amplification of polymorphic DNA fingerprinting has revealed the presence of two subtypes. However, subtype 1 contains both virulent and avirulent strains which are genetically identical. The purpose of this study was to determine the proteomic mediators of virulence in A. viridans. Quantitative proteomic mapping of these two strains has revealed 29 differentially expressed protein spots, seven of which are only expressed in the virulent strain and could act as virulence factors. One protein, chaperonin 60 (Cpn60), is uniquely expressed in the virulent strain and has been shown to act as a virulence factor in many other bacteria. The proteomic mapping strategy employed in this study is the first to show phenotypic differences between virulent and avirulent strains. Cpn60 expression represents a potentially useful tool for identifying the virulent strains of A. viridans in epidemiological studies.

  3. Temporal Profile of Biofilm Formation, Gene Expression and Virulence Analysis in Candida albicans Strains.

    PubMed

    de Barros, Patrícia Pimentel; Rossoni, Rodnei Dennis; De Camargo Ribeiro, Felipe; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso

    2017-04-01

    The characterization of Candida albicans strains with different degrees of virulence became very useful to understand the mechanisms of fungal virulence. Then, the objective of this study was to assess and compare the temporal profiles of biofilms formation, gene expression of ALS1, ALS3, HWP1, BCR1, EFG1, TEC1, SAP5, PLB2 and LIP9 and virulence in Galleria mellonella of C. albicans ATCC18804 and a clinical sample isolated from an HIV-positive patient (CA60). Although the CFU/mL counting was higher in biofilms formed in vitro by ATCC strain, the temporal profile of the analysis of the transcripts of the C. albicans strains was elevated to Ca60 compared to strain ATCC, especially in the genes HWP1, ALS3, SAP5, PLB2 and LIP9 (up regulation). Ca60 was more pathogenic for G. mellonella in the survival assay (p = 0.0394) and hemocytes density (p = 0.0349), agreeing with upregulated genes that encode the expression of hyphae and hydrolase genes of Ca60. In conclusion, the C. albicans strains used in this study differ in the amount of biofilm formation, virulence in vivo and transcriptional profiles of genes analyzed that can change factors associated with colonization, proliferation and survival of C. albicans at different niches. SAP5 and HWP1 were the genes more expressed in the formation of biofilm in vitro.

  4. Expression of the Salmonella virulence plasmid gene spvB in cultured macrophages and nonphagocytic cells.

    PubMed Central

    Fierer, J; Eckmann, L; Fang, F; Pfeifer, C; Finlay, B B; Guiney, D

    1993-01-01

    Certain serotypes of salmonellae carry virulence plasmids that greatly enhance the pathogenicity of these bacteria in experimentally infected mice. This phenotype is largely attributable to the 8-kb spv regulon. However, spv genes are not expressed while bacteria grow in vitro. We now show that spvB, which is required for virulence, is expressed rapidly after Salmonella dublin is ingested by cultured J774 and murine peritoneal macrophages and that expression is not affected by the alkalinization of intracellular vesicles. The level of induction of spvB is reduced when macrophages are pretreated with gamma interferon. spvB is also expressed in human and canine epithelial cell lines and a human hepatoma cell line. In all cases, spvB expression is dependent on the spvR gene, just as it is in stationary-phase cultures in vitro. These data suggest that spv virulence genes are expressed by intracellular salmonellae in vivo in response to a signal that is common to the intracellular compartments of cells that are invaded by salmonellae. PMID:8225598

  5. 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

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

    PubMed

    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.

  7. The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon.

    PubMed

    Fernández de Marco, María del Mar; Alejo, Alí; Hudson, Paul; Damon, Inger K; Alcami, Antonio

    2010-05-01

    Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.

  8. Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

    PubMed

    Kasper, Stephen H; Bonocora, Richard P; Wade, Joseph T; Musah, Rabi Ann; Cady, Nathaniel C

    2016-04-15

    The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy.

  9. Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum.

    PubMed

    Smulian, A George; Gibbons, Reta S; Demland, Jeffery A; Spaulding, Deborah T; Deepe, George S

    2007-11-01

    The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

  10. Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation

    PubMed Central

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

    2014-01-01

    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. PMID:25136970

  11. Deregulation of Listeria monocytogenes virulence gene expression by two distinct and semi-independent pathways.

    PubMed

    Milenbachs Lukowiak, Andrea; Mueller, Kimberly J; Freitag, Nancy E; Youngman, Philip

    2004-02-01

    Expression of the major virulence cluster in Listeria monocytogenes is positively regulated by the transcription factor PrfA and is influenced by several environmental factors, including the presence of readily metabolized carbohydrates such as cellobiose and glucose. Although little is understood about the mechanisms through which environmental factors influence expression of the PrfA regulon, evidence for structural and functional similarities of PrfA to the CRP-FNR family of regulatory proteins suggests the possibility that PrfA activity could be modulated by a small molecule ligand. The identity of components of the PrfA-associated regulatory pathway was sought through the isolation of mutants that exhibit high levels of PrfA-controlled gene expression in the presence of cellobiose or glucose. Here are described the properties and preliminary genetic analysis in two different genetic loci, gcr and csr, both unlinked by general transduction to the major virulence cluster. A mutation in gcr deregulates the expression of PrfA-controlled genes in the presence of several repressing sugars and other environmental conditions, a phenotype similar to that of a G145S substitution in PrfA itself. A mutation in the csr locus, within csrA, results in a cellobiose-specific defect in virulence gene regulation. Gene products encoded by the csr locus share homology with proteins involved in the sensing and transport of beta-glucosides in other bacteria. Mutations in both gcr and csr are required for full relief of cellobiose-mediated repression of the PrfA regulon. These results suggest the existence of two semi-independent pathways for cellobiose-mediated repression and further reconcile conflicting reports in previous literature concerning the repressive effects of carbohydrates on virulence gene expression in L. monocytogenes.

  12. The FTF gene family regulates virulence and expression of SIX effectors in Fusarium oxysporum.

    PubMed

    Niño-Sánchez, Jonathan; Casado-Del Castillo, Virginia; Tello, Vega; De Vega-Bartol, José J; Ramos, Brisa; Sukno, Serenella A; Díaz Mínguez, José María

    2016-09-01

    The FTF (Fusarium transcription factor) gene family comprises a single copy gene, FTF2, which is present in all the filamentous ascomycetes analysed, and several copies of a close relative, FTF1, which is exclusive to Fusarium oxysporum. An RNA-mediated gene silencing system was developed to target mRNA produced by all the FTF genes, and tested in two formae speciales: F. oxysporum f. sp. phaseoli (whose host is common bean) and F. oxysporum f. sp. lycopersici (whose host is tomato). Quantification of the mRNA levels showed knockdown of FTF1 and FTF2 in randomly isolated transformants of both formae speciales. The attenuation of FTF expression resulted in a marked reduction in virulence, a reduced expression of several SIX (Secreted In Xylem) genes, the best studied family of effectors in F. oxysporum, and lower levels of SGE1 (Six Gene Expression 1) mRNA, the presumptive regulator of SIX expression. Moreover, the knockdown mutants showed a pattern of colonization of the host plant similar to that displayed by strains devoid of FTF1 copies (weakly virulent strains). Gene knockout of FTF2 also resulted in a reduction in virulence, but to a lesser extent. These results demonstrate the role of the FTF gene expansion, mostly the FTF1 paralogues, as a regulator of virulence in F. oxysporum and suggest that the control of effector expression is the mechanism involved. © 2016 The Authors Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd.

  13. Differences in virulence gene expression between atypical enteropathogenic Escherichia coli strains isolated from diarrheic and healthy ruminants

    PubMed Central

    Horcajo, Pilar; Domínguez-Bernal, Gustavo; Carrión, Javier; De La Fuente, Ricardo; Ruiz-Santa-Quiteria, José A.; Orden, José A.

    2013-01-01

    Differences in the pathogenicity of atypical enteropathogenic Escherichia coli (EPEC) strains may be due, at least partially, to different expression patterns of some virulence genes. To investigate this hypothesis, the virulence gene expression patterns of 6 atypical EPEC strains isolated from healthy and diarrheic ruminants were compared using quantitative real-time reverse transcription polymerase chain reaction after growing the bacteria in culture medium alone or after binding it to HeLa epithelial cells. Some virulence genes in strains from diarrheic animals were upregulated relative to their expression in strains from healthy animals. When bacteria were cultured in the presence of HeLa cells, the ehxA and efa1/lifA genes, previously associated with the production of diarrhea, were expressed at higher levels in strains from diarrheic animals than in strains from healthy animals. Thus, the expression levels of some virulence genes may help determine which atypical EPEC strains cause diarrhea in ruminants. PMID:24082409

  14. Differences in virulence gene expression between atypical enteropathogenic Escherichia coli strains isolated from diarrheic and healthy ruminants.

    PubMed

    Horcajo, Pilar; Domínguez-Bernal, Gustavo; Carrión, Javier; De La Fuente, Ricardo; Ruiz-Santa-Quiteria, José A; Orden, José A

    2013-04-01

    Differences in the pathogenicity of atypical enteropathogenic Escherichia coli (EPEC) strains may be due, at least partially, to different expression patterns of some virulence genes. To investigate this hypothesis, the virulence gene expression patterns of 6 atypical EPEC strains isolated from healthy and diarrheic ruminants were compared using quantitative real-time reverse transcription polymerase chain reaction after growing the bacteria in culture medium alone or after binding it to HeLa epithelial cells. Some virulence genes in strains from diarrheic animals were upregulated relative to their expression in strains from healthy animals. When bacteria were cultured in the presence of HeLa cells, the ehxA and efa1/lifA genes, previously associated with the production of diarrhea, were expressed at higher levels in strains from diarrheic animals than in strains from healthy animals. Thus, the expression levels of some virulence genes may help determine which atypical EPEC strains cause diarrhea in ruminants.

  15. Siderophore Biosynthesis Governs the Virulence of Uropathogenic Escherichia coli by Coordinately Modulating the Differential Metabolism.

    PubMed

    Su, Qiao; Guan, Tianbing; He, Yan; Lv, Haitao

    2016-04-01

    Urinary tract infections impose substantial health burdens on women worldwide. Urinary tract infections often incur a high risk of recurrence and antibiotic resistance, and uropathogenic E. coli accounts for approximately 80% of clinically acquired cases. The diagnosis of, treatment of, and drug development for urinary tract infections remain substantial challenges due to the complex pathogenesis of this condition. The clinically isolated UPEC 83972 strain was found to produce four siderophores: yersiniabactin, aerobactin, salmochelin, and enterobactin. The biosyntheses of some of these siderophores implies that the virulence of UPEC is mediated via the targeting of primary metabolism. However, the differential modulatory roles of siderophore biosyntheses on the differential metabolomes of UPEC and non-UPEC strains remain incompletely understood. In the present study, we sought to investigate how the differential metabolomes can be used to distinguish UPEC from non-UPEC strains and to determine the associated regulatory roles of siderophore biosynthesis. Our results are the first to demonstrate that the identified differential metabolomes strongly differentiated UPEC from non-UPEC strains. Furthermore, we performed metabolome assays of mutants with different patterns of siderophore deletions; the data revealed that the mutations of all four siderophores exerted a stronger modulatory role on the differential metabolomes of the UPEC and non-UPEC strains relative to the mutation of any single siderophore and that this modulatory role primarily involved amino acid metabolism, oxidative phosphorylation in the carbon fixation pathway, and purine and pyrimidine metabolism. Surprisingly, the modulatory roles were strongly dependent on the type and number of mutated siderophores. Taken together, these results demonstrated that siderophore biosynthesis coordinately modulated the differential metabolomes and thus may indicate novel targets for virulence-based diagnosis

  16. Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection.

    PubMed

    Gröschel, Matthias I; Sayes, Fadel; Shin, Sung Jae; Frigui, Wafa; Pawlik, Alexandre; Orgeur, Mickael; Canetti, Robin; Honoré, Nadine; Simeone, Roxane; van der Werf, Tjip S; Bitter, Wilbert; Cho, Sang-Nae; Majlessi, Laleh; Brosch, Roland

    2017-03-14

    Recent insights into the mechanisms by which Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is recognized by cytosolic nucleotide sensors have opened new avenues for rational vaccine design. The only licensed anti-tuberculosis vaccine, Mycobacterium bovis BCG, provides limited protection. A feature of BCG is the partial deletion of the ESX-1 type VII secretion system, which governs phagosomal rupture and cytosolic pattern recognition, key intracellular phenotypes linked to increased immune signaling. Here, by heterologously expressing the esx-1 region of Mycobacterium marinum in BCG, we engineered a low-virulence, ESX-1-proficient, recombinant BCG (BCG::ESX-1(Mmar)) that induces the cGas/STING/TBK1/IRF-3/type I interferon axis and enhances AIM2 and NLRP3 inflammasome activity, resulting in both higher proportions of CD8(+) T cell effectors against mycobacterial antigens shared with BCG and polyfunctional CD4(+) Th1 cells specific to ESX-1 antigens. Importantly, independent mouse vaccination models show that BCG::ESX-1(Mmar) confers superior protection relative to parental BCG against challenges with highly virulent M. tuberculosis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. MicroRNA expression and virulence in pandemic influenza virus-infected mice.

    PubMed

    Li, Yu; Chan, Eric Y; Li, Jiangning; Ni, Chester; Peng, Xinxia; Rosenzweig, Elizabeth; Tumpey, Terrence M; Katze, Michael G

    2010-03-01

    The worst known H1N1 influenza pandemic in history resulted in more than 20 million deaths in 1918 and 1919. Although the underlying mechanism causing the extreme virulence of the 1918 influenza virus is still obscure, our previous functional genomics analyses revealed a correlation between the lethality of the reconstructed 1918 influenza virus (r1918) in mice and a unique gene expression pattern associated with severe immune responses in the lungs. Lately, microRNAs have emerged as a class of crucial regulators for gene expression. To determine whether differential expression of cellular microRNAs plays a role in the host response to r1918 infection, we compared the lung cellular "microRNAome" of mice infected by r1918 virus with that of mice infected by a nonlethal seasonal influenza virus, A/Texas/36/91. We found that a group of microRNAs, including miR-200a and miR-223, were differentially expressed in response to influenza virus infection and that r1918 and A/Texas/36/91 infection induced distinct microRNA expression profiles. Moreover, we observed significant enrichment in the number of predicted cellular target mRNAs whose expression was inversely correlated with the expression of these microRNAs. Intriguingly, gene ontology analysis revealed that many of these mRNAs play roles in immune response and cell death pathways, which are known to be associated with the extreme virulence of r1918. This is the first demonstration that cellular gene expression patterns in influenza virus-infected mice may be attributed in part to microRNA regulation and that such regulation may be a contributing factor to the extreme virulence of the r1918.

  18. Human Serum Promotes Candida albicans Biofilm Growth and Virulence Gene Expression on Silicone Biomaterial

    PubMed Central

    Samaranayake, Yuthika Hemamala; Cheung, Becky P. K.; Yau, Joyce Y. Y.; Yeung, Shadow K. W.; Samaranayake, Lakshman P.

    2013-01-01

    Objectives Systemic candidal infections are a common problem in hospitalized patients due to central venous catheters fabricated using silicone biomaterial (SB). We therefore evaluated the effect of human serum on C. albicans biofilm morphology, growth, and the expression of virulence-related genes on SB in vitro. Methods We cultivated C. albicans SC5314 (wild-type strain, WT) and its derivative HLC54 (hyphal mutant, HM) for 48 h in various conditions, including the presence or absence of SB discs, and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points by a tetrazolium salt reduction assay and by scanning electron microscopy. We also analyzed by RT-PCR its expression of the virulence-related genes ALS3, HWP1, EAP1, ECE1, SAP1 - SAP10, PLB1, PLB2, PLC and PLD. Results At each time point, planktonic cells of WT strain cultured in yeast nitrogen base displayed a much higher expression of EAP1 and HWP1, and a moderately higher ALS3 expression, than HM cells. In planktonic cells, expression of the ten SAP genes was higher in the WT strain initially, but were highly expressed in the HM strain by 48 h. Biofilm growth of both strains on SB was promoted in the presence of human serum than in its absence. Significant upregulation of ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6 - SAP10, PLB1, PLB2 and PLC was observed for WT biofilms grown on serum-treated SB discs for at least one time point, compared with biofilms on serum-free SB discs. Conclusions Human serum stimulates C. albicans biofilm growth on SB discs and upregulates the expression of virulence genes, particularly adhesion genes ALS3 and HWP1, and hydrolase-encoding genes SAP, PLB1 and PLB2. This response is likely to promote the colonization of this versatile pathogen within the human host. PMID:23704884

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

    PubMed

    Sakai, Fuminori; Talekar, Sharmila J; Lanata, Claudio F; Grijalva, Carlos G; 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 ≥10(4) 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.

  20. 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

  1. 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.

  2. 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.

  3. Leishmania promotes its own virulence by inducing expression of the host immune inhibitory ligand CD200

    PubMed Central

    Cortez, Mauro; Huynh, Chau; Fernandes, Maria Cecilia; Kennedy, Kathleen A.; Aderem, Alan; Andrews, Norma W.

    2011-01-01

    Summary Leishmania parasites infect macrophages, cells normally involved in innate defense against pathogens. L. amazonensis and L. major cause severe or mild disease, respectively, consistent with each parasite’s ability to survive within activated macrophages. The mechanisms underlying increased virulence of L. amazonensis are mostly unknown. We show that L. amazonensis promotes its own survival by inducing expression of CD200, an immunoregulatory molecule that inhibits macrophage activation. L. amazonensis does not form typical non-healing lesions in CD200−/− mice and cannot replicate in CD200−/− macrophages, an effect reversed by exogenous administration of soluble CD200-Fc. The less virulent L. major does not induce CD200 expression and forms small, self-healing lesions in both wild type and CD200−/− mice. Notably, CD200-Fc injection transforms the course of L. major infection to one resembling L. amazonensis, with large, non-healing lesions. CD200-dependent iNOS inhibition allows parasite growth in macrophages, identifying a mechanism for the increased virulence of L. amazonensis. PMID:21669395

  4. Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection.

    PubMed

    Holtsmark, Ingrid; Takle, Gunnhild W; Brurberg, May Bente

    2008-02-01

    The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.

  5. Probiotics Affect Virulence-Related Gene Expression in Escherichia coli O157:H7▿

    PubMed Central

    Medellin-Peña, Maira Jessica; Wang, Haifeng; Johnson, Roger; Anand, Sanjeev; Griffiths, Mansel W.

    2007-01-01

    The attachment of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) to host intestinal epithelial cells is essential for the development of hemorrhagic colitis and hemolytic-uremic syndrome in humans. Genes involved in attachment are carried within a pathogenicity island named the locus of enterocyte effacement (LEE), known to be directly activated by quorum sensing (QS). In the present study, we investigated autoinducer-2 (AI-2) production and the expression of several virulence-related genes in EHEC O157 grown in the absence and presence of a Lactobacillus acidophilus-secreted molecule(s). Transcription of important EHEC O157 virulence-related genes was studied by constructing promoter-reporter fusions and reverse transcriptase PCR. Shiga toxin (Stx) production was assayed by an enzyme immunoassay. When EHEC O157 was grown in the presence of chromatographically selected fractions of L. acidophilus La-5 cell-free spent medium, we observed a significant reduction of both extracellular AI-2 concentration and the expression of important virulence-related genes, although no significant difference in Stx production was observed. We show here that L. acidophilus La-5 secretes a molecule(s) that either acts as a QS signal inhibitor or directly interacts with bacterial transcriptional regulators, controlling the transcription of EHEC O157 genes involved in colonization. PMID:17496132

  6. Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence.

    PubMed

    Tamura, Y; Kijima-Tanaka, M; Aoki, A; Ogikubo, Y; Takahashi, T

    1995-03-01

    Clostridium chauvoei strain Okinawa produced spontaneous non-motile variants at an unusually high rate (approx. 10(-4) per generation) under normal conditions without mutagen. Revertants of non-motile variants were detected at a rate of approximately 10(-3). Biochemically, every variant corresponded well with the parental strain. By transmission electron microscopy, three of nine non-motile variants of strain Okinawa were found to be flagellate, while the other six were found to be aflagellate. These phenotypes were confirmed by Western blot analysis using monoclonal antibodies directed against the flagella of C. chauvoei. Moreover, the parental flagellate strain and non-motile flagellate variants were significantly more virulent in mice than non-motile, aflagellate variants. Our results demonstrated that phase variation in motility and flagellation occurs in C. chauvoei, and that the flagella are associated with the full expression of virulence.

  7. Molecular Mechanisms Governing IL-24 Gene Expression

    PubMed Central

    Sahoo, Anupama

    2012-01-01

    Interleukin-24 (IL-24) belongs to the IL-10 family of cytokines and is well known for its tumor suppressor activity. This cytokine is released by both immune and nonimmune cells and acts on non-hematopoietic tissues such as skin, lung and reproductive tissues. Apart from its ubiquitous tumor suppressor function, IL-24 is also known to be involved in the immunopathology of autoimmune diseases like psoriasis and rheumatoid arthritis. Although the cellular sources and functions of IL-24 are being increasingly investigated, the molecular mechanisms of IL-24 gene expression at the levels of signal transduction, epigenetics and transcription factor binding are still unclear. Understanding the specific molecular events that regulate the production of IL-24 will help to answer the remaining questions that are important for the design of new strategies of immune intervention involving IL-24. Herein, we briefly review the signaling pathways and transcription factors that facilitate, induce, or repress production of this cytokine along with the cellular sources and functions of IL-24. PMID:22536164

  8. Quorum sensing and the regulation of virulence gene expression in pathogenic bacteria.

    PubMed

    Winzer, K; Williams, P

    2001-05-01

    For many pathogens, the outcome of the interaction between host and bacterium is strongly affected by the bacterial population size. Coupling the production of virulence factors with cell population density ensures that the mammalian host lacks sufficient time to mount an effective defence against consolidated attack. Such a strategy depends on the ability of an individual bacterial cell to sense other members of the same species and in response, differentially express specific sets of genes. Such cell-cell communication is called "quorum sensing" and involves the direct or indirect activation of a response regulator by a small diffusible signal molecule. A number of chemically distinct quorum-sensing signal molecules have been described including the N-acyl-L-homoserine lactones (AHLs) in Gram-negative bacteria and post-translationally modified peptides in Gram-positive bacteria. For example, the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus employ AHLs and peptides, respectively, to control the expression of multiple virulence genes in concert with cell population density. Apart from their role in signal transduction, certain quorum-sensing signal molecules, notably N-(3-oxododecanoyl)homoserine lactone, possess intrinsic pharmacological and immunomodulatory activities such that they may function as virulence determinants per se. While quorum-sensing signal molecules have been detected in tissues in experimental animal model and human infections, the mutation of genes involved in either quorum-sensing signal generation or signal transduction frequently results in the attenuation of virulence. Thus, interference with quorum sensing represents a promising strategy for the therapeutic or prophylactic control of infection.

  9. Emodin affects biofilm formation and expression of virulence factors in Streptococcus suis ATCC700794.

    PubMed

    Yang, Yan-Bei; Wang, Shuai; Wang, Chang; Huang, Quan-Yong; Bai, Jing-Wen; Chen, Jian-Qing; Chen, Xue-Ying; Li, Yan-Hua

    2015-12-01

    Streptococcus suis (S. suis) is a swine pathogen and also a zoonotic agent. In this study, the effects of subinhibitory concentrations (sub-MICs) of emodin on biofilm formation by S. suis ATCC700794 were evaluated. As quantified by crystal violet staining, biofilm formation by S. suis ATCC700794 was dose-dependently decreased after growth with 1/2 MIC, 1/4 MIC, or 1/8 MIC of emodin. By scanning electron microscopy, the structural architecture of the S. suis ATCC700794 biofilms was examined following growth in culture medium supplemented with 1/2 MIC, 1/4 MIC, 1/8 MIC, or 1/16 MIC of emodin. Scanning electron microscopy analysis revealed the potential effect of emodin on biofilm formation by S. suis ATCC700794. The expression of luxS gene and virulence genes in S. suis ATCC700794 was investigated by quantitative RT-PCR. It was found that sub-MICs of emodin significantly decreased the expression of gapdh, sly, fbps, ef, and luxS. However, it was found that sub-MICs of emodin significantly increased the expression of cps2J, mrp, and gdh. These findings showed that sub-MICs of emodin could cause the difference in the expression level of the virulence genes.

  10. The expression of virulence for a mixed-mode transmitted parasite in a diapausing host.

    PubMed

    Sheikh-Jabbari, Elham; Hall, Matthew D; Ben-Ami, Frida; Ebert, Dieter

    2014-07-01

    Many parasites survive harsh periods together with their hosts. Without the possibility of horizontal transmission during host diapause, parasite persistence depends entirely on host survival. We therefore hypothesize that a parasite should be avirulent during its host's diapausing stage. In contrast, the parasite may express higher virulence, i.e. parasite-induced fitness reduction of the host, during host life stages with good opportunities for horizontal transmission. Here we study the effects of a vertically and horizontally transmitted microsporidium parasite, Hamiltosporidium tvaerminnensis, on the quantity and survival of resting eggs of its host Daphnia magna. We find that the parasite did not affect egg volume, hatching success and time to hatching of the Daphnia's resting eggs, although it did strongly reduce the number of resting eggs produced by infected females, revealing high virulence during the non-diapause phase of the host's life cycle. These results also explain another aspect of this system - namely the strong decline in natural population prevalence across diapause. This decline is not caused by mortality in infected resting stages, as was previously hypothesized, but because infected female hosts produce lower rates of resting eggs. Together, these results help explain the epidemiological dynamics of a microsporidian disease and highlight the adaptive nature of life stage-dependent parasite virulence.

  11. 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.

  12. Redox pathway sensing bile salts activates virulence gene expression in Vibrio cholerae.

    PubMed

    Xue, Yuanyuan; Tu, Fei; Shi, Mengting; Wu, Chun-Qin; Ren, Guoping; Wang, Xiaojie; Fang, Weihuan; Song, Houhui; Yang, Menghua

    2016-12-01

    Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, has evolved signal transduction systems to control co-ordinately the expression of virulence determinants. It was previously shown that the presence of the bile salts glycocholate and taurocholate in the small intestine causes dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulphide bonds in the TcpP periplasmic domain. In this study, they further investigated the mechanism of how taurocholate affects V. cholerae virulence determinants. In vitro assay of TcpP oxidation by VcDsbA showed that VcDsbA induced TcpP dimerization in the presence of taurocholate. Taurocholate bound to VcDsbA with a KD of 40 ± 2.5 μM, and also bound other Dsb proteins, including EcDsbA, EcDsbC and VcDsbC. Taurocholate inhibited VcDsbA reductase activity without affecting VcDsbA secondary structure or thermostability. VcDsbA and its substrates were more extensively reduced in the presence of taurocholate, as compared with their redox state in the absence of taurocholate. The data presented here not only provide new insights into the mechanism by which bile salts induce V. cholerae virulence but also suggest a means by which to develop inhibitors against DsbA. © 2016 John Wiley & Sons Ltd.

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

    PubMed Central

    Rosso, Marie-Laure; Chauvaux, Sylvie; Dessein, Rodrigue; Laurans, Caroline; Frangeul, Lionel; Lacroix, Céline; Schiavo, Angèle; Dillies, Marie-Agnès; Foulon, Jeannine; Coppée, Jean-Yves; Médigue, Claudine; Carniel, Elisabeth; Simonet, Michel; Marceau, Michaël

    2008-01-01

    Background In man, infection by the Gram-negative enteropathogen Yersinia pseudotuberculosis is usually limited to the terminal ileum. However, in immunocompromised patients, the microorganism may disseminate from the digestive tract and thus cause a systemic infection with septicemia. Results To gain insight into the metabolic pathways and virulence factors expressed by the bacterium at the blood stage of pseudotuberculosis, we compared the overall gene transcription patterns (the transcriptome) of bacterial cells cultured in either human plasma or Luria-Bertani medium. The most marked plasma-triggered metabolic consequence in Y. pseudotuberculosis was the switch to high glucose consumption, which is reminiscent of the acetogenic pathway (known as "glucose overflow") in Escherichia coli. However, upregulation of the glyoxylate shunt enzymes suggests that (in contrast to E. coli) acetate may be further metabolized in Y. pseudotuberculosis. Our data also indicate that the bloodstream environment can regulate major virulence genes (positively or negatively); the yadA adhesin gene and most of the transcriptional units of the pYV-encoded type III secretion apparatus were found to be upregulated, whereas transcription of the pH6 antigen locus was strongly repressed. Conclusion Our results suggest that plasma growth of Y. pseudotuberculosis is responsible for major transcriptional regulatory events and prompts key metabolic reorientations within the bacterium, which may in turn have an impact on virulence. PMID:19055764

  14. The relative impact of bacterial virulence and host genetic background on cytokine expression during Mycobacterium avium infection of mice.

    PubMed Central

    Castro, A G; Minóprio, P; Appelberg, R

    1995-01-01

    Resistance to Mycobacterium avium depends on both genetically encoded macrophage functions and acquired T-cell immunity. Cytokines may play a role in either type of resistance. We studied the expression of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) in naturally susceptible BALB/c (Bcgs) and naturally resistant C.D2 (Bcgr) congenic mice infected with two strains of M. avium (one highly virulent and another of low virulence). We observed that cytokine expression patterns correlated better with the virulence of the micro-organism than with the genetic background of the host. The control of the infection by the low virulence strain in either mouse strain was associated with an increased expression of IFN-gamma and IL-2. Only Bcgs mice infected with a virulent strain of M. avium were unable to restrict bacterial growth. An increased expression of IL-4, early during infection, was detected in the course of the latter infection but played no role in determining the susceptibility to infection. Neutralization of IFN-gamma or IL-2 with specific monoclonal antibodies led to an exacerbation of the infection in Bcgr mice by the two strains of M. avium and in Bcgs mice infected with the low virulence strain of M. avium. PMID:7558149

  15. Iron is a signal for Stenotrophomonas maltophilia biofilm formation, oxidative stress response, OMPs expression, and virulence

    PubMed Central

    García, Carlos A.; Alcaraz, Eliana S.; Franco, Mirta A.; Passerini de Rossi, Beatriz N.

    2015-01-01

    Stenotrophomonas maltophilia is an emerging nosocomial pathogen. In many bacteria iron availability regulates, through the Fur system, not only iron homeostasis but also virulence. The aim of this work was to assess the role of iron on S. maltophilia biofilm formation, EPS production, oxidative stress response, OMPs regulation, quorum sensing (QS), and virulence. Studies were done on K279a and its isogenic fur mutant F60 cultured in the presence or absence of dipyridyl. This is the first report of spontaneous fur mutants obtained in S. maltophilia. F60 produced higher amounts of biofilms than K279a and CLSM analysis demonstrated improved adherence and biofilm organization. Under iron restricted conditions, K279a produced biofilms with more biomass and enhanced thickness. In addition, F60 produced higher amounts of EPS than K279a but with a similar composition, as revealed by ATR-FTIR spectroscopy. With respect to the oxidative stress response, MnSOD was the only SOD isoenzyme detected in K279a. F60 presented higher SOD activity than the wt strain in planktonic and biofilm cultures, and iron deprivation increased K279a SOD activity. Under iron starvation, SDS-PAGE profile from K279a presented two iron-repressed proteins. Mass spectrometry analysis revealed homology with FepA and another putative TonB-dependent siderophore receptor of K279a. In silico analysis allowed the detection of potential Fur boxes in the respective coding genes. K279a encodes the QS diffusible signal factor (DSF). Under iron restriction K279a produced higher amounts of DSF than under iron rich condition. Finally, F60 was more virulent than K279a in the Galleria mellonella killing assay. These results put in evidence that iron levels regulate, likely through the Fur system, S. maltophilia biofilm formation, oxidative stress response, OMPs expression, DSF production and virulence. PMID:26388863

  16. Iron is a signal for Stenotrophomonas maltophilia biofilm formation, oxidative stress response, OMPs expression, and virulence.

    PubMed

    García, Carlos A; Alcaraz, Eliana S; Franco, Mirta A; Passerini de Rossi, Beatriz N

    2015-01-01

    Stenotrophomonas maltophilia is an emerging nosocomial pathogen. In many bacteria iron availability regulates, through the Fur system, not only iron homeostasis but also virulence. The aim of this work was to assess the role of iron on S. maltophilia biofilm formation, EPS production, oxidative stress response, OMPs regulation, quorum sensing (QS), and virulence. Studies were done on K279a and its isogenic fur mutant F60 cultured in the presence or absence of dipyridyl. This is the first report of spontaneous fur mutants obtained in S. maltophilia. F60 produced higher amounts of biofilms than K279a and CLSM analysis demonstrated improved adherence and biofilm organization. Under iron restricted conditions, K279a produced biofilms with more biomass and enhanced thickness. In addition, F60 produced higher amounts of EPS than K279a but with a similar composition, as revealed by ATR-FTIR spectroscopy. With respect to the oxidative stress response, MnSOD was the only SOD isoenzyme detected in K279a. F60 presented higher SOD activity than the wt strain in planktonic and biofilm cultures, and iron deprivation increased K279a SOD activity. Under iron starvation, SDS-PAGE profile from K279a presented two iron-repressed proteins. Mass spectrometry analysis revealed homology with FepA and another putative TonB-dependent siderophore receptor of K279a. In silico analysis allowed the detection of potential Fur boxes in the respective coding genes. K279a encodes the QS diffusible signal factor (DSF). Under iron restriction K279a produced higher amounts of DSF than under iron rich condition. Finally, F60 was more virulent than K279a in the Galleria mellonella killing assay. These results put in evidence that iron levels regulate, likely through the Fur system, S. maltophilia biofilm formation, oxidative stress response, OMPs expression, DSF production and virulence.

  17. Verticillium dahliae transcription factor VdFTF1 regulates the expression of multiple secreted virulence factors and is required for full virulence in cotton.

    PubMed

    Zhang, Wen-Qi; Gui, Yue-Jing; Short, Dylan P G; Li, Ting-Gang; Zhang, Dan-Dan; Zhou, Lei; Liu, Chun; Bao, Yu-Ming; Subbarao, Krishna V; Chen, Jie-Yin; Dai, Xiao-Feng

    2017-05-18

    Fungal transcription factors (TFs) implicated in the regulation of virulence gene expression have been identified in a number of plant pathogens. In Verticillium dahliae, despite its agricultural importance, few regulators of transcription have been characterized. In this study, a T-DNA insertion mutant with significantly reduced virulence towards cotton was identified. The T-DNA was traced to VdFTF1, a gene encoding a TF containing a Fungal_trans domain. Transient expression in onion epidermal cells indicated that VdFTF1 is localized to the nucleus. The VdFTF1-deletion strains displayed normal vegetative growth, mycelial pigmentation and conidial morphology, but exhibited significantly reduced virulence on cotton, suggesting that VdFTF1 is required exclusively for pathogenesis. Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes, 233 of which were associated with catalytic processes. These genes encoded 69 potentially secreted proteins, 43 of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton. Targeted gene deletion of one VdFTF1-regulated gene resulted in significantly impaired vascular colonization, as measured by quantitative polymerase chain reaction, as well as aggressiveness and symptom severity in cotton. In conclusion, VdFTF1, which encodes a TF containing a Fungal_trans domain, regulates the gene expression of plant cell wall degradation enzymes in V. dahliae, which are required for full virulence on cotton. © 2017 BSPP AND JOHN WILEY & SONS LTD.

  18. Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence

    PubMed Central

    Bauer, Beth A.; Stevens, Marla K.; Hansen, Eric J.

    1998-01-01

    The lipooligosaccharide (LOS) present in the outer membrane of Haemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA (isn) gene of Haemophilus influenzae. In H. influenzae type b, inactivation of the gmhA gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396–402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficient Escherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a cat cartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. The H. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When the H. ducreyi gmhA gene was present in trans in gmhA mutant 35000.252, expression of the gmhA gene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen. PMID:9712780

  19. In Vivo Expression Technology Identifies a Novel Virulence Factor Critical for Borrelia burgdorferi Persistence in Mice

    PubMed Central

    Ellis, Tisha Choudhury; Jain, Sunny; Linowski, Angelika K.; Rike, Kelli; Bestor, Aaron; Rosa, Patricia A.; Halpern, Micah; Kurhanewicz, Stephanie; Jewett, Mollie W.

    2013-01-01

    Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice. PMID:24009501

  20. Excess labile carbon promotes the expression of virulence factors in coral reef bacterioplankton.

    PubMed

    Cárdenas, Anny; Neave, Matthew J; Haroon, Mohamed Fauzi; Pogoreutz, Claudia; Rädecker, Nils; Wild, Christian; Gärdes, Astrid; Voolstra, Christian R

    2017-09-12

    Coastal pollution and algal cover are increasing on many coral reefs, resulting in higher dissolved organic carbon (DOC) concentrations. High DOC concentrations strongly affect microbial activity in reef waters and select for copiotrophic, often potentially virulent microbial populations. High DOC concentrations on coral reefs are also hypothesized to be a determinant for switching microbial lifestyles from commensal to pathogenic, thereby contributing to coral reef degradation, but evidence is missing. In this study, we conducted ex situ incubations to assess gene expression of planktonic microbial populations under elevated concentrations of naturally abundant monosaccharides (glucose, galactose, mannose, and xylose) in algal exudates and sewage inflows. We assembled 27 near-complete (>70%) microbial genomes through metagenomic sequencing and determined associated expression patterns through metatranscriptomic sequencing. Differential gene expression analysis revealed a shift in the central carbohydrate metabolism and the induction of metalloproteases, siderophores, and toxins in Alteromonas, Erythrobacter, Oceanicola, and Alcanivorax populations. Sugar-specific induction of virulence factors suggests a mechanistic link for the switch from a commensal to a pathogenic lifestyle, particularly relevant during increased algal cover and human-derived pollution on coral reefs. Although an explicit test remains to be performed, our data support the hypothesis that increased availability of specific sugars changes net microbial community activity in ways that increase the emergence and abundance of opportunistic pathogens, potentially contributing to coral reef degradation.The ISME Journal advance online publication, 12 September 2017; doi:10.1038/ismej.2017.142.

  1. Staphylococcus aureus Virulence Expression Is Impaired by Lactococcus lactis in Mixed Cultures▿ †

    PubMed Central

    Even, Sergine; Charlier, Cathy; Nouaille, Sébastien; Ben Zakour, Nouri L.; Cretenet, Marina; Cousin, Fabien J.; Gautier, Michel; Cocaign-Bousquet, Muriel; Loubière, Pascal; Le Loir, Yves

    2009-01-01

    Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen. PMID:19429556

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

    PubMed Central

    Mansson, Maria; Nielsen, Anita; Kjærulff, Louise; Gotfredsen, Charlotte H.; Wietz, Matthias; Ingmer, Hanne; Gram, Lone; Larsen, Thomas O.

    2011-01-01

    During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium. PMID:22363239

  3. The Virulence Regulator Rns Activates the Expression of CS14 Pili

    PubMed Central

    Bodero, Maria Del Rocio; Munson, George Patrick

    2016-01-01

    Although many viral and bacterial pathogens cause diarrhea, enterotoxigenic E. coli (ETEC) is one of the most frequently encountered in impoverished regions where it is estimated to kill between 300,000 and 700,000 children and infants annually. Critical ETEC virulence factors include pili which mediate the attachment of the pathogen to receptors in the intestinal lumen. In this study we show that the ETEC virulence regulator Rns positively regulates the expression of CS14 pili. Three Rns binding sites were identified upstream of the CS14 pilus promoter centered at −34.5, −80.5, and −155.5 relative to the Rns-dependent transcription start site. Mutagenesis of the promoter proximal site significantly decreased expression from the CS14 promoter. In contrast, the contribution of Rns bound at the promoter distal site was negligible and largely masked by occupancy of the promoter proximal site. Unexpectedly, Rns bound at the site centered at −80.5 had a slight but statistically significant inhibitory effect upon the pilin promoter. Nevertheless, this weak inhibitory effect was not sufficient to overcome the substantial promoter activation from Rns bound to the promoter proximal site. Thus, CS14 pili belong to a group of pili that depend upon Rns for their expression. PMID:27941642

  4. Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

    PubMed Central

    Green, Jeffrey; Rolfe, Matthew D; Smith, Laura J

    2014-01-01

    Molecular oxygen (O2) and nitric oxide (NO) are diatomic gases that play major roles in infection. The host innate immune system generates reactive oxygen species and NO as bacteriocidal agents and both require O2 for their production. Furthermore, the ability to adapt to changes in O2 availability is crucial for many bacterial pathogens, as many niches within a host are hypoxic. Pathogenic bacteria have evolved transcriptional regulatory systems that perceive these gases and respond by reprogramming gene expression. Direct sensors possess iron-containing co-factors (iron–sulfur clusters, mononuclear iron, heme) or reactive cysteine thiols that react with O2 and/or NO. Indirect sensors perceive the physiological effects of O2 starvation. Thus, O2 and NO act as environmental cues that trigger the coordinated expression of virulence genes and metabolic adaptations necessary for survival within a host. Here, the mechanisms of signal perception by key O2- and NO-responsive bacterial transcription factors and the effects on virulence gene expression are reviewed, followed by consideration of these aspects of gene regulation in two major pathogens, Staphylococcus aureus and Mycobacterium tuberculosis. PMID:25603427

  5. Identification of Genes Preferentially Expressed by Highly Virulent Piscine Streptococcus agalactiae upon Interaction with Macrophages

    PubMed Central

    Guo, Chang-Ming; Chen, Rong-Rong; Kalhoro, Dildar Hussain; Wang, Zhao-Fei; Liu, Guang-Jin; Lu, Cheng-Ping; Liu, Yong-Jie

    2014-01-01

    Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood–brain barrier (BBB). The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB. PMID:24498419

  6. 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

  7. Evaluation of Approaches to Monitor Staphylococcus aureus Virulence Factor Expression during Human Disease

    PubMed Central

    Rozemeijer, Wouter; Fink, Pamela; Rojas, Eduardo; Jones, C. Hal; Pavliakova, Danka; Giardina, Peter; Murphy, Ellen; Liberator, Paul; Jiang, Qin; Girgenti, Douglas; Peters, Remco P. H.; Savelkoul, Paul H. M.; Jansen, Kathrin U.; Anderson, Annaliesa S.; Kluytmans, Jan

    2015-01-01

    Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine. PMID:25719409

  8. Host response and bacterial virulence factor expression in Pseudomonas aeruginosa and Streptococcus pneumoniae corneal ulcers.

    PubMed

    Karthikeyan, Rajapandian SivaGanesa; Priya, Jeganathan Lakshmi; Leal, Sixto M; Toska, Jonida; Rietsch, Arne; Prajna, Venkatesh; Pearlman, Eric; Lalitha, Prajna

    2013-01-01

    P. aeruginosa and S. pneumoniae are major bacterial causes of corneal ulcers in industrialized and in developing countries. The current study examined host innate immune responses at the site of infection, and also expression of bacterial virulence factors in clinical isolates from patients in south India. Corneal ulcer material was obtained from 49 patients with confirmed P. aeruginosa and 27 patients with S. pneumoniae, and gene expression of Toll Like Receptors (TLR), cytokines and inflammasome proteins was measured by quantitative PCR. Expression of P. aeruginosa type III secretion exotoxins and S. pneumoniae pneumolysin was detected by western blot analysis. We found that neutrophils comprised >90% cells in corneal ulcers, and that there was elevated expression of TLR2, TLR4, TLR5 and TLR9, the NLRP3 and NLRC4 inflammasomes and the ASC adaptor molecule. IL-1α IL-1β and IFN-γ expression was also elevated; however, there was no significant difference in expression of any of these genes between corneal ulcers from P. aeruginosa and S. pneumoniae infected patients. We also show that 41/49 (84%) of P. aeruginosa clinical isolates expressed ExoS and ExoT, whereas 5/49 (10%) of isolates expressed ExoS, ExoT and ExoU with only 2/49 isolates expressing ExoT and ExoU. In contrast, all 27 S. pneumoniae clinical isolates produced pneumolysin. Taken together, these findings demonstrate that ExoS/T expressing P. aeruginosa and pneumolysin expressing S. pneumoniae predominate in bacterial keratitis. While P. aeruginosa strains expressing both ExoU and ExoS are usually rare, these strains actually outnumbered strains expressing only ExoU in the current study. Further, as neutrophils are the predominant cell type in these corneal ulcers, they are the likely source of cytokines and of the increased TLR and inflammasome expression.

  9. 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

  10. Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

    PubMed

    Freires, Irlan A; Avilés-Reyes, Alejandro; Kitten, Todd; Simpson-Haidaris, P J; Swartz, Michael; Knight, Peter A; Rosalen, Pedro L; Lemos, José A; Abranches, Jacqueline

    2017-01-02

    In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

  11. DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium▿

    PubMed Central

    Balbontín, Roberto; Rowley, Gary; Pucciarelli, M. Graciela; López-Garrido, Javier; Wormstone, Yvette; Lucchini, Sacha; García-del Portillo, Francisco; Hinton, Jay C. D.; Casadesús, Josep

    2006-01-01

    Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam− mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam− mutants in the mouse model may be caused by altered patterns of gene expression. PMID:16997949

  12. Decreased cruzipain and gp85/trans-sialidase family protein expression contributes to loss of Trypanosoma cruzi trypomastigote virulence.

