The Nramp Family

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MOLECULAR BIOLOGY INTELLIGENCE UNIT

Mathieu Cellier and Philippe Gros CELLIER • GROS MBIU

The Nramp Family

The Nramp Family


The Nramp Family Mathieu Cellier, Ph.D. Institut National de la Recherche Scientifique INRS-Institut Armand-Frappier Laval, Québec, Canada

Philippe Gros, Ph.D. Department of Biochemistry McGill University Montréal, Québec, Canada

LANDES BIOSCIENCE / EUREKAH.COM GEORGETOWN, TEXAS U.S.A.

KLUWER ACADEMIC / PLENUM PUBLISHERS NEW YORK, NEW YORK U.S.A.

To Mireille and Catherine

CONTENTS Preface ................................................................................................ xiii 1. Mouse Natural Resistance Associated Macrophage Protein 1 (Nramp1): A Key Player in Host Innate Immunity against Infections .................................................................. 1 Nada Jabado, Steven Lam-Yuk-Tseung, John R. Forbes and Philippe Gros Why Mouse Models? ............................................................................. 2 Nramp1: A Mouse Host Resistance Locus ............................................. 2 Pathogens Under Nramp1 Control ....................................................... 3 Nramp1 Gene and Protein Expression in Macrophages ......................... 4 The Nramp Gene Family ...................................................................... 4 Divalent Cation Transport by Nramp Proteins ..................................... 5 Nramp1 Dependent Divalent Cations Transport at the Phagosomal Membrane ........................................................... 6 Structure/Function Studies by Site Directed Mutagenesis ..................... 7 Divalent Cation Transport and Resistance to Infections ........................ 8 Nramp1 and Phagosomal Maturation ................................................... 9 2. Genetic Susceptibility to Infectious Diseases Linked to NRAMP1 Gene in Farm Animals ..................................................... 16 Judith Caron, Danielle Malo, Christopher Schutta, Joe W. Templeton and L. Garry Adams Comparative Genomics ....................................................................... 17 Nramp1 .............................................................................................. 17 Chicken Genomics, Salmonella Infection and NRAMP1 ..................... 18 Bovine Genomics, Brucella abortus Infection and NRAMP1 ................ 21 3. The NRAMP Genes and Human Susceptibility to Common Diseases............................................................................ 29 Audrey Poon and Erwin Schurr NRAMP1 Gene Location and Structure .............................................. 30 NRAMP1 Polymorphisms ................................................................... 30 NRAMP1 Function ............................................................................. 32 NRAMP1 and Infectious Diseases Susceptibility .................................. 32 NRAMP1 and Autoimmune Diseases Susceptibility ............................ 36 NRAMP2 Location and Genomic Structure ........................................ 38 NRAMP2 Functions ............................................................................ 38 NRAMP2 Polymorphisms ................................................................... 38 NRAMP2 Polymorphisms and Human Diseases Susceptibility ............ 39

4. Pleiotropic Effects of Nramp (Bcg/Lsh/Ity) Gene Expression on Macrophage Functions .................................................................... 44 Luis F. Barrera and Martin Olivier Resistance to Three Pathogens Controlled by One Gene ..................... 45 Bcg/Lsh/Ity Gene Expression in Macrophage ........................................ 45 Bcg/Lsh/Ity Gene Pleiotropic Effects on Macrophage Functions ........... 45 Nramp, a Candidate Gene for Bcg/Lsh/Ity Gene ................................... 47 Impact of Nramp Gene Expression on Macrophage Signaling Activity and Functions Regulation ................................... 48 5. Role of Nramp Family in Pro-Inflammatory Diseases .......................... 53 Jenefer M. Blackwell, Hui-Rong Jiang and Jacqueline K. White Genetic Associations with Pro-Inflammatory Autoimmune Disease in Man ................................................................................ 54 Evidence for Gene x Environment Interactions? .................................. 54 Searching for the Functional Polymorphisms in SLC11A1 .................. 56 Relating Disease Phenotypes to Pleiotropic Effects of SLC11A1 .......... 57 A Direct Role for Iron in SLC11A1 Regulated Autoimmune Disease Phenotypes? ........................................................................ 58 Mouse Models to Study Slc11a1 Regulation of Pro-Inflammatory Diseases .......................................................... 59 6. Role of Nramp2 (DMT1) in Iron Homeostasis .................................... 65 Nancy C. Andrews Mammalian Iron Metabolism .............................................................. 65 Roles of Nramp2 (DMT1) in Iron Homeostasis .................................. 66 Nramp2 (DMT1) in Iron Disorders .................................................... 68 7. Molecular Physiology of the H+-Coupled Iron Transporter DMT1 .............................................................................. 73 Bryan Mackenzie and Matthias A. Hediger Molecular Mechanisms of DMT1 ....................................................... 73 Substrate Profile .................................................................................. 75 Structure-Function Analysis of DMT1 ................................................ 76 The Role of DMT1 in the Biology of Iron Transport .......................... 77 Multiple Splice Forms Reveal Discrete Expression Control Mechanisms ....................................................................... 77 DMT1 and Its Association with Human Disease ................................. 79 8. Cellular and Tissue Expression of Rat DMT1 / Nramp 2 .................... 82 Evan H. Morgan Gastrointestinal Tract .......................................................................... 84 Liver .................................................................................................... 86 Erythroid Tissue .................................................................................. 88 Brain ................................................................................................... 91 Kidney ................................................................................................ 92 Placenta ............................................................................................... 93

9. Tissue Distribution and Subcellular Localization of Nramp Proteins ............................................................................... 96 François Canonne-Hergaux and Philippe Gros NRAMP1: Discovery of The Nramp1 Gene ....................................... 96 Tissue and Cellular Expression of Mouse and Human Nramp1 mRNA .......................................................... 97 Cellular and Subcellular Expression of the Nramp1 Protein in Macrophages and in Neutrophils ................................................. 97 NRAMP2: Nramp2/DMT1 Gene, RNA and Protein Isoforms ........... 99 Topology Model of Nramp2/DMT1 Protein .................................... 100 Nramp2/DMT1: Ubiquitous and Cell Specific Expression ............... 101 Nramp2/DMT1 in Epithelial Cells ................................................... 104 Nramp2/DMT1 in Peripheral Tissues ............................................... 106 Nramp2/DMT1 Studies: Rich but Controversial Literature .............. 109 10. Plant Metal Transporters with Homology to Proteins of the NRAMP Family ....................................................................... 113 Sebastien Thomine and Julian I. Schroeder Genomic Analysis of the NRAMP Family in Plant Species ................ 114 Functional Characterization of NRAMP Metal Transport Properties in Heterologous Expression Systems ............................. 115 NRAMP Gene Expression Pattern and Regulation in Plants ............. 118 Analysis of NRAMP Functions in Plants ........................................... 119 Conclusions and Perspectives for the Analysis of Plant NRAMP Functions ....................................................................... 121 11. The Role of Yeast Nramp Metal Transporters in Manganese and Iron Homeostasis ........................................................................ 124 Edward Luk, Laran Jensen and Valeria Culotta Historical Perspective: Why the Name SMF? .................................... 125 The Function of S. cerevisiae Smf1p .................................................. 125 Smf2p as a Manganese Transporter ................................................... 127 Smf3p as an Iron Transporter ............................................................ 127 Using Yeast as a Model to Study Nramp Metal Transport from Diverse Species ..................................................................... 129 Post-Translation Regulation of SMFs by Manganese ......................... 129 Transcriptional Regulation of SMF3 by Fe ........................................ 131 12. Metal-Ion Transporters: From Yeast to Human Diseases ................... 135 Adiel Cohen, Hannah Nelson and Nathan Nelson Discovery of the Yeast Smf1p as a Metal-Ion Transporter Revealed that Metal Ions Function through NRAMP in Resistance and Sensitivity to Bacterial Infection ........................ 136 A Glimpse into the Mechanism of Metal-Ion Uptake ........................ 139 Expression of Heterologous Metal-Ion Transporters in Yeast Cells ................................................................................. 141 The Involvement of NRAMP in Diseases .......................................... 142

13. Regulation of Bacterial MntH Genes ................................................. 146 John D. Helmann Mycobacterium tuberculosis ................................................................. 147 Bacillus subtilis ................................................................................... 147 Staphylococcus aureus .......................................................................... 148 Escherichia coli and Salmonella enterica serovar Typhimurium ........... 150 14. Manganese and Iron Transport by Prokaryotic Nramp Family Transporters ....................................... 154 Krisztina M. Papp, David G. Kehres and Michael E. Maguire Nramp Proteins in Prokaryotes ......................................................... 155 Physiological Cation Transport ......................................................... 161 Transport Mechanism ....................................................................... 162 Regulation of MntH ......................................................................... 163 Other Mn2+ Transporters ................................................................... 163 Mn2+ in Pathogenesis ......................................................................... 165 15. Role of the Nramp Orthologue, MntH, in the Virulence of Mycobacterium tuberculosis ............................................................ 172 Pilar Domenech and Stewart T. Cole M. tuberculosis Is an Intracellular Pathogen ........................................ 172 Murine Nramp1 and the M. tuberculosis Phagosome ......................... 173 M. tuberculosis MntH Is Constitutively Expressed ............................. 173 M. tuberculosis MntH Protein As a Cation Transporter ..................... 174 Role of the M. tuberculosis MntH Protein in Virulence ...................... 174 16. Molecular Evolutionary Analysis of the Nramp Family ...................... 178 Etienne Richer, Pascal Courville and Mathieu Cellier Early Gene Duplication in Eukaryotes Gave the Outparologs ‘Prototype’ and ‘Archetype’ Nramp ..................... 179 Characterization of Bacterial Nramp Orthologs: MntH A and B .............................................................................. 182 Study of MntH Xenologs: MntH Cα, Cβ, Cγ .................................. 183 Proposed Evolutionary Pathway of Bacterial Nramp Genes ............... 189 Significance and Implications in the Context of Bacterial Infection ..................................................................... 191 Index .................................................................................................. 195

EDITORS Mathieu Cellier, Ph.D. Institut National de la Recherche Scientifique INRS-Institut Armand-Frappier Laval, Québec, Canada Chapter 16

Philippe Gros, Ph.D. Department of Biochemistry McGill University Montréal, Québec, Canada Chapter 1, 9

CONTRIBUTORS L. Garry Adams Department of Veterinary Pathobiology College of Veterinary Medicine Texas A&M University College Station, Texas, U.S.A. [email protected]

François Canonne-Hergaux Faculté de Médecine Xavier Bichat INSERM, Genetic and Molecular Pathology of Hematopoiesis Paris, Cedex, France [email protected]

Chapter 2

Chapter 9

Nancy C. Andrews Division of Hematology and Oncology Children’s Hospital Boston Boston, Massachusetts, U.S.A. [email protected]

Judith Caron Department of Human Genetics McGill University Health Center Center for the Study of Host Resistance Montréal General Hospital Montréal, Québec, Canada

Chapter 6

Chapter 2

Luis F. Barrera Facultad de Medicina Universidad de Antioquia Medellin, Colombia Chapter 4

Jenefer M. Blackwell Cambridge Institute for Medical Research University of Cambridge Cambridge, U.K. [email protected] Chapter 5

Adiel Cohen Department of Biochemistry George S. Wise Faculty of Life Sciences Tel Aviv University Tel Aviv, Israel Chapter 12

Stewart T. Cole UGMB Institut Pasteur Paris, Cedex, France [email protected] Chapter 15

Pascal Courville INRS-Institut Armand-Frappier Laval, Québec, Canada Chapter 16

Nada Jabado Department of Pediatrics Montreal Children’s Hospital McGill University Montréal, Québec, Canada

Valeria Culotta Departments of Environmental Health Sciences, Biochemistry and Molecular Biology Division of Toxicological Sciences Bloomberg School of Public Health Johns Hopkins University Baltimore, Maryland, U.S.A. [email protected]

Chapter 1

Chapter 11

Chapter 11

Pilar Domenech TBRS, LIG, NIAID Rockville, Maryland, U.S.A. [email protected]

Hui-Rong Jiang Cambridge Institute for Medical Research University of Cambridge Cambridge, U.K.

Chapter 15

Laran Jensen Departments of Environmental Health Sciences, Biochemistry and Molecular Biology Bloomberg School of Public Health Johns Hopkins University Baltimore, Maryland, U.S.A.

