scholarly journals Leishmania infection triggers hepcidin-mediated proteasomal degradation of Nramp1 resulting in increased phagolysosomal iron availability

2019 ◽  
Author(s):  
Sourav Banerjee ◽  
Rupak Datta
Keyword(s):  
Leishmania Major ◽  
Peptide Hormone ◽  
Parasite Growth ◽  
Natural Resistance ◽  
Intracellular Pathogens ◽  
Iron Availability ◽  
Regulatory Peptide ◽  
Ubiquitin Proteasome ◽  
Macrophage Protein ◽  
Iron Pool

AbstractNatural resistance associated macrophage protein 1 (Nramp1) was discovered as a genetic determinant of resistance against multiple intracellular pathogens, including Leishmania. It encodes a transmembrane protein of the phago-endosomal vesicles, where it functions as an iron transporter. But how Nramp1 expression is regulated in an infected macrophage is unknown. Its role in controlling iron availability to the intracellular pathogens and in determining the final outcome of an infection also remains to be fully deciphered. Here we report that Nramp1 protein abundance undergoes temporal changes in Leishmania major infected macrophages. At 12 hours post infection, there was drastic lowering of Nramp1 level accompanied by increased phagolysosomal iron availability and enhanced parasite growth. Leishmania infection-induced downregulation of Nramp1 was found to be caused by ubiquitin-proteasome degradation pathway. In fact, blocking of Nramp1 degradation with proteasome inhibitor resulted in depletion of phagolysosomal iron pool with significant reduction in the number of intracellular parasites. Further, we uncovered that this degradation process is mediated by the iron regulatory peptide hormone hepcidin that binds to Nramp1. Interestingly, Nramp1 protein level was restored to normalcy after 30 hours of infection with a concomitant drop in the phagolysosomal iron level, which is suggestive of a host counter defense strategy to deprive the pathogen of this essential micronutrient. Taken together, our study implicates Nramp1 as a central player in the host-pathogen battle for iron. It also unravels Nramp1 as a novel partner for hepcidin. The hitherto unidentified ‘hepcidin-Nramp1 axis’ may have a broader role in regulating macrophage iron homeostasis.ImportanceLeishmania parasites are the causative agents of a group of neglected tropical diseases called leishmaniasis. They reside within the phagolysosomes of mammalian macrophages. Since iron is an essential micronutrient for survival and virulence, intracellular Leishmania must acquire it from the tightly regulated macrophage iron pool. How this challenging task is accomplished remains a fundamental question in Leishmania biology. We report here that Leishmania major infection caused ubiquitin-proteasome-mediated degradation of natural resistance associated macrophage protein 1 (Nramp1). Nramp1 being an iron exporter at the phago-endosomal membrane, its degradation resulted in increased phagolysosomal iron availability thereby stimulating parasite growth. We also uncovered that Nramp1 degradation is controlled by the iron regulatory peptide hormone hepcidin. Interestingly, at a later stage of infection, Nramp1 protein level was restored to normalcy with simultaneous depletion of phagolysosomal iron. Collectively, our study implicates Nramp1 as a central player in the host-pathogen struggle for acquiring iron.

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 Infection with Mycobacterium aviumDifferentially Regulates the Expression of Iron Transport Protein mRNA in Murine Peritoneal Macrophages

2001 ◽  
Vol 69 (11) ◽  
pp. 6618-6624 ◽  
Author(s):  
Wangjian Zhong ◽  
William P. Lafuse ◽  
Bruce S. Zwilling
Keyword(s):  
Host Defense ◽  
Transferrin Receptor ◽  
Iron Transport ◽  
Peritoneal Macrophages ◽  
Natural Resistance ◽  
Intracellular Pathogens ◽  
Mrna Levels ◽  
Iron Availability ◽  
Iron Transporter ◽  
Murine Peritoneal Macrophages