    PubMed

    San Francisco, Juan; Barría, Iván; Gutiérrez, Bessy; Neira, Iván; Muñoz, Christian; Sagua, Hernán; Araya, Jorge E; Andrade, Juan Carlos; Zailberger, Anibal; Catalán, Alejandro; Remonsellez, Francisco; Vega, José Luis; González, Jorge

    2017-01-01

    Two cell lines derived from a single Trypanosoma cruzi clone by long-term passaging generated a highly virulent (C8C3hvir) and a low virulent (C8C3lvir) cell line. The C8C3hvir cell line was highly infective and lethal to Balb/c mice, and the C8C3lvir cell line was three- to five-fold less infective to mouse cardiomyocytes than C8C3hvir. The highly virulent T. cruzi cell line abundantly expressed the major cysteine proteinase cruzipain (Czp), complement regulatory protein (CRP) and trans-sialidase (TS), all of which are known to act as virulence factors in this parasite. The in vitro invasion capacity and in vivo Balb/c mouse infectiveness of the highly virulent strain was strongly reduced by pre-treatment with antisense oligonucleotides targeting TS or CRP or with E64d. Based on these results, we conclude that decreased levels of TS, CRP and Czp expression could contribute to loss of T. cruzi trypomastigote virulence.

  13. The Pseudomonas aeruginosa Vfr regulator controls global virulence factor expression through cyclic AMP-dependent and -independent mechanisms.

    PubMed

    Fuchs, Erin L; Brutinel, Evan D; Jones, Adriana K; Fulcher, Nanette B; Urbanowski, Mark L; Yahr, Timothy L; Wolfgang, Matthew C

    2010-07-01

    Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments.

  14. 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.

  15. Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

    PubMed Central

    Bojer, Martin S.; Zhao, Yu; Friberg, Cathrine; Ifrah, Dan; Glasser Heede, Nina; Larsen, Thomas O.; Frøkiær, Hanne; Frees, Dorte; Zhang, Lixin; Dai, Huanqin

    2016-01-01

    Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than viability. Previously we have shown that norlichexanthone, a small non-reduced tricyclic polyketide produced by fungi and lichens, reduces expression of hla encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum sensing system in S. aureus 8325–4. The aim of the present study was to further characterise the mode of action of norlichexanthone and its effect on biofilm formation. We find that norlichexanthone reduces expression of both hla and RNAIII also in strain USA300. Structurally, norlichexanthone resembles ω-hydroxyemodin that recently was shown to bind the agr two component response regulator, AgrA, which controls expression of RNAIII and the phenol soluble modulins responsible for human neutrophil killing. We show that norlichexanthone reduces S. aureus toxicity towards human neutrophils and interferes directly with AgrA binding to its DNA target. In contrast to ω-hydroxyemodin however, norlichexanthone reduces staphylococcal biofilm formation. Transcriptomic analysis revealed that genes regulated by the SaeRS two-component system are repressed by norlichexanthone when compared to untreated cells, an effect that was mitigated in strain Newman carrying a partially constitutive SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation. PMID:28005941

  16. Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae

    PubMed Central

    Häse, Claudia C.; Mekalanos, John J.

    1999-01-01

    The expression of several virulence factors of Vibrio cholerae is coordinately regulated by the ToxT molecule and the membrane proteins TcpP/H and ToxR/S, which are required for toxT transcription. To identify proteins that negatively affect toxT transcription, we screened transposon mutants of V. cholerae carrying a chromosomally integrated toxT∷lacZ reporter construct for darker blue colonies on media containing 5-bromo-4-chlor-3-indolyl β-d galactoside (X-gal). Two mutants had transposon insertions in a region homologous to the nqr gene cluster of Vibrio alginolyticus, encoding a sodium-translocating NADH–ubiquinone oxidoreductase (NQR). In V. alginolyticus, NQR is a respiration-linked Na+ extrusion pump generating a sodium motive force that can be used for solute import, ATP synthesis, and flagella rotation. Inhibition of NQR enzyme function in V. cholerae by the specific inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) resulted in elevated toxT∷lacZ activity. Increased toxT∷lacZ expression in an nqr mutant strain compared with the parental strain was observed when the TcpP/H molecules alone were strongly expressed, suggesting that the negative effect of the NQR complex on toxT transcription is mediated through TcpP/H. However, the ability of the TcpP/H proteins to activate the toxT∷lacZ reporter construct was greatly diminished in the presence of high NaCl concentrations in the growth medium. The flagellar motor of V. cholerae appears to be driven by a sodium motive force, and modulation of flagella rotation by inhibitory drugs, high media viscosity, or specific mutations resulted in increases of toxT∷lacZ expression. Thus, the regulation of the main virulence factors of V. cholerae appears to be modulated by endogenous and exogenous sodium levels in a complex way. PMID:10077658

  17. Metabolic genetic screens reveal multidimensional regulation of virulence gene expression in Listeria monocytogenes, and an aminopeptidase that is critical for PrfA protein activation.

    PubMed

    Friedman, Sivan; Linsky, Marika; Lobel, Lior; Rabinovich, Lev; Sigal, Nadejda; Herskovits, Anat A

    2017-04-10

    Listeria monocytogenes is an environmental saprophyte and intracellular bacterial pathogen. Upon invading mammalian cells, the bacterium senses abrupt changes in its metabolic environment, which are rapidly transduced to regulation of virulence gene expression. To explore the relationship between L. monocytogenes metabolism and virulence, we monitored virulence gene expression dynamics across a library of genetic mutants grown under two metabolic conditions known to activate the virulent state: charcoal-treated rich medium containing glucose 1-phosphate and minimal defined medium containing limiting concentrations of branched-chain amino acids (BCAAs). We identified over 100 distinct mutants that exhibit aberrant virulence gene expression profiles, the majority of which mapped to non-essential metabolic genes. Mutants displayed enhanced, decreased, early and late virulence gene expression profiles as well as persistent levels, demonstrating a high plasticity in virulence gene regulation. Among the mutants, one was noteworthy for its particularly low virulence gene expression level and mapped to an x-prolyl aminopeptidase (PepP). We show that this peptidase plays a role in posttranslational activation of the major virulence regulator, PrfA. Specifically, PepP mediates recruitment of PrfA to the cytoplasmic membrane, a step identified as critical for PrfA protein activation. This study establishes a novel step in the complex mechanism of PrfA activation, and further highlights the cross regulation of metabolism and virulence.

  18. Activation of CpxRA in Haemophilus ducreyi primarily inhibits the expression of its targets, including major virulence determinants.

    PubMed

    Gangaiah, Dharanesh; Zhang, Xinjun; Fortney, Kate R; Baker, Beth; Liu, Yunlong; Munson, Robert S; Spinola, Stanley M

    2013-08-01

    Haemophilus ducreyi causes chancroid, a genital ulcer disease that facilitates the transmission of human immunodeficiency virus type 1. In humans, H. ducreyi is surrounded by phagocytes and must adapt to a hostile environment to survive. To sense and respond to environmental cues, bacteria frequently use two-component signal transduction (2CST) systems. The only obvious 2CST system in H. ducreyi is CpxRA; CpxR is a response regulator, and CpxA is a sensor kinase. Previous studies by Hansen and coworkers showed that CpxR directly represses the expression of dsrA, the lspB-lspA2 operon, and the flp operon, which are required for virulence in humans. They further showed that CpxA functions predominantly as a phosphatase in vitro to maintain the expression of virulence determinants. Since a cpxA mutant is avirulent while a cpxR mutant is fully virulent in humans, CpxA also likely functions predominantly as a phosphatase in vivo. To better understand the role of H. ducreyi CpxRA in controlling virulence determinants, here we defined genes potentially regulated by CpxRA by using RNA-Seq. Activation of CpxR by deletion of cpxA repressed nearly 70% of its targets, including seven established virulence determinants. Inactivation of CpxR by deletion of cpxR differentially regulated few genes and increased the expression of one virulence determinant. We identified a CpxR binding motif that was enriched in downregulated but not upregulated targets. These data reinforce the hypothesis that CpxA phosphatase activity plays a critical role in controlling H. ducreyi virulence in vivo. Characterization of the downregulated genes may offer new insights into pathogenesis.

  19. Differences in cell morphometry, cell wall topography and gp70 expression correlate with the virulence of Sporothrix brasiliensis clinical isolates.

    PubMed

    Castro, Rafaela A; Kubitschek-Barreira, Paula H; Teixeira, Pedro A C; Sanches, Glenda F; Teixeira, Marcus M; Quintella, Leonardo P; Almeida, Sandro R; Costa, Rosane O; Camargo, Zoilo P; Felipe, Maria S S; de Souza, Wanderley; Lopes-Bezerra, Leila M

    2013-01-01

    Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.

  20. Allele-Dependent Differences in Quorum-Sensing Dynamics Result in Variant Expression of Virulence Genes in Staphylococcus aureus

    PubMed Central

    Geisinger, Edward; Chen, John

    2012-01-01

    Agr is an autoinducing, quorum-sensing system that functions in many Gram-positive species and is best characterized in the pathogen Staphylococcus aureus, in which it is a global regulator of virulence gene expression. Allelic variations in the agr genes have resulted in the emergence of four quorum-sensing specificity groups in S. aureus, which correlate with different strain pathotypes. The basis for these predilections is unclear but is hypothesized to involve the phenomenon of quorum-sensing interference between strains of different agr groups, which may drive S. aureus strain isolation and divergence. Whether properties intrinsic to each agr allele directly influence virulence phenotypes within S. aureus is unknown. In this study, we examined group-specific differences in agr autoinduction and virulence gene regulation by utilizing congenic strains, each harboring a unique S. aureus agr allele, enabling a dissection of agr locus-dependent versus genotype-dependent effects on quorum-sensing dynamics and virulence factor production. Employing a reporter fusion to the principal agr promoter, P3, we observed allele-dependent differences in the timing and magnitude of agr activation. These differences were mediated by polymorphisms within the agrBDCA genes and translated to significant variations in the expression of a key transcriptional regulator, Rot, and of several important exoproteins and surface factors involved in pathogenesis. This work uncovers the contribution of divergent quorum-sensing alleles to variant expression of virulence determinants within a bacterial species. PMID:22467783

  1. Differential expression pattern of Vago in bumblebee (Bombus terrestris), induced by virulent and avirulent virus infections.

    PubMed

    Niu, Jinzhi; Meeus, Ivan; Smagghe, Guy

    2016-09-29

    Viruses are one of the main drivers of the decline of domesticated and wild bees but the mechanisms of antiviral immunity in pollinators are poorly understood. Recent work has suggested that next to the small interfering RNA (siRNA) pathway other immune-related pathways play a role in the defense of the bee hosts against viral infection. In addition, Vago plays a role in the cross-talk between the innate immune pathways in Culex mosquito cells. Here we describe the Vago orthologue in bumblebees of Bombus terrestris, and investigated its role upon the infection of two different bee viruses, the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that BtVago was downregulated upon the infection of IAPV that killed all bumblebees, but not with SBPV where the workers survived the virus infection. Thus, for the first time, Vago/Vago-like expression appears to be associated with the virulence of virus and may act as a modulator of antiviral immunity.

  2. Surface expression of ferripyochelin-binding protein is required for virulence of Pseudomonas aeruginosa.

    PubMed Central

    Sokol, P A

    1987-01-01

    Pseudomonas aeruginosa mutants which do not express ferripyochelin-binding protein (FBP) on the cell surface have previously been isolated. These mutants were used to assess the role of FBP in virulence in an acute and a systemic animal infection model. In a mouse corneal infection model, the pathology of eyes infected with the mutant strains was significantly less than that of eyes infected with the parent strain. The mutants were also cleared more rapidly from the eye. In a burn infection model, the mortality rate in mice infected with mutant FBP-28 was much less than that of mice infected with the parent strain at an inoculum of 10(2) CFU. At higher inocula (10(4) CFU), the mortality rate was not significantly different but the survival time was dramatically longer with the mutant strain. Quantitative bacteriology of blood and tissue homogenates revealed that P. aeruginosa PAO could multiply in the skin and could also be cultured from the blood, livers, and spleens of infected mice. FBP-28 could only be cultured from the skin. Therefore, this mutant could colonize the skin but could not disseminate. These data indicate that functional, exposed FBP is required for virulence of PAO. PMID:3114141

  3. Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen.

    PubMed

    Zhang, Tao; Zhao, Yun-Long; Zhao, Jian-Hua; Wang, Sheng; Jin, Yun; Chen, Zhong-Qi; Fang, Yuan-Yuan; Hua, Chen-Lei; Ding, Shou-Wei; Guo, Hui-Shan

    2016-09-26

    Plant pathogenic fungi represent the largest group of disease-causing agents on crop plants, and are a constant and major threat to agriculture worldwide. Recent studies have shown that engineered production of RNA interference (RNAi)-inducing dsRNA in host plants can trigger specific fungal gene silencing and confer resistance to fungal pathogens(1-7). Although these findings illustrate efficient uptake of host RNAi triggers by pathogenic fungi, it is unknown whether or not such an uptake mechanism has been evolved for a natural biological function in fungus-host interactions. Here, we show that in response to infection with Verticillium dahliae (a vascular fungal pathogen responsible for devastating wilt diseases in many crops) cotton plants increase production of microRNA 166 (miR166) and miR159 and export both to the fungal hyphae for specific silencing. We found that two V. dahliae genes encoding a Ca(2+)-dependent cysteine protease (Clp-1) and an isotrichodermin C-15 hydroxylase (HiC-15), and targeted by miR166 and miR159, respectively, are both essential for fungal virulence. Notably, V. dahliae strains expressing either Clp-1 or HiC-15 rendered resistant to the respective miRNA exhibited drastically enhanced virulence in cotton plants. Together, our findings identify a novel defence strategy of host plants by exporting specific miRNAs to induce cross-kingdom gene silencing in pathogenic fungi and confer disease resistance.

  4. Differential expression pattern of Vago in bumblebee (Bombus terrestris), induced by virulent and avirulent virus infections

    PubMed Central

    Niu, Jinzhi; Meeus, Ivan; Smagghe, Guy

    2016-01-01

    Viruses are one of the main drivers of the decline of domesticated and wild bees but the mechanisms of antiviral immunity in pollinators are poorly understood. Recent work has suggested that next to the small interfering RNA (siRNA) pathway other immune-related pathways play a role in the defense of the bee hosts against viral infection. In addition, Vago plays a role in the cross-talk between the innate immune pathways in Culex mosquito cells. Here we describe the Vago orthologue in bumblebees of Bombus terrestris, and investigated its role upon the infection of two different bee viruses, the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that BtVago was downregulated upon the infection of IAPV that killed all bumblebees, but not with SBPV where the workers survived the virus infection. Thus, for the first time, Vago/Vago-like expression appears to be associated with the virulence of virus and may act as a modulator of antiviral immunity. PMID:27680717

  5. Subinhibitory concentrations of punicalagin reduces expression of virulence-related exoproteins by Staphylococcus aureus.

    PubMed

    Mun, Su-Hyun; Kong, Ryong; Seo, Yun-Soo; Zhou, Tian; Kang, Ok-Hwa; Shin, Dong-Won; Kwon, Dong-Yeul

    2016-11-01

    Staphylococcus aureus produces a number of virulence factors. The major virulence factors exhibited by S aureus include various antigens, enzymes, cytotoxins and exotoxins (e.g. hemolysins, enterotoxins and toxic shock syndrome toxin). In this report, we show the influence of punicalagin on the secretion of exoprotein from S aureus by western blotting, tumor necrosis factor (TNF) release assay and quantitative RT-PCR. When added to S aureus cultures at an OD600 of 0.9, graded subinhibitory concentrations of punicalagin reduced the production of α-toxin, SEA and SEB in methicillin-resistant Staphylococcus aureus in a dose-dependent manner. Consistently, punicalagin reduced TNF-inducing activity by S aureus culture supernatants. Here, the transcriptional level of agr (accessory gene regulator) in S aureus was inhibited by punicalagin, suggesting that the reduced transcription may affect the secretion of exotoxins. These findings suggest that the expression of α-toxin and enterotoxins in S aureus is sensitive to the action of punicalagin, which may be an advantageous candidate in the treatment of toxigenic staphylococcal disease.

  6. Expression of a Toll Signaling Regulator Serpin in a Mycoinsecticide for Increased Virulence

    PubMed Central

    Yang, Linzhi; Keyhani, Nemat O.; Tang, Guirong; Tian, Chuang; Lu, Ruipeng; Wang, Xin; Pei, Yan

    2014-01-01

    Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD50) for the wild-type parent, the LD50 of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ∼3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ∼24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin > laminarin > lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassiana-derived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy. PMID:24837378

  7. Relationship between oviposition, virulence gene expression and parasitism success in Cotesia typhae nov. sp. parasitoid strains.

    PubMed

    Benoist, R; Chantre, C; Capdevielle-Dulac, C; Bodet, M; Mougel, F; Calatayud, P A; Dupas, S; Huguet, E; Jeannette, R; Obonyo, J; Odorico, C; Silvain, J F; Le Ru, B; Kaiser, L

    2017-09-22

    Studying mechanisms that drive host adaptation in parasitoids is crucial for the efficient use of parasitoids in biocontrol programs. Cotesia typhae nov. sp. (Fernández-Triana) (Hymenoptera: Braconidae) is a newly described parasitoid of the Mediterranean corn borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae). Braconidae are known for their domesticated bracovirus, which is injected with eggs in the host larva to overcome its resistance. In this context, we compared reproductive success traits of four Kenyan strains of C. typhae on a French and a Kenyan populations of its host. Differences were found between the four strains and the two most contrasted ones were studied more thoroughly on the French host population. Parasitoid offspring size was correlated with parasitism success and the expression of bracovirus virulence genes (CrV1 and Cystatin) in the host larva after parasitism. Hybrids between these two parasitoid strains showed phenotype and gene expression profiles similar to the most successful parental strain, suggesting the involvement of dominant alleles in the reproductive traits. Ovary dissections revealed that the most successful strain injected more eggs in a single host larva than the less successful one, despite an equal initial ovocyte number in ovaries. It can be expected that the amount of viral particles increase with the number of eggs injected. The ability to bypass the resistance of the allopatric host may in consequence be related to the oviposition behaviour (eggs allocation). The influence of the number of injected eggs on parasitism success and on virulence gene expression was evaluated by oviposition interruption experiments.

  8. Leucine metabolism regulates TRI6 expression and affects deoxynivalenol production and virulence in Fusarium graminearum.

    PubMed

    Subramaniam, Rajagopal; Narayanan, Swara; Walkowiak, Sean; Wang, Li; Joshi, Manisha; Rocheleau, Hélène; Ouellet, Thérèse; Harris, Linda J

    2015-11-01

    TRI6 is a positive regulator of the trichothecene gene cluster and the production of trichothecene mycotoxins [deoxynivalenol (DON)] and acetylated forms such as 15-Acetyl-DON) in the cereal pathogen Fusarium graminearum. As a global transcriptional regulator, TRI6 expression is modulated by nitrogen-limiting conditions, sources of nitrogen and carbon, pH and light. However, the mechanism by which these diverse environmental factors affect TRI6 expression remains underexplored. In our effort to understand how nutrients affect TRI6 regulation, comparative digital expression profiling was performed with a wild-type F. graminearum and a Δtri6 mutant strain, grown in nutrient-rich conditions. Analysis showed that TRI6 negatively regulates genes of the branched-chain amino acid (BCAA) metabolic pathway. Feeding studies with deletion mutants of MCC, encoding methylcrotonyl-CoA-carboxylase, one of the key enzymes of leucine metabolism, showed that addition of leucine specifically down-regulated TRI6 expression and reduced 15-ADON accumulation. Constitutive expression of TRI6 in the Δmcc mutant strain restored 15-ADON production. A combination of cellophane breach assays and pathogenicity experiments on wheat demonstrated that disrupting the leucine metabolic pathway significantly reduced disease. These findings suggest a complex interaction between one of the primary metabolic pathways with a global regulator of mycotoxin biosynthesis and virulence in F. graminearum.

  9. Salmonella grows vigorously on seafood and expresses its virulence and stress genes at different temperature exposure.

    PubMed

    Kumar, Rakesh; Datta, Tirtha K; Lalitha, Kuttanappilly V

    2015-11-03

    Seafood is not considered the natural habitat of Salmonella except the river fish, but still, the incidence of Salmonella in seafood is in a steady rise. By extending our understanding of Salmonella growth dynamics and pathogenomics in seafood, we may able to improve seafood safety and offer better strategies to protect the public health. The current study was thus aimed to assess the growth and multiplication of non-typhoidal and typhoidal Salmonella serovars on seafood and further sought to evaluate their virulence and stress genes expression while in contact with seafood at varying temperature exposure. Salmonella enterica Weltevreden and Salmonella enterica Typhi were left to grow on fish fillets at -20, 4, room temperature (RT) and 45 °C for a period of one week. Total RNA from both Salmonella serovars were extracted and qRT-PCR based relative gene expression approach was used to detect the expression of rpoE, invA, stn and fimA genes at four different temperature conditions studied on incubation days 0, 1, 3, 5 and 7. Salmonella Weltevreden growth on seafood was increased ~4 log10 at RT and 45 °C, nevertheless, nearly 2 and >4 log 10 reduction was observed in cell count stored at 4 and -20 °C on seafood, respectively. Growth pattern of Salmonella Typhi in seafood has shown identical pattern at RT and 45 °C, however, growth was sharply reduced at 4 and -20 °C as compared to the Salmonella Weltevreden. Total RNA of Salmonella Weltevreden was in the range from 1.3 to 17.6 μg/μl and maximum concentration was obtained at 45 °C on day 3. Similarly, RNA concentration of Salmonella Typhi was ranged from 1.2 to 11.8 μg/μl and maximum concentration was obtained at 45 °C on day 3. The study highlighted that expression of invA and stn genes of Salmonella Weltevreden was >8-fold upregulated at RT, whereas, fimA gene was increasingly down regulated at room temperature. Storage of Salmonella Weltevreden at 45 °C on seafood resulted in an increased expression

  10. Expression of Vibrio cholerae virulence genes in response to environmental signals.

    PubMed

    Peterson, Kenneth M

    2002-09-01

    Vibrio cholerae, the causative agent of Asiatic cholera, is a gram-negative motile bacterial species acquired via oral ingestion of contaminated food or water sources. The O1 serogroup of V. cholerae is responsible for pandemic cholera and is divided into two biotypes, classical and El Tor (Butterton and Calderwood, 1995; Mekalanos, 1985). The El Tor biotype is responsible for the current cholera pandemic. In the absence of disease, the vibrio life cycle consists of a free-swimming phase in marine and estuarine environments in association with zooplankton, crustaceans, insects, and water plants. Vibrios interact with various surfaces found in the environment to generate biofilms which may promote survival (Watnick etaL, 1999). Within the host the motile vibrios must evade the innate host defense mechanisms, penetrate the mucus layer covering the intestinal villi, adhere to and colonize the epithelial surface of the small intestine, assume a non-motile phase, replicate and cause disease by secreting numerous exoproteins at the site of infection (Oliver and Kaper, 1997). The voluminous diarrhea associated with cholera infection leads to the dissemination of the vibrios back into a watery environment and thus a continuation of the environmental phase of the life cycle. The host phase of the vibrio life cycle is only possible through the action of a group of virulence genes (ToxR-regulon) controlled by a complex and incompletely understood regulatory cascade. The ToxR regulon colonization and toxin genes are coordinately expressed in response to specific host signals that have yet to be completely defined (Skorupsky and Taylor 1997). Although little is known regarding the host signals that impact the ToxR regulatory cascade, it is clear that these intraintestinal signals play an important role in maximizing the ability of the vibrios to survive and multiply within the host. Key to understanding the complex events involved in the pathogenesis of V. cholerae will be

  11. Entamoeba histolytica: expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain.

    PubMed

    López-Vancell, R; Arreguín Espinosa, R; González-Canto, A; Néquiz Avendaño, M; García de León, M C; Olivos-García, A; López-Vancell, D; Pérez-Tamayo, R

    2010-07-01

    We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG's anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.

  12. Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq

    PubMed Central

    Wilson, J. W.; Ott, C. M.; zu Bentrup, K. Höner; Ramamurthy, R.; Quick, L.; Porwollik, S.; Cheng, P.; McClelland, M.; Tsaprailis, G.; Radabaugh, T.; Hunt, A.; Fernandez, D.; Richter, E.; Shah, M.; Kilcoyne, M.; Joshi, L.; Nelman-Gonzalez, M.; Hing, S.; Parra, M.; Dumars, P.; Norwood, K.; Bober, R.; Devich, J.; Ruggles, A.; Goulart, C.; Rupert, M.; Stodieck, L.; Stafford, P.; Catella, L.; Schurr, M. J.; Buchanan, K.; Morici, L.; McCracken, J.; Allen, P.; Baker-Coleman, C.; Hammond, T.; Vogel, J.; Nelson, R.; Pierson, D. L.; Stefanyshyn-Piper, H. M.; Nickerson, C. A.

    2007-01-01

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight 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 with identical ground control cultures. Global microarray and proteomic analyses revealed that 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. Space flight 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 space flight missions and provide novel therapeutic options on Earth. PMID:17901201

  13. 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.; hide

    2007-01-01

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

  14. Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq.

    PubMed

    Wilson, J W; Ott, C M; Höner zu Bentrup, K; Ramamurthy, R; Quick, L; Porwollik, S; Cheng, P; McClelland, M; Tsaprailis, G; Radabaugh, T; Hunt, A; Fernandez, D; Richter, E; Shah, M; Kilcoyne, M; Joshi, L; Nelman-Gonzalez, M; Hing, S; Parra, M; Dumars, P; Norwood, K; Bober, R; Devich, J; Ruggles, A; Goulart, C; Rupert, M; Stodieck, L; Stafford, P; Catella, L; Schurr, M J; Buchanan, K; Morici, L; McCracken, J; Allen, P; Baker-Coleman, C; Hammond, T; Vogel, J; Nelson, R; Pierson, D L; Stefanyshyn-Piper, H M; Nickerson, C A

    2007-10-09

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight 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 with identical ground control cultures. Global microarray and proteomic analyses revealed that 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. Space flight 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 space flight missions and provide novel therapeutic options on Earth.

  15. RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae.

    PubMed

    Weber, Gregor G; Kortmann, Jens; Narberhaus, Franz; Klose, Karl E

    2014-09-30

    Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.

  16. RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae

    PubMed Central

    Weber, Gregor G.; Kortmann, Jens; Narberhaus, Franz; Klose, Karl E.

    2014-01-01

    Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5′ UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine. PMID:25228776

  17. Nectin-4 Interactions Govern Measles Virus Virulence in a New Model of Pathogenesis, the Squirrel Monkey (Saimiri sciureus).

    PubMed

    Delpeut, Sébastien; Sawatsky, Bevan; Wong, Xiao-Xiang; Frenzke, Marie; Cattaneo, Roberto; von Messling, Veronika

    2017-06-01

    In addition to humans, only certain nonhuman primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys (Saimiri sciureus), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques (Macaca fascicularis) infected with the same virus, the squirrel monkeys developed more-severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with an MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild-type and nectin-4-blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild-type virus was found in tracheal lavage fluids and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and better-performing S. sciureus model of measles pathogenesis.IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here, we report that infection of squirrel monkeys (Saimiri sciureus) fulfills these requirements. Comparative infection with wild-type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies. Copyright © 2017 American Society for Microbiology.

  18. Real-Time Characterization of Virulence Factor Expression in Yersinia pestis Using a Green Fluorescent Protein Reporter System

    SciTech Connect

    Forde, C; Rocco, J; Fitch, J P; McCutchen-Maloney, S

    2004-06-09

    A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopK, yopT, yscN, and lcrE/yopN, which are all components of the Type III secretion virulence mechanism of Y. pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time. Basal expression levels observed for the Y. pestis promoters, expressed as percentages of the positive control with GFP under the control of the lac promoter, were: yopE (15%), sycE (15%), yopK (13%), yopT (4%), lcrE (3.3%) and yscN (0.8%). The yopE reporter showed the strongest gene induction following temperature transition from 26 C to 37 C. The induction levels of the other virulence factors, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11 fold), yscN (7 fold), yopK (6 fold), lcrE (3 fold), yopT (2 fold), and sycE (2 fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y. pestis virulence factors in real time, in living cells.

  19. 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

  20. Iron concentration limits growth rate and the expression of virulence factors in hrp-inducing minimal medium with Pseudomonas syringae

    USDA-ARS?s Scientific Manuscript database

    Although chemically-defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen, Pseudomonas syringae, it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is th...

  1. Artemisia princeps Inhibits Biofilm Formation and Virulence-Factor Expression of Antibiotic-Resistant Bacteria

    PubMed Central

    Choi, Na-Young; Kang, Sun-Young; Kim, Kang-Ju

    2015-01-01

    In this study, we used ethanol extract of A. princeps and investigated its antibacterial effects against MRSA. Ethanol extract of A. princeps significantly inhibited MRSA growth and organic acid production during glucose metabolism at concentrations greater than 1 mg/mL (P < 0.05). MRSA biofilm formation was observed using scanning electron microscopy (SEM) and safranin staining. A. princeps extract was found to inhibit MRSA biofilm formation at concentrations higher than 2 mg/mL significantly (P < 0.05). Bactericidal effects of the A. princeps were observed using confocal laser microscopy, which showed that A. princeps was bactericidal in a dose-dependent manner. Using real-time PCR, expression of mecA, an antibiotic-resistance gene of MRSA, was observed, along with that of sea, agrA, and sarA. A. princeps significantly inhibited mecA, sea, agrA, and sarA, mRNA expression at the concentrations greater than 1 mg/mL (P < 0.05). The phytochemical analysis of A. princeps showed a relatively high content of organic acids and glycosides. The results of this study suggest that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract. PMID:26247012

  2. Canine distemper viruses expressing a hemagglutinin without N-glycans lose virulence but retain immunosuppression.

    PubMed

    Sawatsky, Bevan; von Messling, Veronika

    2010-03-01

    Paramyxovirus glycoproteins are posttranslationally modified by the addition of N-linked glycans, which are often necessary for correct folding, processing, and cell surface expression. To establish the contribution of N glycosylation to morbillivirus attachment (H) protein function and overall virulence, we first determined the use of the potential N-glycosylation sites in the canine distemper virus (CDV) H proteins. Biochemical characterization revealed that the three sites conserved in all strains were N glycosylated, whereas only two of the up to five additional sites present in wild-type strains are used. A wild-type virus with an H protein reproducing the vaccine strain N-glycosylation pattern remained lethal in ferrets but with a prolonged course of disease. In contrast, introduction of the vaccine H protein in the wild-type context resulted in complete attenuation. To further characterize the role of N glycosylation in CDV pathogenesis, the N-glycosylation sites of wild-type H proteins were successively deleted, including a nonstandard site, to ultimately generate a nonglycosylated H protein. Despite reduced expression levels, this protein remained fully functional. Recombinant viruses expressing N-glycan-deficient H proteins no longer caused disease, even though their immunosuppressive capacities were retained, indicating that reduced N glycosylation contributes to attenuation without affecting immunosuppression.

  3. Cloning and expression of Staphylococcus saprophyticus urease gene sequences in Staphylococcus carnosus and contribution of the enzyme to virulence.

    PubMed Central

    Gatermann, S; Marre, R

    1989-01-01

    The urease gene of Staphylococcus saprophyticus CCM883 was cloned and expressed in Staphylococcus carnosus TM300. In vitro translation of the cloned DNA sequences revealed six polypeptides (of 70, 47, 29, 27, 20, and 17 kilodaltons) that were associated with enzyme activity. Introduction of the cloned genes into a urease-negative mutant of S. saprophyticus restored the virulence of this strain, confirming our previous suggestion (S. Gatermann, J. John, and R. Marre, Infect. Immun. 57:110-116, 1989) that this enzyme is a major virulence factor of the organism and contributes mainly to cystopathogenicity. Images PMID:2777370

  4. Staphylococcus aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression.

    PubMed

    Bronesky, Delphine; Wu, Zongfu; Marzi, Stefano; Walter, Philippe; Geissmann, Thomas; Moreau, Karen; Vandenesch, François; Caldelari, Isabelle; Romby, Pascale

    2016-09-08

    Staphylococcus aureus RNAIII is one of the main intracellular effectors of the quorum-sensing system. It is a multifunctional RNA that encodes a small peptide, and its noncoding parts act as antisense RNAs to regulate the translation and/or the stability of mRNAs encoding transcriptional regulators, major virulence factors, and cell wall metabolism enzymes. In this review, we explain how regulatory proteins and RNAIII are embedded in complex regulatory circuits to express virulence factors in a dynamic and timely manner in response to stress and environmental and metabolic changes.

  5. Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection.

    PubMed

    Cornelis, Karen; Maes, Tania; Jaziri, Mondher; Holsters, Marcelle; Goethals, Koen

    2002-04-01

    The phytopathogenic bacterium Rhodococcus fascians provokes shoot meristem formation and malformations on aerial plant parts, mainly at the axils. The interaction is accompanied by bacterial colonization of the plant surface and tissues. Upon infection, the two bacterial loci required for full virulence, fas and att, were expressed only at the sites of symptom development, although their expression profiles differed both spatially and temporally. The att locus was expressed principally in bacteria located on the plant surface at early stages of infection. Expression of the fas locus occurred throughout infection, mainly in bacteria that were penetrating, or had penetrated, the plant tissues and coincided with sites of meristem initiation and proliferation. The implications for the regulation of virulence genes of R. fascians during plant infection are discussed.

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

    PubMed

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

    2012-07-01

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

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

    PubMed

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

    2013-01-01

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

  8. Increased expression of virulence attributes in oral Candida albicans isolates from human immunodeficiency virus-positive individuals.

    PubMed

    Mane, Arati; Gaikwad, Shraddha; Bembalkar, Shilpa; Risbud, Arun

    2012-02-01

    Oral candidiasis caused by Candida albicans is recognized as one of the most frequent opportunistic infections in human immunodeficiency virus (HIV)-infected individuals. The overall severity and chronicity of oral candidiasis has been attributed exclusively to the HIV-induced immune deficiency of the affected individuals but not to the virulence factors of the pathogen, i.e. C. albicans. However, genotypic and phenotypic studies have suggested that HIV infection might be associated with preferential selection of C. albicans strains with altered virulence determinants, leading to colonization with Candida populations that are better able to cause disease in these immunologically compromised hosts. If this process of selection is indeed related to pathogenicity, it may be possible to measure alterations in different virulence factors produced by C. albicans in HIV-infected patients. To evaluate this hypothesis, the present work was undertaken to determine simultaneously the expression of five virulence factors in oral C. albicans isolates colonizing and infecting HIV-positive and -negative individuals. The significance of genotypes in the pathogenesis of oral candidiasis was also elucidated. Oral swabs were collected from 335 consecutive individuals (210 HIV-positive and 125 HIV-negative). Virulence factors and genotypes were determined for all the C. albicans strains isolated. The results showed significantly increased expression of proteinase, phospholipase and haemolytic activities, as well as a greater ability to adhere, in isolates from HIV-positive compared with HIV-negative individuals (P<0.05). However, no significant differences in virulence factor expression in isolates colonizing or infecting HIV-positive individuals were seen. Genotype A was the predominant type (71.3 %); however, a relationship could not be established between the genotypes and the virulence factors, or with clinical infection. These data support the concept of preferential C. albicans

  9. Ectopic expression of Ralstonia solanacearum effector protein PopA early in invasion results in loss of virulence.

    PubMed

    Kanda, Ayami; Yasukohchi, Masahiko; Ohnishi, Kouhei; Kiba, Akinori; Okuno, Tetsuro; Hikichi, Yasufumi

    2003-05-01

    Ralstonia solanacearum OE1-1 (OE1-1) is pathogenic to tobacco. The type III-secreted effector protein popA of OE1-1 showed 97.6% identity to popA of R. solanacearum GMI1000, which is not pathogenic to tobacco. Reverse transcription-polymerase chain reaction analysis showed that popA in OE1-1 was expressed at 3 h after inoculation (HAI), but not before, in infiltrated-tobacco leaves. Pathogenicity analysis using a popABC operon-deleted mutant of OE1-1 (deltaABC) showed that popABC is not directly involved in the pathogenicity of OE1-1. When Papa, which constitutively expresses popA, was infiltrated into tobacco leaves, popA was expressed by 0.5 HAI. Papa could no longer multiply or spread in tobacco leaves and was no longer virulent. Moreover, the hypersensitive response (HR) and expression of HR-related genes were not induced in Papa-infiltrated leaves. Papa was also avirulent in a tobacco root-dipping inoculation assay. These results suggest that the expression of popA in Papa immediately after invasion triggers the suppression of bacterial proliferation and movement, resulting in loss of virulence. However, Papa retained its virulence when directly inoculated into xylem vessels. This result suggests that tobacco plants can recognize PopA when it is expressed early in disease development, and respond with an effective defense in the intercellular spaces.

  10. Paralogous Outer Membrane Proteins Mediate Uptake of Different Forms of Iron and Synergistically Govern Virulence in Francisella tularensis tularensis*

    PubMed Central

    Ramakrishnan, Girija; Sen, Bhaswati; Johnson, Richard

    2012-01-01

    Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a 55Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host. PMID:22661710

  11. Induction of Virulence Gene Expression in Staphylococcus aureus by Pulmonary Surfactant

    PubMed Central

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

    2014-01-01

    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. PMID:24452679

  12. 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.

  13. Virulence gene regulation by CvfA, a putative RNase: the CvfA-enolase complex in Streptococcus pyogenes links nutritional stress, growth-phase control, and virulence gene expression.

    PubMed

    Kang, Song Ok; Caparon, Michael G; Cho, Kyu Hong

    2010-06-01

    Streptococcus pyogenes, a multiple-auxotrophic human pathogen, regulates virulence gene expression according to nutritional availability during various stages in the infection process or in different infection sites. We discovered that CvfA influenced the expression of virulence genes according to growth phase and nutritional status. The influence of CvfA in C medium, rich in peptides and poor in carbohydrates, was most pronounced at the stationary phase. Under these conditions, up to 30% of the transcriptome exhibited altered expression; the levels of expression of multiple virulence genes were altered, including the genes encoding streptokinase, CAMP factor, streptolysin O, M protein (more abundant in the CvfA(-) mutant), SpeB, mitogenic factor, and streptolysin S (less abundant). The increase of carbohydrates or peptides in media restored the levels of expression of the virulence genes in the CvfA(-) mutant to wild-type levels (emm, ska, and cfa by carbohydrates; speB by peptides). Even though the regulation of gene expression dependent on nutritional stress is commonly linked to the stringent response, the levels of ppGpp were not altered by deletion of cvfA. Instead, CvfA interacted with enolase, implying that CvfA, a putative RNase, controls the transcript decay rates of virulence factors or their regulators according to nutritional status. The virulence of CvfA(-) mutants was highly attenuated in murine models, indicating that CvfA-mediated gene regulation is necessary for the pathogenesis of S. pyogenes. Taken together, the CvfA-enolase complex in S. pyogenes is involved in the regulation of virulence gene expression by controlling RNA degradation according to nutritional stress.

  14. 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.

  15. Differences in Cell Morphometry, Cell Wall Topography and Gp70 Expression Correlate with the Virulence of Sporothrix brasiliensis Clinical Isolates

    PubMed Central

    Castro, Rafaela A.; Kubitschek-Barreira, Paula H.; Teixeira, Pedro A. C.; Sanches, Glenda F.; Teixeira, Marcus M.; Quintella, Leonardo P.; Almeida, Sandro R.; Costa, Rosane O.; Camargo, Zoilo P.; Felipe, Maria S. S.; de Souza, Wanderley; Lopes-Bezerra, Leila M.

    2013-01-01

    Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes. PMID:24116065

  16. Helicobacter pylori virulence factors affecting gastric proton pump expression and acid secretion.

    PubMed

    Hammond, Charles E; Beeson, Craig; Suarez, Giovanni; Peek, Richard M; Backert, Steffen; Smolka, Adam J

    2015-08-01

    Acute Helicobacter pylori infection of gastric epithelial cells and human gastric biopsies represses H,K-ATPase α subunit (HKα) gene expression and inhibits acid secretion, causing transient hypochlorhydria and supporting gastric H. pylori colonization. Infection by H. pylori strains deficient in the cag pathogenicity island (cag PAI) genes cagL, cagE, or cagM, which do not transfer CagA into host cells or induce interleukin-8 secretion, does not inhibit HKα expression, nor does a cagA-deficient strain that induces IL-8. To test the hypothesis that virulence factors other than those mediating CagA translocation or IL-8 induction participate in HKα repression by activating NF-κB, AGS cells transfected with HKα promoter-Luc reporter constructs containing an intact or mutated NF-κB binding site were infected with wild-type H. pylori strain 7.13, isogenic mutants lacking cag PAI genes responsible for CagA translocation and/or IL-8 induction (cagA, cagζ, cagε, cagZ, and cagβ), or deficient in genes encoding two peptidoglycan hydrolases (slt and cagγ). H. pylori-induced AGS cell HKα promoter activities, translocated CagA, and IL-8 secretion were measured by luminometry, immunoblotting, and ELISA, respectively. Human gastric biopsy acid secretion was measured by microphysiometry. Taken together, the data showed that HKα repression is independent of IL-8 expression, and that CagA translocation together with H. pylori transglycosylases encoded by slt and cagγ participate in NF-κB-dependent HKα repression and acid inhibition. The findings are significant because H. pylori factors other than CagA and IL-8 secretion are now implicated in transient hypochlorhydria which facilitates gastric colonization and potential triggering of epithelial progression to neoplasia.

  17. H-NS Plays a Role in Expression of Acinetobacter baumannii Virulence Features

    PubMed Central

    Eijkelkamp, Bart A.; Stroeher, Uwe H.; Hassan, Karl A.; Elbourne, Liam D. H.; Paulsen, Ian T.

    2013-01-01

    Acinetobacter baumannii has become a major problem in the clinical setting with the prevalence of infections caused by multidrug-resistant strains on the increase. Nevertheless, only a limited number of molecular mechanisms involved in the success of A. baumannii as a human pathogen have been described. In this study, we examined the virulence features of a hypermotile derivative of A. baumannii strain ATCC 17978, which was found to display enhanced adherence to human pneumocytes and elevated levels of lethality toward Caenorhabditis elegans nematodes. Analysis of cellular lipids revealed modifications to the fatty acid composition, providing a possible explanation for the observed changes in hydrophobicity and subsequent alteration in adherence and motility. Comparison of the genome sequences of the hypermotile variant and parental strain revealed that an insertion sequence had disrupted an hns-like gene in the variant. This gene encodes a homologue of the histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes seen in the autotransporter Ata, a type VI secretion system, and a type I pilus cluster. Interestingly, isolation and analysis of a second independent hypermotile ATCC 17978 variant revealed a mutation to a residue within the DNA binding region of H-NS. Taken together, these mutants indicate that the phenotypic and transcriptomic differences seen are due to loss of regulatory control effected by H-NS. PMID:23649094

  18. Phase-variable Expression of Lipopolysaccharide Contributes to the Virulence of Legionella pneumophila

    PubMed Central

    Lüneberg, Edeltraud; Zähringer, Ulrich; Knirel, Yuriy A.; Steinmann, Dorothee; Hartmann, Maike; Steinmetz, Ivo; Rohde, Manfred; Köhl, Jörg; Frosch, Matthias

    1998-01-01

    With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625–positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625–positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila. This is the first description of phase-variable expression of Legionella LPS. PMID:9653083

  19. H-NS plays a role in expression of Acinetobacter baumannii virulence features.

    PubMed

    Eijkelkamp, Bart A; Stroeher, Uwe H; Hassan, Karl A; Elbourne, Liam D H; Paulsen, Ian T; Brown, Melissa H

    2013-07-01

    Acinetobacter baumannii has become a major problem in the clinical setting with the prevalence of infections caused by multidrug-resistant strains on the increase. Nevertheless, only a limited number of molecular mechanisms involved in the success of A. baumannii as a human pathogen have been described. In this study, we examined the virulence features of a hypermotile derivative of A. baumannii strain ATCC 17978, which was found to display enhanced adherence to human pneumocytes and elevated levels of lethality toward Caenorhabditis elegans nematodes. Analysis of cellular lipids revealed modifications to the fatty acid composition, providing a possible explanation for the observed changes in hydrophobicity and subsequent alteration in adherence and motility. Comparison of the genome sequences of the hypermotile variant and parental strain revealed that an insertion sequence had disrupted an hns-like gene in the variant. This gene encodes a homologue of the histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes seen in the autotransporter Ata, a type VI secretion system, and a type I pilus cluster. Interestingly, isolation and analysis of a second independent hypermotile ATCC 17978 variant revealed a mutation to a residue within the DNA binding region of H-NS. Taken together, these mutants indicate that the phenotypic and transcriptomic differences seen are due to loss of regulatory control effected by H-NS.

  20. Pallial mucus of the oyster Crassostrea virginica regulates the expression of putative virulence genes of its pathogen Perkinsus marinus.

