PROJECT SUMMARY Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection both domestically and globally, with the rate of cases per 100,000 increasing every year. However, infected individuals often fail to seek treatment largely due to the asymptomatic nature of approximately 70% of infections. Unchecked, chronic infection can lead to numerous sequelae, including ectopic pregnancy and tubal factor infertility in women or epididymitis and sterility in men, and those who do receive treatment are typically prescribed broad spectrum antibiotics. Given the steadily increasing case rate of infections, the risk of chronic issues, and the limited options for antibiotic stewardship, a more comprehensive understanding of chlamydial biology to produce a more targeted therapeutic treatment is critical. Despite having a highly reduced genome, Chlamydia undergoes a complex developmental cycle in which the bacteria differentiate between two functionally and morphologically distinct forms: the infectious, non- replicative elementary body (EB) and the non-infectious, replicative reticulate body (RB). An EB initiates infection by binding to and inducing uptake into a host cell. Within the cell, the EB undergoes primary differentiation into an RB, which will then give rise to a population of RBs. At an unknown signal mid-developmental cycle, newly formed RBs will undergo secondary differentiation from RB to EB. The transitions between EBs and RBs are not mediated by division events that re-distribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require protein turnover. As such, we hypothesize that ClpX plays a critical role during chlamydial differentiation through targeted protein degradation. To test this hypothesis, we generated numerous constructs for overexpression of various mutant ClpX isoforms in Chlamydia. The overarching goal of this proposal is to delineate a mechanism of ClpX-mediated degradation of substrates through adaptor dependent or independent means, which may uncover a network of targets that contribute to chlamydial differentiation. Previous in vitro characterization of these mutations from other labs studying other bacterial ClpX paralogs allows us to take a more targeted assessment of ClpX function rather than standard alanine scanning. Aim 1 will determine in which facets of chlamydial biology ClpX plays a role by observing the effects that these mutant proteins exert on chlamydial development, differentiation state, and morphology. The proposed experiments of Aim 2 will identify targets of chlamydial ClpX, which will provide significant insight into its substrate recognition and processing. We have developed a chemical crosslinking approach combined with Click-iT labeling of chlamydial proteins, specifically, to maintain complex stability when performing affinity purification of ClpX and its adaptors and substrates. Collectively, these experiments w...