PROJECT SUMMARY The overarching goal of this collaborative project is to identify the underlying mechanisms for Chlamydia evasion of cell-autonomous immune defenses. The Hybiske lab has developed a library of ~1000 unique chimeras bearing genomic material derived from the human-tropic pathogen Chlamydia trachomatis (Ct) and the rodent-tropic pathogen Chlamydia muridarum (Cm). The Coers lab previously demonstrated that the mechanisms of host-driven ubiquitination of intracellular pathogens varies significantly between humans and mice. Cm blocks the host-driven delivery of ubiquitin and members of the guanylate binding protein (GBP) family to the chlamydial parasitophorous vacuole (termed the inclusion) in mouse but not in human cells. In a reciprocal process, Ct inhibits the delivery of ubiquitin and GBPs to its inclusion in human cells but fails to do so in mouse cells. These processes lead to host-adapted survival by chlamydial species in their appropriate host cells, and the hypothesis that one or more Ct loci specifically interfere with human GBP/ubiquitin functions whereas one or more Cm loci interfere with mouse-specific GBP/ubiquitin functions. The identities of these critical chlamydial factors and the corresponding mechanisms they interface with in host cells are not known. To identify the discrete Ct and Cm loci responsible for host-specific immune evasion, we will pursue two independent aims. In Aim 1, we will screen our CtCm chimera library in human and mouse cells to identify chlamydial loci that mediate the evasion of IFN?-mediated growth restriction. In Aim 2, we will define the host- driven mechanism of inclusion ubiquitination and determine the loci responsible for inhibition of GBP and ubiquitin recruitment to inclusion membranes. These chimeric genomes are predominantly Ct L2 with unique regions of Cm genes recombined into them. Growth screens will identify chimeras that have lost the ability to subvert human cell-autonomous defenses as well as those which gain the ability to resist IFN?-mediated defenses in mouse cells. Genetic analyses of these fully genome-sequenced chimeras will reveal genes from both species that are critical for their evasion of these otherwise deleterious pathways. We will additionally characterize the chimeric strains for susceptibility or resistance to host defense effector functions occurring downstream of GBP/ubiquitin inclusion tagging, and test whether the virulence of chimeric strains susceptible to host-dependent cell-autonomous immunity is restored in cells deficient for individual GBPs or clusters of GBP genes. Defining both the nature of chlamydial counter defenses and the corresponding host-specific innate immune responses will advance our understanding of Chlamydia pathogenesis, and facilitate the broader goal of developing novel therapeutics and an effective Ct vaccine.