    PubMed

    Pales Espinosa, Emmanuelle; Corre, Erwan; Allam, Bassem

    2014-04-01

    Perkinsus marinus is a pathogen responsible for severe mortalities of the eastern oyster Crassostrea virginica along the East and Gulf coasts of the United States. When cultivated, the pathogenicity of this microorganism decreases significantly, hampering the study of its virulence factors. Recent investigations have shown a significant increase of the in vivo virulence of P. marinus exposed to oyster pallial mucus. In the current study, we investigated the effect of pallial mucus on P. marinus gene expression compared with cultures supplemented with oyster digestive extracts or with un-supplemented cultures. In parallel, parasite cells cultured under these three conditions were used to challenge oysters and to assess virulence in vivo. Perkinsus marinus mRNA sequencing was performed on an Illumina GAIIX sequencer and data were analysed using the Tuxedo RNAseq suite for mapping against the draft P. marinus genome and for differential expression analysis. Results showed that exposure of P. marinus to mucus induces significant regulation of nearly 3,600 transcripts, many of which are considered as putative virulence factors. Pallial mucus is suspected to mimic internal host conditions, thereby preparing the pathogen to overcome defense factors before invasion. This hypothesis is supported by significant regulation in several antioxidant proteins, heat shock proteins, protease inhibitors and proteasome subunits. In addition, mucus exposure induced the modulation of several genes known to affect immunity and apoptosis in vertebrates and invertebrates. Several proteases (proteolysis) and merozoite surface proteins (cell recognition) were also modulated. Overall, these results provide a baseline for targeted, in depth analysis of candidate virulence factors in P. marinus.

  1. 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

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

    PubMed

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

    2015-01-01

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

  3. Effects of the ERES pathogenicity region regulator Ralp3 on Streptococcus pyogenes serotype M49 virulence factor expression.

    PubMed

    Siemens, Nikolai; Fiedler, Tomas; Normann, Jana; Klein, Johannes; Münch, Richard; Patenge, Nadja; Kreikemeyer, Bernd

    2012-07-01

    Streptococcus pyogenes (group A streptococcus [GAS]) is a highly virulent Gram-positive bacterium. For successful infection, GAS expresses many virulence factors, which are clustered together with transcriptional regulators in distinct genomic regions. Ralp3 is a central regulator of the ERES region. In this study, we investigated the role of Ralp3 in GAS M49 pathogenesis. The inactivation of Ralp3 resulted in reduced attachment to and internalization into human keratinocytes. The Δralp3 mutant failed to survive in human blood and serum, and the hyaluronic acid capsule was slightly decreased. In addition, the mutant showed a lower binding capacity to human plasminogen, and the SpeB activity was significantly decreased. Complementation of the Δralp3 mutant restored the wild-type phenotype. The transcriptome and quantitative reverse transcription-PCR analysis of the serotype M49 GAS strain and its isogenic Δralp3 mutant identified 16 genes as upregulated, and 43 genes were found to be downregulated. Among the downregulated genes, there were open reading frames encoding proteins involved in metabolism (e.g., both lac operons and the fru operon), genes encoding lantibiotics (e.g., the putative salivaricin operon), and ORFs encoding virulence factors (such as the whole Mga core regulon and further genes under Mga control). In summary, the ERES region regulator Ralp3 is an important serotype-specific transcriptional regulator for virulence and metabolic control.

  4. Epigenetic Control of Virulence Gene Expression in Pseudomonas aeruginosa by a LysR-Type Transcription Regulator

    PubMed Central

    Turner, Keith H.; Vallet-Gely, Isabelle; Dove, Simon L.

    2009-01-01

    Phenotypic variation within an isogenic bacterial population is thought to ensure the survival of a subset of cells in adverse conditions. The opportunistic pathogen Pseudomonas aeruginosa variably expresses several phenotypes, including antibiotic resistance, biofilm formation, and the production of CupA fimbriae. Here we describe a previously unidentified bistable switch in P. aeruginosa. This switch controls the expression of a diverse set of genes, including aprA, which encodes the secreted virulence factor alkaline protease. We present evidence that bistable expression of PA2432, herein named bexR (bistable expression regulator), which encodes a LysR-type transcription regulator, controls this switch. In particular, using DNA microarrays, quantitative RT–PCR analysis, chromatin immunoprecipitation, and reporter gene fusions, we identify genes directly under the control of BexR and show that these genes are bistably expressed. Furthermore, we show that bexR is itself bistably expressed and positively autoregulated. Finally, using single-cell analyses of a GFP reporter fusion, we present evidence that positive autoregulation of bexR is necessary for bistable expression of the BexR regulon. Our findings suggest that a positive feedback loop involving a LysR-type transcription regulator serves as the basis for an epigenetic switch that controls virulence gene expression in P. aeruginosa. PMID:20041030

  5. The alternative sigma factor B modulates virulence gene expression in a murine Staphylococcus aureus infection model but does not influence kidney gene expression pattern of the host.

    PubMed

    Depke, Maren; Burian, Marc; Schäfer, Tina; Bröker, Barbara M; Ohlsen, Knut; Völker, Uwe

    2012-01-01

    Infections caused by Staphylococcus aureus are associated with significant morbidity and mortality and are an increasing threat not only in hospital settings. The expression of the staphylococcal virulence factor repertoire is known to be affected by the alternative sigma factor B (SigB). However, its impact during infection still is a matter of debate. Kidney tissues of controls or mice infected with S. aureus HG001 or its isogenic sigB mutant were analyzed by transcriptome profiling to monitor the host response, and additionally expression of selected S. aureus genes was monitored by RT-qPCR. Direct transcript analysis by RT-qPCR revealed significant SigB activity in all mice infected with the wild-type strain, but not in its isogenic sigB mutant (p<0.0001). Despite a clear-cut difference in the SigB-dependent transcription pattern of virulence genes (clfA, aur, and hla), the host reaction to infection (either wild type or sigB mutant) was almost identical. Despite its significant activity in vivo, loss of SigB did neither have an effect on the outcome of infection nor on murine kidney gene expression pattern. Thus, these data support the role of SigB as virulence modulator rather than being a virulence determinant by itself.

  6. Biological variation among african trypanosomes: I. Clonal expression of virulence is not linked to the variant surface glycoprotein or the variant surface glycoprotein gene telomeric expression site.

    PubMed

    Inverso, Jill A; Uphoff, Timothy S; Johnson, Scott C; Paulnock, Donna M; Mansfield, John M

    2010-05-01

    The potential association of variant surface glycoprotein (VSG) gene expression with clonal expression of virulence in African trypanosomes was addressed. Two populations of clonally related trypanosomes, which differ dramatically in virulence for the infected host, but display the same apparent VSG surface coat phenotype, were characterized with respect to the VSG genes expressed as well as the chromosome telomeric expression sites (ES) utilized for VSG gene transcription. The VSG gene sequences expressed by clones LouTat 1 and LouTat 1A of Trypanosoma brucei rhodesiense were identical, and gene expression in both clones occurred precisely by the same gene conversion events (duplication and transposition), which generated an expression-linked copy (ELC) of the VSG gene. The ELC was present on the same genomic restriction fragments in both populations and resided in the telomere of a 330-kb chromosome; a single basic copy of the LouTat 1/1A VSG gene, present in all variants of the LouTat 1 serodeme, was located at an internal site of a 1.5-Mb chromosome. Restriction endonuclease mapping of the ES telomere revealed that the VSG ELC of clones LouTat 1 and 1A resides in the same site. Therefore, these findings provide evidence that the VSG gene ES and, potentially, any cotranscribed ES-associated genes do not play a role in the clonal regulation of virulence because trypanosome clones LouTat 1 and 1A, which differ markedly in their virulence properties, both express identical VSG genes from the same chromosome telomeric ES.

  7. Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis.

    PubMed

    Kerr, P J; Perkins, H D; Inglis, B; Stagg, R; McLaughlin, E; Collins, S V; Van Leeuwen, B H

    2004-06-20

    Rabbit IL-4 was expressed in the virulent standard laboratory strain (SLS) and the attenuated Uriarra (Ur) strain of myxoma virus with the aim of creating a Th2 cytokine environment and inhibiting the development of an antiviral cell-mediated response to myxomatosis in infected rabbits. This allowed testing of a model for genetic resistance to myxomatosis in wild rabbits that have undergone 50 years of natural selection for resistance to myxomatosis. Expression of IL-4 significantly enhanced virulence of both virulent and attenuated virus strains in susceptible (laboratory) and resistant (wild) rabbits. SLS-IL-4 completely overcame genetic resistance in wild rabbits. The pathogenesis of SLS-IL-4 was compared in susceptible and resistant rabbits. The results support a model for resistance to myxomatosis of an enhanced innate immune response controlling virus replication and allowing an effective antiviral cell-mediated immune response to develop in resistant rabbits. Expression of IL-4 did not overcome immunity to myxomatosis induced by immunization.

  8. 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

  9. Medicinal plants extracts affect virulence factors expression and biofilm formation by the uropathogenic Escherichia coli.

    PubMed

    Wojnicz, Dorota; Kucharska, Alicja Z; Sokół-Łętowska, Anna; Kicia, Marta; Tichaczek-Goska, Dorota

    2012-12-01

    Medicinal plants are an important source for the therapeutic remedies of various diseases including urinary tract infections. This prompted us to perform research in this area. We decided to focus on medicinal plants species used in urinary tract infections prevention. The aim of our study was to determine the influence of Betula pendula, Equisetum arvense, Herniaria glabra, Galium odoratum, Urtica dioica, and Vaccinium vitis-idaea extracts on bacterial survival and virulence factors involved in tissue colonization and biofilm formation of the uropathogenic Escherichia coli rods. Qualitative and quantitative analysis of plant extracts were performed. Antimicrobial assay relied on the estimation of the colony forming unit number. Hydrophobicity of cells was established by salt aggregation test. Using motility agar, the ability of bacteria to move was examined. The erythrocyte hemagglutination test was used for fimbriae P screening. Curli expression was determined using YESCA agar supplemented with congo red. Quantification of biofilm formation was carried out using a microtiter plate assay and a spectrophotometric method. The results of the study indicate significant differences between investigated extracts in their antimicrobial activities. The extracts of H. glabra and V. vitis-idaea showed the highest growth-inhibitory effects (p < 0.05). Surface hydrophobicity of autoaggregating E. coli strain changed after exposure to all plant extracts, except V. vitis-idaea (p > 0.05). The B. pendula and U. dioica extracts significantly reduced the motility of the E. coli rods (p < 0.05). All the extracts exhibited the anti-biofilm activity.

  10. Expressed sequence tags reveal genetic diversity and putative virulence factors of the pathogenic oomycete Pythium insidiosum.

    PubMed

    Krajaejun, Theerapong; Khositnithikul, Rommanee; Lerksuthirat, Tassanee; Lowhnoo, Tassanee; Rujirawat, Thidarat; Petchthong, Thanom; Yingyong, Wanta; Suriyaphol, Prapat; Smittipat, Nat; Juthayothin, Tada; Phuntumart, Vipaporn; Sullivan, Thomas D

    2011-07-01

    Oomycetes are unique eukaryotic microorganisms that share a mycelial morphology with fungi. Many oomycetes are pathogenic to plants, and a more limited number are pathogenic to animals. Pythium insidiosum is the only oomycete that is capable of infecting both humans and animals, and causes a life-threatening infectious disease, called "pythiosis". In the majority of pythiosis patients life-long handicaps result from the inevitable radical excision of infected organs, and many die from advanced infection. Better understanding P. insidiosum pathogenesis at molecular levels could lead to new forms of treatment. Genetic and genomic information is lacking for P. insidiosum, so we have undertaken an expressed sequence tag (EST) study, and report on the first dataset of 486 ESTs, assembled into 217 unigenes. Of these, 144 had significant sequence similarity with known genes, including 47 with ribosomal protein homology. Potential virulence factors included genes involved in antioxidation, thermal adaptation, immunomodulation, and iron and sterol binding. Effectors resembling pathogenicity factors of plant-pathogenic oomycetes were also discovered, such as, a CBEL-like protein (possible involvement in host cell adhesion and hemagglutination), a putative RXLR effector (possibly involved in host cell modulation) and elicitin-like (ELL) proteins. Phylogenetic analysis mapped P. insidiosum ELLs to several novel clades of oomycete elicitins (ELIs), and homology modeling predicted that P. insidiosum ELLs should bind sterols. Most of the P. insidiosum ESTs showed homology to sequences in the genome or EST databases of other oomycetes, but one putative gene, with unknown function, was found to be unique to P. insidiosum. The EST dataset reported here represents the first steps in identifying genes of P. insidiosum and beginning transcriptome analysis. This genetic information will facilitate understanding of pathogenic mechanisms of this devastating pathogen. Copyright © 2011 The

  11. A subdose of fluconazole alters the virulence of Cryptococcus gattii during murine cryptococcosis and modulates type I interferon expression.

    PubMed

    Fontes, Alide Caroline Lima; Bretas Oliveira, Danilo; Santos, Juliana Ribeiro Alves; Carneiro, Hellem Cristina Silva; Ribeiro, Noelly de Queiroz; Oliveira, Lorena Vívien Neves de; Barcellos, Vanessa Abreu; Paixão, Tatiane Alves; Abrahão, Jonatas Santos; Resende-Stoianoff, Maria Aparecida; Vainstein, Marilene Henning; Santos, Daniel Assis

    2017-02-01

    Cryptococcosis is an invasive infection caused by yeast-like fungus of the genera Cryptococcus spp. The antifungal therapy for this disease provides some toxicity and the incidence of infections caused by resistant strains increased. Thus, we aimed to assess the consequences of fluconazole subdoses during the treatment of cryptococcosis in the murine inflammatory response and in the virulence factors of Cryptococcus gattii. Mice infected with Cryptococcus gattii were treated with subdoses of fluconazole. We determined the behavior of mice and type 1 interferon expression during the treatment; we also studied the virulence factors and susceptibility to fluconazole for the colonies recovered from the animals. A subdose of fluconazole prolonged the survival of mice, but the morbidity of cryptococcosis was higher in treated animals. These data were linked to the increase in: (i) fluconazole minimum inhibitory concentration, (ii) capsule size and (iii) melanization of C. gattii, which probably led to the increased expression of type I interferons in the brains of mice but not in the lungs. In conclusion, a subdose of fluconazole altered fungal virulence factors and susceptibility to this azole, leading to an altered inflammatory host response and increased morbidity.

  12. Trichothecenes and aspinolides produced by Trichoderma arundinaceum regulate expression of Botrytis cinerea genes involved in virulence and growth.

    PubMed

    Malmierca, Mónica G; Izquierdo-Bueno, Inmaculada; McCormick, Susan P; Cardoza, Rosa E; Alexander, Nancy J; Barua, Javier; Lindo, Laura; Casquero, Pedro A; Collado, Isidro G; Monte, Enrique; Gutiérrez, Santiago

    2016-11-01

    Trichoderma arundinaceum (Ta37) and Botrytis cinerea (B05.10) produce the sesquiterpenoids harzianum A (HA) and botrydial (BOT), respectively. TaΔTri5, an HA non-producer mutant, produces high levels of the polyketide compounds aspinolides (Asp) B and C. We analyzed the role of HA and Asp in the B. cinerea-T. arundinaceum interaction, including changes in BOT production as well as transcriptomic changes of BcBOT genes involved in BOT biosynthesis, and also of genes associated with virulence and ergosterol biosynthesis. We found that exogenously added HA up-regulated the expression of the BcBOT and all the virulence genes analyzed when B. cinerea was grown alone. However, a decrease in the amount of BOT and a down-regulation of BcBOT gene expression was observed in the interaction zone of B05.10-Ta37 dual cultures, compared to TaΔTri5. Thus, the confrontation with T. arundinaceum results in an up-regulation of most of the B. cinerea genes involved in virulence yet the presence of T. arundinaceum secondary metabolites, HA and AspC, act separately and together to down-regulate the B. cinerea genes analyzed. The present work emphasizes the existence of a chemical cross-regulation between B. cinerea and T. arundinaceum and contributes to understanding how a biocontrol fungus and its prey interact with each other.

  13. Virulent Shigella flexneri Affects Secretion, Expression, and Glycosylation of Gel-Forming Mucins in Mucus-Producing Cells

    PubMed Central

    Sperandio, Brice; Fischer, Natalie; Chevalier-Curt, Marie Joncquel; Rossez, Yannick; Roux, Pascal; Robbe Masselot, Catherine

    2013-01-01

    Mucin glycoproteins are secreted in large amounts by the intestinal epithelium and constitute an efficient component of innate immune defenses to promote homeostasis and protect against enteric pathogens. In this study, our objective was to investigate how the bacterial enteropathogen Shigella flexneri, which causes bacillary dysentery, copes with the mucin defense barrier. We report that upon in vitro infection of mucin-producing polarized human intestinal epithelial cells, virulent S. flexneri manipulates the secretion of gel-forming mucins. This phenomenon, which is triggered only by virulent strains, results in accumulation of mucins at the cell apical surface, leading to the appearance of a gel-like structure that favors access of bacteria to the cell surface and the subsequent invasion process. We identify MUC5AC, a gel-forming mucin, as a component of this structure. Formation of this gel does not depend on modifications of electrolyte concentrations, induction of trefoil factor expression, endoplasmic reticulum stress, or response to unfolded proteins. In addition, transcriptional and biochemical analyses of infected cells reveal modulations of mucin gene expression and modifications of mucin glycosylation patterns, both of which are induced by virulent bacteria in a type III secretion system-dependent manner. Thus, S. flexneri has developed a dedicated strategy to alter the mucus barrier by targeting key elements of the gel-forming capacity of mucins: gene transcription, protein glycosylation, and secretion. PMID:23876800

  14. Effect of salt and acidic pH on the stability of virulence plasmid (pYV) in Yersinia enterocolitica and expression of virulence-associated characteristics

    USDA-ARS?s Scientific Manuscript database

    The stability of the Yersinia enterocolitica virulence plasmid (pYV) under different NaCl concentrations and under acidic pH conditions was investigated. Exposure of five strains representing five serotypes of pYV-bearing virulent Y. enterocolitica to 0.5, 2 and 5% NaCl and under conditions of pH 4...

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. AiiA-mediated quorum quenching does not affect virulence or toxoflavin expression in Burkholderia glumae SL2376.

    PubMed

    Park, J Y; Lee, Y H; Yang, K Y; Kim, Y C

    2010-12-01

    The primary objective of this study was to determine the effects of quorum quenching in the pathogenicity and toxoflavin production of Burkholderia glumae causing bacterial rice grain rot. An acyl-homoserine lactonase (aiiA) gene from Bacillus sp. was expressed in B. glumae under the control of a constitutive promoter. Acyl-homoserine lactone production in the aiiA-transformants was reduced significantly, and the aiiA-expressing B. glumae strain reduced the severity of soft rot when the strain was co-inoculated with a soft-rot pathogen, Pectobacterium carotovorum ssp. carotovorum SCCI. However, the aiiA-transformant still caused rice seedling rot and rice grain rot. The aiiA-expressing strains had wild-type levels of transcription from the genes in the toxoflavin biosynthetic operon, and as well as wild-type levels of toxin production. Our results show that aiiA-mediated quorum quenching does not affect virulence or toxoflavin production in B. glumae. Indirect quorum quenching may prove an ineffective approach to the control of rice grain rot, because it reduces, but does not eliminate entirely homoserine lactones in B. glumae. Virulence of rice grain rot was retained despite reduction in homoserine lactones by the expression of aiiA in B. glumae. © 2010 The Authors. Letters in Applied Microbiology 51, 619-624 © 2010 The Society for Applied Microbiology.

  20. Identification of novel secreted virulence factors from Xylella fastidiosa using a TRV expression system

    USDA-ARS?s Scientific Manuscript database

    Xylella fastidiosa is a bacterium that causes leaf scorch diseases of agriculturally important crops including grapevines and almonds. Little is known about virulence factors that are necessary for X. fastidiosa to grow and cause disease in the xylem vessels of a plant host. Any protein secreted by ...

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

    PubMed

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

    2015-06-01

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

  2. Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for....food

    USDA-ARS?s Scientific Manuscript database

    In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of several virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding...

  3. Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for...food

    USDA-ARS?s Scientific Manuscript database

    In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding (dark-v...

  4. 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

  5. The two-component system CpxR/A represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD.

    PubMed

    De la Cruz, Miguel A; Pérez-Morales, Deyanira; Palacios, Irene J; Fernández-Mora, Marcos; Calva, Edmundo; Bustamante, Víctor H

    2015-01-01

    Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.

  6. The two-component system CpxR/A represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD

    PubMed Central

    De la Cruz, Miguel A.; Pérez-Morales, Deyanira; Palacios, Irene J.; Fernández-Mora, Marcos; Calva, Edmundo; Bustamante, Víctor H.

    2015-01-01

    Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella. PMID:26300871

  7. Coordination of Metabolism and Virulence Factors Expression of Extraintestinal Pathogenic Escherichia coli Purified from Blood Cultures of Patients with Sepsis *

    PubMed Central

    Pettersen, Veronika Kuchařová; Mosevoll, Knut Anders; Lindemann, Paul Christoffer; Wiker, Harald G.

    2016-01-01

    One of the trademarks of extraintestinal pathogenic Escherichia coli is adaptation of metabolism and basic physiology to diverse host sites. However, little is known how this common human pathogen adapts to permit survival and growth in blood. We used label-free quantitative proteomics to characterize five E. coli strains purified from clinical blood cultures associated with sepsis and urinary tract infections. Further comparison of proteome profiles of the clinical strains and a reference uropathogenic E. coli strain 536 cultivated in blood culture and on two different solid media distinguished cellular features altered in response to the pathogenically relevant condition. The analysis covered nearly 60% of the strains predicted proteomes, and included quantitative description based on label-free intensity scores for 90% of the detected proteins. Statistical comparison of anaerobic and aerobic blood cultures revealed 32 differentially expressed proteins (1.5% of the shared proteins), mostly associated with acquisition and utilization of metal ions critical for anaerobic or aerobic respiration. Analysis of variance identified significantly altered amounts of 47 proteins shared by the strains (2.7%), including proteins involved in vitamin B6 metabolism and virulence. Although the proteomes derived from blood cultures were fairly similar for the investigated strains, quantitative proteomic comparison to the growth on solid media identified 200 proteins with substantially changed levels (11% of the shared proteins). Blood culture was characterized by up-regulation of anaerobic fermentative metabolism and multiple virulence traits, including cell motility and iron acquisition. In a response to the growth on solid media there were increased levels of proteins functional in aerobic respiration, catabolism of medium-specific carbon sources and protection against oxidative and osmotic stresses. These results demonstrate on the expressed proteome level that expression of

  8. 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.

  9. Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect.

    PubMed

    Dosunmu, Ejovwoke; Chaudhari, Atul A; Singh, Shree R; Dennis, Vida A; Pillai, Shreekumar R

    2015-01-01

    The antimicrobial activity of silver-coated carbon nanotubes (AgCNTs) and their potential mode of action against mucoid and nonmucoid strains of Pseudomonas aeruginosa was investigated in vitro. The results showed that AgCNTs exhibited antimicrobial activity against both strains with minimum inhibitory concentrations of approximately 8 µg/mL, indicating a high sensitivity of P. aeruginosa to AgCNTs. AgCNTs were also bactericidal against both strains at the same minimum inhibitory concentration. Scanning and transmission electron-microscopy studies further revealed that a majority of the cells treated with AgCNTs transformed from smooth rod-shape morphology to disintegrated cells with broken/damaged membranes, resulting in leakage of cytoplasmic contents to produce ghost cells. The molecular effects of AgCNTs on P. aeruginosa genes involved in virulence and pathogenicity, stress response, and efflux pumps were evaluated for changes in their expression. Quantitative real-time PCR (qRT-PCR) showed that after exposure to AgCNTs, the expression levels of the rpoS, rsmZ, and oprD genes were significantly downregulated in both strains of P. aeruginosa compared to the untreated samples. These results suggest that the mechanism of action of AgCNTs may be attributed to their effect on cell-membrane integrity, downregulation of virulence-gene expression, and induction of general and oxidative stress in P. aeruginosa.

  10. Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect

    PubMed Central

    Dosunmu, Ejovwoke; Chaudhari, Atul A; Singh, Shree R; Dennis, Vida A; Pillai, Shreekumar R

    2015-01-01

    The antimicrobial activity of silver-coated carbon nanotubes (AgCNTs) and their potential mode of action against mucoid and nonmucoid strains of Pseudomonas aeruginosa was investigated in vitro. The results showed that AgCNTs exhibited antimicrobial activity against both strains with minimum inhibitory concentrations of approximately 8 µg/mL, indicating a high sensitivity of P. aeruginosa to AgCNTs. AgCNTs were also bactericidal against both strains at the same minimum inhibitory concentration. Scanning and transmission electron-microscopy studies further revealed that a majority of the cells treated with AgCNTs transformed from smooth rod-shape morphology to disintegrated cells with broken/damaged membranes, resulting in leakage of cytoplasmic contents to produce ghost cells. The molecular effects of AgCNTs on P. aeruginosa genes involved in virulence and pathogenicity, stress response, and efflux pumps were evaluated for changes in their expression. Quantitative real-time PCR (qRT-PCR) showed that after exposure to AgCNTs, the expression levels of the rpoS, rsmZ, and oprD genes were significantly downregulated in both strains of P. aeruginosa compared to the untreated samples. These results suggest that the mechanism of action of AgCNTs may be attributed to their effect on cell-membrane integrity, downregulation of virulence-gene expression, and induction of general and oxidative stress in P. aeruginosa. PMID:26346483

  11. Role of Staphylococcus aureus global regulators sae and sigmaB in virulence gene expression during device-related infection.

    PubMed

    Goerke, Christiane; Fluckiger, Ursula; Steinhuber, Andrea; Bisanzio, Vittoria; Ulrich, Martina; Bischoff, Markus; Patti, Joseph M; Wolz, Christiane

    2005-06-01

    The ability of Staphylococcus aureus to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators sae and sigmaB of S. aureus and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of sae, asp23, hla, clfA, coa, and fnbA was determined in strain Newman and its isogenic saeS/R and sigB mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators. sae had a dominant effect on target gene expression during device-related infection. SigmaB seemed to be less active throughout the infection than under induced conditions in vitro.

  12. Expression profiling of virulence and pathogenicity genes of Xanthomonas axonopodis pv. citri.

    PubMed

    Astua-Monge, Gustavo; Freitas-Astua, Juliana; Bacocina, Gisele; Roncoletta, Juliana; Carvalho, Sérgio A; Machado, Marcos A

    2005-02-01

    DNA macroarrays of 279 genes of Xanthomonas axonopodis pv. citri potentially associated with pathogenicity and virulence were used to compare the transcriptional alterations of this bacterium in response to two synthetic media. Data analysis indicated that 31 genes were up-regulated by synthetic medium XVM2, while only 7 genes were repressed. The results suggest that XVM2 could be used as an in vitro system to identify candidate genes involved in pathogenesis of X. axonopodis pv. citri.

  13. The Burkholderia pseudomallei BpeAB-OprB Efflux Pump: Expression and Impact on Quorum Sensing and Virulence

    PubMed Central

    Chan, Ying Ying; Chua, Kim Lee

    2005-01-01

    BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen Burkholderia pseudomallei and is responsible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of bpeAB-oprB is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the bpeAB-oprB operon. bpeR expression was similarly induced at stationary phase but lagged behind the induction of bpeAB-oprB expression. The induction of bpeAB-oprB expression could be advanced to the early exponential phase by exogenous addition of the B. pseudomallei autoinducers N-octanoyl-homoserine lactone (C8HSL) and N-decanoyl-homoserine lactone (C10HSL), suggesting that the bpeAB-oprB operon may be quorum regulated. On the other hand, acyl-homoserine lactone (acyl-HSL) production was undetectable in the bpeAB-null mutant and strains which overexpress bpeR. The failure of these strains to produce acyl-HSLs seemed to be at the level of synthesis of acyl-HSLs, as growth-phase-dependent expression of the autoinducer synthase BpsI was abolished in the bpeAB-null mutant. bpsI expression remained growth phase dependent in the bpeR mutant which had functional BpeAB-OprB. BpeAB-OprB function is likewise necessary for optimal production of quorum-sensing-controlled virulence factors such as siderophore and phospholipase C and for biofilm formation. Cell invasion and cytotoxicity towards human lung epithelial (A549) and human macrophage (THP-1) cells were also significantly attenuated in both the bpeAB mutant and bpeR-overexpressing strains, thus suggesting the possibility of attenuating B. pseudomallei virulence using inhibitors of the BpeAB-OprB efflux pump. PMID:15995185

  14. The Burkholderia pseudomallei BpeAB-OprB efflux pump: expression and impact on quorum sensing and virulence.

    PubMed

    Chan, Ying Ying; Chua, Kim Lee

    2005-07-01

    BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen Burkholderia pseudomallei and is responsible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of bpeAB-oprB is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the bpeAB-oprB operon. bpeR expression was similarly induced at stationary phase but lagged behind the induction of bpeAB-oprB expression. The induction of bpeAB-oprB expression could be advanced to the early exponential phase by exogenous addition of the B. pseudomallei autoinducers N-octanoyl-homoserine lactone (C8HSL) and N-decanoyl-homoserine lactone (C10HSL), suggesting that the bpeAB-oprB operon may be quorum regulated. On the other hand, acyl-homoserine lactone (acyl-HSL) production was undetectable in the bpeAB-null mutant and strains which overexpress bpeR. The failure of these strains to produce acyl-HSLs seemed to be at the level of synthesis of acyl-HSLs, as growth-phase-dependent expression of the autoinducer synthase BpsI was abolished in the bpeAB-null mutant. bpsI expression remained growth phase dependent in the bpeR mutant which had functional BpeAB-OprB. BpeAB-OprB function is likewise necessary for optimal production of quorum-sensing-controlled virulence factors such as siderophore and phospholipase C and for biofilm formation. Cell invasion and cytotoxicity towards human lung epithelial (A549) and human macrophage (THP-1) cells were also significantly attenuated in both the bpeAB mutant and bpeR-overexpressing strains, thus suggesting the possibility of attenuating B. pseudomallei virulence using inhibitors of the BpeAB-OprB efflux pump.

  15. 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.; hide

    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

  16. Inhibitory Effects of Chrysanthemum boreale Essential Oil on Biofilm Formation and Virulence Factor Expression of Streptococcus mutans

    PubMed Central

    Kim, Beom-Su; Park, Sun-Ju; Kim, Myung-Kon; Kim, Young-Hoi; Lee, Sang-Bong; Lee, Kwang-Hee; Lee, Young-Rae; Lee, Young-Eun; You, Yong-Ouk

    2015-01-01

    The aim of the study was to evaluate the antibacterial activity of essential oil extracted from Chrysanthemum boreale (C. boreale) on Streptococcus mutans (S. mutans). To investigate anticariogenic properties, and bacterial growth, acid production, biofilm formation, bacterial adherence of S. mutans were evaluated. Then gene expression of several virulence factors was also evaluated. C. boreale essential oil exhibited significant inhibition of bacterial growth, adherence capacity, and acid production of S. mutans at concentrations 0.1–0.5 mg/mL and 0.25–0.5 mg/mL, respectively. The safranin staining and scanning electron microscopy results showed that the biofilm formation was also inhibited. The result of live/dead staining showed the bactericidal effect. Furthermore, real-time PCR analysis showed that the gene expression of some virulence factors such as gtfB, gtfC, gtfD, gbpB, spaP, brpA, relA, and vicR of S. mutans was significantly decreased in a dose dependent manner. In GC and GC-MS analysis, seventy-two compounds were identified in the oil, representing 85.42% of the total oil. The major components were camphor (20.89%), β-caryophyllene (5.71%), α-thujone (5.46%), piperitone (5.27%), epi-sesquiphellandrene (5.16%), α-pinene (4.97%), 1,8-cineole (4.52%), β-pinene (4.45%), and camphene (4.19%). These results suggest that C. boreale essential oil may inhibit growth, adhesion, acid tolerance, and biofilm formation of S. mutans through the partial inhibition of several of these virulence factors. PMID:25763094

  17. 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

  18. Virulence gene expression, adhesion and invasion of Campylobacter jejuni exposed to oxidative stress (H2O2).

    PubMed

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

    2016-03-02

    Studies were undertaken to investigate the effect of oxidative stress conditions (exposure to hydrogen peroxide, H2O2) on [1] the expression of 14 Campylobacter jejuni virulence-associated genes associated with motility and/or invasion (flaA, flaB, flhA, flhB, ciaB, iamA), adhesion (cadF), cytotoxin production (cdtA, cdtB, cdtC) as well as some of the regulators of these genes (rpoN, fliA, luxS, cj1000), in 10 C. jejuni strains (5 poultry and 5 human) and [2] the ability of these cells to adhere to and invade Caco-2 cells. Using 16S rRNA as the reference gene (preliminary research demonstrated that this gene was stably expressed), the expression of the 14 virulence associated genes was investigated under normal and oxidative stress conditions using reverse transcription PCR. A Caco-2 cell tissue culture assay was used to examine adhesion and invasion. The response to oxidative stress was strain-dependent. Two strains showed significant (p<0.05) up or down regulation in 7 of the 14 genes tested, while only 1-2 genes were affected in the remaining strains. Expression of cadF was significantly (p<0.05) changed in all strains, cdt B in 4 strains and cj1000 in 3 strains. Expression of the remaining genes was either unaffected or significantly altered in 1-2 strains. NCTC 11168 completely lost the ability to adhere to and invade Caco-2 cells. One other strain also demonstrated reduced adherence while two others were unable to invade Caco-2 cells after exposure to oxidative stress conditions. In contrast strain 7, a poultry isolate, showed increased invasion. It was concluded that oxidative stress affects expression of C. jejuni virulence genes in a strain-dependent manner, CadF may have a secondary survival function and the cdtB gene may have a different promoter than cdtA and cdtC.

  19. Expression of STM4467-encoded arginine deiminase controlled by the STM4463 regulator contributes to Salmonella enterica serovar Typhimurium virulence.

    PubMed

    Choi, Younho; Choi, Jeongjoon; Groisman, Eduardo A; Kang, Dong-Hyun; Shin, Dongwoo; Ryu, Sangryeol

    2012-12-01

    Arginine deiminase (ADI), carbamate kinase (CK), and ornithine transcarbamoylase (OTC) constitute the ADI system. In addition to metabolic functions, the ADI system has been implicated in the virulence of certain pathogens. The pathogenic intracellular bacterium Salmonella enterica serovar Typhimurium possesses the STM4467, STM4466, and STM4465 genes, which are predicted to encode ADI, CK, and OTC, respectively. Here we report that the STM4467 gene encodes an ADI and that ADI activity plays a role in the successful infection of a mammalian host by S. Typhimurium. An STM4467 deletion mutant was defective for replication inside murine macrophages and was attenuated for virulence in mice. We determined that a regulatory protein encoded by the STM4463 gene functions as an activator for STM4467 expression. The expression of the ADI pathway genes was enhanced inside macrophages in a process that required STM4463. Lack of STM4463 impaired the ability of S. Typhimurium to replicate within macrophages. A mutant defective in STM4467-encoded ADI displayed normal production of nitric oxide by macrophages.

  20. Expression of STM4467-Encoded Arginine Deiminase Controlled by the STM4463 Regulator Contributes to Salmonella enterica Serovar Typhimurium Virulence

    PubMed Central

    Choi, Younho; Choi, Jeongjoon; Groisman, Eduardo A.; Kang, Dong-Hyun

    2012-01-01

    Arginine deiminase (ADI), carbamate kinase (CK), and ornithine transcarbamoylase (OTC) constitute the ADI system. In addition to metabolic functions, the ADI system has been implicated in the virulence of certain pathogens. The pathogenic intracellular bacterium Salmonella enterica serovar Typhimurium possesses the STM4467, STM4466, and STM4465 genes, which are predicted to encode ADI, CK, and OTC, respectively. Here we report that the STM4467 gene encodes an ADI and that ADI activity plays a role in the successful infection of a mammalian host by S. Typhimurium. An STM4467 deletion mutant was defective for replication inside murine macrophages and was attenuated for virulence in mice. We determined that a regulatory protein encoded by the STM4463 gene functions as an activator for STM4467 expression. The expression of the ADI pathway genes was enhanced inside macrophages in a process that required STM4463. Lack of STM4463 impaired the ability of S. Typhimurium to replicate within macrophages. A mutant defective in STM4467-encoded ADI displayed normal production of nitric oxide by macrophages. PMID:23006851

  1. 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.

  2. Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

    PubMed Central

    Burnside, Kellie; Lembo, Annalisa; de los Reyes, Melissa; Iliuk, Anton; BinhTran, Nguyen-Thao; Connelly, James E.; Lin, Wan-Jung; Schmidt, Byron Z.; Richardson, Anthony R.; Fang, Ferric C.; Tao, Weiguo Andy; Rajagopal, Lakshmi

    2010-01-01

    Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. PMID:20552019

  3. Quorum sensing transcriptional regulator QseA is essential for the expression of multiple virulence regulons of enterohemorrhagic Escherichia coli O157:H7

    USDA-ARS?s Scientific Manuscript database

    Introduction and Objectives: QseA is one of several transcriptional regulators that regulates the virulence gene expression in enterohemorrhagic Escherichia coli (EHEC) O157:H7 through quorum sensing. QseA has been shown to regulate the expression of the locus of enterocyte effacement (LEE), non-LEE...

  4. Quorum sensing transcriptional regulator QseA is essential for the expression of multiple virulence regulons of enterohemorrhagic Escherichia coli O157:H7

    USDA-ARS?s Scientific Manuscript database

    Introduction and Objectives: QseA is one of several transcriptional regulators that regulates the virulence gene expression in enterohemorrhagic Escherichia coli (EHEC) O157:H7 through quorum sensing. QseA has been shown to regulate the expression of the locus of enterocyte effacement (LEE), non-LEE...

  5. Pyrokinin β-Neuropeptide Affects Necrophoretic Behavior in Fire Ants (S. invicta), and Expression of β-NP in a Mycoinsecticide Increases Its Virulence

    PubMed Central

    Fan, Yanhua; Pereira, Roberto M.; Kilic, Engin; Casella, George; Keyhani, Nemat O.

    2012-01-01

    Fire ants are one of the world's most damaging invasive pests, with few means for their effective control. Although ecologically friendly alternatives to chemical pesticides such as the insecticidal fungus Beauveria bassiana have been suggested for the control of fire ant populations, their use has been limited due to the low virulence of the fungus and the length of time it takes to kill its target. We present a means of increasing the virulence of the fungal agent by expressing a fire ant neuropeptide. Expression of the fire ant (Solenopsis invicta) pyrokinin β -neuropeptide (β-NP) by B. bassiana increased fungal virulence six-fold towards fire ants, decreased the LT50, but did not affect virulence towards the lepidopteran, Galleria mellonella. Intriguingly, ants killed by the β-NP expressing fungus were disrupted in the removal of dead colony members, i.e. necrophoretic behavior. Furthermore, synthetic C-terminal amidated β-NP but not the non-amidated peptide had a dramatic effect on necrophoretic behavior. These data link chemical sensing of a specific peptide to a complex social behavior. Our results also confirm a new approach to insect control in which expression of host molecules in an insect pathogen can by exploited for target specific augmentation of virulence. The minimization of the development of potential insect resistance by our approach is discussed. PMID:22238569

  6. Governance.

    ERIC Educational Resources Information Center

    Moran, K. D.

    The author notes that two trends appear to be developing in litigation over the governance of the public schools. One trend is increasing participation of organized groups in suits against the schools. The other is a greater volume of litigation dealing with open meeting laws and freedom of information acts. Reflecting the second trend, the…

  7. Governance.

    ERIC Educational Resources Information Center

    Moran, K. D.

    This chapter summarized and analyzes all state supreme court and federal court decisions as well as other significant court decisions affecting the realm of school governance. The cases discussed are generally limited to those decided during 1974 and reported in the General Digest on or before March 1, 1975. Because of its unusual significance,…

  8. Influence of Tigecycline on Expression of Virulence Factors in Biofilm-Associated Cells of Methicillin-Resistant Staphylococcus aureus▿

    PubMed Central

    Smith, Karen; Gould, Katherine A.; Ramage, Gordon; Gemmell, Curtis G.; Hinds, Jason; Lang, Sue

    2010-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) infections are complicated by the ability of the organism to grow in surface-adhered biofilms on a multitude of abiotic and biological surfaces. These multicellular communities are notoriously difficult to eradicate with antimicrobial therapy. Cells within the biofilm may be exposed to a sublethal concentration of the antimicrobial due to the metabolic and phenotypic diversity of the biofilm-associated cells or the protection offered by the biofilm structure. In the present study, the influence of a sublethal concentration of tigecycline on biofilms formed by an epidemic MRSA-16 isolate was investigated by transcriptome analysis. In the presence of the drug, 309 genes were upregulated and 213 genes were downregulated by more than twofold in comparison to the levels of gene regulation detected for the controls not grown in the presence of the drug. Microarray data were validated by real-time reverse transcription-PCR and phenotypic assays. Tigecycline altered the expression of a number of genes encoding proteins considered to be crucial for the virulence of S. aureus. These included the reduced expression of icaC, which is involved in polysaccharide intercellular adhesin production and biofilm development; the upregulation of fnbA, clfB, and cna, which encode adhesins which attach to human proteins; and the downregulation of the cap genes, which mediate the synthesis of the capsule polysaccharide. The expression of tst, which encodes toxic shock syndrome toxin 1 (TSST-1), was also significantly reduced; and an assay performed to quantify TSST-1 showed that the level of toxin production by cells treated with tigecycline decreased by 10-fold (P < 0.001) compared to the level of production by untreated control cells. This study suggests that tigecycline may reduce the expression of important virulence factors in S. aureus and supports further investigation to determine whether it could be a useful adjunct to therapy

  9. Molecular architecture of the regulatory Locus sae of Staphylococcus aureus and its impact on expression of virulence factors.

    PubMed

    Steinhuber, Andrea; Goerke, Christiane; Bayer, Manfred G; Döring, Gerd; Wolz, Christiane

    2003-11-01

    We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

  10. Influence of tigecycline on expression of virulence factors in biofilm-associated cells of methicillin-resistant Staphylococcus aureus.

    PubMed

    Smith, Karen; Gould, Katherine A; Ramage, Gordon; Gemmell, Curtis G; Hinds, Jason; Lang, Sue

    2010-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) infections are complicated by the ability of the organism to grow in surface-adhered biofilms on a multitude of abiotic and biological surfaces. These multicellular communities are notoriously difficult to eradicate with antimicrobial therapy. Cells within the biofilm may be exposed to a sublethal concentration of the antimicrobial due to the metabolic and phenotypic diversity of the biofilm-associated cells or the protection offered by the biofilm structure. In the present study, the influence of a sublethal concentration of tigecycline on biofilms formed by an epidemic MRSA-16 isolate was investigated by transcriptome analysis. In the presence of the drug, 309 genes were upregulated and 213 genes were downregulated by more than twofold in comparison to the levels of gene regulation detected for the controls not grown in the presence of the drug. Microarray data were validated by real-time reverse transcription-PCR and phenotypic assays. Tigecycline altered the expression of a number of genes encoding proteins considered to be crucial for the virulence of S. aureus. These included the reduced expression of icaC, which is involved in polysaccharide intercellular adhesin production and biofilm development; the upregulation of fnbA, clfB, and cna, which encode adhesins which attach to human proteins; and the downregulation of the cap genes, which mediate the synthesis of the capsule polysaccharide. The expression of tst, which encodes toxic shock syndrome toxin 1 (TSST-1), was also significantly reduced; and an assay performed to quantify TSST-1 showed that the level of toxin production by cells treated with tigecycline decreased by 10-fold (P < 0.001) compared to the level of production by untreated control cells. This study suggests that tigecycline may reduce the expression of important virulence factors in S. aureus and supports further investigation to determine whether it could be a useful adjunct to therapy

  11. The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression

    PubMed Central

    Crosby, Heidi A.; Schlievert, Patrick M.; Merriman, Joseph A.; King, Jessica M.; Salgado-Pabón, Wilmara; Horswill, Alexander R.

    2016-01-01

    Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins. PMID:27144398

  12. Expression of stress and virulence genes in Escherichia coli O157:H7 heat shocked in fresh dairy compost.

    PubMed

    Singh, Randhir; Jiang, Xiuping

    2015-01-01

    The purpose of this study was to determine the gene expression of Escherichia coli O157:H7 heat shocked in dairy compost. A two-step real-time PCR assay was used to evaluate the expression of stress and virulence genes in E. coli O157:H7 heat shocked in compost at 47.5°C for 10 min. Heat-shocked E. coli O157:H7 in compost was isolated by using an immunomagnetic bead separation method, followed by total RNA extraction, which was then converted to cDNA by using a commercial kit. E. coli O157:H7 heat shocked in broth served as the media control. In compost, heat shock genes (clpB, dnaK, and groEL) and the alternative sigma factor (rpoH) of E. coli O157:H7 were upregulated (P < 0.05), whereas the expression of trehalose synthesis genes did not change. Virulence genes, such as stx1 and fliC, were upregulated, while genes stx2, eaeA, and hlyA were downregulated. In the toxin-antitoxin (TA) system, toxin genes, mazF, hipA, and yafQ were upregulated, whereas among antitoxin genes, only dinJ was upregulated (P < 0.05). In tryptic soy broth, all heat shock genes (rpoH, clpB, dnaK, and groEL) were upregulated (P < 0.05), and most virulence genes (stx1, stx2, hlyA, and fliC) and TA genes (mazF-mazE, hipA-hipB, and yafQ-dinJ and toxin gene chpS) were down-regulated. Our results revealed various gene expression patterns when E. coli O157:H7 inoculated in compost was exposed to a sublethal temperature. Clearly, induction of the heat shock response is one of the important protective mechanisms that prolongs the survival of pathogens during the composting process. In addition, other possible mechanisms (such as the TA system) operating along with heat shock response may be responsible for the extended survival of pathogens in compost.

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

    PubMed Central

    2011-01-01

    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

  14. 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.

