Project Summary Bacterial infections remain a leading cause of morbidity and mortality worldwide and a critical health issue due to increasing antibiotic resistance and limited vaccines. Non-typhoidal Salmonella (NTS) serovars such as Salmonella enterica serovar Typhimurium (STm) are a leading cause of inflammatory diarrhea in otherwise healthy individuals, causing infections that are usually localized to the gut. Among immunocompromised individuals, children, and elderly, STm can cause a potentially lethal bacteremia that requires antibiotic treatment. Pivotal to innate host defense in preventing the spread of bacteria from the gastrointestinal tract to the bloodstream are antimicrobial peptides, complement system proteins, and phagocytic cells including macrophages and neutrophils. Pathogenic strains of STm can subvert the phagolysosome to survive intracellularly in macrophages, whereupon the autophagy system emerges as a critical step for killing intracellular STm. PgtE, a STm outer membrane protease, has been previously described to cleave over a dozen mammalian protein substrates in vitro, including complement protein C3, but these activities have only been observed with avirulent mutant strains possessing a defective outer membrane. In recent years, a newly emergent clade of invasive STm has been responsible for an epidemic of bacteremia in sub-Saharan Africa, with several studies suggesting an increased expression of PgtE as an important virulence mechanism although the mechanism remains unknown. Our preliminary studies suggest that PgtE plays an important role during infection in both the initial mucosal colonization and in the systemic phase with a virulent STm. Our central hypothesis is that after STm has been phagocytosed, STm utilizes PgtE to protect the disrupted outer membrane from antimicrobial peptides, complement, and autophagy killing, thereby promoting its survival in the host. In Aim 1, I will determine the role of PgtE in enabling STm to evade IL-22-mediated gut epithelial defenses after STm is phagocytosed in the lumen. In Aim 2, I will determine how PgtE subverts complement component C3 signaling and macrophage autophagic killing. This work will provide a framework on how pathogens utilize proteases to evade the immune system with potential novel therapeutic targets.