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Chlamydia trachomatis is the most common infectious cause of blindness and a prevalent bacterial agent of sexually transmitted infections, with an annual incidence exceeding 130 million cases. The current therapeutic approach to Chlamydia infections relies on broad-spectrum antibiotics. However, while generally effective, these antibiotics carry the risk of substantial collateral damage, for instance by promoting resistance in bystander pathogens and by adversely affecting commensal microbes. Hence, the development of a more sustainable, narrow-spectrum treatment would be advantageous. In this context, the bacterium’s highly specialized obligate intracellular lifestyle could offer a wealth of unique targets for intervention. This thesis specifically investigates the potential of harnessing the protective power of cell-autonomous immunity in our battle against Chlamydia.

It is envisaged that the therapeutic exploitation of cell-autonomous immunity will initially necessitate three pivotal steps. Firstly, it will require identifying protective host cellular defense programs and counteracting virulence factors, which could serve as potential molecular targets. Secondly, it will be crucial to determine the molecular mechanisms by which the pathogen’s virulence factors suppress the host cellular defenses. Thirdly, pharmacological means to target the identified virulence factors or host cellular defense programs will need to be identified. This thesis outlines three independent projects, executed concurrently, to advance our knowledge at these three steps.

The first project involved the implementation of an innovative molecular genetic screening approach, which was devised to reveal host cellular defense mechanisms that could effectively restrict the growth of C. trachomatis provided they were not actively suppressed by the pathogen. This investigation culminated in the discovery of a mutant C. trachomatis strain that lacks the ability to effectively evade xenophagy. Overall, this finding highlighted Chlamydia’s ability to evade this defense mechanism as a potential novel target for therapeutic intervention.

The second project encompassed the molecular characterization of CpoS, a C. trachomatis virulence factor previously identified to counteract cell-autonomous immunity by inhibiting induction of type-I interferon responses and premature host cell death. The analyses revealed that CpoS manipulates host cellular membrane trafficking and suppresses host cellular type-Iinterferon responses through its interactions with the host factor Rab35.

The third project involved a compound screening campaign that identified several novel selective anti-chlamydial compounds. Interestingly, one molecule exhibited reduced activity in xenophagy-deficient cells, implying a potential involvement of xenophagy in its mechanism of action.

In summary, this research pinpointed xenophagy as a potential defensive mechanism against C. trachomatis, offered in-depth understanding of the operational mode of the virulence factor CpoS, and discovered new selective therapeutic alternatives, which in part utilize xenophagyin their mechanism of action. Consequently, this thesis provides a comprehensive overview of the transition from fundamental research to the more application-oriented domain of drug discovery and may inspire the development of more sustainable therapeutic strategies for the clinical handling of Chlamydia infections.

Chlamydia trachomatis is the most prevalent bacterial cause of sexually transmitted infections, with ~128 million new cases reported annually. In addition, it is the agent of trachoma, making it also the leading infectious cause of blindness worldwide. Current treatment relies on broad-spectrum antibiotics, which are usually effective but can disrupt the microbiota and contribute to resistance development. These limitations underscore the urgent need for narrow-spectrum therapeutic strategies that exploit the pathogen’s unique obligate intracellular lifestyle and vulnerabilities.

This thesis investigated how C. trachomatis maintains its parasitophorous vacuole, the inclusion, and how host cells respond when inclusion integrity fails, focusing on the pathogen’s dependence on host-derived sphingolipids and the interplay with autophagy to identify mechanisms that could be harnessed for therapeutic targeting.

The first chapter details the identification of the inclusion membrane protein CpoS – a secreted C. trachomatis effector that inserts into the vacuole membrane – as a multifunctional virulence factor. Specifically, CpoS was found to organize inclusion membrane microdomains, modulate the host cytoskeleton, and interact with Rab GTPases, thereby manipulating membrane trafficking while also suppressing STING-dependent interferon responses and a defensive form of premature host cell death.

The second chapter presents findings from a genome-wide CRISPR screen and validations using novel microscopic inclusion damage reporters, collectively revealing that CpoS stabilizes inclusions by mediating acquisition of sphingolipids. Pharmacologic inhibition of sphingolipid synthesis further destabilized CpoS-deficient inclusions, resulting in infection clearance and host cell survival rather than death.

The third and final chapter describes how the application of inclusion damage reporters in electron microscopy and live cell imaging revealed that destabilization of CpoS-deficient inclusions initially involved small inclusion membrane breaks, followed by inclusion rupture and host cell death. Bacteria released from damaged inclusions were targeted by the xenophagic machinery. However, xenophagic degradation was incomplete due to the rapid progression toward cell death.

Collectively, this thesis established novel spatiotemporal imaging tools, elucidated the role of the inclusion in the evasion of host cell-autonomous immunity, and laid the groundwork for potential future therapeutic strategies focused on destabilizing the inclusion to combat C. trachomatis infection.