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A mechanistic understanding of how intracellular pathogens evade the intrinsic defenses of their host cells could open up intriguing therapeutic opportunities. Here, we applied a genome-wide genetic screening approach to investigate the nature of the defensive host cell death response suppressed by the membrane trafficking modulator CpoS, an effector protein secreted by the obligate intracellular bacterial pathogen Chlamydia trachomatis. Initially, this work revealed a CpoS-deficient mutant to exhibit a markedly increased dependence on host cellular synthesis of ceramides, the precursors of complex sphingolipids. Using novel microscopic reporters, we then established CpoS' role in defense evasion to occur by preserving the integrity of Chlamydia's parasitophorous vacuole (the inclusion) via ensuring an adequate sphingolipid supply. More specifically, we observed CpoS deficiency to destabilize inclusions, initially characterized by a release of individual bacteria into the host cell cytosol, then followed by inclusion rupture concomitant with host cell death. Exogenous addition of sphingosine stabilized CpoS-deficient inclusions, whereas disruption of host cellular ceramide synthesis destabilized wild-type inclusions. In combination, CpoS deficiency and impaired ceramide synthesis – presumably disrupting both Chlamydia's vesicular and non-vesicular sphingolipid supply routes – destabilized inclusions even earlier, resulting in infection clearance and host cell survival rather than host cell death. Overall, this study highlights how the vacuolar pathogen C. trachomatis maintains vacuole integrity by ensuring a steady sphingolipid supply, potentially offering inspiration and directions for future therapeutic strategies targeting parasitophorous vacuoles.

The obligate intracellular bacterium Chlamydia trachomatis inserts a family of inclusion membrane (Inc) proteins into the membrane of its vacuole (the inclusion). The Inc CpoS is a critical suppressor of host cellular immune surveillance, but the underlying mechanism remained elusive. By complementing a cpoS mutant with various natural orthologs and variants of CpoS, we linked distinct molecular interactions of CpoS to distinct functions. Unexpectedly, we found CpoS to be essential for the formation of inclusion membrane microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. While the function of CpoS in microdomains was uncoupled from its role in the suppression of host cellular defenses, we found the ability of CpoS to interact with Rab GTPases to be required not only for the manipulation of membrane trafficking, such as to mediate transport of ceramide-derived lipids (sphingolipids) to the inclusion, but also for the inhibition of Stimulator of interferon genes (STING)-dependent type I interferon responses. Indeed, depletion of Rab35 phenocopied the exacerbated interferon responses observed during infection with CpoS-deficient mutants. Overall, our findings highlight the role of Inc-Inc interactions in shaping the inclusion microenvironment and the modulation of membrane trafficking as a pathogenic immune evasion strategy.

IMPORTANCE: Chlamydia trachomatis is a prevalent bacterial pathogen that causes blinding ocular scarring and urogenital infections that can lead to infertility and pregnancy complications. Because Chlamydia can only grow within its host cell, boosting the intrinsic defenses of human cells may represent a novel strategy to fight pathogen replication and survival. Hence, CpoS, a Chlamydia protein known to block host cellular defenses, or processes regulated by CpoS, could provide new opportunities for therapeutic intervention. By revealing CpoS as a multifunctional virulence factor and by linking its ability to block host cellular immune signaling to the modulation of membrane trafficking, the present work may provide a foundation for such rationale targeting and advances our understanding of how intracellular bacteria can shape and protect their growth niche.