Mapping CqsS Binding and Activation in Vibrio Cholerae

Abstract

Quorum sensing (QS) is a process of bacterial cell-cell communication involving the production, release, and detection of extracellular signal molecules called autoinducers. The bacterium responsible for the acute intestinal disease cholera, Vibrio cholerae, uses QS to regulate virulence factor expression. This research focuses on the CAI-1 autoinducer and CqsS phosphorelay pathway of V. cholerae. Specifically, the objective of this research is to define the binding properties of agonists to the twocomponent histidine sensor kinase, CqsS, in order to investigate its influence on transmembrane signaling. The CqsS agonist denoted #92, a pro-QS molecule, was identified in a screen, and is more potent than the natural autoinducer CAI-1 in agonizing the CqsS receptor. Interestingly, a related molecule, #92-Me, which differs only in the absence of one methyl group, exhibits a 20 fold decreased potency relative to #92. To define the binding pocket of CqsS and the mechanism of the subsequent transduction of the ligand induced signal, CqsS mutants were screened and characterized for differential #92-Me activity. Two mutants, CqsS G60E and CqsS H120Q, were identified as having an improved response to CqsS agonists compared to WT, while CqsS mutants at F91 mutants were deficient across all molecules. Multiple CqsS F91 mutants were only able to respond to CqsS agonists containing a pyrrolidine moiety, which highlights the importance of this structure in CqsS agonists and also reveals the orientation of CqsS agonist binding.