CHARACTERIZATION OF THE CHLAMYDIA TRACHOMATIS CLP PROTEASE SYSTEM

Author

• Prajakta Prasad Mahajan, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2026

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Fisher, Derek

Abstract

Chlamydia is the most prevalent sexually transmitted bacterial infection worldwide and in the United States. Infections frequently remain asymptomatic and can cause complications like infertility and pelvic inflammatory disease. We lack a vaccine for chlamydia, and current treatment relies on broad spectrum antibiotics that disrupt the microbiome and can promote resistance of drug development in bystander bacteria. C. trachomatis undergoes a biphasic developmental cycle alternating between two main forms, the infectious elementary body (EB) and the replicative reticulate body (RB). The uniqueness of the chlamydial developmental cycle makes it an attractive target for Chlamydia-specific therapeutic approaches. We hypothesize that proteome remodeling is necessary for EB and RB differentiation events, and our published data supports that a Caseinolytic protease (Clp) system consisting of ClpX/ClpP2/ClpP1 and ClpC/ClpP2/ClpP1 are specifically involved in RB to EB conversion. In the Clp system, ClpP2 and ClpP1 form the proteolytic component while ClpX and ClpC act as adaptor proteins identifying and delivering proteins to the ClpP2/ClpP1 protease for degradation. Note that there are also adaptors for ClpX and ClpC. There is a significant gap in knowledge for Chlamydia regarding the substrates for ClpX and ClpC and adaptors for these proteins. These molecular details are key for determining how the Clp system regulates and/or enables differentiation and could reveal a novel Achilles’ heel of this important pathogen. Here, we sought to develop approaches for characterizing ClpX and ClpC partner interactions and to begin mapping those interactions. We report purification of ClpX from Escherichia coli, and chlamydial AtoS, ClpC wild type and mutant constructs, and McsA/McsB for use in in vitro assays. We also constructed a large collection of vectors for use in the bacterial adenylate cyclase two-hybrid (BACTH) assay and used BACTH to identify interactions between ClpX and AtoS, AtoC, ChxR, and Trigger factor along with interactions between ClpC and McsA. Collectively, we have developed tools and approaches that bring us one step closer to obtaining mechanistic insights into the function of ClpX and ClpC in chlamydial proteostasis and development.