PROJECT SUMMARY Chlamydia trachomatis is the leading cause of sexually transmitted diseases, non-congenital infertility, and the blinding eye disease called trachoma. This obligate intracellular bacterium replicates within a membrane bound compartment, the inclusion, where it utilizes a specialized protein export system called the type III secretion system to translocate effector proteins into the cytoplasm of the host cell. A unique subfamily of type III effector proteins the Inclusion membrane proteins (Incs), is inserted into the inclusion membrane, with its N- and C- termini ideally poised at the host-pathogen interface to interact with host proteins and organelles. However, our understanding of the function of Incs has been limited, as they share little homology to each other or to other known proteins, and genetic manipulation of Chlamydia only recently become possible. To define the C. trachomatis Inc-human protein interactome, the Engel lab performed a high-throughput affinity-purification mass spectroscopy screen of 58 predicted Incs. Remarkably, a subset of Incs was identified to target components of the host ubiquitin (Ub) machinery, which prompts the hypothesis that a subset of Chlamydia Incs may modulate host ubiqutylation during infection and that these changes may be critical to the pathogenesis of C. trachomatis infection. Ubiquitylation, which involves the covalent attachment of Ub to a target protein by an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub ligase, alters the function, stability, or location of the Ub-modified protein within the cell. Modulating protein ubiquitylation during infection is an emerging theme for intracellular pathogens. Indeed, previous studies have established that C. trachomatis infection globally alters the host protein stability; however, how Inc-Ub machinery interactions reshape the host proteome is unknown. The long-term goal of this project is to understand how interactions between specific Incs and the host Ub machinery alters the host Ub landscape and promotes Chlamydia pathogenesis. In aim 1, a newly developed MS-based ubiquitin remnant profiling (URP) approach and bioinformatics will be used to globally define C. trachomatis-induced changes in the host ubiquinome. These changes will be compared to data sets for other intracellular pathogens obtained by a similar pipeline to identify changes in protein ubiquitylation that are specific to Chlamydia infections as well as protein ubiquitylation events that are common targets of intracellular pathogens. In aim 2, a combination of URP, host and bacterial genetics, and cell and molecular biology will be used to identify changes in the host ubiquinome that are dependent on the Inc (CT383) and its interactions with components of host Ub machinery. Together, these studies will provide critical new insights on the mechanisms by which C. trachomatis reprograms the host cell through ubiquitylation to promote pathogenesis. !