scholarly journals Host Resistance to Intracellular Infection: Mutation of Natural Resistance-associated Macrophage Protein 1 (Nramp1) Impairs Phagosomal Acidification

1998 ◽  
Vol 188 (2) ◽  
pp. 351-364 ◽  
Author(s):  
David J. Hackam ◽  
Ori D. Rotstein ◽  
Wei-jian Zhang ◽  
Samantha Gruenheid ◽  
Philippe Gros ◽  
...  
Keyword(s):  
Integral Membrane Protein ◽  
Natural Resistance ◽  
Wild Type ◽  
Intracellular Parasites ◽  
Late Endosomes ◽  
Latex Beads ◽  
Buffering Power ◽  
Ratio Imaging ◽  
Macrophage Protein ◽  
Phagosomal Membrane

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 ± 0.06 versus pH 6.6 ± 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton “leak”, as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase–containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.

scholarly journals Natural Resistance to Infection with Intracellular Pathogens: The Nramp1 Protein Is Recruited to the Membrane of the Phagosome

1997 ◽  
Vol 185 (4) ◽  
pp. 717-730 ◽  
Author(s):  
Samantha Gruenheid ◽  
Elhanan Pinner ◽  
Michel Desjardins ◽  
Philippe Gros
Keyword(s):  
Membrane Protein ◽  
Structural Characteristics ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Natural Resistance ◽  
Intracellular Parasites ◽  
Biochemical Fractionation ◽  
Macrophage Protein ◽  
Endocytic Vesicles ◽  
Phagosomal Membrane

The Nramp1 (natural-resistance-associated macrophage protein 1) locus (Bcg, Ity, Lsh) controls the innate resistance or susceptibility of mice to infection with a group of unrelated intracellular parasites which includes Salmonella, Leishmania, and Mycobacterium. Nramp1 is expressed exclusively in professional phagocytes and encodes an integral membrane protein that shares structural characteristics with ion channels and transporters. Its function and mechanism of action remain unknown. The intracellular localization of the Nramp1 protein was analyzed in control 129/sv and mutant Nramp1−/− macrophages by immunofluorescence and confocal microscopy and by biochemical fractionation. In colocalization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment. Double immunofluorescence studies and direct purification of latex bead–containing phagosomes demonstrated that upon phagocytosis, Nramp1 is recruited to the membrane of the phagosome and remains associated with this structure during its maturation to phagolysosome. After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5. The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.

scholarly journals Localisation of Nramp1 in macrophages: modulation with activation and infection

1998 ◽  
Vol 111 (19) ◽  
pp. 2855-2866 ◽  
Author(s):  
S. Searle ◽  
N.A. Bright ◽  
T.I. Roach ◽  
P.G. Atkinson ◽  
C.H. Barton ◽  
...  
Keyword(s):  
Macrophage Activation ◽  
Leishmania Major ◽  
Polyclonal Antibodies ◽  
Natural Resistance ◽  
Activated Macrophages ◽  
Wild Type ◽  
Late Endosomes ◽  
Gold Labelling ◽  
Macrophage Protein ◽  
Phagosome Fusion

The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.

scholarly journals Natural Resistance to Intracellular Infections

2000 ◽  
Vol 192 (9) ◽  
pp. 1237-1248 ◽  
Author(s):  
Nada Jabado ◽  
Andrzej Jankowski ◽  
Samuel Dougaparsad ◽  
Virginie Picard ◽  
Sergio Grinstein ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Fluorescence Emission ◽  
Integral Membrane Protein ◽  
Peritoneal Macrophages ◽  
Natural Resistance ◽  
Susceptibility To Infection ◽  
The Difference ◽  
Macrophage Protein ◽  
Phagosomal Membrane ◽  
Intracellular Infections

Mutations at the natural resistance–associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan–FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn2+ was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (−/−) macrophages. Phagosomes from Nramp1+/+ mice extrude Mn2+ faster than their Nramp−/− counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H+ dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

Transcriptional control of Nramp1: a paradigm for the repressive action of c-Myc

2004 ◽  
Vol 32 (6) ◽  
pp. 1084-1086 ◽  
Author(s):  
A.S. Lapham ◽  
E.S. Phillips ◽  
C.H. Barton
Keyword(s):  
Transcriptional Control ◽  
Cell Activation ◽  
Zinc Finger Protein ◽  
Specific Binding ◽  
Divalent Cations ◽  
Natural Resistance ◽  
Finger Protein ◽  
Late Endosomes ◽  
Solute Carrier ◽  
Macrophage Protein

Slc11a1/Nramp1 (solute carrier family 11 member a1/murine natural resistance-associated macrophage protein 1 gene) encodes a divalent cation transporter that resides within lysosomes/late endosomes of macrophages. Nramp1 modulates the cellular distribution of divalent cations in response to cell activation by intracellular pathogens. Nramp1 expression is repressed and activated by the proto-oncogene c-Myc and Miz-1 (c-Myc-interacting zinc finger protein 1) respectively. Here we demonstrate, using a c-Myc mutant (V394D, Val394→Asp) that is incapable of binding Miz-1, that c-Myc repression of Nramp1 transcription is dependent on its interaction with Miz-1. An oligo pull-down assay demonstrates specific binding of recombinant Miz-1 to the Nramp1 Miz-1-binding site or initiator element(s), and Miz-1-dependent c-Myc recruitment.

