Slc11 Synapomorphy: A Conserved 3D Framework Articulating Carrier Conformation Switch.

Transmembrane carriers of the Slc11 family catalyze proton (H+)-dependent uptake of divalent metal ions (Me2+) such as manganese and iron-vital elements coveted during infection. The Slc11 mechanism of high-affinity Me2+ cell import is selective and conserved between prokaryotic (MntH) and eukaryotic (Nramp) homologs, though processes coupling the use of the proton motive force to Me2+ uptake evolved repeatedly. Adding bacterial piracy of Nramp genes spread in distinct environmental niches suggests selective gain of function that may benefit opportunistic pathogens. To better understand Slc11 evolution, Alphafold (AF2)/Colabfold (CF) 3D predictions for bacterial sequences from sister clades of eukaryotic descent (MCb and MCg) were compared using both native and mutant templates. AF2/CF model an array of native MCb intermediates spanning the transition from outwardly open (OO) to inwardly open (IO) carriers. In silico mutagenesis targeting (i) a set of (evolutionarily coupled) sites that may define Slc11 function (putative synapomorphy) and (ii) residues from networked communities evolving during MCb transition indicates that Slc11 synapomorphy primarily instructs a Me2+-selective conformation switch which unlocks carrier inner gate and contributes to Me2+ binding site occlusion and outer gate locking. Inner gate opening apparently proceeds from interaction between transmembrane helix (h) h5, h8 and h1a. MCg1 xenologs revealed marked differences in carrier shape and plasticity, owing partly to an altered intramolecular H+ network. Yet, targeting Slc11 synapomorphy also converted MCg1 IO models to an OO state, apparently mobilizing the same residues to control gates. But MCg1 response to mutagenesis differed, with extensive divergence within this clade correlating with MCb-like modeling properties. Notably, MCg1 divergent epistasis marks the emergence of the genus Bordetella-Achromobacter. Slc11 synapomorphy localizes to the 3D areas that deviate least among MCb and MCg1 models (either IO or OO) implying that it constitutes a 3D network of residues articulating a Me2+-selective carrier conformation switch which is maintained in fast-evolving clades at the cost of divergent epistatic interactions impacting carrier shape and dynamics.

CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infection.

Salmonella presents a global public health concern. Central to Salmonella pathogenicity is an ability to subvert host defences through strategically targeting host proteins implicated in restricting infection. Therefore, to gain insight into the host-pathogen interactions governing Salmonella infection, we performed an in vivo genome-wide mutagenesis screen to uncover key host defence proteins. This revealed an uncharacterized role of CYRI (FAM49B) in conferring host resistance to Salmonella infection. We show that CYRI binds to the small GTPase RAC1 through a conserved domain present in CYFIP proteins, which are known RAC1 effectors that stimulate actin polymerization. However, unlike CYFIP proteins, CYRI negatively regulates RAC1 signalling, thereby attenuating processes such as macropinocytosis, phagocytosis and cell migration. This enables CYRI to counteract Salmonella at various stages of infection, including bacterial entry into non-phagocytic and phagocytic cells as well as phagocyte-mediated bacterial dissemination. Intriguingly, to dampen its effects, the bacterial effector SopE, a RAC1 activator, selectively targets CYRI following infection. Together, this outlines an intricate host-pathogen signalling interplay that is crucial for determining bacterial fate. Notably, our study also outlines a role for CYRI in restricting infection mediated by Mycobacterium tuberculosis and Listeria monocytogenes. This provides evidence implicating CYRI cellular functions in host defence beyond Salmonella infection.

Developmental Control of NRAMP1 (SLC11A1) Expression in Professional Phagocytes.

