Graduation Date

Spring 5-6-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Scot P. Ouellette, PhD

Abstract

Chlamydia trachomatis, an obligate intracellular human pathogen, is the etiological agent of the most commonly reported bacterial sexually transmitted infection and the leading cause of preventable infectious blindness worldwide. In adapting to an intracellular niche, Chlamydia has evolved to eliminate superfluous genes from its chromosome. Despite its highly reduced genome, Chlamydia undergoes a complex developmental cycle in which the bacteria differentiate between two functionally and morphologically distinct forms: the infectious, non-replicative elementary body (EB) and the non-infectious, replicative reticulate body (RB). An EB initiates infection by binding to and inducing uptake into a host cell. Within the cell, the EB undergoes primary differentiation into an RB, which then serves as the progenitor for a population of RBs. At a currently unknown signal mid-developmental cycle, nascent RBs undergo secondary differentiation from RB to EB. Once this process is initiated, the cascade continues irreversibly until completion. However, the transition from RB to EB is not mediated by division events that re-distribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require protein turnover. As such, we hypothesize that ClpX, a member of the ATP-dependent Clp protease family, plays a critical role during chlamydial differentiation through targeted protein degradation. Our initial studies using basic genetic approaches demonstrated the importance of the Clp protease system to chlamydial development, specifically leading to our focus primarily on ClpX. We determined that, through overexpression of an RKH mutant ClpX isoform, tag-independent substrate recognition of substrates by ClpX likely serves as a negative regulator of secondary differentiation. We corroborated this by utilizing CRISPR interference coupled with complementation of tmRNA, the driving factor of trans-translation, in reciprocal experiments to demonstrate that the accumulation of SsrA-tagged substrates is crucial for the triggering of secondary differentiation. We then utilized ATPase-deficient ClpX isoforms to trap substrates, affinity purified these protein complexes, and identified co-precipitating proteins using mass spectrometry. Functional analyses of the mass spectrometry hits revealed a diverse network of pathways, suggesting extensive modulation of energy and cofactor biosynthesis by ClpX. Moreover, our studies highlighted the central role of ClpX during the triggering event of secondary differentiation. Taken together, our data support a model where regulated proteolysis serves as a driving force for developmental cycle progression through mediation of key biological events.

Comments

2023 Copyright, the authors

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