  15. Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus

    PubMed Central

    Ericson, Megan E.; Subramanian, Chitra; Frank, Matthew W.

    2017-01-01

    ABSTRACT The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus, but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. PMID:28765222

  16. Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice.

    PubMed

    Bakala N'Goma, Jean Claude; Le Moigne, Vincent; Soismier, Nathalie; Laencina, Laura; Le Chevalier, Fabien; Roux, Anne-Laure; Poncin, Isabelle; Serveau-Avesque, Carole; Rottman, Martin; Gaillard, Jean-Louis; Etienne, Gilles; Brosch, Roland; Herrmann, Jean-Louis; Canaan, Stéphane; Girard-Misguich, Fabienne

    2015-02-01

    Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

  17. The C-terminal extension of PrhG impairs its activation of hrp expression and virulence in Ralstonia solanacearum.

    PubMed

    Zhang, Yong; Luo, Feng; Hikichi, Yasufumi; Kiba, Akinori; Yasuo, Igarashi; Ohnishi, Kouhei

    2015-04-01

    Ralstonia solanacearum is the second most destructive bacterial plant pathogens worldwide and HrpG is the master regulator of its pathogenicity. PrhG is a close paralogue of HrpG and both belong to OmpR/PhoB family of two-component response regulators. Despite a high similarity (72% global identity and 96% similarity in helix-loop-helix domain), they display distinct roles in pathogenicity. HrpG is necessary for the bacterial growth in planta and pathogenicity, while PrhG is dispensable for bacterial growth in planta and contributes little to pathogenicity. The main difference between HrpG and PrhG is the 50-amino-acid-long C-terminal extension in PrhG (amino-acid residues 230-283), which is absent in HrpG. When this extension is deleted, truncated PrhGs (under the control of its native promoter) allowed complete recovery of bacterial growth in planta and wild-type virulence of hrpG mutant. This novel finding demonstrates that the extension region in PrhG is responsible for the functional difference between HrpG and PrhG, which may block the binding of PrhG to target promoters and result in impaired activation of hrp expression by PrhG and reduced virulence of R. solanacearum. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Decreased in vivo virulence and altered gene expression by a Brucella melitensis light-sensing histidine kinase mutant.

    PubMed

    Gourley, Christopher R; Petersen, Erik; Harms, Jerome; Splitter, Gary

    2015-03-01

    Brucella species utilize diverse virulence factors. Previously, Brucella abortus light-sensing histidine kinase was identified as important for cellular infection. Here, we demonstrate that a Brucella melitensis LOV-HK (BM-LOV-HK) mutant strain has strikingly different gene expression than wild type. General stress response genes including the alternative sigma factor rpoE1 and its anti-anti-sigma factor phyR were downregulated, while flagellar, quorum sensing (QS), and type IV secretion system genes were upregulated in the ΔBM-LOV-HK strain vs. wild type. Contextually, expression results agree with other studies of transcriptional regulators involving ΔrpoE1, ΔphyR, ΔvjbR, and ΔblxR (ΔbabR) Brucella strains. Additionally, deletion of BM-LOV-HK decreases virulence in mice. During C57BL/6 mouse infection, the ΔBM-LOV-HK strain had 2 logs less CFUs in the spleen 3 days postinfection, but similar levels 6 days post infection compared to wild type. Infection of IRF-1(-/-) mice more specifically define ΔBM-LOV-HK strain attenuation with fewer bacteria in spleens and significantly increased survival of mutant vs. wild-type infected IRF-1(-/-) mice. Upregulation of flagella, QS, and VirB genes, along with downregulation of rpoE1 and related sigma factor, rpoH2 (BMEI0280) suggest that BM-LOV-HK modulates both QS and general stress response regulatory components to control Brucella gene expression on a global level.

  19. Molecular Cooperativity Governs Diverse and Monoallelic Olfactory Receptor Expression

    NASA Astrophysics Data System (ADS)

    Xing, Jianhua; Tian, Xiaojun; Zhang, Hang; Sannerud, Jens

    Multiple-objective optimization is common in biological systems. In the mammalian olfactory system, each sensory neuron stochastically expresses only one out of up to thousands of olfactory receptor (OR) gene alleles; at organism level the types of expressed ORs need to be maximized. The molecular mechanism of this Nobel-Prize winning puzzle remains unresolved after decades of extensive studies. Existing models focus only on monoallele activation, and cannot explain recent observations in mutants, especially the reduced global diversity of expressed ORs in G9a/GLP knockouts. In this work we integrated existing information on OR expression, and proposed an evolutionarily optimized three-layer regulation mechanism, which includes zonal segregation, epigenetic and enhancer competition coupled to a negative feedback loop. This model not only recapitulates monoallelic OR expression, but also elucidates how the olfactory system maximizes and maintains the diversity of OR expression. The model is validated by several experimental results, and particularly underscores cooperativity and synergy as a general design principle of multi-objective optimization in biology. The work is supported by the NIGMS/DMS Mathematical Biology program.

  20. Subinhibitory Concentrations of Perilla Oil Affect the Expression of Secreted Virulence Factor Genes in Staphylococcus aureus

    PubMed Central

    Luo, Mingjing; Li, Hongen; Dong, Jing; Wang, Jianfeng; Leng, Bingfeng; Wang, Xiaoliang; Feng, Haihua; Ren, Wenzhi; Deng, Xuming

    2011-01-01

    Background The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins. Methodology/Principal Findings A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Conclusions/Significance The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins. PMID:21283822

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

    PubMed Central

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

    2016-01-01

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

  2. 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

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

    PubMed

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

    2015-10-05

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

  4. Identification of genomic islands in Chilean Piscirickettsia salmonis strains and analysis of gene expression involved in virulence.

    PubMed

    Lagos, F; Cartes, C; Vera, T; Haussmann, D; Figueroa, J

    2017-10-01

    Piscirickettsia salmonis, an agent of Piscirickettsiosis, is the cause of major losses in the Chilean salmon industry. We identified, characterized and bioinformatically analysed genomic islands in field strains of P. Salmonis, using the bioinformatic software PIPS, that uses the characteristics of the islands of pathogenicity to identify them. We analysed nine partially sequenced genomes in different new field strains, and compared them with the LF-89 (Type strain) genome, selecting a genomic island present in all of them. We then evaluated the relative expression of three genes present in that island. From the obtained results, we conclude that the expression of the tcf gene is directly proportional to the cytopathogenicity in vitro of the bacteria; the product of the dnsa gene could contribute to its pathogenicity, but would be potentiated by one or more factors. The product of the gene liso is necessary for the virulence process and could have functions in early stages of infection. Regarding the strains, the IBM-040 strain showed a significant increase in the expression of all the genes in the study. Contrarily, LF-89 only presented a significant increase in expression of the gene liso, which correlates with the cytopathogenicity in vitro observed in the SHK-1 cells. © 2017 John Wiley & Sons Ltd.

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

    PubMed

    Palmieri, Ana Carolina Basílio; do Amaral, Alexandre Morais; Homem, Rafael Augusto; Machado, Marcos Antonio

    2010-04-01

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

  6. cis-Acting Elements That Control Expression of the Master Virulence Regulatory Gene atxA in Bacillus anthracis

    PubMed Central

    Dale, Jennifer L.; Raynor, Malik J.; Dwivedi, Prabhat

    2012-01-01

    Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5′ and 3′ deletion analysis and site-directed mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A+T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between −13 and +36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax. PMID:22636778

  7. Region between the canine distemper virus M and F genes modulates virulence by controlling fusion protein expression.

    PubMed

    Anderson, Danielle E; von Messling, Veronika

    2008-11-01

    Morbilliviruses, including measles and canine distemper virus (CDV), are nonsegmented, negative-stranded RNA viruses that cause severe diseases in humans and animals. The transcriptional units in their genomes are separated by untranslated regions (UTRs), which contain essential transcription and translation signals. Due to its increased length, the region between the matrix (M) protein and fusion (F) protein open reading frames is of particular interest. In measles virus, the entire F 5' region is untranslated, while several start codons are found in most other morbilliviruses, resulting in a long F protein signal peptide (Fsp). To characterize the role of this region in morbillivirus pathogenesis, we constructed recombinant CDVs, in which either the M-F UTR was replaced with that between the nucleocapsid (N) and phosphoprotein (P) genes, or 106 Fsp residues were deleted. The Fsp deletion alone had no effect in vitro and in vivo. In contrast, substitution of the UTR was associated with a slight increase in F gene and protein expression. Animals infected with this virus either recovered completely or experienced prolonged disease and death due to neuroinvasion. The combination of both changes resulted in a virus with strongly increased F gene and protein expression and complete attenuation. Taken together, our results provide evidence that the region between the morbillivirus M and F genes modulates virulence through transcriptional control of the F gene expression.

  8. Expression of Xylella fastidiosa RpfF in citrus disrupts signaling in Xanthomonas citri subsp. citri and thereby its virulence.

    PubMed

    Caserta, R; Picchi, S C; Takita, M A; Tomaz, J P; Pereira, W E L; Machado, M A; Ionescu, M; Lindow, S; De Souza, A A

    2014-11-01

    Xylella fastidiosa and Xanthomonas citri subsp. citri, that cause citrus variegated chlorosis (CVC) and citrus canker diseases, respectively, utilize diffusible signal factor (DSF) for quorum sensing. DSF, produced by RpfF, are similar fatty acids in both organisms, although a different set of genes is regulated by DSF in each species. Because of this similarity, Xylella fastidiosa DSF might be recognized and affect the biology of Xanthomonas citri. Therefore, transgenic Citrus sinensis and Carrizo citrange plants overexpressing the Xylella fastidiosa rpfF were inoculated with Xanthomonas citri and changes in symptoms of citrus canker were observed. X. citri biofilms formed only at wound sites on transgenic leaves and were thicker; however, bacteria were unable to break through the tissue and form pustules elsewhere. Although abundant growth of X. citri occurred at wound sites on inoculated transgenic leaves, little growth was observed on unwounded tissue. Genes in the DFS-responsive core in X. citri were downregulated in bacteria isolated from transgenic leaves. DSF-dependent expression of engA was suppressed in cells exposed to xylem sap from transgenic plants. Thus, altered symptom development appears to be due to reduced expression of virulence genes because of the presence of antagonists of DSF signaling in X. citri in rpfF-expressing plants.

  9. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

    PubMed Central

    2010-01-01

    Background Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model. Results HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model. Conclusions Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed

  10. Association Between Helicobacter pylori cagA, babA2 Virulence Factors and Gastric Mucosal Interleukin-33 mRNA Expression and Clinical Outcomes in Dyspeptic Patients.

    PubMed

    Shahi, Heshmat; Reiisi, Somayeh; Bahreini, Rasol; Bagheri, Nader; Salimzadeh, Loghman; Shirzad, Hedayatollah

    2015-01-01

    Helicobacter pylori (H. pylori) infection has been reported in more than half of the world human population. It is associated with gastric inflammation and noticeable infiltration of the immune cells to the stomach mucosa by several cytokines secretion. IL-1β, IL-18 have been shown to contribute to H. pylori induced gastritis, but the details of inflammation and association of virulence factors remain unclear. IL-1 cytokine family has a new additional cytokine, Interleukin-33 (IL-33), which is contemplated to have an important role for host defense against microorganisms. H. pylori virulence factors important in gastritis risk are the cag pathogenicity island (cag-PAI) and babA. This study evaluated IL-33 mucosal mRNA expression levels in infected and uninfected patients and its relationship with bacterial virulence factors cagA, babA2 and type of gastritis. Total RNA was extracted from gastric biopsies of 79 H. pylori-infected patients and 51 H. pylori-negative patients. Mucosal IL-33 mRNA expression levels in gastric biopsies were assessed using real-time PCR. Existence of virulence factors were detected by PCR. IL-33 mRNA expression was significantly higher in biopsies of H. pylori-infected patients compared to H. pylori-uninfected patients (P<0.0001). Also there was a direct relationship between virulence factor bab-A2 and enhancement in IL-33 mRNA expression. Furthermore, IL-33 mRNA expression level was significantly lower in chronic gastritis patients compared with patients with active gastritis (P<0.001). IL-33 may play a crucial role in the inflammatory response and induction of the chronic gastritis and severity of inflammatory changes in the gastric mucosa.

  11. Association Between Helicobacter pylori cagA, babA2 Virulence Factors and Gastric Mucosal Interleukin-33 mRNA Expression and Clinical Outcomes in Dyspeptic Patients

    PubMed Central

    Shahi, Heshmat; Reiisi, Somayeh; Bahreini, Rasol; Bagheri, Nader; Salimzadeh, Loghman; Shirzad, Hedayatollah

    2015-01-01

    Helicobacter pylori (H. pylori) infection has been reported in more than half of the world human population. It is associated with gastric inflammation and noticeable infiltration of the immune cells to the stomach mucosa by several cytokines secretion. IL-1β, IL-18 have been shown to contribute to H. pylori induced gastritis, but the details of inflammation and association of virulence factors remain unclear. IL-1 cytokine family has a new additional cytokine, Interleukin-33 (IL-33), which is contemplated to have an important role for host defense against microorganisms. H. pylori virulence factors important in gastritis risk are the cag pathogenicity island (cag-PAI) and babA. This study evaluated IL-33 mucosal mRNA expression levels in infected and uninfected patients and its relationship with bacterial virulence factors cagA, babA2 and type of gastritis. Total RNA was extracted from gastric biopsies of 79 H. pylori-infected patients and 51 H. pylori-negative patients. Mucosal IL-33 mRNA expression levels in gastric biopsies were assessed using real-time PCR. Existence of virulence factors were detected by PCR. IL-33 mRNA expression was significantly higher in biopsies of H. pylori-infected patients compared to H. pylori-uninfected patients (P<0.0001). Also there was a direct relationship between virulence factor bab-A2 and enhancement in IL-33 mRNA expression. Furthermore, IL-33 mRNA expression level was significantly lower in chronic gastritis patients compared with patients with active gastritis (P<0.001). IL-33 may play a crucial role in the inflammatory response and induction of the chronic gastritis and severity of inflammatory changes in the gastric mucosa. PMID:27014647

  12. Fur is required for the activation of virulence gene expression through the induction of the sae regulatory system in Staphylococcus aureus.

    PubMed

    Johnson, Miranda; Sengupta, Mrittika; Purves, Joanne; Tarrant, Emma; Williams, Peter H; Cockayne, Alan; Muthaiyan, Arunachalam; Stephenson, Robert; Ledala, Nagender; Wilkinson, Brian J; Jayaswal, Radheshyam K; Morrissey, Julie A

    2011-01-01

    Our previous studies showed that both Sae and Fur are required for the induction of eap and emp expression in low iron. In this study, we show that expression of sae is also iron-regulated, as sae expression is activated by Fur in low iron. We also demonstrate that both Fur and Sae are required for full induction of the oxidative stress response and expression of non-covalently bound surface proteins in low-iron growth conditions. In addition, Sae is required for the induced expression of the important virulence factors isdA and isdB in low iron. Our studies also indicate that Fur is required for the induced expression of the global regulators Agr and Rot in low iron and a number of extracellular virulence factors such as the haemolysins which are also Sae- and Agr-regulated. Hence, we show that Fur is central to a complex regulatory network that is required for the induced expression of a number of important S. aureus virulence determinants in low iron.

  13. Mycobacterium abscessus Phospholipase C Expression Is Induced during Coculture within Amoebae and Enhances M. abscessus Virulence in Mice

    PubMed Central

    Bakala N'Goma, Jean Claude; Le Moigne, Vincent; Soismier, Nathalie; Laencina, Laura; Le Chevalier, Fabien; Roux, Anne-Laure; Poncin, Isabelle; Serveau-Avesque, Carole; Rottman, Martin; Gaillard, Jean-Louis; Etienne, Gilles; Brosch, Roland; Canaan, Stéphane

    2014-01-01

    Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria. PMID:25486995

  14. Vru (Sub0144) controls expression of proven and putative virulence determinants and alters the ability of Streptococcus uberis to cause disease in dairy cattle

    PubMed Central

    Egan, Sharon A.; Ward, Philip N.; Watson, Michael; Field, Terence R.

    2012-01-01

    The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696). PMID:22383474

  15. AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000

    USDA-ARS?s Scientific Manuscript database

    Plant pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an ECF sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and is active while the bacteria are associa...

  16. 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.

  17. Relatedness of Streptococcus suis Isolates of Various Serotypes and Clinical Backgrounds as Evaluated by Macrorestriction Analysis and Expression of Potential Virulence Traits

    PubMed Central

    Allgaier, Achim; Goethe, Ralph; Wisselink, Henk J.; Smith, Hilde E.; Valentin-Weigand, Peter

    2001-01-01

    We evaluated the genetic diversity of Streptococcus suis isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes SmaI and ApaI allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the S. suis population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains. PMID:11158088

  18. Mycobacterium bovis-infected macrophages from resistant and susceptible cattle exhibited a differential pro-inflammatory gene expression profile depending on strain virulence.

    PubMed

    Alfonseca-Silva, Edgar; Hernández-Pando, Rogelio; Gutiérrez-Pabello, José A

    2016-08-01

    Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular bacterium that normally persists inside host macrophages. However, the influence of bacterial virulence and host resistance on the final outcome in this interaction is not well known. In this study, we infected macrophages isolated from natural disease resistant (R) and susceptible (S) cattle donors with M. bovis strains characterized as attenuated and virulent to assess pro-inflammatory cytokine (TNFα, IL-12, IL-18, IL-1β, IL-6), chemokine (MCP-1, MCP-2, MIP-1), macrophage receptor (MSR1, TLR2, TLR4, MMR) and iNOS mRNA expression levels. Our findings identified a pro-inflammatory gene expression profile as a common feature after M. bovis infection regardless of bacterial virulence, however in S macrophages a superior expression was induced by the attenuated strain, whereas in R macrophages it was accomplished by the virulent M. bovis. A macrophage pro-inflammatory profile is intended to control M. bovis intracellular growth; however the host resistant phenotype plays a determinant role in it, since R macrophages had better intracellular bacterial control than S cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. 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. © 2014 Eur J Oral Sci.

  20. TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae.

    PubMed

    Häse, C C; Mekalanos, J J

    1998-01-20

    The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

  1. 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

  2. The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype-specific targets.

    PubMed

    Danger, Jessica L; Cao, Tram N; Cao, Tran H; Sarkar, Poulomee; Treviño, Jeanette; Pflughoeft, Kathryn J; Sumby, Paul

    2015-04-01

    Bacterial pathogens commonly show intra-species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T-antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT-types), and (c) the streptokinase-encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype-specific manner. Parental, fasX mutant and complemented derivatives of serotype M1 (ska-2, FCT-2), M2 (ska-1, FCT-6), M6 (ska-2, FCT-1) and M28 (ska-1, FCT-4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT-region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen-expressing mice. Our data are the first to identify and characterize serotype-specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.

  3. Comparative proteome profiling of bovine and human Staphylococcus epidermidis strains for screening specifically expressed virulence and adaptation proteins.

    PubMed

    Siljamäki, Pia; Varmanen, Pekka; Kankainen, Matti; Pyörälä, Satu; Karonen, Taru; Iivanainen, Antti; Auvinen, Petri; Paulin, Lars; Laine, Pia K; Taponen, Suvi; Simojoki, Heli; Sukura, Antti; Nyman, Tuula A; Savijoki, Kirsi

    2014-08-01

    The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 (http://proteomecentral.proteomexchange.org/dataset/PXD000404).

  4. 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

  5. Global selection of Plasmodium falciparum virulence antigen expression by host antibodies

    PubMed Central

    Abdi, Abdirahman I.; Warimwe, George M.; Muthui, Michelle K.; Kivisi, Cheryl A.; Kiragu, Esther W.; Fegan, Gregory W.; Bull, Peter C.

    2016-01-01

    Parasite proteins called PfEMP1 that are inserted on the surface of infected erythrocytes, play a key role in the severe pathology associated with infection by the Plasmodium falciparum malaria parasite. These proteins mediate binding of infected cells to the endothelial lining of blood vessels as a strategy to avoid clearance by the spleen and are major targets of naturally acquired immunity. PfEMP1 is encoded by a large multi-gene family called var. Mutually-exclusive transcriptional switching between var genes allows parasites to escape host antibodies. This study examined in detail the patterns of expression of var in a well-characterized sample of parasites from Kenyan Children. Instead of observing clear inverse relationships between the expression of broad sub-classes of PfEMP1, we found that expression of different PfEMP1 groups vary relatively independently. Parasite adaptation to host antibodies also appears to involve a general reduction in detectable var gene expression. We suggest that parasites switch both between different PfEMP1 variants and between high and low expression states. Such a strategy could provide a means of avoiding immunological detection and promoting survival under high levels of host immunity. PMID:26804201

  6. The Riboflavin analog roseoflavin targets an FMN-riboswitch and blocks Listeria monocytogenes growth, but also stimulates virulence gene-expression and infection

    PubMed Central

    Mansjö, Mikael

    2011-01-01

    During recent years, riboswitches have emerged as potential targets for novel antibacterial substances. In this study, we investigated how one flavin analog, roseoflavin, affected the gene-expression, growth and infectivity of the human bacterial pathogen Listeria monocytogenes to determine the potential of this analog to function as an antibacterial substance. The results indicate that roseoflavin has a profound inhibiting effect on the growth of L. monocytogenes at very low concentrations. Also, expression of the gene located downstream of the FMN riboswitch, a riboflavin transporter, was blocked by the addition of roseoflavin. Base-substitution mutations in the FMN riboswitch allowed the bacteria to grow in the presence of roseoflavin, showing that roseoflavin targeted the FMN riboswitch directly. Surprisingly, we found that roseoflavin stimulated L. monocytogenes virulence gene expression and infection abilities in a mechanism independent of the FMN riboswitch. Our results suggest that roseoflavin can block growth but also enhance Listeria virulence. PMID:21593602

  7. Natural plant products inhibits growth and alters the swarming motility, biofilm formation, and expression of virulence genes in enteroaggregative and enterohemorrhagic Escherichia coli.

    PubMed

    García-Heredia, Alam; García, Santos; Merino-Mascorro, José Ángel; Feng, Peter; Heredia, Norma

    2016-10-01

    The purpose of this study was to determine the effects of plant products on the growth, swarming motility, biofilm formation and virulence gene expression in enterohemorrhagic Escherichia coli O157:H7 and enteroaggregative E. coli strain 042 and a strain of O104:H4 serotype. Extracts of Lippia graveolens and Haematoxylon brassiletto, and carvacrol, brazilin were tested by an antimicrobial microdilution method using citral and rifaximin as controls. All products showed bactericidal activity with minimal bactericidal concentrations ranging from 0.08 to 8.1 mg/ml. Swarming motility was determined in soft LB agar. Most compounds reduced swarming motility by 7%-100%; except carvacrol which promoted motility in two strains. Biofilm formation studies were done in microtiter plates. Rifaximin inhibited growth and reduced biofilm formation, but various concentrations of other compounds actually induced biofilm formation. Real time PCR showed that most compounds decreased stx2 expression. The expression of pic and rpoS in E. coli 042 were suppressed but in E. coli O104:H4 they varied depending on compounds. In conclusion, these extracts affect E. coli growth, swarming motility and virulence gene expression. Although these compounds were bactericidal for pathogenic E. coli, sublethal concentrations had varied effects on phenotypic and genotypic traits, and some increased virulence gene expression.

  8. A hemoglobin-binding outer membrane protein is involved in virulence expression by Haemophilus ducreyi in an animal model.

    PubMed Central

    Stevens, M K; Porcella, S; Klesney-Tait, J; Lumbley, S; Thomas, S E; Norgard, M V; Radolf, J D; Hansen, E J

    1996-01-01

    Haemophilus ducreyi exhibits a requirement for exogenously supplied heme for aerobic growth in vitro. Nine of ten wild-type isolates of H. ducreyi were shown to contain a readily detectable hemoglobin-binding activity. Spontaneous hemoglobin-binding-negative mutants of two of these wild-type isolates lost the ability to express an outer membrane protein with an apparent molecular mass of approximately 100 kDa. Similarly, the single wild-type isolate that lacked the ability to bind hemoglobin also appeared to lack expression of this same 100-kDa protein. A monoclonal antibody (5A9) to this 100-kDa protein was used to identify a recombinant clone which possessed an H. ducreyi chromosomal fragment containing the gene encoding the 100-kDa protein; this protein was designated hemoglobin utilization protein A (HupA). Nucleotide sequence analysis of the hupA gene revealed that the predicted protein, with a calculated molecular mass of 108 kDa, was similar to TonB-dependent outer membrane proteins of other bacteria. Increasing the concentration of heme in the growth medium resulted in decreased expression of the HupA protein. Mutant analysis was used to prove that the HupA protein was essential for the utilization by H. ducreyi of both hemoglobin and hemoglobin-haptoglobin as sources of heme in vitro. In addition, it was found that an isogenic hupA mutant was less virulent than the wild-type parent strain in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi. PMID:8613384

  9. Virulence gene expression by Staphylococcus epidermidis biofilm cells exposed to antibiotics.

    PubMed

    Gomes, Fernanda; Teixeira, Pilar; Cerca, Nuno; Ceri, Howard; Oliveira, Rosário

    2011-06-01

    Staphylococcus epidermidis have become important causes of nosocomial infections, as its pathogenesis is correlated with the ability to form biofilms on polymeric surfaces. Production of poly-N-acetylglucosamine (PNAG) is crucial for S. epidermidis biofilm formation and is synthesized by the gene products of the icaADBC gene cluster. Production of PNAG/polysaccharide intercellular adhesin and biofilm formation are regulated by the alternative sigma factor, σ(B), and is influenced by a variety of environmental conditions including disinfectants and other antimicrobial substances. The susceptibility of five S. epidermidis strains to antibiotics alone and in double combination was previously tested. Our results demonstrated that some combinations are active and present a general broad spectrum against S. epidermidis biofilms, namely rifampicin-clindamycin and rifampicin-gentamicin. In the present study, it was investigated whether the combination of rifampicin with clindamycin and gentamicin and these antibiotics alone influence the expression of specific genes (icaA and rsbU) of S. epidermidis within biofilms using real-time polymerase chain reaction. The data showed that in most cases the expression of both genes tested significantly increased after exposure to antimicrobial agents alone and in combination. Besides having a similar antimicrobial effect, rifampicin combined with clindamycin and gentamicin induced a lower expression of biofilm-related genes relatively to rifampicin alone. Associated with the advantage of combinatorial therapy in avoiding the emergence of antibiotic resistance, this study demonstrated that it can also cause a lower genetic expression of icaA and rsbU genes, which are responsible for PNAG/polysaccharide intercellular adhesin production, and consequently reduce biofilm formation recidivism, relatively to rifampicin alone.

  10. Evaluation of three plant extracts against biofilm formation and expression of quorum sensing regulated virulence factors in Pseudomonas aeruginosa.

    PubMed

    Karbasizade, Vajihe; Dehghan, Parichehr; Sichani, Maryam Mohammadi; Shahanipoor, Kahin; Jafari, Reyhaneh; Yousefian, Rozita

    2017-03-01

    Following the increasing antibiotic resistance of pathogenic bacteria, the use of medicinal herbs as antibacterial agents has attracted growing attention. Pseudomonas aeruginosa is a human opportunistic pathogen that uses quorum sensing for regulating virulence gene expression (pyocyanin, protease, and elastase production and biofilm formation). This study examined the anti-quorum sensing activity of Quercus infectoria, Zataria multiflora and Trachyspermum copticum extracts on standard P. aeruginosa strain. The minimum inhibitory concentration (MIC) of Q. infectoria, Z. multiflora and T. copticum extracts for standard P. aeruginosa strain was determined through micro dilution. Microtiter plates were used to evaluate the anti-quorum sensing effects of the three extracts (at a sub-MIC concentration) on pyocyanin, protease, and elastase production and biofilm formation. The acetone extract of Q. infectoria showed the highest anti-quorum sensing activity and reduced the pyocyanin, protease, and elastase production and biofilm formation by 89.1%, 78%, 73.3%, and 70.1%, respectively. The corresponding values were 88.2%, 72.1%, 69%, and 61.1% for the methanol extract of Z. multiflora and 70.6%, 63.42%, 60.1%, and 59.1% for the methanol extract of T. copticum. Considering the high anti-quorum sensing activity of the studied extracts, especially the acetone extract of Q. infectoria, these herbs can be used as antipathogenic drugs.

  11. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants

    PubMed Central

    Sinha, Deepak K.; Chandran, Predeesh; Timm, Alicia E.; Aguirre-Rojas, Lina; Smith, C. Michael

    2016-01-01

    The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion. PMID:26815857

  12. Effects of different promoters on the virulence and immunogenicity of a HIV-1 Env-expressing recombinant vaccinia vaccine.

    PubMed

    Isshiki, Mao; Zhang, Xianfeng; Sato, Hirotaka; Ohashi, Takashi; Inoue, Makoto; Shida, Hisatoshi

    2014-02-07

    Previously, we developed a vaccination regimen that involves priming with recombinant vaccinia virus LC16m8Δ (rm8Δ) strain followed by boosting with a Sendai virus-containing vector. This protocol induced both humoral and cellular immune responses against the HIV-1 envelope protein. The current study aims to optimize this regimen by comparing the immunogenicity and safety of two rm8Δ strains that express HIV-1 Env under the control of a moderate promoter, p7.5, or a strong promoter, pSFJ1-10. m8Δ-p7.5-JRCSFenv synthesized less gp160 but showed significantly higher growth potential than m8Δ-pSFJ-JRCSFenv. The two different rm8Δ strains induced antigen-specific immunity; however, m8Δ-pSFJ-JRCSFenv elicited a stronger anti-Env antibody response whereas m8Δ-p7.5-JRCSFenv induced a stronger Env-specific cytotoxic T lymphocyte response. Both strains were less virulent than the parental m8Δ strain, suggesting that they would be safe for use in humans. These findings indicate the vaccine can be optimized to induce favorable immune responses (either cellular or humoral), and forms the basis for the rational design of an AIDS vaccine using recombinant vaccinia as the delivery vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. [Construction and Identification of Transgenic Strain of Toxoplasma gondii High-expressing Virulence Factor ROP18].

    PubMed

    Jiang, Zong-ru; An, Ran; Yang, Jie; Wan, Li-juan; Du, Jan

    2015-02-01

    To construct a transgenic strain of Toxoplasma gondii high-expressing ROP18. The gene sequence of encoding ROP18 was amplified by RT-PCR with RNA of T. gondii RH strain. The purified PCR product was subcloned into pCR-Blunt II-Top vector to construct pROP18. The gene sequence of encoding ROP18 was amplified from pROP18, and subcloned into pTUB8-mycGFPPftail-Ty1. The recombinant plasmid pTUB8-ROP18-Ty1 was electroporated into T. gondii RH strain. Stable transgenic cells were selected in the presence of 25 µg/ml mycophenolic acid and 50 µg/ml xanthine, and parasite clones were isolated by limiting dilution after drug selection. The expression of ROP18 in transgenic parasites was detected by immunofluorescence analysis and Western blotting. Giemsa assay was used to detect the proliferation rate of RH strain and the transgenic strain of T. gondii high-expressing ROP18. Twenty mice were divided into two groups. Each mouse in RH strain group was intraperitoneally injected with 1 x 10(3) RH strain, and that of transgenic strain group received 1 x 10(3) transgenic high-expressing ROP18 strain. The full-length sequence of ROP18 gene (1,665 bp) was amplified by RT-PCR. The recombinant plasmid pTUB8-ROP18-Ty1 was identified by restriction enzyme digestion and sequencing methods. Western blotting analysis showed that the transgenic high-expressing ROP18 strain expressed ROP18-Ty1 (Mr 56,000). Immunofluorescence assay showed that ROP18-Ty1 was localized in the rhoptry of T. gondii. Giemsa assay confirmed that ROP18 protein enhanced the proliferation of T. gondii. On the 6th, 12th, and 24th hour after HFF cells infected with T. gondii, the number of tachyzoites in transgenic strain group was 100.0 ± 16.9, 476.0 ± 31.1, and 860.0 ± 52.3, respectively, higher than that of RH strain group (88.0 ± 16.9, 300.0 ± 11.3, 675.0 ± 35.4) (P < 0.05). In RH strain group, all the mice were survival on the 8th day post-infection, the survival rate on the 14th day was 30% (3/10), and

  14. 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

  15. 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

  16. Identification and characterization of msa (SA1233), a gene involved in expression of SarA and several virulence factors in Staphylococcus aureus.

    PubMed

    Sambanthamoorthy, Karthik; Smeltzer, Mark S; Elasri, Mohamed O

    2006-09-01

    The staphylococcal accessory regulator (sarA) plays a central role in the regulation of virulence in Staphylococcus aureus. To date, studies involving sarA have focused on its activity as a global regulator that modulates transcription of a wide variety of genes (>100) and its role in virulence. However, there is also evidence to suggest the existence of accessory elements that modulate SarA production and/or function. A reporter system was developed to identify such elements, and a new gene, msa (SA1233), mutation of which results in reduced expression of SarA, was identified and characterized. Additionally, it was shown that mutation of msa resulted in altered transcription of the accessory gene regulator (agr) and the genes encoding several virulence factors including alpha toxin (hla) and protein A (spa). However, the impact of mutating msa was different in the laboratory strain RN6390 and the clinical isolate UAMS-1. For instance, mutation of msa caused a decrease in spa and hla transcription in RN6390 but had a different effect in UAMS-1. The strain-dependent effects of the msa mutation were similar to those observed previously, which suggests that msa may modulate the production of specific virulence factors through its impact on sarA. Interestingly, sequence analysis of Msa suggests that it is a putative membrane protein with three membrane-spanning regions, indicating that Msa might interact with the environment. The findings show that msa is involved in the expression of SarA and several virulence factors.

  17. Interplay between Fur and HNS in controlling virulence gene expression in Salmonella typhimurium.

    PubMed

    Prajapat, Mahendra Kumar; Saini, Supreet

    2012-11-01

    Salmonella enterica is responsible for a large number of diseases in a wide-range of hosts. Two of the global regulators involved in controlling gene expression during the infection cycle of the bacterium are Fur and HNS. In this paper, we demonstrate computationally that Fur and HNS have disproportionately high density of binding sites in the Pathogenicity Islands on the Salmonella chromosome. Moreover, the frequency of binding sites for the two proteins is correlated throughout the genome of the organism. These results indicate a complex interplay between Fur and HNS in regulating cellular global behavior. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Identification by microarray of a common pattern of gene expression in intact intestine and cultured intestinal cells exposed to virulent Aeromonas hydrophila isolates.

    PubMed

    Hayes, Samuel L; Lye, Bethany R; Lye, Dennis J; Rodgers, Mark R; Stelma, Gerard; Malard, Joel M; Vandewalle, Alain; Vesper, Stephen J

    2006-09-01

    The genus Aeromonas comprises known virulent and avirulent isolates and has been implicated in waterborne disease. A common infection model of human gastroenteritis associated with A. hydrophila uses neonatal mice. The goal of this research was to evaluate whether a murine small intestinal cell line could provide comparable results to the gene expression changes in the neonatal mouse model. Changes in mRNA expression in host cell cultures and intestinal tissues were measured after exposure to virulent Aeromonas hydrophila strains. A. hydrophila caused the up-regulation of more than 200 genes in neonates and over 50 genes in cell culture. Twenty-six genes were found to be in common between the two models, of which the majority are associated with the innate immune response.

  19. Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana.

    PubMed

    Fang, Weiguo; Feng, Jin; Fan, Yanhua; Zhang, Yongjun; Bidochka, Michael J; Leger, Raymond J St; Pei, Yan

    2009-10-01

    Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT(50), without a reduction in LC(50). The LT(50) of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC(50), more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.

  20. Trans-Cinnamaldehyde, Carvacrol, and Eugenol Reduce Campylobacter jejuni Colonization Factors and Expression of Virulence Genes in Vitro

    PubMed Central

    Upadhyay, Abhinav; Arsi, Komala; Wagle, Basanta R.; Upadhyaya, Indu; Shrestha, Sandip; Donoghue, Ann M.; Donoghue, Dan J.

    2017-01-01

    Campylobacter jejuni is a major foodborne pathogen that causes severe gastroenteritis in humans characterized by fever, diarrhea, and abdominal cramps. In the human gut, Campylobacter adheres and invades the intestinal epithelium followed by cytolethal distending toxin mediated cell death, and enteritis. Reducing the attachment and invasion of Campylobacter to intestinal epithelium and expression of its virulence factors such as motility and cytolethal distending toxin (CDT) production could potentially reduce infection in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentration not inhibiting bacterial growth) of three GRAS (generally recognized as safe) status phytochemicals namely trans-cinnamaldehyde (TC; 0.005, 0.01%), carvacrol (CR; 0.001, 0.002%), and eugenol (EG; 0.005, 0.01%) in reducing the attachment, invasion, and translocation of C. jejuni on human intestinal epithelial cells (Caco-2). Additionally, the effect of these phytochemicals on Campylobacter motility and CDT production was studied using standard bioassays and gene expression analysis. All experiments had duplicate samples and were replicated three times on three strains (wild type S-8, NCTC 11168, 81–176) of C. jejuni. Data were analyzed using ANOVA with GraphPad ver. 6. Differences between the means were considered significantly different at P < 0.05. The majority of phytochemical treatments reduced C. jejuni adhesion, invasion, and translocation of Caco-2 cells (P < 0.05). In addition, the phytochemicals reduced pathogen motility and production of CDT in S-8 and NCTC 11168 (P < 0.05). Real-time quantitative PCR revealed that phytochemicals reduced the transcription of select C. jejuni genes critical for infection in humans (P < 0.05). Results suggest that TC, CR, and EG could potentially be used to control C. jejuni infection in humans. PMID:28487683

  1. The micronutrient zinc inhibits EAEC strain 042 adherence, biofilm formation, virulence gene expression, and epithelial cytokine responses benefiting the infected host

    PubMed Central

    Medeiros, Pedro; Bolick, David T; Roche, James K; Noronha, Francisco; Pinheiro, Caio; Kolling, Glynis L; Lima, Aldo; Guerrant, Richard L

    2013-01-01

    Enteroaggregative Escherichia coli (EAEC) is a major pathogen worldwide, associated with diarrheal disease in both children and adults, suggesting the need for new preventive and therapeutic treatments. We investigated the role of the micronutrient zinc in the pathogenesis of an E. coli strain associated with human disease. A variety of bacterial characteristics—growth in vitro, biofilm formation, adherence to IEC-6 epithelial cells, gene expression of putative EAEC virulence factors as well as EAEC-induced cytokine expression by HCT-8 cells—were quantified. At concentrations (≤ 0.05 mM) that did not alter EAEC growth (strain 042) but that are physiologic in serum, zinc markedly decreased the organism’s ability to form biofilm (P < 0.001), adhere to IEC-6 epithelial cells (P < 0.01), and express putative EAEC virulence factors (aggR, aap, aatA, virK) (P < 0.03). After exposure of the organism to zinc, the effect on virulence factor generation was prolonged (>3 h). Further, EAEC-induced IL-8 mRNA and protein secretion by HCT-8 epithelial cells were significantly reduced by 0.05 mM zinc (P < 0.03). Using an in vivo murine model of diet-induced zinc-deficiency, oral zinc supplementation (0.4 µg/mouse daily) administered after EAEC challenge (1010 CFU/mouse) significantly abrogated growth shortfalls (by >90%; P < 0.01); furthermore, stool shedding was reduced (days 9–11) but tissue burden of organisms in the intestine was unchanged. These findings suggest several potential mechanisms whereby physiological levels of zinc alter pathogenetic events in the bacterium (reducing biofilm formation, adherence to epithelium, virulence factor expression) as well as the bacterium’s effect on the epithelium (cytokine response to exposure to EAEC) to alter EAEC pathogenesis in vitro and in vivo. These effects may help explain and extend the benefits of zinc in childhood diarrhea and malnutrition. PMID:23958904

  2. Serum influences the expression of Pseudomonas aeruginosa quorum-sensing genes and QS-controlled virulence genes during early and late stages of growth

    PubMed Central

    Kruczek, Cassandra; Qaisar, Uzma; Colmer-Hamood, Jane A; Hamood, Abdul N

    2014-01-01

    In response to diverse environmental stimuli at different infection sites, Pseudomonas aeruginosa, a serious nosocomial pathogen, coordinates the production of different virulence factors through a complicated network of the hierarchical quorum-sensing (QS) systems including the las, rhl, and the 2-alkyl-4-quinolone-related QS systems. We recently showed that at early stages of growth serum alters the expression of numerous P. aeruginosa genes. In this study, we utilized transcriptional analysis and enzyme assays to examine the effect of serum on the QS and QS-controlled virulence factors during early and late phases of growth of the P. aeruginosa strain PAO1. At early phase, serum repressed the transcription of lasI, rhlI, and pqsA but not lasR or rhlR. However, at late phase, serum enhanced the expression of all QS genes. Serum produced a similar effect on the synthesis of the autoinducers 3OC12-HSL, C4-HSL, and HHQ/PQS. Additionally, serum repressed the expression of several QS-controlled genes in the early phase, but enhanced them in the late phase. Furthermore, serum influenced the expression of different QS-positive (vqsR, gacA, and vfr) as well as QS-negative (rpoN, qscR, mvaT, and rsmA) regulatory genes at either early or late phases of growth. However, with the exception of PAOΔvfr, we detected comparable levels of lasI/lasR expression in PAO1 and PAO1 mutants defective in these regulatory genes. At late stationary phase, serum failed to enhance lasI/lasR expression in PAOΔvfr. These results suggest that depending on the phase of growth, serum differentially influenced the expression of P. aeruginosa QS and QS-controlled virulence genes. In late phase, serum enhanced the expression of las genes through vfr. PMID:24436158

  3. RNA- and protein-mediated control of Listeria monocytogenes virulence gene expression

    PubMed Central

    Lebreton, Alice; Cossart, Pascale

    2017-01-01

    ABSTRACT The model opportunistic pathogen Listeria monocytogenes has been the object of extensive research, aiming at understanding its ability to colonize diverse environmental niches and animal hosts. Bacterial transcriptomes in various conditions reflect this efficient adaptability. We review here our current knowledge of the mechanisms allowing L. monocytogenes to respond to environmental changes and trigger pathogenicity, with a special focus on RNA-mediated control of gene expression. We highlight how these studies have brought novel concepts in prokaryotic gene regulation, such as the ‘excludon’ where the 5′-UTR of a messenger also acts as an antisense regulator of an operon transcribed in opposite orientation, or the notion that riboswitches can regulate non-coding RNAs to integrate complex metabolic stimuli into regulatory networks. Overall, the Listeria model exemplifies that fine RNA tuners act together with master regulatory proteins to orchestrate appropriate transcriptional programmes. PMID:27217337

  4. Recombinant Probiotic Expressing Listeria Adhesion Protein Attenuates Listeria monocytogenes Virulence In Vitro

    PubMed Central

    Koo, Ok Kyung; Amalaradjou, Mary Anne Roshni; Bhunia, Arun K.

    2012-01-01

    Background Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. Methodology/Principal Findings The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (LbpLAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to LbpLAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, LbpLAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. LbpLAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, LbpLAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. Conclusions/Significance Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, LbpLAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant

  5. The vapA co-expressed virulence plasmid gene vcgB (orf10) of the intracellular actinomycete Rhodococcus equi.

    PubMed

    Miranda-Casoluengo, Raúl; Miranda-Casoluengo, Aleksandra A; O'Connell, Enda P; Fahey, Ruth J; Boland, Clara A; Vázquez-Boland, Jose A; Meijer, Wim G

    2011-08-01

    The virulence plasmid of the pathogenic actinomycete Rhodococcus equi is essential for proliferation of this pathogen in macrophages and the development of disease. The pathogenicity island of this plasmid encodes a family of virulence-associated proteins (Vap), one of which (VapA) is a virulence factor. This paper describes the vcgAB operon (vapA co-expressed gene), located upstream of the vapA operon. Transcription of the vcgAB operon gave rise to transcripts with a half-life similar to those determined for other virulence plasmid genes (1.8 min). Transcription started at a promoter similar to the vapA promoter, and proceeded through an inefficient terminator into the downstream vcgC gene. In addition, vcgC is also transcribed from a promoter downstream of vcgB. The vcgAB and vapA operons were coordinately regulated by temperature and pH in a synergistic manner. The latter parameter only affected transcription at higher growth temperatures, indicating that temperature is the dominant regulatory signal. Transcription of the vcgAB operon increased 10-fold during the late exponential and stationary growth phases. Transcription was also upregulated during the initial hours following phagocytosis by phagocytic cells. In contrast to vcgA and vcgC, the vcgB gene is conserved in the porcine VapB-encoding plasmid, as well as in pathogenic mycobacteria. The coordinated regulation of vcgB and vapA, transcription of vcgB following phagocytosis and conservation of vcgB in pathogenic mycobacteria indicate a role for vcgB and the vcg genes in the virulence of R. equi.

  6. The global response regulator ExpA controls virulence gene expression through RsmA-mediated and RsmA-independent pathways in Pectobacterium wasabiae SCC3193.

