Strain AM-19226 is a pathogenic non-O1/non-O139 serogroup Vibrio cholerae strain that does not encode the toxin-coregulated pilus or cholera toxin but instead causes disease using a type three secretion system (T3SS). Two genes within the T3SS pathogenicity island, herein named vttR A (locus tag A33_1664) and vttR B (locus tag A33_1675), are predicted to encode proteins that show similarity to the transcriptional regulator ToxR, which is found in all strains of V. cholerae. Strains with a deletion of vttR A or vttR B showed attenuated colonization in vivo, indicating that the T3SS-encoded regulatory proteins play a role in virulence. lacZ transcriptional reporter fusions to intergenic regions upstream of genes encoding the T3SS structural components identified growth in the presence of bile as a condition that modulates gene expression. Under this condition, VttR A and VttR B were necessary for maximal gene expression. In contrast, growth in bile did not substantially alter the expression of a reporter fusion to the vopF gene, which encodes an effector protein. Increased vttR B reporter fusion activity was observed in a ⌬vttR B strain background, suggesting that VttR B may regulate its own expression. The collective results are consistent with the hypothesis that T3SS-encoded regulatory proteins are essential for pathogenesis and control the expression of selected T3SS genes.

Numerous virulence factors have been associated with pathogenic non-O1/non-O139 serogroup strains of Vibrio cholerae. Among them are the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH), which share amino acid similarities to the TDH and TRH proteins of Vibrio parahaemolyticus, where they have been shown to contribute to pathogenesis. Although TDH and TRH homologs can be encoded on extrachromosomal elements in V. cholerae, type III secretion system (T3SS)-positive strains, such as AM-19226, carry a copy of trh within the T3SS genomic island. Transcriptional fusion analysis showed that in strain AM-19226, trh expression is regulated in a bile-dependent manner by a family of transmembrane transcriptional regulators that includes VttR A , VttR B , and ToxR. Genes encoding T3SS structural components are expressed under similar conditions, suggesting that within the T3SS genomic island, genes encoding proteins unrelated to the T3SS and loci involved in T3SS synthesis are coregulated. Despite similar in vitro expression patterns, however, TRH is not required for AM-19226 to colonize the infant mouse intestine, nor does it contribute to bile-mediated cytotoxicity when strain AM-19226 is cocultured with the mammalian cell line Caco2-BBE. Instead, we found that a functional T3SS is essential for AM-19226 to induce bile-mediated cytotoxicity in vitro. Collectively, the results are consistent with a more minor role for the V. cholerae TRH in T3SS-positive strains compared to the functions attributed to the V. parahaemolyticus TDH and TRH proteins.