YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells

Yersinia spp. inject effector proteins ( Y ersinia o uter p roteins, Yop s ) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro . To investigate the intracellular, ' in vivo ' targets of YopE we generated a Yersinia enterocolitica strain WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia .