Title: Small molecules as novel probes for understanding Vibrio cholerae virulence mechanisms and regulation in vitro and in vivo
Description: The etiological agent of cholera is the environmental bacterium Vibrio cholerae. While over 200 serogroups of the pathogen are known, most cause only sporadic disease and are typically found in the marine environment as free living bacteria. The majority of epidemic disease is caused by V. cholerae of two serogroups, O1 and O139, which are solely responsible for the last eight pandemics of cholera. Upon entering the host, the bacterium elaborates two major virulence factors, Cholera Toxin (CT) and the Toxin Co-regulated Pilus (TCP). A small molecule which inhibits V. cholerae virulence gene expression in vitro was identified in a chemical genetics screen of 50,000 compounds and called virstatin. We demonstrated that virstatin specifically inhibits TCP-dependent, but not TCP-independent colonization of the infant mouse small intestine. Administration of virstatin to mice with an established infection reduced colonization by several logs. Virstatin was shown to target the activity of the transcriptional activator, ToxT. Using virstatin as a tool, we demonstrated that it inhibits the dimerization of ToxT and that this dimerization is crucial to the activity of the activator at the CT and TCP promoters.

Next, virstatin was used in an in vivo chemical genetics screen of a panel of non-O1/non-O139 strains of Vibrio cholerae, which as a group are capable of causing sporadic disease. Several strains were identified which are able to colonize in a virstatin-resistant manner, suggesting that they contain an alternative virulence mechanism. Of these, five contain a divergent toxT allele, which was defined as being resistant to virstatin in vitro and required for efficient colonization. The remaining strains contain an epidemic strain toxT allele and must thereby colonize using an alternate virulence mechanism. Of these strains, we demonstrated that one, 623-39, has the ability to invade epithelial cells, an unusual phenotype for Vibrio cholerae. A LuxR-type modulator of invasion, LmvI, was identified and its activity was shown to be dependent on phosphorylation. Finally, a novel Type Three Secretion Island was discovered in the strain by shotgun sequencing, and determined to be necessary for colonization of infant mice. This work contributes to the field of Vibrio cholerae pathogenesis and opens up multiple new avenues of research both with newly developed tools and methods as well as the identification of new pathways and strains of interest.