PROJECT SUMMARY The risk of sexually transmitted infection (STI) in humans depends on multiple biological factors, among which the occurrence of a cervicovaginal microbiota that is ‘permissive’ to STI stands out. The microbial composition of a STI-permissive microbiota is similar to that observed in association with the syndrome of bacterial vaginosis, a condition that is generally defined by a high pH (>4.5), the absence of Lactobacillus spp. and an array of strict and facultative anaerobes such as Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp., and Prevotella spp. In contrast, a typical ‘non-permissive’ microbiota is dominated by one of several species of Lactobacillus, a unique feature of the human cervicovaginal microbiota. The mechanism(s) by which a non-permissive cervicovaginal microbiota provides protection against STIs remains poorly understood, as no animal or cell culture model system developed to date satisfactorily reproduces the cervicovaginal mucosa in its natural environment as a target for experimental infection. As a consequence, our knowledge of the pathogenesis of STIs is incomplete, particularly as it pertains to the critical role of the human cervicovaginal microbiota. We have established extensive preliminary data that support the scientific premise of Project 1 and states that a non- permissive indigenous microbiota interacts with the cervicovaginal epithelium to establish a homeostatic state that blocks STI and/or reduces disease severity. Conversely, a permissive microbiota disrupts host cell homeostasis, thereby allowing STI to progress. Innovative and advanced 3D biomimetic models of the cervicovaginal mucosa that eliminate the inherent ethical and biological limitations of existing models will be used to test this hypothesis, using the most prevalent agent of STIs worldwide, Chlamydia trachomatis. Project 1 aims to develop a better understand of the host-microbiota interactions and how they modulate the fate of C. trachomatis infection. In this project, the biomimetic models will be exploited to ask specific questions about how different types of microbiota (permissive and non-permissive) modulate epithelial cell functioning (Aim 1) in relationship to their susceptibility/resistance to chlamydial infection (Aim 2). Project 1 will also assess the preclinical potential of the model for testing preventive (Aim 2) and therapeutic (Aim 3) interventions against STIs. For these studies, Project 1 will leverage the full force of an array of state-of-the-art omics methodologies, including metatranscriptomics and RNA-sequencing to identify and characterize alterations of microbiota and host cellular homeostasis, metagenomics and metataxonomics to define the composition of carefully reconstructed microbiota or from human donors on the biomimetic models, and glycomics to analyze mucus structural features.