    PubMed

    Broberg, M; Lee, G W; Nykyri, J; Lee, Y H; Pirhonen, M; Palva, E T

    2014-03-01

    ExpA (GacA) is a global response regulator that controls the expression of major virulence genes, such as those encoding plant cell wall-degrading enzymes (PCWDEs) in the model soft rot phytopathogen Pectobacterium wasabiae SCC3193. Several studies with pectobacteria as well as related phytopathogenic gammaproteobacteria, such as Dickeya and Pseudomonas, suggest that the control of virulence by ExpA and its homologues is executed partly by modulating the activity of RsmA, an RNA-binding posttranscriptional regulator. To elucidate the extent of the overlap between the ExpA and RsmA regulons in P. wasabiae, we characterized both regulons by microarray analysis. To do this, we compared the transcriptomes of the wild-type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray data for selected virulence-related genes were confirmed through quantitative reverse transcription (qRT-PCR). Subsequently, assays were performed to link the observed transcriptome differences to changes in bacterial phenotypes such as growth, motility, PCWDE production, and virulence in planta. An extensive overlap between the ExpA and RsmA regulons was observed, suggesting that a substantial portion of ExpA regulation appears to be mediated through RsmA. However, a number of genes involved in the electron transport chain and oligogalacturonide metabolism, among other processes, were identified as being regulated by ExpA independently of RsmA. These results suggest that ExpA may only partially impact fitness and virulence via RsmA.

  7. Terpenoids from Platostoma rotundifolium (Briq.) A. J. Paton Alter the Expression of Quorum Sensing-Related Virulence Factors and the Formation of Biofilm in Pseudomonas aeruginosa PAO1

    PubMed Central

    Rasamiravaka, Tsiry; Ngezahayo, Jérémie; Pottier, Laurent; Oliveira Ribeiro, Sofia; Souard, Florence; Hari, Léonard; Stévigny, Caroline; El Jaziri, Mondher; Duez, Pierre

    2017-01-01

    Platostoma rotundifolium (Briq.) A. J. Paton aerial parts are widely used in Burundi traditional medicine to treat infectious diseases. In order to investigate their probable antibacterial activities, crude extracts from P. rotundifolium were assessed for their bactericidal and anti-virulence properties against an opportunistic bacterial model, Pseudomonas aeruginosa PAO1. Whereas none of the tested extracts exert bacteriostatic and/or bactericidal proprieties, the ethyl acetate and dichloromethane extracts exhibit anti-virulence properties against Pseudomonas aeruginosa PAO1 characterized by an alteration in quorum sensing gene expression and biofilm formation without affecting bacterial viability. Bioguided fractionation of the ethyl acetate extract led to the isolation of major anti-virulence compounds that were identified from nuclear magnetic resonance and high-resolution molecular spectroscopy spectra as cassipourol, β-sitosterol and α-amyrin. Globally, cassipourol and β-sitosterol inhibit quorum sensing-regulated and -regulatory genes expression in las and rhl systems without affecting the global regulators gacA and vfr, whereas α-amyrin had no effect on the expression of these genes. These terpenoids disrupt the formation of biofilms at concentrations down to 12.5, 50 and 50 µM for cassipourol, β-sitosterol and α-amyrin, respectively. Moreover, these terpenoids reduce the production of total exopolysaccharides and promote flagella-dependent motilities (swimming and swarming). The isolated terpenoids exert a wide range of inhibition processes, suggesting a complex mechanism of action targeting P. aeruginosa virulence mechanisms which support the wide anti-infectious use of this plant species in traditional Burundian medicine. PMID:28613253

  8. Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients.

    PubMed Central

    Ollert, M W; Wende, C; Görlich, M; McMullan-Vogel, C G; Borg-von Zepelin, M; Vogel, C W; Korting, H C

    1995-01-01

    The increased prevalence and the severity of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients are attributed exclusively to the virus-induced immune deficiency of the host. The present study was aimed at answering the question of whether Candida albicans secretory proteinase, a putative virulence factor of the opportunistic C. albicans yeast, has any potential influence on the clinical manifestation of oropharyngeal candidiasis in HIV-positive patients. We measured the secretory proteinase activities of clinical C. albicans isolates from the oropharynges of either HIV-positive individuals (n = 100) or a control group (n = 122). The mean secretory proteinase activity of C. albicans isolates from the HIV-positive group (4,255 +/- 2,372 U/liter) was significantly higher compared with that of isolates from the control group (2,324 +/- 1,487 U/liter) (P < 0.05). The higher level of secretory proteinase activity in the culture supernatants of individual C. albicans isolates correlated with the increased level of proteinase expression on the cell surface, as revealed by cytofluorometry, and with higher levels of secretion of the immunodetectable protein, as shown by Western blotting (immunoblotting). Proteinase activity within the population of C. albicans isolates from HIV-positive individuals was independent of the patient's clinical disease stage and the CD4+/CD8+ cell numbers. Furthermore, no correlation of the proteinase activities with the C. albicans serotype was found, although C. albicans serotype B was significantly more frequent in the HIV-positive group (40%) compared with that in the control group (12%). However, a positive correlation of proteinase activity to antifungal susceptibility was evident.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8567880

  9. Virulence factors of Helicobacter pylori vacA increase markedly gastric mucosal TGF-β1 mRNA expression in gastritis patients.

    PubMed

    Rahimian, Ghorbanali; Sanei, Mohammad Hosein; Shirzad, Hedayatollah; Azadegan-Dehkordi, Fatemeh; Taghikhani, Afshin; Salimzadeh, Loghman; Hashemzadeh-Chaleshtori, Morteza; Rafieian-Kopaei, Mahmoud; Bagheri, Nader

    2014-01-01

    Helicobacter pylori (H. pylori) infection is the main cause of gastric inflammation. Regulatory T cells (Treg cells) suppress the activation and proliferation of antigen-specific T cells and mediate immunologic tolerance. TGF-β1 was shown to be secreted in a subset of Treg cells known as 'Th3 cells'. These cells have not been sufficiently studied in context to H. pylori-induced inflammation in human gastric mucosa. In this study we therefore, aimed to investigate the expression of TGF-β1 in the context of H. pylori colonization in chronic gastritis, to examine the relationship between it and histopathologic findings and to compare it with virulence factors. Total RNA was extracted from gastric biopsies of 48 H. pylori-infected patients and 38 H. pylori-negative patients with gastritis. Mucosal TGF-β1 mRNA expression in H. pylori-infected and uninfected gastric biopsies was determined by real-time PCR. Presence of vacA, cagA, iceA, babA2 and oipA virulence factors was evaluated using PCR. TGF-β1 mRNA expression was significantly increased in biopsies of H. pylori-infected patients compared to H. pylori-uninfected patients. There was association between virulence factors and TGF-β1 mRNA expression. TGF-β1 mRNA expression in mucosa was significantly higher in patients with vacA s1 and s1m1. TGF-β1 may play an important role in the inflammatory response and promote the chronic and persistent inflammatory changes in the gastric. This may ultimately influence the outcome of H. pylori-associated diseases that arise within the context of gastritis and vacA may suffice to induce expression of TGF-β1 mRNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Virulence Determination

    USDA-ARS?s Scientific Manuscript database

    This chapter reviews the in vitro and in vivo assays that are available for determination of pathogenic potential of Listeria monocytogenes bacteria, highlighting the value of using multiplex PCR for rapid and accurate assessment of listerial virulence....

  11. Classification of e-government documents based on cooperative expression of word vectors

    NASA Astrophysics Data System (ADS)

    Fu, Qianqian; Liu, Hao; Wei, Zhiqiang

    2017-03-01

    The effective document classification is a powerful technique to deal with the huge amount of e-government documents automatically instead of accomplishing them manually. The word-to-vector (word2vec) model, which converts semantic word into low-dimensional vectors, could be successfully employed to classify the e-government documents. In this paper, we propose the cooperative expressions of word vector (Co-word-vector), whose multi-granularity of integration explores the possibility of modeling documents in the semantic space. Meanwhile, we also aim to improve the weighted continuous bag of words model based on word2vec model and distributed representation of topic-words based on LDA model. Furthermore, combining the two levels of word representation, performance result shows that our proposed method on the e-government document classification outperform than the traditional method.

  12. Listeria monocytogenes and Salmonella enterica enteritidis biofilms susceptibility to different disinfectants and stress-response and virulence gene expression of surviving cells.

    PubMed

    Rodrigues, Diana; Cerca, Nuno; Teixeira, Pilar; Oliveira, Rosário; Ceri, Howard; Azeredo, Joana

    2011-06-01

    Disinfection of food contact surfaces is a challenging task, aggravated by bacteria's capacity to survive and/or resist antimicrobials by means of mechanisms not yet completely understood. This work evaluated the susceptibility of Listeria monocytogenes and Salmonella enterica biofilms to four disinfectants, and analyzed how those chemical agents influenced stress-response and virulence genes expression by surviving cells. Three strains of each bacterial species mentioned were used, and their biofilms were treated with sodium hypochlorite, benzalkonium chloride, hydrogen peroxide, and triclosan using the Calgary Biofilm Device. Expression of L. monocytogenes and S. enterica stress-response genes cplC and ropS, and virulence genes prfA and avrA, respectively, was analyzed through quantitative real-time polymerase chain reaction. Results showed sodium hypochlorite to have the lowest minimum biofilm eradication concentration values (3.125 μg/ml), whereas triclosan had the worst performance since no S. enterica biofilm eradication was achieved even at the maximum concentration used (4,000 μg/ml). L. monocytogenes stress-response gene and S. enterica virulence gene were significantly upregulated in surviving cells compared with controls. In general, this work points out sodium hypochlorite as the most effective disinfectant against biofilms of both species used, and L. monocytogenes biofilms to be more susceptible to disinfection than S. enterica biofilms. Moreover, it was found that disinfection surviving biofilm cells seem to develop a stress response and/or become more virulent, which may compromise food safety and potentiate public health risk.

  13. The cyclic AMP receptor protein, CRP, is required for both virulence and expression of the minimal CRP regulon in Yersinia pestis biovar microtus.

    PubMed

    Zhan, Lingjun; Han, Yanping; Yang, Lei; Geng, Jing; Li, Yingli; Gao, He; Guo, Zhaobiao; Fan, Wei; Li, Gang; Zhang, Lianfeng; Qin, Chuan; Zhou, Dongsheng; Yang, Ruifu

    2008-11-01

    The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a >15,000-fold loss of virulence after subcutaneous infection but a <40-fold increase in the 50% lethal dose by intravenous inoculation. Therefore, CRP is required for the virulence of Y. pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge

  14. The Haemophilus ducreyi Fis protein is involved in controlling expression of the lspB-lspA2 operon and other virulence factors.

    PubMed

    Labandeira-Rey, Maria; Dodd, Dana A; Brautigam, Chad A; Fortney, Kate R; Spinola, Stanley M; Hansen, Eric J

    2013-11-01

    Expression of the lspB-lspA2 operon encoding a virulence-related two-partner secretion system in Haemophilus ducreyi 35000HP is directly regulated by the CpxRA regulatory system (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, we show that this secretion system is also regulated by the small nucleoid-associated protein Fis. Inactivation of the H. ducreyi fis gene resulted in a reduction in expression of both the H. ducreyi LspB and LspA2 proteins. DNA microarray experiments showed that a H. ducreyi fis deletion mutant exhibited altered expression levels of genes encoding other important H. ducreyi virulence factors, including DsrA and Flp1, suggesting a possible global role for Fis in the control of virulence in this obligate human pathogen. While the H. ducreyi Fis protein has a high degree of sequence and structural similarity to the Fis proteins of other bacteria, its temporal pattern of expression was very different from that of enterobacterial Fis proteins. The use of a lacZ-based transcriptional reporter provided evidence which indicated that the H. ducreyi Fis homolog is a positive regulator of gyrB, a gene that is negatively regulated by Fis in enteric bacteria. Taken together, the Fis protein expression data and the observed regulatory effects of Fis in H. ducreyi suggest that this small DNA binding protein has a regulatory role in H. ducreyi which may differ in substantial ways from that of other Fis proteins.

  15. The Ability of Proteus mirabilis To Sense Surfaces and Regulate Virulence Gene Expression Involves FliL, a Flagellar Basal Body Protein

    PubMed Central

    Belas, Robert; Suvanasuthi, Rooge

    2005-01-01

    Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL− mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL+ in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL+ was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression. PMID:16166542

  16. Association and virulence gene expression vary among serotype III group B streptococcus isolates following exposure to decidual and lung epithelial cells.

    PubMed

    Korir, Michelle L; Knupp, David; LeMerise, Kathryn; Boldenow, Erica; Loch-Caruso, Rita; Aronoff, David M; Manning, Shannon D

    2014-11-01

    Group B Streptococcus (GBS) causes severe disease in neonates, the elderly, and immunocompromised individuals. GBS species are highly diverse and can be classified by serotype and multilocus sequence typing. Sequence type 17 (ST-17) strains cause invasive neonatal disease more frequently than strains of other STs. Attachment and invasion of host cells are key steps in GBS pathogenesis. We investigated whether four serotype III strains representing ST-17 (two strains), ST-19, and ST-23 differ in their abilities to attach to and invade both decidual cells and lung epithelial cells. Virulence gene expression following host cell association and exposure to amnion cells was also tested. The ST-17 strains differed in their abilities to attach to and invade decidual cells, whereas there were no differences with lung epithelial cells. The ST-19 and ST-23 strains, however, attached to and invaded decidual cells less than both ST-17 strains. Although the ST-23 strain attached to lung epithelial cells better than ST-17 and -19 strains, none of the strains effectively invaded the lung epithelial cells. Notably, the association with host cells resulted in the differential expression of several virulence genes relative to basal expression levels. Similar expression patterns of some genes were observed regardless of cell type used. Collectively, these results show that GBS strains differ in their abilities to attach to distinct host cell types and express key virulence genes that are relevant to the disease process. Enhancing our understanding of pathogenic mechanisms could aid in the identification of novel therapeutic targets or vaccine candidates that could potentially decrease morbidity and mortality associated with neonatal infections.

  17. Characterization of Pectobacterium carotovorum proteins differentially expressed during infection of Zantedeschia elliotiana in vivo and in vitro which are essential for virulence.

    PubMed

    Wang, Huan; Yang, Zhongling; Du, Shuo; Ma, Lin; Liao, Yao; Wang, Yujie; Toth, Ian; Fan, Jiaqin

    2016-09-27

    The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' (in vitro) and in plant tissues (in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which (clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors.

  18. Differential Expression of Virulence and Stress Fitness Genes between Escherichia coli O157:H7 Strains with Clinical or Bovine-Biased Genotypes▿ †

    PubMed Central

    Vanaja, Sivapriya Kailasan; Springman, Amber C.; Besser, Thomas E.; Whittam, Thomas S.; Manning, Shannon D.

    2010-01-01

    Escherichia coli O157:H7 strains can be classified into different genotypes based on the presence of specific Shiga toxin-encoding bacteriophage insertion sites. Certain O157:H7 genotypes predominate among human clinical cases (clinical genotypes), while others are more frequently found in bovines (bovine-biased genotypes). To determine whether inherent differences in gene expression explain the variation in infectivity of these genotypes, we compared the expression patterns of clinical genotype 1 strains with those of bovine-biased genotype 5 strains using microarrays. Important O157:H7 virulence factors, including locus of enterocyte effacement genes, the enterohemolysin, and several pO157 genes, showed increased expression in the clinical versus bovine-biased genotypes. In contrast, genes essential for acid resistance (e.g., gadA, gadB, and gadC) and stress fitness were upregulated in bovine-biased genotype 5 strains. Increased expression of acid resistance genes was confirmed functionally using a model stomach assay, in which strains of bovine-biased genotype 5 had a 2-fold-higher survival rate than strains of clinical genotype 1. Overall, these results suggest that the increased prevalence of O157:H7 illness caused by clinical genotype 1 strains is due in part to the overexpression of key virulence genes. The bovine-biased genotype 5 strains, however, are more resistant to adverse environmental conditions, a characteristic that likely facilitates O157:H7 colonization of bovines. PMID:19880650

  19. Patterns of Cellular Gene Expression in Swine Macrophages Infected with Highly Virulent Classical Swine Fever Virus Strain Brescia

    USDA-ARS?s Scientific Manuscript database

    Experimental exposure of swine to highly virulent Classical Swine Fever Virus (CSFV) strain Brescia causes an invariably fatal disease of all infected animals by 8 to 14 days post-infection. Host mechanisms involved in this severe outcome of infection have not been clearly established. To understa...

  20. The primary transcriptome of the Escherichia coli O104:H4 pAA plasmid and novel insights into its virulence gene expression and regulation

    PubMed Central

    Berger, Petya; Knödler, Michael; Förstner, Konrad U.; Berger, Michael; Bertling, Christian; Sharma, Cynthia M.; Vogel, Jörg; Karch, Helge; Dobrindt, Ulrich; Mellmann, Alexander

    2016-01-01

    Escherichia coli O104:H4 (E. coli O104:H4), which caused a massive outbreak of acute gastroenteritis and hemolytic uremic syndrome in 2011, carries an aggregative adherence fimbriae I (AAF/I) encoding virulence plasmid, pAA. The importance of pAA in host-pathogen interaction and disease severity has been demonstrated, however, not much is known about its transcriptional organization and gene regulation. Here, we analyzed the pAA primary transcriptome using differential RNA sequencing, which allows for the high-throughput mapping of transcription start site (TSS) and non-coding RNA candidates. We identified 248 TSS candidates in the 74-kb pAA and only 21% of them could be assigned as TSS of annotated genes. We detected TSS for the majority of pAA-encoded virulence factors. Interestingly, we mapped TSS, which could allow for the transcriptional uncoupling of the AAF/I operon, and potentially regulatory antisense RNA candidates against the genes encoding dispersin and the serine protease SepA. Moreover, a computational search for transcription factor binding sites suggested for AggR-mediated activation of SepA expression, which was additionally experimentally validated. This work advances our understanding of the molecular basis of E. coli O104:H4 pathogenicity and provides a valuable resource for further characterization of pAA virulence gene regulation. PMID:27748404

  1. Differential adherence and expression of virulence traits by Candida albicans and Candida parapsilosis in mono- and dual-species cultures in artificial saliva.

    PubMed

    Seabra, C L; Botelho, C M; Henriques, M; Oliveira, R

    2013-08-01

    To evaluate specific virulence factors of Candida albicans and Candida parapsilosis clinical oral isolates in mono- and dual-species culture in the presence of artificial saliva. Two of the strains used in this study were isolated from co-infection (C. albicans AM and C. parapsilosis AM2), and the other two were isolated from single infection (C. albicans AC and C. parapsilosis AD). The number of adhered yeast cells was measured and their enzymatic activity was determined simultaneously. In mono-species culture, C. parapsilosis strains adhered to a higher extent to the surface in comparison with the C. albicans strains. In dual-species culture, the C. parapsilosis strains adhered more in the presence of C. albicans AM. Interestingly, C. albicans AM and C. parapsilosis AD adhered to a higher extent when compared with all other co-cultures. In dual-species culture, the enzymatic activity of C. parapsilosis strains in the presence of C. albicans AC was higher than in the presence of C. albicans AM. The virulence factors of C. albicans and C. parapsilosis differ from strain to strain and are influenced by the presence of other species in culture. To understand the expression of virulence factors in Candida dual-species systems.

  2. Modulation of behaviour and virulence of a high alginate expressing Pseudomonas aeruginosa strain from cystic fibrosis by oral commensal bacterium Streptococcus anginosus

    PubMed Central

    Qureshi, Muhammad R.

    2017-01-01

    Cystic fibrosis (CF) airways harbour complex and dynamic polymicrobial communities that include many oral bacteria. Despite increased knowledge of CF airway microbiomes the interaction between established CF pathogens and other resident microbes and resulting impact on disease progression is poorly understood. Previous studies have demonstrated that oral commensal streptococci of the Anginosus group (AGS) can establish chronic pulmonary infections and become numerically dominant in CF sputa indicating that they play an important role in CF microbiome dynamics. In this study a strain of Pseudomonas aeruginosa (DWW2) of the mucoid alginate overproducing phenotype associated with chronic CF airway infection and a strain of the oral commensal AGS species Streptococcus anginosus (3a) from CF sputum were investigated for their ability to co-exist and their responses to biofilm co-culture. Bacteria in biofilms were quantified, pyocyanin expression by DWW2 was measured and the effect of AGS strain 3a on reversion of DWW2 to a non-mucoidal phenotype investigated. The virulence of DWW2, 3a and colony variant phenotypes of DWW2 in mono- and co-culture were compared in a Galleria mellonella infection model. Co-culture biofilms were formed in normoxic, hypercapnic (10% CO2) and anoxic atmospheres with the streptococcus increasing in number in co-culture, indicating that these bacteria would be able to co-exist and thrive within the heterogeneous microenvironments of the CF airway. The streptococcus caused increased pyocyanin expression by DWW2 and colony variants by stimulating reversion of the mucoid phenotype to the high pyocyanin expressing non-mucoid phenotype. The latter was highly virulent in the infection model with greater virulence when in co-culture with the streptococcus. The results of this study demonstrate that the oral commensal S. anginosus benefits from interaction with P. aeruginosa of the CF associated mucoid phenotype and modulates the behaviour of the

  3. Modulation of behaviour and virulence of a high alginate expressing Pseudomonas aeruginosa strain from cystic fibrosis by oral commensal bacterium Streptococcus anginosus.

    PubMed

    Waite, Richard D; Qureshi, Muhammad R; Whiley, Robert A

    2017-01-01

    Cystic fibrosis (CF) airways harbour complex and dynamic polymicrobial communities that include many oral bacteria. Despite increased knowledge of CF airway microbiomes the interaction between established CF pathogens and other resident microbes and resulting impact on disease progression is poorly understood. Previous studies have demonstrated that oral commensal streptococci of the Anginosus group (AGS) can establish chronic pulmonary infections and become numerically dominant in CF sputa indicating that they play an important role in CF microbiome dynamics. In this study a strain of Pseudomonas aeruginosa (DWW2) of the mucoid alginate overproducing phenotype associated with chronic CF airway infection and a strain of the oral commensal AGS species Streptococcus anginosus (3a) from CF sputum were investigated for their ability to co-exist and their responses to biofilm co-culture. Bacteria in biofilms were quantified, pyocyanin expression by DWW2 was measured and the effect of AGS strain 3a on reversion of DWW2 to a non-mucoidal phenotype investigated. The virulence of DWW2, 3a and colony variant phenotypes of DWW2 in mono- and co-culture were compared in a Galleria mellonella infection model. Co-culture biofilms were formed in normoxic, hypercapnic (10% CO2) and anoxic atmospheres with the streptococcus increasing in number in co-culture, indicating that these bacteria would be able to co-exist and thrive within the heterogeneous microenvironments of the CF airway. The streptococcus caused increased pyocyanin expression by DWW2 and colony variants by stimulating reversion of the mucoid phenotype to the high pyocyanin expressing non-mucoid phenotype. The latter was highly virulent in the infection model with greater virulence when in co-culture with the streptococcus. The results of this study demonstrate that the oral commensal S. anginosus benefits from interaction with P. aeruginosa of the CF associated mucoid phenotype and modulates the behaviour of the

  4. Generation of Recombinant Viral Hemorrhagic Septicemia Virus (rVHSV) Expressing Two Foreign Proteins and Effect of Lengthened Viral Genome on Viral Growth and In Vivo Virulence.

    PubMed

    Kim, Min Sun; Lee, Su Jin; Kim, Dong Soo; Kim, Ki Hong

    2016-04-01

    In this study, a new recombinant VHSV (rVHSV-Arfp-Bgfp) was generated by insertion of a red fluorescent protein (RFP) gene between N and P genes, a green fluorescent protein (GFP) gene between P and M genes of VHSV genome, the expression of each heterologous gene in infected cells, and effects of the lengthened recombinant VHSV's genome on the replication ability and in vivo virulence to olive flounder (Paralichthys olivaceus) fingerlings were compared with previously generated rVHSVs (rVHSV-wild, rVHSV-Arfp, and rVHSV-Brfp). The expression of RFP and GFP in cells infected with rVHSV-Arfp-Bgfp was verified through fluorescent microscopy and FACS analysis. In the viral growth analysis, rVHSV-Arfp and rVHSV-Brfp showed significantly lower viral titers than rVHSV-wild, and the replication of rVHSV-Arfp-Bgfp was significantly decreased compared to that of even rVHSV-Arfp or rVHSV-Brfp. These results suggest that the genome length is a critical factor for the determination of rVHSVs replication efficiency. In the in vivo virulence experiment, the cumulative mortalities of olive flounder fingerlings infected with each rVHSV were inversely proportional to the length of the viral genome, suggesting that decreased viral growth rate due to the lengthened viral genome is accompanied with the decrease of in vivo virulence of rVHSVs. Recombinant viruses expressing multiple foreign antigens can be used for the development of combined vaccines. However, as the present rVHSV-Arfp-Bgfp still possesses an ability to kill hosts (although very weakened), researches on the producing more attenuated viruses or propagation-deficient replicon particles are needed to solve safety-related problems.

  5. Surface expression of Kv1 channels is governed by a C-terminal motif.

    PubMed

    Li, D; Takimoto, K; Levitan, E S

    2000-04-21

    Voltage-gated K(+) channel subunits must reach the plasma membrane to repolarize action potentials. Yet the efficiency of cell surface targeting varies among Kv subunits with some requiring auxiliary subunits for optimal expression. Here we identify a conserved motif located in the variable C-terminal region of Kv1 channels that controls the efficiency of functional channel expression. Variations among wild type channels in the optimal sequence VXXSL produce differences in distribution and the requirement for auxiliary subunits. Furthermore, deletion of this motif decreases subunit glycosylation and surface localization but does not prohibit subunit multimerization. Finally, the action of the essential sequence is shown to be independent of the chaperone effect of Kvbeta subunits. Thus, the newly identified C-terminal motif governs processing and cell surface expression of Kv1 voltage-gated K(+) channels.

  6. Expression of Amoebapores Is Required for Full Expression of Entamoeba histolytica Virulence in Amebic Liver Abscess but Is Not Necessary for the Induction of Inflammation or Tissue Damage in Amebic Colitis

    PubMed Central

    Zhang, Xiaochun; Zhang, Zhi; Alexander, Diane; Bracha, Rivka; Mirelman, David; Stanley, Samuel L.

    2004-01-01

    Entamoeba histolytica trophozoites produce amoebapores, a family of small amphipathic peptides capable of insertion into bacterial or eukaryotic membranes and causing cellular lysis. Recently, E. histolytica trophozoites that are totally deficient in the production of amoebapore-A were created through a gene silencing mechanism (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). Here we tested the virulence of amoebapore A(−) trophozoites in models of the two major forms of amebic disease: amebic liver abscess and amebic colitis. We demonstrate that amoebapore expression is required for full virulence in the SCID mouse model of amebic liver abscess, but E. histolytica trophozoites that do not express amoebapore-A can still cause inflammation and tissue damage in infected human colonic xenografts. These data are consistent with the concept that tissue damage may proceed by different mechanisms in amebic liver abscess compared to amebic colitis. PMID:14742508

  7. Insights into Entamoeba histolytica virulence modulation.

    PubMed

    Padilla-Vaca, F; Anaya-Velázquez, F

    2010-08-01

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

  8. Gene expression changes governing extreme dehydration tolerance in an Antarctic insect.

    PubMed

    Teets, Nicholas M; Peyton, Justin T; Colinet, Herve; Renault, David; Kelley, Joanna L; Kawarasaki, Yuta; Lee, Richard E; Denlinger, David L

    2012-12-11

    Among terrestrial organisms, arthropods are especially susceptible to dehydration, given their small body size and high surface area to volume ratio. This challenge is particularly acute for polar arthropods that face near-constant desiccating conditions, as water is frozen and thus unavailable for much of the year. The molecular mechanisms that govern extreme dehydration tolerance in insects remain largely undefined. In this study, we used RNA sequencing to quantify transcriptional mechanisms of extreme dehydration tolerance in the Antarctic midge, Belgica antarctica, the world's southernmost insect and only insect endemic to Antarctica. Larvae of B. antarctica are remarkably tolerant of dehydration, surviving losses up to 70% of their body water. Gene expression changes in response to dehydration indicated up-regulation of cellular recycling pathways including the ubiquitin-mediated proteasome and autophagy, with concurrent down-regulation of genes involved in general metabolism and ATP production. Metabolomics results revealed shifts in metabolite pools that correlated closely with changes in gene expression, indicating that coordinated changes in gene expression and metabolism are a critical component of the dehydration response. Finally, using comparative genomics, we compared our gene expression results with a transcriptomic dataset for the Arctic collembolan, Megaphorura arctica. Although B. antarctica and M. arctica are adapted to similar environments, our analysis indicated very little overlap in expression profiles between these two arthropods. Whereas several orthologous genes showed similar expression patterns, transcriptional changes were largely species specific, indicating these polar arthropods have developed distinct transcriptional mechanisms to cope with similar desiccating conditions.

  9. Gene expression changes governing extreme dehydration tolerance in an Antarctic insect

    PubMed Central

    Teets, Nicholas M.; Peyton, Justin T.; Colinet, Herve; Renault, David; Kelley, Joanna L.; Kawarasaki, Yuta; Lee, Richard E.; Denlinger, David L.

    2012-01-01

    Among terrestrial organisms, arthropods are especially susceptible to dehydration, given their small body size and high surface area to volume ratio. This challenge is particularly acute for polar arthropods that face near-constant desiccating conditions, as water is frozen and thus unavailable for much of the year. The molecular mechanisms that govern extreme dehydration tolerance in insects remain largely undefined. In this study, we used RNA sequencing to quantify transcriptional mechanisms of extreme dehydration tolerance in the Antarctic midge, Belgica antarctica, the world’s southernmost insect and only insect endemic to Antarctica. Larvae of B. antarctica are remarkably tolerant of dehydration, surviving losses up to 70% of their body water. Gene expression changes in response to dehydration indicated up-regulation of cellular recycling pathways including the ubiquitin-mediated proteasome and autophagy, with concurrent down-regulation of genes involved in general metabolism and ATP production. Metabolomics results revealed shifts in metabolite pools that correlated closely with changes in gene expression, indicating that coordinated changes in gene expression and metabolism are a critical component of the dehydration response. Finally, using comparative genomics, we compared our gene expression results with a transcriptomic dataset for the Arctic collembolan, Megaphorura arctica. Although B. antarctica and M. arctica are adapted to similar environments, our analysis indicated very little overlap in expression profiles between these two arthropods. Whereas several orthologous genes showed similar expression patterns, transcriptional changes were largely species specific, indicating these polar arthropods have developed distinct transcriptional mechanisms to cope with similar desiccating conditions. PMID:23197828

  10. Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins.

    PubMed

    Santiago-Mateo, Kristina; Zhao, Mojun; Lin, Jun; Zhang, Weiping; Francis, David H

    2012-10-12

    Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.

  11. The Link between Morphotype Transition and Virulence in Cryptococcus neoformans

    PubMed Central

    Wang, Linqi; Zhai, Bing; Lin, Xiaorong

    2012-01-01

    Cryptococcus neoformans is a ubiquitous human fungal pathogen. This pathogen can undergo morphotype transition between the yeast and the filamentous form and such morphological transition has been implicated in virulence for decades. Morphotype transition is typically observed during mating, which is governed by pheromone signaling. Paradoxically, components specific to the pheromone signaling pathways play no or minimal direct roles in virulence. Thus, the link between morphotype transition and virulence and the underlying molecular mechanism remain elusive. Here, we demonstrate that filamentation can occur independent of pheromone signaling and mating, and both mating-dependent and mating-independent morphotype transition require the transcription factor Znf2. High expression of Znf2 is necessary and sufficient to initiate and maintain sex-independent filamentous growth under host-relevant conditions in vitro and during infection. Importantly, ZNF2 overexpression abolishes fungal virulence in murine models of cryptococcosis. Thus, Znf2 bridges the sex-independent morphotype transition and fungal pathogenicity. The impacts of Znf2 on morphological switch and pathogenicity are at least partly mediated through its effects on cell adhesion property. Cfl1, a Znf2 downstream factor, regulates morphogenesis, cell adhesion, biofilm formation, and virulence. Cfl1 is the first adhesin discovered in the phylum Basidiomycota of the Kingdom Fungi. Together with previous findings in other eukaryotic pathogens, our findings support a convergent evolution of plasticity in morphology and its impact on cell adhesion as a critical adaptive trait for pathogenesis. PMID:22737071

  12. Expression of scorpion toxin LqhIT2 increases the virulence of Metarhizium acridum towards Locusta migratoria manilensis.

    PubMed

    Peng, Guoxiong; Xia, Yuxian

    2014-11-01

    LqhIT2 is an insect-specific neurotoxin from the venom of scorpion. In this study, the LqhIT2 gene was introduced into the entomopathogenic fungus, Metarhizium acridum. The virulence of the genetically modified strain MaLqhIT2 was then evaluated against locusts (Locusta migratoria manilensis). Compared with the wild-type strain, the median lethal cell density (LC50) for MaLqhIT2 was a 22.6-fold lower, and the median times to death (LT50) for MaLqhIT2 were reduced by 30.3 and 29.6 %, respectively, after topical inoculation and injection. MaLqhIT2 also grew significantly faster in the hemolymph than wild-type strain. There were no significant differences in germination, appressorium formation and sporulation in locust carcasses between the MaLqhIT2 and wild-type strain. These results indicate that LqhIT2 increased the virulence of M. acridum towards locusts by shortening the in vivo infection period, without affecting cuticle penetration or conidia formation in the carcasses. LqhIT2 thus shows considerable potential for increasing fungal virulence against locusts.

  13. Surface-Associated Lipoproteins Link Enterococcus faecalis Virulence to Colitogenic Activity in IL-10-Deficient Mice Independent of Their Expression Levels.

    PubMed

    Ocvirk, Soeren; Sava, Irina G; Lengfelder, Isabella; Lagkouvardos, Ilias; Steck, Natalie; Roh, Jung H; Tchaptchet, Sandrine; Bao, Yinyin; Hansen, Jonathan J; Huebner, Johannes; Carroll, Ian M; Murray, Barbara E; Sartor, R Balfour; Haller, Dirk

    2015-06-01

    The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2±1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (ΔepaB score: 4.7±2.3; p<0.05) and was almost completely abrogated in E. faecalis deficient for lipoproteins (Δlgt score: 2.3±2.3; p<0.0001). Consistently both E. faecalis mutants showed significantly impaired virulence in Galleria mellonella and Caenorhabditis elegans. Loss of E-cadherin in the epithelium was shown for all bacterial strains in inflamed IL-10-/- but not wild type mice. Inactivation of epaB in E. faecalis reduced microcolony and biofilm formation in vitro, altered bacterial adhesion to intestinal epithelium of germ-free Manduca sexta larvae

  14. Surface-Associated Lipoproteins Link Enterococcus faecalis Virulence to Colitogenic Activity in IL-10-Deficient Mice Independent of Their Expression Levels

    PubMed Central

    Lengfelder, Isabella; Lagkouvardos, Ilias; Steck, Natalie; Roh, Jung H.; Tchaptchet, Sandrine; Bao, Yinyin; Hansen, Jonathan J.; Huebner, Johannes; Carroll, Ian M.; Murray, Barbara E.; Sartor, R. Balfour; Haller, Dirk

    2015-01-01

    The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2±1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (ΔepaB score: 4.7±2.3; p<0.05) and was almost completely abrogated in E. faecalis deficient for lipoproteins (Δlgt score: 2.3±2.3; p<0.0001). Consistently both E. faecalis mutants showed significantly impaired virulence in Galleria mellonella and Caenorhabditis elegans. Loss of E-cadherin in the epithelium was shown for all bacterial strains in inflamed IL-10-/- but not wild type mice. Inactivation of epaB in E. faecalis reduced microcolony and biofilm formation in vitro, altered bacterial adhesion to intestinal epithelium of germ-free Manduca sexta larvae

  15. Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism

    PubMed Central

    Machuca, Alvaro; Martinez, Victor

    2016-01-01

    The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence. PMID:28033422

  16. Inactivation and Gene Expression of a Virulent Wastewater Escherichia coli Strain and the Nonvirulent Commensal Escherichia coli DSM1103 Strain upon Solar Irradiation.

    PubMed

    Al-Jassim, Nada; Mantilla-Calderon, David; Wang, Tiannyu; Hong, Pei-Ying

    2017-04-04

    This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

  17. Expression of the Streptococcus agalactiae virulence-associated protease CspA in a soluble, active form utilizing the Gram-positive host, Lactococcus lactis.

    PubMed

    Shelver, Daniel; Bryan, Joshua D

    2008-09-10

    The heterologous expression of streptococcal genes in common Gram-negative hosts may be complicated by low-level expression, toxicity to the host, formation of inclusion bodies, and mislocalization of the encoded proteins. Biochemical study of the Streptococcus agalactiae virulence-associated cell-envelope protease (CEP) CspA, as well as other CEPs, has been limited by the lack of effective expression systems. In this study, we present a simple strategy to express cspA as a catalytically active exoprotein. A recombinant allele of cspA, cspADeltaCWA, was engineered to eliminate the dispensable cell-wall anchor. The cspADeltaCWA allele was expressed in the Gram-positive organism, Lactococcus lactis, using an established, plasmid-based, nisin-inducible expression system. After induction, nearly all of the exoprotein observable by SDS-PAGE corresponded to CspADeltaCWA. CspADeltaCWA-containing medium exhibited similar fibrinolytic activity as whole cells of GBS, indicating the recombinant protein was active. Characterization of CspADeltaCWA indicated that like some other CEPs, it is N-terminally processed, exists predominantly as a dimer, and has the ability to cleave itself at its C-terminus. Taken together, this work presents an efficient strategy for expression of cspA that could be applied to other streptococcal proteins that are not amenable to expression using common Gram-negative hosts.

  18. A Novel Antisense RNA from the Salmonella Virulence Plasmid pSLT Expressed by Non-Growing Bacteria inside Eukaryotic Cells

    PubMed Central

    Rico-Pérez, Gadea; Pucciarelli, M. Graciela; García-del Portillo, Francisco

    2013-01-01

    Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the S. Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the PSLT047- PSLT046 (mig5) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for ‘Intracellular-expressed-sRNA-1′. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200–300 fold) following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the PSLT047-iesR-1-PSLT046 (mig5) region support a cis-acting regulatory mechanism of IesR-1 as antisense RNA over the PSLT047 transcript involving interaction at their respective 3′ ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5′ and 3′ regions. Taken together, these data reveal that S. Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may

  19. A novel antisense RNA from the Salmonella virulence plasmid pSLT expressed by non-growing bacteria inside eukaryotic cells.

    PubMed

    Gonzalo-Asensio, Jesús; Ortega, Alvaro D; Rico-Pérez, Gadea; Pucciarelli, M Graciela; García-Del Portillo, Francisco

    2013-01-01

    Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the S. Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the PSLT047- PSLT046 (mig5) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for 'Intracellular-expressed-sRNA-1'. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200-300 fold) following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the PSLT047-iesR-1-PSLT046 (mig5) region support a cis-acting regulatory mechanism of IesR-1 as antisense RNA over the PSLT047 transcript involving interaction at their respective 3' ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5' and 3' regions. Taken together, these data reveal that S. Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may contribute to

  20. H-NS Nucleoid Protein Controls Virulence Features of Klebsiella pneumoniae by Regulating the Expression of Type 3 Pili and the Capsule Polysaccharide

    PubMed Central

    Ares, Miguel A.; Fernández-Vázquez, José L.; Rosales-Reyes, Roberto; Jarillo-Quijada, Ma. Dolores; von Bargen, Kristine; Torres, Javier; González-y-Merchand, Jorge A.; Alcántar-Curiel, María D.; De la Cruz, Miguel A.

    2016-01-01

    Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections. Main virulence determinants of K. pneumoniae are pili, capsular polysaccharide, lipopolysaccharide, and siderophores. The histone-like nucleoid-structuring protein (H-NS) is a pleiotropic regulator found in several gram-negative pathogens. It has functions both as an architectural component of the nucleoid and as a global regulator of gene expression. We generated a Δhns mutant and evaluated the role of the H-NS nucleoid protein on the virulence features of K. pneumoniae. A Δhns mutant down-regulated the mrkA pilin gene and biofilm formation was affected. In contrast, capsule expression was derepressed in the absence of H-NS conferring a hypermucoviscous phenotype. Moreover, H-NS deficiency affected the K. pneumoniae adherence to epithelial cells such as A549 and HeLa cells. In infection experiments using RAW264.7 and THP-1 differentiated macrophages, the Δhns mutant was less phagocytized than the wild-type strain. This phenotype was likely due to the low adherence to these phagocytic cells. Taken together, our data indicate that H-NS nucleoid protein is a crucial regulator of both T3P and CPS of K. pneumoniae. PMID:26904512

  1. H-NS Nucleoid Protein Controls Virulence Features of Klebsiella pneumoniae by Regulating the Expression of Type 3 Pili and the Capsule Polysaccharide.

    PubMed

    Ares, Miguel A; Fernández-Vázquez, José L; Rosales-Reyes, Roberto; Jarillo-Quijada, Ma Dolores; von Bargen, Kristine; Torres, Javier; González-y-Merchand, Jorge A; Alcántar-Curiel, María D; De la Cruz, Miguel A

    2016-01-01

    Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections. Main virulence determinants of K. pneumoniae are pili, capsular polysaccharide, lipopolysaccharide, and siderophores. The histone-like nucleoid-structuring protein (H-NS) is a pleiotropic regulator found in several gram-negative pathogens. It has functions both as an architectural component of the nucleoid and as a global regulator of gene expression. We generated a Δhns mutant and evaluated the role of the H-NS nucleoid protein on the virulence features of K. pneumoniae. A Δhns mutant down-regulated the mrkA pilin gene and biofilm formation was affected. In contrast, capsule expression was derepressed in the absence of H-NS conferring a hypermucoviscous phenotype. Moreover, H-NS deficiency affected the K. pneumoniae adherence to epithelial cells such as A549 and HeLa cells. In infection experiments using RAW264.7 and THP-1 differentiated macrophages, the Δhns mutant was less phagocytized than the wild-type strain. This phenotype was likely due to the low adherence to these phagocytic cells. Taken together, our data indicate that H-NS nucleoid protein is a crucial regulator of both T3P and CPS of K. pneumoniae.

  2. Identification of Unconventional Intestinal Pathogenic Escherichia coli Isolates Expressing Intermediate Virulence Factor Profiles by Using a Novel Single-Step Multiplex PCR▿

    PubMed Central

    Müller, Daniel; Greune, Lilo; Heusipp, Gerhard; Karch, Helge; Fruth, Angelika; Tschäpe, Helmut; Schmidt, M. Alexander

    2007-01-01

    Intestinal pathogenic Escherichia coli represents a global health problem for mammals, including humans. At present, diarrheagenic E. coli bacteria are grouped into seven major pathotypes that differ in their virulence factor profiles, severity of clinical manifestations, and prognosis. In this study, we developed and evaluated a one-step multiplex PCR (MPCR) for the straightforward differential identification of intestinal pathotypes of E. coli. The specificity of this novel MPCR was validated by using a subset of reference strains and further confirmed by PCR-independent pheno- and genotypic characterization. Moreover, we tested 246 clinical E. coli isolates derived from diarrhea patients from several distinct geographic regions. Interestingly, besides strains belonging to the defined and well-described pathotypes, we identified five unconventional strains expressing intermediate virulence factor profiles. These strains have been further characterized and appear to represent intermediate strains carrying genes and expressing factors associated with enteropathogenic E. coli, Shiga toxin-producing E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli alike. These strains represent further examples of the extraordinary plasticity of the E. coli genome. Moreover, this implies that the important identification of specific pathotypes has to be based on a broad matrix of indicator genes. In addition, the presence of intermediate strains needs to be accounted for. PMID:17400780

  3. Two overlapping two-component systems in Xanthomonas oryzae pv. oryzae contribute to full fitness in rice by regulating virulence factors expression

    PubMed Central

    Zheng, Dehong; Yao, Xiaoyan; Duan, Meng; Luo, Yufeng; Liu, Biao; Qi, Pengyuan; Sun, Ming; Ruan, Lifang

    2016-01-01

    Two-component signal transduction systems (TCSs) are widely used by bacteria to adapt to the environment. In the present study, StoS (stress tolerance-related oxygen sensor) and SreKRS (salt response kinase, regulator, and sensor) were found to positively regulate extracellular polysaccharide (EPS) production and swarming in the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). Surprisingly, the absence of stoS or sreKRS did not attenuate virulence. To better understand the intrinsic functions of StoS and SreKRS, quantitative proteomics isobaric tags for relative and absolute quantitation (iTRAQ) was employed. Consistent with stoS and sreK mutants exhibiting a similar phenotype, the signalling circuits of StoS and SreKRS overlapped. Carbohydrate metabolism proteins and chemotaxis proteins, which could be responsible for EPS and swarming regulation, respectively, were reprogrammed in stoS and sreK mutants. Moreover, StoS and SreKRS demonstrated moderate expression of the major virulence factor, hypersensitive response and pathogenicity (Hrp) proteins through the HrpG-HrpX circuit. Most importantly, Xoo equipped with StoS and SreKRS outcompetes strains without StoS or SreKRS in co-infected rice and grows outside the host. Therefore, we propose that StoS and SreKRS adopt a novel strategy involving the moderation of Hrp protein expression and the promotion of EPS and motility to adapt to the environment. PMID:26957113

  4. flrA, flrB and flrC regulate adhesion by controlling the expression of critical virulence genes in Vibrio alginolyticus

    PubMed Central

    Luo, Gang; Huang, Lixing; Su, Yongquan; Qin, Yingxue; Xu, Xiaojin; Zhao, Lingmin; Yan, Qingpi

    2016-01-01

    Adhesion is an important virulence trait of Vibrio alginolyticus. Bacterial adhesion is influenced by environmental conditions; however, the molecular mechanism underlying this effect remains unknown. The expression levels of flrA, flrB and flrC were significantly downregulated in adhesion-deficient V. alginolyticus strains cultured under Cu2+, Pb2+, Hg2+ and low-pH stresses. Silencing these genes led to deficiencies in adhesion, motility, flagellar assembly, biofilm formation and exopolysaccharide (EPS) production. The expression levels of fliA, flgH, fliS, fliD, cheR, cheV and V12G01_22158 (Gene ID) were significantly downregulated in all of the RNAi groups, whereas the expression levels of toxT, ctxB, acfA, hlyA and tlh were upregulated in flrA- and flrC-silenced groups. These genes play a key role in the virulence mechanisms of most pathogenic Vibrio species. Furthermore, the expression of flrA, flrB and flrC was significantly influenced by temperature, salinity, starvation and pH. These results indicate that (1) flrA, flrB and flrC are important for V. alginolyticus adhesion; (2) flrA, flrB and flrC significantly influence bacterial adhesion, motility, biofilm formation and EPS production by controlling expression of key genes involved in those phenotypes; and (3) flrA, flrB and flrC regulate adhesion in the natural environment with different temperatures, pH levels, salinities and starvation time. PMID:27485498

  5. Host cell contact induces expression of virulence factors and VieA, a cyclic di-GMP phosphodiesterase, in Vibrio cholerae.

