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 this project 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. Advanced 3D organotypic 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. We aim to develop a better understanding of the host-microbiota interactions and how they modulate the fate of C. trachomatis infection. In this project, 3D organotypic models colonized with different types of reconstructed microbiota 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). We will also assess the preclinical potential of the model for testing preventive (Aim 2) and therapeutic (Aim 3) interventions against STIs. For these studies, we will leverage the full force of state-of-the-art microscopic and RNA-sequencing methodologies to identify and characterize microbiota specific alterations of structural and host epithelial homeostasis and expand our understanding of the triangular relationship between the host, the microbiota and C. trachomatis.