Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool.

Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7-TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7-TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nramp2 isoform II can also transport Co(2+) and Cd(2+) but not Mg(2+) into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein.

The Nramp1 protein and its role in resistance to infection and macrophage function.

Susceptibility to infectious diseases is under genetic control in humans. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. The Nramp1 gene was isolated by positional cloning the host resistance locus Bcg/Ity/Lsh, and mutations at this locus impair the resistance of mice to infections with intracellular parasites, such as Salmonella, Leishmania, and Mycobacterium. Allelic variants at the human Nramp1 homologue have recently been found to be associated with susceptibility to tuberculosis and leprosy in humans. The Nramp1 protein is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication. Although the biochemical mechanism of action of Nramp1 at that site remains unknown, Nramp homologues have been identified in many other animal species and actually define a protein family conserved from bacteria to humans. Some of these homologues have been shown to be divalent cation transporters. Recently, a second member of the mammalian Nramp family, Nramp2, was discovered and shown to be mutated in animal models of iron deficiency. The Nramp2 protein was subsequently shown to be the major transferrin-independent iron uptake system of the intestine. Together, these results suggest that Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space.

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 protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome 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 thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing 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, increased 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 indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

Macrophage NRAMP1 and its role in resistance to microbial infections.

The identification and characterization of genetic factors influencing natural susceptibility to infectious diseases in humans and in model organisms, such as the laboratory mouse, can provide new insight into the basic mechanisms of host defense against infections. In the mouse, resistance or susceptibility to infection with intracellular pathogens such as Salmonella, Mycobacterium and Leishmnania is controlled by the Natural resistance associated macrophage protein (Nramp1) gene on chromosome 1, which influences the rate of intracellular replication of these parasites in macrophages. Nramp1 codes for an integral membrane protein, which is expressed exclusively in macrophage/monocytes and polymorphonuclear leukocytes. The protein is localized to the endosomal/lysosomal compartment of the macrophage and is rapidly recruited to the membrane of the particle-containing phagosome upon phagocytosis. Nramp defines a novel family of functionally related membrane proteins including Nramp2, which was recently shown to be the major transferrin-independent uptake system of the intestine in mammals. This observation supports the hypothesis that the phagocyte-specific Nramp1 protein may regulate the intraphagosomal replication of antigenically unrelated bacteria by controlling divalent cation concentrations at that site. Recent genetic studies have found that allelic variants at the human NRAMP1 locus are associated with susceptibility to leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis) and possibly with the onset of rheumatoid arthritis.

Cell-specific and inducible Nramp1 gene expression in mouse macrophages in vitro and in vivo.

Mutations in the Nramp1 gene abolish natural resistance to infections with many unrelated intracellular parasites in vivo. Global cDNA amplification was used to analyse Nramp1 mRNA expression in bone marrow-derived cell colonies corresponding to either undifferentiated progenitors or to mature lymphoid, erythroid, and myeloid lineages. Nramp1 mRNA was detected in mature myeloid colonies expressing molecular markers for either the monocyte/macrophage or granulocytic lineages. Having established constitutive expression of Nramp1 in phagocytic cells, the parameters of inducible Nramp1 expression by cytokines and lipopolysaccharide (LPS) were studied in the mouse macrophage cell line RAW 264.7. LPS caused up-regulation of Nramp1 expression in a time- and dose-dependent fashion. This induction required de novo protein synthesis and was abrogated by treatment with cycloheximide. Treatment with interferon-gamma (IFN-gamma) also caused a modest but reproducible twofold induction of Nramp1 mRNA expression. In addition, maximum Nramp1 mRNA induction in RAW 264.7 cells was observed after pretreatment with IFN-gamma followed by LPS exposure. In vivo, Nramp1 mRNA expression could be up-regulated in macrophage populations by intraperitoneal injection of either LPS or thioglycollate. Together these results indicate that Nramp1 is expressed in professional phagocytes of the myeloid lineage and can be further up-regulated during macrophage activation in response to infectious, inflammatory, or cytokine stimuli. Finally, the patterns of constitutive and inducible expression of Nramp1 have been compared to those of the inducible nitric oxide synthase (iNOS) gene in the same cells.

The Bcg/Ity/Lsh locus: genetic transfer of resistance to infections in C57BL/6J mice transgenic for the Nramp1 Gly169 allele.