    PubMed

    Dey, Amit K; Bhagat, Abha; Chowdhury, Rukhsana

    2013-05-01

    Vibrio cholerae, a noninvasive bacterium, colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes the severe fluid loss characteristic of the disease cholera. In this study, we demonstrate that adherence of V. cholerae to the intestinal epithelial cell line INT 407 strongly induces the expression of the major virulence genes ctxAB and tcpA and the virulence regulatory gene toxT. No induction of toxR and tcpP, which encode transcriptional activators of toxT, was observed in adhered bacteria, and the adherence-dependent upregulation of toxT expression was independent of ToxR and TcpP. A sharp increase in the expression of the vieA gene, which encodes a cyclic di-GMP (c-di-GMP) phosphodiesterase, was observed in INT 407-adhered V. cholerae immediately after infection. Induction of toxT, ctxAB, and tcpA in INT 407-adhered vieA mutant strain O395 ΔvieA was consistently lower than in the parent strain, although no effect was observed in unadhered bacteria, suggesting that VieA has a role in the upregulation of toxT expression specifically in host cell-adhered V. cholerae. Furthermore, though VieA has both a DNA binding helix-turn-helix domain and an EAL domain conferring c-di-GMP phosphodiesterase activity, the c-di-GMP phosphodiesterase activity of VieA is necessary and sufficient for the upregulation of toxT expression.

  6. The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms.

    PubMed

    Kovacikova, Gabriela; Lin, Wei; Taylor, Ronald K; Skorupski, Karen

    2017-04-01

    FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadR-binding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadR-mediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms.IMPORTANCE Fatty acids (FAs) play important roles in membrane lipid homeostasis and energy metabolism in all organisms. In Vibrio cholerae, the causative agent of the acute intestinal disease cholera, they also influence virulence by binding into an N-terminal pocket of the master virulence regulator, ToxT, and modulating its activity. FadR is a transcription factor

  7. Effect of Ethanol on Differential Protein Production and Expression of Potential Virulence Functions in the Opportunistic Pathogen Acinetobacter baumannii

    PubMed Central

    Nwugo, Chika C.; Arivett, Brock A.; Zimbler, Daniel L.; Gaddy, Jennifer A.; Richards, Ashley M.; Actis, Luis A.

    2012-01-01

    Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments. PMID:23284824

  8. Effect of ethanol on differential protein production and expression of potential virulence functions in the opportunistic pathogen Acinetobacter baumannii.

    PubMed

    Nwugo, Chika C; Arivett, Brock A; Zimbler, Daniel L; Gaddy, Jennifer A; Richards, Ashley M; Actis, Luis A

    2012-01-01

    Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments.

  9. RegA, an AraC-like protein, is a global transcriptional regulator that controls virulence gene expression in Citrobacter rodentium.

    PubMed

    Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J; Frankel, Gad; Hartland, Elizabeth L; Robins-Browne, Roy M

    2008-11-01

    Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

  10. RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium▿

    PubMed Central

    Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J.; Frankel, Gad; Hartland, Elizabeth L.; Robins-Browne, Roy M.

    2008-01-01

    Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization. PMID:18765720

  11. Cryptococcus neoformans growth and protection from innate immunity are dependent on expression of a virulence-associated DEAD-box protein, Vad1.

    PubMed

    Qiu, Jin; Olszewski, Michal A; Williamson, Peter R

    2013-03-01

    The fungus Cryptococcus neoformans has emerged as a major cause of meningoencephalitis worldwide. Host response to the fungus involves both innate and adaptive immunity, but fungal genes that modulate these processes are poorly understood. Previous studies demonstrated attenuated virulence of a mutant of a virulence-associated DEAD-box protein (VAD1) in mice, despite normal growth at host temperatures, suggesting modulation of the immune response. In the present study, the Δvad1 mutant demonstrated progressive clearance from lung and was unable to induce pathological lesions or to cause extrapulmonary disease, despite retaining its ability to grow in mouse serum and a J774.16 macrophage cell line. Pulmonary clearance occurred with a minimal cellular infiltrate, marked by reduced CD4 cells, CD11b(+) Ly6C(high) monocytes, and F4/80(+) macrophages, but the mutant strain retained recruitment of CD8 cells, compared to infections with wild-type fungi. Adaptive cytokine responses were reduced, including Th1, Th2, and Th17 cytokines; however, early gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) responses were retained while nonprotective interleukin 4 (IL-4) and IL-5 were diminished. Furthermore, the Δvad1 mutant was controlled in lungs despite CD4/CD8 cell depletion. These data, along with improved phagocytosis by macrophages and increases in early/innate IL-1α, IFN-γ, and chemokines elicited in the lungs within 3 days of infection with the Δvad1 mutant, indicate that VAD1 expression reduces innate recognition of C. neoformans, rendering the yeast resistant to elimination by the innate mechanisms of host defense. Thus, our studies define a novel role of the cryptococcal Vad1 protein as a central regulator of cryptococcal virulence and illustrate that Vad1 promotes microbe resistance to innate host defenses.

  12. A Pectate Lyase-Coding Gene Abundantly Expressed during Early Stages of Infection Is Required for Full Virulence in Alternaria brassicicola.

    PubMed

    Cho, Yangrae; Jang, Mina; Srivastava, Akhil; Jang, Jae-Hyuk; Soung, Nak-Kyun; Ko, Sung-Kyun; Kang, Dae-Ook; Ahn, Jong Seog; Kim, Bo Yeon

    2015-01-01

    Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development.

  13. A Pectate Lyase-Coding Gene Abundantly Expressed during Early Stages of Infection Is Required for Full Virulence in Alternaria brassicicola

    PubMed Central

    Cho, Yangrae; Jang, Mina; Srivastava, Akhil; Jang, Jae-Hyuk; Soung, Nak-Kyun; Ko, Sung-Kyun; Kang, Dae-Ook; Ahn, Jong Seog; Kim, Bo Yeon

    2015-01-01

    Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development. PMID:25996954

  14. Y14 governs p53 expression and modulates DNA damage sensitivity

    PubMed Central

    Lu, Chia-Chen; Lee, Chi-Chieh; Tseng, Ching-Tzu; Tarn, Woan-Yuh

    2017-01-01

    Y14 is a core component of the exon junction complex (EJC), while it also exerts cellular functions independent of the EJC. Depletion of Y14 causes G2/M arrest, DNA damage and apoptosis. Here we show that knockdown of Y14 induces the expression of an alternative spliced isoform of p53, namely p53β, in human cells. Y14, in the context of the EJC, inhibited aberrant exon inclusion during the splicing of p53 pre-mRNA, and thus prevent p53β expression. The anti-cancer agent camptothecin specifically suppressed p53β induction. Intriguingly, both depletion and overexpression of Y14 increased overall p53 protein levels, suggesting that Y14 governs the quality and quantity control of p53. Moreover, Y14 depletion unexpectedly reduced p21 protein levels, which in conjunction with aberrant p53 expression accordingly increased cell sensitivity to genotoxic agents. This study establishes a direct link between Y14 and p53 expression and suggests a function for Y14 in DNA damage signaling. PMID:28361991

  15. Homeobox transcription factor Six7 governs expression of green opsin genes in zebrafish.

    PubMed

    Ogawa, Yohey; Shiraki, Tomoya; Kojima, Daisuke; Fukada, Yoshitaka

    2015-08-07

    Colour discrimination in vertebrates requires cone photoreceptor cells in the retina, and high-acuity colour vision is endowed by a set of four cone subtypes expressing UV-, blue-, green- and red-sensitive opsins. Previous studies identified transcription factors governing cone photoreceptor development in mice, although loss of blue and green opsin genes in the evolution of mammals make it difficult to understand how high-acuity colour vision was organized during evolution and development. Zebrafish (Danio rerio) represents a valuable vertebrate model for studying colour vision as it retains all the four ancestral vertebrate cone subtypes. Here, by RT-qPCR and in situ hybridization analysis, we found that sine oculis homeobox homolog 7 (six7), a transcription factor widely conserved in ray-finned fish, is expressed predominantly in the cone photoreceptors in zebrafish at both the larval and the adult stages. TAL effector nuclease-based six7 knock-out revealed its roles in expression of green, red and blue cone opsin genes. Most prominently, the six7 deficiency caused a loss of expression of all the green opsins at both the larval and adult stages. six7 is indispensable for the development and/or maintenance of the green cones. © 2015 The Author(s).

  16. 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.

    PubMed

    Hayes, S L; Rodgers, M R; Lye, D J; Stelma, G N; McKinstry, C A; Malard, J M; Vesper, S J

    2007-10-01

    To assess the virulence of Aeromonas spp. using two models, a neonatal mouse assay and a mouse intestinal cell culture. 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. 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. Having the ability to determine virulence of waterborne pathogens quickly would potentially assist public health officials to rapidly assess exposure risks.

  17. The equine herpesvirus-1 (EHV-1) IR3 transcript downregulates expression of the IE gene and the absence of IR3 gene expression alters EHV-1 biological properties and virulence

    PubMed Central

    Ahn, Byung Chul; Zhang, Yunfei; O’Callaghan, Dennis J.

    2011-01-01

    The IR3 transcript of equine herpesvirus-1 (EHV-1) harbors 117 nts antisense to the immediate-early (IE) mRNA, suggesting it plays a regulatory role. Here, we show that the IR3 transcript downregulates IE gene expression and that the absence of IR3 expression altered EHV-1 biological properties and virulence in mice. Reporter assays revealed that the IR3/IE overlapping sequences [IR3(+226/+342)] and an additional IR3(+343/+433) region are necessary for the IR3 RNA to downregulate IE expression. Experiments with the ΔIR3 EHV-1 showed that the IR3 gene is dispensable for EHV-1 replication. Protein expression of the IE and representative EHV-1 genes was increased in cells infected with ΔIR3 EHV-1 as compared to that of cells infected with wt EHV-1. The ΔIR3 EHV-1 exhibited increased virulence in mice as compared to the parent virus. The finding that the IR3 transcript affects IE gene expression extends the role of RNA as a regulatory molecule in alphaherpesvirus infection. PMID:20417949

  18. The equine herpesvirus-1 (EHV-1) IR3 transcript downregulates expression of the IE gene and the absence of IR3 gene expression alters EHV-1 biological properties and virulence.

    PubMed

    Ahn, Byung Chul; Zhang, Yunfei; O'Callaghan, Dennis J

    2010-07-05

    The IR3 transcript of equine herpesvirus-1 (EHV-1) harbors 117 nts antisense to the immediate-early (IE) mRNA, suggesting it plays a regulatory role. Here, we show that the IR3 transcript downregulates IE gene expression and that the absence of IR3 expression altered EHV-1 biological properties and virulence in mice. Reporter assays revealed that the IR3/IE overlapping sequences [IR3(+226/+342)] and an additional IR3(+343/+433) region are necessary for the IR3 RNA to downregulate IE expression. Experiments with the DeltaIR3 EHV-1 showed that the IR3 gene is dispensable for EHV-1 replication. Protein expression of the IE and representative EHV-1 genes was increased in cells infected with DeltaIR3 EHV-1 as compared to that of cells infected with wt EHV-1. The DeltaIR3 EHV-1 exhibited increased virulence in mice as compared to the parent virus. The finding that the IR3 transcript affects IE gene expression extends the role of RNA as a regulatory molecule in alphaherpesvirus infection. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3.

    PubMed

    Leskinen, Katarzyna; Varjosalo, Markku; Skurnik, Mikael

    2015-02-01

    YbeY was recently recognized as an endoribonuclease playing a role in ribosome biosynthesis. In Escherichia coli it functions as a single-strand-specific RNase that processes the 3' end of the 16S rRNA and is crucial for the late-stage 70S ribosome quality control system. Here we report that YbeY is not essential in Yersinia enterocolitica serotype O:3, yet its absence strongly compromised the bacterium. The lack of YbeY resulted in misprocessing of 16S rRNA and a severe decrease of growth rate with complete growth arrest observed at elevated temperatures. Moreover, a ybeY mutation severely disturbed regulation of the Yersinia virulence plasmid (pYV) genes and affected the expression of regulatory small RNA species. Transcription of the pYV genes was upregulated in the ybeY mutant at 22 °C; the same genes were repressed in the wild-type bacterium. Furthermore, ybeY inactivation impaired many virulence-related features, such as resistance to elevated temperature and acid, and hindered utilization of different carbohydrates. In addition, the ybeY mutant strain showed decreased infectivity in a tissue culture infection model, especially at the stage of cell adhesion. Taken together, this study demonstrates the crucial role of YbeY in Y. enterocolitica O:3 physiology and pathogenicity.

  20. Large Deletions in the pAtC58 Megaplasmid of Agrobacterium tumefaciens Can Confer Reduced Carriage Cost and Increased Expression of Virulence Genes

    PubMed Central

    Morton, Elise R.; Merritt, Peter M.; Bever, James D.; Fuqua, Clay

    2013-01-01

    The accessory plasmid pAtC58 of the common laboratory strain of Agrobacterium tumefaciens confers numerous catabolic functions and has been proposed to play a role in virulence. Genomic sequencing of evolved laboratory strains of A. tumefaciens revealed the presence of multiple deletion events in the At plasmid, with reductions in plasmid size ranging from 25% to 30% (115–194 kb). Flanking both ends of the sites of these deletions is a short-nucleotide repeat sequence that is in a single copy in the deleted plasmids, characteristic of a phage- or transposon-mediated deletion event. This repeat sequence is widespread throughout the C58 genome, but concentrated on the At plasmid, suggesting its frequency to be nonrandom. In this study, we assess the prevalence of the larger of these deletions in multiple C58 derivatives and characterize its functional significance. We find that in addition to elevating virulence gene expression, this deletion is associated with a significantly reduced carriage cost to the cell. These observations are a clear demonstration of the dynamic nature of the bacterial genome and suggest a mechanism for genetic plasticity of these costly but otherwise stable plasmids. Additionally, this phenomenon could be the basis for some of the dramatic recombination events so ubiquitous within and among megaplasmids. PMID:23783172

  1. The effect of sub-inhibitory concentrations of rifaximin on urease production and on other virulence factors expressed by Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus.

    PubMed

    Ricci, Annalisa; Coppo, Erika; Barbieri, Ramona; Debbia, Eugenio A; Marchese, Anna

    2017-04-01

    Rifaximin, a topical derivative of rifampin, inhibited urease production and other virulence factors at sub-MIC concentrations in strains involved in hepatic encephalopathy and the expression of methicillin resistance in Staphylococcus aureus. In particular, urease production was affected in all Proteus mirabilis and Klebsiella pneumoniae strains as well as in all tested Pseudomonas aeruginosa isolates. Other exotoxins, synthesized by P. aeruginosa, such as protease, gelatinase, lipase, lecithinase and DNAse were also not metabolized in the presence of rifaximin. This antibiotic inhibited pigment production in both P. aeruginosa and Chromobacterium violaceum, a biosensor control strain. Lastly, rifaximin affected haemolysin production in S. aureus and was able to restore cefoxitin susceptibility when the strain was cultured in the presence of sub-MICs of the drug. The present findings confirm and extend previous observations about the beneficial effects of rifaximin for the treatment of gastrointestinal diseases, since in this anatomic site, it reaches a large array of concentrations which prevents enterobacteria from thriving and/or producing their major virulence factors.

  2. Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects virulence-associated regulators and growth in Acanthamoeba castellanii.

    PubMed

    Forsbach-Birk, Vera; McNealy, Tamara; Shi, Chunwei; Lynch, Damien; Marre, Reinhard

    2004-07-01

    Legionella bacteria have a developmental cycle in which they go from existing in the aquatic environment to replicating inside eukaryotic host cells. The adaptation to the new environment requires an efficient regulatory system. Overexpression of CsrA, a global regulatory protein found in a variety of gram-negative bacteria has been shown to suppress virulence-associated traits in Legionella pneumophila. Since evidence resulting only from overproduction may not be sufficient to validate the role of a regulatory protein, a csrA mutant strain, CsrA(-), with a drastically reduced production of CsrA, was created. Using RNA slot blots and Western blotting it was shown that fliA and flaA, genes which contribute to flagellation, were expressed early in the mutant. Additionally, in CsrA(-) the levels of the stationary-phase sigma factor, RpoS, and a recently described regulator of virulence traits, LetE, were increased. Growth curves of CsrA(-) bacteria were delayed with pigment production occurring at the same OD578 but at reduced levels in the mutant. Replication ability of the CsrA(-) mutant in amoebae was also affected. Based on these results, we could show that CsrA is involved in the regulation of the bacterial switch from the replicative to the transmissible form.

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

    PubMed

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

    2015-06-05

    There is an urgent need to develop new antibacterial agents because of multidrug resistance by bacteria and fungi. Schiff bases (aldehyde or ketone-like compounds) exhibit intense antibacterial characteristics, and are therefore, promising candidates as antibacterial agents. To investigate the mechanism of action of newly designed benzaldehyde Schiff bases, a series of high-yielding benzaldehyde Schiff bases were synthesized, and their structures were determined by NMR and MS spectra data. The structure-microbicidal activity relationship of derivatives was investigated, and the antibacterial mechanisms were investigated by gene assays for the expression of functional genes in vitro using Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The active compounds were selective for certain active groups. The polar substitution of the R2 group of the amino acids in the Schiff bases, affected the antibacterial activity against E. coli and S. aureus; specific active group at the R3 or R4 groups of the acylhydrazone Schiff bases could improve their inhibitory activity against these three tested organisms. The antibacterial mechanism of the active benzaldehyde Schiff bases appeared to regulate the expression of metabolism-associated genes in E. coli, hemolysis-associated genes in B. subtilis, and key virulence genes in S. aureus. Some benzaldehyde Schiff bases were bactericidal to all the three strains and appeared to regulate gene expression associated with metabolism, hemolysis, and virulence, in vitro. The newly designed benzaldehyde Schiff bases possessed unique antibacterial activity and might be potentially useful for prophylactic or therapeutic intervention of bacterial infections. Copyright © 2015. Published by Elsevier Masson SAS.

  4. Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito

    PubMed Central

    2013-01-01

    Background Adult and larval mosquitoes regulate food digestion in their gut with trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by the ovaries and the neuroendocrine system. TMOF is currently being developed as a mosquitocide, however, delivery of the peptide to the mosquito remains a significant challenge. Entomopathogenic fungi offer a means for targeting mosquitoes with TMOF. Findings The efficacy of wild type and transgenic Beauveria bassiana strains expressing Aedes aegypti TMOF (Bb-Aa1) were evaluated against larvae and sugar- and blood-fed adult Anopheles gambiae mosquitoes using insect bioassays. Bb-Aa1 displayed increased virulence against larvae, and sugar and blood fed adult A. gambiae when compared to the wild type parent strain. Median lethal dose (LD50) values decreased by ~20% for larvae, and ~40% for both sugar and blood-fed mosquitoes using Bb-Aa1 relative to the wild type parent. Median lethal time (LT50) values were lower for blood-fed compared to sugar-fed mosquitoes in infections with both wild type and Bb-Aa1. However, infection using Bb-Aa1 resulted in 15% to 25% reduction in LT50 values for sugar- and blood fed mosquitoes, and ~27% for larvae, respectively, relative to the wild type parent. In addition, infection with Bb-Aa1 resulted in a dramatic reduction in fecundity of the target mosquitoes. Conclusions B. bassiana expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae compared to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease. PMID:23336669

  5. Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito.

    PubMed

    Kamareddine, Layla; Fan, Yanhua; Osta, Mike A; Keyhani, Nemat O

    2013-01-21

    Adult and larval mosquitoes regulate food digestion in their gut with trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by the ovaries and the neuroendocrine system. TMOF is currently being developed as a mosquitocide, however, delivery of the peptide to the mosquito remains a significant challenge. Entomopathogenic fungi offer a means for targeting mosquitoes with TMOF. The efficacy of wild type and transgenic Beauveria bassiana strains expressing Aedes aegypti TMOF (Bb-Aa1) were evaluated against larvae and sugar- and blood-fed adult Anopheles gambiae mosquitoes using insect bioassays. Bb-Aa1 displayed increased virulence against larvae, and sugar and blood fed adult A. gambiae when compared to the wild type parent strain. Median lethal dose (LD50) values decreased by ~20% for larvae, and ~40% for both sugar and blood-fed mosquitoes using Bb-Aa1 relative to the wild type parent. Median lethal time (LT50) values were lower for blood-fed compared to sugar-fed mosquitoes in infections with both wild type and Bb-Aa1. However, infection using Bb-Aa1 resulted in 15% to 25% reduction in LT50 values for sugar- and blood fed mosquitoes, and ~27% for larvae, respectively, relative to the wild type parent. In addition, infection with Bb-Aa1 resulted in a dramatic reduction in fecundity of the target mosquitoes. B. bassiana expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae compared to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease.

  6. Transcriptional Profiling of the Iron Starvation Response in Bordetella pertussis Provides New Insights into Siderophore Utilization and Virulence Gene Expression ▿ §

    PubMed Central

    Brickman, Timothy J.; Cummings, Craig A.; Liew, Sin-Yee; Relman, David A.; Armstrong, Sandra K.

    2011-01-01

    Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes. PMID:21742863

  7. Expression of Five Endopolygalacturonase Genes and Demonstration that MfPG1 Overexpression Diminishes Virulence in the Brown Rot Pathogen Monilinia fructicola

    PubMed Central

    Yu, Pei-Ling; Ho, Jia-Fang; Bostock, Richard M.; Chung, Kuang-Ren; Huang, Jenn-Wen; Lee, Miin-Huey

    2015-01-01

    Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG) genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR) revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the β-glucuronidase (GUS) reporter gene fused with MfPG1 (MfPG1-GUS). The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS) on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus–M. fructicola interactions. PMID:26120831

  8. Differential expression of virulence genes and role of gyrA mutations in quinolone resistant and susceptible strains of Salmonella Weltevreden and Newport isolated from seafood.

    PubMed

    Deekshit, V K; Kumar, B K; Rai, P; Karunasagar, I; Karunasagar, I

    2015-10-01

    To investigate the differential expression of virulence genes and role of gyrA mutations in quinolone resistant and susceptible strains of Salmonella isolated from seafood. Forty Salmonella isolates from seafood were tested for antibiotic sensitivity. Minimal inhibitory concentration (MIC) was determined and two nalidixic acid-resistant isolates, viz Salmonella Weltevreden (SW9) and Salmonella Newport (SN36) were selected for identifying the mechanism of resistance. SW9 showed mutation in the gyrA gene at codon 83 (Ser to Tyr) while SN36 presented at codon 87 (Asp to Asn). Experimental induction of resistance to a sensitive Salm. Newport (SN71) showed point mutation at codon 87 (Asp to Gly) in the gyrA gene, and was designated SN71R. All the isolates resistant to nalidixic acid had a single mutation at different positions in the gyrA gene. However, induction of resistance to a sensitive Salm. Weltevreden (SW30) was exceptional in that it did not show any mutation in the gyrA region. Use of Phe-Arg-β-naphthylamide (PAβN) also could not reduce MIC below the Clinical and Laboratory Standards Institute guidelines revealing the absence of efflux mediated resistance. Thus, the resistance mechanism in SW30R is unknown. The growth rate of quinolone resistant isolates was slower than the susceptible ones. The resistant isolates showed decreased epithelial cell invasion and intracellular replication. The mRNA expression levels of some of the genes were significantly (P < 0·005) reduced in SN71R compared to the sensitive strain (SN71). Nalidixic acid-resistant Salmonella strains are associated with lower virulence and pathogenicity than the sensitive strains. This study provided valuable information on the difference in the growth, cytotoxicity, infectivity and expression of virulence genes in resistant and susceptible strains. Furthermore, the gyrA mutation was shown to be the main mechanism of quinolone resistance in Salmonella other than the overexpression of efflux

  9. Functional characterisation of cis-regulatory elements governing dynamic Eomes expression in the early mouse embryo.

    PubMed

    Simon, Claire S; Downes, Damien J; Gosden, Matthew E; Telenius, Jelena; Higgs, Douglas R; Hughes, Jim R; Costello, Ita; Bikoff, Elizabeth K; Robertson, Elizabeth J

    2017-02-07

    The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable and only the VPE is required for optimal Eomes expression in vivo Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions embryonic stem cells (ESC), prior to gene activation. The locus resides in a large (500kb) pre-formed compartment in ESC and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a, and four additional putative enhancers display increased chromatin accessibility in DE associated with Smad2/3 binding coincident with transcriptional activation. In contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.

  10. EVALUATING VIRULENCE OF WATERBORNE AND CLINCIAL AEROMONAS ISOLATES USING GENE EXPRESSION AND MORTALITY IN NEONATAL MICE FOLLOWED BY ASSESSING CELL CULTURE'S ABILITY TO PREDICT VIRULENCE BASED ON TRANSCRIPTIONAL RESPONSE

    EPA Science Inventory

    The virulence of multiple Aeromonas spp. were assessed using two models, a neonatal mouse assay and a mouse intestinal cell culture. Transcriptional responses to both infection models were assessed using microarrays. After artificial infection with a variety of Aeromonas spp., ...

  11. EVALUATING VIRULENCE OF WATERBORNE AND CLINCIAL AEROMONAS ISOLATES USING GENE EXPRESSION AND MORTALITY IN NEONATAL MICE FOLLOWED BY ASSESSING CELL CULTURE'S ABILITY TO PREDICT VIRULENCE BASED ON TRANSCRIPTIONAL RESPONSE

    EPA Science Inventory

    The virulence of multiple Aeromonas spp. were assessed using two models, a neonatal mouse assay and a mouse intestinal cell culture. Transcriptional responses to both infection models were assessed using microarrays. After artificial infection with a variety of Aeromonas spp., ...

  12. Patterns of virulence factor expression and antimicrobial resistance in Achromobacter xylosoxidans and Achromobacter ruhlandii isolates from patients with cystic fibrosis.

    PubMed

    Pereira, R H V; Leão, R S; Carvalho-Assef, A P; Albano, R M; Rodrigues, E R A; Firmida, M C; Folescu, T W; Plotkowski, M C; Bernardo, V G; Marques, E A

    2017-02-01

    Achromobacter spp. are opportunistic pathogens increasingly recovered from adult patients with cystic fibrosis (CF). We report the characterization of 122 Achromobacter spp. isolates recovered from 39 CF patients by multilocus sequence typing, virulence traits, and susceptibility to antimicrobials. Two species, A. xylosoxidans (77%) and A. ruhlandii (23%) were identified. All isolates showed a similar biofilm formation ability, and a positive swimming phenotype. By contrast, 4·3% and 44·4% of A. xylosoxidans and A. ruhlandii, respectively, exhibited a negative swarming phenotype, making the swimming and swarming abilities of A. xylosoxidans significantly higher than those of A. ruhlandii. A. xylosoxidans isolates from an outbreak clone also exhibited significantly higher motility. Both species were generally susceptible to ceftazidime, ciprofloxacin, imipenem and trimethoprim/sulphamethoxazole and there was no significant difference in susceptibility between isolates from chronic or sporadic infection. However, A. xylosoxidans isolates from chronic and sporadic cases were significantly more resistant to imipenem and ceftazidime than isolates of the outbreak clone.

  13. Deletion of AS87_03730 gene changed the bacterial virulence and gene expression of Riemerella anatipestifer

    PubMed Central

    Wang, Xiaolan; Yue, Jiaping; Ding, Chan; Wang, Shaohui; Liu, Beibei; Tian, Mingxing; Yu, Shengqing

    2016-01-01

    Riemerella anatipestifer is an important pathogen of waterfowl, which causes septicemia anserum exsudativa in ducks. In this study, an AS87_03730 gene deletion R. anatipestifer mutant Yb2ΔAS87_03730 was constructed to investigate the role of AS87_03730 on R. anatipestifer virulence and gene regulation. By deleting a 708-bp fragment from AS87_03730, the mutant Yb2ΔAS87_03730 showed a significant decreased growth rate in TSB and invasion capacity in Vero cells, compared to wild-type strain Yb2. Moreover, the median lethal dose (LD50) of Yb2ΔAS87_03730 was 1.24 × 107 colony forming units (CFU), which is about 80-fold attenuated than that of Yb2 (LD50 = 1.53 × 105 CFU). Furthermore, RNA-Seq analysis and Real-time PCR indicated 19 up-regulated and two down-regulated genes in Yb2ΔAS87_03730. Functional analysis revealed that 12 up-regulated genes were related to “Translation, ribosomal structure and biogenesis”, two were classified into “Cell envelope biogenesis, outer membrane”, one was involved in “Amino acid transport and metabolism”, and the other four had unknown functions. Polymerase chain reaction and sequence analysis indicated that the AS87_03730 gene is highly conserved among R. anatipestifer strains, as the percent sequence identity was over 93.5%. This study presents evidence that AS87_03730 gene is involved in bacterial virulence and gene regulation of R. anatipestifer. PMID:26928424

  14. Expression at mRNA level of cytokines and A238L gene in porcine blood-derived macrophages infected in vitro with African swine fever virus (ASFV) isolates of different virulence.

    PubMed

    Gil, S; Spagnuolo-Weaver, M; Canals, A; Sepúlveda, N; Oliveira, J; Aleixo, A; Allan, G; Leitão, A; Martins, C L V

    2003-11-01

    Porcine macrophage cultures were infected with two ASFV isolates of variable virulence and mRNA levels of several relevant macrophage-derived cytokines were quantified by real time PCR. At six hours post infection, a clear enhancement of mRNA expression of TNFalpha, IL6, IL12 and IL15 was observed in macrophages infected with the low virulent ASFV/NH/P68 (NHV) when compared to those infected with the highly virulent ASFV/L60 (L60). The sequence of the A238L gene homologue to the cellular IkappaB was found identical in both viral isolates and its expression at mRNA level was higher in macrophages infected with NHV when compared to macrophages infected with L60. Furthermore our results suggest a negative correlation between the mRNA expression of A238L gene and the mRNA expression of the above mentioned cytokines (with the exception of IL10) in L60 infected macrophages in opposition to the positive correlation (with exception of the IL1) suggested in NHV infection. Overall, our data strongly emphasize that virulence of ASFV isolates may depend on their capacity to regulate the expression of macrophage-derived cytokines relevant for the development of host protective responses by yet unknown mechanisms triggered by the virus at early stages of the cellular infection.

  15. Quorum sensing in bacterial virulence.

    PubMed

    Antunes, L Caetano M; Ferreira, Rosana B R; Buckner, Michelle M C; Finlay, B Brett

    2010-08-01

    Bacteria communicate through the production of diffusible signal molecules termed autoinducers. The molecules are produced at basal levels and accumulate during growth. Once a critical concentration has been reached, autoinducers can activate or repress a number of target genes. Because the control of gene expression by autoinducers is cell-density-dependent, this phenomenon has been called quorum sensing. Quorum sensing controls virulence gene expression in numerous micro-organisms. In some cases, this phenomenon has proven relevant for bacterial virulence in vivo. In this article, we provide a few examples to illustrate how quorum sensing can act to control bacterial virulence in a multitude of ways. Several classes of autoinducers have been described to date and we present examples of how each of the major types of autoinducer can be involved in bacterial virulence. As quorum sensing controls virulence, it has been considered an attractive target for the development of new therapeutic strategies. We discuss some of the new strategies to combat bacterial virulence based on the inhibition of bacterial quorum sensing systems.

  16. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses.

    PubMed

    Billecocq, Agnès; Gauliard, Nicolas; Le May, Nicolas; Elliott, Richard M; Flick, Ramon; Bouloy, Michèle

    2008-09-01

    Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.

  17. Expression of Listeria monocytogenes key virulence genes during growth in liquid medium, on rocket and melon at 4, 10 and 30 °C.

    PubMed

    Hadjilouka, Agni; Molfeta, Christina; Panagiotopoulou, Olga; Paramithiotis, Spiros; Mataragas, Marios; Drosinos, Eleftherios H

    2016-05-01

    The aim of the present study was to assess the expression of key virulence genes, during growth of a Listeria monocytogenes isolate in liquid medium, on melon and rocket at different temperatures and time. For that purpose, BHI broth, rocket and melon were inoculated at 7.0-7.5 log CFU mL(-1) or g(-1)and stored at 4, 10 and 30 °C. Sampling took place upon inoculation and after 0.5, 6 and 24 h of incubation. The RNA was stabilized and the expression of hly, plcA, plcB, sigB, inlA, inlB, inlC, inlJ, lmo2672 and lmo2470 was assessed by RT-qPCR. The results obtained were summarized into two observations; the first one referring to the interactive effect of incubation temperature and type of substrate and the second one to the effect of time on gene expression. Regarding the latter, nearly all genes were regulated upon inoculation and exhibited differential expression in the subsequent sampling times indicating the existence of additional regulatory mechanisms yet to be explored. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

    PubMed Central

    Zheng, Yuqiang; Zhang, Xuemei; Wang, Xiaofang; Wang, Libin; Zhang, Jinghui; Yin, Yibing

    2017-01-01

    The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComED58E) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level. PMID

  19. ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae.

    PubMed

    Zheng, Yuqiang; Zhang, Xuemei; Wang, Xiaofang; Wang, Libin; Zhang, Jinghui; Yin, Yibing

    2017-01-01

    The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComE(D58E)) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level.

  20. In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis

    PubMed Central

    Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey C.; Velineni, Sridhar; Timoney, John F.

    2015-01-01

    Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection. PMID:26607308

  1. Multiple promoter elements govern expression of the human ornithine decarboxylase gene in colon carcinoma cells.

    PubMed Central

    Moshier, J A; Osborne, D L; Skunca, M; Dosescu, J; Gilbert, J D; Fitzgerald, M C; Polidori, G; Wagner, R L; Friezner Degen, S J; Luk, G D

    1992-01-01

    Overexpression of the ornithine decarboxylase (ODC) gene may be important to the development and maintenance of colonic neoplasms, as well as tumors in general. In this study, we examined the promoter elements governing constitutive expression of the human ODC gene in HCT 116 human colon carcinoma cells and, for comparison, K562 human erythro-leukemia cells. It was determined by functional analysis that the promoter elements responsible reside within the 378 bp immediately upstream from the transcription start site. Within this sequence, there are at least three regions that modulate the efficiency of the ODC promoter cooperatively. Both DNA bandshift and footprint assays demonstrated all three regions to be rich in sites that bind to nuclear proteins isolated from HCT 116 and K562 cells; the protein binding pattern of non-transformed, diploid fibroblasts was found to be much less complex. Several of the protein binding sequences have little or no homology to common regulatory elements. We suggest that the constitutive activity of the ODC gene in HCT 116 colon carcinoma cells, and perhaps transformed cells in general, involves a complex interaction of multiple regulatory sequences and their associated nuclear proteins. Finally, the saturation of the promoter in these transformed cell lines suggests that high levels of protein binding in the ODC promoter may contribute to elevated constitutive expression of this gene. Images PMID:1598217

  2. Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence.

    PubMed

    Feng, Wenchao; Minor, Dylan; Liu, Mengyao; Li, Jinquan; Ishaq, Suzanne L; Yeoman, Carl; Lei, Benfang

    2017-01-01

    Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [Sse(A+)] and normal SpeB activity [SpeB(A+)]). Sse(A+) SpeB(A+) variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse(A+) SpeB(A+) isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse(A+) SpeB(A+) isolates also had nonsense mutations or small indels in rocA RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence. Copyright © 2016 American Society for Microbiology.

  3. The RNA chaperone Hfq is involved in stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression.

    PubMed

    Fantappiè, Laura; Metruccio, Matteo M E; Seib, Kate L; Oriente, Francesca; Cartocci, Elena; Ferlicca, Francesca; Giuliani, Marzia M; Scarlato, Vincenzo; Delany, Isabel

    2009-05-01

    The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis, we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored.

  4. A family of genus-specific RNAs in tandem with DNA-binding proteins control expression of the badA major virulence factor gene in Bartonella henselae.

    PubMed

    Tu, Nhan; Carroll, Ronan K; Weiss, Andy; Shaw, Lindsey N; Nicolas, Gael; Thomas, Sarah; Lima, Amorce; Okaro, Udoka; Anderson, Burt

    2017-04-01

    Bartonella henselae is a gram-negative zoonotic bacterium that causes infections in humans including endocarditis and bacillary angiomatosis. B. henselae has been shown to grow as large aggregates and form biofilms in vitro. The aggregative growth and the angiogenic host response requires the trimeric autotransporter adhesin BadA. We examined the transcriptome of the Houston-1 strain of B. henselae using RNA-seq revealing nine novel, highly-expressed intergenic transcripts (Bartonella regulatory transcript, Brt1-9). The Brt family of RNAs is unique to the genus Bartonella and ranges from 194 to 203 nucleotides with high homology and stable predicted secondary structures. Immediately downstream of each of the nine RNA genes is a helix-turn-helix DNA-binding protein (transcriptional regulatory protein, Trp1-9) that is poorly transcribed under the growth conditions used for RNA-seq. Using knockdown or overexpressing strains, we show a role of both the Brt1 and Trp1 in the regulation of badA and also in biofilm formation. Based on these data, we hypothesize that Brt1 is a trans-acting sRNA that also serves as a cis-acting riboswitch to control the expression of badA. This family of RNAs together with the downstream Trp DNA-binding proteins represents a novel coordinated regulatory circuit controlling expression of virulence-associated genes in the bartonellae. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  5. RNA-seq comparative analysis of Peking ducks spleen gene expression 24 h post-infected with duck plague virulent or attenuated virus.

    PubMed

    Liu, Tian; Cheng, Anchun; Wang, Mingshu; Jia, Renyong; Yang, Qiao; Wu, Ying; Sun, Kunfeng; Zhu, Dekang; Chen, Shun; Liu, Mafeng; Zhao, XinXin; Chen, Xiaoyue

    2017-09-13

    Duck plague virus (DPV), a member of alphaherpesvirus sub-family, can cause significant economic losses on duck farms in China. DPV Chinese virulent strain (CHv) is highly pathogenic and could induce massive ducks death. Attenuated DPV vaccines (CHa) have been put into service against duck plague with billions of doses in China each year. Researches on DPV have been development for many years, however, a comprehensive understanding of molecular mechanisms underlying pathogenicity of CHv strain and protection of CHa strain to ducks is still blank. In present study, we performed RNA-seq technology to analyze transcriptome profiling of duck spleens for the first time to identify differentially expressed genes (DEGs) associated with the infection of CHv and CHa at 24 h. Comparison of gene expression with mock ducks revealed 748 DEGs and 484 DEGs after CHv and CHa infection, respectively. Gene pathway analysis of DEGs highlighted valuable biological processes involved in host immune response, cell apoptosis and viral invasion. Genes expressed in those pathways were different in CHv infected duck spleens and CHa vaccinated duck spleens. The results may provide valuable information for us to explore the reasons of pathogenicity caused by CHv strain and protection activated by CHa strain.

  6. Infections of virulent and avirulent viruses differentially influenced the expression of dicer-1, ago-1, and microRNAs in Bombus terrestris

    PubMed Central

    Niu, Jinzhi; Meeus, Ivan; De Coninck, Dieter IM; Deforce, Dieter; Etebari, Kayvan; Asgari, Sassan; Smagghe, Guy

    2017-01-01

    The microRNA (miRNA) pathway is well established to be involved in host-pathogen interactions. As key insect pollinators, bees are suffering from widely spreading viruses, especially honeybees and bumblebees. In order to better understand bee-virus interaction, we comparatively analyzed the involvement of the bumblebee miRNA pathway upon infection by two different viruses. In our setup, an avirulent infection is induced by slow bee paralysis virus (SBPV) and a virulent infection is induced by Israeli acute paralysis virus (IAPV). Our results showed the increased expressions of dicer-1 and ago-1 upon SBPV infection. There were 17 and 12 bumblebee miRNAs differentially expressed upon SBPV and IAPV infections, respectively. These results may indicate the involvement of the host miRNA pathway in bumblebee-virus interaction. However, silencing of dicer-1 did not influence the genome copy number of SBPV. Target prediction for these differentially expressed miRNAs showed their possible involvement in targeting viral genomic RNA and in the regulation of networks in bumblebee. Our study opens a new insight into bee-virus interaction meditated by host miRNAs. PMID:28374846

  7. Infections of virulent and avirulent viruses differentially influenced the expression of dicer-1, ago-1, and microRNAs in Bombus terrestris.

    PubMed

    Niu, Jinzhi; Meeus, Ivan; De Coninck, Dieter Im; Deforce, Dieter; Etebari, Kayvan; Asgari, Sassan; Smagghe, Guy

    2017-04-04

    The microRNA (miRNA) pathway is well established to be involved in host-pathogen interactions. As key insect pollinators, bees are suffering from widely spreading viruses, especially honeybees and bumblebees. In order to better understand bee-virus interaction, we comparatively analyzed the involvement of the bumblebee miRNA pathway upon infection by two different viruses. In our setup, an avirulent infection is induced by slow bee paralysis virus (SBPV) and a virulent infection is induced by Israeli acute paralysis virus (IAPV). Our results showed the increased expressions of dicer-1 and ago-1 upon SBPV infection. There were 17 and 12 bumblebee miRNAs differentially expressed upon SBPV and IAPV infections, respectively. These results may indicate the involvement of the host miRNA pathway in bumblebee-virus interaction. However, silencing of dicer-1 did not influence the genome copy number of SBPV. Target prediction for these differentially expressed miRNAs showed their possible involvement in targeting viral genomic RNA and in the regulation of networks in bumblebee. Our study opens a new insight into bee-virus interaction meditated by host miRNAs.

  8. The Ustilago maydis b mating type locus controls hyphal proliferation and expression of secreted virulence factors in planta.

    PubMed

    Wahl, Ramon; Zahiri, Alexander; Kämper, Jörg

    2010-01-01

    Sexual development in fungi is controlled by mating type loci that prevent self-fertilization. In the phytopathogenic fungus Ustilago maydis, the b mating type locus encodes two homeodomain proteins, termed bE and bW. After cell fusion, a heterodimeric bE/bW complex is formed if the proteins are derived from different alleles. The bE/bW complex is required and sufficient to initiate pathogenic development and sexual reproduction; for the stages of pathogenic development succeeding plant penetration, however, its role was unclear. To analyse b function during in planta development, we generated a temperature-sensitive bE(ts) protein by exchange of a single amino acid. bE(ts) strains are stalled in pathogenic development at restrictive temperature in planta, and hyphae develop enlarged, bulbous cells at their tips that contain multiple nuclei, indicating a severe defect in the control and synchronization of cell cycle and cytokinesis. DNA array analysis of bE(ts) mutant strains in planta revealed a b-dependent regulation of genes encoding secreted proteins that were shown to influence fungal virulence. Our data demonstrate that in U. maydis the b heterodimer is not only essential to establish the heterodikaryon after mating of two compatible sporidia and to initiate fungal pathogenicity, but also to sustain in planta proliferation and ensure sexual reproduction.

