Date of Award
Fisher, Derek J
Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for sexually transmitted infections and the ocular infection trachoma. Chlamydia undergo a distinctive developmental cycle transitioning between the infectious elementary body form and the replicative reticulate body form. Each developmental form has a distinct proteome reflecting its different physiological needs. We are interested in studying how protein degradation impacts proteomic patterns throughout the developmental cycle. Chlamydia encode a pan-bacterial Clp protease system consisting of five genes: clpX, clpC, two clpP paralogs (P1 and P2), and clpB. ClpX and ClpC are chaperone proteins that unfold protein substrates and feed them into the oligomeric ClpP protease; ClpB is a deaggregase. We hypothesize that the Clp protease system plays an essential role in proteomic turnover between the developmental forms. As a first test of our hypothesis, we initially sought to confirm and characterize the protease activity of the putative ClpP paralogs. Escherichia coli was used to produce the chlamydial ClpP1 and ClpP2 proteins with C-terminal 6x His-tags. Proteins were then purified using immobilized metal (cobalt) affinity chromatography. Function was assessed through analysis of oligomerization along with testing of protease activity using casein and fluorescent-peptide substrates. We also explored the effects of ClpP-binding small-molecule compounds on in vitro ClpP1/2 activity and growth of C. trachomatis in a cell culture infection model. Our results indicate that ClpP2, but not ClpP1, is able to form a mature oligomer and possesses protease activity under the in vitro conditions tested. In addition, ClpP2 activity was stimulated in the presence of various ClpP-binding molecules and these molecules reduced growth of C. trachomatis in cells. Collectively, our findings support the presence of a functional Clp protease system in Chlamydia and suggest that the system is essential for growth making it an intriguing target for the development of novel anti-chlamydial drugs.
Available for download on Friday, May 10, 2019