PROJECT SUMMARY/ABSTRACT The vector-borne spirochetes that cause relapsing fever are transmitted to humans by either ticks or human body lice. Despite identification of the etiological agents of relapsing fever over 100 years ago, very little information exists regarding their pathogenesis. Although relapsing fever is more common in developing countries, tickborne relapsing fever (TBRF) occurs in areas of the U.S. where Ixodes and Ornithodoros species of ticks, the vectors for TBRF spirochetes, are endemic. During their natural enzootic cycle, vector-borne spirochetes exist in two distinct niches found within the arthropod vector and the vertebrate. It is well established that Lyme disease spirochetes must undergo significant changes in global gene expression to allow them to adapt to these two diverse environments. Cyclic dinucleotide second messengers (e.g., c-di-GMP and c-di-AMP) play key roles during the enzootic cycle of Borrelia burgdorferi, but their regulatory contributions in TBRF spirochetes have not been investigated. We will test the role of the c-di-AMP signaling pathway in promoting adaptation of TBRF spirochetes to the different host environments encountered during the bacterial natural lifecycle. Because the c-di-AMP signaling pathway is found in all pathogenic Borrelia, findings from this work also has the potential to provide insight into the function of this system in Lyme disease Borrelia. In Specific Aim 1, we will inactivate individual components c-di-AMP signaling system in the TBRF spirochete, Borrelia turicatae, and define their roles in messenger molecule production and general spirochetal physiology. We will also study the regulation of individual pathway components and c-di-AMP synthesis. Specific Aims 2 and 3 will elucidate the impact of the c-di-AMP signaling system on global regulation, pathogenesis, and vector colonization/transmission. These aims will provide critical knowledge regarding the regulatory networks that control B. turicatae adaptation during transmission and infection and identify virulence determinants required by the bacteria for host-pathogen and vector-pathogen interaction(s). Regulators and virulence factors identified in this project represent potential targets against which future therapeutic interventions and/or diagnostics for TBRF could be developed. Molecular characterization of the c-di-AMP-dependent signaling system and c-di-AMP-regulated virulence determinants will be the focus of future R01 grant proposals.