Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Chlamydia trachomatis is a gram negative, obligate intracellular pathogen with a highly reduced genome of ~1 Mbp. It is the leading cause of the reportable sexually transmitted infection known as chlamydia in the United States and is the leading cause of preventable blindness (trachoma) worldwide. While treatment of infections is possible, weaknesses of current approaches include treatment failure, antibiotic-induced dysbiosis, and resistance development of bystander bacteria during chlamydial treatment. These weaknesses support the need for improved therapeutic approaches. C. trachomatis undergoes a biphasic developmental cycle with two forms, the infectious elementary body (EB) and replicative reticulate body (RB), that have unique protein profiles. Due to the differing proteomes of each developmental form, we hypothesized that mechanisms that facilitate protein turnover will be essential for progression of C. trachomatis through the developmental cycle making them ideal drug targets. This study focused on characterization of two caseinolytic protease (Clp) systems: the ClpX/P2/P1 system and the ClpC/P1/P2 / McsAB system. We predicted that ClpP1 and ClpP2 come together to form the proteolytic component, that ClpX and ClpC are unfoldases that unfold and linearize large substrates in an ATP dependent manner for ClpP-dependent proteolysis, and that McsAB are adaptor proteins with McsA activating the kinase McsB to tag proteins for degradation by the ClpC/P1/P2 complex. The Clp system has been the focus of numerous studies as a target for novel antimicrobials and we hypothesized that the chlamydial Clp system would also be a druggable target. To assess the functionality of the Clp system, we successfully purified all components except McsB for use in vitro assays. Using oligomerization, peptide and protein degradation assays, and ATP hydrolysis assays, we characterized the activity of the ClpP1, ClpP2, and ClpX components individually and in complexes. We also measured the activity of a collection of ClpX mutants. In addition, we assessed the activity of ClpP-targeted activating compounds that were potent in vivo inhibitors of C. trachomatis. We demonstrated that ClpP2/P1 can form hetero-oligomers and degrade peptides and that ClpX has ATPase activity, can oligomerize, and can degrade an SsrA-tagged GFP when complexed with ClpP2/P1. While the activator studies did not support interactions with ClpP2/P1 under the conditions tested, assays were developed for further analysis of Clp-targeted compounds. Our in vitro results support that C. trachomatis possesses a functional Clp system. In addition, in vivo expression of ClpX mutants confirmed to lack activity in our in vitro assays led to reduced chlamydial fitness and alterations in development supporting our hypothesis that the Clp system is required for chlamydial development. Collectively, our results indicate that the Clp system is critical to C. trachomatis survival in cells and suggests that drugs altering Clp-function could be a novel approach for anti-chlamydial therapeutics.
This thesis is Open Access and may be downloaded by anyone.