Chapter 5

John R. Forbes Department of Biochemistry McGill University Montréal, Québec, Canada Chapter 1

Matthias A. Hediger Membrane Biology Program and Renal Division Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts, U.S.A. [email protected]

David G. Kehres Department of Pharmacology School of Medicine Case Western Reserve University Cleveland, Ohio, U.S.A. Chapter 14

Steven Lam-Yuk-Tseung Department of Biochemistry McGill University Montréal, Québec, Canada Chapter 1

Chapter 7

John D. Helmann Department of Microbiology Cornell University Ithaca, New York, U.S.A. [email protected] Chapter 13

Edward Luk Departments of Environmental Health Sciences, Biochemistry and Molecular Biology Bloomberg School of Public Health Johns Hopkins University Baltimore, Maryland, U.S.A. Chapter 11

Bryan Mackenzie Membrane Biology Program and Renal Division Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts, U.S.A. Chapter 7

Michael E. Maguire Department of Pharmacology School of Medicine Case Western Reserve University Cleveland, Ohio, U.S.A. Chapter 14

Danielle Malo Department of Human Genetics and Medicine McGill University Health Center Center for the Study of Host Resistance Montréal General Hospital Montréal, Québec, Canada [email protected]

Martin Olivier Departments of Medicine, Microbiology and Immunology McGill University Health Centre Research Institute Centre for the Study of Host Resistance Montréal, Québec, Canada [email protected] Chapter 4

Krisztina M. Papp Department of Pharmacology School of Medicine Case Western Reserve University Cleveland, Ohio, U.S.A. [email protected] Chapter 14

Audrey Poon McGill University Health Centre Research Institute Montréal General Hospital Montréal, Québec, Canada

Chapter 2

Chapter 3

Evan H. Morgan Department of Physiology School of Biomedical and Chemical Sciences University of Western Australia Crawley, Western Australia, Australia [email protected]

Etienne Richer INRS-Institut Armand-Frappier Laval, Québec, Canada [email protected]

Chapter 8

Hannah Nelson Department of Biochemistry George S. Wise Faculty of Life Sciences Tel Aviv University Tel Aviv, Israel

Chapter 16

Julian I. Schroeder Cell and Developmental Biology Section Division of Biology Center for Molecular Genetics University of California, San Diego La Jolla, California, U.S.A. [email protected] Chapter 10

Chapter 12

Nathan Nelson Department of Biochemistry George S. Wise Faculty of Life Sciences Tel Aviv University Tel Aviv, Israel [email protected] Chapter 12

Erwin Schurr McGill University Health Centre Research Institute Montréal General Hospital Montréal, Québec, Canada [email protected] Chapter 3

Christopher Schutta Department of Veterinary Pathobiology College of Veterinary Medicine Texas A&M University College Station, Texas, U.S.A.

Sebastien Thomine Institut des Sciences du Vegetal Gif-sur-Yvette, Cedex, France [email protected] Chapter 10

Chapter 2

Joe W. Templeton Department of Veterinary Pathobiology College of Veterinary Medicine Texas A&M University College Station, Texas, U.S.A. Chapter 2

Jacqueline K. White Cambridge Institute for Medical Research University of Cambridge Cambridge, U.K. Chapter 5

PREFACE A genetic component for susceptibility to typhoid first came to light in mice following the development of inbred mouse strains. The pioneering work of Webster, Schott, and others in the 1920s and 1930s identified important differences in the survival of inbred strains to a lethal challenge with Salmonella. This laid the groundwork for subsequent genetic analyses that demonstrated that susceptibility to S. typhimurium was indeed under simple genetic control in mice. In the early 1980s, a major locus controlling S. typhimurium replication in vivo was mapped to the proximal portion of mouse chromosome 1 and was designated Ity. Ity appeared not only to control bacterial replication in target tissues and survival in the context of an acute infection, but also appeared to affect the ability of mice to mount a protective immune response following vaccination with avirulent isolates. Independently, it was discovered that Lsh, a locus responsible for controlling the replication of Leishmania donovani in liver, also maps to the vicinity of Ity and studies in recombinant inbred mouse strains suggested that they were indeed the same locus. Finally, while studying the genetic control of replication of small inoculum of avirulent Mycobacteria (M. bovis, BCG) in spleen of inbred strains, we mapped a locus (Bcg) to the same region of chromosome 1. Similarities in phenotypic expression of resistance/susceptibility in the three infection models together with common map locations suggested that Ity, Lsh and Bcg were most likely the same gene affecting the replication of unrelated intracellular infectious agents. Studies in vivo and studies in explanted cell populations ex vivo showed that the macrophage was the cell type affected by Ity/Lsh/Bcg, and it was proposed that the locus probably regulates an important bacteriostatic or bactericidal function of these cells towards antigenically and taxonomically unrelated intracellular pathogens. In the early 1990s, the locus was isolated by positional cloning. The gene was identified on the basis of its genetic and physical location, the specific expression of its mRNA in macrophages and the presence of sequence polymorphism associated with susceptibility to infection in inbred strains. The gene was designated Nramp1 for “Natural Resistance Associated Macrophage Protein 1”and was found to encode a predicted hydrophobic peptide possibly corresponding to an integral membrane protein. This book picks up the story from there. It summarizes recent work by several laboratories on the characterization of the Nramp1 protein (recently re-named Slc11a1) and of the role it plays in resistance to infection and in macrophage function, including its biochemical mechanism of action. It also explores the function of the close mammalian homolog Nramp2 (also named Divalent Metal Transporter1, DMT1 and Slc11A2) and the critical role it performs in iron metabolism. This book further contains a series of chapters that illustrate the astonishing degree of conservation of this protein family throughout evolution and that analyze the role of these proteins in divalent metals metabolism not only in mammals but also in plants, fungi and bacteria.

The mouse Nramp1 protein is expressed at the membrane of lysosomes and late endosomes of macrophages and is targeted to the membrane of phagosomes soon after phagocytosis. The proposed functions of Nramp1 at the phagosomal membrane are reviewed along with the substrates and proposed mechanisms of transport at that site. The effect of Nramp1 transport on the ability of internalized bacteria to influence their intracellular fate in mouse macrophages is also addressed (Chapter 1). Comparative genomic approaches have led to the characterization of the NRAMP1 orthologs in farm animals and have provided evidence that these genes contribute to host resistance to bacterial infections (Chapter 2). In humans, polymorphic variants within or near the human NRAMP1 ortholog have been identified and have been found associated with susceptibility to infections such as tuberculosis and leprosy both in regions of endemic disease and in the outbreak situation (Chapter 3). Likewise, certain NRAMP1 polymorphisms have been associated with certain inflammatory conditions, related to the pleitotropic effects of Nramp1 on macrophage functions (Chapter 4), and to pro-inflammatory responses typical of human pathologies (Chapter 5). Vertebrates also possess a closely related homolog Nramp2 gene that was initially identified by cross hybridization. Nramp2 turned out to be a key protein in iron metabolism that has been extensively studied. Much of what we now understand about Nramp1 is derived in part from parallel studies of Nramp2. Nramp2 protein mediates transferrin-independent iron uptake at the intestinal brush border but is also responsible for transport of transferrin-iron across the membrane of acidified endosomes in peripheral tissues. A mutation in Nramp2 (Dmt1) was shown to be responsible for microcytic anemia in the mk mouse and in the Belgrade rat which were identified in the 1960s as animals with natural and radiation-induced erythroid defects (Chapter 6). Based on differential expression in the proximal intestine in response to iron deprivation, Nramp2 mRNA was functionally cloned and characterized in Xenopus oocytes as a pH-dependent iron uptake system (Chapter 7). Much work has been done to characterize the organ, cellular and sub-cellular distribution of Nramp2 mRNA transcripts and proteins, along with their regulation by iron in normal and mutant animals (Chapter 8). The transport function of Nramp2 in iron metabolism and its role in the iron regulatory networks of the body will also be reviewed with special emphasis on the tissue, cellular and subcellular localization of Nramp 2 protein compared to Nramp1 (Chapter 9). A great diversity of organisms express conserved Nramp homologs. In all the species where they have been studied, Nramp homologs were shown to function as pH-dependent divalent metal transporters, indicating that the remarkable sequence conservation throughout evolution reflects both Nramp substrate ubiquity and similar transport function. Green plants in-

cluding Arabidopsis thaliana possess several Nramp homologs. Plant Nramps are involved in metal uptake from soil, distribution to other tissues and subcellular metabolism; their number provided the opportunity to compare substrate specificities and regulation of expression by different environmental metal conditions (Chapter 10). There exist three homologs in the yeast Saccharomyces cerevisiae (named SMF1, 2 and 3) that have complementary roles in manganese and iron homeostasis, based on the elucidation of their subcellular localization and metal-dependent regulation of expression (Chapter 11). The first hints of Nramp protein family function came from the study of yeast Smf genes. Initially identified as high-copy suppressors of the mitochondrial import factor mif mutants (smf ), the SMF1 and 2 proteins were shown to mediate uptake of divalent metals in yeast cells and have been studied by electrophysiology in Xenopus oocytes (Chapter 12). The functional conservation in the Nramp family is illustrated in eukaryotes by the fact that mammalian and plant Nramp proteins can complement yeast smf mutants. Last but not least, increasing numbers of Nramp-related genes (encoding proton-dependent manganese transporters, MntH) have been identified in a substantial fraction of genomes of Bacteria (vs. Archaea) which sequence have recently become available. The bacterial sequences belong to three phylogenetic groups; MntH homologs from groups A and C have been functionally characterized in their respective hosts. Their mechanisms of regulation by metal availability will be reviewed (Chapter 13); transport properties of MntH A proteins and their possible impact in virulence for a mammalian host have been analyzed in selected Gram negative (Chapter 14) and Gram positive bacteria (Chapter 15). MntH groups differ in both their taxonomic and phylogenetic patterns, including intriguing examples of horizontal transmission and resemblance to eukaryotic Nramp. These studies suggest an evolutionary pathway for the emergence of eukaryotic Nramp in possible relation to host defense (Chapter 16). Mathieu Cellier Philippe Gros

CHAPTER 1

Mouse Natural Resistance Associated Macrophage Protein 1 (Nramp1): A Key Player in Host Innate Immunity against Infections Nada Jabado, Steven Lam-Yuk-Tseung, John R. Forbes and Philippe Gros

Abstract

N

ramp1 is one of the few host resistance genes that have been well characterized at the molecular and functional level. This protein is an integral transmembrane protein expressed in the lysosomal compartment of phagocytic cells and is recruited to the phagosomal membrane where it affects pathogen replication. Nramp1 is part of a large gene family conserved through evolution that codes for divalent cation transporters. In this chapter, we will summarize studies carried out on this gene in the mouse model, and will review recent work on the protein and its mechanism of action at the membrane of phagosomes formed in phagocytes. We propose that Nramp1 is a pH-dependent divalent cation efflux pump at the phagosomal membrane. It affects microbial replication by modulating divalent cation content in the phagosomal space.

Introduction Infectious diseases remain a major health problem worldwide, accounting for 33% of world mortality. The spreading of Human Immunodeficiency Virus (HIV) made tuberculosis and other mycobacterial infections undergo a dramatic comeback in many western as well as in third world countries and it is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis. The widespread emergence of multi-drug resistance in these mycobacterial infections, as in almost all infectious diseases, has increased the severity of this health problem and has prompted the search for alternative strategies for intervention. It is widely recognized that genetic factors are important in the response to infectious diseases and that, in the majority of infections, only a proportion of individuals exposed to the same pathogen will develop a clinically active disease. For example, in tuberculosis only around 10% of individuals who become infected will in fact develop clinical disease. These individual differences in susceptibility are influenced both by environmental factors, in particular those affecting exposure and virulence status of the bacterium, but also host-derived such as genetic predisposition, and type, and efficiency of immune response. On the pathogen side, microbes have developed sophisticated virulence mechanisms to either block or subvert normal host cellular processes contributing to increased pathogenicity. Identification of genes/proteins involved in host defense mechanisms or in increased microbial pathogenicity will likely improve our understanding of infectious diseases and may provide much needed novel targets for pharmacological intervention. Nramp1 is one of at least 11 genes/loci identified to date and affecting host susceptibility/resistance to mycobacterial infections.1 This gene plays a pivotal role in macrophage function, and the mechanism of action of the encoded protein is now fairly The Nramp Family, edited by Mathieu Cellier and Philippe Gros. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers.

