Date of Award

5-1-2019

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Fisher, Derek

Abstract

Chlamydia are obligate intracellular bacterial pathogens that are responsible for infectious blindness, sexually transmitted infections, and acute respiratory disease in humans. These pathogens undergo an essential biphasic developmental cycle differentiating between two functionally distinct forms known as the infectious elementary body (EB) and the replicative reticulate body (RB). Identifying the signals and regulatory mechanisms that enable Chlamydia to establish infection, differentiate between the two developmental forms, and survive within the host cell is critical to understanding chlamydial pathogenesis and developing future therapeutic strategies. In pathogenic bacteria, serine, threonine, and tyrosine (Ser/Thr/Tyr) protein kinases and phosphatases are critical for development, metabolism, and virulence. Chlamydia encode two validated protein kinases (pkn1 and pknd), a putative protein phosphatase (ctl0511; CppA), and appear capable of global phosphorylation that differs between the developmental forms. While these findings support a role for protein phosphorylation in chlamydial pathogenesis, a validated cognate protein phosphatase for Pkn1 and PknD mediating reversible phosphorylation was lacking. We hypothesized that CppA is the partner phosphatase for the chlamydial protein kinases, and in this study we validated and characterized CppA as a broad specificity protein phosphatase type 2C. Using in vivo and in vitro approaches we demonstrated that CppA acts on P-Ser/Thr/Tyr residues and can dephosphorylate multiple chlamydial protein substrates including PknD and the FHA 2 domain of CdsD, a component of the type 3 secretion apparatus. The importance of CppA for chlamydial growth and development was determined using a chemical “knock-out” approach and study of CppA missense mutations identified in slow growing C. trachomatis L2 chemical mutants. Treatment of C. trachomatis L2, C. trachomatis D, and C. muridarum with CppA inhibitors significantly reduced progeny levels and inclusion size in a time dependent manner with more significant growth inhibition in the first 12 hours post infection. Collectively, our findings support that CppA works in conjunction with PknD, and likely Pkn1, to mediate reversible phosphorylation of multiple protein substrates leading to changes in chlamydial physiology that appear to be key for early steps in development.

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