Novel targets and a cofactor for the Vibrio cholerae quorum sensing transcriptional regulator, HapR

Vibrio cholerae, which causes the severely dehydrating diarrheal disease, cholera, undergoes drastic changes in living conditions as it moves between its aquatic reservoir and its human host. These different living conditions require different gene expression profiles. One method that V. cholerae uses to sense changes in the environment, particularly changes in cell density, is quorum sensing. The bacteria produce small molecules called autoinducers that accumulate with increasing cell density and trigger intracellular signaling pathways to alter gene expression. These intracellular signaling pathways regulate the expression level of the transcriptional regulator, HapR, which controls many phenotypes including virulence factor production and biofilm formation. The studies presented here provide insight into the phenotypes that HapR regulates and the mechanism by which HapR provides this regulation. A bioinformatics-based approach was used to determine consensus binding sequences for HapR and to identify novel targets that are directly regulated by HapR. Many new HapR-regulated promoters were found, and some were activated while others were repressed by HapR. This study led us to investigate HapR's regulation of a hemolysin, HlyA, that had previously been shown to be a potential virulence factor. HapR was found to repress HlyA on two levels: one by directly binding to and repressing transcription from its promoter and another by inducing the transcription of a protease, HapA, which degrades the HlyA protein. HapR is known to repress V. cholerae's two main virulence factors, cholera toxin and the toxin-coregulated pilus, and so its regulation of HlyA represents another mechanism by which quorum sensing represses virulence. The third study identifies a two-component system, VarS/VarA, that is required for full HapR function. A previous report showed that VarS and VarA regulate HapR expression through the quorum sensing system, but we show that in addition to this, VarS and VarA also modulate HapR activity. Since VarS and VarA affect HapR's regulation of its targets in two ways, it is likely that the integration of VarS/VarA signaling with quorum sensing is important. We propose that this enables V. cholerae to combine cell density and nutrient availability information to more appropriately control gene expression.