The Nramp Family

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well understood. Here, we will summarize studies carried out on this gene in the mouse model, and will review recent work on the protein and its mechanism of action at the membrane of phagosomes formed in phagocytes.

Why Mouse Models? Although a genetic component for susceptibility to infectious diseases such as tuberculosis and leprosy has been well-established in humans,2 the study of individual genes involved is difficult due to the multifactorial nature of disease susceptibility, the complexity of the genetic control, the intrinsic heterogeneity of human populations, and the difficulty of obtaining repeated biological samples for in vitro analysis.3 In addition, variations in pathogen-specific virulence determinants that ultimately reveal host susceptibility, further complicate genetic analyses in humans. Such complex interactions can be teased out (including the identification of relevant genes) in appropriate animal models of infections where the basic pathophysiology of the human disease is respected in the animal host. The laboratory mouse is the experimental model of choice for this type of genetic analysis for the following reasons: 1. Large numbers of well characterized and carefully maintained inbred, recombinant inbred and mutant stocks are available to study disease genes. 2. Mouse is the best genetically characterized mammal with high-density coverage of informative markers for linkage studies. 3. Informative segregating animals can be generated easily and quickly in big numbers. 4. A complete transcript map and genome assembly are available. 5. Candidate genes can be targeted for inactivation in the germ line by homologous recombination, and gain-of-function alleles can be created by reintroducing normal variants on mutated background either as single genes or as group of genes clustered on BAC clones (functional complementation in vivo).

Nramp1: A Mouse Host Resistance Locus In the early 70’s, the first reports describing a mouse locus possibly controlling intracellular replication of taxonomically different pathogens appeared in the literature. A survey of inbred mouse strains showed that they segregated into 2 groups, being either resistant (i.e., with an LD100>104-105 bacteria) or susceptible (i.e., with an LD10010 µM) and well above the physiologically relevant free Fe2+ concentration. Mn2+ has subsequently been implicated as a physiologically relevant substrate for mammalian Nramp1. Bacterial mntH loci appear to be regulated by Mn2+ through MntR, a Mn2+-dependent repressor belonging to the DtxR class of repressors. Fe2+ can also regulate mntH expression through Fur while OxyR alters expression in response to peroxide challenge. Mutation of mntH does not confer any strong growth or other Mn2+-dependent phenotype, suggesting that alternative routes for Mn2+ uptake exist. In Salmonella enterica serovar Typhimurium, this function is fulfilled by SitABCD, an ABC-class ATPase transporter. SitABCD transport has Mn2+ transport and cation selectivity properties very similar to MntH with the major exception being Zn2+ which is a much more potent inhibitor of SitABCD than MntH. Mutation of mntH does not affect the ability of S. Typhimurium to invade macrophages, nor survive within them. Upon invasion mntH expression is markedly induced. Oral inoculation of mntH S. Typhimurium in a Nramp1-/- BALB/c mouse shows no attenuation. In contrast, tail vein injection in Nramp1+/+ 129/SvJ mice showed marked attenuation for sitABCD but not mntH mutants; however complete attenuation was seen with mntH sitABCD double mutants. Oral inoculation in the Nramp1+/+ C3H strain showed marked attenuation in a mntH mutant strain. These results suggest strongly that there is an interaction between a homologous Bacterial and host gene during infection and demonstrate than Mn2+ acquisition, although for currently unknown reasons, is important for Bacterial virulence.

Introduction

More than 25 years ago, Plant and Glynn1-3 made the initial discovery that a host gene can convey resistance to a bacterial pathogen. The presence of the wild type locus referred to as ity renders the mouse less susceptible to infection from Salmonella enterica serovar Typhimurium (S. Typhimurium). The locus was eventually mapped to mouse chromosome one. Succeeding studies generated further links confirming that this locus influences host susceptibility to other infections, including Leishmania donovani (lsh) and Mycobacterium bovis (bcg). The ity, lsh, and bcg loci were all independently mapped to mouse chromosome one strongly indicating The Nramp Family, edited by Mathieu Cellier and Philippe Gros. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers.

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that the same gene was involved in the host resistance to pathogens. Interestingly, this same locus is also associated with increased resistance to Toxoplasma gondii infection.4 In 1993, using exon trapping, Vidal et al5 identified six potential candidate genes for bcg. RNA expression studies indicated one of the genes, now referred to as Nramp1 (Natural Resistance Associated Macrophage Protein 1), was primarily expressed in macrophages in the spleen and liver. Nramp1 is predicted to encode an integral membrane protein of 12 putative transmembrane domains with homology to both prokaryotic and eukaryotic transport systems.5 Further work confirmed that bcg, ity, and lsh were identical to Nramp1. A single amino acid difference, Gly to Asp at residue 169 within transmembrane domain 4 of Nramp1, was found in susceptible mice compared to resistant mice. When infected with bacteria, mice carrying the wild type isoform had fewer bacteria in their spleen and liver than those with the Asp169 allele. Macrophages that express the wild type G169 Nramp1 respond to interferon-γ or lipopolysaccharide stimulation with a substantial respiratory burst, increased formation of reactive oxygen species, and increased acidification of the phagosome, all of which would greatly assist host efforts to kill invading bacterial pathogens.6 In 1995, a second Nramp class gene, Nramp 2, mapping to chromosome 15, was discovered using a cross-hybridization approach.7-10 Unlike Nramp1, Nramp2 is expressed in multiple tissues with levels highest in the duodenum and kidney.11 The Nramp2 locus is regulated by dietary iron levels, is a crucial mediator of intestinal iron absorption, and is involved in transfer and storage of iron in the endosome.12,13 Strains of mice and rats carrying a Gly to Arg mutation in the Nramp2 residue equivalent to Gly169 of Nramp1 have been isolated. The mutation leads to microcytic anemia in both species due to iron deficiency.10,12-20 This same mutation generates symptoms of Mn2+ deficiency as well.21,22 These observations of links to cation homeostasis coupled with the protein’s localization in the membrane obviously suggested that Nramp proteins transported cations. In 1997, this was confirmed for Nramp2. Initially referred to in the rat as DCT1, Nramp2 was shown to be a highly electrogenic proton and divalent cation cotransporter with a broad substrate range that included Fe2+, Mn2+, Zn2+, Co2+, Cd2+, Cu2+, Ni2+, and Pb2+.23 Subsequent studies in mammalian systems have focused almost exclusively on iron as the physiologically relevant cation.10,12,13,15,18,24-34 More recently however, stimulated largely by work in several labs on prokaryotic Nramp homologs, Mn2+ has been implicated as a physiologically relevant substrate for Nramp1. Characterization of Nramp homologs in the Bacteria will be the focus of this review.

Nramp Proteins in Prokaryotes Initially identified in mice, Nramp homologs have been found in a wide range of organisms, including Homo sapiens, Drosophila melanogaster, Saccharomyces cerevisiae, Caenorhabditis elegans, and plants (Oryza sativa).35 Similarly, Nramp homologs are widespread in the Bacteria. A representative phylogenetic tree for Bacterial Nramps (Table 1 and Fig. 1) is strikingly congruent with that of the Bacteria as a whole, suggesting that this structural class of proteins is of ancient origin. Interestingly, there are few Nramp homologs in the Archaea based on current genomic sequence data.

Phylogenetics There are no other proteins convincingly related to Nramps in Bacterial genomes. BLAST analysis indicates that the closest prokaryotic relatives of Nramps are two small and weakly related families of hydrophobic proteins of unknown function (Table 1 and Fig. 1). One is a family of four proteins with single members in two Proteobacteria that lack Nramps and two Firmicutes that possess Nramps (labeled “Type II” in Figure 1. The other is a family of seven proteins with two isoforms in an α-Proteobacterium that thus far lacks Nramps, and one or two isoforms in three Firmicutes that possess Nramps (labeled “Type III” in Figure 1. Weaker homology with the most similar proteins of known function —branched-chain amino acid carriers in the sodium-substrate symporter family— is not sufficiently convincing to suggest analogous structures or transport mechanisms. Thus, while widespread, Bacterial Nramps have

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Table 1. Representative Nramp proteins and putative homologs

Protein1

Species or Common Name

% Identity to Human Nramp14

MTGDKGPQRL…EEDQKGETSG MISDKSPPRL…EEQGGVQGSG MSGDTGPPKQ…EDQEKGRTSG MSGDTGPPKQ…EDQEKGRTSG MSGDTGPSKQ…EDQEKGRTSG MSGDTGTPNQ…EDQEEGRTSG MPGDMGPPKQ…EDQENGRTSG MTGDTDPPKQ…HQHFLYGLLE MSGSGPAMAS…ADVPGLAGPH MVLGPEQKMS…TEEATRGYVK MVLDPKEKMP…SEDTSGGNIK MVLDPEEKIP…TVDAVSLVSR AFTGPGFLMS…VCAINMYFVV FPANNETLEV…SVIVYVTDLN

34 33 33 33 33 33 34 32 32 30 31 31 33 27

100 88 90 90 89 89 90 87 68 64 64 64 71 65

gi|746574 gi|746573 AA445891 SWAMCA1730SK gi|1346609|sp|P49283 ACC:AF047331 PID:g2921865

MASSNNDGPI…SAVHDNAGYQ MSIAYLDPGN…ETFVVNDVYF SGFSFRKLWA…IYLVLEAGFL MSSNEAYHEP…AIMGEFVNGL MALNEGYHNK…CADCNSDVLP

30 29 36 33 33

47 51 61 59 60

gi|2642450 AL035526 NID:g4539378 PID:g5231117 GI:5231117

MPQLENNEPL…PWPFKAESSH MTGSTVSRQE…MSERVVSTET MENDVKENLE…LPKRVSVSNS

33 31 32

43 45 45

First 10 and Last 10 Amino Acids of Sequence3

gi|1352521|sp|P49279 gi|730193|sp|P41251 gi|2499218|sp|Q27981 gi|3024227|sp|Q95102 gi|3024223|sp|Q2794 gi|2232291 gi|2232289 gi|3258616 gi|1709350|sp|P51027 ACC:BAA24933 PID:g2911112 gi|2921274 gi|2921272 gb|AF048761|AF048761 gb|AI588597.1|fb97b06.y1

Table contined on next page

The Nramp Family

VERTEBRATA: >HS1 (Nramp1) Human >MM1 (Nramp1) Mouse >BT1 (Nramp1) Bovine >BB1 (Nramp1) Bison >BA1 (Nramp1) Water buffalo >OA1 (Nramp1) Sheep >CL1 (Nramp1) Red deer >SS1 (Nramp1) Pig >GG1 (Nramp1) Chicken >HS2 (Nramp2) Human >MM2 (Nramp2) Mouse >RN2 (Nramp2) Rat >OM1 Rainbow trout >DA15 Zebrafish METAZOA: >CE1 C. elegans >CE2 C. elegans >BM15 Brugia malayi >DM1 D. melanogaster >DH1 D. heteroneura PLANTS: (all MAGNOLIOPHYTA) >AT1 A. thaliana >AT2 A. thaliana >AT3 A. thaliana

% Identity to S. Typhimurium MntH4

Accession or Contig Number2

Protein1



MEAEIVNVRP…KNVTAYGSLG VLQSVQIPFA…LVIVINGYLL MGVTKAEAVA…ADEDSKEPPV MRPAFSWRKL…RLRSAMTKST IPGPGFLISI…NGAAHKFKTR

(23) (37) 34 33 32

18 35 34 50 38

ACC#:Z74864 Y13140 ACC#:U00062 U00093 ACC#:Z73206 Y13138 C.albicans_Con4-2733 C.albicans_Con4-3075 C.albicans_Con4-3072 ACC#:Z69368 GI:1182037

MVNVGPSHAA…QLGMSHGDIS MTSQEYEPIQ…GFTTGKEVHL MRSYMQILQK…SYLLGADIHF MENKVSEISE…QMAVSGVTGS* MGFLTNFTNF…SMLLGYDVPL* MNQQQQQPLL…IIAFANGADI MSSQSYYMND…IVWLGMGVSF

29 31 31 30 31 31 32

26 27 28 26 27 25 26

gi|5670176|AF161317_1 gnl|Sanger_601|S.tContig367 gi|5670178|AF161318_1 gnl|Sanger_632|Y.p_Contg669 gi|5670180|AF161319_1 gi|5670182|AF161320_1 gi|5670184|AF161321_1 RRC02893 A1_32844_33512

