PROJECT SUMMARY With over 130 million new Chlamydia trachomatis infections each year, the development of innovative strategies to prevent these infections is a global public health priority. A number of prospective studies have reported an association between bacterial vaginosis and increased risk of C. trachomatis acquisition. However, the precise nature of the association between vaginal bacterial species and C. trachomatis susceptibility, and the biologic mechanisms driving these associations, are not well understood. Emerging data from in vitro studies suggest that metabolites and enzymes produced by specific BV-associated bacteria impact urogenital C. trachomatis growth. However, data from in vivo studies assessing this relationship are sparse. To address this important knowledge gap, we propose a multidisciplinary approach that combines epidemiologic and laboratory studies to assess the impact of vaginal bacteria, and their metabolites, on C. trachomatis acquisition. This resubmission will utilize data and samples collected from women participating in the Mombasa Cohort study, an ongoing NIH- sponsored open cohort study. We will conduct a nested case-control study using both broad-range and quantitative PCR to determine if detection and concentrations of BV-associated species are associated with increased risk of C. trachomatis infection (Aim 1). In Aim 2, we will test vaginal samples collected as part of the case-control study to evaluate whether key metabolites used by C. trachomatis are associated with increased risk of C. trachomatis infection. In Aim 3, we will use a systems biology approach to integrate data from Aims 1 & 2 to identify taxonomic drivers of functional shifts in the vaginal metabolome that lead to increased C. trachomatis susceptibility. The proposed studies represent a novel approach to understanding how the vaginal microbiome and metabolome mediate C. trachomatis susceptibility. Findings from the proposed research will identify critical targets that enhance C. trachomatis susceptibility and inform development of innovative C. trachomatis prevention strategies that seek to disrupt or eliminate bacterial or metabolomics targets that facilitate C. trachomatis growth, thus reducing C. trachomatis infection.