Regulation by ToxR-Like Proteins Converges on vttR B Expression To Control Type 3 Secretion System-Dependent Caco2-BBE Cytotoxicity in Vibrio cholerae

Genes carried on the type 3 secretion system (T3SS) pathogenicity island of Vibrio cholerae non-O1/non-O139 serogroup strain AM-19226 must be precisely regulated in order for bacteria to cause disease. Previously reported results showed that both T3SS function and the presence of bile are required to cause Caco2-BBE cell cytotoxicity during coculture with strain AM-19226. We therefore investigated additional parameters affecting in vitro cell death, including bacterial load and the role of three transmembrane transcriptional regulatory proteins, VttRA, VttRB, and ToxR. VttRA and VttRB are encoded on the horizontally acquired T3SS genomic island, whereas ToxR is encoded on the ancestral chromosome. While strains carrying deletions in any one of the three transcriptional regulatory genes are unable to cause eukaryotic cell death, the results of complementation studies point to a hierarchy of regulatory control that converges on vttRB expression. The data suggest both that ToxR and VttRA act upstream of VttRB and that modifying the level of either vttRA or vttRB expression can strongly influence T3SS gene expression. We therefore propose a model whereby T3SS activity and, hence, in vitro cytotoxicity are ultimately regulated by vttRB expression.