Tuberculosis is a major source of concern in North America due to the increased prevalence and devastating effect of this infection in AIDS patients, and the emergence of highly virulent and multidrug resistant strains of M. tuberculosis. The host mechanisms of defense against this infection, and the bacterial mechanisms underlying long-term survival within the host phagocytes remain poorly understood. Elucidating and understanding the molecular basis of genetic differences in natural susceptibility to mycobacterial infections observed in humans and in experimental animals, should provide new insight into the mechanisms of host defense against these infections. In the mouse, natural resistance to infection with mycobacteria is controlled by the Bcg/Ity/Lsh gene. We have cloned a candidate gene for Bcg designated nramp (natural resistance associated macrophage protein) which codes for a novel macrophage specific membrane transport protein. The two major goals if this proposal are the formal demonstration that nramp and Bcg are the same gene, and the comprehensive genetic and biochemical analysis of the Nramp protein, with the aim of understanding its biological role in the macrophage. The creation of a mutant mouse strain carrying a null allele at the nramp locus generated by homologous recombination in embryonal stem cells will be used to determine 1) if nramp and Bcg are the same gene, and 2) to further analyze in vivo the role of nramp in macrophage function. As an alternative approach, we will use transgenic animals to introduce a cloned copy of the Bcg' allele of nramp (129/sv, dominant) onto a Bcgs mouse background (C57BL/6J, recessive), and determine if it can reverse susceptibility to infection in C57BL/6J mice. A structural and functional characterization of the nramp gene is proposed, with special emphasis on the identification of cis-acting sequences and trans- acting factors responsible for constitutive or inducible macrophage specific expression of this gene. We will also determine if novel mutations in nramp and associated with disease susceptibility can be identified in additional inbred mouse strains, but also in the human NRAMP homolog in individuals from familial pedigrees segregating for susceptibility to tuberculosis. A biochemical analysis of the Nramp protein, including the production of specific antibodies, and the identification of its cellular and subcellular localization, putative post-translational modifications, membrane associated transport functions, in particular with respect to reactive nitrogen intermediates, are also proposed. Together, these studies should help understand the role of nramp in macrophage mediated resistance to mycobacterial infections, perhaps providing a new target for therapeutic intervention in the modulation of host defenses against tuberculosis.
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