| Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation. This process, known as quorum sensing, relies on the secretion and detection of signaling molecules called autoinducers. At low cell density V. cholerae activates the expression of virulence factors and forms biofilms. At high cell density the accumulation of two quorum-sensing autoinducers represses these traits. These two autoinducers, cholerae autoinducer-1 (CAI-1) and autoinducer-2 (AI-2), function synergistically to control gene regulation, although CAI-1 is the stronger of the two signals. V. cholerae AI-2 is the furanosyl borate diester (2S,4 S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. This thesis describes the purification of CAI-1 and identification of the molecule as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. The activities of both the R and S isomers of CAI-1 as well as simple homologues are evaluated. Synthetic (S )-3-hydroxytridecan-4-one functions as effectively as natural CAI-1 in repressing production of the canonical virulence factor TCP (toxin co-regulated pilus).;CAI-1 biosynthesis depends upon the CqsA enzyme. The mechanism of CqsA is investigated and its substrates are identified as (S)-2-aminobutyrate and decanoyl-coenzyme A. The product of CqsA activity is 3-aminotridecane-4-one (amino-CAI-1). Crystallographic analysis combined with spectral studies show that CqsA produces amino-CAI-1 by a pyridoxal phosphate (PLP)-dependent aminotransferase reaction. Though the mechanism is unknown, preliminary studies indicate that bacterial cultures are capable of converting amino-CAI-1 into CAI-1. Both molecules are detected in cell-free culture fluids, with amino-CAI-1 present at < 1% of CAI-1 levels. The findings presented in this thesis suggest that CAI-1 or a CAI-1-analogue could be used as a therapy to prevent cholera infection and, furthermore, that strategies to manipulate bacterial quorum sensing hold promise in the clinical arena. |
