A CRISPR knockout screen revealed modulators of Chlamydia trachomatis parasitophorous vacuole stability

Chlamydia trachomatis is a prevalent bacterial cause of urogenital and ocular infections. The pathogen uses the effector CpoS to suppress a host defense response that aborts intracellular bacterial growth by inducing host cell death. We conducted a CRISPR knockout screen to identify host genes contributing to this response, thereby revealing modulators of C. trachomatis parasitophorous vacuole stability. In brief, we transduced HeLa cells, a human cervical epithelial cell line, with a genome-wide knockout library. More specifically, we used the Brunello sgRNA library (which targets 19,114 genes and comprises a total of 77,441 sgRNAs, including about four sgRNAs per gene and 1000 non-targeting control sgRNAs). An aliquot of the transduced cells was collected to determine the composition of the pre-selection cell population (= sample “Pre”). In the selection procedure, we infected transduced cells with C. trachomatis L2/434/Bu, either wildtype (CTL2) or a strain carrying an insertional disruption of cpoS (CTL2-cpoS::cat). Later, we collected cells resistant to infection-mediated killing, that is, cells resistant to late-stage lytic death in the case of CTL2 or premature death in the case of CTL2-cpoS::cat. Hence, we included four distinct conditions: uninfected cells and cells infected with CTL2-cpoS::cat collected at 30 hours post infection (samples “UI30h” and “KO30h”), and uninfected cells and cells infected with CTL2 collected at 60 hours post infection (samples “UI60h” and “WT60h”). The screen was performed in two independent replicates (R1+R2).