MTDNRVENSS…LLVGTVMGLS MTDXRVENSS…LLVGTVMGLS MTNYRVESSS…WLLVGTALGL LMGPAFIAAI…ILGKLVVLIV MDPGNWATAI…NLTLLYFWFG MSAKDTPAPQ…LQLLADFAFG WATDIQAGSQ…LALGIVGATI MIHENSMSGR…IIGATVMPHA

100 95 93 80 46 40 40 49

33 31 33 34 34 37 40 35

gi|3025117|sp|P96593 MMNKDITAQS…NVFLIVDTFR* gnl|TIGR_1280_2|S.a_2653+3345 AQLAIIATDI…ILNVYLIVQT

50 34

32 33


AF141203 GI:5231114 gb|AI441171 sa52b11.y1 gi|2231132|gb|AAB62273.1 gi|2231149|gb|AAB61961.1 gi|1698582|gb|AAC49720.1


157

>ATEIN2A5 A. thaliana >GM15 Soybean >OS1 Oryza sativa >OS2 Oryza sativa >OS3 Oryza sativa FUNGI: (all ASCOMYCOTA) >SC1 S. cerevesiae >SC2 S. cerevesiae >SC3 S. cerevesiae >CB1 C. albicans >CB2 C. albicans >CB3 C. albicans >SP1 S. pombe BACTERIA:6 Proteobacteria: >ST1 S. Typhimurium >SI1 S. typhi >EC1 E. coli >YP1 Y. pestis >PA1 P. aeruginosa >PA2 P. aeruginosa >BC15 B. cepacia >RC1 R. capsulata Firmicutes: >BS1 B. subtilis >SA1 S. aureus




Table 1. Continued

158

Table 1. Continued

Protein1



MDKLSIREDN…LLYLTFTGQT* MRKINIFKGK…LNIALLVSYF* MSFWKTLFAY…AMEQQVEEVK* LAQRTQGSLK…MVLIWLTVTG LAQDTRTSLK…VMLIYLTVTG* MSHPFRLGFW…VVLIYLTVTS*

44 28 32 43 44 41

29 26 29 30 29 26

gnl|TIGR_1097|C.t_gct_3

MAVSFSKLIQ…LLSTTVTGDS*

37

30

gnl|TIGR_1299|D.r_8793

MDSRSPSLPD…GYLLWELLGG*

51

32

gb|L42023|L42023 gnl|Sanger_65699|N.m_NM.seq gi|2632707|embl|Z99106.1 gnl|TIGR_1280_2|S.a_2229

RNAVLGAAFL…FVVIIMAILS RNALIGAAFL…IVVGLMAVLS LMGAAFLMAT…ALVVIVMAVM ILLSIIIDIG…DAVLVTILTG

17 17 15 14

15 15 14 15

gnl|TIGR_920|t_f_218 gnl|TIGR_920|t_f_199 gnl|TIGR_1764|M.avium_5456 gnl|TIGR_1764|M.avium_5540 emb|AL035300|MLCB1886 gnl|GTC_1488|C.a_AE001437 gnl|GTC_1488|C.a_AE001437

MSVIPELHGA…YGYTVVFPSA* MSAAPAARNI…YGGLNSIPGW* ITGPGLIVMV…WCLVLLSLAL LAIVGPGIIV…YERADTVLGA MTYISTDAIS…PVRRIGLLML LGIIGPGLIT…IIILSLILFV SIVGPGLITA…IILTILAFAG

21 21 20 20 18 18 19

17 19 16 18 16 20 20


gi|4027858|AE001438 gnl|GTC_1488|C.a_AE001437 gnl|TIGR_1351|gef_6353 gnl|TIGR_1764|M.avium_5712 gi|3097212|embl|-AJ005699.1 gb|U15184|MLU15184


The Nramp Family

>CA1 C. acetobutylicum >CA2 C. acetobutylicum >EF1 E. faecalis >MA1 M. avium >MB17 M. bovis BCG >ML1 M. leprae Green sulfur bacteria: >CT1 C. tepidum Thermus/Deinococcus group: >DR1 D. radiodurans Type II Homologs: >HIH21 H. influenzae >NMH21 N. meningiditis >BSH21 B. subtilis >SAH21 S. aureus Type III Homologs: >TFH31 T. ferrooxidans >TFH32 T. ferrooxidans >MAH31 M. avium >MAH32 M. avium >MLH31 M. leprae >CAH31 C. acetobutylicum >CAH32 C. acetobutylicum




159

Table 1. Legend 1Protein abbreviations correspond to those used in Figure 1. 2For proteins with DNA or amino acid

sequence in GenBank, accession numbers are indicated. For proteins derived from incomplete genome projects, the most recent sequence or contig number at the time or writing is indicated. 3As sequence identifiers in incomplete genome projects may change between releases, the first and last 10 amino acids of each sequence in this table are indicated to facilitate retrieval. 4Percent identities are based on a CLUSTALW alignment of all 63 sequences in this Table. 5Nramp sequences of these organisms are incomplete. Percent identities are given for common regions. For ATEIN2A from Arabidopsis, percent identity reflects only the N-terminal portion of the protein that is homologous to Nramps. 6The following Bacterial species have complete or nearly complete genomic sequences and lack an Nramp homolog: Aquificales: Aquifex aeolicus; Chlamydiales: Chlamydia trachomatis; Cyanobacteria: Synechocystis ssp. PCC6803; Cytophagales: Porphyromonas gingivalis; Firmicutes: Streptococcus sp., Mycoplasma sp.; α-Proteobacteria: Rickettsia prowezekii; β-Proteobacteria: Bordetella sp., Neisseria sp.; γ-Proteobacteria: Haemophilus influenzae, Vibrio cholerae, A. actinomycetemcomitans; ε-Proteobacteria: Campylobacter jejuni, Helicobacter pylori; Spirochaetales: Borrelia burgdorferi, Treponema pallidum; Thermotogales: Thermotoga maritima; 7The amino acid sequence of M. tuberculosis Nramp is identical to that of M. bovis BCG 40.

not undergone large scale amplification and divergence comparable to that of the ABC and P-type ATPase transporter families. Bacterial Nramps are as similar to Eukaryotic Nramps as the latter are collectively to one other. Nramps are found in every major branch of the crown group Eukaryotes for which there is representative sequence, i.e., vertebrates, worms, insects, plants and fungi. As with Bacteria, individual Eukaryotes appear to have relatively few Nramp genes (one to three per genome), and phylogenetic relationships among them mirror those of their respective organisms. Identity among Nramps from divergent multicellular Eukaryotes ranges from 25% to 60%, identity of these to the fungal Nramps is 25% to 30%. Identity between any Eukaryotic and Bacterial Nramp is comparable to the identity between Bacterial subclasses themselves, about 30%-40% (Table 1). As within the prokaryotes, there are no other proteins within the Eukarya with similarity to the Nramps with the exception of the EIN2 protein of Arabidopsis thaliana, involved in the ethylene signaling pathway.36 Only the N-terminus of this protein shows similarity to Nramps, and the significance of this relationship is unknown.

Membrane Topology Topology predictions suggest that all Nramps, Bacterial and Eukaryotic, share a conserved core extending at least from the first transmembrane segment through the hydrophilic region following the eighth transmembrane segment, and in most cases extending through the eleventh transmembrane segment. Eukaryotic Nramps however appear to have an additional twelfth transmembrane segment that is absent in all Bacterial Nramps.

Transport Functionality of the Bacterial homologs for cation transport has been demonstrated in Mycobacterium tuberculosis, Mycobacterium leprae, Escherichia coli, S. Typhimurium, and Bacillus subtilis. In the Enterobacteriaciae, Nramp genes have been identified though not yet characterized in Salmonella paratyphi, Yersinia pestis and Klebsiella pneumoniae among others.11,35,37,38 Excepting K. pneumoniae, the Enterobacteriaciae share a gene arrangement in which Nramp and nupC (a nucleoside permease) are transcribed divergently with about 300 base pairs between the open reading frames.37 Currently, the transport function of the presumed Nramp transporters has been characterized in depth only in E. coli, S. Typhimurium,37-39 and Mycobacterium tuberculosis.40

160

The Nramp Family

Figure 1. Phylogenetic tree of bacterial NRAMPs and related proteins. Phylogenetic tree of bacterial Nramps and related proteins. A CLUSTALW alignment was performed using two human Nramps, three S. cerevisiae Nramps, 18 Bacterial Nramps and 11 Type II and Type III putative bacterial Nramp homologs indexed in (Table 1), along with an outlier group composed of 6 Bacterial branched chain amino acid transporters. Accession numbers for these latter sequences are: ECB1 (E. coli) = PID:g1786601, PAB1 (P. aeruginosa) = gi|115118|sp|P19072, SAB1 (S. aureus) = gnl|TIGR_1280|S.aureus_4348, SNB1 (S. pneumoniae) = gnl|TIGR_1313|S.pneumoniae_sp_21 , EFB1 (E. faecalis) = gnl|TIGR_1351|gef_6271 , and CP1 (C. perfringens) = gi|1220104|gnl|PID|d1009231. The CLUSTALW multiple alignment program74 was used to align Nramp sequences, to derive an unrooted phylogenetic tree using the PHYLIP neighbor-joining program75 and to assign confidence level estimates to individual tree branches using a multiple random resampling (bootstrap) program76 with 1000 resamplings as described previously.77 The results were converted to a dendrogram using a Windows 95® version of NJPlot (http://pbil.univ-lyon1.fr/software/ njplot.html). Branches shown are the most probable in all cases; the numbers on each branch of the dendrogram are the number of times in 1000 resamplings that the corresponding node occurred. Numbers above 200 are generally considered to be significant indication of a valid branching.


161

As will be detailed below, proton-stimulated divalent cation transport has been demonstrated for two bacterial Nramp proteins, distinctively Mn2+ transport, which is H+-stimulated. Consequently, Bacterial Nramp homologs have been named mntH. Makui et al38 determined that the yfeP gene (renamed mntH) of the E. coli K-12 genome was an Nramp family member and showed uptake of Fe2+ and Mn2+. Concomitantly, Kehres et al37 cloned Nramp homologs from S. Typhimurium, Pseudomonas aeruginosa, and Burkholderia cepacia, in addition to that of E. coli K-12. E. coli and S. Typhimurium each have a single Nramp homolog, and each was shown to transport divalent cations. Interestingly, P. aeruginosa and B. cepacia have two and three Nramp homologs respectively.

Mutant Phenotypes Disruption of mntH in E. coli and S. Typhimurium does not affect bacterial growth under aerobic conditions in minimal or rich medium, implying that MntH is not crucial for growth in normal laboratory conditions.37,38 The lack of a strong growth phenotype suggests either that Mn2+ is not critical for growth or that other transporters in the bacteria can compensate for the loss of MntH-mediated uptake. Phenotypes other than growth however were described by both laboratories. Of most direct relevance to the cation transport function of MntH, Makui et al38 demonstrated that overexpression of mntH from a plasmid would restore growth to the temperature sensitive hflB1 mutant, which requires high intracellular metal ion concentrations to grow at nonpermissive temperatures.41 This result suggests that transport of metal ions by MntH enables the mutant to grow at nonpermissive temperatures. In addition, when MntH was overexpressed, Mn2+, Cd2+, Co2+, Fe2+, Zn2+, Ni2+, and Cu2+ (in decreasing order) were cytotoxic to cells. Since Mn2+ was the most cytotoxic, MntH was suggested to have a higher affinity for Mn2+.38 Likewise, Kehres et al. confirmed that overexpression of mntH renders both E. coli and S. Typhimurium more sensitive to growth inhibition by Mn2+ and Cd2+, and in addition showed that the loss of mntH rendered S. Typhimurium and E. coli more sensitive to hydrogen peroxide but not superoxide.37

