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A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells. YopE is an important virulence factor that is translocated into mammalian cells via a plasmid-encoded type III system in Yersinia spp. YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis. It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases. YopE contains an ‘arginine finger’ motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins. We show here that a GST–YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras. Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST–YopE. Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GAP function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA-V14) prevented the disruption of actin filaments and cell rounding by YopE. Production of an activated form of Rac1 (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with YopE antiphagocytic activity. These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.