2026-10-10T17:33:11Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33995986 2026-09-25T15:43:50Z category_4 category_8 category_12 category_13 category_14 category_16 category_46 category_64 category_132 portal_63 item_type_3 month_year_09_2026

ExoU-Mediated Host-Cell Cytotoxicity is Associated with Phosphorylation-Dependent Regulation of DDX3Y during Pseudomonas aeruginosa Infection Cheng Nong (13952865) Derong Dai (23891816) Yibo Zhu (4350361) Yunjiao Li (9130394) Jiajin Chenghuang (21382904) Mingming Gao (147782) Jianping Ying (24668230) Yuxiao Gui (25110787) Rui Bao (165955) Biochemistry Microbiology Cell Biology Genetics Molecular Biology Physiology Immunology Cancer Infectious Diseases potent phospholipase a2 derived a549 cells causes rapid host findings identify myh6 restored cytotoxicity relative relatively weak association tagged infection model pseudomonas aeruginosa infection including altered association associated host factors >- s142a infection exou catalytic activity deficient ddx3y mutants ddx3y rip analyses reduced infection mediated host identified myh6 induced cytotoxicity cell cytotoxicity altered exou vitro pull rna sequencing results extend positive p nfkbiz </ multisite regulation il6st </ endogenously flag dependent regulation current understanding combined s101a cellular response cell injury bound transcriptome associated rnas 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 <i>exoU</i>-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 <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. 2026-09-25T00:00:00Z Dataset Dataset 10.1021/acsinfecdis.6c00279.s007 https://figshare.com/articles/dataset/ExoU-Mediated_Host-Cell_Cytotoxicity_is_Associated_with_Phosphorylation-Dependent_Regulation_of_DDX3Y_during_Pseudomonas_aeruginosa_Infection/33995986 CC BY-NC 4.0