The human pathogen Salmonella Typhi (STy) causes more than 20 million infections and 200,000 deaths each year. Our laboratory has developed a novel model of typhoid fever that uses humanized mice engrafted with human hematopoietic stem cells obtained from umbilical cord blood. This is the first small animal model to recapitulate the characteristic pathology and lethality of acute STy infection. We have also obtained evidence to suggest that STy persists in human macrophages by promoting an alternative pathway of activation and shown that humanized mice can be immunized with a live attenuated STy vaccine strain to engender protective adaptive immune responses. This application builds upon our preliminary observations to test the central hypothesis that Salmonella Typhi evades human innate immunity by promoting alternative macrophage activation and requires adaptive immunity for clearance. The specific aims of the application are to: 1. Determine the mechanisms that allow STy to persist in human macrophages. We have found that STy persists in cultured human macrophages whereas STm causes rapid cell death. This is an important clue to understanding differences in the pathogenesis of typhoidal and non-typhoidal Salmonella infections. Cell surface markers, signaling pathways, and cytokines/chemokines associated with macrophage polarization and apoptosis will be assayed in human macrophages to understand serovar-specific differences in Salmonella interaction with human macrophages. 2. Analyze STy genetic loci required for virulence in humanized mice. A high-complexity transposon mutant library has identified bacterial genes required for STy proliferation in humanized mice. The screen has identified both known and novel virulence determinants including genes involved in Vi capsular polysaccharide production and export, transcriptional regulation, central metabolism, O-antigen modification, and type IV pilus biosynthesis. In-depth analysis will determine the roles of a counter-silencing transcriptional regulator, a novel permease complex, iron uptake systems and type IV pili in typhoid pathogenesis. 3. Characterize protective adaptive immune responses to STy vaccines in humanized mice. We have shown that humanized mice develop protective responses following vaccination with live attenuated STy. Detailed immunological characterization will analyze adaptive immune responses to vaccination of humanized mice and determine the mechanism of vaccine-elicited protection. Relevance: These studies will advance our understanding of typhoid pathogenesis and establish a new research platform for the evaluation of candidate typhoid vaccines.