Project Summary: Characterizing the Function of the Periplasmic Protease Tsp in Chlamydial Secondary Differentiation Chlamydia is an obligate intracellular bacterial pathogen that causes a range of serious diseases in humans. In developed countries, Chlamydia trachomatis is the primary cause of bacterial sexually transmitted infections (STI). Indeed, recent reports from the Centers for Disease Control highlight the increasing incidence of STIs, with chlamydia infections consistently outpacing all other types. In developing countries, C. trachomatis is not only a significant cause of STI, but it is also responsible for the primary cause of infectious preventable blindness, trachoma. The major concern of chlamydial infections is that they are often asymptomatic and undiagnosed, which can lead to chronic sequelae. These include pelvic inflammatory disease, tubal factor infertility, and reactive arthritis for C. trachomatis. Consequently, chlamydial diseases remain a significant burden on health care systems around the world. In adapting to obligate intracellular growth, Chlamydia has significantly reduced its genome size and eliminated genes from various pathways as it relies on the host cell for its metabolic needs. This pathogen has also adapted to alternate between different functional and morphological forms during its normal growth, also referred to as its developmental cycle. These observations, combined with its obligate intracellular dependence, makes Chlamydia a difficult organism with which to work. However, recent development of genetic tools to study chlamydiae mechanistically have significantly enhanced our understanding of this pathogen. This proposal applies a combination of these new genetic techniques and classical biochemical studies to evaluate the role of a conserved periplasmic protease system in chlamydial growth and pathogenesis. The hypothesis of the proposed work is that Chlamydia uses this periplasmic protease to effect secondary differentiation from the replicative to the infectious form of the organism by degrading cell division- related proteins. This prevents further division of the replicative form as the bacteria differentiate. Major goals of this proposal are (i) to characterize the function of the periplasmic protease Tsp both in vitro and in vivo and (ii) to identify and validate substrates of Tsp. Results will advance our understanding of this important pathogen and lead to the design of novel therapeutic agents that are specific for Chlamydia. This in turn will allow for minimal effects on normal flora for patients receiving treatment for this highly prevalent disease.