Serine-71 phosphorylation of Rac1/Cdc42 diminishes the pathogenic effect of Clostridium difficile toxin A

Clostridium difficile toxin A and B (TcdA/TcdB) are glucosyltransferases that glucosylate GTPases of the Rho family. The epidermal growth factor (EGF) positively modulates C. difficile toxin-induced disturbance of the intestinal barrier function by an unknown mechanism. We found that EGF-treated CaCo-2 monolayers were less susceptible to TcdA-catalysed glucosylation of Rac1 but not of RhoA, which correlated with phosphorylation of Rac1 at Ser-71. Phospho-Rac1/phospho-Cdc42 (Ser-71) still bound to the PAK-CRIB domain indicating an active state. A more detailed characterization of phospho-Rac1 was performed using the phosphomimetic mutant Rac1 S71E. Ectopic expression of Rac1 S71E induced a specific phenotype of cells showing an increase in filopodial structures that were also induced by EGF. Rac1 S71E (and Cdc42 S71E) but not Rac1 S71A was at least fivefold weaker substrate for TcdA-catalysed glucosylation compared with wild type Rac1. The protective effect was checked in transfection experiments where Rac1 S71E and, to a lesser extent, Cdc42 S71E reduced the TcdA-induced cytopathic effect. Thus, Ser-71 phosphorylation of Rac1 might be interesting for modulation of microbial pathogenesis where Rho GTPases, especially Rac1 and Cdc42, are involved. In addition, this is the first description of a specific functional outcome of Rac1 phosphorylation at Ser-71.