Project Summary Lyme disease (LD) is a multisystem infectious disorder caused by the spirochete Borrelia burgdorferi (Bb). With an estimated 476,000 cases diagnosed and treated annually, LD is easily the most prevalent arthropod-borne infection in the United States. Transit of Bb between vector and reservoir host involves an intricate and poorly understood coordination of gene expression with control of motility. Differential gene expression in Bb is orchestrated, in large part, by two global regulatory networks – the RpoN/RpoS pathway and the Hk1/Rrp1 two component system (TCS). The RpoS pathway is activated by the bloodmeal during transmission, remains ON throughout mammalian infection, and rapidly turns OFF during larval acquisition. The Hk1/Rrp1 pathway signals via the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) synthesized by Rrp1, a diguanylate cyclase, in response to unidentified molecules generated during the larval and nymphal blood meals. c-di-GMP exerts pleiotropic effects in LD spirochetes. Bb lacking Hk1 or Rrp1 are destroyed in ticks during acquisition and transmission but are fully virulent in mice. Δrrp1 Bb display aberrant motility in vitro, indicating that c-di-GMP also impacts flagellar rotation. c-di-GMP-dependent responses often are mediated by binding to proteins with a PilZ domain. Accordingly, efforts to unravel c-di-GMP signaling in Bb have centered about PlzA, the sole PilZ domain protein in most LD spirochetes. In feeding ticks, ΔplzA Bb phenocopy Δhk1 and Δrrp1 mutants. Paradoxically, in mice, where the Hk1/Rrp1 TCS is OFF, PlzA deficiency markedly attenuates infectivity, implying a c-di-GMP independent function for PlzA in mammals. In other bacteria, PilZ proteins (e.g., YcgR) slow motility by directly interfering with the flagellar motor. Surprisingly, inhibition of Bb motility by c-di-GMP appears to be PlzA-independent. This proposal is intended to achieve an integrated conceptual and mechanistic framework for c-di-GMP signaling and PlzA functions across the entire enzootic cycle. Experiments in Aim 1, are designed to elucidate how PlzA functions as a c-di-GMP biosensor to promote both tick and mammalian host-adaptation by Borrelia burgdorferi. In Subaim 1.1, we will investigate the ability of liganded- PlzA to bind to a c-di-GMP regulated promoter (glp operon) using EMSAs and DNA-protein interaction analysis, and generate PlzA point mutants to identify residues responsible for DNA-binding. In Subaim 1.2, we use pull- down and site-directed mutagenesis to gain insight into the protein-protein interactions that enable unliganded PlzA to fulfill its c-di-GMP-independent, mammalian host-specific functions. Aim 2 is designed to deconvolute the contribution(s) of c-di-GMP to spirochete motility and biphasic dissemination at the tick-mammal interface. In Subaim 2.1, we will use gelatin matrices as an ex vivo tick midgut surrogate to monitor spirochete motility in response to the blood me...