Project Summary/Abstract Chlamydia infections are important causes of human disease for which no vaccine exists. Their extraordinary prevalence, associated morbidity, health care costs, and link to various chronic disease states make them public concerns of critical importance. Although infections can be treated with antibiotics, no drug is cost-effective enough for widespread elimination of disease. An important gap in our knowledge is how this obligate intracellular vacuolar bacterium establishes a privileged niche--a membrane bound compartment termed the inclusion--and avoids the host innate immune response. Chlamydia encode a distinctive family of secreted effectors, the Incs (Inclusion membrane proteins), which are translocated from the bacteria through the type III secretion system and inserted into the inclusion membrane. We hypothesize that some of these effectors, by virtue of their position at the host-pathogen interface, modulate the innate immune response. However, since tractable genetic tools have only recently been developed for Chlamydia, the specific function of the majority of Incs remains unknown. We pioneered using a high throughput affinity purification-mass spectroscopy (AP-MS) strategy in conjunction with transfection to identify putative host binding partners for 2/3 of the C. trachomatis Incs. In recent unpublished work, we have discovered unexpected roles for several of these effectors that suggests that not only can Incs function as proteins scaffolds, but that they may establish higher order novel protein complexes at the inclusion. Nonetheless, our “transfection” interactome is potentially limited by the non-native presentation of Incs or the requirement that some Inc-host protein-protein interactions (PPIs) require multiple Incs. In aim 1, we propose a high throughput strategy to overcome these limitations by adapting our AP-MS screen to rapidly identify and validate host interacting partners in the context of infection and to identify new Inc-host PPIs that may have been missed in our initial screen. In preliminary data, we validate this “infection” interactome approach, which has enabled us to prioritize the further study of two fascinating effectors, CT226 and CT224, that may influence the host innate immune response to C. trachomatis infections, an understudied area in general for intracellular vacuolar human pathogens. We have discovered that the predicted coiled-coil domain in the C-terminus of CT226 binds to a complex comprised of Flightless-1 and LRRFIP1 and/or LRRFIP2 (L/F complex). The function of this complex is incompletely understood but has been reported to modulate inflammasome as well as IRF3 and NFkB signaling in mammalian cells. We have discovered that the predicted coiled-coil domain in the C- terminus of CT224 binds to TRAF7, a unique member of the TNF receptor associated factors that modulates NFkB signaling, cytokine production, and apoptosis. We hypothesize that the CT226:L/F and the CT224:TRAF...