CHARACTERIZATION OF THE CHLAMYDIAL PARTNER SWITCHING MECHANISM USING IN VITRO, IN VIVO, AND IN SILICO APPROACHES

Author

Evan Landers, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2018

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Fisher, Derek

Abstract

Chlamydia trachomatis is a Gram-negative, obligate intracellular pathogen that is the causative agent of sexually transmitted infections and the ocular disease trachoma. Chlamydia trachomatis undergoes a biphasic developmental cycle differentiating between the infectious elementary body (EB) and the replicative reticulate body (RB). Under certain stress conditions, C. trachomatis can stall its developmental cycle and enter an aberrant state termed persistence. While in a persistent state, C. trachomatis is refractory toward antibiotics, can evade the host immune response, and becomes undetectable using standard clinical detection methods. Environmental and other pathogenic microbes are known to utilize partner switching mechanisms (PSM) to regulate sigma factors used to initiate a stress response. For this reason, this study focuses on the chlamydial PSM, its role in regulating the availability of the housekeeping sigma factor σ66, and its role in the developmental cycle and stress response of C. trachomatis. The chlamydial PSM is composed of five known proteins: the anti-sigma factor RsbW, two anti-anti-sigma factors RsbV1 and RsbV2, a regulatory phosphatase RsbU, and a second phosphatase-like protein CTL0852. In order to test the role of the PSM in the chlamydial stress response, a panel of C. trachomatis rsbV1 mutants were generated, persistence inducing iron starvation and tryptophan starvation cell culture conditions were optimized, and growth of the rsbV1 mutants under iron starvation conditions were assayed. No significant differences were seen between rsbV1 mutants under iron starvation nor recovery conditions as determined by progeny production and inclusion size analysis. Furthermore, this study generated PSM protein producing Escherichia coli strains for in vitro protein work and performed operon mapping of the PSM genes of C. trachomatis to help aid in future studies of the chlamydial PSM by facilitating the development of new chlamydial PSM mutants. This study gives phylogenetic support to the classification of ctl0852 as a chlamydial PSM gene by comparing relative mutations rates of PSM genes across chlamydial species.