PROJECT SUMMARY/ABSTRACT A major challenge in bacterial pathogenesis is the prevention and treatment of polymicrobial diseases, as microbe-microbe interactions alter the initiation of disease and its susceptibility to antimicrobial treatment. For example, bacterial vaginosis (BV) occurs within the vaginal microbial community, and the factors that drive its initiation, treatment, and high levels of recurrence (>50%) are not understood. BV is the most common condition of the female genital tract in reproductive-aged women, impacting over 29% of women in the United States with an estimated annual economic burden of $5 billion in North America. Importantly, in addition to vaginal pain, odor, and discharge, BV causes increased susceptibility to sexually transmitted infections, including HIV, and is associated with preterm birth and cervical cancer. Research has sought to understand BV pathogenesis through studying differences in community composition at the species, genus, and phylum levels and through identifying genomic and phenotypic differences across BV microbes. However, this previous work only partially explains differences across women in the development of BV and the response to treatment. This proposal investigates single-cell genomic and transcriptomic diversity as two underexplored areas that will transform our understanding of the underlying factors that control BV progression. The goal of this proposal is to identify aspects of diversity that impact BV and provide insights into new strategies for prevention, treatment, and diagnosis. Further, these findings will have broad applicability to polymicrobial infections. In this study, the overarching hypothesis is that intrapersonal strain-level genomic diversity and transcriptomic heterogeneity alter the stability of communities, their interactions with the host, and their susceptibility to treatment. First, this proposal tests the importance of intrapersonal strain-level diversity in pre-clinical models and clinical specimens (Objective 1). Across microbiome sites and polymicrobial infections, including in the vaginal microbiome, previous work has identified that individuals can concurrently harbor multiple strains of the same species, but the functional implications of this diversity in disease is unknown. This objective will demonstrate how variation in intrapersonal strain-level diversity impacts bacterial physiology and BV progression. Second, this proposal will characterize the role of single-cell transcriptomic heterogeneity in microbial interactions related to BV and responses to antimicrobials through applying the new approach, bacterial single-cell RNA-seq, to in vitro vaginal communities and clinical specimens (Objective 2). This innovative analysis will result in the identification of transcriptomic subpopulations with altered traits, including pathogenesis and antimicrobial susceptibility, that can be targeted to promote vaginal health. Overall, this proposed work will have broa...