Project Summary Chlamydia trachomatis (C.t.) is the leading cause of non-congenital blindness and the most prevalent sexually transmitted bacterial infection in the world, which if left untreated can result in severe consequences. All chlamydiae are obligate intracellular bacteria and thus gaining entry into a host cell is essential for the pathogen to complete its replicative cycle and proliferate. Despite the critical nature of the invasion process, the molecular mechanisms and details regarding how C.t. forces its way into non-phagocytic cells remains a large knowledge gap. A major premise of the current model suggests that invasion is facilitated by delivery of prepackaged conventional type III secretion system (cT3SS) effector proteins into the host cell prior to pathogen entry. We hypothesize that a subset of these cT3SS effectors coordinate active subversion of the host cytoskeleton through direct manipulation of key regulators of actin dynamics. New data from our laboratory indicates that the cT3SS effector protein TmeA binds to the nucleation promoting factor N-WASP, both of which we show are essential for invasion. In Aim 1, we will mechanistically determine how TmeA regulates N-WASP and determine whether this interaction promotes key membrane features that likely aid in bacterial entry such as; membrane ruffling, pedestal formation, and filopodial dynamics. Given the essential nature of invasion to bacterial proliferation and survival, C.t. likely employs multiple measures for invasion. In Aim 2, we will evaluate the complex interplay between TarP, TepP, and TmeA and reveal the ultimate molecular effects of their effector function and describe how the seemingly disparate pathways targeted by these effectors converge to assure chlamydial invasion. Furthermore, we will determine whether other cT3SS effectors, only expressed in the invasive EB form of chlamydia, are necessary for invasion of non-phagocytic cells. Detailed characterization of the bacterial and host proteins required to promote reorganization of the actin cytoskeleton during C.t. invasion will provide a holistic view of how intracellular pathogens, such as C.t., coordinate subversion of cytoskeletal regulators to invade host cells.