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

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was rec ently shown to possess GAP activity towards the Rho GTPases RhoA, Pac and C DC42 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 st imulated with: (i) bradykinin to induce actin microspikes followed by ruffl es as an assay for CDC42 activity followed by CDC42 stimulated Pac activity ; (ii) sphingosine-1-phosphate to form ruffles by direct Pac activation; or (iii) thrombin to generate actin stress fibres through Rho activation, In WA(pYLCR + E)-infected HUVEC microspike formation stimulated with bradykini n remained intact but the subsequent development of ruffles was abolished, furthermore, ruffle formation after stimulation with sphingosine-l-phosphat e or thrombin induced production of stress fibres was unaltered in the infe cted cells, These data suggest that YopE is able to inhibit Rac- but not Rh o- or CDC42-regulated actin structures and, more specifically that YopE is capable of blocking CDC42Hs dependent Pac activation but not direct Pac act ivation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Pac-mediated signalling pathways in primary target c ells of Yersinia.