NRAMP1 (SLC11A1) is a professional phagocyte membrane importer of divalent metals that contributes to iron recycling at homeostasis and to nutritional immunity against infection. Analyses of data generated by several consortia and additional studies were integrated to hypothesize mechanisms restricting NRAMP1 expression to mature phagocytes. Results from various epigenetic and transcriptomic approaches were collected for mesodermal and hematopoietic cell types and compiled for combined analysis with results of genetic studies associating single nucleotide polymorphisms (SNPs) with variations in NRAMP1 expression (eQTLs). Analyses establish that NRAMP1 is part of an autonomous topologically associated domain delimited by ubiquitous CCCTC-binding factor (CTCF) sites. NRAMP1 locus contains five regulatory regions: a predicted super-enhancer (S-E) key to phagocyte-specific expression; the proximal promoter; two intronic areas, including 3' inhibitory elements that restrict expression during development; and a block of upstream sites possibly extending the S-E domain. Also the downstream region adjacent to the 3' CTCF locus boundary may regulate expression during hematopoiesis. Mobilization of the locus 14 predicted transcriptional regulatory elements occurs in three steps, beginning with hematopoiesis; at the onset of myelopoiesis and through myelo-monocytic differentiation. Basal expression level in mature phagocytes is further influenced by genetic variation, tissue environment, and in response to infections that induce various epigenetic memories depending on microorganism nature. Constitutively associated transcription factors (TFs) include CCAAT enhancer binding protein beta (C/EBPb), purine rich DNA binding protein (PU.1), early growth response 2 (EGR2) and signal transducer and activator of transcription 1 (STAT1) while hypoxia-inducible factors (HIFs) and interferon regulatory factor 1 (IRF1) may stimulate iron acquisition in pro-inflammatory conditions. Mouse orthologous locus is generally conserved; chromatin patterns typify a de novo myelo-monocytic gene whose expression is tightly controlled by TFs Pu.1, C/ebps and Irf8; Irf3 and nuclear factor NF-kappa-B p 65 subunit (RelA) regulate expression in inflammatory conditions. Functional differences in the determinants identified at these orthologous loci imply that species-specific mechanisms control gene expression.

Suppression of hepcidin expression and iron overload mediate Salmonella susceptibility in ankyrin 1 ENU-induced mutant.

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1(Ity16/Ity16) mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1(Ity16/Ity16) mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1(Ity16/Ity16), the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1(+/Ity16) mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1(Ity16/Ity16) and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1(+/Ity16) mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1(+/Ity16) heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.

Cell-Type Specific Determinants of NRAMP1 Expression in Professional Phagocytes.

The Natural resistance-associated macrophage protein 1 (Nramp1 or Solute carrier 11 member 1, Slc11a1) transports divalent metals across the membrane of late endosomes and lysosomes in professional phagocytes. Nramp1 represents an ancient eukaryotic cell-autonomous defense whereas the gene duplication that yielded Nramp1 and Nramp2 predated the origin of Sarcopterygians (lobe-finned fishes and tetrapods). SLC11A1 genetic polymorphisms associated with human resistance to tuberculosis consist of potential regulatory variants. Herein, current knowledge of the regulation of SLC11A1 gene expression is reviewed and comprehensive analysis of ENCODE data available for hematopoietic cell-types suggests a hypothesis for the regulation of SLC11A1 expression during myeloid development and phagocyte functional polarization. SLC11A1 is part of a 34.6 kb CTCF-insulated locus scattered with predicted regulatory elements: a 3' enhancer, a large 5' enhancer domain and four elements spread around the transcription start site (TSS), including several C/EBP and PU.1 sites. SLC11A1 locus ends appear mobilized by ETS-related factors early during myelopoiesis; activation of both 5' and 3' enhancers in myelo-monocytic cells correlate with transcription factor binding at the TSS. Characterizing the corresponding cis/trans determinants functionally will establish the mechanisms involved and possibly reveal genetic variation that impacts susceptibility to infectious or immune diseases.

Nramp: from sequence to structure and mechanism of divalent metal import.

