ABSTRACTToxR is a major virulence gene regulator inVibrio cholerae. Although constitutively expressed under many laboratory conditions, our previous work demonstrated that the level of ToxR increases significantly when cells are grown in the presence of the 4 amino acids asparagine, arginine, glutamate, and serine (NRES). We show here that the increase in ToxR production in response to NRES requires the Var/Csr global regulatory circuit. The VarS/VarA two-component system controls the amount of active CsrA, a small RNA-binding protein involved in the regulation of a wide range of cellular processes. Our data show that avarAmutant, which is expected to overproduce active CsrA, had elevated levels of ToxR in the absence of the NRES stimulus. Conversely, specific amino acid substitutions in CsrA were associated with defects in ToxR production in response to NRES. These data indicate that CsrA is a positive regulator of ToxR levels. Unlike previously described effects of CsrA on virulence gene regulation, the effects of CsrA on ToxR were not mediated through quorum sensing and HapR. CsrA is likely essential inV. cholerae, since a complete deletion ofcsrAwas not possible; however, point mutations in CsrA were tolerated well. The CsrA Arg6His mutant had wild-type growthin vitrobut was severely attenuated in the infant mouse model ofV. choleraeinfection, showing that CsrA is critical for pathogenesis. This study has broad implications for our understanding of howV. choleraeintegrates its response to environmental cues with the regulation of important virulence genes.IMPORTANCEIn order to colonize the human host,Vibrio choleraemust sense and respond to environmental signals to ensure appropriate expression of genes required for pathogenesis. Uncovering howV. choleraesenses its environment and activates its virulence gene repertoire is critical for our understanding of howV. choleraetransitions from its natural aquatic habitat to the human host. Here we demonstrate a previously unknown link between the global regulator CsrA and the majorV. choleraevirulence gene regulator ToxR. The role of CsrA in the cell is to receive input from the environment and coordinate an appropriate cellular response. By linking environmental sensing to the ToxR regulon, CsrA effectively acts as a switch that controls pathogenesis in response to specific signals. We demonstrate that CsrA is critical for virulence in the infant mouse model ofV. choleraeinfection, consistent with its role as anin vivoregulator of virulence gene expression.
Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model
