Polymorphism of Slc11a1 (Nramp1) gene and canine leishmaniasis in a case-control study

The prevalence of canine leishmaniasis infection in an endemic area such as the Mediterranean basin (67%) is higher than the prevalence of the disease (10%), suggesting a role of host genetics related to the outcome of the disease. Because Slc11a1 gene affects susceptibility and clinical outcome of autoimmune and infectious diseases, we analyzed five polymorphisms of the Slc11a1 gene in a case-control study with 97 dogs: three new single nucleotide polymorphisms and a G-stretch in the promoter and a microsatellite in intron 1. Haplotype frequency distributions showed significant differences between case and control populations (P = .01), most likely owing to the single nucleotide polymorphisms in the promoter region that were associated to case dogs. The most frequent haplotypes included TAG-8-141, which was present in all the breeds, in both case and control animals; and TAG-9-145, which was overrepresented in the control population and mostly found in boxer dogs. Within the boxer breed, 81% of the healthy dogs were homozygous TAG-9-145, whereas TAG-8-141 was significantly associated to case boxers (P = .02). The special genotype distribution for the Slc11a1 polymorphism associated with the prevalence of the illness in the boxer breed emphasizes the potential importance that breed genetic background has in canine leishmaniasis susceptibility.     

The natural resistance-associated macrophage protein 1 Slc11a1 (formerly Nramp1) and iron metabolism in macrophages

Slc11a1 (solute carrier family 11 member 1) (formerly Nramp1) modulation of iron metabolism in macrophages plays an important role in early phase macrophage activation, and therefore host innate immunity. This review focuses on the role of Nramp1 in intramacrophage iron metabolism, with emphasis on the two prevailing mechanisms of Nramp1 modulation of iron metabolism in macrophages.     

Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.     

Genetic determinants of acute inflammation regulate Salmonella infection and modulate Slc11a1 gene (formerly Nramp1) effects in selected mouse lines

Two lines of mice selected to produce maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactions (AIR) differ in their susceptibility to infection by Salmonella enterica serotype Typhimurium (S. Typhimurium). The LD(50) for AIRmax mice is 1000 times higher than that observed for AIRmin mice, and higher frequencies of Slc11a1 alleles (known to confer either resistance (R) or high susceptibility (S) to S. Typhimurium) were consistently found in AIRmax and AIRmin mouse lines, respectively. In order to evaluate the effect of the quantitative trait loci (QTL) segregated in AIRmax and AIRmin mice on Slc11a1 dependent susceptibility to S. Typhimurium, the R and S alleles were fixed in homozygosity in AIRmax and AIRmin backgrounds by genotype assisted breedings. These new lines were named AIRmax(RR), AIRmax(SS), AIRmin(RR), and AIRmin(SS). Acute inflammation of Slc11a1(RR) animals was more severe in comparison to their Slc11a1(SS) counterparts, implicating Slc11a1 (or other linked genes) in AIR regulation. The LD(50) of S. Typhimurium was 800-times higher for AIRmax(SS) than for AIRmin(SS), demonstrating that AIR QTL can act as modifiers of the Slc11a1(SS) susceptibility gene. Four microsatellite markers for S. Typhimurium susceptibility QTL described in other mouse lines showed specific allele fixation in AIRmax or AIRmin mice, suggesting that these chromosomal regions also segregate with inflammatory phenotypes.     

Identification of a tyrosine-based motif (YGSI) in the amino terminus of Nramp1 (Slc11a1) that is important for lysosomal targeting

In macrophages, Nramp1 (Slc11a1) is expressed in lysosomes and restricts replication of intracellular pathogens by removing divalent metals (Mn2+ and Fe2+) from the phagolysosome. Nramp2 (DMT1, Slc11a2) is expressed both at the duodenal brush border where it mediates uptake of dietary iron and ubiquitously at the plasma membrane/recycling endosomes of many cell types where it transports transferrin-associated iron across the endosomal membrane. In Nramp2, a carboxyl-terminal cytoplasmic motif ((555)YLLNT(559)) is critical for internalization and recycling of the transporter from the plasma membrane. Here we studied the subcellular trafficking properties of Nramp1 and investigated the cis-acting sequences responsible for targeting to lysosomes. For this, we constructed and studied Nramp1/Nramp2 chimeric proteins where homologous domains of each protein were exchanged. Chimeras exchanging the amino-(upstream TM1) and carboxyl-terminal (downstream TM12) cytoplasmic segments of both transporters were stably expressed in porcine LLC-PK1 kidney cells and were studied with respect to expression, maturation, stability, cell surface targeting, transport activity, and subcellular localization. An Nramp2 isoform II chimera bearing the amino terminus of Nramp1 was not expressed at the cell surface but was targeted to lysosomes. This lysosomal targeting was abolished by single alanine substitutions at Tyr15 and Ile18 of a (15)YGSI(18) motif present in the amino terminus of Nramp1. These results identify YGSI as a tyrosine-based sorting signal responsible for lysosomal targeting of Nramp1.     

Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.     

Comparative analysis of two slc11 (Nramp) loci in Takifugu rubripes

To study the evolution of the solute carrier family 11 (slc11; formerly Nramp) protein, we isolated and characterized two paralogs from the pufferfish Takifugu rubripes (Fugu). These teleost genes, designated Fugu slc11a-a and Fugu slc11a-b, comprise open reading frames of 1743 nucleotides (581 amino acids) and 1662 nt (554 aa), respectively. The proteins are 81% similar, and both exhibit signature features of the slc11 family of proteins including 12 transmembrane domains, a conserved transport motif and a glycosylated loop. Both Fugu paralogs are more Slc11a2-like based on sequence homology and phylogenetic studies. Analysis of gene environment placed both in the proximity of multiple loci syntenic to human chromosome 12q13, that is, within a SLC11A2 gene environment. However, Fugu slc11a-a also gave one match with chromosome 2q35, where human SLC11A1 resides. Functional diversification was suggested by differences in tissue distribution and subcellular localization. Fugu slc11a-a exhibits a restricted expression profile and a complex subcellular localization, including LAMP1 positive late endosomes/lysosomes in transiently transfected mouse macrophages. Fugu slc11a-b is expressed ubiquitously and localizes solely to late endosomes/lysosomes. This comparative analysis extends our understanding of the evolution and function of this important family of divalent cation transporters. [Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AJ496547/8/9 and AJ496550.]     

Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene

 Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4. Received: 15 May 1999 / Revised: 25 September 1999     

Structure and topology of Slc11a1(164-191) with G169D mutation in membrane-mimetic environments

Solute carrier family 11 member 1 (Slc11a1) is a proton-mediated divalent metal cation transporter with 12 putative transmembrane domains. Variation in it reveals alterations in host resistance against intracellular pathogens. A naturally occurring glycine to aspartic acid mutation at position 169 (G169D) in the putative transmembrane domain 4 (TM4) makes mice susceptible to Salmonella typhimurim, Leishmania donovani, and Mycobacterium bovis. In this work, a 28-residue peptide corresponding to Slc11a1(164-191), including TM4 of Slc11a1, with G169D mutation is characterized using CD and NMR methods in 2,2,2-trifluoroethanol solvent and SDS micelles and the results of present study on the G169D peptide are compared with those of previous study on the wild-type peptide. Similarly to the wild-type peptide, the G169D peptide forms a predominantly alpha-helical structure and is totally embedded in SDS micelles as a homologous assembly. However, the G169D mutation changes the local conformation near the mutation site, the cooperative manner in proton binding of the residue Asp located in the center of SDS micelles and the interaction strength of this residue with Mn2+ ions.     

IL-1 Receptor antagonist as a positional candidate gene in a murine model of allergic asthma

Interleukin-1 receptor antagonist (IL-1ra) is an inhibitor of the proinflammatory IL-1. The IL-1ra gene (Il1rn) maps near the allergen-induced bronchial hyper-responsiveness-1 locus, Abhr1, which we previously mapped to murine chromosome 2 using A/J (asthma susceptible) and C3H/HeJ (asthma resistant) mice. We evaluated the role of Il1rn in our mouse model by comparing its genomic sequence between A/J and C3H/HeJ mice as well as assessing strain-specific RNA and protein production in response to allergen. We identified no functional sequence variations in the Il1rn gene between A/J and C3H/HeJ mice. Il1rn mRNA and protein were induced by ovalbumin (OVA) exposure in both strains, but to a greater extent in A/J mice at the earlier time points. We examined other IL-1 family members (Il1a, Il1b, Il1f9, and Il1r2) and found OVA-induced expression increases at 6 h, yet only Il1b and Il1f9 had strain-specific differences. Of these, only Il1f9 is located within Abhr1, and we found several non-coding polymorphisms in the Il1f9 gene between A/J and C3H/HeJ mice. Our results exclude Il1rn as the gene for Abhr1 and indicate that Il1f9 warrants further investigation based on genetic and expression differences observed in our mouse model of allergic asthma.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Role of MAP kinase activation in Nramp1 mRNA stability in RAW264.7 macrophages expressing Nramp1(Gly169)

