Toxin-coregulated Pilus Production Research Articles

Genetic and mutational analysis of virulence traits and their modulation in an environmental toxigenic Vibrio cholerae non-O1/non-O139 strain, VCE232.

Vibrio cholerae O1 and O139 isolates deploy cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause the diarrhoeal disease cholera. The ctxAB and tcpA genes encoding CT and TCP are part of two acquired genetic elements, the CTX phage and Vibrio pathogenicity island-1 (VPI-1), respectively. ToxR and ToxT proteins are the key regulators of virulence genes of V. cholerae O1 and O139. V. cholerae isolates belonging to serogroups other than O1/O139, called non-O1/non-O139, are usually devoid of virulence-related elements and are non-pathogenic. Here, we have analysed the available whole genome sequence of an environmental toxigenic V. cholerae non-O1/non-O139 strain, VCE232, carrying the CTX phage and VPI-1. Extensive bioinformatics and phylogenetic analyses indicated high similarity of the VCE232 genome sequence with the genome of V. cholerae O1 strains, including organization of the VPI-1 locus, ctxAB, tcpA and toxT genes, and promoters. We established that the VCE232 strain produces an optimal amount of CT at 30 °C under AKI conditions. To investigate the role of ToxT and ToxR in the regulation of virulence factors, we constructed ΔtoxT, ΔtoxR and ΔtoxTΔtoxR deletion mutants of VCE232. Extensive genetic analyses of these mutants indicated that the toxT and toxR genes of VCE232 are crucial for CT and TCP production. However, unlike O1 isolates, the presence of either toxT or toxR gene is sufficient for optimal CT production in VCE232. In addition, the VCE232 ΔtoxR mutant showed differential regulation of the major outer membrane proteins, OmpT and OmpU. This is the first attempt to explore the regulation of expression of major virulence genes and regulators in an environmental toxigenic V. cholerae non-O1/non-O139 strain.

The Cpx system regulates virulence gene expression in Vibrio cholerae.

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.

Mechanism for inhibition of Vibrio cholerae ToxT activity by the unsaturated fatty acid components of bile.

The Gram-negative curved bacillus Vibrio cholerae causes the severe diarrheal illness cholera. During host infection, a complex regulatory cascade results in production of ToxT, a DNA-binding protein that activates the transcription of major virulence genes that encode cholera toxin (CT) and toxin-coregulated pilus (TCP). Previous studies have shown that bile and its unsaturated fatty acid (UFA) components reduce virulence gene expression and therefore are likely important signals upon entering the host. However, the mechanism for the bile-mediated reduction of TCP and CT expression has not been clearly defined. There are two likely hypotheses to explain this reduction: (i) UFAs decrease DNA binding by ToxT, or (ii) UFAs decrease dimerization of ToxT. The work presented here elucidates that bile or UFAs directly affect DNA binding by ToxT. UFAs, specifically linoleic acid, can enter V. cholerae when added exogenously and are present in the cytoplasm, where they can then interact with ToxT. Electrophoretic mobility shift assays (EMSAs) with ToxT and various virulence promoters in the presence or absence of UFAs showed a direct reduction in ToxT binding to DNA, even in promoters with only one ToxT binding site. Virstatin, a synthetic ToxT inhibitor, was previously shown to reduce ToxT dimerization. Here we show that virstatin affects DNA binding only at ToxT promoters with two binding sites, unlike linoleic acid, which affects ToxT binding promoters having either one or two ToxT binding sites. This suggests a mechanism in which UFAs, unlike virstatin, do not affect dimerization but affect monomeric ToxT binding to DNA. Vibrio cholerae must produce the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause cholera. CT and TCP production depends on ToxT, the major virulence transcription activator. ToxT activity is negatively regulated by unsaturated fatty acids (UFAs) present in the lumen of the upper small intestine. This study investigated the mechanism for inhibition of ToxT activity by UFAs and found that UFAs directly reduce specific ToxT binding to DNA at virulence promoters and subsequently reduce virulence gene expression. UFAs inhibit ToxT monomers from binding DNA. This differs from the inhibitory mechanism of a synthetic ToxT inhibitor, virstatin, which inhibits ToxT dimerization. Understanding the mechanisms for inhibition of virulence could lead to better cholera therapeutics.

Vibrio cholerae ToxR Downregulates Virulence Factor Production in Response to Cyclo(Phe-Pro)

ABSTRACTVibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue.

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus.

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.

Identifying New Variables During Infection: Proximity to the Host Epithelium and Epigenetic Programs Alter the Expression of Virulence Factors in Vibrio Cholerae

Identifying New Variables During Infection: Proximity to the Host Epithelium and Epigenetic Programs Alter the Expression of Virulence Factors in Vibrio Cholerae

The Cyclic Dipeptide Cyclo(Phe-Pro) Inhibits Cholera Toxin and Toxin-Coregulated Pilus Production in O1 El Tor Vibrio cholerae

Cyclo(Phe-Pro) is a cyclic dipeptide produced by multiple Vibrio species. In this work, we present evidence that cyclo(Phe-Pro) inhibits the production of the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) in O1 El Tor Vibrio cholerae strain N16961 during growth under virulence gene-inducing conditions. The cyclo(Phe-Pro) inhibition of CT and TCP production correlated with reduced transcription of the virulence regulator tcpPH and was alleviated by overexpression of tcpPH.

