The Vibrio cholerae trh gene is coordinately regulated in vitro with type III secretion system genes by VttR(A)/VttR(B) but does not contribute to Caco2-BBE cell cytotoxicity.

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.