Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7

Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes. Nramp1 encodes an integral membrane prote in abundantly expressed in the endosomal-lysosomal compartment of macrophag es and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagoso me to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) allele and t hus are permissive to replication of specific intracellular parasites). Rec ombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia e nterocolitica containing phagosomes. Evaluation of the antibacterial activi ty of RAW264.7 transfectants showed that expression of the recombinant Nram p1 protein abrogated intracellular replication of S. typhimurium. Studies w ith a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expressio n was specific, since (i) it was not seen in RAW264.7 transfectants overexp ressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells , Nramp1, and Nramp2 transfectants could all efficiently kill a temperature -sensitive, replication-defective mutant of S. typhimurium. Finally, increa sed antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele, Together, these results i ndicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell l ine can be used as a direct assay to study both Nramp1 function and mechani sm of action as well as to identify structure-function relationships in thi s protein.