Mn and Fe are important for energy metabolism and oxidative stress resistance and cells maintain adequate stores for survival and prevention of toxicity. Membrane permeases of the natural resistance-associated macrophage protein (Nramp) family importing protons and divalent metals are conserved from bacteria to man. Nramp hydrophobic core relates structurally to a superfamily of cation-driven carriers with inverted symmetry. Molecular phylogeny and sequence features support Nramp pseudo-symmetric three-dimensional (3D) model, and remote ancestry to the LeuT superfamily. Genetic analyses suggest conservation of Nramp sequence marks the transition from a phylogenetic out-group and may relate to divalent metal selectivity. Three phylogroups of bacterial proton-dependent manganese transporters (MntH) demonstrate specific patterns of sequence conservation suggesting functional constraints linked to ecological or taxonomical distributions, which may contribute to bacterial virulence. Nramp 3D model is supported experimentally by transmembrane topology and structure-function studies of Escherichia coli and mouse homologs as well as peptide structure analyses. Eukaryotic Nramps are required for Mn and Fe homeostasis, contributing in multicellular organisms to subcellular and systemic metal traffic and intercellular signaling. Nramps are subjected to elaborate regulation including developmental control of gene expression, protein subcellular targeting, dynamic metallo-dependent control of messenger RNA and protein stability and trafficking. Several human pathologies may result from defects in Nramp-dependent Fe(2+) or Mn(2+) transport, including iron overload, neurodegenerative diseases and innate susceptibility to infectious diseases.

Nutritional immunity: homology modeling of Nramp metal import.

The Natural resistance-associated macrophage proteins (Nramp1 and 2) are proton-dependent solute carriers of divalent metals such as Fe(2+) and Mn(2+) (Slc11a1 and 2). Their expression in both resting and microbicidal macrophages which metabolize iron differently, raises questions about Nramp mechanism of Me(2+) transport and its impact in distinct phenotypic contexts. We developed a low resolution 3D model for Slc11 based on detailed phylogeny and remote homology threading using Escherichia coli Nramp homolog (proton-dependent Mn(2+) transporter, MntH) as experimental system. The predicted fold is consistent with determinations of transmembrane topology and activity; it indicates Slc11 carriers are part of the LeuT superfamily. Homology implies that inverted structural symmetry facilitates Slc11 H(+)-driven Me(2+) import and provides a 3D framework to test structure-activity relationships in macrophages and study functional evolution of MntH/Nramp (Slc11) carriers.

A novel metal transporter mediating manganese export (MntX) regulates the Mn to Fe intracellular ratio and Neisseria meningitidis virulence.

Neisseria meningitidis (Nm) and N. gonorrhoeae (Ng) are adapted to different environments within their human host. If the basis of this difference has not yet been fully understood, previous studies (including our own data) have reported that, unlike Ng, Nm tolerates high manganese concentrations. As transition metals are essential regulators of cell growth and host pathogen interactions, we aimed to address mechanisms of Nm Mn²âº tolerance and its pathogenic consequences. Using bioinformatics, gene deletion and heterologous expression we identified a conserved bacterial manganese resistance factor MntX (formerly YebN). The predicted structure suggests that MntX represents a new family of transporters exporting Mn. In the Neisseria genus, this exporter is present and functional in all Nm isolates but it is mutated in a majority of Ng strains and commonly absent in nonpathogenic species. In Nm, Mn²âº export via MntX regulates the intracellular Mn/Fe ratio and protects against manganese toxicity that is exacerbated in low iron conditions. MntX is also important for N. meningitidis to resist killing by human serum and for survival in mice blood during septicemia. The present work thus points to new clues about Mn homeostasis, its interplay with Fe metabolism and the influence on N. meningitidis physiology and pathogenicity.

The natural resistance-associated macrophage protein from the protozoan parasite Perkinsus marinus mediates iron uptake.

Microbial pathogens succeed in acquiring essential metals such as iron and manganese despite their limited availability because of the host's immune response. The eukaryotic natural resistance-associated macrophage proteins mediate uptake of divalent metals and, during infection, may compete directly for metal acquisition with the pathogens' transporters. In this study, we characterize the Nramp gene family of Perkinsus marinus, an intracellular parasite of the eastern oyster, and through yeast complementation, we demonstrate for the first time for a protozoan parasite that Nramp imports environmental Fe. Three PmNramp isogenes differ in their exon-intron structures and encode transcripts that display a trans splicing leader at the 5' end. The protein sequences share conserved properties predicted for the Nramp/Solute carrier 11 (Slc11) family, such as 12-transmembrane segment (TMS) topology (N- and C-termini cytoplasmic) and preferential conservation of four TMS predicted to form a pseudosymmetric proton/metal symport pathway. Yeast fet3fet4 mutant complementation assays showed iron transport activity for PmNramp1 and a fusion chimera of the PmNramp3 hydrophobic core and PmNramp1 N- and C-termini. PmNramp1 site-directed mutagenesis demonstrated that Slc11 invariant and predicted pseudosymmetric motifs (TMS1 Asp-Pro-Gly and TMS6 Met-Pro-His) are key for transport function. PmNramp1 TMS1 mutants D76E, G78A, and D76E/G78A prevented membrane protein expression, while TMS6 M250A, H252Y, and M250A/H252Y specifically abrogated Fe uptake; the TMS6 H252Y mutation also correlates with divergence from Nramp specificity for divalent metals.

