Vibrio cholerae is a genetically diverse species, and pathogenic strains can encode different virulence factors that mediate colonization and secretory diarrhea. Although the toxin-coregulated pilus (TCP) is the primary colonization factor in epidemic-causing V. cholerae strains, other strains do not encode the TCP and instead promote colonization via the activity of a type 3 secretion system (T3SS). Using the infant mouse model and T3SS-positive O39 serogroup strain AM-19226, we sought to determine which of 12 previously identified, T3SS-translocated proteins (Vops) are important for host colonization. We constructed inframe deletions in each of the 12 loci in strain AM-19226 and identified five Vop deletion strains, including ⌬VopM, which were severely attenuated for colonization. Interestingly, a subset of deletion strains was also incompetent for effector protein transport. Our collective data therefore suggest that several translocated proteins may also function as components of the structural apparatus or translocation machinery and indicate that while VopM is critical for establishing an infection, the combined activities of other effectors may also contribute to the ability of T3SS-positive strains to colonize host epithelial cell surfaces. The toxin-coregulated pilus (TCP) is the major colonization factor encoded by all pathogenic O1 and O139 serogroup Vibrio cholerae strains, which cause epidemic cholera. In contrast, most clinically isolated non-O1/non-O139 serogroup strains do not encode TCP and thus must employ other mechanisms to effectively colonize the human intestinal epithelium and cause sporadic, cholera-like disease (1-3). Genome sequence analysis of a clinically isolated O39 serogroup strain, named AM-19226, identified a pathogenicity island on the large chromosome that encodes the structural proteins for a type 3 secretion system (T3SS) (4). T3SSs function as principal virulence mechanisms in many Gram-negative bacterial pathogens (e.g., Escherichia, Salmonella, Pseudomonas, Shigella, and Yersinia spp.), and in vivo studies using different animal models confirmed that the V. cholerae T3SS is essential for causing disease (5-7). In addition, numerous groups have identified T3SS-positive V. cholerae strains in laboratory collections, from patients, and from endemic environments, suggesting that a subset of non-O1/non-O139 serogroup strains depends on T3SS activity for virulence (1,3,4,(8)(9)(10).The V. cholerae T3SS is most closely related to the Vibrio parahaemolyticus T3SS2. T3SS2 is associated with pandemic V. parahaemolyticus strains, whereas T3SS1 is present in all strains (11). Comparison of the T3SS genomic islands in V. cholerae strain AM-19226 and V. parahaemolyticus strain RIMD2210633 reveals synteny within a conserved, central "core" region, flanked by 5= and 3= regions of greater coding diversity between clades and species (4, 10, 12-14). The core region encodes proteins that form the T3SS structural apparatus and is transcriptionally organized into four main operons in V. cholera...

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, VttR A , VttR B , and ToxR. VttR A and VttR B 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 vttR B expression. The data suggest both that ToxR and VttR A act upstream of VttR B and that modifying the level of either vttR A or vttR B expression can strongly influence T3SS gene expression. We therefore propose a model whereby T3SS activity and, hence, in vitro cytotoxicity are ultimately regulated by vttR B expression. IMPORTANCEIn contrast to O1 and O139 serogroup V. cholerae strains that cause cholera using two main virulence factors (toxin-coregulated pilus [TCP] and cholera toxin [CT]), O39 serogroup strain AM-19226 uses a type 3 secretion system as its principal virulence mechanism. Although the regulatory network governing TCP and CT expression is well understood, the factors influencing T3SS-associated virulence are not. Using an in vitro mammalian cell model to investigate the role of three ToxR-like transmembrane transcriptional activators in causing T3SS-dependent cytotoxicity, we found that expression levels and a hierarchical organization were important for promoting T3SS gene expression. Furthermore, our results suggest that horizontally acquired, ToxR-like proteins act in concert with the ancestral ToxR protein to orchestrate T3SS-mediated pathogenicity. P athogenic bacteria must effectively control the expression of virulence factors in order to achieve productive infection. As a waterborne pathogen, Vibrio cholerae senses and responds to signals from two distinct environments, the aquatic reservoir and the human host, requiring appropriate temporospatial changes in gene expression. Epidemic-causing O1 and O139 serogroup strains use the ToxR-ToxT regulatory hierarchy to modulate the expression of genes encoding factors responsible for colonization (toxin-coregulated pilus [TCP]) and diarrhea (cholera toxin [CT]) (1, 2). Whereas toxR is an ancestral gene found in all V. cholerae strains, toxT is carried on horizontally acquired Vibrio pathogenicity island 1 (VPI-1), which encodes the TCP. ToxR can directly activate transcription from the toxT promoter (3, 4). VPI-1 also encodes the TcpPH proteins, which can increase toxT transcription and are required for...