The murine Bcg/Ity/Lsh locus determines the susceptibilities of inbred strains to infection with unrelated intracellular parasites, such as Mycobacterium bovis, Salmonella typhimurium, and Leishmania donovani. A candidate for Bcg/Ity/Lsh, designated Nramp1, has been recently identified and shown to encode a novel integral membrane protein that is expressed exclusively in professional phagocytes but whose function remains unknown. In inbred strains, the susceptibility to infection is associated with a single glycine-to-aspartic acid substitution at position 169 (G169D) in the predicted TM4 of the protein. To confirm the candidacy of Nramp1 as Bcg/Ity/Lsh and to determine the importance of the G169D mutation on Nramp1 function, we constructed transgenic mice in which the G169 allele of Nramp1 was transferred onto the background of a homozygous D169 allele. These transgenic mice were analyzed for their sensitivity to infections under the control of Bcg/Ity/Lsh. The transgene constructed for these studies contained the entire Nramp1G169 gene together with approximately 5 kb of sequences upstream of the transcription initiation site of this gene. We observed that these sequences were sufficient to direct Nramp1G169 expression in transgenic macrophages, resulting in the appearance of a mature protein of 90 to 100 kDa over a background of Nramp1G169 characterized by the complete absence of the mature Nramp1 polypeptide. The appearance of the Nramp1G169-encoded protein in transgenic macrophages was concomitant with the emergence of resistance to infection by M. bovis BCG, as measured by the extent of bacteria] replication in the spleen, and by S. typhimurium, as measured by survival after an intravenous challenge. The gain of function detected in transgenic Nramp1G169 animals establishes unambiguously that Nramp1 and Bcg/Ity/Lsh are allelic.

The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.

In mice, natural resistance or susceptibility to infection with intracellular parasites is determined by a locus or group of loci on chromosome 1, designated Bcg, Lsh, and Ity, which controls early microbial replication in reticuloendothelial organs. We have identified by positional cloning a candidate gene for Bcg, Nramp1, which codes for a novel macrophage-specific membrane transport protein. We have created a mouse mutant bearing a null allele at Nramp1, and we have analyzed the effect of such a mutation on natural resistance to infection. Targeted disruption of Nramp1 has pleiotropic effects on natural resistance to infection with intracellular parasites, as it eliminated resistance to Mycobacterium bovis, Leishmania donovani, and lethal Salmonella typhimurium infection, establishing that Nramp1, Bcg, Lsh, and Ity are the same locus. Comparing the profiles of parasite replication in control and Nramp1-/- mice indicated that the Nramp1Asp169 allele of BcgS inbred strains is a null allele, pointing to a critical role of this residue in the mechanism of action of the protein. Despite their inability to control parasite growth in the early nonimmune phase of the infection, Nramp1-/- mutants can overcome the infection in the late immune phase, suggesting that Nramp1 plays a key role only in the early part of the macrophage-parasite interaction and may function by a cytocidal or cytostatic mechanism distinct from those expressed by activated macrophages.

Genomic structure, promoter sequence, and induction of expression of the mouse Nramp1 gene in macrophages.

A candidate gene for the mouse chromosome 1 host resistance locus Bcg/Ity/Lsh was recently cloned and designated Nramp (natural resistance-associated macrophage protein). Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. Primer extension and cDNA cloning were used to isolate the complete 5' end of the Nramp1 mRNA. Analysis of genomic cosmid and bacteriophage clones overlapping the complete Nramp1 gene revealed that the gene was composed of 15 exons and spanned 11.5 kb of genomic DNA. Positioning of introns on the coding portion of the mRNA revealed a modular relationship between coding exons and predicted structural domains of the protein, with 8 of the 12 transmembrane (TM) domains encoded by individual exons. Northern blotting analysis indicated that Nramp1 expression was restricted to J774A.1 and RAW 264.7 macrophage lines and was dramatically increased by treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Primer extension and S1 nuclease mapping experiments were used to locate the transcription initiation site of Nramp1 and revealed the presence of one major and several minor initiation sites. Nucleotide sequencing of the corresponding region failed to detect classical TATA and CAAT elements, but identified two putative initiator sequences located near the major initiation site. Consensus sequences for binding of the macrophage and B-cell-specific transcription factor PU.1, as well as several LPS (NF-IL6) and IFN-gamma response elements, were also identified.

Human natural resistance-associated macrophage protein: cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression.

Natural resistance to infection with unrelated intracellular parasites such as Mycobacteria, Salmonella, and Leishmania is controlled in the mouse by a single gene on chromosome 1, designated Bcg, Ity, or Lsh. A candidate gene for Bcg, designated natural resistance-associated macrophage protein (Nramp), has been isolated and shown to encode a novel macrophage-specific membrane protein, which is altered in susceptible animals. We have cloned and characterized cDNA clones corresponding to the human NRAMP gene. Nucleotide and predicted amino acid sequence analyses indicate that the human NRAMP polypeptide encodes a 550-amino acid residue membrane protein with 10-12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif. Identification of genomic clones corresponding to human NRAMP indicates that the gene maps to chromosome 2q35 within a group of syntenic loci conserved with proximal mouse 1. The gene is composed of at least 15 exons, with several exons encoding discrete predicted structural domains of the protein. These studies have also identified an alternatively spliced exon encoded by an Alu element present within intron 4. Although this novel exon was found expressed in vivo, it would introduce a termination codon in the downstream exon V, resulting in a severely truncated protein. Northern blot analyses indicate that NRAMP mRNA expression is tightly controlled in a tissue-specific fashion, with the highest sites of expression being peripheral blood leukocytes, lungs, and spleen. Additional RNA expression studies in cultured cells identified the macrophage as a site of expression of human NRAMP and indicated that increased expression was correlated with an advanced state of differentiation of this lineage.