ABSTRACT Iron is an important element for the growth of microorganisms as well as in the defense of the host by serving as a catalyst for the generation of free radicals via the Fenton/Haber-Weiss reactions. The iron transporter natural resistance-associated macrophage protein 1 (Nramp1) confers resistance to the growth of a variety of intracellular pathogens including Mycobacterium avium. Recently several other proteins that are involved in iron transport, including the highly homologous iron transporter Nramp2 and the transferrin receptor-associated protein HFE (hereditary hemochromatosis protein), have been described. The relationship of these proteins to host defense and to the growth of intracellular pathogens is not known. Here, we report that infection with M. avium differentially regulates mRNA expression of the proteins associated with iron transport in murine peritoneal macrophages. Both Nramp1 and Nramp2 mRNA levels increase following infection, while the expression of transferrin receptor mRNA decreases. The level of expression of HFE mRNA remains unchanged. The difference in the expression of the mRNA of these proteins following infection or cytokine stimulation suggests that they may play an important role in host defense by maintaining a delicate balance between iron availability for host defense and at the same time limiting iron availability for microbial growth.

Functional analysis of bovine Nramp1 and production of transgenic cloned embryosin vitro

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Xiang Cheng ◽  
Xiaoli Yu ◽  
Yajun Liu ◽  
Jie Deng ◽  
Xiaoling Ma ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Formation Rate ◽  
Natural Resistance ◽  
Intracellular Bacteria ◽  
Intracellular Pathogens ◽  
Qinchuan Cattle ◽  
Polymerase Chain ◽  
Macrophage Protein ◽  
Rectal Palpation

SummaryNatural resistance-associated macrophage protein 1 (Nramp1) plays an important role in restraining the growth of intracellular pathogens within macrophages. In this study,Nramp1cDNA was cloned from Qinchuan cattle and its anti-bacterial activity was demonstrated as being able to significantly inhibit the growth ofSalmonella abortusovisandBrucella abortusin macrophages. Calf fibroblasts stably transfected with pSP–NRAMP1–HA vector were used to reconstruct bovine embryos by somatic cell nuclear transfer (SCNT). Reconstructed embryos were maturatedin vitroand the blastocyst formation rate (14.0%) was similar to that of control embryos (14.5%). Transgenic blastocysts were transplanted into 43 recipient cattle, of which 14 recipients became pregnant as evidenced by non-return estrus and by rectal palpation. One fetus was aborted after 6½ months of pregnancy and transgene integration was confirmed by semi-quantitative polymerase chain reaction. Together, this study showed that bovine Nramp1 retains biological function against the growth of intracellular bacteria and can be used to reconstruct embryos and produceNramp1transgenic cattle, which may benefit the animal and enhance their ability to prevent attack by intracellular pathogens.

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.

Both Nramp1 and Dmt1 Are Necessary for Efficient Macrophage Iron Recycling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1994-1994
Author(s):  
Shan Soe-Lin ◽  
Sameer S Apte ◽  
Marc R Mikhael ◽  
Lidia Kayembe ◽  
Guangjun Nie ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Natural Resistance ◽  
Heme Oxygenase 1 ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
Labile Iron Pool ◽  
Raw264.7 Macrophages ◽  
Macrophage Protein ◽  
Iron Pool

Abstract Abstract 1994 Poster Board I-1016 Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process. Wild-type RAW264.7 macrophages (Nramp1-/-), and those stably transfected with Nramp1 (Nramp1+/+) were treated with either DMT1-siRNA, or with ebselen, a selective inhibitor of DMT1. While macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis. These results suggest that while the loss of either Nramp1 or DMT1 transport ability results in minor impairment following erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Disruption of the Gene Homologous to Mammalian Nramp1 in Mycobacterium tuberculosis Does Not Affect Virulence in Mice

2002 ◽  
Vol 70 (8) ◽  
pp. 4124-4131 ◽  
Author(s):  
Neio Boechat ◽  
Béatrice Lagier-Roger ◽  
Stéphanie Petit ◽  
Yann Bordat ◽  
Jean Rauzier ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Natural Resistance ◽  
Mouse Bone Marrow ◽  
Iron Availability ◽  
Macrophage Cell ◽  
Growth Impairment ◽  
Allelic Exchange ◽  
Mutant Strains ◽  
Macrophage Protein ◽  
Growth Of Bacteria

ABSTRACT Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis. Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions. However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability. The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1+/+ and Nramp1 −/− congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate that Mramp does not contribute to the virulence of M. tuberculosis in mice.

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