Physiological Cation Transport Studies in eukaryotes have shown that Nramp homologs transport a variety of divalent metals with some apparent selectivity for Fe2+ and Mn2+.12,23,42 Unfortunately, no investigators have comprehensively determined affinities of eukaryotic Nramps for potential substrate or inhibitory cations, especially Fe2+ versus Mn2+. The only values extant in the literature appear to be a demonstration of a 2 µM affinity of Fe2+ for rat Nramp2 (DCT1)43 and a Zn2+ affinity of 0.6 µM for mouse and human Nramp1.24 It is not possible without further data to determine whether mammalian Nramp1 and Nramp2 are general divalent cation transporters or are more or less selective for Fe2+, Mn2+, Zn2+ or another metal. In prokaryotes however, more information is available. Makui et al38 indicated, on the basis of a 10-fold difference in total cation uptake, that E. coli MntH preferred Mn2+ over Fe2+ at the concentrations tested. Kehres et al. directly determined cation affinities for the S. Typhimurium and E. coli MntH transporters37 either by direct kinetic analysis or by inhibition of54 Mn2+ uptake with other cations. Mg2+ and Ca2+ do not inhibit54 Mn2+ uptake in either species. E. coli MntH exhibits an affinity (Ka) of 1 µM for Mn2+ while S. Typhimurium exhibits a Ka of 0.1 µM (Fig. 2). Both affinities are independent of pH; however, for both species, the transport rate (Vmax) increases with decreasing pH, suggesting proton dependence of transport. In contrast, the Ki for inhibition by other cations, with the exception of Fe3+, is pH dependent with acid pH increasing affinities three to ten-fold. In S. Typhimurium, Ni2+, Cu2+ and Zn2+ inhibit uptake via MntH with Ki’s greater than 100 µM while Co2+ inhibits with a Ki of 20 µM. Fe3+ and Pb2+ also inhibit weakly, exhibiting Ki’s of 100 µM or greater. Cd2+ is the most potent inhibitor with a Ki of 1 µM. Fe2+ inhibition of Mn2+ uptake is pH dependent with a Ki of 10 µM at pH 5.5, rising to 100 µM at pH 7.5 (Fig. 2). The E. coli MntH had a similar inhibition profile, except that Ki’s were three- to ten-fold higher for all cations. The pattern of affinities suggests that, at least for E. coli and S. Typhimurium, the physiologically relevant

162

The Nramp Family

Figure 2. Cation inhibition of MntH transport. A S. Typhimurium strain carrying mntH on a high copy number plasmid was incubated with 0.1 µM54Mn2+ containing different concentrations of potential competitors. Uptake at different inhibitor concentrations was normalized to uptake in the absence of inhibitor. Details of this transport assay and additional cation inhibition data have been published.37 τ= Mn2+; υ= Cd2+; ν= Fe2+; σ= Fe3+; λ= Zn2+

cation transported by MntH is Mn2+. While MntH is capable of transporting Fe2+, the Ka for Fe2+ is too high to be physiological since free Fe2+ concentrations in biological systems are at least two orders of magnitude lower than the apparent Ka. In contrast, the Ka for Mn2+ is on the same order as the concentrations found in biological systems.37,39

Transport Mechanism Initial studies in eukaryotes showed that divalent metal transport through Nramp was proton-coupled (Gunshin et al, 1997)23 involving cotransport of H+ and divalent cation with a 1:1 stoichiometry. Most unusually for a transporter, subsequent work has reported that the stoichiometry can apparently be variable.34,44-46 Strikingly, while mammalian Nramp2 clearly cotransports proton and cation, murine and human Nramp1 can function as a H+ and divalent cation antiporter.24 Mechanistic studies in prokaryotes are not nearly as advanced although what data there is suggests, like eukaryotic Nramp2, cotransport of proton and divalent cation. Treatment of E.


163

coli with a protonophore markedly inhibited MntH transport while depletion of ATP did not38 suggesting proton coupling. As already noted, the Vmax for both E. coli and S. Typhimurium Mn2+ influx is pH sensitive while the Km is independent of pH, consistent with proton coupling.37 While these experiments are not conclusive, the mechanism of MntH transport appears similar to mammalian Nramp transport.23

Regulation of MntH Initial observations indicated that expression of a mntH::lacZ reporter construct could be regulated by reactive oxygen species, as well as divalent cations including Mn2+ and Fe2+. Inspection of the promoters of E. coli and S. Typhimurium mntH suggested at least three possible regulatory sites, one for Fur, one for OxyR, and a novel inverted repeat encompassing 22 bp.37 In Gram-negative and Gram-positive bacteria with low GC content, proteins involved in iron uptake are repressed by Fur (ferric uptake regulator) in the presence of adequate iron.47-49 Since MntH transports iron, it would appear at first glance logical for the locus to be regulated by Fur. Likewise, both Mn2+ and Fe2+ are involved in defense against reactive oxygen and nitrogen species, making regulation by OxyR reasonable since this repressor controls numerous genes important for response to reactive oxygen.50,51 Both of these suppositions are correct; Fur (Fig. 3) and OxyR repress mntH in the presence of Fe2+ and peroxide, respectively.37,52 Mn2+ clearly regulates mntH expression but does not mediate that repression via Fur although Mn2+ is known to bind to Fur to regulate some genes.52 Additional Fe2+ and Mn2+ regulated repressors have been identified, one of which is the diphtheria toxin repressor, DtxR, which regulates toxin production in an iron-dependent manner.53 DtxR homologs have since been found in many Gram-positive bacteria. However, DtxR homologs have a much broader role. DtxR homologs are preferentially regulated by Mn2+ rather than Fe2+ in Treponema pallidum,54 Streptococcus gordoni, 55 B. subtilis,56 and Staphylococcus aureus.57 These repressors have generally been named MntR. DtxR homologs also have been shown to bind to an inverted repeat promoter sequence.58,59 Consequently, when the genomic sequence of E. coli and S. Typhimurium indicated a weak DtxR/MntR homolog in both species (15-20% identity), it was hypothesized that it encoded a Mn2+ dependent repressor that bound to the inverted repeat in the mntH promoter. Patzer and Hantke in E. coli and this laboratory in S. Typhimurium confirmed this hypothesis (Fig. 3).60,52 In E. coli Patzer and Hantke also directly demonstrated Mn2+-dependent repressor binding to the inverted repeat sequence using DNA footprinting.60 In both species, MntR regulates expression of mntH in response to Mn2+. In S. Typhimurium at least, MntR can also repress mntH expression when bound with iron albeit at a concentration higher than iron’s affinity for Fur.52 Fur and MntR have been shown to account for all cation regulatory effects at the mntH locus.52,60 Since mntH is regulated (in part) by OxyR and since Mn2+ is an obligate cofactor for sodA, encoding a cytosolic Mn2+ superoxide dismutase, a relationship between Mn2+ and toxicity from reactive oxygen species might be expected. E. coli K-12 and S. Typhimurium LT2 have been tested for reactive oxygen sensitivity.37 Mutation of mntH rendered both species significantly more sensitive to killing by peroxide than wild type cells. Overexpression of mntH did not alter resistance to peroxide or superoxide. Interestingly, although Mn2+ is a cofactor for one of the organism’s superoxide dismutases, an mntH mutation did not alter sensitivity to superoxide.37 Further, since S. Typhimurium LT2 is rpoS- and E. coli is rpoS+, identical responses in both species to reactive oxygen suggests that RpoS, a sigma factor active in stress responses, is not involved in regulation of mntH. Thus, one role for MntH may be to provide Mn2+ as one component of the cell’s defenses against reactive oxygen, whether in the general environment or specifically within the host cell during an infection.

Other Mn2+ Transporters

Since loss of MntH does not result in a significant Mn2+-dependent phenotype and elicits no obvious growth deficiency attributable to Mn2+, the cell presumably must have other Mn2+ transporters that can compensate for its loss. Sequence analysis of the S. Typhimurium genome

164

The Nramp Family

Figure 3. Control of iron and manganese repression by Fur and MntR. Cultures of wild type or S. Typhimurium or S. Typhimurium strains carrying various mutations were grown overnight with no added metal, with 10 µM MnSO4 or with 10 µM FeSO4. For each strain the β-galactosidase activity of metal-challenged cultures was normalized to that of the same strain grown in the absence of added metal. Panel A shows the effect of mutation of fur or the Fur motif of the mntH promoter on repression by Mn2+ and Fe2+. Panel B shows the effect of mutation of mntR or the MntR motif of the mntH promoter on repression by Mn2+ and Fe2+. The data indicate that both Fe2+ and Mn2+ can mediate repression through MntR, but that under the conditions of this assay, only Fe2+ can mediate repression through Fur. Additional data extending these results has been published.52

determined that sitABCD, an ABC cassette-ATPase type transporter previously considered to be a ferrous iron transporter,61 is the only other gene in the S. Typhimurium genome that possesses the palindromic sequence bound by MntR.39 Subsequent analysis of SitABCD’s transport properties and regulation indicated that SitABCD is not a physiologically relevant Fe2+ transporter but like MntH is highly selective for Mn2+. Its Mn2+ affinity of 100 nM is equivalent to that of MntH (Fig. 4). Also similar to MntH, SitABCD’s affinity for Mn2+ is not dependent on pH while the Ki for inhibitory cations is pH dependent. As with MntH, Co2+, Cu2+, Ni2+, Fe2+, and Fe3+ all inhibited Mn2+ uptake via SitABCD. The Ki of Fe2+ for SitABCD


165

Figure 4. Cation inhibition of SitABCD transport. A S. Typhimurium strain carrying sitABCD on a high copy number plasmid was incubated with 0.1 µM54Mn2+ containing different concentrations of potential competitors. Uptake at different inhibitor concentrations was normalized to uptake in the absence of inhibitor. Details of this transport assay and additional cation inhibition data have been published.39 τ= Mn2+; υ= Zn2+; n= Fe2+; σ= Fe3+.

is slightly better than that for MntH but still above physiological free Fe2+ concentrations. Unlike MntH, Zn2+ inhibited SitABCD with a significantly better affinity than MntH and was not sensitive to pH (Fig. 4). Interestingly, SitABCD transport, while pH dependent like MntH, is maximal at alkaline pH rather than the acid pH preferred by MntH. Thus uptake through SitABCD increases with increasing pH, whereas uptake through MntH decreases with increasing pH implying that the organism may use MntH and SitABCD under different growth conditions.

Mn2+ in Pathogenesis S. typhimurium is an intracellular pathogen. Upon phagocytosis by a macrophage, bacteria reside in a subcellular compartment in the mammalian cell referred to as Salmonella-containing vacuole (SCV).62,63 Residence within the SCV has at least two consequences relevant to Mn2+. First, essential ions and nutrients needed for bacterial growth and survival are low in the SCV.64 Second, invasion by a bacterium elicits multiple responses from the host cell, among which generation of reactive oxygen and nitrogen species are vital. Thus Mn2+ could be necessary to the bacterium for both nutrient and defense purposes. The invasion of S. Typhimurium carrying a mutation in mntH has been studied using a murine macrophage-like cell line, RAW264.7 cells. Upon bacterial invasion of the cells, ex-

166

The Nramp Family

Figure 5. Induction of mntH transcription after invasion of RAW264.7 macrophages. S. Typhimurium strain 14028s was transformed with the single copy mntH::lacZYA operon fusion plasmid pMLZ10437 and used to infect RAW264.7 cell lines with or without a functional Nramp1 allele65 β-Galactosidase activity was measured using a chemiluminescent assay.

pression of mntH increased significantly, reaching a maximum level three hours after infection (Fig. 5). However, neither invasion efficiency nor survival of the bacteria was affected by mutation of mntH.37 Similar results have been obtained after S. Typhimurium invasion of epithelial cells (M.L. Zaharik and B.B. Finlay, personal communication). Although mntH induction occurred in the macrophage cells (Fig. 5), the degree was modest, usually three- to four-fold.37 However, RAW264.7 cells do not express Nramp1 as they carry a gene with the inactivating Gly169Asp mutation. An Nramp1 expressing cell can be generated however by stable transfection of RAW264.7 cells.65 When this Nramp1+/+ line is infected with S. Typhimurium, expression of mntH is markedly induced thirty-fold or more (Fig. 5), an order of magnitude greater than in Nramp1-/- cells. This remarkable result shows that expression of a bacterial gene is influenced by the presence or absence of its ortholog in the host cell. Such results suggest that MntH and Mn2+ might be important in the pathogenic process. Currently available literature does not provide extensive information on the regulation of sitABCD expression. The operon contains both a Fur and MntR site, but unlike mntH it has no OxyR site. Thus its expression is controlled primarily by Mn2+ and Fe2+ (A. Janakiraman and J.M. Slauch, personal communication). As with mntH, expression of sitABCD is induced upon invasion of the macrophage-like RAW264.7 cell line, and the degree of expression is greater in cells carrying a functional Nramp1 (M.L. Zaharik, D.G. Kehres, M.E. Maguire and B.B. Finlay, manuscript in preparation).


