This thesis presents work toward understanding how the obligate intracellular pathogen, Chlamydia trachomatis, modulates the host cell to establish a protected replicative niche. In order to evade host-cell innate immune surveillance, internalized Chlamydia develop within a membrane-bound compartment referred to as the inclusion. Given that C. trachomatis relies on host-cell derived nutrients and energy, this bacterial pathogen must avoid globally inhibiting host-cell function while building what is essentially a novel organelle. Through strategic deployment of effectors into the host cytosol and inclusion membrane, C. trachomatis actively remodels host-cell structures from within the inclusion. This enables the bacteria to obtain the required metabolites and regulate specific organelle functions. This work focuses on: understanding which host proteins and cellular structures are repositioned around the growing inclusion; identifying which bacterial effectors are responsible for these modifications; and elucidating the mechanisms by which C. trachomatis calibrates organelle function to divert specific resources to the replicating bacteria while maintaining host viability.

Until recently, the host targets of only a few Incs had been identified. We utilized high-throughput affinity purification-mass spectrometry to comprehensively define the Inc-human protein interactome, and discovered putative binding partners for 38/58 of the predicted C. trachomatis Incs. Using confocal immunofluorescence microscopy, we screened ~200 of the 335 identified high-confidence Inc-human protein-protein interactions for localization at the inclusion membrane, and we identified the recruitment of many host proteins involved in host processes consistent with Chlamydia’s intracellular life cycle. Thus, Chlamydia effectors recruit distinct subsets of host proteins to the inclusion, and mediate precise changes to the landscape of the host cell.

In the first project, we characterized an interaction between the host dynactin complex, and the C. trachomatis Inc CT192, hereafter named Dre1 for Dynactin Recruiting Effector 1 (Chapter 2). In eukaryotes, dynactin is a ubiquitous and multifunctional protein complex that modulates the activity of the microtubule motor, dynein, at many different cellular structures. Using a combination of confocal immunofluorescence microscopy and biochemistry, we show that dynactin is recruited to the inclusion by Dre1 and that the Dre1:dynactin interaction modulates the positioning of specific host organelles, including the centrosome, mitotic spindle, and Golgi Apparatus around the inclusion. Deletion of Dre1 resulted in decreased C. trachomatis fitness in cell-based assays and in a mouse model of infection. By targeting particular functions of the host dynactin complex, Dre1 facilitates re-arrangement of specific organelles around the growing inclusion. Thus, C. trachomatis employs a single effector to evoke large-scale changes in host cell organelle organization.

In the second project, I describe my pilot work using cross-linking mass spectrometry and cryo-electron microscopy to physically map the interaction between Dre1 and the host dynactin complex (Chapter 3). We report preliminary evidence that Dre1 binds along the Arp1 filament of the dynactin complex. Typically, the Arp1 filament mediates interactions with dynein and various adaptors that enhance processivity or specify cargo-binding of this tripartite complex. Given that Dre1 binds at this same position, we believe that further structural resolution will reveal the mechanism by which Dre1 is able to target particular functions or regulatory states of this omnipresent and versatile protein complex.

In the third project, I describe my contributions to identifying a novel inhibitor of the type III secretion system, and show that C. trachomatis’ ability to assemble secretion machinery is essential for virulence (Chapter 4). The type III secretion system is a highly conserved, needle-like apparatus that many diverse pathogens use to inject effector proteins into the host cytosol. First identified through its inhibition of the Yersinia pestis type III secretion system, 4EpDN is a compound that appears to have broad efficacy against evolutionarily distant injectisome type III secretion systems. This compound does not, however, target the Salmonella flagellar type III secretion system, indicating that 4EpDN specifically inhibits injectisome type III machinery. As an obligate intracellular pathogen, C. trachomatis is absolutely reliant on its injectisome type III secretion system to build the inclusion within the hostile environment of the host cytosol. We show that 4EpDN inhibited C. trachomatis progeny production and reinfection, but not initial inclusion formation. This suggests that 4EpDN prevents assembly of de novo type III secretion machinery over the course of the Chlamydial intracellular life cycle, but does not inhibit secretion by the assembled type III injectisomes that were pre-packaged in the bacteria prior to exposure to 4EpDN. We then utilized Chlamydia’s requirement for the type III secretion system to apply strong selective pressure to bacteria passaged in the presence of 4EpDN at MIC90 and isolate resistant mutants. We are currently sequencing mutants that escaped inhibition to determine the genes involved in resistance and to elucidate the mechanism by which 4EpDN targets injectisome type III secretion systems. As antibiotic-resistant bacteria are an emerging global health threat, new antimicrobials are urgently needed. Understanding how this compound targets the injectisome of multiple pathogens may help with the generation of novel therapeutics to treat recalcitrant infections.

Together, this thesis work illustrates the importance of C. trachomatis effectors in constructing and maintaining infection in host cells, as well as the importance of Chlamydia’s ability to specifically modulate host functions that support bacterial growth without globally altering host fitness. The interdisciplinary nature of this work, which spans high-resolution microscopy, cell biology, chemistry, biochemistry, genetics, microbiology, and structural analysis, has revealed exciting insights into how C. trachomatis successfully establishes its intracellular replicative niche.