Graduation Date

Spring 5-4-2024

Document Type


Degree Name

Doctor of Philosophy (PhD)


Pathology & Microbiology

First Advisor

Elizabeth A. Rucks


Chlamydia trachomatis is the pathogen responsible for the majority of infectious blindness and sexually transmitted bacterial infections worldwide. As an obligate intracellular pathogen, it establishes a niche within a pathogen-specific vacuole, termed the chlamydial inclusion. C. trachomatis modifies the inclusion membrane by type III secretion and insertion of effectors known as inclusion membrane proteins, or Incs. These Inc proteins are critical for chlamydial pathogenesis via their interactions with host proteins and their capacity to stabilize the inclusion membrane. Previous studies from our lab demonstrated the localization of two eukaryotic proteins, Flightless 1 (FLI1) and its binding partner leucine-rich repeat interacting protein of FLI1 (LRRF1), to the inclusion membrane during chlamydial infection. Furthermore, the Inc protein Ct226 was shown to directly interact with LRRF1 by co-immunoprecipitation. These host proteins are involved in a variety of functions in eukaryotic cells, including cytoskeleton modulation, transcriptional regulation, and regulation of the innate immune response, but their mechanism of recruitment and their function at the inclusion membrane remains unknown. As such, we hypothesized that C. trachomatis uses Inc effectors to actively recruit FLI1 and LRRF1 to the inclusion membrane to alter their endogenous function in order to support chlamydial pathogenesis. Co-immunoprecipitation studies demonstrated that FLI1 co-immunoprecipitates with Ct226, but this interaction was dependent on the presence of LRRF1, and we further showed that both FLI1 and LRRF1 could localize independently of each other. To better understand relationship between Ct226 and FLI1/LRRF1 localization to the inclusion, we developed and characterized a series of CRISPR interference (CRISPRi) knockdown and complementation strains in C. trachomatis serovar L2 that target ct226 and co-transcribed candidate Incs, ct225 and ct224. Our results demonstrated that knockdown of ct226 and ct225, but not ct224, negatively impacted localization of both FLI1 and LRRF1, though only complementation of ct226 rescued their inclusion localization. We further demonstrated that ct225, which had been annotated as an inclusion membrane protein, in fact localizes to the bacterial membrane and is potentially involved in regulating Ct226 secretion to the inclusion membrane. Further, we explored two possible functions for FLI1 and LRRF1 at the inclusion membrane, relating to modulation of the actin cytoskeleton and their activity as regulators of host transcription. Our results indicated that actin polymerization is not required for FLI1 localization, though we could not definitively conclude if its presence contributed to actin polymerization at the inclusion membrane during infection. By examining transcription of their purported eukaryotic gene targets, we also demonstrated that their localization to the inclusion does not impact transcription levels. In all, our findings point to an alternative mechanism for FLI1 and LRRF1 during chlamydial infection. In summary, we further characterized the chlamydial components necessary for FLI1 and LRRF1 localization to the inclusion during infection and demonstrated that Ct226 is critical for their localization. We also showed that LRRF1 and FLI1 are recruited independently of each other, and our data suggest an alternative Inc may also be responsible for FLI1 localization, which has implications for their function at the inclusion membrane. Thus, we developed critical tools using CRISPRi in order to study Inc-host protein interactions and laid the groundwork for future studies into the mechanism of host proteins FLI1 and LRRF1 during chlamydial pathogenesis.


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Available for download on Monday, March 16, 2026