Graduation Date

Spring 5-10-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Elizabeth A. Rucks

Abstract

Chlamydia trachomatis is a Gram-negative, obligate intracellular bacterial pathogen of humans. It is the most common bacterial sexually transmitted infection. As an obligate intracellular pathogen, it grows within a eukaryotic host cell. Within the host cell, it resides within a membrane-derived vacuole known as the inclusion. The inclusion integrates itself within the host cell membrane transport system, selectively localizing eukaryotic proteins and lipids to the IM. The inclusion membrane (IM) functions as the interface between the bacteria and host cell. The bacteria actively modify the IM by inserting type III secretion effectors into the IM, known as Incs. Incs interact with various host cell proteins. The cytokine, IFN-γ, is known to induce tryptophan catabolism, thus depleting host cell tryptophan. Since C. trachomatis is a tryptophan auxotroph, the bacteria enter a persistence phenotype, in which they are viable but non-replicative. We hypothesized that C. trachomatis would maintain its usual recruitment of eukaryotic proteins and lipids to the IM during persistence. For the most part, the bacteria were still capable of recruiting host lipids and proteins to the IM during persistence. Inc protein expression during persistence depended on multiple factors, such as Inc tryptophan content, ability to be secreted, and stability on the IM. Separately, prior studies had demonstrated that a eukaryotic SNARE protein, VAMP3, transiently interacted with multiple Incs. We hypothesized that proper Inc protein expression would be necessary for localization of VAMP3 to the IM. To test this, we developed and characterized novel inducible CRISPRi strains to knock down transcription of the first Incs that interact with VAMP3, IncF and IncG. Using these strains, we showed that VAMP3 recruitment to the IM is a complex process, in some cases supporting the idea that these Incs are important for VAMP3 localization to the IM and in other cases suggesting that they are dispensable. Finally, we showed that IncF and IncG interact with one another on the IM, contextualizing Inc-Inc interactions to help understand the mechanisms behind VAMP3 recruitment to the IM.

Comments

2025 Copyright, the authors

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