Graduation Date

Summer 8-9-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Scot P. Ouellette

Abstract

Chlamydia trachomatis (Ctr) is the leading cause of bacterial sexually transmitted infections. Ctr, a Gram-negative, obligate intracellular bacterium, develops within a membrane-bound vacuole termed an inclusion. This protective, nutrient rich, intracellular environment has allowed Ctr to undergo extensive reductive evolution – resulting in a streamlined genome of approximately 1 Mbp and the loss or fragmentation of several biosynthetic pathways, including the ability to synthesize tryptophan (Trp) de novo. During infection, the host immune system releases the cytokine IFNγ. Upon binding to a host cell, IFNγ stimulates the expression of indoleamine-2,3-dioxygenase, an enzyme capable of catabolizing cytosolic Trp, thereby starving Ctr of this essential amino acid. In response, Ctr enters an abnormal developmental state termed persistence. Ctr remains viable, but halts replication and cannot complete development to the point of acquiring infectivity. Once IFNγ levels dissipate and Trp levels return to normal, Ctr can convert back to its competent developmental state and continue with the infection cycle. The molecular mechanisms governing the entry into, maintenance of, and exit from persistence are widely unknown. There are, however, several interesting observations that have been made – such as the disjointing of transcription and translation, a global increase in transcription, early termination of transcription of genes encoding Trp, and the apparent disproportionate upregulation of those same genes encoding Trp. To further investigate the molecular underpinnings of chlamydial persistence, we first developed an experimental model capable of inducing a comparable persistent state in an IFNγ free environment. Given the multitude of downstream effects caused by IFNγ exposure, an IFNγ free persistence model served to minimize confounding variables. Our next objective was to utilize our models to further investigate the transcriptional phenomena on a genome wide scale. RNA sequencing was performed on each of our persistence models to verify disproportionate transcription of genes encoding Trp as well as early termination of those same genes. One hypothesis for why genes destined for termination would be upregulated was that translational defects resulted in dysregulated sigma factors. To investigate this further, we characterized the indirect regulons of both minor sigma factors in Ctr.

Comments

2023 Copyright, the authors

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