  9. Burkholderia pseudomallei Evades Nramp1 (Slc11a1)- and NADPH Oxidase-Mediated Killing in Macrophages and Exhibits Nramp1-Dependent Virulence Gene Expression

    PubMed Central

    Muangsombut, Veerachat; Withatanung, Patoo; Srinon, Varintip; Chantratita, Narisara; Stevens, Mark P.; Blackwell, Jenefer M.; Korbsrisate, Sunee

    2017-01-01

    Bacterial survival in macrophages can be affected by the natural resistance-associated macrophage protein 1 (Nramp1; also known as solute carrier family 11 member a1 or Slc11a1) which localizes to phagosome membranes and transports divalent cations, including iron. Little is known about the role of Nramp1 in Burkholderia infection, in particular whether this differs for pathogenic species like Burkholderia pseudomallei causing melioidosis or non-pathogenic species like Burkholderia thailandensis. Here we show that transfected macrophages stably expressing wild-type Nramp1 (Nramp1+) control the net replication of B. thailandensis, but not B. pseudomallei. Control of B. thailandensis was associated with increased cytokine responses, and could be abrogated by blocking NADPH oxidase-mediated production of reactive oxygen species but not by blocking generation of reactive nitrogen species. The inability of Nramp1+ macrophages to control B. pseudomallei was associated with rapid escape of bacteria from phagosomes, as indicated by decreased co-localization with LAMP1 compared to B. thailandensis. A B. pseudomallei bipB mutant impaired in escape from phagosomes was controlled to a greater extent than the parent strain in Nramp1+ macrophages, but was also attenuated in Nramp1− cells. Consistent with reduced escape from phagosomes, B. thailandensis formed fewer multinucleated giant cells in Nramp1+ macrophages at later time points compared to B. pseudomallei. B. pseudomallei exhibited elevated transcription of virulence-associated genes of Type VI Secretion System cluster 1 (T6SS-1), the Bsa Type III Secretion System (T3SS-3) and the bimA gene required for actin-based motility in Nramp1+ macrophages. Nramp1+ macrophages were found to contain decreased iron levels that may impact on expression of such genes. Our data show that B. pseudomallei is able to evade Nramp1- and NADPH oxidase-mediated killing in macrophages and that expression of virulence-associated genes by

  10. Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

    PubMed Central

    Sarabhai, Sajal; Sharma, Prince; Capalash, Neena

    2013-01-01

    Background Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors. Methods and Results Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C12HSL and C4HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C4HSL. F7 also showed antagonistic activity against 3-oxo-C12HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract. Conclusions This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced

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

    PubMed

    Sarabhai, Sajal; Sharma, Prince; Capalash, Neena

    2013-01-01

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

  12. Regulation of Bacterial Virulence by Csr (Rsm) Systems

    PubMed Central

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

    2015-01-01

    SUMMARY Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5′ untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens. PMID:25833324

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

    PubMed

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

    2015-06-01

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

  14. Trans-cinnamaldehyde decreases attachment and invasion of uropathogenic Escherichia coli in urinary tract epithelial cells by modulating virulence gene expression.

    PubMed

    Amalaradjou, Mary Anne Roshni; Narayanan, Amoolya; Venkitanarayanan, Kumar

    2011-04-01

    Uropathogenic Escherichia coli is the primary bacterium causing urinary tract infection in humans. Attachment and invasion of urinary tract epithelial cells by UPEC is the first critical step in establishing a successful urinary tract infection. We investigated the efficacy of subinhibitory concentrations of trans-cinnamaldehyde to inhibit uropathogenic E. coli attachment and invasion of human uroepithelial cells. We also determined the trans-cinnamaldehyde effect on uropathogenic E. coli genes encoding virulence factors critical for uroepithelial cell bacterial attachment and invasion. Polystyrene 24-well plates seeded with uroepithelial cells were inoculated with uropathogenic E. coli (about 6.0 log cfu) and subinhibitory concentrations of trans-cinnamaldehyde (0, 325, 560 and 750 μM), and incubated for 60 minutes at 37C. Uroepithelial cells were washed and lysed to enumerate adhered uropathogenic E. coli populations. For the invasion assay uroepithelial cells were treated with gentamicin after incubation and lysed to enumerate invaded uropathogenic E. coli. Also, the trans-cinnamaldehyde effect on uropathogenic E. coli genes encoding attachment and invasion associated virulence factors was determined by real-time quantitative polymerase chain reaction. Trans-cinnamaldehyde significantly decreased uroepithelial cell attachment and invasion by uropathogenic E. coli (p <0.05). Real-time quantitative polymerase chain reaction revealed that trans-cinnamaldehyde significantly decreased the expression of major genes involved in uropathogenic E. coli attachment and invasion of host tissue (p <0.05). The down-regulating effect of trans-cinnamaldehyde on these genes potentially translated into decreased ability of uropathogenic E. coli to attach and invade bladder cells. Trans-cinnamaldehyde may potentially be used as a safe, effective antimicrobial to control uropathogenic E. coli infection. Followup studies in animal models are warranted. Copyright © 2011 American

  15. RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

    PubMed

    Stefanovic, Branko

    2013-01-01

    Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

    PubMed Central

    2012-01-01

    Background Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). Results Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. Conclusions Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds

  17. Telomerase governs immunomodulatory properties of mesenchymal stem cells by regulating FAS ligand expression

    PubMed Central

    Chen, Chider; Akiyama, Kentaro; Yamaza, Takayoshi; You, Yong-Ouk; Xu, Xingtian; Li, Bei; Zhao, Yimin; Shi, Songtao

    2014-01-01

    Bone marrow mesenchymal stem cells (BMMSCs) are capable of differentiating into multiple cell types and regulating immune cell response. However, the mechanisms that govern the immunomodulatory properties of BMMSCs are still not fully elucidated. Here we show that telomerase-deficient BMMSCs lose their capacity to inhibit T cells and ameliorate the disease phenotype in systemic sclerosis mice. Restoration of telomerase activity by telomerase reverse transcriptase (TERT) transfection in TERT−/− BMMSCs rescues their immunomodulatory functions. Mechanistically, we reveal that TERT, combined with β-catenin and BRG1, serves as a transcriptional complex, which binds the FAS ligand (FASL) promoter to upregulate FASL expression, leading to an elevated immunomodulatory function. To test the translational value of these findings in the context of potential clinical therapy, we used aspirin treatment to upregulate telomerase activity in BMMSCs, and found a significant improvement in the immunomodulatory capacity of BMMSCs. Taken together, these findings identify a previously unrecognized role of TERT in improving the immunomodulatory capacity of BMMSCs, suggesting that aspirin treatment is a practical approach to significantly reduce cell dosage in BMMSC-based immunotherapies. Subject Categories Stem Cells; Immunology PMID:24401839

  18. Fur Negatively Regulates hns and Is Required for the Expression of HilA and Virulence in Salmonella enterica Serovar Typhimurium▿ †

    PubMed Central

    Troxell, Bryan; Sikes, Michael L.; Fink, Ryan C.; Vazquez-Torres, Andres; Jones-Carson, Jessica; Hassan, Hosni M.

    2011-01-01

    Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns. PMID:21075923

  19. Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium.

    PubMed

    Troxell, Bryan; Sikes, Michael L; Fink, Ryan C; Vazquez-Torres, Andres; Jones-Carson, Jessica; Hassan, Hosni M

    2011-01-01

    Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.

  20. ChIP-seq analysis of the LuxR-type regulator VjbR reveals novel insights into the Brucella virulence gene expression network

    PubMed Central

    Sycz, Gabriela; Bonomi, Hernán R.; Rodríguez, Romina M.; Zorreguieta, Angeles

    2017-01-01

    Abstract LuxR-type transcription factors control diverse physiological functions necessary for bacterial adaptation to environmental changes. In the intracellular pathogen Brucella, the LuxR homolog VjbR has been shown to regulate the expression of virulence factors acting at early stages of the intracellular infection and, directly or indirectly, hundreds of additional genes. However, the precise determination of VjbR direct targets has so far proved elusive. Here, we performed chromatin immunoprecipitation of VjbR followed by next-generation sequencing (ChIP-seq). We detected a large amount of VjbR-binding sites distributed across the Brucella genome and determined a markedly asymmetric binding consensus motif, an unusual feature among LuxR-type regulators. RNA-seq analysis performed under conditions mimicking the eukaryotic intracellular environment revealed that, among all loci associated to VjbR-binding, this regulator directly modulated the expression of only a subset of genes encoding functions consistent with an intracellular adaptation strategy for survival during the initial stages of the host cell infection. Other VjbR-binding events, however, showed to be dissociated from transcription and may require different environmental signals to produce a transcriptional output. Taken together, our results bring new insights into the extent and functionality of LuxR-type-related transcriptional networks. PMID:28334833

  1. Inhibition of Expression in Escherichia coli of a Virulence Regulator MglB of Francisella tularensis Using External Guide Sequence Technology

    PubMed Central

    Xiao, Gaoping; Lundblad, Eirik W.; Izadjoo, Mina; Altman, Sidney

    2008-01-01

    External guide sequences (EGSs) have successfully been used to inhibit expression of target genes at the post-transcriptional level in both prokaryotes and eukaryotes. We previously reported that EGS accessible and cleavable sites in the target RNAs can rapidly be identified by screening random EGS (rEGS) libraries. Here the method of screening rEGS libraries and a partial RNase T1 digestion assay were used to identify sites accessible to EGSs in the mRNA of a global virulence regulator MglB from Francisella tularensis, a Gram-negative pathogenic bacterium. Specific EGSs were subsequently designed and their activities in terms of the cleavage of mglB mRNA by RNase P were tested in vitro and in vivo. EGS73, EGS148, and EGS155 in both stem and M1 EGS constructs induced mglB mRNA cleavage in vitro. Expression of stem EGS73 and EGS155 in Escherichia coli resulted in significant reduction of the mglB mRNA level coded for the F. tularensis mglB gene inserted in those cells. PMID:19005569

  2. Two Stable Variants of Burkholderia pseudomallei Strain MSHR5848 Express Broadly Divergent in vitro Phenotypes Associated with their Virulence Differences

    DTIC Science & Technology

    2016-11-21

    Refs are in Endnote file] Burkholderia pseudomallei (Bp) causes melioidosis and is a CDC Tier 1 bacterial pathogen. Bp is a saprophytic, free...expression of various alternate forms of an antigen on the bacterial surface). Either form of colony morphotype expression can result from genetic...Vial et al. 2010; Bernier et al., 2008). Such bacterial -host models may provide insights useful in the analysis of Bp strain MSHR5848

  3. The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of Salmonella enterica serovar Infantis in mouse vs. avian hosts

    PubMed Central

    Mikhlin, Svetlana; Cohen, Helit; Vitman Zilber, Shaul; Grassl, Guntram A.

    2017-01-01

    Salmonella enterica serovar Infantis is one of the prevalent Salmonella serovars worldwide. Different emergent clones of S. Infantis were shown to acquire the pESI virulence-resistance megaplasmid affecting its ecology and pathogenicity. Here, we studied two previously uncharacterized pESI-encoded chaperone-usher fimbriae, named Ipf and Klf. While Ipf homologs are rare and were found only in S. enterica subspecies diarizonae and subspecies VII, Klf is related to the known K88-Fae fimbria and klf clusters were identified in seven S. enterica subspecies I serovars, harboring interchanging alleles of the fimbria major subunit, KlfG. Regulation studies showed that the klf genes expression is negatively and positively controlled by the pESI-encoded regulators KlfL and KlfB, respectively, and are activated by the ancestral leucine-responsive regulator (Lrp). ipf genes are negatively regulated by Fur and activated by OmpR. Furthermore, induced expression of both klf and ipf clusters occurs under microaerobic conditions and at 41°C compared to 37°C, in-vitro. Consistent with these results, we demonstrate higher expression of ipf and klf in chicks compared to mice, characterized by physiological temperature of 41.2°C and 37°C, respectively. Interestingly, while Klf was dispensable for S. Infantis colonization in the mouse, Ipf was required for maximal colonization in the murine ileum. In contrast to these phenotypes in mice, both Klf and Ipf contributed to a restrained infection in chicks, where the absence of these fimbriae has led to moderately higher bacterial burden in the avian host. Taken together, these data suggest that physiological differences between host species, such as the body temperature, can confer differences in fimbriome expression, affecting Salmonella colonization and other host-pathogen interplays. PMID:28817673

  4. The low-virulent African swine fever virus (ASFV/NH/P68) induces enhanced expression and production of relevant regulatory cytokines (IFNalpha, TNFalpha and IL12p40) on porcine macrophages in comparison to the highly virulent ASFV/L60.

    PubMed

    Gil, S; Sepúlveda, N; Albina, E; Leitão, A; Martins, C

    2008-01-01

    The impact of infection by the low-virulent ASFV/NH/P68 (NHV) and the highly virulent ASFV/L60 (L60) isolates on porcine macrophages was assessed through the quantification of IFNalpha, TNFalpha, IL12p40, TGFbeta and ASFV genes by real-time PCR at 2, 4 and 6 h post-infection. Increased IFNalpha, TNFalpha and IL12p40 expression was found in infection with NHV, in which expression of TGFbeta was lower than in infection with L60. Principal component analysis showed a positive interaction of cytokines involved in cellular immune mechanisms, namely IFNalpha and IL12p40 in the NHV infection. Quantification by ELISA confirmed higher production of IFNalpha, TNFalpha and IL12p40 in the NHV-infected macrophages. Overall, our studies reinforce and clarify the effect of the NHV infection by targeting cellular and cellular-based immune responses relevant for pig survival against ASFV infection.

  5. Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression

    PubMed Central

    Zusman, Tal; Gal-Mor, Ohad; Segal, Gil

    2002-01-01

    To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression. PMID:11741845

  6. Analysis of Expressed Genes of the Bacterium ‘Candidatus Phytoplasma Mali’ Highlights Key Features of Virulence and Metabolism

    PubMed Central

    Siewert, Christin; Luge, Toni; Duduk, Bojan; Seemüller, Erich; Büttner, Carmen; Sauer, Sascha; Kube, Michael

    2014-01-01

    ‘Candidatus Phytoplasma mali’ is a phytopathogenic bacterium of the family Acholeplasmataceae assigned to the class Mollicutes. This causative agent of the apple proliferation colonizes in Malus domestica the sieve tubes of the plant phloem resulting in a range of symptoms such as witches’- broom formation, reduced vigor and affecting size and quality of the crop. The disease is responsible for strong economical losses in Europe. Although the genome sequence of the pathogen is available, there is only limited information on expression of selected genes and metabolic key features that have not been examined on the transcriptomic or proteomic level so far. This situation is similar to many other phytoplasmas. In the work presented here, RNA-Seq and mass spectrometry shotgun techniques were applied on tissue samples from Nicotiana occidentalis infected by ‘Ca. P. mali’ strain AT providing insights into transcriptome and proteome of the pathogen. Data analysis highlights expression of 208 genes including 14 proteins located in the terminal inverted repeats of the linear chromosome. Beside a high portion of house keeping genes, the recently discussed chaperone GroES/GroEL is expressed. Furthermore, gene expression involved in formation of a type IVB and of the Sec-dependent secretion system was identified as well as the highly expressed putative pathogenicity–related SAP11-like effector protein. Metabolism of phytoplasmas depends on the uptake of spermidine/putescine, amino acids, co-factors, carbohydrates and in particular malate/citrate. The expression of these transporters was confirmed and the analysis of the carbohydrate cycle supports the suggested alternative energy-providing pathway for phytoplasmas releasing acetate and providing ATP. The phylogenetic analyses of malate dehydrogenase and acetate kinase in phytoplasmas show a closer relatedness to the Firmicutes in comparison to Mycoplasma species indicating an early divergence of the

  7. Proteomic Characterization of Yersinia pestis Virulence

    SciTech Connect

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

    2005-01-05

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

  8. Molecular insight into the activity of LasR protein from Pseudomonas aeruginosa in the regulation of virulence gene expression by this organism.

    PubMed

    Chowdhury, Nilkanta; Bagchi, Angshuman

    2016-04-10

    Pseudomonas aeruginosa is an opportunistic human pathogen. This organism attacks human patients suffering from diseases like AIDS, cancer, cystic fibrosis, etc. One of the important virulent factors produced by this organism is Hydrogen Cyanide. This is expressed from the genes encoded by the hcnABC operon. The expressions of the genes encoded by hcnABC operon are mediated mainly by the interactions of LasR protein with the corresponding promoter region of the hcnABC operon. The LasR protein acts as a dimer and binds to the promoter DNA with the help of an autoinducer ligand. However, till date the detailed molecular mechanism of how the LasR protein interacts with the promoter DNA is not clearly known. Therefore, in this work, an attempt has been made to analyze the mode of interactions of the LasR protein with the promoter DNA region of the hcnABC operon. We analyzed the three dimensional structure of the LasR protein from Pseudomonas aeruginosa and docked the protein with the autoinducer ligand. We then docked the ligand-bound-LasR-protein as well the LasR-protein-without-the-autoinducer-ligand on to the promoter DNA region of hcnABC operon. We analyzed the details of the interaction profiles of LasR protein with the autoinducer ligand. We also deciphered the details of the LasR promoter-DNA interactions. We compared the modes of DNA bindings by the LasR protein in presence and absence of the autoinducer ligand and tried to analyze the molecular details of the binding of LasR protein with the promoter DNA region of hcnABC operon during hcnABC gene expression. This study may therefore pave the pathway for future experiments to determine the relative effects of the amino acid residues of LasR protein in DNA binding during the transcription of hcnABC operon.

  9. Ocular infection of mice with an avirulent recombinant HSV-1 expressing IL-4 and an attenuated HSV-1 strain generates virulent recombinants in vivo.

    PubMed

    Mott, Kevin R; Wechsler, Steven L; Ghiasi, Homayon

    2010-10-26

    To assess the relative impact of overexpression of interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN-γ) expressing recombinant herpes simplex virus type 1 (HSV-1) on altering immune responses in ocularly infected mice. BALB/c mice were co-infected ocularly with avirulent HSV-1 strain KOS and avirulent recombinant HSV-1 expressing murine IL-4 (HSV-IL-4). Controls mice were co-infected with KOS+HSV-IL-2 or KOS+HSV-IFNγ. Following ocular infection, virus replication in the eye, corneal scarring (CS), and survival were determined. We also isolated recombinant viruses from eye and trigeminal ganglia of KOS+HSV-IL-4 infected mice. In this study we found that ocular infection of BALB/c mice with a mixture of HSV-IL-4 and KOS resulted in increased death and increased eye disease. In contrast, when mice were infected in one eye with KOS and the other eye with HSV-IL-4 no death or eye disease was seen. Intraperitoneal co-infection of mice with KOS and HSV-IL-4 also did not result in HSV-1 induced death. Interestingly, ocular infection of mice with a mixture of HSV-IL-2 and KOS did not have any effect on severity of the disease in infected mice. We isolated recombinant viruses from KOS+HSV-IL-4 infected mice eye and trigeminal ganglia. Some of the isolated viruses were more neurovirulent then either parental virus. Infection of macrophages with IL-4 expressing virus down-regulated IL-12 production by macrophages. These results suggest a role for IL-4 in suppression of immune response and generation of virulent viruses in vivo.

  10. In vivo virulence of viral haemorrhagic septicaemia virus (VHSV) in rainbow trout Oncorhynchus mykiss correlates inversely with in vitro Mx gene expression.

    PubMed

    Cano, Irene; Collet, Bertrand; Pereira, Clarissa; Paley, Richard; Aerle, Ronny van; Stone, David; Taylor, Nick G H

    2016-05-01

    The in vitro replication of viral haemorrhagic septicaemia virus (VHSV) isolates from each VHSV genotype and the associated cellular host Mx gene expression were analysed. All the isolates were able to infect RTG-2 cells and induce increased Mx gene expression (generic assay detecting isoforms 1 and 3 [Mx1/3]). A trout pathogenic, genotype Ia isolate (J167), showing high replication in RTG-2 cells (by infective titre and N gene expression) induced lower Mx1/3 gene expression than observed in VHSV isolates known to be non-pathogenic to rainbow trout: 96-43/8, 96-43/10 (Ib); 1p49, 1p53 (II); and MI03 (IVb). Paired co-inoculation assays were analysed using equal number of plaque forming units per ml (PFU) of J167 (Ia genotype) with other less pathogenic VHSV genotypes. In these co-inoculations, the Mx1/3 gene expression was significantly lower than for the non-pathogenic isolate alone. Of the three rainbow trout Mx isoforms, J167 did not induce Mx1 up-regulation in RTG-2 or RTgill-W1 cells. Co-inoculating isolates resulted in greater inhibition of Mx in both rainbow trout cell lines studied. Up-regulation of sea bream Mx in SAF-1 cells induced by 96-43/8 was also lower in co-inoculation assays with J167. The RTG-P1 cell line, expressing luciferase under the control of the interferon-induced Mx rainbow trout gene promoter, showed low luciferase activity when inoculated with pathogenic strains: J167, DK-5131 (Ic), NO-A-163/68 (Id), TR-206239-1, TR-22207111 (Ie), 99-292 (IVa), and CA-NB00-01 (IVc). Co-inoculation assays showed a J167-dose dependent inhibition of the luciferase activity. The data suggest that virulent VHSV isolates may interfere in the interferon pathways, potentially determining higher pathogenicity. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  11. Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

    PubMed Central

    Zhao, Jinlei

    2015-01-01

    Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site. PMID:26637603

  12. Microbiota-Derived Short-Chain Fatty Acids Modulate Expression of Campylobacter jejuni Determinants Required for Commensalism and Virulence

    PubMed Central

    Luethy, Paul M.; Huynh, Steven; Ribardo, Deborah A.

    2017-01-01

    ABSTRACT Campylobacter jejuni promotes commensalism in the intestinal tracts of avian hosts and diarrheal disease in humans, yet components of intestinal environments recognized as spatial cues specific for different intestinal regions by the bacterium to initiate interactions in either host are mostly unknown. By analyzing a C. jejuni acetogenesis mutant defective in converting acetyl coenzyme A (Ac-CoA) to acetate and commensal colonization of young chicks, we discovered evidence for in vivo microbiota-derived short-chain fatty acids (SCFAs) and organic acids as cues recognized by C. jejuni that modulate expression of determinants required for commensalism. We identified a set of C. jejuni genes encoding catabolic enzymes and transport systems for amino acids required for in vivo growth whose expression was modulated by SCFAs. Transcription of these genes was reduced in the acetogenesis mutant but was restored upon supplementation with physiological concentrations of the SCFAs acetate and butyrate present in the lower intestinal tracts of avian and human hosts. Conversely, the organic acid lactate, which is abundant in the upper intestinal tract where C. jejuni colonizes less efficiently, reduced expression of these genes. We propose that microbiota-generated SCFAs and lactate are cues for C. jejuni to discriminate between different intestinal regions. Spatial gradients of these metabolites likely allow C. jejuni to locate preferred niches in the lower intestinal tract and induce expression of factors required for intestinal growth and commensal colonization. Our findings provide insights into the types of cues C. jejuni monitors in the avian host for commensalism and likely in humans to promote diarrheal disease. PMID:28487428

  13. Identification of Differentially Expressed Proteins from Leishmania amazonensis Associated with the Loss of Virulence of the Parasites

    PubMed Central

    Magalhães, Rubens D. M.; Duarte, Mariana C.; Mattos, Eliciane C.; Martins, Vivian T.; Lage, Paula S.; Chávez-Fumagalli, Miguel A.; Lage, Daniela P.; Menezes-Souza, Daniel; Régis, Wiliam C. B.; Manso Alves, Maria J.; Soto, Manuel; Tavares, Carlos A. P.; Nagen, Ronaldo A. P.; Coelho, Eduardo A. F.

    2014-01-01

    Background The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach. Methodology/Principal Findings Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified. Conclusions/Significance The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis

  14. The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression

    PubMed Central

    Luzader, Deborah H.; Willsey, Graham G.; Wargo, Matthew J.

    2016-01-01

    Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome throughout the world. A defining feature of EHEC pathogenesis is the formation of attaching and effacing (AE) lesions on colonic epithelial cells. Most of the genes that code for AE lesion formation, including a type three secretion system (T3SS) and effectors, are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). In this study, we report that a putative regulator, which is encoded in the cryptic E. coli type three secretion system 2 (ETT2) locus and herein renamed EtrB, plays an important role in EHEC pathogenesis. The etrB gene is expressed as a monocistronic transcript, and EtrB autoregulates expression. We provide evidence that EtrB directly interacts with the ler regulatory region to activate LEE expression and promote AE lesion formation. Additionally, we mapped the EtrB regulatory circuit in EHEC to determine a global role for EtrB. EtrB is regulated by the transcription factor QseA, suggesting that these proteins comprise a regulatory circuit important for EHEC colonization of the gastrointestinal tract. PMID:27324484

  15. Genome-Wide Gene Expression Analysis Identifies the Proto-oncogene Tyrosine-Protein Kinase Src as a Crucial Virulence Determinant of Infectious Laryngotracheitis Virus in Chicken Cells

    PubMed Central

    Li, Hai; Wang, Fengjie; Han, Zongxi; Gao, Qi; Li, Huixin; Shao, Yuhao; Sun, Nana

    2015-01-01

    ABSTRACT Given the side effects of vaccination against infectious laryngotracheitis (ILT), novel strategies for ILT control and therapy are urgently needed. The modulation of host-virus interactions is a promising strategy to combat the virus; however, the interactions between the host and avian ILT herpesvirus (ILTV) are unclear. Using genome-wide transcriptome studies in combination with a bioinformatic analysis, we identified proto-oncogene tyrosine-protein kinase Src (Src) to be an important modulator of ILTV infection. Src controls the virulence of ILTV and is phosphorylated upon ILTV infection. Functional studies revealed that Src prolongs the survival of host cells by increasing the threshold of virus-induced cell death. Therefore, Src is essential for viral replication in vitro and in ovo but is not required for ILTV-induced cell death. Furthermore, our results identify a positive-feedback loop between Src and the tyrosine kinase focal adhesion kinase (FAK), which is necessary for the phosphorylation of either Src or FAK and is required for Src to modulate ILTV infection. To the best of our knowledge, we are the first to identify a key host regulator controlling host-ILTV interactions. We believe that our findings have revealed a new potential therapeutic target for ILT control and therapy. IMPORTANCE Despite the extensive administration of live attenuated vaccines starting from the mid-20th century and the administration of recombinant vaccines in recent years, infectious laryngotracheitis (ILT) outbreaks due to avian ILT herpesvirus (ILTV) occur worldwide annually. Presently, there are no drugs or control strategies that effectively treat ILT. Targeting of host-virus interactions is considered to be a promising strategy for controlling ILTV infections. However, little is known about the mechanisms governing host-ILTV interactions. The results from our study advance our understanding of host-ILTV interactions on a molecular level and provide experimental

  16. The Klebsiella pneumoniae YfgL (BamB) lipoprotein contributes to outer membrane protein biogenesis, type-1 fimbriae expression, anti-phagocytosis, and in vivo virulence.

    PubMed

    Hsieh, Pei-Fang; Hsu, Chun-Ru; Chen, Chun-Tang; Lin, Tzu-Lung; Wang, Jin-Town

    2016-07-03

    Klebsiella pneumoniae is an opportunistic pathogen that causes several kinds of infections, including pneumonia, bacteremia, urinary tract infection and community-acquired pyogenic liver abscess (PLA). Adhesion is the critical first step in the infection process. Our previous work demonstrated that the transcellular translocation is exploited by K. pneumoniae strains to migrate from the gut flora into other tissues, resulting in systemic infections. However, the initial stages of K. pneumoniae infection remain unclear. In this study, we demonstrated that a K. pneumoniae strain deleted for yfgL (bamB) exhibited reduced adherence to and invasion of host cells; changed biogenesis of major β-barrel outer membrane proteins; decreased transcriptional expression of type-1 fimbriae; and increased susceptibility to vancomycin and erythromycin. The yfgL deletion mutant also had reduced ability to against neutrophil phagocytosis; exhibited decreased induction of host IL-6 production; and was profoundly attenuated for virulence in a K. pneumoniae model of bacteremia. Thus, the K. pneumoniae YfgL lipoprotein mediates in outer membrane proteins biogenesis and is crucial for anti-phagocytosis and survival in vivo. These data provide a new insight for K. pneumoniae attachment and such knowledge could facilitate preventive therapies or alternative therapies against K. pneumoniae.

  17. Expression of parasite genetic variation changes over the course of infection: implications of within-host dynamics for the evolution of virulence

    PubMed Central

    Clerc, Melanie; Ebert, Dieter; Hall, Matthew D.

    2015-01-01

    How infectious disease agents interact with their host changes during the course of infection and can alter the expression of disease-related traits. Yet by measuring parasite life-history traits at one or few moments during infection, studies have overlooked the impact of variable parasite growth trajectories on disease evolution. Here we show that infection-age-specific estimates of host and parasite fitness components can reveal new insight into the evolution of parasites. We do so by characterizing the within-host dynamics over an entire infection period for five genotypes of the castrating bacterial parasite Pasteuria ramosa infecting the crustacean Daphnia magna. Our results reveal that genetic variation for parasite-induced gigantism, host castration and parasite spore loads increases with the age of infection. Driving these patterns appears to be variation in how well the parasite maintains control of host reproduction late in the infection process. We discuss the evolutionary consequences of this finding with regard to natural selection acting on different ages of infection and the mechanism underlying the maintenance of castration efficiency. Our results highlight how elucidating within-host dynamics can shed light on the selective forces that shape infection strategies and the evolution of virulence. PMID:25761710

  18. Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen.

    PubMed

    Chen, Tsung-Hsien; Chen, Ten-Hong; Hu, Chung-Chi; Liao, Jia-Teh; Lee, Chin-Wei; Liao, Jiunn-Wang; Lin, Maw-Yeong; Liu, Hung-Jen; Wang, Min-Ying; Lin, Na-Sheng; Hsu, Yau-Heiu

    2012-06-01

    Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. DotU expression is highly induced during in vivo infection and responsible for virulence and Hcp1 secretion in avian pathogenic Escherichia coli

    PubMed Central

    Wang, Shaohui; Dai, Jianjun; Meng, Qingmei; Han, Xiangan; Han, Yue; Zhao, Yichao; Yang, Denghui; Ding, Chan; Yu, Shengqing

    2014-01-01

    Type VI secretion systems (T6SSs) contribute to pathogenicity in many pathogenic bacteria. Three distinguishable T6SS loci have been discovered in avian pathogenic Escherichia coli (APEC). The sequence of APEC T6SS2 locus is highly similar to the sequence of the newborn meningitis Escherichia coli (NMEC) RS218 T6SS locus, which might contribute to meningitis pathogenesis. However, little is known about the function of APEC T6SS2. We showed that the APEC T6SS2 component organelle trafficking protein (DotU) could elicit antibodies in infected ducks, suggesting that DotU might be involved in APEC pathogenicity. To investigate DotU in APEC pathogenesis, mutant and complemented strains were constructed and characterized. Inactivation of the APEC dotU gene attenuated virulence in ducks, diminished resistance to normal duck serum, and reduced survival in macrophage cells and ducks. Furthermore, deletion of the dotU gene abolished hemolysin-coregulated protein (Hcp) 1 secretion, leading to decreased interleukin (IL)-6 and IL-8 gene expression in HD-11 chicken macrophages. These functions were restored for the complementation strain. Our results demonstrated that DotU plays key roles in the APEC pathogenesis, Hcp1 secretion, and intracellular host response modulation. PMID:25426107

  20. The knockdown of each component of the cysteine proteinase-adhesin complex of Entamoeba histolytica (EhCPADH) affects the expression of the other complex element as well as the in vitro and in vivo virulence.

    PubMed

    Ocádiz-Ruiz, Ramón; Fonseca, Wendy; Linford, Alicia S; Yoshino, Timothy P; Orozco, Esther; Rodríguez, Mario A

    2016-01-01

    Entamoeba histolytica is the protozoan parasite causative of human amoebiasis, disease responsible for 40 000-100 000 deaths annually. The cysteine proteinase-adhesin complex of this parasite (EhCPADH) is a heterodimeric protein formed by a cysteine protease (EhCP112) and an adhesin (EhADH) that plays an important role in the cytopathic mechanism of this parasite. The coding genes for EhCP112 and EhADH are adjacent in the E. histolytica genome, suggesting that their expression may be co-regulated, but this hypothesis has not yet been confirmed. Here, we performed the knockdown of EhCP112 and EhADH using gene-specific short-hairpin RNAs (shRNA), and the effect of these knockdowns on the expression of both complex components as well as on the in vitro and in vivo virulence was analysed. Results showed that the knockdown of one of the EhCPADH components produced a simultaneous downregulation of the other protein. Accordingly, a concomitant reduction in the overall expression of the complex was observed. The downregulation of each component also produced a significant decrease in the in vitro and in vivo virulence of trophozoites. These results demonstrated that the expression of EhCP112 and EhADH is co-regulated and confirmed that the EhCPADH complex plays an important role in E. histolytica virulence.

  1. Global Regulator of Virulence A (GrvA) Coordinates Expression of Discrete Pathogenic Mechanisms in Enterohemorrhagic Escherichia coli through Interactions with GadW-GadE

    PubMed Central

    Morgan, Jason K.; Harro, Carly M.; Vendura, Khoury W.; Shaw, Lindsey N.

    2015-01-01

    ABSTRACT Global regulator of virulence A (GrvA) is a ToxR-family transcriptional regulator that activates locus of enterocyte effacement (LEE)-dependent adherence in enterohemorrhagic Escherichia coli (EHEC). LEE activation by GrvA requires the Rcs phosphorelay response regulator RcsB and is sensitive to physiologically relevant concentrations of bicarbonate, a known stimulant of virulence systems in intestinal pathogens. This study determines the genomic scale of GrvA-dependent regulation and uncovers details of the molecular mechanism underlying GrvA-dependent regulation of pathogenic mechanisms in EHEC. In a grvA-null background of EHEC strain TW14359, RNA sequencing analysis revealed the altered expression of over 700 genes, including the downregulation of LEE- and non-LEE-encoded effectors and the upregulation of genes for glutamate-dependent acid resistance (GDAR). Upregulation of GDAR genes corresponded with a marked increase in acid resistance. GrvA-dependent regulation of GDAR and the LEE required gadE, the central activator of GDAR genes and a direct repressor of the LEE. Control of gadE by GrvA was further determined to occur through downregulation of the gadE activator GadW. This interaction of GrvA with GadW-GadE represses the acid resistance phenotype, while it concomitantly activates the LEE-dependent adherence and secretion of immune subversion effectors. The results of this study significantly broaden the scope of GrvA-dependent regulation and its role in EHEC pathogenesis. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) is an intestinal human pathogen causing acute hemorrhagic colitis and life-threatening hemolytic-uremic syndrome. For successful transmission and gut colonization, EHEC relies on the glutamate-dependent acid resistance (GDAR) system and a type III secretion apparatus, encoded on the LEE pathogenicity island. This study investigates the mechanism whereby the DNA-binding regulator GrvA coordinates activation of the LEE with

  2. Inhibition of PbGP43 Expression May Suggest that gp43 is a Virulence Factor in Paracoccidioides brasiliensis

    PubMed Central

    Torres, Isaura; Hernandez, Orville; Tamayo, Diana; Muñoz, Jose F.; Leitão, Natanael P.; García, Ana M.; Restrepo, Angela

    2013-01-01

    Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80–85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ−10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis. PMID:23874627

  3. Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties.

    PubMed

    Asmat, Tauseef M; Klingbeil, Katharina; Jensch, Inga; Burchhardt, Gerhard; Hammerschmidt, Sven

    2012-03-01

    Lactococcus lactis is a non-pathogenic bacterium that is used in the food industry but is also used as a heterologous host to reveal protein functions of pathogenic bacteria. The adhesin PspC from Streptococcus pneumoniae is a choline-binding protein that is non-covalently anchored to the bacterial cell wall. To assess the exclusive impact of pneumococcal surface protein C (PspC) on the interplay with its host we generated recombinant L. lactis producing a nisin-inducible and covalently anchored variant of PspC on the lactococcal cell surface. A translational fusion of the 5'-end of pspC3.4 with the 3'-end of hic (pspC11.4) was designed to decorate the surface of L. lactis with a chimeric PspC. The PspC3.4 part comprises the first 281 aa residues of PspC3.4, while the Hic sequence consists of the proline-rich and sortase-anchored domain. The results demonstrated that PspC is sufficient for adhesion and subsequent invasion of host epithelial cells expressing the human polymeric Ig receptor (hpIgR). Moreover, invasion via hpIgR was even more pronounced when the chimeric PspC was produced by lactococci compared with pneumococci. This study shows also for the first time that PspC plays no significant role during phagocytosis by macrophages. In contrast, recruitment of Factor H via the PspC chimer has a dramatic effect on phagocytosis of recombinant but not wild-type lactococci, as Factor H interacts specifically with the amino-terminal part of PspC and mediates the contact with phagocytes. Furthermore, L. lactis expressing PspC increased intracellular calcium levels in pIgR-expressing epithelial cells, thus resembling the effect of pneumococci, which induced release of Ca(2+) from intracellular stores via the PspC-pIgR mechanism. In conclusion, expression of the chimeric PspC confers adhesive properties to L. lactis and indicates the potential of L. lactis as a suitable host to study the impact of individual bacterial factors on their capacity to interfere with the

  4. Ciclopirox Olamine Treatment Affects the Expression Pattern of Candida albicans Genes Encoding Virulence Factors, Iron Metabolism Proteins, and Drug Resistance Factors

    PubMed Central

    Niewerth, Markus; Kunze, Donika; Seibold, Michael; Schaller, Martin; Korting, Hans Christian; Hube, Bernhard

    2003-01-01

    The hydroxypyridone ciclopirox olamine belongs to the antimycotic drugs used for the treatment of superficial mycoses. In contrast to the azoles and other antimycotic drugs, its specific mode of action is only poorly understood. To investigate the mode of action of ciclopirox olamine on fungal viability, pathogenicity, and drug resistance, we examined the expression patterns of 47 Candida albicans genes in cells grown in the presence of a subinhibitory concentration (0.6 μg/ml) of ciclopirox olamine by reverse transcription-PCR. In addition, we used suppression-subtractive hybridization to further identify genes that are up-regulated in the presence of ciclopirox olamine. The expression of essential genes such as ACT1 was not significantly modified in cells exposed to ciclopirox olamine. Most putative and known virulence genes such as genes encoding secreted proteinases or lipases had no or only moderately reduced expression levels. In contrast, exposure of cells to ciclopirox olamine led to a distinct up- or down-regulation of genes encoding iron permeases or transporters (FTR1, FTR2, FTH1), a copper permease (CCC2), an iron reductase (CFL1), and a siderophore transporter (SIT1); these effects resembled those found under iron-limited conditions. Addition of FeCl3 to ciclopirox olamine-treated cells reversed the effect of the drug. Addition of the iron chelator bipyridine to the growth medium induced similar patterns of expression of distinct iron-regulated genes (FTR1, FTR2). While serum-induced yeast-to-hyphal phase transition of C. albicans was not affected in ciclopirox olamine-treated cells in the presence of subinhibitory conditions, a dramatic increase in sensitivity to oxidative stress was noted, which may indicate the reduced activities of iron-containing gene products responsible for detoxification. Although the Candida drug resistance genes CDR1 and CDR2 were up-regulated, no change in resistance or increased tolerance could be observed even after an

  5. Ciclopirox olamine treatment affects the expression pattern of Candida albicans genes encoding virulence factors, iron metabolism proteins, and drug resistance factors.

    PubMed

    Niewerth, Markus; Kunze, Donika; Seibold, Michael; Schaller, Martin; Korting, Hans Christian; Hube, Bernhard

    2003-06-01

    The hydroxypyridone ciclopirox olamine belongs to the antimycotic drugs used for the treatment of superficial mycoses. In contrast to the azoles and other antimycotic drugs, its specific mode of action is only poorly understood. To investigate the mode of action of ciclopirox olamine on fungal viability, pathogenicity, and drug resistance, we examined the expression patterns of 47 Candida albicans genes in cells grown in the presence of a subinhibitory concentration (0.6 micro g/ml) of ciclopirox olamine by reverse transcription-PCR. In addition, we used suppression-subtractive hybridization to further identify genes that are up-regulated in the presence of ciclopirox olamine. The expression of essential genes such as ACT1 was not significantly modified in cells exposed to ciclopirox olamine. Most putative and known virulence genes such as genes encoding secreted proteinases or lipases had no or only moderately reduced expression levels. In contrast, exposure of cells to ciclopirox olamine led to a distinct up- or down-regulation of genes encoding iron permeases or transporters (FTR1, FTR2, FTH1), a copper permease (CCC2), an iron reductase (CFL1), and a siderophore transporter (SIT1); these effects resembled those found under iron-limited conditions. Addition of FeCl(3) to ciclopirox olamine-treated cells reversed the effect of the drug. Addition of the iron chelator bipyridine to the growth medium induced similar patterns of expression of distinct iron-regulated genes (FTR1, FTR2). While serum-induced yeast-to-hyphal phase transition of C. albicans was not affected in ciclopirox olamine-treated cells in the presence of subinhibitory conditions, a dramatic increase in sensitivity to oxidative stress was noted, which may indicate the reduced activities of iron-containing gene products responsible for detoxification. Although the Candida drug resistance genes CDR1 and CDR2 were up-regulated, no change in resistance or increased tolerance could be observed even after

  6. The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus.

    PubMed

    Tan, Honglue; Peng, Zhaoxiang; Li, Qingtian; Xu, Xiaofen; Guo, Shengrong; Tang, Tingting

    2012-01-01

    also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis.

  7. The Daiokanzoto (TJ-84) Kampo Formulation Reduces Virulence Factor Gene Expression in Porphyromonas gingivalis and Possesses Anti-Inflammatory and Anti-Protease Activities

    PubMed Central

    Fournier-Larente, Jade; Azelmat, Jabrane; Yoshioka, Masami; Hinode, Daisuke; Grenier, Daniel

    2016-01-01

    Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest. PMID:26859747

  8. Phosphate Limitation Induces Drastic Physiological Changes, Virulence-Related Gene Expression, and Secondary Metabolite Production in Pseudovibrio sp. Strain FO-BEG1

    PubMed Central

    González, José M.; Bondarev, Vladimir

    2015-01-01

    Phosphorus is a vital nutrient for living organisms and is obtained by bacteria primarily via phosphate uptake. However, phosphate is often scarcely accessible in nature, and there is evidence that in many areas of the ocean, its concentration limits bacterial growth. Surprisingly, the phosphate starvation response has been extensively investigated in different model organisms (e.g., Escherichia coli), but there is a dearth of studies on heterotrophic marine bacteria. In this work, we describe the response of Pseudovibrio sp. strain FO-BEG1, a metabolically versatile alphaproteobacterium and potential symbiont of marine sponges, to phosphate limitation. We compared the physiology, protein expression, and secondary metabolite production under phosphate-limited conditions to those under phosphate surplus conditions. We observed that phosphate limitation had a pleiotropic effect on the physiology of the strain, triggering cell elongation, the accumulation of polyhydroxyalkanoate, the degradation of polyphosphate, and the exchange of membrane lipids in favor of phosphorus-free lipids such as sulfoquinovosyl diacylglycerols. Many proteins involved in the uptake and degradation of phospho-organic compounds were upregulated, together with subunits of the ABC transport system for phosphate. Under conditions of phosphate limitation, FO-BEG1 secreted compounds into the medium that conferred an intense yellow coloration to the cultures. Among these compounds, we identified the potent antibiotic tropodithietic acid. Finally, toxin-like proteins and other proteins likely involved in the interaction with the eukaryotic host were also upregulated. Altogether, our data suggest that phosphate limitation leads to a pronounced reorganization of FO-BEG1 physiology, involving phosphorus, carbon, and sulfur metabolism; cell morphology; secondary metabolite production; and the expression of virulence-related genes. PMID:25769826

  9. Expression of the LspA1 and LspA2 proteins by Haemophilus ducreyi is required for virulence in human volunteers.

    PubMed

    Janowicz, Diane M; Fortney, Kate R; Katz, Barry P; Latimer, Jo L; Deng, Kaiping; Hansen, Eric J; Spinola, Stanley M

    2004-08-01

    Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HP Omega 12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm(2)) than at parent sites (mean size, 39.1 mm(2)) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

  10. Alteration of the Microbiota and Virulence Gene Expression in E. coli O157:H7 in Pig Ligated Intestine with and without AE Lesions

    PubMed Central

    Yu, Hai; Feng, Yanni; Ying, Xin; Gong, Joshua; Gyles, Carlton L.

    2015-01-01

    Background Previously we found that E. coli O157:H7 inoculated into ligated pig intestine formed attaching and effacing (AE) lesions in some pigs but not in others. The present study evaluated changes in the microbial community and in virulence gene expression in E. coli O157:H7 in ligated pig intestine in which the bacteria formed AE lesions or failed to form AE lesions. Methodology/Principal Findings The intestinal microbiota was assessed by RNA-based denaturing gradient gel electrophoresis (DGGE) analysis. The DGGE banding patterns showed distinct differences involving two bands which had increased intensity specifically in AE-negative pigs (AE- bands) and several bands which were more abundant in AE-positive pigs. Sequence analysis revealed that the two AE- bands belonged to Veillonella caviae, a species with probiotic properties, and Bacteroides sp. Concurrent with the differences in microbiota, gene expression analysis by quantitative PCR showed that, compared with AE negative pigs, E. coli O157:H7 in AE positive pigs had upregulated genes for putative adhesins, non-LEE encoded nleA and quorum sensing qseF, acid resistance gene ureD, and genes from the locus of enterocyte effacement (LEE). Conclusions/Significance The present study demonstrated that AE-positive pigs had reduced activities or populations of Veillonella caviae and Bacterioides sp. compared with AE-negative pigs. Further studies are required to understand how the microbiota was changed and the role of these organisms in the control of E. coli O157:H7. PMID:26090813

  11. The Sensor Histidine Kinase RgfC Affects Group B Streptococcal Virulence Factor Expression Independent of Its Response Regulator RgfA

    PubMed Central

    Gendrin, Claire; Lembo, Annalisa; Whidbey, Christopher; Burnside, Kellie; Berry, Jessica; Ngo, Lisa; Banerjee, Anirban; Xue, Liang; Arrington, Justine; Doran, Kelly S.; Tao, W. Andy

    2015-01-01

    Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutant increased induction of proinflammatory signaling pathways in vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis. PMID:25561709

  12. [Diffusible signal factor production and virulence expression in deltarpfFxoo, deltarpfCxoo and deltarpfGxoo, the gene deletion mutants of DSF/Rpf signaling proteins of Xanthomonas oryzae pv. oryzae].

