The ability to invade non-phagocytic host cells is essential for Salmonella virulence. SipA, SipC, SopB, SopE, and SopE2 are five type III effectors that are known to promote Salmonella invasion by modulating host actin dynamics both directly and indirectly. SipA and SipC regulate actin dynamics directly by mimicking functions of host actin-binding proteins. Indirectly, SopB, SopE, and SopE2 subvert host cell signal transduction pathways to induce actin cytoskeletal rearrangements and membrane ruffling. The concerted actions of these bacterial effector proteins eventually lead to the efficient uptake of the bacterium. SipA, SipC, SopB are multifunctional effectors also involved in effector translocation and in inducing host inflammatory responses. Despite improved understanding of these effectors and their individual roles in Salmonella-induced actin cytoskeleton rearrangements, little has been done to separate their multifunctional activities and to examine how these activities functionally interact to promote Salmonella invasion. In this study, we propose to investigate the individual contributions of their actin-modulating activities and how they are functionally coordinated during bacterial invasion. Our preliminary data showed that SipC exerts its actin-nucleation activity through transient dimer formation and that SipA and SipC may functionally cooperate to promote bacterial entry. We also demonstrated SopB-dependent VAMP8 recruitment to Salmonella-induced membrane ruffles and that VAMP8 is required for efficient bacterial invasion. We further determined that SipA and SipC may mediate SopB- and SopE-independent Salmonella entry. We hypothesize that SipA, SipC, SopB, and SopE function both cooperatively and independently to regulate host actin dynamics to promote Salmonella entry. To test this hypothesis, we will: (i) characterize the contributions of SipA, SipC actin-modulating activities in Salmonella invasion;(ii) define the molecular mechanism by which SopB promotes actin cytoskeleton rearrangements and Salmonella invasion;(iii) study how SipA, SipC, SopB, and SopE coordinate their actin-modulating activities to promote Salmonella invasion. Results from this study will provide a molecular basis for how SipA, SipC and SopB exploit the host actin cytoskeleton both independently and cooperatively to promote Salmonella entry. Project Narrative: Despite the understanding gained by our long-standing interest in studying Salmonella, salmonellosis continues to pose worldwide medical concerns and remains the number one cause of food-borne diseases even in developed countries. The identification of host cellular targets of Salmonella virulence proteins will aid clinical therapeutic drug designs and treatment of multidrug-resistant bacteria. Results from this study will not only broaden our understanding of Salmonella pathogenesis but will also provide vital insights into basic host cell biology.