Solute carrier 11a1 (Slc11a1; formerly Nramp1) regulates metabolism and release of iron acquired by phagocytic, but not transferrin-receptor-mediated, iron uptake

2002 ◽  
Vol 363 (1) ◽  
pp. 89-94 ◽  
Author(s):  
Victoriano MULERO ◽  
Susan SEARLE ◽  
Jenefer M. BLACKWELL ◽  
Jeremy H. BROCK
Keyword(s):  
Iron Uptake ◽  
Interferon Γ ◽  
Natural Resistance ◽  
Null Alleles ◽  
Iron Release ◽  
Wild Type ◽  
Bivalent Cation ◽  
Insoluble Form ◽  
Solute Carrier ◽  
Macrophage Protein

Solute carrier 11a1 (Slc11a1; formerly Nramp1; where Nramp stands for natural-resistance-associated macrophage protein) is a proton/bivalent cation antiporter that localizes to late endosomes/lysosomes and controls resistance to pathogens. In the present study the role of Slc11a1 in iron turnover is examined in macrophages transfected with Slc11a1Gly169 (wild-type) or Slc11a1Asp169 (mutant = functional null) alleles. Following direct acquisition of transferrin (Tf)-bound iron via the Tf receptor, iron uptake and release was equivalent in wild-type and mutant macrophages and was not influenced by interferon-γ/lipopolysaccharide activation. Following phagocytosis of [59Fe]Tf—anti-Tf immune complexes, iron uptake was equivalent and up-regulated similarly with activation, but intracellular distribution was markedly different. In wild-type macrophages most iron was in the soluble (60%) rather than insoluble (12%) fraction, with 28% ferritin (Ft)-bound. With activation, the soluble component increased to 82% at the expense of Ft-bound iron (< 5%). In mutant macrophages, 40–50% of iron was in insoluble form, 50–60% was soluble and < 5% was Ft-bound. Western-blot analysis confirmed failure of mutant macrophages to degrade complexes 24h after phagocytic uptake. Confocal microscopy showed that complexes were within lysosome-associated membrane protein 1-positive vesicles in wild-type and mutant macrophages at 30min and 24h, implying failure in the degradative process in mature phagosomes in mutant macrophages. NO-mediated iron release was 2.4-fold higher in activated wild-type macrophages compared with mutant macrophages. Overall, our data suggest that iron acquired by phagocytosis and degradation is retained within the phagosomal compartment in wild-type macrophages, and that NO triggers iron release by direct secretion of phagosomal contents rather than via the cytoplasm.

c-Myc represses the murine Nrampl promoter

2002 ◽  
Vol 30 (4) ◽  
pp. 774-777 ◽  
Author(s):  
H. Bowen ◽  
T. E. Biggs ◽  
S. T. Baker ◽  
E. Phillips ◽  
V. H. Perry ◽  
...  
Keyword(s):  
Binding Sites ◽  
Natural Resistance ◽  
Intracellular Pathogens ◽  
Flanking Sequence ◽  
Inhibitory Effects ◽  
Late Endosomes ◽  
E Box ◽  
Macrophage Protein ◽  
Iron Pool ◽  
Divalent Cation Transporter

The Nrampl (natural resistance-associated macrophage protein 1) gene modulates the growth of intracellular pathogens and encodes a divalent cation transporter within lysosomes/late endosomes of macrophages. Nrampl modulates the cytoplasmic iron pool. Wu, Polack and Dalla-Favera [(1999) Science 283, 676–679] showed reciprocal control of H-ferritin and IRP2 by c-Myc, and suggest that c-Myc regulates genes to increase cytoplasmic iron. A role for c-Myc in Nrampl regulation was evaluated. Co-transfection studies show that c-Myc represses Nrampl promoter function. Five non-canonical Myc-max binding sites (E-box) identified within the Nrampl 5′-flanking sequence are not responsible for the inhibitory effects of c-Myc on Nrampl expression. An initiator(s) adjacent to the transcriptioninitiation site is a candidate for the inhibition observed. Results are consistent with a role for Nrampl removing iron from the cytosol and antagonizing c-Myc function.

scholarly journals Role of SLC11A1 gene in disease resistance

2012 ◽  
Vol 28 (1) ◽  
pp. 99-106 ◽  
Author(s):  
N. Thomas ◽  
S. Joseph
Keyword(s):  
Disease Resistance ◽  
Genetic Resistance ◽  
Integral Membrane Protein ◽  
Natural Resistance ◽  
Human Beings ◽  
Economic Traits ◽  
Slc11a1 Gene ◽  
Breeding Programmes ◽  
Macrophage Protein

