The thrust of our proposed research is to examine the typhoid model of mouse infection using animals carrying the nramp resistant allele. In this experimental model of infection, we propose to examine well-characterized bacterial mutants, as well as focusing on the location of the bacteria during the acute phases of infection and during chronic carriage. Because persistent infection is asymptomatic, we rely upon a non-invasive, semi-quantitative photonic detection system to identify infected animals. We will employ bacterial strains carrying fluorescent reporter molecules to follow the location of the bacteria in the tissue, as well as using fluorescence activated sorting (FACS) of infected tissue to locate the host cell populations interacting with the invading bacteria. Using immunohistochemical methods, in-situ hybridization, confocal microscopy with antibody tagged reporter molecules, as well as biochemical assays of tissue, we will determine the precise nature of the host cells persistently infected by Salmonella. We have constructed a spotted DNA microarray of the entire S. typhimurium genome, as well as a 40000-element mouse DNA microarray. We plan to use these experimental tools to monitor both the bacterial response and the host response to infection with particular emphasis on the transition by both the microbe and the host from an acute infectious process to a systemic and eventually persistent infection. Finally, we will employ genetic screening methods based on signature tagged transposon analysis, as well as a new novel DNA microarray-based screen, to identify bacterial genes that are essential for persistent infection and bacterial transmission. ? ?
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