Effect of the efflux inhibitors 1-(1-naphthylmethyl)-piperazine and phenyl-arginine- -naphthylamide on antimicrobial susceptibility and virulence factor production in Vibrio cholerae

The aim of the study was to test the hypothesis that the efflux pump inhibitors (EPIs) 1-(1-naphthylmethyl)-piperazine (NMP) and phenyl-arginine-beta-naphthylamide (PAbetaN) can inhibit the Vibrio cholerae resistance-nodulation-division (RND) family efflux systems, and thereby render V. cholerae susceptible to antimicrobial agents and inhibit the production of the virulence factors cholera toxin (CT) and the toxin coregulated pilus (TCP). The susceptibility of V. cholerae to antimicrobial compounds was determined in the presence or absence of NMP and PAbetaN. Transcriptional reporters were used to assess the effects of NMP and PAbetaN on the expression of the genes encoding the virulence factor regulators TcpP and ToxT, whereas CT and TCP production were determined by ELISA using GM1 ganglioside-coated microtitre plates and TcpA Western immunoblotting, respectively. NMP and PAbetaN potentiated antimicrobial compounds that were substrates for the V. cholerae RND efflux systems. PAbetaN exhibited complete inhibition of the RND efflux systems for Triton X-100 and deoxycholate, but partial inhibition of the efflux systems for cholate and erythromycin. NMP exhibited partial inhibition for all compounds tested except for SDS. The presence of NMP reduced the MIC of SDS to a level that was lower than that observed in an RND efflux-deficient strain, whereas the SDS MIC was unaffected by the presence of PAbetaN. Neither EPI potentiated polymyxin B, penicillin, ampicillin or chloramphenicol. Both NMP and PAbetaN inhibited the production of CT and the TCP and appeared to have additional virulence gene repressing activity independent of RND efflux inhibition. RND efflux inhibitors represent potential novel therapeutics for the treatment of cholera.

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1.

BT23, a Vibrio cholerae O1 E1 Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.

Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae.

The Vibrio cholerae genome contains a 5.4-kb pil gene cluster that resembles the Aeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designated pilABCD and yacE, based on the nomenclature of related genes from Pseudomonas aeruginosa and Escherichia coli K-12. This cluster is present in both classical and El Tor biotypes, and the pilA and pilD genes are 100% conserved. The pilA gene encodes a putative type IV pilus subunit. However, deletion of pilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that pilA does not encode the primary subunit of a pilus essential for these processes. The pilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of the pilD gene showed that pilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. Some pilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in trans, indicating that the defect may not be directly due to a loss of pilD. Taken together, these data demonstrate the effectiveness of the V. cholerae genome project for rapid identification and characterization of potential virulence factors.

Rfb mutations in Vibrio cholerae do not affect surface production of toxin-coregulated pili but still inhibit intestinal colonization.

The toxin-coregulated pilus (TCP) of Vibrio cholerae is essential for colonization. It was recently reported that rfb mutations in V. cholerae 569B cause the translocation arrest of the structural subunit of TCP, raising the possibility that the colonization defects of lipopolysaccharide mutants are due to effects on TCP biogenesis. However, an rfbB gene disruption in either V. cholerae O395 or 569B has no apparent effect on surface TCP production as assessed by immunoelectron microscopy and CTX phage transduction, and an rfbD::Tn5lac mutant of O395 also shows no defect in TCP expression. We conclude that the colonization defect associated with rfb mutations is unrelated to defects in TCP assembly.

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

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.

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1

BT23, a Vibrio cholerae O1 El Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.

Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression.

Motility is thought to contribute to the virulence of Vibrio cholerae, but the role it plays in pathogenesis is not completely understood. To investigate the influence of motility on virulence gene expression and intestinal colonization, we have isolated mutants with altered swarming abilities in soft agar medium. Both spontaneous hyperswarmer (exhibiting faster swarm rates) and spontaneous or transposon-induced nonmotile mutants of strain 0395 were obtained. Surprisingly, we found that two of three classes of hyperswarmer mutants were defective in autoagglutination, a phenotype associated with expression of toxin-coregulated pili (TCP), an essential ToxR-regulated colonization factor of V. cholerae. In contrast, nonmotile mutants exhibited autoagglutination under growth conditions that normally repress this phenotype. Further characterization of mutant strains revealed differences in the expression of other virulence determinants. Class I hyperswarmer mutants were defective in production of TCP, cholera toxin, and a cell-associated hemolysin but showed increased levels of protease and fucose-sensitive hemagglutinin. All nonmotile mutants examined, including those with insertions in a sequence homologous to motB, exhibited increased expression of TCP pilin, cholera toxin, and cell-associated hemolysin but dramatically decreased levels of fucose-sensitive hemagglutinin and HEp-2 adhesins. In general, nonmotile mutants displayed few or no defects in intestinal colonization, while class I hypermotile mutants were highly defective in colonization. These results suggest that the motility phenotype of V. cholerae is tightly coupled to the expression of multiple ToxR-regulated and non-ToxR-regulated virulence determinants.