Nramp1 (natural resistance-associated macrophage protein 1) is a phagosomal iron transport molecule. In addition to its anti-microbial activity, Nramp1 exerts a wide range of pleiotropic effects, including increased stability of Nramp1 mRNA and a variety of other mRNA species. Previously, we showed that the increased stability of Nramp1 mRNA is regulated by an oxidant-generated signaling pathway that requires PKC. In the current study, we show that inhibition of ERK1,2 and p38 MAP kinase activities decreases Nramp1 mRNA stability in Mycobacterium avium infected RAW264.7 cells expressing Nramp1(Gly169) but not in RAW264.7-Nramp1(Asp169) cells. Phosphorylation of ERK1,2 and p38 MAP kinases, which could be inhibited by the anti-oxidant BHA and a protein kinase C inhibitor, was higher in M. avium infected RAW264.7-Nramp1(Gly169) cells than in RAW26.47-Nramp1(Asp169) cells. These results suggest that generation of oxidants by Nramp1 iron transport activates MAP kinase signaling cascades that result in stabilization of Nramp1 mRNA.     

Structure and transmembrane topology of Slc11a1 TMD1-5 in lipid membranes

The importance of solute carrier family 11 (Slc11) in divalent metal-ion transport has been well established. The core domains TMD1-5 and TMD6-10 of the proteins were modeled as a symmetric but inversely orientated arrangement with respect to membrane normal. In this article, the structures and transmembrane topologies of TMD1-5 of Slc11a1 incorporated with phospholipids 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG), and POPC/POPG (3:1) were explored using circular dichroism, fluorescence, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies. The segments TMD2-5 were inserted in lipid membranes mainly as an α-helix with orientations of helices along membrane normal. The tilt angles of the helices were in an order of TMD3 > TMD4 > TMD2 > TMD5 in these membranes. In contrast, TMD1 was partly inserted in membranes, leaving partial segment at membrane surface. The amount of the lipid component with negatively charged headgroups had an effect on both the helicity and orientation of the transmembrane domains (TMDs). Nevertheless, the helices maintained similar topologies in various membranes.     

Study on structure and assembly of the third transmembrane domain of Slc11a1

The structure and self‐assembly of the peptide corresponding to the third transmembrane domain (TMD3) of Slc11a1 and its E139A mutant are studied in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) aqueous solution by NMR and CD experiments. Slc11a1 is an integral membrane protein with 12 putative TMDs and functions as a pH‐coupled divalent metal cation transporter. Glu139 of Slc11a1 is highly conserved within predicted TMD3 of the Slc11 protein family and function‐associated. Here, we provide the first direct experimental evidence for the structural features of two 24‐residue peptides corresponding to TMD3 of Slc11a1 and its E139A mutant in 60% HFIP‐d₂ aqueous solution using CD and NMR spectroscopies. Our study shows that the membrane‐spanning peptide folds as a typical amphipathic α‐helix structure from Ile5 to Met20 with hydrophilic residues Glu12 (Glu139 in Slc11a1) and Asp19 lying on the same side of the helix. The substitution of Glu139 by an alanine residue has little effect on the structure of the peptide, but increases hydrophobicity and facilitates self‐assembly of the peptide. Although the wildtype peptide is monomeric in HFIP aqueous solution, the E139A mutant forms a dimer. The increase in hydrophobicity of the membrane‐spanning peptide and/or change in the interactions between transmembrane segments induced by E139A mutation may affect the metal ion transport of the protein. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG)

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.     

The Phosphate Transporter PiT1 (Slc20a1) Revealed As a New Essential Gene for Mouse Liver Development

Background

PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na⁺-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Methodology/Principal Findings

To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5–E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.

Conclusion/Significance

This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.