CRISPR-Cas9 mutagenesis of A549 cells for variants resistant to the ExoU toxin from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading cause of nosocomial infection and is responsible for severe acute and chronic infections such as pneumonia, urinary tract infections and lung infections in cystic fibrosis patients. The bacterium possesses a type 3 secretion system whose expression is linked with poor outcomes in both animal models and human patients. In particular, this syringe-like apparatus injects ExoU, the most toxic T3SS effector. However, the mechanism of ExoU in P. aeruginosa's virulence remains elusive compare to the other T3SS toxins. It was previously suggested that the toxin uses a host trafficking system to reach the plasma membrane and to induce cell necrosis. In this work, we demonstrated that once injected into the cell cytoplasm, ExoU binds to DNAJC5+ vesicles to rapidly reach the plasma membrane. Overall design: PA14 is a P. aeruginosa strain expressing ExoU, as well ExoT and ExoY T3SS effectors. The virulence of this strain has been associated with ExoU injection, as ExoU induces rapid cell death in different cell types (epithelial or endothelial cells and macrophages). A549 cells are adenocarcinomic human alveolar basal epithelial cells and were selected for this study, as they possess characteristics from pneumocytic cells, which are in contact with P. aeruginosa in lung infection. PA14 causes A549 cell death in an ExoU-dependent manner. A549 cells were transduced with a lentiviral library expressing guide-RNAs with the endonuclease Cas9 in the same plasmid. This one-component library contains 71,090 total gRNAs targeting 18,053 genes (4 gRNAs per gene). The gRNA library-expressing A549 cells (or A549-CRISPR cells) were infected for 90 min with PA14 at MOI of 10. Remaining cells (20-30%) were expanded with antibiotics to approximately 70% confluence and were subjected to a second and third round of infection. The gRNA sequences from surviving cells were identified via next-generation sequencing (NGS) and candidate genes ranked based on the number of unique gRNAs versus NGS reads according to MAGeCK method. DNAJC5 was the only gene to stand out with the most significant gRNAs compared to uninfected cells. DNAJC5 is an ubiquitous cytoplasmic protein, mostly located at the surface of late endosomes. It functions as a co-chaperone with Hsc70/Hsp70 proteins in protein homeostasis and is involved in an unconventional protein secretion pathway named MAPS (Misfoded-Associated Protein Secretion) allowing the transportation of misfolded proteins from the perinuclear region to the plasma membrane.