Abstract

Vibrio cholerae, the cause of an often fatal infectious diarrhea, remains a large global public health threat. Little is known about the challenges V. cholerae encounters during colonization of the intestines, which genes are important for overcoming these challenges, and how these genes are regulated. In this study, we examined the V. cholerae response to nitric oxide (NO), an antibacterial molecule derived during infection from various sources, including host inducible NO synthase (iNOS). We demonstrate that the regulatory protein NorR regulates the expression of NO detoxification genes hmpA and nnrS, and that all three are critical for resisting low levels of NO stress under microaerobic conditions in vitro. We also show that prxA, a gene previously thought to be important for NO detoxification, plays no role in NO resistance under microaerobic conditions and is upregulated by H(2)O(2), not NO. Furthermore, in an adult mouse model of prolonged colonization, hmpA and norR were important for the resistance of both iNOS- and non-iNOS-derived stresses. Our data demonstrate that NO detoxification systems play a critical role in the survival of V. cholerae under microaerobic conditions resembling those of an infectious setting and during colonization of the intestines over time periods similar to that of an actual V. cholerae infection. IMPORTANCE: Little is known about what environmental stresses Vibrio cholerae, the etiologic agent of cholera, encounters during infection, and even less is known about how V. cholerae senses and counters these stresses. Most prior studies of V. cholerae infection relied on the 24-h infant mouse model, which does not allow the analysis of survival over time periods comparable to that of an actual V. cholerae infection. In this study, we used a sustained mouse colonization model to identify nitric oxide resistance as a function critical for the survival of V. cholerae in the intestines and further identified the genes responsible for sensing and detoxifying this stress.

Abstract

The human pathogen Vibrio cholerae uses quorum sensing to regulate the expression of a number of phenotypes, including virulence factor production, in response to changes in cell density. It produces small molecules called autoinducers that increase in concentration as cell density increases, and these autoinducers bind to membrane sensors once they reach a certain threshold. This binding leads to signalling through a downstream phosphorelay pathway to alter the expression of the transcriptional regulator HapR. Previously, it was shown that the VarS/VarA two-component system acts on a component of the phosphorelay pathway upstream of HapR to regulate HapR expression levels. Here, we show that in addition to this mechanism of regulation, VarS and VarA also indirectly modulate HapR protein activity. This modulation is mediated by the small RNA CsrB but is independent of the known quorum-sensing system that links the autoinducers to HapR. Thus, the VarS/VarA two-component system intersects with the quorum-sensing network at two levels. In both cases, the effect of VarS and VarA on quorum sensing is dependent on the Csr small RNAs, which regulate carbon metabolism, suggesting that V. cholerae may integrate nutrient status and cell density sensory inputs to tailor its gene expression profile more precisely to surrounding conditions.

Abstract

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, C(235), is critical for oxygen responsiveness, as the AphB(C235S) 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 C(235) 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.

Abstract

BACKGROUND:

Vibrio cholerae is the causative agent of cholera. Extensive studies reveal that complicated regulatory cascades regulate expression of virulence genes, the products of which are required for V. cholerae to colonize and cause disease. In this study, we investigated the expression of the key virulence regulator ToxR under different conditions.

RESULTS:

We found that compared to that of wild type grown to stationary phase, the toxR expression was lower in an aphB mutant strain. AphB has been previously shown to be a key virulence regulator that is required to activate the expression of tcpP. When expressed constitutively, AphB is able to activate the toxR promoter. Furthermore, gel shift analysis indicates that AphB binds toxR promoter region directly. We also characterize the effect of AphB on the levels of the outer membrane porins OmpT and OmpU, which are known to be regulated by ToxR.

CONCLUSIONS:

Our data indicate that V. cholerae possesses an additional regulatory loop that use AphB to activate the expression of two virulence regulators, ToxR and TcpP, which together control the expression of the master virulence regulator ToxT.

Metabolism of bile salts in mice influences spore germination in Clostridium difficile.

Giel JL, Sorg JA, Sonenshein AL, Zhu J.

PLoS One. 2010 Jan 15;5(1):e8740.