    PubMed

    Sun, Lei; Wu, Maosen; Chen, Huamin; He, Chenyang

    2010-06-01

    To better elucidate the functions of RpfFxoo, RpfCxoo and RpfGxoo, 3 proteins of diffusible signal factor (DSF)-dependent cell-cell signaling system in regulation of virulence expression of Xanthomonas oryzae pv. oryzae (Xoo). Deltarpfxoo, the gene deletion mutants were generated from PXO99(A), the wild-type strain of Xoo via marker-exchanging and DSF biosynthesis and extracellular polysaccharide production and virulence to rice of the mutants were assayed. rpfFxoo,rpfCxoo and rpfGxoo were cloned from the genomic DNA of PXO99(A) and the relative single or double mutants were constructed. Compared to PXO99(A), DSF production was deficient in deltarpfFxoo, deltarpfFCxoo and deltarpfFGxoo, while DSF was overproduced in deltarpfCxoo and reduced in deltarpfGxoo. DSF production of deltarpfFxcc, deltarpfCxcc and deltarpfGxcc, the mutants of X. campestris pv. campestris can be restored as XC1, the wild-type strain by in trans complementation of rpfFxoo, rpfCxoo and rpfGxoo. All the mutants except deltarpfFxoo were remarkably deficient in production All the mutants significantly exhibited the reduced bacterial virulence to rice. of extracellular polysaccharide. DSF signaling proteins RpfFxoo, RpfCxoo and RpfGxoo might function in regulation of DSF biogenesis and EPS production and bacterial virulence.

  13. Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico

    PubMed Central

    2014-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors. Methods We implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE). Results In this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination. Conclusion These findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections. PMID:24405688

  14. Cloning, Expression, and Functional Characterization of Serine Protease Aprv2 from Virulent Isolate Dichelobacter nodosus of Indian Origin.

    PubMed

    Wani, Aasim Habib; Sharma, Mandeep; Salwan, Richa; Singh, Geetanjali; Chahota, Rajesh; Verma, Subhash

    2016-10-01

    A gene encoding an extracellular protease from Dichelobacter nodosus was characterized and expressed in E. coli rosetta-gami (DE3). The nucleotide sequence analysis revealed an ORF of 1427 bp ecoding 475 amino acids long protein of calculated molecular weight 50.6 kDa and pI value 6.09. The phylogenetic analysis showed relatedness to subtilisin-like serine proteases of peptidase S8 family. The amino acid sequence analysis showed presence of N-terminal pre-peptide (1-23 aa), pro-peptide (24-160 aa), peptidase S8 domain (161-457 aa), and a C-terminal extension (458-475 aa). The gene harboring native signal peptide was expressed in pET-22b(+) for production of AprV2 recombinant protein. SDS-PAGE revealed the highest production of IPTG induced recombinant protein ∼37 kDa at 16 °C after 16 h. The purified protein after Ni-NTA affinity chromatography showed single protein band of ∼37 kDa which was also confirmed by the detection of blue coloured band of same size in Western blotting. The recombinant protein showed activity over broad temperature and pH range with optimum at 35 °C and pH 7.0. Similarly, the enzyme was stable over broad range 15-65 °C and 4-10 pH with maximum stability at 25 °C and pH 6. The activity of purified enzyme was also stimulated in the presence of Ca(2+). The purified enzyme showed highest activity towards casein as compared to gelatin and BSA. These findings suggest AprV2 as an important candidate for industrial applications such as pharmaceuticals.

  15. Bacterial proteases and virulence.

    PubMed

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host.

  16. Expression of the Salmonella Spp. Virulence Factor SifA in Yeast Alters Rho1 Activity on Peroxisomes

    PubMed Central

    Vinh, Dani B. N.; Ko, Dennis C.; Rachubinski, Richard A.; Aitchison, John D.

    2010-01-01

    The Salmonella typhimurium effector protein SifA regulates the assembly and tubulation of the Salmonella phagosome. SifA localizes to the phagosome and interacts with the membrane via its prenylated tail. SifA is a structural homologue of another bacterial effector that acts as a GTP-exchange factor for Rho family GTPases and can bind GDP-RhoA. When coexpressed with a bacterial lipase that is activated by RhoA, SifA can induce tubulation of mammalian endosomes. In an effort to develop a genetic system to study SifA function, we expressed SifA and characterized its activity in yeast. GFP-SifA predominantly localized to yeast peroxisomal membranes. Under peroxisome-inducing conditions, GFP-SifA reduced the number of free peroxisomes and promoted the formation of large peroxisomes with membrane invaginations. GFP-SifA activity depended on the recruitment to peroxisomes of wild-type Rho1p and Pex25p, a receptor for Rho1p. GFP-SifA could also rescue the actin organization defects in pex25Δ and rho1 mutants, suggesting that SifA may recruit and potentiate Rho1p activity. We reexamined the distribution of GFP-SifA in mammalian cells and found the majority colocalizing with LAMP1-positive compartment and not with the peroxisomal marker PMP70. Together, these data suggest that SifA may use a similar mode of action via Rho proteins to alter yeast peroxisomal and mammalian endosomal membranes. Further definition of SifA activity on yeast peroxisomes could provide more insight into its role in regulating host membrane dynamics and small GTPases. PMID:20739463

  17. Generation of a recombinant viral hemorrhagic septicemia virus (VHSV) expressing olive flounder (Paralichthys olivaceus) interferon-γ and its effects on type I interferon response and virulence.

    PubMed

    Kwak, Jun Soung; Kim, Min Sun; Kim, Ki Hong

    2017-09-01

    Rhabdoviruses including viral hemorrhagic septicemia virus (VHSV) are highly susceptible to type I interferon (IFN) responses, and IFN-γ that is belonging to the type II IFN has been known to enhance type I IFN responses in mammals. In this study, we generated a recombinant VHSV that can express olive flounder IFN-γ (rVHSV-A-IFNγ) using reverse genetics technology, and analyzed the effect of rVHSV-A-IFNγ infection on type I IFN response in Epithelioma papulosum cyprini (EPC) cells. Furthermore, the virulence of rVHSV-A-IFNγ was evaluated by infection to olive flounder (Paralichthys olivaceus). Using a recombinant VHSV full genome vector in which the olive flounder IFN-γ ORF was inserted between N and P genes, rVHSV-A-IFNγ was successfully rescued, and the recombinant virus was grown well in EPC cells. On the other hand, the growth of rVHSV-A-IFNγ rescued from EPC cells was severely retarded when infected into hirame natural embryo (HINAE) cells that were originated from olive flounder. These results indicate that the EPC cell's IFN-γ receptor could not bind to olive flounder IFN-γ, but the species-specific binding of IFN-γ in HINAE cells induced antiviral responses. The expression of Mx1 gene in EPC cells infected with rVHSV-A-IFNγ was not greatly different from cells infected with rVHSV-Arfp (a recombinant VHSV harboring red fluorescent protein gene between N and P genes of the genome), however, in HINAE cells, rVHSV-A-IFNγ infection induced distinctively higher Mx1 gene expression compared to other recombinant viruses. These results suggest that olive flounder IFN-γ produced from rVHSV-A-IFNγ effectively enhanced type I IFN response in HINAE cells. In the present study, the lowest mortality of olive flounder fingerlings was recorded in the group of fish challenged with rVHSV-A-IFNγ, suggesting that the recombinant VHSV was attenuated by production of IFN-γ by itself. However, although rVHSV-A-IFNγ induced significantly lower mortality, the

  18. Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat.

    PubMed

    Marshall, Rosalind; Kombrink, Anja; Motteram, Juliet; Loza-Reyes, Elisa; Lucas, John; Hammond-Kosack, Kim E; Thomma, Bart P H J; Rudd, Jason J

    2011-06-01

    Secreted effector proteins enable plant pathogenic fungi to manipulate host defenses for successful infection. Mycosphaerella graminicola causes Septoria tritici blotch disease of wheat (Triticum aestivum) leaves. Leaf infection involves a long (approximately 7 d) period of symptomless intercellular colonization prior to the appearance of necrotic disease lesions. Therefore, M. graminicola is considered as a hemibiotrophic (or necrotrophic) pathogen. Here, we describe the molecular and functional characterization of M. graminicola homologs of Ecp6 (for extracellular protein 6), the Lysin (LysM) domain-containing effector from the biotrophic tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum, which interferes with chitin-triggered immunity in plants. Three LysM effector homologs are present in the M. graminicola genome, referred to as Mg3LysM, Mg1LysM, and MgxLysM. Mg3LysM and Mg1LysM genes were strongly transcriptionally up-regulated specifically during symptomless leaf infection. Both proteins bind chitin; however, only Mg3LysM blocked the elicitation of chitin-induced plant defenses. In contrast to C. fulvum Ecp6, both Mg1LysM and Mg3LysM also protected fungal hyphae against plant-derived hydrolytic enzymes, and both genes show significantly more nucleotide polymorphism giving rise to nonsynonymous amino acid changes. While Mg1LysM deletion mutant strains of M. graminicola were fully pathogenic toward wheat leaves, Mg3LysM mutant strains were severely impaired in leaf colonization, did not trigger lesion formation, and were unable to undergo asexual sporulation. This virulence defect correlated with more rapid and pronounced expression of wheat defense genes during the symptomless phase of leaf colonization. These data highlight different functions for MgLysM effector homologs during plant infection, including novel activities that distinguish these proteins from C. fulvum Ecp6.

  19. Regulation and secretion of Xanthomonas virulence factors.

    PubMed

    Büttner, Daniela; Bonas, Ulla

    2010-03-01

    Plant pathogenic bacteria of the genus Xanthomonas cause a variety of diseases in economically important monocotyledonous and dicotyledonous crop plants worldwide. Successful infection and bacterial multiplication in the host tissue often depend on the virulence factors secreted including adhesins, polysaccharides, LPS and degradative enzymes. One of the key pathogenicity factors is the type III secretion system, which injects effector proteins into the host cell cytosol to manipulate plant cellular processes such as basal defense to the benefit of the pathogen. The coordinated expression of bacterial virulence factors is orchestrated by quorum-sensing pathways, multiple two-component systems and transcriptional regulators such as Clp, Zur, FhrR, HrpX and HpaR. Furthermore, virulence gene expression is post-transcriptionally controlled by the RNA-binding protein RsmA. In this review, we summarize the current knowledge on the infection strategies and regulatory networks controlling secreted virulence factors from Xanthomonas species.

  20. ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm

    PubMed Central

    Ellis, Pamela S.; Burbridge, Sarah; Soubes, Sandrine; Ohyama, Kyoji; Ben-Haim, Nadav; Chen, Canhe; Dale, Kim; Shen, Michael M.; Constam, Daniel; Placzek, Marysia

    2015-01-01

    The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed Nodal. Exposure of prechordal mesoderm microcultures to Nodal-conditioned medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, targeted electroporation of Fgfr3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal-FGFR3 signalling and pSmad1/5/8, and show that proNodal-FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated. PMID:26417042

  1. Comparison of virulence factors and expression of specific genes between uropathogenic Escherichia coli and avian pathogenic E. coli in a murine urinary tract infection model and a chicken challenge model.

    PubMed

    Zhao, Lixiang; Gao, Song; Huan, Haixia; Xu, Xiaojing; Zhu, Xiaoping; Yang, Weixia; Gao, Qingqing; Liu, Xiufan

    2009-05-01

    Avian pathogenic Escherichia coli (APEC) and uropathogenic E. coli (UPEC) establish infections in extraintestinal habitats of different hosts. As the diversity, epidemiological sources and evolutionary origins of extraintestinal pathogenic E. coli (ExPEC) are so far only partially defined, in the present study,100 APEC isolates and 202 UPEC isolates were compared by their content of virulence genes and phylogenetic groups. The two groups showed substantial overlap in terms of their serogroups, phylogenetic groups and virulence genotypes, including their possession of certain genes associated with large transmissible plasmids of APEC. In a chicken challenge model, both UPEC U17 and APEC E058 had similar LD(50), demonstrating that UPEC U17 had the potential to cause significant disease in poultry. To gain further information about the similarities between UPEC and APEC, the in vivo expression of 152 specific genes of UPEC U17 and APEC E058 in both a murine urinary tract infection (UTI) model and a chicken challenge model was compared with that of these strains grown statically to exponential phase in rich medium. It was found that in the same model (murine UTI or chicken challenge), various genes of UPEC U17 and APEC E058 showed a similar tendency of expression. Several iron-related genes were upregulated in the UTI model and/or chicken challenge model, indicating that iron acquisition is important for E. coli to survive in blood or the urinary tract. Based on these results, the potential for APEC to act as human UPEC or as a reservoir of virulence genes for UPEC should be considered. Further, this study compared the transcriptional profile of virulence genes among APEC and UPEC in vivo.

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

    PubMed

    Keeney, Kristie M; Finlay, B Brett

    2013-02-04

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

  3. Modulation of Shigella virulence in response to available oxygen in vivo

    PubMed Central

    Marteyn, Benoit; West, Nicholas; Browning, Douglas; Cole, Jeffery; Shaw, Jonathan; Palm, Fredrik; Mounier, Joelle; Prévost, Marie-Christine; Sansonetti, Philippe; Tang, Christoph

    2013-01-01

    Bacteria co-ordinate expression of virulence determinants in response to localised microenvironments in their hosts. Here we show that Shigella flexneri, which causes dysentery, encounters varying oxygen concentrations in the gastrointestinal (GI) tract, which govern activity of its type three secretion system (T3SS); the T3SS is essential for cell invasion and virulence1. In anaerobic environments (e.g. the GI tract lumen), Shigella expresses extended T3SS needles while reducing Ipa (Invasion plasmid antigen) effector secretion. This is mediated by FNR, a regulator of anaerobic metabolism that represses transcription of spa32 and spa33, virulence genes that the switch in secretion through the T3SS. We demonstrate there is a zone of relative oxygenation adjacent to the GI tract mucosa, caused by diffusing from the capillary network at the tips of villi. This would reverse the anaerobic block of Ipa secretion, allowing T3SS activation at its precise site of action, enhancing invasion and virulence. PMID:20436458

  4. Gene duplication, silencing and expression alteration govern the molecular evolution of PRC2 genes in plants.

    PubMed

    Furihata, Hazuka Y; Suenaga, Kazuya; Kawanabe, Takahiro; Yoshida, Takanori; Kawabe, Akira

    2016-10-13

    PRC2 genes were analyzed for their number of gene duplications, dN/dS ratios and expression patterns among Brassicaceae and Gramineae species. Although both amino acid sequences and copy number of the PRC2 genes were generally well conserved in both Brassicaceae and Gramineae species, we observed that some rapidly evolving genes experienced duplications and expression pattern changes. After multiple duplication events, all but one or two of the duplicated copies tend to be silenced. Silenced copies were reactivated in the endosperm and showed ectopic expression in developing seeds. The results indicated that rapid evolution of some PRC2 genes is initially caused by a relaxation of selective constraint following the gene duplication events. Several loci could become maternally expressed imprinted genes and acquired functional roles in the endosperm.

  5. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

    PubMed Central

    Teoh-Fitzgerald, ML; Fitzgerald, MP; Zhong, W; Askeland, RW; Domann, FE

    2013-01-01

    Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging

  6. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis.

    PubMed

    Teoh-Fitzgerald, M L; Fitzgerald, M P; Zhong, W; Askeland, R W; Domann, F E

    2014-01-16

    Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging

  7. Comparison on virulence and immunogenicity of two recombinant vaccinia vaccines, Tian Tan and Guang9 strains, expressing the HIV-1 envelope gene.

    PubMed

    Zhu, Rong; Huang, Weijin; Wang, Wenbo; Liu, Qiang; Nie, Jianhui; Meng, Shufang; Yu, Yongxin; Wang, Youchun

    2012-01-01

    The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT. To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E. Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.

  8. Comparison on Virulence and Immunogenicity of Two Recombinant Vaccinia Vaccines, Tian Tan and Guang9 Strains, Expressing the HIV-1 Envelope Gene

    PubMed Central

    Zhu, Rong; Huang, Weijin; Wang, Wenbo; Liu, Qiang; Nie, Jianhui; Meng, Shufang; Yu, Yongxin; Wang, Youchun

    2012-01-01

    Background The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT. Methodology/Principal Findings To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E. Conclusions/Significance Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development. PMID:23139778

  9. The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule

    PubMed Central

    Mégroz, Marianne; Kleifeld, Oded; Wright, Amy; Powell, David; Harrison, Paul; Adler, Ben; Harper, Marina

    2016-01-01

    The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin. PMID:26883595

  10. Evolution of virulence when transmission occurs before disease.

    PubMed

    Osnas, Erik E; Dobson, Andrew P

    2010-08-23

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

  11. Pseudomonas aeruginosa Virulence and Pathogenesis Issues

    USDA-ARS?s Scientific Manuscript database

    Regulation of gene expression can occur through cell-cell communication or quorum sensing (QS) via the production of small molecules called autoinducers. QS is known to control expression of a number of virulence factors. Another form of gene regulation which allows the bacteria to rapidly adapt t...

  12. Exopolyphosphatase of Pseudomonas aeruginosa is essential for the production of virulence factors, and its expression is controlled by NtrC and PhoB acting at two interspaced promoters.

    PubMed

    Gallarato, Lucas A; Sánchez, Diego G; Olvera, Leticia; Primo, Emiliano D; Garrido, Mónica N; Beassoni, Paola R; Morett, Enrique; Lisa, Angela T

    2014-02-01

    The exopolyphosphatase (Ppx) of Pseudomonas aeruginosa is encoded by the PA5241 gene (ppx). Ppx catalyses the hydrolysis of inorganic polyphosphates to orthophosphate (Pi). In the present work, we identified and characterized the promoter region of ppx and its regulation under environmental stress conditions. The role of Ppx in the production of several virulence factors was demonstrated through studies performed on a ppx null mutant. We found that ppx is under the control of two interspaced promoters, dually regulated by nitrogen and phosphate limitation. Under nitrogen-limiting conditions, its expression was controlled from a σ(54)-dependent promoter activated by the response regulator NtrC. However, under Pi limitation, the expression was controlled from a σ(70) promoter, activated by PhoB. Results obtained from the ppx null mutant demonstrated that Ppx is involved in the production of virulence factors associated with both acute infection (e.g. motility-promoting factors, blue/green pigment production, C6-C12 quorum-sensing homoserine lactones) and chronic infection (e.g. rhamnolipids, biofilm formation). Molecular and physiological approaches used in this study indicated that P. aeruginosa maintains consistently proper levels of Ppx regardless of environmental conditions. The precise control of ppx expression appeared to be essential for the survival of P. aeruginosa and the occurrence of either acute or chronic infection in the host.

  13. Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin*♦

    PubMed Central

    Samuel, Premila P.; Smith, Lucian P.; Phillips, George N.; Olson, John S.

    2015-01-01

    Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. PMID:26205820

  14. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    SciTech Connect

    Ye, Fuzhou; Wang, Chao; Fu, Qinqin; Zhang, Lian-hui; Gao, Yong-gui

    2015-08-25

    The crystallization of the novel virulence factors SghA and SghR is reported. Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction.

  15. Evaluation of the roles of four Candida albicans genes in virulence by using gene disruption strains that express URA3 from the native locus.

    PubMed

    Cheng, Shaoji; Nguyen, M Hong; Zhang, Zongde; Jia, Hongyan; Handfield, Martin; Clancy, Cornelius J

    2003-10-01

    Reintroducing URA3 to its native locus in Candida albicans not5, not3, bur2, and kel1 disruption mutants enabled us to directly compare strains with control strain CAI-12. We showed that URA3 position affected orotidine 5'-monophosphate decarboxylase activity, hyphal morphogenesis, adherence, and mortality in murine disseminated candidiasis. After URA3 was reintroduced to its native locus, only NOT5 could be conclusively ascribed a role in virulence.

  16. Evaluation of the Roles of Four Candida albicans Genes in Virulence by Using Gene Disruption Strains That Express URA3 from the Native Locus

    PubMed Central

    Cheng, Shaoji; Nguyen, M. Hong; Zhang, Zongde; Jia, Hongyan; Handfield, Martin; Clancy, Cornelius J.

    2003-01-01

    Reintroducing URA3 to its native locus in Candida albicans not5, not3, bur2, and kel1 disruption mutants enabled us to directly compare strains with control strain CAI-12. We showed that URA3 position affected orotidine 5′-monophosphate decarboxylase activity, hyphal morphogenesis, adherence, and mortality in murine disseminated candidiasis. After URA3 was reintroduced to its native locus, only NOT5 could be conclusively ascribed a role in virulence. PMID:14500538

  17. Mechanism Governing Human Kappa-Opioid Receptor Expression under Desferrioxamine-Induced Hypoxic Mimic Condition in Neuronal NMB Cells

    PubMed Central

    Babcock, Jennifer; Herrera, Alberto; Coricor, George; Karch, Christopher; Liu, Alexander H.; Rivera-Gines, Aida; Ko, Jane L.

    2017-01-01

    Cellular adaptation to hypoxia is a protective mechanism for neurons and relevant to cancer. Treatment with desferrioxamine (DFO) to induce hypoxia reduced the viability of human neuronal NMB cells. Surviving/attached cells exhibited profound increases of expression of the human kappa-opioid receptor (hKOR) and hypoxia inducible factor-1α (HIF-1α). The functional relationship between hKOR and HIF-1α was investigated using RT-PCR, Western blot, luciferase reporter, mutagenesis, siRNA and receptor-ligand binding assays. In surviving neurons, DFO increased HIF-1α expression and its amount in the nucleus. DFO also dramatically increased hKOR expression. Two (designated as HIFC and D) out of four potential HIF response elements of the hKOR gene (HIFA–D) synergistically mediated the DFO response. Mutation of both elements completely abolished the DFO-induced effect. The CD11 plasmid (containing HIFC and D with an 11 bp spacing) produced greater augmentation than that of the CD17 plasmid (HIFC and D with a 17 bp-spacing), suggesting that a proper topological interaction of these elements synergistically enhanced the promoter activity. HIF-1α siRNA knocked down the increase of endogenous HIF-1α messages and diminished the DFO-induced increase of hKOR expression. Increased hKOR expression resulted in the up-regulation of hKOR protein. In conclusion, the adaptation of neuronal hKOR under hypoxia was governed by HIF-1, revealing a new mechanism of hKOR regulation. PMID:28117678

  18. Cbfβ governs osteoblast-adipocyte lineage commitment through enhancing β-catenin signaling and suppressing adipogenesis gene expression.

    PubMed

    Wu, Mengrui; Wang, Yiping; Shao, Jian-Zhong; Wang, Jue; Chen, Wei; Li, Yi-Ping

    2017-09-19

    The mechanism underlying how transcription factors regulate mesenchymal stem cell lineage commitment remains unclear. To determine the role of core-binding factor subunit beta (Cbfβ) in osteoblast lineage commitment, we generated three mouse models by deleting Cbfβ at different osteoblast lineage stages. We demonstrated that the Cbfβ(f/f)Prx1-Cre, Cbfβ(f/f)Col2α1-Cre, and Cbfβ(f/f)Osx-Cre mice exhibited severe osteoporosis with substantial accumulation of marrow adipocytes resembling aged bone from enhanced adipogenesis, indicating that mesenchymal stem cells and osteoblasts can be programed and reprogramed, respectively, into adipocytes. Consistently, Cbfβ-deficient calvarial cells and bone marrow mesenchymal stem cells displayed strong adipogenic potential, with 5- to ∼70-fold increased adipocyte gene expression, which can be rescued by Cbfβ overexpression. Canonical Wnt signaling was impeded in the Cbfβ-deficient cells, with ∼80% decrease of Wnt10b expression. Accordingly, ChIP and luciferase assays demonstrated that Cbfβ/RUNX2 binds to Wnt10b promoter driving Wnt10b expression. Furthermore, Wnt3a suppressed adipogenesis but did not rescue osteoblastogenesis in Cbfβ-deficient cells. Notably, mixing culture of Cbfβ-deficient with normal cells demonstrates that Cbfβ functions not only through WNT paracrine pathway but also through endogenous signaling. Further analysis shows that Cbfβ/RUNX2 inhibits c/ebpα expression at transcriptional level. Our results show that, besides its osteogenic role, Cbfβ governs osteoblast-adipocyte lineage commitment both cell nonautonomously through enhancing β-catenin signaling and cell autonomously through suppressing adipogenesis gene expression to maintain osteoblast lineage commitment, indicating Cbfβ may be a therapeutic target for osteoporosis.

  19. Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human β-defensin, and cytokine expression by engineered human oral mucosa.

    PubMed

    Semlali, A; Leung, K P; Curt, S; Rouabhia, M

    2011-05-01

    We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human β-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammatory cytokine secretion (IL-1β and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent.

  20. Transformed tobacco (Nicotiana tabacum) plants over-expressing a peroxisome proliferator-activated receptor gene from Xenopus laevis (xPPARα) show increased susceptibility to infection by virulent Pseudomonas syringae pathogens.

    PubMed

    Valenzuela-Soto, José Humberto; Iruegas-Bocardo, Fernanda; Martínez-Gallardo, Norma Angélica; Molina-Torres, Jorge; Gómez-Lim, Miguel Angel; Délano-Frier, John Paul

    2011-03-01

    Transgenic tobacco plants capable of over-expressing Xenopus PPARα (xPPARα), a transcription factor known to be required for peroxisome proliferation in animals, were recently generated. These plants (herewith referred to as PPAR-OE) were found to have increased peroxisome abundance, higher peroxisomal acyl-CoA oxidase and catalase activity and modified fatty acid metabolism. Further characterization of PPAR-OE plants revealed a higher susceptibility to virulent and a partial loss of resistance to avirulent Pseudomonas syringae pathogens, whereas the basal resistance response remained unaffected. Biochemical- and defense-related gene expression analyses showed that increased susceptibility to bacterial invasion coincided with the generalized reduction in H(2)O(2) and salicylic acid (SA) levels observed within the first 24 h of bacterial contact. Decreased H(2)O(2) levels were correlated with modified activity levels of catalase and other antioxidant enzymes. A correspondence between a rapid (within 1-24 hpi; ACCO and AOC) and sustained increase (up to 6 days pi; ACCO) in the expression levels of ethylene (ACCO) and jasmonic acid (AOC) biosynthetic genes and a higher susceptibility to virulent bacterial invasion was also observed in PPAR-OE plants. Conversely, no apparent differences in the short- and/or long-term expression levels of markers for the hypersensitive-response, oxidative burst and systemic-acquired resistance were observed between wild type and PPAR-OE plants. The results suggest that peroxisome proliferation could lead to increased susceptibility to bacterial pathogens in tobacco by altering the redox balance of the plant and the expression pattern of key defense signaling pathway genes.

  1. Spaceflight Effects on Virulence of Pseudomonas Aeruginosa

    NASA Astrophysics Data System (ADS)

    Broadway, S.; Goins, T.; Crandell, C.; Richards, C.; Patel, M.; Pyle, B.

    2008-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen found in the environment. It is known to infect the immunocompromised. The organism has about 25 virulence genes that play different roles in disease processes. Several exotoxin proteins may be produced, including ExoA, ExoS, ExoT and ExoY, and other virulence factors. In spaceflight, possible increased expression of P. aeruginosa virulence proteins could increase health risks for spaceflight crews who experience decreased immunity. Cultures of P. aeruginosa strains PA01 and PA103 grown on orbit on Shuttle Endeavour flight STS-123 vs. static ground controls were used for analysis. The production of ETA was quantitated using an ELISA procedure. Results showed that while flight cultures of PA103 produced slightly more ETA than corresponding ground controls, the opposite was found for PA01. While it appears that spaceflight has little effect on ETA, stimulation of other virulence factors could cause increased virulence of this organism in space flight. Similar increased virulence in spaceflight has been observed for other bacteria. This is important because astronauts may be more susceptible to opportunistic pathogens including P. aeruginosa.

  2. Extent of flow recirculation governs expression of atherosclerotic and thrombotic biomarkers in arterial bifurcations

    PubMed Central

    Martorell, Jordi; Santomá, Pablo; Kolandaivelu, Kumaran; Kolachalama, Vijaya B.; Melgar-Lesmes, Pedro; Molins, José J.; Garcia, Lawrence; Edelman, Elazer R.; Balcells, Mercedes

    2014-01-01

    Aims Atherogenesis, evolution of plaque, and outcomes following endovascular intervention depend heavily on the unique vascular architecture of each individual. Patient-specific, multiscale models able to correlate changes in microscopic cellular responses with relevant macroscopic flow, and structural conditions may help understand the progression of occlusive arterial disease, providing insights into how to mitigate adverse responses in specific settings and individuals. Methods and results Vascular architectures mimicking coronary and carotid bifurcations were derived from clinical imaging and used to generate conjoint computational meshes for in silico analysis and biocompatible scaffolds for in vitro models. In parallel with three-dimensional flow simulations, geometrically realistic scaffolds were seeded with human smooth muscle cells (SMC) or endothelial cells and exposed to relevant, physiological flows. In vitro surrogates of endothelial health, atherosclerotic progression, and thrombosis were locally quantified and correlated best with an quantified extent of flow recirculation occurring within the bifurcation models. Oxidized low-density lipoprotein uptake, monocyte adhesion, and tissue factor expression locally rose up to three-fold, and phosphorylated endothelial nitric oxide synthase and Krüppel-like factor 2 decreased up to two-fold in recirculation areas. Isolated testing in straight-tube idealized constructs subject to static, oscillatory, and pulsatile conditions, indicative of different recirculant conditions corroborated these flow-mediated dependencies. Conclusions Flow drives variations in vascular reactivity and vascular beds. Endothelial health was preserved by arterial flow but jeopardized in regions of flow recirculation in a quasi-linear manner. Similarly, SMC exposed to flow were more thrombogenic in large recirculating regions. Health, thrombosis, and atherosclerosis biomarkers correlate with the extent of recirculation in vascular

  3. A functionally conserved Zn2 Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat.

    PubMed

    Rybak, Kasia; See, Pao Theen; Phan, Huyen T T; Syme, Robert A; Moffat, Caroline S; Oliver, Richard P; Tan, Kar-Chun

    2017-04-01

    The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best-characterized effector-sensitivity gene systems are SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora tritici-repentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.

  4. Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein).

    PubMed

    Mondal, Shankar P; Cook, R Frank; Chelvarajan, R Lakshman; Henney, Pamela J; Timoney, Peter J; Balasuriya, Udeni B R

    2016-04-01

    Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3(+) T cells and CD14(+) monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques.

  5. Virulence factors in Escherichia coli urinary tract infection.

    PubMed Central

    Johnson, J R

    1991-01-01

    Uropathogenic strains of Escherichia coli are characterized by the expression of distinctive bacterial properties, products, or structures referred to as virulence factors because they help the organism overcome host defenses and colonize or invade the urinary tract. Virulence factors of recognized importance in the pathogenesis of urinary tract infection (UTI) include adhesins (P fimbriae, certain other mannose-resistant adhesins, and type 1 fimbriae), the aerobactin system, hemolysin, K capsule, and resistance to serum killing. This review summarizes the virtual explosion of information regarding the epidemiology, biochemistry, mechanisms of action, and genetic basis of these urovirulence factors that has occurred in the past decade and identifies areas in need of further study. Virulence factor expression is more common among certain genetically related groups of E. coli which constitute virulent clones within the larger E. coli population. In general, the more virulence factors a strain expresses, the more severe an infection it is able to cause. Certain virulence factors specifically favor the development of pyelonephritis, others favor cystitis, and others favor asymptomatic bacteriuria. The currently defined virulence factors clearly contribute to the virulence of wild-type strains but are usually insufficient in themselves to transform an avirulent organism into a pathogen, demonstrating that other as-yet-undefined virulence properties await discovery. Virulence factor testing is a useful epidemiological and research tool but as yet has no defined clinical role. Immunological and biochemical anti-virulence factor interventions are effective in animal models of UTI and hold promise for the prevention of UTI in humans. Images PMID:1672263

  6. Regulation of virulence: The rise and fall of gastrointestinal pathogens

    PubMed Central

    Kitamoto, Sho; Nagao-Kitamoto, Hiroko; Kuffa, Peter; Kamada, Nobuhiko

    2015-01-01

    Colonization resistance by the commensal microbiota is a key defense against infectious pathogens in the gastrointestinal tract. The microbiota directly competes with incoming pathogens by occupying the colonization niche, depleting nutrients in the gut lumen as well as indirectly inhibiting the growth of pathogens through activation of host immunity. Enteric pathogens have evolved strategies to cope with microbiota-mediated colonization resistance. Pathogens utilize a wide array of virulence factors to outcompete their commensal rivals in the gut. However, since the expression of virulence factors is costly to maintain and reduces bacterial fitness, pathogens need to regulate their virulence properly in order to maximize their fitness. To this end, most pathogens use environmental cues to regulate their virulence gene expression. Thus, a dynamic regulation of virulence factor expression is a key invasion strategy utilized by enteric pathogens. On the other hand, host immunity selectively targets virulent pathogens in order to counter infection in the gut. The host immune system is generally tolerant of harmless microorganisms, such as the commensal microbiota. Moreover, the host relies on its commensal microbiota to contribute, in concert with its immune system, to the elimination of pathogens. Collectively, regulation of virulence determines the fate of enteric pathogens, from the establishment of infection to the eventual elimination. Here, we will review the dynamics of virulence and its role in infection. PMID:26553054

  7. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens.

    PubMed

    Ye, Fuzhou; Wang, Chao; Fu, Qinqin; Zhang, Lian-hui; Gao, Yong-gui

    2015-09-01

    Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen-host interaction.

  8. Cryptosporidium Pathogenicity and Virulence

    PubMed Central

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

    2013-01-01

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

  9. Parasitoid wasp virulence

    PubMed Central

    Mortimer, Nathan T

    2013-01-01

    In nature, larvae of the fruit fly Drosophila melanogaster are commonly infected by parasitoid wasps. Following infection, flies mount an immune response termed cellular encapsulation in which fly immune cells form a multilayered capsule that covers and kills the wasp egg. Parasitoids have thus evolved virulence factors to suppress cellular encapsulation. To uncover the molecular mechanisms underlying the antiwasp response, we and others have begun identifying and functionally characterizing these virulence factors. Our recent work on the Drosophila parasitoid Ganaspis sp.1 has demonstrated that a virulence factor encoding a SERCA-type calcium pump plays an important role in Ganaspis sp.1 virulence. This venom SERCA antagonizes fly immune cell calcium signaling and thereby prevents the activation of the encapsulation response. In this way, the study of wasp virulence factors has revealed a novel aspect of fly immunity, namely a role for calcium signaling in fly immune cell activation, which is conserved with human immunity, again illustrating the marked conservation between fly and mammalian immune responses. Our findings demonstrate that the cellular encapsulation response can serve as a model of immune cell function and can also provide valuable insight into basic cell biological processes. PMID:24088661

  10. Virulence Plasmid (pYV)-Associated Expression of Phenotypic Virulent Determinants in Pathogenic Yersinia Species: A Convenient Method for Monitoring the Presence of pYV under Culture Conditions and Its Application for Isolation/Detection of Yersinia pestis in Food

    PubMed Central

    Bhaduri, Saumya; Smith, James L.

    2011-01-01

    In Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica, phenotypic expression of virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low-calcium response (Lcr, pinpoint colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding (dark-violet colony), Congo Red (CR) uptake (red pinpoint colony, size = 0.36 mm), autoagglutination (AA = cells agglutinate), and hydrophobicity (HP = clumping of cells). Y. pseudotuberculosis is chromosomally closely related to Y. pestis; whereas, Y. enterocolitica is chromosomally more distantly related to Y. pestis and Y. pseudotuberculosis. All three species demonstrate Lcr, CV binding, and CR uptake. The colony morphology/size, AA, and HP characteristics are expressed in both Y. pseudotuberculosis and Y. enterocolitica but not in Y. pestis. Congo red uptake in Y. pestis was demonstrated only on calcium-deficient CR magnesium oxalate tryptic soy agar (CR-MOX), whereas this phenotype was expressed on both CR-MOX and low-calcium agarose media in Y. pseudotuberculosis and Y. enterocolitica. These phenotypes were detectable at 37°C within 24 h in Y. enterocolitica and Y. pseudotuberculosis but did not appear until 48 h in Y. pestis due to its slower growth rate at 37°C. The pYV is unstable (i.e., easily lost under a variety of culture conditions) in all three species but is more unstable in Y. pestis. The specific CR uptake by Y. pestis in CR-MOX and the delayed time interval to express Lcr and CR uptake provide a means to differentiate Y. pestis from Y. enterocolitica and Y. pseudotuberculosis. These differences in pYV expression in Y. pestis can be used for its isolation and detection in food. PMID:22567340

  11. Salmonella promotes virulence by repressing cellulose production

    PubMed Central

    Pontes, Mauricio H.; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A.

    2015-01-01

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission. PMID:25848006

  12. Genetically manipulated virulence of Yersinia enterocolitica.

    PubMed Central

    Heesemann, J; Algermissen, B; Laufs, R

    1984-01-01

    Mobilizable virulence plasmids of Yersinia enterocolitica of serotypes O:3 and O:9 were constructed by cointegration of a mobilizable vector into the virulence plasmids. The obtained cointegrates were mobilized into plasmidless Y. enterocolitica strains of serotypes O:3, O:5, O:8, and O:9. The transfer experiments revealed the existence of two different subgroups of plasmid-associated traits. (i) Animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains (serotypes O:3, O:8, and O:9), but they were not transferable to Y. enterocolitica antigen reference strains (serotypes O:3 and O:8) or to a plasmidless clinical isolate of serotype O:5. A further striking result was that a serotype O:8 strain regained the mouse lethality trait after receipt of a plasmid from a strain not lethal to mice. These results demonstrate that plasmid-mediated animal virulence functions are not uniformly expressed within Y. enterocolitica. (ii) The second subgroup of plasmid-mediated traits (calcium dependency, surface agglutinogens, HEp-2 cell adherence, and protein release) were transferable to all Y. enterocolitica recipient strains tested (serotypes O:3, O:5, O:8, and O:9 of different origin). For the first time HEp-2 cell adherence and temperature-induced release of five major protein species are described as transferable traits. Images PMID:6480101

  13. Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI-1 and SPI-2 virulence regulons through HilD.

    PubMed

    Martínez, Luary C; Yakhnin, Helen; Camacho, Martha I; Georgellis, Dimitris; Babitzke, Paul; Puente, José L; Bustamante, Víctor H

    2011-06-01

    Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) play key roles in the pathogenesis of Salmonella enterica. Previously, we showed that when Salmonella grows in Luria-Bertani medium, HilD, encoded in SPI-1, first induces the expression of hilA, located in SPI-1, and subsequently of the ssrAB operon, located in SPI-2. These genes code for HilA and the SsrA/B two-component system, the positive regulators of the SPI-1 and SPI-2 regulons respectively. In this study, we demonstrate that CsrA, a global regulatory RNA binding protein, post-transcriptionally regulates hilD expression by directly binding near the Shine-Dalgarno and translation initiation codon sequences of the hilD mRNA, preventing its translation and leading to its accelerated turnover. Negative regulation is counteracted by the global SirA/BarA two-component system, which directly activates the expression of CsrB and CsrC, two non-coding regulatory RNAs that sequester CsrA, thereby preventing it from binding to its target mRNAs. Our results illustrate the integration of global and specific regulators into a multifactorial regulatory cascade controlling the expression of virulence genes acquired by horizontal transfer events.

  14. Identification of gyrB and rpoB gene mutations and differentially expressed proteins between a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain and its virulent parent strain.

    PubMed

    Pridgeon, J W; Yildirim-Aksoy, M; Klesius, P H; Kojima, K; Mobley, J A; Srivastava, K K; Reddy, P G

    2013-10-25

    A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23 kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection.

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

    SciTech Connect

    Hong-geller, Elizabeth; Valdez, Yolanda E; Shou, Yulin; Yoshida, Thomas M; Marrone, Babetta L; Dunbar, John

    2009-01-01

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

  16. Systemic Approach to Virulence Gene Network Analysis for Gaining New Insight into Cryptococcal Virulence

    PubMed Central

    Malachowski, Antoni N.; Yosri, Mohamed; Park, Goun; Bahn, Yong-Sun; He, Yongqun; Olszewski, Michal A.

    2016-01-01

    Cryptococcus neoformans is pathogenic yeast, responsible for highly lethal infections in compromised patients around the globe. C. neoformans typically initiates infections in mammalian lung tissue and subsequently disseminates to the central nervous system where it causes significant pathologies. Virulence genes of C. neoformans are being characterized at an increasing rate, however, we are far from a comprehensive understanding of their roles and genetic interactions. Some of these reported virulence genes are scattered throughout different databases, while others are not yet included. This study gathered and analyzed 150 reported virulence associated factors (VAFs) of C. neoformans. Using the web resource STRING database, our study identified different interactions between the total VAFs and those involved specifically in lung and brain infections and identified a new strain specific virulence gene, SHO1, involved in the mitogen-activated protein kinase signaling pathway. As predicted by our analysis, SHO1 expression enhanced C. neoformans virulence in a mouse model of pulmonary infection, contributing to enhanced non-protective immune Th2 bias and progressively enhancing fungal growth in the infected lungs. Sequence analysis indicated 77.4% (116) of total studied VAFs are soluble proteins, and 22.7% (34) are transmembrane proteins. Motifs involved in regulation and signaling such as protein kinases and transcription factors are highly enriched in Cryptococcus VAFs. Altogether, this study represents a pioneering effort in analysis of the virulence composite network of C. neoformans using a systems biology approach. PMID:27833589

  17. Analysis of mRNA expression for genes associated with regulatory T lymphocytes (CD25, FoxP3, CTLA4, and IDO) after experimental infection with bovine viral diarrhea virus of low or high virulence in beef calves.

    PubMed

    Palomares, Roberto A; Hurley, David J; Woolums, Amelia R; Parrish, Jacqueline E; Brock, Kenny V

    2014-12-01

    Immunosuppression caused by bovine viral diarrhea virus (BVDV) has been associated with lymphocyte depletion, leukopenia and impairment of leukocyte function; however, no work has been done on the relationship between BVDV and regulatory T lymphocytes (Tregs). The objective of this study was to compare the mRNA expression of genes associated with Tregs (CD25, FoxP3, CTLA4, and IDO), after experimental infection of beef calves with low (LV) or high (HV) virulence BVDV. Thirty BVDV-naïve calves were randomly assigned to three groups. Calves were intra-nasally inoculated with LV (n=10, strain SD-1) or HV (n=10, strain 1373) BVDV or BVDV-free cell culture medium (control, n=10). Quantitative RT-PCR was used to determine the expression of target genes in tracheo-bronchial lymph nodes and spleen on day 5 post-infection. The mRNA expression of CD25 was up-regulated in tracheo-bronchial lymph nodes of LV (P<0.05), but not in HV compared to the control group. The expression of FoxP3 and CTLA4 was not increased in tracheo-bronchial lymph nodes of either of the BVDV-inoculated groups. A dramatic up-regulation of IDO mRNA was observed in tracheo-bronchial lymph nodes of LV (P<0.05), but not HV compared to the control calves. In conclusion, experimental infection with BVDV did not provide evidence of Treg activation based on expression of FoxP3 and CTL4. Differential expression of CD25 and IDO mRNA on day 5 post-infection with HV or LV BVDV might reflect temporal differences in transcription occurring during the immune response elicited by these viral strains, or differences in viral infectivity of the host cells.

  18. Membrane lipid composition and stress/virulence related gene expression of Salmonella Enteritidis cells adapted to lactic acid and trisodium phosphate and their resistance to lethal heat and acid stress.

    PubMed

    Yang, Yishan; Kadim, Mellissa Irlianti; Khoo, Wei Jie; Zheng, Qianwang; Setyawati, Magdiel Inggrid; Shin, Yu-Jin; Lee, Seung-Cheol; Yuk, Hyun-Gyun

    2014-11-17

    This study evaluated the acid and heat resistance of Salmonella Enteritidis in simulated gastric fluid (pH 2.0) and during thermal treatment (54-60 °C), respectively, after adaptation to lactic acid (LA) or trisodium phosphate (TSP) at various pHs (pH 5.3-9.0). The changes in membrane lipid composition and expression levels of RpoS and RpoH were examined to elucidate their roles in bacterial stress resistance. Transcriptional profile of several virulence-related genes was also analyzed. Results showed that LA-adapted cells at pH 5.3 and 6.3 had higher acid and heat resistance than control cells and cells adapted to TSP at pH 8.3 and 9.0. LA-adapted cells had the lowest ratio of unsaturated to saturated fatty acids, indicating that they might possess a less fluid membrane. It was observed that the expression levels of RpoH and RpoS were upregulated in TSP-adapted cells but not in LA-adapted cells. Thus, these results indicate that the increased acid and heat resistance of LA-adapted S. Enteritidis was possibly due to the decreased membrane fluidity instead of the upregulation of RpoS and RpoH. About 6.0, 2.1, and 2.46-fold upregulation of spvR, avrA, and hilA were observed in cells adapted to TSP at pH 9.0, except sefA that had its highest expression level in the control cells, indicating that the expression of these virulence genes highly depends on environmental conditions. This is the first study to show that the alteration in the cytoplasmic membrane rather than RpoS and RpoH plays a more crucial role in conferring greater acid and heat resistance on LA-adapted S. Enteritidis, thus providing a better understanding on the bacterial stress response to acidic conditions.

  19. Virulence evolution at the front line of spreading epidemics.

    PubMed

    Griette, Quentin; Raoul, Gaël; Gandon, Sylvain

    2015-11-01

    Understanding and predicting the spatial s