A genome-wide genetic screen identified targets for destabilizing the parasitophorous vacuole of Chla­mydia trachomatis. In this study, we have employed the power of the just recently established molecular genetic toolbox for Chlamydia to develop the first tool able to specifically detect early forms of membrane damage in the parasitophorous vacuole of Chlamydia (the inclusion) and to localize individual Chlamydia entering the host cell cytosol. Critical­ly, this advance enabled us to conduct a thorough mechanistic follow-up of results from a  genome-wide CRISPR knockout screen, which un­covered that the pathogen employs the secreted ef­fector CpoS, a modulator of membrane trafficking, to stabilize its vacuole by ensuring ade­quate sphingolipid supply. Notably, we observed that a simultaneous blockade of two lipid transport routes to the inclusion destabilized the vacuole early, leading to infection clear­ance without damaging the host cell. Thereby, this work can provide a foundation for a future therapeutic target­ing of the Chlamydia inclusion. The project was primarily driven by Mohammed Rizwan Babu Sait and Lana Jachmann, two members of the Sixt lab, yet was a team effort with key contributions from several former lab members and project students, as well as our collaborators from the Karolinska Institut, the Freie Universität Berlin, and the microscopy facilities at Umeå University.