167

The experiments described above are suggestive of a role for mntH and Mn2+ in pathogenesis but do not directly assess involvement. More direct evidence has recently been provided in an elegant series of experiments by Jabado et al66 showing that the eukaryotic Nramp1 transporters actively modify Mn2+ content of the SCV. These authors used a Mn2+-binding dye coupled to a porous particle to assess Mn2+ movement within the macrophage during phagocytosis. Particles with coupled dye were ingested by macrophages, and fluorescence quenching of the dye was followed by digital fluorescence imaging. Nramp1 is known to be recruited to the phagosome after particle ingestion or bacterial invasion.67-70 The results indicated that Nramp1+/+ macrophages appear to extrude Mn2+ from the phagosome much faster than Nramp1-/- macrophages. Extrusion was dependent on acidification of the phagosome, consistent with H+-dependent Mn2+ transport by Nramp1. Thus, Mn2+ transport by Nramp1 appears to be directly involved in phagocytosis. Dyes or other methodologies to detect movement of other transition metal divalent cations are not available however. Therefore, whether this effect of Nramp1 is completely Mn2+ selective will have to await the detailed characterization of cation affinities for Nramp1 transport. Nonetheless, these experiments give strong support to the hypothesis that Mn2+ is important for pathogenesis. These results with Nramp1 imply that Mn2+ transport would be important for the bacterium also. Previous work37 suggested that mutation of mntH alone had minimal effect upon oral infection of BALB/c mice in that mortality was not reduced but only delayed by about two days. However, BALB/c mice are Nramp1-/-. As mice congenic except for the Nramp1 locus were not available to us, we chose to test C3H mice which are Nramp1+/+. As shown in Figure 6, the effect of mutation in mntH is significant in C3H mice. The dose used is about two logs above the S. Typhimurium LD50 for an Nramp1+/+ mouse, so the attenuation is marked. The double mutant is extremely attenuated. These data are suggestive of an interaction between orthologous genes in host and pathogen. Nonetheless, the experiment in Figure 6 is compromised, because the C3H and BALB/c mice are not congenic thus making it possible that another mouse locus contributes to the observed phenotype. Significantly more convincing evidence that mntH and sitABCD and thus Mn2+ are important for pathogenicity has recently been published by Boyer and colleagues.71 These authors used strains of S. Typhimurium with combinations of mutations in mntH, sitABCD and feoB (ferrous iron transporter).72,73 Transport of Mn2+ by SitABCD was shown in agreement with our results.39 Using tail vein injection of S. Typhimurium as a model of typhoid disease, these authors reported that the Nramp1+/+ 129/SvJ strain of mice could clear both wild type bacteria and all combinations of mutant strains without mortality. This contrasts with our results that mntH mutants are attenuated in C3H Nramp1+/+ mice. In the Nramp-/- 129/SvJ mice, Boyer at al. reported that mutation of mntH alone had no effect on virulence, consistent with the results of Kehres et al. using oral inoculation of Nramp1-/- BALB/c mice.37 However, mutation of sitABCD alone significantly attenuated virulence, increasing mean time to initial morbidity and ultimately reducing overall mortality. The mntH sitABCD double mutant was completely avirulent. A mutation in feoB also resulted in decreased mortality though less so than with a sitABCD mutation. Although mntH mutation alone did not diminish virulence, its combination with a feoB mutation also led to complete attenuation. (Strains deficient in feoB and sitABCD were also avirulent as was the triple mutant strain.71) These data clearly show that acquisition of both Mn2+ and iron by the pathogen is important for virulence. It is of interest to note also the differences (as far as they can be compared) between these results and our unpublished data in the Nramp1+/+ C3H mice. In the Nramp1+/+ 129/SvJ strain, bacteria injected into the tail vein are readily cleared whether or not mutations in mntH or sitABCD are present. In contrast, in the Nramp1+/+ C3H strain, attenuation of a mntH mutant is clearly seen after oral inoculation. Whether this is a difference in mouse strains used, a difference in route of inoculation or both will have to be sorted out by further experiments. Either answer would be of interest however. If the difference is due primarily to a strain difference, it implies interaction with another locus in addition to Nramp1. If the difference is due to route of inoculation, this clearly has implications for where and why Mn2+ is important. For

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Figure 6. Effect of mutation of mntH on virulence in BALB/c and C3H mice. Mice were orally inoculated with 3-5 108 bacteria of the indicated strain and monitored twice daily for morbidity and mortality. No difference in induced morbid symptoms was observed with wild type versus mntH strains.

example, it may be that Mn2+ acquisition is important while S. Typhimurium is within the macrophage SCV and being distributed throughout the host organism. However, once within the circulation, Mn2+ acquisition is not crucial to pathogen survival.

Conclusions The MntH proteins, the Bacterial Nramp homologs, like their mammalian counterparts are transition metal divalent cation transporters. To date, the Enterobacterial MntH transporters are quite selective for Mn2+ although they can transport Fe2+ and probably Cd2+. Their transport of Fe2+ does not appear physiologically relevant for the bacterium. Nonetheless, it is important to remember that the selectivity observed in E. coli and S. Typhimurium for Mn2+ may not be present in MntH orthologs from other Bacteria. Indeed, it is hard to understand why organisms like P. aeruginosa express two and B. cepacia three transporters unless the cation selectivity and thus physiological relevance of each transporter differs in some manner. It will be important to thoroughly investigate factors such as cation selectivity in each new system to ensure that data are interpreted with regard to the physiologically relevant cation. Just as Fe2+ cannot be assumed to be the physiologically relevant ligand for a transporter solely on the basis of homology or growth phenotype, homology and phenotype cannot define a Mn2+ transporter in the absence of actual transport data. Iron has long been thought to be the primary divalent transition metal cation important for virulence. Characterization of Nramp transport in eukaryotes has led to investigation of Nramp function in the Bacterial and the demonstration in some Enterobacterial species that Mn2+ transport via an Nramp class transporter is important for bacterial pathogenicity. The role of Mn2+ in prokaryotic metabolism and bacterial pathogenesis will clearly be a very active field of investigation for some time to come.


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References 1. Plant J, Glynn AA. Natural resistance to Salmonella infection, delayed hypersensitivity and Ir genes in different strains of mice. Nature 1974; 248(446):345-347. 2. Plant J, Glynn AA. Genetics of resistance to infection with Salmonella typhimurium in mice. J Infect Dis 1976; 133(1):72-78. 3. Glynn AA, Bradley DJ, Blackwell JM et al. Genetics of resistance to infection. Lancet 1982; 2(8290):151. 4. Blackwell JM, Roberts CW, Roach TI et al. Influence of macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) on Toxoplasma gondii infection in mice. Clin Exp Immunol 1994; 97(1):107-112. 5. Vidal SM, Malo D, Vogan K et al. Natural resistance to infection with intracellular parasites: Isolation of a candidate for Bcg. Cell 1993; 73(3):469-485. 6. Barton CH, Whitehead SH, Blackwell JM. Nramp transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: Influence on oxidative burst and nitric oxide pathways. Mol Med 1995; 1(3):267-279. 7. Gruenheid S, Cellier M, Vidal S et al. Identification and characterization of a second mouse Nramp gene. Genomics 1995; 25(2):514-525. 8. Vidal S, Belouchi AM, Cellier M et al. Cloning and characterization of a second human NRAMP gene on chromosome 12q13. Mamm Genome 1995; 6(4):224-230. 9. Cellier M, Prive G, Belouchi A et al. Nramp defines a family of membrane proteins. Proc Natl Acad Sci USA 1995; 92(22):10089-10093. 10. Canonne-Hergaux F, Gruenheid S, Ponka P et al. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood 1999; 93(12):4406-4417. 11. Forbes JR, Gros P. Divalent-metal transport by NRAMP proteins at the interface of host- pathogen interactions. Trends Microbiol 2001; 9(8):397-403. 12. Fleming MD, Romano MA, Su MA et al. Nramp2 is mutated in the anemic Belgrade (b ) rat: Evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci USA 1998; 95(3):1148-1153. 13. Fleming MD, Trenor CC, Su MA et al. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 1997; 16(4):383-386. 14. Oates PS, Morgan EH. Defective iron uptake by the duodenum of Belgrade rats fed diets of different iron contents. Am J Physiol 1996; 270(5 Pt 1):G826-G832. 15. Garrick MD, Scott D, Kulju D et al. Evidence for and consequences of chronic heme deficiency in Belgrade rat reticulocytes. Biochim Biophys Acta 1999; 1449(2):125-136. 16. Pallarés I, Lisbona F, Lopez Aliaga I et al. Effect of iron deficiency on the digestive utilization of iron, phosphorus, calcium and magnesium in rats. Br J Nutr 1993; 70:609-620. 17. Al Younes HM, Rudel T, Brinkmann V et al. Low iron availability modulates the course of Chlamydia pneumoniae infection. Cell Microbiol 2001; 3(6):427-437. 18. Su MA, Trenor CC, Fleming JC et al. The G185R mutation disrupts function of the iron transporter Nramp2. Blood 1998; 92(6):2157-2163. 19. Fleming MD, Andrews NC. Mammalian iron transport: An unexpected link between metal homeostasis and host defense. J Lab Clin Med 1998; 132(6):464-468. 20. Sheth S, Brittenham GM. Genetic disorders affecting proteins of iron metabolism: Clinical implications. Annu Rev Med 2000; 51:443-64.443-464. 21. Chua AC, Morgan EH. Manganese metabolism is impaired in the Belgrade laboratory rat. J Comp Physiol [B] 1997; 167(5):361-369. 22. Chua AC, Morgan EH. Effects of iron deficiency and iron overload on manganese uptake and deposition in the brain and other organs of the rat. Biol Trace Elem Res 1996; 55(1-2):39-54. 23. Gunshin H, Mackenzie B, Berger UV et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388(6641):482-488. 24. Goswami T, Bhattacharjee A, Babal P et al. Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter. Biochem J 2001; 354(Pt 3):511-519. 25. Kuhn DE, Baker BD, Lafuse WP et al. Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169. J Leukoc Biol 1999; 66(1):113-119. 26. Zoller H, Pietrangelo A, Vogel W et al. Duodenal metal-transporter (DMT-1, NRAMP-2) expression in patients with hereditary haemochromatosis. Lancet 1999; 353(9170):2120-2123. 27. Atkinson PG, Barton CH. High level expression of Nramp1G169 in RAW264.7 cell transfectants: Analysis of intracellular iron transport. Immunology 1999; 96(4):656-662.

