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

Abstract

Vibrio 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.

The Cpx System Regulates Virulence Gene Expression in Vibrio cholerae

“…This observation is in apparent contradiction to our finding that when cpxR is overexpressed in V. cholerae El Tor strain C6706, it upregulates the expression of the virulence regulator ToxR (54), an activator of ctxA and tcpA expression (16,56). In this regard, a recent study showed for the first time that ToxR can negatively regulate the production of CT and TCP in response to cyclic dipeptides (CDPs) (89). Bacterial production of cyclo(Phe-Pro) leads to ToxR-mediated activation of the LysR transcription factor LeuO, which in turn represses CT production (89).…”

“…In this regard, a recent study showed for the first time that ToxR can negatively regulate the production of CT and TCP in response to cyclic dipeptides (CDPs) (89). Bacterial production of cyclo(Phe-Pro) leads to ToxR-mediated activation of the LysR transcription factor LeuO, which in turn represses CT production (89). At this point, we cannot say whether Cpx-mediated activation of ToxR production is involved in the inhibition of CT and TCP via CDPs or some other mechanism (Fig.…”

Cyclo(valine–valine) inhibits Vibrio cholerae virulence gene expression

“…The resulting analysis showed that cVV exhibited an IC 50 of~166 mM, which confirmed that cVV exhibited more potent antivirulence activity relative to cFP, which exhibited an IC 50 of~300 mM (Bina & Bina, 2010;Bina et al, 2013).…”

“…We therefore cultured the respective mutants (Dlrp : : Cm, DvpsR, DtoxRS and DleuO) and JB58 under AKI conditions in the presence and absence of 0.5 mM cVV and quantified aphA expression by qRT-PCR. The toxRS mutant was included to test whether cVV activity was dependent on ToxR as was observed with cFP (Bina et al, 2013). The results showed that cVV exhibited inhibitory activity in the lrp, vpsR and leuO deletion backgrounds, as indicated by the cVV-dependent inhibition of aphA expression in each strain (Fig.…”

“…Effect of cVV on genes that are upstream of aphA in the ToxR regulon Lrp, VpsR and LeuO have been shown to regulate aphA expression (Bina et al, 2013;Lin et al, 2007). We therefore examined whether cVV activity was dependent on any of these genes.…”