Pseudomonas aeruginosa ExoS ADP‐ribosyltransferase inhibits ERM phosphorylation

Summary Pseudomonas aeruginosa causes life‐threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96–219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234–453) encodes a 14‐3‐3‐dependent ADP‐ribosyltransferase domain which transfers ADP‐ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high‐affinity substrates for ADP‐ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP‐ribosyltransferase activity. MALDI‐MS and site‐directed mutagenesis studies determined that ExoS ADP‐ribosylated moesin at three C‐terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP‐ribosylated‐moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP‐ribosylation directly inhibited ERM phosphorylation. Expression of dominant active‐moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP‐ribosylation. These data explain how the expression of the ADP‐ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton.