Interactions of the effector ExoU from Pseudomonas aeruginosa with short-chain phosphatidylinositides provide insights into ExoU targeting to host membranes

Pseudomonas aeruginosa is an opportunistic multidrug-resistant pathogen and a common cause of infection in cystic fibrosis and ventilator-associated pneumonia, and in burn and wound patients. P. aeruginosa uses its Type III secretion system (T3SS) to secrete various effector proteins directly into mammalian host cells. ExoU is a potent T3SS effector that after secretion localizes to the inner cytoplasmic membrane of eukaryotic cells where it exerts its phospholipase A2 activity upon interacting with ubiquitin and/or ubiquitinated proteins. In this study, we used site-directed spin labeling (SDSL) EPR spectroscopy to examine the interaction of ExoU with soluble analogs of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P-2). We found that dioctanoyl (diC8) PI(4,5)P-2 binds to and induces conformational changes in a C-terminal four-helix bundle (4HB) domain of ExoU previously implicated in membrane binding. Other soluble phosphoinositides also interacted with the 4HB, but less effectively. Molecular modeling and ligand docking studies indicated the potential for numerous hydrogen-bond interactions within and between interhelical loops of the 4HB and suggested several potential interaction sites for PI(4,5)P-2. Site-directed mutagenesis experiments confirmed that the side chains of Gln-623 and Arg-661 play important roles in mediating PI(4,5)P-2-induced conformational changes in ExoU. These results support a mechanism in which direct interactions with phosphatidylinositol-containing lipids play an essential role in targeting ExoU to host membrane bilayers. Molecules or peptides that block this interaction may prove useful in preventing the cytotoxic effects of ExoU to mitigate the virulence of P. aeruginosa strains that express this potent phospholipase toxin.