ExoU-Mediated Host-Cell Cytotoxicity is Associated with Phosphorylation-Dependent Regulation of DDX3Y during Pseudomonas aeruginosa 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 <i>IL6ST</i> and <i>NFKBIZ</i> 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.