Characterization of receptors of the bacterial signaling molecule autoinducer-2

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DESCRIPTION (provided by applicant): Bacteria communicate through a process known as quorum sensing, in which small chemical signals are produced and released into the environment. As the population increases, these signals (called autoinducers) accumulate and, when they reach sufficient concentration, are detected by other bacteria. Bacteria modulate gene expression in response to these signals, and thus are able to regulate their behavior in a population-wide manner [1-8]. Many important behaviors are regulated by quorum sensing, including virulence, motility, and biofilm formation [9-11, 13]. While most autoinducers mediate communication within a given bacterial species, a novel signal molecule, termed autoinducer-2 (AI-2), appears to be a universal signal molecule, facilitating signaling between species [6, 19]. More than 55 species of bacteria are known to carry the gene for LuxS, the AI-2 synthase, including pathogens such as Salmonella typhimurium, Bacillus anthracis, Yersinia pestis, and Vibrio cholerae [20]. Thus, AI-2 mediated quorum sensing offers a possible mechanism for controlling bacterial behavior of significant human pathogens [29, 30, 50-53]. Knowledge of the chemical identity of AI-2 is essential for the design of agonists and antagonists of quorum sensing, but previous work has shown that two different bacterial species recognize chemically distinct forms of AI-2 [24, 32]. Thus, it is crucial to identify AI-2 receptors in a given species and characterize AI-2 in the context of its receptor. This work seeks to both extend the set of known AI-2 receptors and determine the chemical form of AI-2 recognized by biomedically relevant bacterial species. Candidate AI-2 receptors will be identified in several bacterial species, including Bacillus cereus, Enterobacter sp. 638, Sinorhizobium meliloti and Pseudomonas aeruginosa, through genomic analysis including sequence similarity searches and fold prediction. These candidate proteins will be cloned, overexpressed, purified, and assayed for AI-2 binding via a Vibrio harveyi bioluminescence assay. Once AI-2 receptors have been identified biochemically, they will be further purified and crystallized. X-ray structures will be determined for AI-2/receptor complexes, allowing characterization of the chemical form of AI- 2 recognized by a particular species.