Identification of Substrates for, and Redox Regulation of the Chlamydia trachomatis Tail-specific Protease

Author

• Jonathan Davis Brooks, 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 trachomatis is an obligate intracellular gram-negative bacterium responsible for the most prevalent reportable bacterial sexually transmitted infection as well as the leading cause of preventable blindness worldwide. Discovering new therapeutic targets is important for lowering the global burden of this significant human pathogen. C. trachomatis undergoes a biphasic development cycle where the infectious, oxidized elementary bodies (EBs) differentiate into the replicative, reduced reticulate bodies (RBs) (primary differentiation) that asynchronously convert into EBs (secondary differentiation) before exiting the host cell. Protein turnover is imperative for differentiation between developmental morphologies and can be coordinated through chaperones and proteases. Tail-specific proteases (Tsp) are a family of periplasmic serine proteases conserved in many gram-negative bacteria and are often associated with peptidoglycan (PG) homeostasis. Chlamydial Tsp is distinct from other constitutively expressed Tsp homologs in that it is temporally expressed alongside secondary differentiation when PG is degraded/absent within C. trachomatis. In addition, Tsp contains four cysteine residues not conserved in other gram-negative bacteria (homologs typically contain no cysteines). Previously, Tsp has been shown to be necessary for production of infectious EBs, although the exact role of Tsp within secondary differentiation is still unknown. In addition, conversion between the RB and EB morphologies has been correlated to a change in internal redox state, suggestive of cysteine-facilitated redox regulation of Ct_Tsp. To better understand the role of Tsp, we set out to identify and validate Tsp substrates using far-western blotting and targeted degradation analyses with purified recombinant Tsp and potential substrates. Results have identified multiple substrates related to disulfide catalysis and peptidoglycan turnover, in addition to several hypothetical proteins. Additionally, we investigated the effect of oxidative stress on Tsp proteolytic activity and stability and discovered that degradation of the model substrate casein is partially inhibited in the presence of hydrogen peroxide. Cysteine to alanine substitutions in Tsp maintained Tsp activity, increased stability of full-length Tsp, and rendered Tsp less susceptible to hydrogen peroxide inactivation. By better understanding how Tsp impacts the development of C. trachomatis, we gain insight into potential drug targets that could impede the chlamydial development cycle.