Date of Award
12-1-2024
Degree Name
Master of Science
Department
Molecular Biology Microbiology and Biochemistry
First Advisor
Fisher, Derek
Abstract
The obligate intracellular bacterium Chlamydia trachomatis is the causative agent of chlamydia, the most reported bacterial sexually-transmitted infection in the United States. Currently, there is no commercially-available vaccine. Infection with C. trachomatis is treatable with broad-spectrum antibiotics; however, these treatments can cause dysbiosis in patients and contribute to antibiotic resistance in other bacterial species. Improvements to drug therapies would reduce side-effects of treatment and increase efficacy. The developmental cycle of C. trachomatis involves a transition between two forms, the infectious elementary body (EB) and the replicative reticulate body (RB). As these two forms have unique protein profiles, we posit that significant remodeling of the proteome occurs during differentiation. We speculate that proteases promote chlamydial development through proteomic turnover and thus, we reason that these protease systems would make appealing drug targets that could be chlamydial specific. One of the proteases that C. trachomatis possesses is the caseinolytic protease (Clp) system, comprised of two proteolytic heptamers, ClpP1 and ClpP2, as well as two adaptors: ClpC and ClpX. Our prior research supports a role for ClpX in development, and we seek to define the molecular mechanism(s) by which ClpX enables EB production. To this end, our goal was to identify ClpX adaptors and substrates. To enable a screen for substrates/adaptors, we attempted to develop a medium throughput in vivo GFP-degradation based assay using Escherichia coli that expressed the chlamydial ClpP2/P1/X. When the screening assay failed, we switched to a targeted approach focusing on potential adaptors and substrates including: the histidine kinase and cognate response regulator AtoS/AtoC, the transcriptional regulator ChxR, and CTL0300, a putative Single-Stranded DNA Binding protein (SSB). We established a variety of targeted in vitro assays for probing ClpX-adaptor/substrate interactions including ATPase, immunoprecipitation, protein cross-linking, Native-PAGE, and electrophoretic mobility shift assays (EMSA). We also validated the annotated function of CTL0300 as an SSB through EMSA and tryptophan quenching assays. Collectively, the stage is now set to rigorously evaluate ClpX substrates and adaptors allowing for molecular insights into the role of ClpX in chlamydial development.
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