Investigating and modulating bacterial quorum sensing in Vibrio harveyi and Vibrio cholerae using small molecules

Quorum sensing is a process by which groups of bacteria control collective behaviors according to population density as perceived through the secretion and detection of small organic molecules called autoinducers. By coordinating behaviors across a colony, quorum sensing allows bacteria, which are single-celled organisms, to achieve a measure of multicellularity. A number of different classes of autoinducers have been identified. Gram-negative bacteria typically utilize acylated-homoserine lactones (AHL's) as autoinducers, while Gram-positive species typically utilize oligopeptides (AIP's). AHL's and AIP's are used to facilitate System One quorum sensing, defined as intraspecies communication within a particular population of bacteria. A second quorum sensing system, System Two, facilitates interspecies communication among bacteria utilizing the autoinducer AI-2. Thus, through quorum sensing an individual bacterium can determine both the number of its own kind and the ratio of self-to-other in a heterogeneous community of many bacterial species.;Bacteria use the information derived from quorum sensing to control behavior, usually those behaviors that are most beneficial to the population when synchronously undertaken by a group. Virulence factor expression is often controlled using this mechanism. Therefore, quorum sensing circuits are attractive targets for the development of new anti-microbial drugs: small molecules that interfere with cell-to-cell communication between bacteria to prevent the expression of virulence genes. A particularly appealing attribute of this method of drug therapy is a decreased selective pressure towards resistance development, since interference with communication mechanisms does not directly jeopardize bacterial survival.;Here, three methods to control bacterial quorum sensing using small molecules are investigated: two focus on the manipulation of interspecies communication through System Two quorum sensing, and one focuses on intraspecies communication through the System One circuit of Vibrio cholerae, the causative agent of the disease cholera. Efforts to modulate System Two quorum sensing consist of the development of AI-2 analogs to take the place of the native signal in the bacterial environment and the inhibition of the LuxS protein to prevent the biosynthesis of AI-2. AI-2 analogs were developed using rational design; however, none of the compounds synthesized acted as either agonists or antagonists of quorum sensing. A high-throughput screening approach taken to identify LuxS inhibitors successfully identified a lead structure, 2-(3-(biphenyl-2-yloxy)propylthio)-6-methylpyrimidin-4-ol, upon which modifications will be made to develop a more potent inhibitor. Studies into quorum sensing in V. cholerae led to the identification of CAI-1 as (3S)-hydroxytridecan-4-one, the first example of a novel class of System One autoinducers: alpha-hydroxyketones. Various classes of CAI-1 analogs were synthesized to develop an understanding of the specificity of the CAI-1 sensor, CqsS. Of these analogs, 3-aminotridecan-4-one was exceptionally potent, which initiated studies of the enzymatic mechanism of the CAI-1 synthase, CqsA, a putative aminotransferase.