 

Abstract

Vibrio cholerae is the agent of the severe diarrheal disease cholera, and it perpetuates in aquatic reservoirs when not in the host. Within the host's intestines, the bacteria execute a complex regulatory pathway culminating with the production of virulence factors that allow colonization and cause disease. The ability of V. cholerae to form biofilms is thought to aid its persistence in the aquatic environment and passage through the gastric acid barrier of the stomach. The transcriptional activators VpsR and VpsT are part of the biofilm formation-regulatory network. In this study, we screened a V. cholerae genomic library in Escherichia coli cells containing a P(vpsT)-luxCDBAE transcriptional fusion reporter and found that a plasmid clone containing the aphA gene activates the expression of vpsT in E. coli. AphA is a master virulence regulator in V. cholerae that is required to activate the expression of tcpP, whose gene products in turn activate all virulence genes including those responsible for the synthesis of the toxin-coregulated pilus (TCP) and cholera toxin through the activation of toxT. AphA has a direct effect on the vpsT promoter, as gel shift experiments demonstrated that AphA binds to the vpsT promoter region. Furthermore, V. cholerae aphA mutants exhibit significantly lower levels of vpsT expression as well as reduced biofilm formation. AphA thus links the expression of virulence and biofilm synthesis genes.

Abstract

Recent work has shown that in addition to cholera toxin (CT) and the toxin-coregulated pilus (TCP), other cytotoxic proteins in Vibrio cholerae also cause disease symptoms, and this is particularly evident in strains lacking CT. One such protein is the hemolysin encoded by hlyA. Here we show that, like CT and TCP, HlyA is repressed by the quorum-sensing-regulated transcription factor HapR. This repression occurs on two levels: one at the transcriptional level that is independent of the metalloprotease HapA and one at the posttranslational level that is mediated by HapA. The transcriptional regulation is significantly more apparent on solid media than in liquid cultures. This is the first time that hemolysis has been shown to be directly regulated by quorum sensing in V. cholerae, and it is interesting that, like other virulence factors, HlyA is also repressed by HapR, which is expressed late in infection.

Abstract

Pathogenic bacteria, such as Vibrio cholerae, must be capable of adapting to diverse living conditions, especially when transitioning from life in environmental reservoirs to life in a host. The abilities to sense arrival at a site suitable for colonization or infection and to respond with appropriate alterations in gene expression are crucial for a pathogen's success. Recently, we have shown that V. cholerae is able to recognize that it has reached its colonization site in the small intestine by sensing breakage of its flagellum as it penetrates the mucosal layer overlaying the intestinal epithelium. Flagellar loss results in the release of the anti-sigma factor FlgM and subsequent activation of the alternative sigma-factor FliA. FliA represses the quorum sensing-controlled transcriptional regulator, HapR, allowing increased expression of virulence factors such as Cholera Toxin (CT) and the Toxin Coregulated Pilus (TCP). In this way, the de-repression of virulence factor expression coincides with the arrival of bacteria at the site of infection at the intestinal mucosa. Our work reveals an interesting interplay between motility and quorum sensing signaling pathways to precisely time virulence gene expression during colonization.

Abstract

The quorum-sensing pathway in Vibrio cholerae controls the expression of the master regulator HapR, which in turn regulates several important processes such as virulence factor production and biofilm formation. While HapR is known to control several important phenotypes, there are only a few target genes known to be transcriptionally regulated by HapR. In this work, we combine bioinformatic analysis with experimental validation to discover a set of novel direct targets of HapR. Our results provide evidence for two distinct binding motifs for HapR-regulated genes in V. cholerae. The first binding motif is similar to the motifs recently discovered for orthologs of HapR in V. harveyi and V. vulnificus. However, our results demonstrate that this binding motif can be of variable length in V. cholerae. The second binding motif shares common elements with the first motif, but is of fixed length and lacks dyad symmetry at the ends. The contributions of different bases to HapR binding for this second motif were demonstrated using systematic mutagenesis experiments. The current analysis presents an approach for systematically expanding our knowledge of the quorum-sensing regulon in V. cholerae and other related bacteria.

