Listeria monocytogenes is a model facultative intracellular pathogen which primarily infects pregnant women and immunocompromised individuals. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO is largely responsible for rupture of the host vacuole which results from phagocytosis. Perfringolysin O (PFO) is a related pore-forming protein which is involved in the pathogenesis of infections by an extracellular pathogen. When expressed by L. monocytogenes, PFO mediates escape from the phagocytic vacuole, but is toxic to the cell. Normally, LLO is continually expressed during infection, but in contrast to PFO, it is proteolytically degraded in the cytosol and presented on the cell surface in association with MHC class I molecules. It is hypothesized that pH optimum and cytosolic processing are essential LLO-encoded determinants which distinguish it from PFO. The focus of the current proposal is to define the precise structural and mechanistic features of LLO which differentiate it from other members of the family of thiol-activated cytolysins, facilitate its intravacuolar activity, and direct its fate in the cytosol. In Aim I, protein sequences responsible for LLO's acidic pH optimum and processing in the host cytosol will be identified. This will be accomplished by domain and sub-domain swapping between LLO and PFO, and modified charged-to-alanine scanning mutagenesis. The LLO/PFO chimeras will be purified from E. coli and characterized biochemically. Next, the chimeras will be introduced into L. monocytogenes and characterized in tissue culture models of infection., In Aim II, the pathway of LLO processing in the host cytosol will be fully evaluated. LLO will be identified by metabolic labeling of L. monocytogenes within infected host cells, followed by immunoprecipitation. Precursor/product relationships will be determined by pulse-chase experiments. Specific inhibitors will be used to evaluate the role of the proteosome and other proteases in degradation. The role of LLO phosphorylation will be evaluated biochemically and genetically. In Aim III, the precise nature of the L. monocytogenes phagosome will be characterized with regard to pH, time of perforation, and markers of the endosome/lysosome pathway of maturation. The role of pH optimum and the L. monocytogenes phospholipases will be evaluated by using mutant and chimeric strains. In Aim IV, the investigators will evaluate the feasibility of using E. coli K12 expressing LLO and a recombinant protein as a novel system to introduce foreign proteins into the mammalian cytosol for antigen presentation.