Host defenses that protect the neonate from infection are varied, complex, and interactive, requiring that individual mechanisms be evaluated in the presence of the contextual influences of the intact living organism. Using noninvasive monitoring, innate host defenses in living animals will be assessed in a murine model of systemic infection. Salmonella typhimurium infections begin in the gastrointestinal (GI) tract, and following penetration of the epithelial barrier, can lead to lethal systemic infections. Nramp1 (natural resistance associated macrophage protein) is critical for limiting infections by Salmonella spp., as well as other intracellular pathogens to the early stages of disease and preventing dissemination. Resistant and sensitive Nramp1 alleles, differing by a single amino acid substitution (G169D), have been identified in mice. Homozygous sensitive mice are more susceptible to systemic salmonellosis than their resistant counterparts. Using noninvasive imaging in live mice, we have demonstrated that Salmonella infections in resistant animals do not extend beyond the GI tract, while sensitive animals demonstrate a disease pattern consistent with systemic infection. Expression of the dominant resistant Nramp1 allele in monocytes/macrophages appears to be required for infection resistance, yet the precise requirement and/or mechanism of Nramp1 action remains enigmatic. Expression of Nramp1 is inducible by IFNgamma and the gene encodes a phosphoglycoprotein with features resembling an ion transporter. The Nramp1 protein localizes to phagosomes and the plasma membrane, appears to be involved in a pathway leading to macrophage activation and antigen presentation, and has been linked to nitric oxide production and apoptosis. To investigate the role of Nramp1 in resistance to infection and the effects of IFNgamma, we propose to assess levels of expression in monocytes obtained from transgenic mice, and cell lines in the presence and absence of bacterial pathogens. Then, the basal levels of expression at various tissue sites in living transgenic mice, at different ages, will be assessed and the location and tempo of activation following oral inoculation of Salmonella determined. This work will involve in vivo monitoring of existing bioluminescent strains of Salmonella in resistant and sensitive strains of mice, as well as engineering and monitoring host promoters fused to a spectrally distinct eukaryotic luciferase in transgenic mice. The different wavelengths of emission permit dual detection allowing the relationship between changes in host gene expression and infection to be evaluated. We will use a luciferase-GFP gene fusion as the reporter such that results from macroscopic detection in living animals can be supported by cell sorting and/or microscopic detection in postmortem tissues. Since homologues of Nramp1 have been found in humans, studying this mode of resistance to microbial infection is significant for understanding disease and minimizing human infections.
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