PROJECT SUMMARY The intracellular bacterium Chlamydia trachomatis is a major cause of sexually transmitted infections and infectious blindness, with over 150 million cases worldwide. Yet, it is still considered a neglected disease pathogen by the World Health Organization. Once inside the cell, Chlamydia replicates within a parasitic compartment called an inclusion, the integrity of which is essential for Chlamydia survival. Inclusion membranes are decorated with ~60 transmembrane Inc proteins that directly interact with host cell components and play a critical role in sustaining Chlamydia’s life cycle. These inclusion proteins include IncA, which is required for the homotypic fusion of chlamydial inclusions. This fusion event is important for pathogenicity as patients infected with non-fusogenic C. trachomatis strains that express mutated forms of IncA display fewer clinical symptoms than those infected with WT C. trachomatis. Yet, how Chlamydia controls homotypic fusion is unknown. While the chlamydial protein IncA is involved, little is known about the molecular mechanism regulating homotypic fusion. Here, we propose to test the hypothesis that the bacterial fusion protein IncA uses a combination of intra- and extra-molecular regulatory mechanisms to control membrane fusion and ensure the proper maturation of the inclusion. Our extensive expertise in membrane fusion and Chlamydia pathogenesis places us in an ideal position to address this significant knowledge gap. Unlike membrane fusion driven by eukaryotic cells and enveloped viruses, which are extensively studied, membrane fusion driven by bacteria is poorly understood. This project will characterize a novel prokaryotic fusion system, thus providing a roadmap to identify similar fusion systems in other bacteria. In parallel, data gathered here will determine the extent to which this homotypic fusion event is essential for C. trachomatis fitness, thus providing new information about disease progression.