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28. Fleming RE, Migas MC, Zhou X et al. Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: Increased duodenal expression of the iron transporter DMT1. Proc Natl Acad Sci USA 1999; 96(6):3143-3148. 29. Gruenheid S, Canonne-Hergaux F, Gauthier S et al. The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes. J Exp Med 1999; 189(5):831-841. 30. Zhong W, Lafuse WP, Zwilling BS. Infection with Mycobacterium avium differentially regulates the expression of iron transport protein mRNA in murine peritoneal macrophages. Infect Immun 2001; 69(11):6618-6624. 31. Picard V, Govoni G, Jabado N et al. Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool. J Biol Chem 2000; 275(46):35738-35745. 32. Gomes MS, Appelberg R. Evidence for a link between iron metabolism and Nramp1 gene function in innate resistance against Mycobacterium avium. Immunology 1998; 95(2):165-168. 33. Thomine S, Wang R, Ward JM et al. Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proc Natl Acad Sci USA 2000; 97(9):4991-4996. 34. Sacher A, Cohen A, Nelson N. Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes. J Exp Biol 2001; 204(Pt 6):1053-1061. 35. Cellier MF, Bergevin I, Boyer E et al. Polyphyletic origins of bacterial Nramp transporters. Trends Genet 2001; 17(7):365-370. 36. Alonso JM, Hirayama T, Roman G et al. EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 1999; 284(5423):2148-2152. 37. Kehres DG, Zaharik ML, Finlay BB et al. The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen. Molec Microbiol 2000; 36:1085-1100. 38. Makui H, Roig E, Cole ST et al. Identification of the Escherichia coli K-12 NRAMP orthologue (MntH) as a selective divalent metal ion transporter. Mol Microbiol 2000; 35(5):1065-1078. 39. Kehres DG, Janakiraman A, Slauch JM et al. SitABCD Is the Alkaline Mn2+ Transporter of Salmonella enterica Serovar Typhimurium. J Bacteriol 2002; 184(12):3159-3166. 40. Agranoff D, Monahan IM, Mangan JA et al. Mycobacterium tuberculosis Expresses a Novel pH-dependent Divalent Cation Transporter Belonging to the Nramp Family. J Exp Med 1999; 190(5):717-724. 41. Herman C, Lecat S, D’Ari R et al. Regulation of the heat-shock response depends on divalent metal ions in an hflB mutant of Escherichia coli. Mol Microbiol 1995; 18(2):247-255. 42. Pinner E, Gruenheid S, Raymond M et al. Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2, a member of the mammalian natural resistance- associated macrophage protein family. J Biol Chem 1997; 272(46):28933-28938. 43. Supek F, Supekova L, Nelson H et al. A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria. Proc Natl Acad Sci USA 1996; 93(10):5105-5110. 44. Reeve I, Hummell D, Nelson N et al. Overexpression, purification, and site-directed spin labeling of the Nramp metal transporter from Mycobacterium leprae. Proc Natl Acad Sci USA 2002; 99(13):8608-8613. 45. Chen XZ, Peng JB, Cohen A et al. Yeast SMF1 mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents. J Biol Chem 1999; 274(49):35089-35094. 46. Nelson N. Metal ion transporters and homeostasis. EMBO J 1999; 18(16):4361-4371. 47. Ratledge C, Dover LG. Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 2000; 54:881-941.881-941. 48. Braun V, Killmann H. Bacterial solutions to the iron-supply problem. Trends Biochem Sci 1999; 24(3):104-109. 49. Hantke K. Iron and metal regulation in bacteria. Curr Opin Microbiol 2001; 4(2):172-177. 50. Storz G, Imlay JA. Oxidative stress. Curr Opin Microbiol 1999; 2(2):188-194. 51. Storz G, Tartaglia LA, Ames BN. The OxyR regulon. Antonie Van Leeuwenhoek 1990; 58:157-161. 52. Kehres DG, Janakiraman A, Slauch JM et al. Regulation of Salmonella enterica Serovar Typhimurium mntH Transcription by H2O2, Fe2+, and Mn2+. J Bacteriol 2002; 184(12):3151-3158. 53. Boyd J, Oza MN, Murphy JR. Molecular cloning and DNA sequence analysis of a diphtheria toxin iron-dependent regulatory element (dtxR) from Corynebacterium. Proc Natl Acad Sci USA 1990; 87(15):5968-5972.


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54. Posey JE, Hardham JM, Norris SJ et al. Characterization of a manganese-dependent regulatory protein, TroR, from Treponema pallidum. Proc Natl Acad Sci USA 1999; 96(19):10887-10892. 55. Jakubovics NS, Smith AW, Jenkinson HF. Expression of the virulence-related Sca (Mn2+) permease in Streptococcus gordonii is regulated by a diphtheria toxin metallorepressor-like protein ScaR. Mol Microbiol 2000; 38(1):140-153. 56. Que Q, Helmann JD. Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol Microbiol 2000; 35(6):1454-1468. 57. Horsburgh MJ, Wharton SJ, Cox AG et al. MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake. Mol Microbiol 2002; 44(5):1269-1286. 58. Tao X, Boyd J, Murphy JR. Specific binding of the diphtheria tox regulatory element DtxR to the tox operator requires divalent heavy metal ions and a 9-base-pair interrupted palindromic sequence. Proc Natl Acad Sci USA 1992; 89(13):5897-5901. 59. Tao X, Murphy JR. Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection. Proc Natl Acad Sci USA 1994; 91(20):9646-9650. 60. Patzer SI, Hantke K. Dual repression by Fe2+-Fur and Mn2+-MntR of the mntH gene, encoding an NRAMP-like Mn2+ transporter in Escherichia coli. J Bacteriol 2001; 183(16):4806-4813. 61. Zhou D, Hardt WD, Galan JE. Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island. Infect Immun 1999; 67(4):1974-1981. 62. Knodler LA, Celli J, Finlay BB. Pathogenic trickery: Deception of host cell processes. Nat Rev Mol Cell Biol 2001; 2(8):578-588. 63. Brumell JH, Tang P, Mills SD et al. Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments. Traffic 2001; 2(9):643-653. 64. Garcia-del Portillo F, Foster JW, Maguire ME et al. Characterization of the micro-environment of Salmonella typhimurium-containing vacuoles within MDCK epithelial cells. Mol Microbiol 1992; 6:3289-3297. 65. Govoni G, Canonne-Hergaux F, Pfeifer CG et al. Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7. Infect Immun 1999; 67(5):2225-2232. 66. Jabado N, Jankowski A, Dougaparsad S et al. Natural resistance to intracellular infections: Natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane. J Exp Med 2000; 192(9):1237-1248. 67. Gruenheid S, Pinner E, Desjardins M et al. Natural resistance to infection with intracellular pathogens: The Nramp1 protein is recruited to the membrane of the phagosome. J Exp Med 1997; 185(4):717-730. 68. Hackam DJ, Rotstein OD, Zhang W et al. Host resistance to intracellular infection: Mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification. J Exp Med 1998; 188(2):351-364. 69. Jabado N, Canonne-Hergaux F, Gruenheid S et al. Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells. Blood 2002; 100(7):2617-2622. 70. Searle S, Bright NA, Roach TI et al. Localisation of Nramp1 in macrophages: Modulation with activation and infection. J Cell Sci 1998; 111(Pt 19):2855-2866. 71. Boyer E, Bergevin I, Malo D et al. Acquisition of Mn(II) in addition to Fe(II) is required for full virulence of Salmonella enterica serovar Typhimurium. Infect Immun 2002; 70(11):6032-6042. 72. Velayudhan J, Hughes NJ, McColm AA et al. Iron acquisition and virulence in Helicobacter pylori: A major role for FeoB, a high-affinity ferrous iron transporter. Mol Microbiol 2000; 37(2):274-286. 73. Hantke K. Ferrous iron transport mutants in Escherichia coli K-12. FEMS Microbiol Lett 1987; 44:53-57. 74. Higgins DG, Thompson JD, Gibson TJ. Using CLUSTAL for multiple sequence alignments. Methods Enzymol 1996; 266:383-402. 75. Felsenstein J. Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods. Methods Enzymol 1996; 266:418-427. 76. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985; 39:783-791. 77. Kehres DG, Lawyer CH, Maguire ME. The CorA magnesium transporter gene family. Microbial & Comparative Genomics 1998; 43:151-169.

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CHAPTER 15

Role of the Nramp Orthologue, MntH, in the Virulence of Mycobacterium tuberculosis Pilar Domenech and Stewart T. Cole

Abstract

M

ycobacterium tuberculosis is the causative agent of tuberculosis. The natural habitat of this pathogen is the alveolar macrophage where it modulates the maturation of the phagosome, inhibiting the fusion of the phago-lysosome. Eukaryotic Nramp1 is localized in late endosomal and lysosomal compartments. Mycobacterium tuberculosis also possesses a Nramp1 orthologue (MntH), suggesting a model in which both Nramp proteins compete for the same kind of cations; inactivation of this transporter may disadvantage the corresponding cell. A M. tuberculosis MntH knock-out strain created by allelic interchange did not show any impairment in intracellular growth irrespective of the host Nramp1 background. Nor was attenuation observed in mice that have the Asp169 mutation inactivating Nramp1. This indicates, that in the model used, M. tuberculosis MntH does not play a detectable role in determining the outcome of infection. Tuberculosis has plagued mankind since prehistoric times and is still an important source of morbidity and mortality.1 The causative agents of this disease are the bacteria comprising the Mycobacterium tuberculosis complex, namely M. tuberculosis. M. africanum, M. canettii, M. bovis, M. microti and the attenuated vaccine strain M. bovis BCG. These are all slow growing mycobacteria, with a replication time of approximately 20 hours. This slow growth complicates microbial diagnostics and contributes to the necessarily long-term drug treatment. Although tuberculosis can manifest in any tissue, the lung represents the main port of entry and the most important site of disease manifestation.

M. tuberculosis Is an Intracellular Pathogen The natural habitat of M. tuberculosis is the alveolar macrophage. After phagocytosis by the macrophage, M. tuberculosis is maintained in vacuoles that fail to fuse with lysosomes and this “non-fusogenic phenotype” correlates with the viability of the infecting organism.2,3 The mycobacterial factors involved in this process have not been identified so far. Acidification of the phagosome is a characteristic of maturation along the endosomallysosomal network. Early endosomes are acidified to pH=6 and late endosomes and lysosomes are acidified to pH=5.5. This process results from delivery of the vacuole proton pump to early and late endosomes and removal of the Na+/K+ -ATPase from the late endosome.4 Mycobacteria-containing phagosomes are less acidic than lysosomes5 with a pH=6.3-6.5.6 This failure to acidify normally is, at least in part, due to the exclusion of the vacuolar proton The Nramp Family, edited by Mathieu Cellier and Philippe Gros. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers.

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pump ATPase.6 Another factor that might affect the lack of acidification could be the presence of the Na+/K+ -ATPase in the mycobacteria containing phagosome, but inhibition of this ATPase with ouabain did not affect the pH of M.bovis BCG-containing phagosomes. 7 Immunoelectron microscopy of murine bone marrow macrophages infected with M. avium or M. tuberculosis indicates that the vacuolar membrane surrounding the bacilli possesses the late endosoma/lysosomal marker, Lamp1 but lacks the vesicular proton-ATPase. 8 The Mycobacterium-containing phagosome is a dynamic structure that retains the ability to fuse with and is accessible to glycosphingolipids from the host cell plasmalemma.9 The presence of MHC class II molecules and the transferrin receptor indicates persistent interaction of the M. tuberculosis phagosome with early endosomes.2 The presence of the Rab5 GTPase, another early endosome marker, but not Rab7 (a late endosome marker) has been detected in M. bovis BCG-containing vacuoles, indicating that interruption of the maturation of mycobacterial phagosome occurs between these two stages.10 Accessibility to exogenously administered transferrin indicates that the Mycobacterium-containing vacuole is arrested within the transferrin recycling pathway of the host cell.11 M. avium containing phagosomes acquire the hydrolase cathepsin D not from the lysosome but from the biosynthetic pathway of the macrophage12,13 This result provides an explanation for the anomalous distribution of lysosomal proteins in Mycobacterium-containing phagosomes which are positive for Lamp1, cathepsin D and transferrin receptor but show low levels of acquisition of the vesicular proton ATPase.

Murine Nramp1 and the M. tuberculosis Phagosome

Eukaryotic Nramp1 is expressed exclusively in professional phagocytes.14 Murine Nramp1 is localized in late endosomal and lysosomal compartments colocalizing with Lamp1 and cathepsins D and L. Double immunofluoresence 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 during the maturation of the phagolysosome.14,15 Nramp1 functions as a pH dependent transporter that modulates the phagosomal space by extruding Mn2+. These divalent cations are essential for microbial function, either as nutrients or as cofactors of enzymatic ativities, and therefore Nramp1 contributes to defense against infections.16 Comparison of the properties of M. bovis BCG infected macrophages from either Nramp1 positive mice or from Nramp1 null mutant mice indicates that Nramp1 plays a role in the events that lead to phagosomal acidification. The pH of phagosomes containing live M. bovis BCG more acidic in nramp1 +/+ cells than in nramp1 -/-. This acidification is associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase-containing late endosomes and/or lysosomes.7 It remains unknown if the Nramp1 protein plays the same role in M. tuberculosis-containing phagosomes. Resolution answering this question might provide valuable insight into the differences between the pathogen M. tuberculosis and the vaccine strain M. bovis BCG. The role of eukaryotic Nramp1 in resistance to M. tuberculosis infection in mice is controversial. While some authors report that Nramp1 is involved in resistance to infections initiated with very small intravenous inocula.17 Others consider that the eukaryotic Nramp1 is of limited importance in resistance to M. tuberculosis infection.18 It has been shown that mouse strains homozygous for the nramp1 resistant allele are equally or even less resistant to M. tuberculosis infections than strains homozygous for the susceptible allele.19-21

M. tuberculosis MntH Is Constitutively Expressed Analysis of the sequence of the M. tuberculosis genome reveals the presence of an extensive group of genes encoding cation transporters such as 11 P-type ATPases and a member of the Nramp family of proteins (mntH).22,23 Nramp orthologues are also present in the genome sequences of other members of the M. tuberculosis complex (M. bovis and M. bovis BCG) and in other pathogenic (M. avium) and saprophytic (M. smegmatis) mycobacteria. The MntH