Transmembrane topology of the mammalian Slc11a2 iron transporter.

The mammalian Slc11a1 and Slc11a2 proteins define a large family of secondary metal transporters. Slc11a1 and Slc11a2 function as pH-dependent divalent cation transporters that play a critical role in host defenses against infections and in Fe2+ homeostasis, respectively. The position and polarity of individual transmembrane domains (TMD) of Slc11a2 were studied by an epitope tagging method based on the insertion of small antigenic hemagglutinin A (HA) peptides (YPYDVPDYAS) in predicted intra- or extracellular loops of the protein. The tagged proteins were expressed in transfected LLC-PK1 kidney cells and tested for transport activity, and the polarity of inserted tags with respect to the plasma membrane was determined by immunofluorescence in intact and permeabilized cells. HA epitope tags were inserted at positions 1, 98, 131, 175, 201, 243, 284, 344, 403, 432, 468, 504, and 561. Insertions at positions 98, 131, 175, 403, and 432 abrogated metal transport by Slc11a2, while insertions at positions 1, 201, 243, 284, 344, 468, 504, and 561 resulted in functional proteins. Topology mapping in functional HA-tagged Slc11a2 proteins indicated that the N-terminus (1), as well as loops delineated by TMD4-5 (201), TMD6-7 (284), and TMD10-11 (468), and C-terminus (561) are intracellular, while loops separating TMD5-6 (243), TMD7-8 (344), and TMD11-12 (504) are extracellular. These results are compatible with a topology of 12 transmembrane domains, with intracellular amino and carboxy termini. Structural models constructed by homology threading support this 12TMD topology and show 2-fold structural symmetry in the arrangement of membrane helices for TM1-5 and TM6-10 (conserved Slc11 hydrophobic core).

Transcription factors Sp1 and C/EBP regulate NRAMP1 gene expression.

The natural resistance-associated macrophage protein 1 (Nramp1), which belongs to a conserved family of membrane metal transporters, contributes to phagocyte-autonomous antimicrobial defense mechanisms. Genetic polymorphisms in the human NRAMP1 gene predispose to susceptibility to infectious or inflammatory diseases. To characterize the transcriptional mechanisms controlling NRAMP1 expression, we previously showed that a 263 bp region upstream of the ATG drives basal promoter activity, and that a 325 bp region further upstream confers myeloid specificity and activation during differentiation of HL-60 cells induced by vitamin D. Herein, the major transcription start site was mapped in the basal region by S1 protection assay, and two cis-acting elements essential for myeloid transactivation were characterized by in vitro DNase footprinting, electrophoretic mobility shift experiments, in vivo transfection assays using linker-mutated constructs, and chromatin immunoprecipitation assays in differentiated monocytic cells. One distal cis element binds Sp1 and is required for NRAMP1 myeloid regulation. Another site in the proximal region binds CCAAT enhancer binding proteins alpha or beta and is crucial for transcription. This study implicates Sp1 and C/EBP factors in regulating the expression of the NRAMP1 gene in myeloid cells.

Solute carrier 11 cation symport requires distinct residues in transmembrane helices 1 and 6.

