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.