Project Summary/Abstract Lyme disease has reached epidemic proportions in the northeastern United States and poses a substantial burden on affected communities. The illness can present with a spectrum of clinical manifestations which vary in severity and duration, including certain complications that persist after antibiotic-therapy, termed post-Lyme syndromes. However, the reasons for this range in disease severity are not clear, and biomarkers to identify patients at greater risk for adverse clinical outcomes are lacking. This presents a challenge for both physicians and patients whose symptoms do not resolve with standard antibiotic therapy. Our recent findings suggest that inappropriate immune responses, which are shaped at least in part by microbial genetics, are an important factor in these outcomes. We previously identified a particularly virulent Borrelia burgdorferi RST1 OspC type A strain which leads to excessive inflammation and more severe disease, including more symptomatic early infection and a greater risk for antibiotic-refractory Lyme arthritis, a post-Lyme complication of this disease. These findings provided a new paradigm for studying inappropriate immune responses and untoward clinical outcomes in Lyme disease. However, specific spirochetal genes associated with RST1 OspC type A strains are not yet know, and knowledge of microbial genetic determinants of immunogenicity and virulence are limited. In this proposal, we are applying our multi-prong approach using cutting-edge methodologies, clinical samples from well-defined patients, and functional studies in cells and tissue to delineate spirochetal genes and variants associated with dysregulated immune responses and adverse clinical outcomes in Lyme disease. We propose 2 specific aims: Aim 1: To identify microbial genes implicated in greater virulence a) by characterizing Bb isolates from patients with mild or severe disease using whole genome sequencing, b) by delineating genes associated with RST1 strains using GWAS, and c) by correlating candidate genes with immune and clinical phenotype in patients. Aim 2: To test the functional impact of Bb RST1 strains on host immune responses a) by assessing transcriptome profiles in skin lesions and synovial tissue from patients infected with an RST1 strain and b) by delineating Bb-sensing pathways that mediate excessive inflammation to RST1 in macrophage, fibroblast, and PBMC. The work proposed here will provide new insights into pathogenesis and is likely to lay the groundwork for novel diagnostic approaches to identify patients at greater risk for severe disease, including post-Lyme syndromes. The ability to identify such patients could help guide more effective treatment strategies such as combination of antibiotics and immunotherapy.