PlzA, cyclic-di-GMP and the enzootic cycle for Lyme disease

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Project summary: Lyme disease is the most common tick-borne disease in the northern hemisphere and its incidence is steadily increasing. The ability of the Lyme disease spirochetes to adapt to changing environmental conditions is dependent in part on regulation mediated by the secondary messenger molecule, cyclic-di-GMP (c-di-GMP). This proposal will identify the critical functional determinants of PlzA, the sole identified c-di-GMP binding protein produced by all Lyme disease spirochete isolates. Based on the recently determined atomic structure of PlzA which revealed that PlzA belongs to the unique xPilZ domain class of c-di-GMP binding proteins and data demonstrating that PlzA has RNA chaperone activities, we will perform a comprehensive analysis to dissect structure-function relationships of both apo and holo forms of the protein. Based on comparative sequence and structural analyses, surface-exposed amino acid residues will be targeted for site-directed mutagenesis. The impact of these mutations on PlzA structure, c-di-GMP binding, RNA winding, RNA unwinding, and protein-protein interactions will be assessed in vitro. Based on these analyses, a series of B. burgdorferi transgenic strains in which wild-type plzA is replaced with plzA genes that encode PlzA proteins with altered activity will be generated. The ability of each strain to infect and transmit between ticks and mammals will be determined. These analyses will define the functional domains and biological mechanisms by which apo and holo PlzA regulate cellular processes required for the completion of the enzootic cycle.