ExoU-Mediated Host-Cell Cytotoxicity is Associated with Phosphorylation-Dependent Regulation of DDX3Y during Pseudomonas aeruginosa Infection

Abstract The Pseudomonas aeruginosa type III secretion system effector ExoU is a potent phospholipase A2 that causes rapid host-cell injury, but how it engages intracellular host networks during infection remains incompletely understood. Here, using an endogenously Flag-tagged infection model in male-derived A549 cells, we identified MYH6 and DDX3Y as ExoU-associated host factors. Knockdown of either protein reduced PA14-induced cytotoxicity, indicating that both contribute to the cellular response to ExoU-positive P. aeruginosa. An in vitro pull-down assay using purified proteins further supported a direct but relatively weak association between ExoU-S142A and DDX3Y. Quantitative phosphoproteomic analysis revealed extensive host phosphorylation changes associated with ExoU catalytic activity and identified multiple phosphorylation sites on DDX3Y. Phosphorylation at S506 was detected following PA14 infection but not following exoU-S142A infection. Phospho-deficient DDX3Y mutants at S101, S141, and S506 showed increased association with ExoU, whereas the combined S101A/S141A/S506A mutant further enhanced this association and reduced infection-induced cytotoxicity. Conversely, the phosphomimetic S506E mutant showed reduced association with ExoU and restored cytotoxicity relative to S506A. RNA sequencing and DDX3Y RIP analyses further showed that ExoU catalytic activity was associated with a broader inflammatory transcriptional response and with changes in the DDX3Y-bound transcriptome, including altered association with IL6ST and NFKBIZ transcripts. Together, these findings identify MYH6 and DDX3Y as distinct ExoU-associated host factors and support a model in which ExoU catalytic activity is linked to multisite regulation of DDX3Y, altered ExoU-DDX3Y association, and changes in DDX3Y-associated RNAs. These results extend the current understanding of ExoU-mediated cytotoxicity beyond direct membrane damage.