Project Summary Chlamydia trachomatis is the causative agent of most commonly reported sexually transmitted disease in the United States. Disease is most severe in women and can cause detrimental effects to reproductive health, such as infertility. Establishment of an intracellular niche within non-immune cells, such as mucosal epithelium, is sufficient to drive immunopathology and disease sequela. C. trachomatis lacking both TmeA and TmeB, type III secretion system effectors, generate fewer infectious progeny in tissue culture and have significantly decreased infectivity in mice. Previous attempts to identify their individual functions utilized fluorescence- reported allelic exchange mutagenesis (FRAEM), but resulted in unclear phenotypes due to cassette-induced polar effects. Recently, I have developed floxed-cassette allelic exchange mutagenesis (FLAEM), to reverse cassette-induced polar effects and generate the first ever markerless C. trachomatis deletion mutant. I hypothesize that TmeA and TmeB are functionally related and important for establishing the inclusion, because they are co-transcribed, share a common chaperone, and are secreted into the host cell at the same time. The proposed work will utilize the novel marker-less C. trachomatis deletion mutant to determine when during chlamydial development TmeA and TmeB are important, and identify their eukaryotic host cell targets. The outcomes of this research will directly contribute to our understanding of basic C. trachomatis biology and how they use secreted effectors to establish an intracellular, which will identify potential drug targets to prevent the spread of infection to millions world-wide.