We have shown that the S. Typhimurium type III secretion system (T3SS) effector SipA is necessary and sufficient for the promotion of active states of intestinal inflammation, a hallmark pathology of salmonellosis (1). SipA is a bi-functional molecule that is not only responsible for the induction of PMN migration across the intestinal epithelium but also plays a role in promoting actin polymerization, a process that facilitates bacterial entry into epithelial cells. We determined that SipA harbors distinct functioal motifs that account for its induction of PMN transcellular signals and the binding to actin (2). In examining the bi-functional properties of SipA, we discovered that SipA contains a caspase-3 (CASP3) recognition and cleavage motif (DEVD) at amino acid position 431, a site precisely located between the two functional domains, termed SipAa (inflammatory domain) and SipAb (actin binding domain). The caspase-3 cleavage motif is physiologically significant, as a single amino acid substitution to a sequence not recognized by CASP3 profoundly attenuates the virulence of this pathogen in both in vitro and in vivo models of salmonellosis. Further analysis of the S. Typhimurium T3SS revealed the presence of CASP3 cleavage motifs in other type three secreted effectors (T3SE) with known bi-functional properties (i.e., SopA, SifA), indicating this phenomenon is not limited to SipA. Based on these observations we speculate that certain effector proteins of the S. Typhimurium T3SS exist in a pro-form, requiring processing by CASP3 to become functional. Thus, the objective of this proposal is to test the novel hypothesis that CASP3 cleavage of T3SS secreted effectors represents a common mechanism by which effector functions are regulated in host cells. We envisage activation of CASP3 cleaves Salmonella effector proteins harboring CASP3 recognition motifs into distinct functional subunits. To test this hypothesis, the aims of this proposal are centered on the mechanism underlying CASP3 cleavage of T3SEs with a particular focus on the temporal, spacial, and biological significance of this novel biological phenomenon. Specific Aim 1 is designed to determine the structure/function relationship of the Salmonella-effector CASP3 motifs. Under this aim, extensive biochemical analysis (using both in vitro and in vivo assays) will be performed to evaluate the extent to which the activity of Salmonella type III secreted effector functional domains requires CASP3 cleavage. In Specific Aim 2, we will begin to evaluate CASP3 activation and its role during infection by S. Typhimurium. In particular, we will employ state-of-the-art imaging techniques combined with sophisticated biochemical and bioinformatics approaches designed to the link temporal induction of CASP3 activity to the cleavage of specific S. Typhimurium effectors. Finally, in Specific Aim 3 we will define the mechanism(s) of activation of CASP3 during infection by S. Typhimurium. Our prior studies have shown that early after infection, the S. Typhimurium effector, SipA, is necessary and sufficient to promote activation of CASP3 but without inducing apoptosis or necrosis. Using a multi- disciplinary approach, involving cell biology, biochemistry and animal modeling, we will explore the molecular mechanism by which SipA triggers the activation of CASP3. Understanding the role that CASP3 plays in controlling T3SS effector function will greatly advance the understanding of Salmonella pathogenesis, and reveal novel insight on co-evolutionary relationships. On a broader spectrum, understanding of this new biological phenomenon will allow us to dissect specific aspects of host-bacterial relationships that have yet to be documented. Speculatively, the ability of the organism to hijack the host CASP3 machinery in order to activate secreted effectors is likely to be a general paradigm employed by enteric pathogens that harbor a T3SS to cause disease.