Bile-mediated ToxS homodimerization provides a model for ToxRS periplasmic interactions

ABSTRACT The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens. These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors. In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxR is a DNA-binding regulator associated in the periplasm with ToxS, a protein of poorly understood function. ToxS modulates the activity of its binding partner ToxR in the presence of bile salts, antimicrobial cholesterol metabolites secreted into the gut. To date, the molecular mechanism underlying this regulation remains unclear. We present crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. ToxSp has a highly conserved hydrophobic core that stabilizes the β-barrel fold, while the binding pocket tolerates substantial variation, consistent with binding hydrophobic ligands. Strikingly, we discovered that Vp-ToxSp binds three molecules of glycocholate and the presence of this bile salt leads to the formation a strand-swapped ToxS homodimer. Finally, modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp homodimer provides a structure-based model for bile salt-mediated heterotetramerization of the ToxRS system. Overall, our study addresses a major longstanding question in the field of Vibrio virulence regulation providing a scenario that could apply to other pathogens that utilize these membrane-bound family transcriptional regulators. AUTHOR SUMMARY The ability to sense the environment is essential for bacterial pathogens to successfully colonize the human host. Many pathogenic bacteria utilize members of the family of co-component transmembrane transcription regulators (coTTRs) to regulate pathogenesis in response to host signals. The ToxRS system is a coTTR regulatory pair that is essential for host colonization and virulence regulation of the enteric pathogens Vibrio cholerae and Vibrio parahaemolyticus. This system encompasses a transcription factor, ToxR, and a binding partner of enigmatic function, ToxS. Here, we solved the structure of the ToxS periplasmic domain V. parahaemolyticus and found that it belongs to a larger family of chaperone-like proteins present in many organisms. Strikingly, we discovered that the presence of the bile salt glycocholate leads to the dimerization of the periplasmic domain of ToxS. We propose that bile salt-induced ToxS dimerization brings two ToxR cognates together forming a heterotetramer activating the regulatory system. Our results and model address a major longstanding question in the fields of Vibrio virulence regulation and could apply to other pathogens that utilize these membrane bound family transcriptional regulators. Competing Interest Statement The authors have declared no competing interest. Footnotes This version has been significantly revised to clarify the results as well as the model. As part of the revision, the number of figures has been reduced and the figure describing the model has been replaced.