Enzymology of Chlamydia Pathogenesis

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DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis is the most common sexually-transmitted pathogen in the developed world, >100 million persons are infected worldwide annually. C. trachomatis infections cause numerous disease-related complications including pelvic inflammatory disease, ectopic pregnancy, infertility, and blindness. Infecting the single columnar layer of epithelial cells of the urogenital tract and conjunctiva, C. trachomatis persists within parasitophorous inclusion vacuoles. During infection, C. trachomatis releases numerous effector molecules into the host cell cytoplasm to interfere with host signaling, which aids in chlamydial cooption of key host cell functions that benefit the infection. Of these effectors, the serine protease chlamydial protease-like activity factor (CPAF) is an enzyme that has emerged as a central virulence factor due to its multivariable roles in modifying host or bacterial proteins involved in lipid and membrane transport, the actin cytoskeleton, microtubule-based motors, lysosomal recognition of the inclusion, ERK/MEK signaling pathways, and the onset of programmed cell death and inflammation signaling. We have developed the first selective and potent cell permeable inhibitors for CPAF, including a newly discovered nonpeptidic small molecule inhibitor, and we have used CPAF inhibitors to establish a critical role for this enzyme as both an antimicrobial and antivirulence target in cell culture models of human chlamydial infections. Inhibition of CPAF activity leads to decreased bacterial replication, destruction of the inclusion vacuole, and bacterial cell death. In addition, CPAF inhibitors reprogrammed infected cells to initiate host immune defense responses such as secretion of pro-inflammatory cytokines and activation of an inflammasome-dependent programmed cell death pathway in the host cell. Based on these results, we hypothesize that CPAF inhibitors may form the basis for a new class of small molecule 'antimicrobial vaccines' that not only kill bacteria, but may promote the development of adaptive and acquired immunity. Since Chlamydiae sp. are resistant to genetic manipulation, we lack an appropriate understanding of the identity of CPAF targets and substrate processing mechanisms in vivo, an appreciation for the scope of the involvement of CPAF in chlamydial pathogenesis, and an understanding of the type and magnitude of antimicrobial and immune defensive responses triggered by CPAF inhibition in animal models of infection. Our long-term goals for this proposal are to understand the role CPAF in C. trachomatis pathogenesis, to gain insight into the molecular level details of CPAF protease function, inhibition and identification o targets, and to determine the extent to which CPAF inhibitors can clear infections, rescue host immune defense activity, and protect against reinfection in a clinically relevant murine animal model of chlamydial genital tract infection.