Abstract

The study of bacterial pathogenesis is in many ways the study of the regulatory mechanisms at work in the microbe during infection. The astonishing flexibility and adaptability of the bacterial cell has enabled many pathogenic species to freely transition between dramatically different environmental conditions. The transcriptional changes that underlie this ability can determine the success of the pathogen in the host. Many techniques have been devised to examine the transcriptional repertoire of bacteria in vivo during infection. Here, we review a class of technologies known as in vivo expression technology (IVET), which use promoter-trapping with a variety of different reporter constructs to allow researchers to probe the transcriptional changes taking place in bacteria under various environmental conditions. Using IVET techniques, researchers have been able to catalogue a wide variety of virulence factors in the host for several important human pathogens, as well as examining the timing of virulence gene regulation. Most recently, IVET techniques have also been used to identify transcriptional repression events in vivo, such as the suppression of anti-colonization factors deleterious to infection. As the array of IVET reporters and promoter-trapping strategies grow, researchers are increasingly able to illuminate the myriad transcriptional activities that allow bacteria to survive and cause disease in the host.

Abstract

The pathogen Vibrio cholerae uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host and establish itself at its preferred colonization site at the host intestinal mucosa. The key regulator in this process is the AraC/XylS family transcription factor, ToxT, which plays critical roles in pathogenesis, including the regulation of two type IV pili, the anticolonization factor mannose-sensitive hemagglutinin and the toxin-coregulated pilus. Previously, it was thought ToxT required dimerization in order to effect transcriptional regulation at its cognate promoters. Here, we present evidence that ToxT directly represses transcription of the msh operon by binding to three promoters within this operon and that dimerization may not be required for transcriptional repression of target promoters by ToxT, suggesting that this regulator uses different mechanisms to modulate the transcriptional repertoire of V. cholerae.

Abstract

To successfully infect a host and cause the diarrheal disease cholera, Vibrio cholerae must penetrate the intestinal mucosal layer and express virulence genes. Previous studies have demonstrated that the transcriptional regulator HapR, which is part of the quorum sensing network in V. cholerae, represses the expression of virulence genes. Here, we show that hapR expression is also modulated by the regulatory network that governs flagellar assembly. Specifically, FliA, which is the alternative sigma-factor (sigma(28)) that activates late-class flagellin genes in V. cholerae, represses hapR expression. In addition, we show that mucin penetration by V. cholerae is sufficient to break flagella and so cause the secretion of FlgM, the anti-sigma factor that inhibits FliA activity. During initial colonization of host intestinal tissue, hapR expression is repressed because of low cell density. However, full repression of hapR expression does not occur in fliA mutants, which results in attenuated colonization. Our results suggest that V. cholerae uses flagellar machinery to sense particular intestinal signals before colonization and enhance the expression of virulence genes by modulating the output of quorum sensing signaling.

Abstract

The pathogen Vibrio cholerae modulates the expression of many genes in order to transition from its environmental reservoir to its niche in the human host. Among these are genes encoding two related Type IV pili, the mannose-sensitive haemagglutinin (MSHA) pilus, which aids V. cholerae persistence in aquatic environments but causes clearance of bacteria by host immune defences, and the toxin co-regulated pilus (TCP) required for colonization. These antagonistic effects are resolved transcriptionally by the regulator ToxT, which represses msh genes while activating tcp genes during infection. We show that these two pili systems are also intertwined post-transcriptionally through the ToxT-regulated pre-pilin peptidase TcpJ. We found that the major MSHA pilin, MshA, was degraded in V. cholerae in a TcpJ-dependent fashion. In a heterologous Escherichia coli system, TcpJ can recognize both MshA and its cognate substrate, the TCP subunit TcpA, but that processing by TcpJ causes the degradation of MshA. Through site-directed mutagenesis and chimeric pilin analysis, we show that this process targets a combination of MshA N-terminal motifs and depends on the proteolytic activity of TcpJ. Moreover, overexpression of tcpJ partially restored the ability of bacteria unable to transcriptionally downregulate msh genes to colonize infant mice. These findings describe co-ordinated proteolysis as a regulatory mechanism in V. cholerae and illustrate this organism's adaptability in the face of dramatic environmental changes.