The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence

A variety of pathogenic bacteria use type III secretion pathways to translo cate virulence proteins into host eukaryotic cells. YopE is an important vi rulence factor that is translocated into mammalian cells via a plasmid-enco ded type III system in Yersinia spp, YopE action in mammalian cells promote s the disruption of actin filaments, cell rounding and blockage of phagocyt osis, It was reported recently that two proteins with sequence similarity t o YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases. YopE contain s an 'arginine finger' motif that is present in SptP, ExoS and other Rho GA Ps and is essential for catalysis by this class of proteins. We show here t hat a GST-YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rad, but not by Pas. Conversion of the esse ntial arginine in the arginine finger motif to alanine (R144A) eliminated t he in vitro GAP activity of GST-YopE. Infection assays carried out with a Y ersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell roundin g and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GA P function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that prod uces a constitutively active form of RhoA (RhoA-V14) prevented the disrupti on of actin filaments and cell rounding by YopE. Production of an activated form of Rad (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with Yo pE antiphagocytic activity. These results demonstrate that YopE functions a s a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.