Ubiquitous solute carriers 11 (SLC11) contribute to metal-ion homeostasis by importing Me(2+) and H(+) into the cytoplasm. To identify residues mediating cation symport, Escherichia coli proton-dependent manganese transporter (MntH) was mutated at five SLC11-specific transmembrane (TM) sites; each mutant activity was compared with wild-type MntH, and the biochemical results were tested by homology threading. Cd(2+) and H(+) uptake kinetics were analyzed in whole cells as a function of pH and temperature, and right-side out membrane vesicles were used to detail energy requirements and to probe site accessibility by Cys replacement and thiol modification. This approach revealed that TM segment 1 (TMS1) residue Asp(34) couples H(+) and Me(2+) symport and contributes to MntH forward transport electrogenicity, whereas the TMS6 His(211) residue mediates pH-dependent Me(2+) uptake; MntH Asn(37), Asn(250), and Asn(401) in TMS1, TMS7, and TMS11 participate in transporter cycling and/or helix packing interactions. These biochemical results fit the LeuT/SLC6 structural fold, which suggests that conserved peptide motifs Asp(34)-Pro-Gly (TMS1) and Met-Pro-His(211) (TMS6) form antiparallel "TM helix/extended peptide" boundaries, lining a "pore" cavity and enabling H(+)-dependent Me(2+) import.

Nramp1 phagocyte intracellular metal withdrawal defense.

Natural resistance-associated macrophage proteins (Nramp) are multispecific symporters facilitating proton-dependent import of divalent metals. Nramp1 restricts microbial access to essential micro-nutrients such as iron and manganese within professional phagosomes. Increased understanding of Nramp1 roles in human phagocytes will be useful for future therapeutic approaches against selected infectious and immune diseases.

Identification of functional amino acids in the Nramp family by a combination of evolutionary analysis and biophysical studies of metal and proton cotransport in vivo.

The natural resistance-associated macrophage protein (Nramp) family is functionally conserved in bacteria and eukarya; Nramp homologues function as proton-dependent membrane transporters of divalent metals. Sequence analyses indicate that five phylogenetic groups comprise the Nramp family, three bacterial and two eukaryotic, which are distinct from a more distantly related group of microbial sequences (Nramp outgroup). The Nramp family and outgroup share many conserved residues, suggesting they derived from a common ancestor and raising the possibility that the residues invariant in the Nramp family that correspond to residues which are different but also conserved in the outgroup represent candidate sites of functional divergence of the Nramp family. Four Nramp family-specific residues were identified within transmembrane domains 1, 6, and 11, and replaced by the corresponding invariant outgroup residues in the Escherichia coli Nramp ortholog (the proton-dependent manganese transporter, MntH of group A, EcoliA). The resulting mutants (Asp(34)Gly, Asn(37)Thr, His(211)Tyr, and Asn(401)Gly) were tested for both divalent metal uptake and proton transport; quasi-simultaneous analyses of uptake of metals and protons revealed for the first time protons and metals cotransport by a bacterial Nramp homologue. Additional mutations were studied for comparison (Asp(34)Asn, Asn(37)Asp and Asn(37)Val, Asn(401)Thr, His(211)Ala, His(216)Ala, and His(216)Arg). EcoliA activity was impaired after each of the Nramp/outgroup substitutions, as well as after more conservative replacements, showing that the tested sites are all important for metal uptake and metal-dependent H(+) transport. It is proposed that co-occurrence of these four Nramp-specific transmembrane residues may have contributed to the emergence of this family of metal and proton cotransporters.

Gene organization and expression of the divalent cation transporter Nramp in the protistan parasite Perkinsus marinus.

Trophozoites of the protistan parasite Perkinsus marinus reside and proliferate inside phagosomelike structures of hemocytes from the host, the eastern oyster Crassostrea virginica. In a murine model, it has been proposed that the outcome of intracellular parasite-host interactions is determined, at least in part, by the activity of the host's divalent cation transporter natural resistance-associated macrophage protein 1 (Nramp1). Although nucleotide sequences from members of the Nramp family in protozoan parasites have recently become available in public databases, little is known about their molecular, structural, and functional aspects that may relate to the parasite's survival of intracellular killing by the host. The complementary DNA (cDNA) sequence of the Nramp from P. marinus (PmNramp) was obtained by polymerase chain reaction amplification with degenerated primers, followed by rapid amplification of cDNA ends. The 2,082-bp cDNA sequence encoded a predicted protein of 558 amino acids. PmNramp is a single-copy gene composed of 7 exons and 6 short introns (44-61 bp) with the canonical splicing signal (GT/AG). A phylogenetic analysis indicates that P. marinus and apicomplexan Nramp genes derive from a common "archetype" Nramp ancestor. However, the apicomplexan Nramps are highly divergent from the P. marinus sequence and the rest of the archetype Nramp group. Preliminary studies suggest that expression of PmNramp in in vitro-cultured P. marinus trophozoites is modulated by metals and by exogenous oxidative stress.

