Grant title: Type III exported effectors of Chlamydia trachomatis Abstract: The human pathogen Chlamydia trachomatis is a significant concern in the United States due to its prevalence and the combined health and socioeconomic impact of acute and chronic disease. Chlamydiae are obligate intracellular pathogens that possess the ability to modulate host-cell functions while sequestered within a membrane-bound parasitophorous vacuole. Although it is established that a type III secretion system (T3SS) represents one mechanism employed to modulate critical host cell pathways, the overall understanding of this process in Chlamydia remains limited. During the past funding cycle, we made significant progress in understanding the role of T3S effectors designated CT694 and CT695. Importantly, we also leveraged novel genetic manipulation techniques to investigate the impact of T3S in chlamydial pathogenesis. Perhaps our most important contribution was the development of a fluorescence-reported allelic exchange mutagenesis (FRAEM) approach to allow, for the first time, deletion of targeted chlamydial genes. We have used this technique to create null mutants for ct694 and ct695, and these strains are attenuated in both tissue-culture and animal-infection models. We propose to elucidate molecular details by which mutant phenotypes are manifested. A balanced combination of genetic, biochemical, cell-biology, and animal-based studies are proposed that will define developmental defects, identify host targets important for effector function(s), and gauge the overall role of effectors and target proteins in promoting Chlamydia pathogenesis. Completion of this work will extend the efficacy of genetically manipulating a challenging pathogen and lead to an enhanced understanding of Chlamydia-mediated disease.