<p>During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease chlamydial protease-like activity factor (CPAF) into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Understanding the role of CPAF in pathogenesis is hampered because Chlamydia are not genetically tractable organisms. As such, chemical biology approaches were used to confirm CPAF function in vitro and in vivo, and to validate it as a virulence target. Here we report the development of assays, investigation of substrate specificity, and establishment of CPAF as a central virulence factor in chlamydial pathogenesis. A system for the expression and purification of CPAF was developed. An in vitro assay would allow for determination of kinetic parameters and aid in understanding the function of this protease. Two in vitro proteolysis assays, a discontinuous HPLC-based assay and a continuous fluorescence quenching assay, were developed for use in kinetic parameter determination and inhibitor discovery. </p><p>CPAF substrate specificity studies were conducted through the use of alanine scanning, proteomic identification of protease cleavage sites (PICS), and quantitative proteomics. Results from these studies showed that CPAF exhibited a preference for glycine, alanine, and serine in position P1, and valine in position P2' of peptide substrates.</p><p>Additionally, we designed and synthesized a zymogen-derived inhibitor peptide with nanomolar affinity that inhibited CPAF activity in vitro and in vivo. Using this, anti-CPAF peptide, we established CPAF as a virulence factor for chlamydial pathogenesis. Furthermore, CPAF inhibition resulted in degradation of the inclusion vacuole, exposing the bacteria and stimulating bacterial killing, thus CPAF inhibition created an antibacterial effect. CPAF inhibition also leads to the stimulation of innate immune defense activation, namely activation of caspase 1. In addition, CPAF was determined to be inhibited by the natural product salinosporamide A, a variant of omuralide, and the active form of the proteasome inhibitor lactacystin. Salinosporamide A and omuralide offer advantages over peptide therapeutics because of their intrinsic resistance to proteolytic degradation and improved oral bioavailability. Toward that end, progress toward CPAF inhibitor derivates from this natural product scaffold is also presented. Collectively this thesis lends support for CPAF as an antivirulence target for Chlamydia.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/4996 |
| Date | January 2011 |
| Creators | Bednar, Maria Michelle |
| Contributors | McCafferty, Dewey G |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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