Disturbances of the human vaginal microbiota can cause severe complications for women and, if pregnant, their neonates. Currently, about one in four women experience vaginal dysbiosis, a condition marked by the absence of the health-associated genus Lactobacillus and the presence of a heterogenous consortium of microbes. Although clinical studies have found associations between Lactobacillus dominant vaginal communities and reduced vaginal infection, the complexity of the role that vaginal microbiota play in the protection against pathobiont outgrowth has not been thoroughly studied. Without an understanding of protective microbial and host factors leading into vaginal dysbiosis, antibiotic therapeutics targeting the symptomatic influx of other taxa have not been effective in preventing the recurrence of infection. Thus, it is necessary to define the initial factors that increase susceptibility to vaginal infection. The overarching goal of this proposal is to understand the capacity at which the vaginal microbiota can directly confer protection against pathobiont outgrowth or indirectly alter the local immune response to inhibit pathobiont colonization. To attribute a reduction of pathobiont colonization to local factors, we will delineate whether the vaginal microbiota or immune profile are shaped by microbial and immunological processes in the gut. We hypothesize that endogenous vaginal microbiota are sufficient to determine susceptibility to pathobiont colonization and shape the local immune profile both in homeostasis and in vaginal dysbiosis. To understand both the vaginal microbiota’s influence on the transition to the susceptible state of vaginal dysbiosis and the ability to enhance immunological protection against pathobiont outgrowth, we will interrogate in vivo and in vitro models. Specifically, we will determine the microbe and immune factors that mediate protection against vaginal dysbiosis-associated pathobionts Group B Streptococcus and Prevotella bivia by utilization of the HMbmice model, which reflects a humanized vaginal tract dominated by Lactobacillus spp. We will describe the vaginal microbiota of HMbmice to understand the community dynamics and compositions through longitudinal 16S rRNA gene sequencing. We will then employ mini- bioreactors to cultivate vaginal microbes in vitro, testing for specific bacteria and metabolites that are capable of limiting vaginal infection. Additionally, we will utilize flow cytometry to elucidate the impact that different vaginal microbiota have on training of the local immune function. Lastly, we will generate gnotobiotic mice to resolve whether the vaginal microbiota and immune system are influenced by the gut, establishing the source of vaginal susceptibility to infection. Ultimately, this proposal seeks to benefit underrepresented groups in science through the therapeutic discoveries for women’s health and the training of the applicant, who will focus on disseminating scientific knowledge ...