Genetic improvement in livestock was achieved earlier by selective breeding of individuals with superior phenotypes. Now due to the advances in molecular genetics and biotechnology candidate genes of economic traits can be included in selection for breeding programmes. Genes responsible for the resistance/susceptibility to infections with various pathogens (Major Histo Compatibility (MHC) genes, Solute Carrier family11 member A1 (SLC11A1) gene, Toll Like Receptor (TLR) genes etc.), have been recently identified and characterized in human beings as well as in many animals. Among these the role of SLC11A1 gene is very important due to its association with resistance/ susceptibility to various intracellular pathogens in human as well as in livestock species. The SLC11A1 gene, formerly known as natural resistance-associated macrophage protein 1 (NRAMP1) encodes an integral membrane protein regulating the activity of macrophages. Genetic resistance/ susceptibility to diseases due to candidate gene polymorphisms could be used in selection and breeding for disease resistance in animals.

scholarly journals Role of Nramp1 Deletion inChlamydia Infection in Mice

2000 ◽  
Vol 68 (8) ◽  
pp. 4831-4833 ◽  
Author(s):  
Sukumar Pal ◽  
Ellena M. Peterson ◽  
Luis M. de la Maza
Keyword(s):  
Chlamydia Trachomatis ◽  
Chlamydial Infection ◽  
Chlamydia Pneumoniae ◽  
Natural Resistance ◽  
Wild Type ◽  
Intranasal Infection ◽  
Macrophage Protein

ABSTRACT Elicited macrophages from 129sv mice with a functional deletion of the natural-resistance-associated macrophage protein 1 gene (Nramp1) were shown to be as susceptible as wild-type mice to infection with the Chlamydia trachomatis mouse pneumonitis and L3 serovars and to Chlamydia pneumoniae. Furthermore, the two groups of mice were shown to be similarly susceptible to an intranasal infection with these microorganisms. In conclusion, the Nramp1 gene does not appear to play a major role in the regulation of the susceptibility of mice to a chlamydial infection.

Genetic analysis of SLC11A1 polymorphisms in multiple sclerosis patients

2004 ◽  
Vol 10 (6) ◽  
pp. 618-620 ◽  
Author(s):  
Manuel Comabella ◽  
Laura Altet ◽  
Francesc Peris ◽  
Pablo Villoslada ◽  
Armand Sánchez ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Natural Resistance ◽  
Bivalent Cation ◽  
Slc11a1 Gene ◽  
Late Endosomes ◽  
Solute Carrier ◽  
Macrophage Protein ◽  
Multiple Sclerosis Patients ◽  
Control Study

Solute carrier 11a1 (SLC11A1; formerly NRAMP1, where NRAMP stands for natural resistance-associated macrophage protein) is a proton/bivalent cation antiporter that localizes to late endosomes/lysosomes. SLC11A1 regulates macrophage functions that are of potential importance in the induction and/or maintenance of autoimmune diseases such as rheumatoid arthritis, type 1 diabetes and Crohn’s disease. We investigated SLC11A1 gene as a candidate gene for genetic susceptibility to multiple sclerosis (MS) in our population. Four SLC11A1 gene polymorphisms (5?GT repeat, D543N, 1729 -55del4 and 1729 -271del4) were analysed in a case-control study of 195 patients with MS and 125 control subjects. We found no evidence of association between SLC11A1 polymorphisms and MS susceptibility in the Spanish population.

The Tonoplast-Localized Transporter OsNRAMP2 is involved in Fe Homeostasis and affects Seed Germination in rice

2021 ◽  
Author(s):  
Yun Li ◽  
Jingjun Li ◽  
Yihong Yu ◽  
Xia Dai ◽  
Changyi Gong ◽  
...  
Keyword(s):  
Seed Germination ◽  
Natural Resistance ◽  
Fe Deficiency ◽  
Shoot Biomass ◽  
Wild Type ◽  
Crop Development ◽  
In The Wild ◽  
Macrophage Protein ◽  
Fe Homeostasis ◽  
Fe Toxicity

Abstract Vacuolar storage of iron (Fe) is important for Fe homeostasis in plants. When sufficient, the excess Fe could be stored in vacuoles for remobilization in case of Fe deficiency. Although the mechanism of Fe remobilization from vacuoles is critical for crop development under low Fe stress, the transporters that mediate vacuolar Fe translocation into the cytosol in rice remains unknown. Here, we showed that under higher Fe 2+ concentrations, the Δccc1 yeast mutant transformed with rice natural resistance-associated macrophage protein 2 (OsNRAMP2) became more sensitive to Fe toxicity. In rice protoplasts and transgenic plants expressing Pro35S: OsNRAMP2-GFP, OsNRAMP2 was localized to tonoplast. Vacuolar Fe contents in osnramp2 knockdown lines were higher than in the wild-type, while the growth of osnramp2 knockdown plants was significantly influenced by Fe deficiency. Furthermore, the germination of osnramp2 knockdown plants was arrested. Inversely, the vacuolar Fe contents of Pro35S: OsNRAMP2-GFP lines were significantly lower than in the wild-type, and overexpression of OsNRAMP2 increased shoot biomass under Fe deficiency. Taken together, we propose that OsNRAMP2 transports Fe from the vacuole to the cytosol and plays a pivotal role in seed germination.

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