Exploring the Enzymology of Chlamydial Pathogenesis: An Investigation of Virulence and Energy Metabolism-Associated Enzymes

Abstract

Chlamydia trachomatis is the obligate intracellular pathogen responsible for the most common bacterial sexually transmitted infection worldwide. While our front-line antibiotics have been historically successful in combatting chlamydial infections, emerging issues including treatment failure and chlamydial persistence necessitate the development of new therapeutic approaches. In this work, an enzyme-focused approach was devised to explore two of the intricate survival strategies of C. trachomatis: virulence and energy metabolism. We sought to employ biochemistry, enzymology, and chemical biology tools to interrogate enzyme functions and inform the design of new antichlamydial agents. To these ends, our efforts focused on characterization of the virulence-associated effector protein chlamydial protease-like activity factor (CPAF) and the futalosine pathway for menaquinone biosynthesis. Mechanistic analyses of CPAF zymogen maturation and peptide hydrolysis were performed that collectively classified CPAF as a serine protease with a catalytic tetrad. Analogs of the natural product salinosporamide A were subsequently explored as CPAF inhibitors. The futalosine pathway was discovered to be a source of novel antichlamydial targets through traditional and chemical genetics analyses in a HeLa cell model of chlamydial infection. The foundation was also established for studying a specific pathway enzyme, CT263, in a continuous coupled enzyme assay. Collectively, this dissertation has progressed our knowledge of several enzymes involved in critical processes for chlamydial pathogenicity and viability. The insights gained on a mechanistic level and in the context of chlamydial infection have laid the groundwork for pursuing virulence and energy metabolism enzymes as antichlamydial targets and for developing inhibitors of their activity, which are much-needed resources to combat this extremely prevalent sexually transmitted infection.