Determination of transmembrane topology of the Escherichia coli natural resistance-associated macrophage protein (Nramp) ortholog.

The natural resistance-associated macrophage protein (Nramp) defines a conserved family of secondary metal transporters. Molecular evolutionary analysis of the Nramp family revealed the early duplication of an ancestral eukaryotic Nramp gene, which was likely derived from a bacterial ortholog and characterized as a proton-dependent manganese transporter MntH (Makui, H., Roig, E., Cole, S. T., Helmann, J. D., Gros, P., and Cellier, M. F. (2000) Mol. Microbiol. 35, 1065-1078). Escherichia coli MntH represents a model of choice to study structure function relationship in the Nramp protein family. Here, we report E. coli MntH transmembrane topology using a combination of in silico predictions, genetic fusion with cytoplasmic and periplasmic reporters, and MntH functional assays. Constructs of the secreted form of beta-lactamase (Blam) revealed extra loops between transmembrane domains 1/2, 5/6, 7/8, and 9/10, and placed the C terminus periplasmically; chloramphenicol acetyltransferase constructs indicated cytoplasmic loops 2/3, 6/7, 8/9, and 10/11. Two intra loops for which no data were produced (N terminus, intra loop 4/5) both display composition bias supporting their deduced localization. The extra loops 5/6 and 6/7 and periplasmic exposure of the C terminus were confirmed by targeted reporter insertion. Three of them preserved MntH function as measured by a disk assay of divalent metal uptake and a fluorescence assay of divalent metal-dependent proton transport, whereas a truncated form lacking transmembrane domain 11 was inactive. These results demonstrate that EcoliA is a type III integral membrane protein with 11 transmembrane domains transporting both divalent metal ions and protons.

Horizontal gene transfer of "prototype" Nramp in bacteria.

Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in Calpha, Cbeta, Cgamma. The proteins from C. tepidum (MntH B) and E. faecalis (MntH Cbeta1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria Wigglesworthia mntH Cbeta gene is linked to an Asn t-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the Cbeta genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcus lactis and Burkholderia mallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii "prototype" Nramp and some MntH Calpha (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other "prototype" Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that "prototype" Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection.

Expression and subcellular localization of NRAMP1 in human neutrophil granules.

Mutations at the Nramp1 gene cause susceptibility to infections with intracellular pathogens. In human blood, polymorphonuclear (PMN) leukocytes are the most abundant site of NRAMP1 messenger RNA (mRNA) expression, suggesting that NRAMP1 plays an important role in the activity of these cells. By Northern blot analysis, NRAMP1 mRNA was only detected in most mature neutrophils from bone marrow (band and segmented cells). A high-affinity polyclonal rabbit antihuman NRAMP1 antibody directed against the amino terminus of the protein was produced and used to study cellular and subcellular localization of the protein in primary human neutrophils. Subcellular fractionation of granule populations together with immunoblotting studies with granule-specific markers indicate that NRAMP1 expression is primarily in tertiary granules. These granules are positive for the matrix enzyme gelatinase and the membrane subunit of the vacuolar H(+)/ATPase and can be recruited for exocytosis by treatment of neutrophils with phorbol myristate acetate. Immunogold studies by cryoelectron microscopy with primary neutrophils confirm that a majority (75%) of NRAMP1-positive granules are also positive for gelatinase, but they also suggest further heterogeneity in this granule population. Presence of NRAMP1 in tertiary granules is in agreement with the late-stage appearance of NRAMP1 mRNA during neutrophil maturation in bone marrow. Finally, immunofluorescence studies of Candida albicans-containing phagosomes formed in neutrophils indicate that NRAMP1 is recruited from tertiary granules to the phagosomal membrane on phagocytosis, supporting a role for NRAMP1 in the antimicrobial defenses of human neutrophils.