The secretion and translocation of T3S effectors into the cytosol of animal or plant cells initiates a biochemical cross talk between pathogen and host (6). allows Gram-negative bacteria to produce and translocate effector proteins into the cytoplasm of host cells. While the T3S system is usually conserved among distantly related pathogens, secreted effectors are pathogen specific (5). The secretion and translocation of T3S effectors into the cytosol of animal or herb cells initiates a biochemical cross talk between pathogen and host (6). Four T3S effectors have been identified in to invade tissue by breaking down physical barriers, damaging host cells, and conferring resistance to phagocytosis and host immune defenses (7, 8). Specifically, ExoS and ExoT are bifunctional effectors that have 76% homology, and both include Rho GTPase-activating (GAP) and ADP-ribosyltransferase (ADPr) activities (9). The GAP activities of ExoS and ExoT function similarly to inhibit internalization by inactivating Rho GTPases, Rho, Rac, and Cdc42, which regulate actin cytoskeleton structure Solcitinib (GSK2586184) (10C15). ExoS ADPr activity targets multiple specific substrates, including Ras family proteins, such as Ras, RalA, Solcitinib (GSK2586184) Rac1, and Rabs, to interrupt cell signaling (16C18). The substrate specificity of ExoT ADPr activity differs from that of ExoS ADPr activity and is limited to Crk-I (CT10 regulator of kinase I) and Crk-II adaptor proteins, which integrate protein tyrosine kinase signal transduction pathways (19C21). ExoU has Solcitinib (GSK2586184) been characterized as a necrotizing toxin with phospholipase activity (22) and has been found to block phagocyte-mediated clearance of contamination (23). ExoY has adenylate cyclase activity and does not appear to play a major role in pathogenesis (24, 25). Rab proteins, including Rab5, Rab7, Rab8, and Rab11, are known to be ADP-ribosylated by ExoS and (26). Rab proteins are a family of small GTP-binding proteins that regulate intracellular membrane trafficking of several pathogens, including serovar Typhimurium (27C29), spp. (30), and (31). Rab5 also functions in the phagocytosis of IgG opsonized particles (32). studies have demonstrated that ExoS ADP-ribosylation of Rab5 diminishes the conversation between Rab5 and early endosome antigen 1 (EEA1) and fluid-phase uptake in intact cells Rab5, and its guanine exchange factors (GEFs), which include Rabex-5, Rin1, and Rap6 (also known as GAPex5) (33C36), play a critical role in intracellular membrane trafficking (37), including phagocytosis of apoptotic cells (38). Although Rab5 was found to be present on phagosomes following phagocytosis of several bacterial pathogens and latex beads, the functional role for Rab5 in phagocytosis of is not fully comprehended. In this study, we demonstrate that Rab5 activity was regulated during early stages of phagocytosis in J774-Eclone macrophages. Expression of wild-type Rab5 (Rab5:WT) or a Rab5:Q79L, a GTP hydrolysis-defective mutant, increased invasion of heat-inactivated but was inactivated during invasion of live on phagocytosis. Inactivation of Rab5 by live was dependent on ExoS ADPr activity, and in J774-Eclone cells, ExoS ADPr activity caused a more severe inhibition of phagocytosis than ExoS GAP activity. Finally, we found that expression of Rin1, a Rab5 GEF, interfered with the ability of live to inactivate Rab5. The ability of live to regulate phagocytosis by altering Rab5 activation provides further insight into how is Solcitinib (GSK2586184) able to manipulate the host during infection. MATERIALS AND METHODS Solcitinib (GSK2586184) Materials. All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. Primary and secondary antibodies used in immunoblotting were purchased from Cell Signaling Technology Inc. (Danvers, MA). Culture supplies were purchased FAM162A from Invitrogen Life Technologies (Carlsbad, CA)..