Event Information
Insights into Chlamydial Protease-Like Activity Factor (CPAF)
- Abstract:
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. Here we report the development assays, investigation of substrate specificity, and establish CPAF as a central virulence factor in chlamydial pathogenesis. We produced or acquired ~20 different plasmids and attempted over 150 growth conditions to produce soluble CPAF. Following purification, two in vitro proteolysis assays, a discontinuous HPLC-based assay and a continuous fluorescence quenching assay, were produced for use in kinetic parameter determination and large scale inhibitor screening.
Additionally, we synthesized a zymogen-derived inhibitor peptide that proved capable of in vivo inactivation of CPAF. Due to the inability to genetically manipulate Chlamydia, this inhibitor peptide allowed for the first testing of specific inhibition of CPAF. Through the use of the inhibitor peptide, we were able to establish CPAF as a virulence factor, and confirm CPAF as an attractive candidate for future drug design.
The elucidation of CPAF substrate specificity elucidation was attempted through the use of alanine scanning, PICS, and quantitative proteomics. The last of these proved to be the most informative, showing a preference for small amino acids in P1 and P2’. Lastly, efforts toward the total synthesis of a phenyl derivative of lactacystin are currently underway.
Ph.D. Dissertation Defense Examination Seminar
Departmental Seminar