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protein is predicted to have 428 amino acids and a molecular mass of 44.9 kD. It contains 11 putative transmembrane domains and shows an identity of 38% with the MntH protein of Escherichia coli and 39% with that of Salmonella typhimurium. MntH mRNA was detected in cultures of M. bovis BCG grown in the macrophage-like cell line THP1 indicating that this gene is constitutively expressed when the bacteria grow intracellularly. In vitro MntH mRNA expression is stimulated (~ 50 fold) in the presence of Fe2+at concentrations between 1 and 48 µM, while addition of Cu2+ in the same range to the culture media results in a 10-fold increase of MntH expression that is maximal at 5 µM.24

M. tuberculosis MntH Protein As a Cation Transporter Some experimental evidence suggesting that the M. tuberculosis MntH protein functions as a cation transporter was provided by Agranoff and coworkers.24 These investigators showed a 20 fold increase in 65Zn2+ and 55Fe2+uptake in Xenopus laevis oocytes that were injected with mRNA encoding the M. tuberculosis MntH protein. This transport was dependent on acidic extracellular pH and was maximal between pH 5.5 and 6.5. They also showed that this transport was abolished when excess Mn2+ and Cu2+was present, suggesting a possible interaction of the M. tuberculosis MntH protein with a broad range of divalent cations. The MntH proteins of E. coli and S. typhimurium act as divalent cation transporters (Fe2+, Mn2+) in a proton-dependent manner, showing the highest affinity for Mn2+.25,26 However, no data have been reported so far regarding the affinities of the M. tuberculosis MntH protein for divalent cations. In B. subtilis, mntH expression is regulated by the MntR protein, a transcriptional regulator related to the Diphteria toxin repressor family of proteins (DtxR) that regulates the expression of mntH and an ABC transporter in a Mn2+-dependent manner.27 By contrast, in E. coli, mntH shows dual repression by Fe2+ via Fur and by Mn2+ via MntR.28 The M. tuberculosis genome sequence revealed more than 100 putative transcriptional regulators,22 reflecting the diversity of environments that this organism faces. Among these transcriptional regulators two (furA and furB) belong to the Fur (Ferric Uptake Regulator) family, whilst IdeR and SirR are metal-dependent regulators of the DtxR family. FurA is involved in the regulation of the catalase-peroxidase gene KatG, and hence is involved in isoniazid sensitivity and virulence.29 The IdeR protein of M. tuberculosis has been found to be a pleiotropic regulator that represses the expression of the genes involved in the synthesis of the salicylate-derived mycobactin siderophores of M. tuberculosis, constituting the main Fe3+-acquisition system in this organism. IdeR also activates expression of the iron storage protein bacterioferritin in an iron-dependent way.30,31 No function has been described for the M. tuberculosis SirR although the predicted protein shows 50 % identity with the Mn2+-dependent transcriptional regulator of Corynebacterium glutamicum. SirR was first identified in Staphylococcus epidermis as a metal-dependent (Mn2+ and Fe2+) regulator of the sitABC operon, an ABC transporter that appears to be involved in iron uptake in Staphylococcus epidermis.32,33 It remains to be seen whether Fur, SirR or IdeR play a role in regulating mntH transcription.

Role of the M. tuberculosis MntH Protein in Virulence The presence of the MntH protein, able to act as a divalent cation transporter, in intracellular pathogens like M. tuberculosis and M. leprae suggests a model in which the mammalian (Nramp1) and the bacterial (MntH) proteins could compete for the same cations present in the phagosome. These cations are essential nutrients for the bacteria and could also play an important role in the defense of the bacteria against the adverse conditions of the macrophage. For example, these cations are cofactors of many enzymatic activities like the SOD (superoxide dismutase), a metalloenzyme that catalyzes the conversion of superoxide anion to hydrogen peroxide and confers resistance in the bacteria to the oxidative stress produced by the macrophage. It has been reported that a SOD-deficient M. tuberculosis H37Rv mutant was attenuated in the mouse model.34


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The role of the MntH protein in the intracellular survival of M. tuberculosis and thereby its virulence has been studied by two different groups of investigators.35,36 In both cases, a M. tuberculosis mntH knock-out strain was generated by allelic exchange mutagenesis. Disruption of the gene did not affect extracellular growth under standard conditions although under iron-deficient conditions, the M. tuberculosis mntH knock-out mutant showed impaired growth. This result indicates that the MntH protein confers an advantage to the wild-type strain over the mutant, for growth under low iron conditions, suggesting a role as divalent cation transporter for the M. tuberculosis MntH protein.36 However, some experiments still need to be done in order to elucidate the physiological role of the M. tuberculosis MntH protein. 65Zn2+, 55 2+ Fe , Mn 2+ and Fe 2+ in vitro uptake experiments with the mntH knock-out mutant and the wild-type strains will reveal whether the absence of MntH is compensated by any of the 11 P-ATPases present in M. tuberculosis. To study the effect of this transporter on intracellular growth, the M. tuberculosis wild-type and the mntH knock-out mutant strains were assayed in different kinds of macrophages. Bone marrow-derived macrophages from mice with intact nramp1 gene (strain 129SV) and mice that have the Asp169 mutation inactivating Nramp1 (strains BALB/c and C57BL/6) were used in these studies 35,36 together with macrophage lines derived from the bone marrow of 129SV mice expressing the wild-type nramp1 gene and from an isogenic mice strain that bears a homozygous null allele at the nramp1 locus.36 There was no impairment in the intracellular growth of the M. tuberculosis mntH knock-out compared to the wild-type in any eukaryotic Nramp1 background tested. These results suggest that, at least in vitro, MntH is dispensable for intracellular growth of M. tuberculosis independently of the presence of a functional eukaryotic Nramp1. The role of the M. tuberculosis MntH protein in virulence was studied by intravenous infection of BALB/c mice with the M. tuberculosis wild-type and the mntH knock-out mutant. Growth kinetics of these strains were followed for periods of more than three months in lung and spleen. In both studies the results were consistent and no significant difference was observed in the growth of both strains either in the initial pre-immune phase of the infection, in which there is an exponential increase in the number of bacilli in both organs, or after acquired immunity had developed and the mice controlled the intracellular multiplication of the bacteria.35,36 Nor were differences observed in the survival of the mice infected with both M. tuberculosis strains, wild-type and mntH knock-out mutant.36 Taken together, these results indicate that the MntH protein of M. tuberculosis is not essential for the bacteria to establish a chronic infection, therefore ruling out the role of the M. tuberculosis MntH protein in virulence in the model used. The mice employed in both of the virulence studies mentioned above harbor the Asp169 mutation that inactivates Nramp1. Boechat and coworkers36 reported no differences in intracellular growth between the mntH knock-out mutant and the wild-type M. tuberculosis strains in resting macrophages of mice with an intact nramp1. Murine Nramp1 expression is upregulated by LPS and IFN-γ administration both in primary macrophages and in macrophages cell lines37,38 in a time and dose-dependent fashion.39 It still remains unknown if the absence of MntH in the M. tuberculosis KO mutant determines the outcome of the infection in the Nramp1 positive mouse model. MntH is not specific to intracellular parasites and is present in saprophytic bacteria, so alternatively, MntH may contribute to the extracellular life of the bacteria. Extracellular growth of the bacteria occurs in the last stage of pulmonary tuberculosis. At that stage, the solid caseous center of the granuloma liquefies. Macrophages do not survive in this liquefied caseum and bacteria grow extracellularly for the first time in the course of the disease. They can reach such large numbers that the cell-mediated immunity, developed in a resistant host, becomes overwhelmed. Cavitation of the bronchial tree is produced by discharging the content of the liquefied lesions and spread of the bacteria to other parts of the lung occurs and to the nearby

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environment. Cavitary tuberculosis is therefore, highly contagious.40,41 No tissue damage, liquefaction or cavitation is produced in the mouse model of tuberculosis contrasting with the necrotic pulmonary lesions and cavitation present in rabbits, guinea pigs and humans.42 It is possible that in this liquefied caseous environment the bacteria need highly controlled metal uptake that allows their survival. This could be provided by cation transporters such as the 11 P-type P-ATPases and MntH. In such a scenario, a M. tuberculosis mntH knock-out mutant could be impaired in the development of disease at that last stage. Testing this hypothesis will require a different experimental model of tuberculosis.

References 1. http://www.who.int/gtb/ GTPWHO. 2. Clemens DL, Horwitz MA. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med 1995; 181(1):257-270. 3. Frehel C, de Chastellier C, Lang T et al. Evidence for inhibition of fusion of lysosomal and prelysosomal compartments with phagosomes in macrophages infected with pathogenic Mycobacterium avium. Infect Immun 1986; 52(1):252-262. 4. Clemens DL. Characterization of the Mycobacterium tuberculosis phagosome. Trends Microbiol 1996; 4(3):113-118. 5. Crowle AJ, Dahl R, Ross E et al. Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Infect Immun 1991; 59(5):1823-1831. 6. Sturgill-Koszycki S, Schlesinger PH, Chakraborty P et al. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 1994; 263(5147):678-681. 7. Hackam DJ, Rotstein OD, Zhang W et al. Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification. J Exp Med 1998; 188(2):351-364. 8. Xu S, Cooper A, Sturgill-Koszycki S et al. Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages. J Immunol 1994; 153(6):2568-2578. 9. Russell DG, Dant J, Sturgill-Koszycki S. Mycobacterium avium- and Mycobacterium tuberculosiscontaining vacuoles are dynamic, fusion-competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma. J Immunol 1996; 156(12):4764-4773. 10. Via LE, Deretic D, Ulmer RJ et al. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J Biol Chem 1997; 272(20):13326-13331. 11. Clemens DL, Horwitz MA. The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin. J Exp Med 1996; 184(4):1349-1355. 12. Sturgill-Koszycki S, Schaible UE, Russell DG. Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. Embo J 1996; 15(24):6960-6968. 13. Ullrich HJ, Beatty WL, Russell DG. Direct delivery of procathepsin D to phagosomes: implications for phagosome biogenesis and parasitism by Mycobacterium. Eur J Cell Biol 1999; 78(10):739-748. 14. Gruenheid S, Pinner E, Desjardins M et al. Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome. J Exp Med 1997; 185(4):717-730. 15. Searle S, Bright NA, Roach TI et al. Localisation of Nramp1 in macrophages: modulation with activation and infection. J Cell Sci 1998; 111(Pt 19):2855-2866. 16. Jabado N, Jankowski A, Dougaparsad S et al. Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane. J Exp Med 2000; 192(9):1237-1248. 17. Brown DH, Miles BA, Zwilling BS. Growth of Mycobacterium tuberculosis in BCG-resistant and -susceptible mice: establishment of latency and reactivation. Infect Immun 1995; 63(6):2243-2247. 18. North RJ, Ryan L, LaCource R et al. Growth rate of mycobacteria in mice as an unreliable indicator of mycobacterial virulence. Infect Immun 1999; 67(10):5483-5485. 19. Medina E, North RJ. Evidence inconsistent with a role for the Bcg gene (Nramp1) in resistance of mice to infection with virulent Mycobacterium tuberculosis. J Exp Med 1996; 183(3):1045-1051.


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20. Medina E, North RJ. Resistance ranking of some common inbred mouse strains to Mycobacterium tuberculosis and relationship to major histocompatibility complex haplotype and Nramp1 genotype. Immunology 1998; 93(2):270-274. 21. Medina E, Rogerson BJ, North RJ. The Nramp1 antimicrobial resistance gene segregates independently of resistance to virulent Mycobacterium tuberculosis. Immunology 1996; 88(4):479-481. 22. Cole ST, Brosch R, Parkhill J et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998; 393(6685):537-544. 23. Tekaia F, Gordon SV, Garnier T et al. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber Lung Dis 1999; 79(6):329-342. 24. Agranoff D, Monahan IM, Mangan JA et al. Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family. J Exp Med 1999; 190(5):717-724. 25. Makui H, Roig E, Cole ST et al. Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter. Mol Microbiol 2000; 35(5):1065-1078. 26. Kehres DG, Zaharik ML, Finlay BB et al. The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen. Mol Microbiol 2000; 36(5):1085-1100. 27. Que Q, Helmann JD. Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol Microbiol 2000; 35(6):1454-1468. 28. Patzer SI, Hantke K. Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli. J Bacteriol 2001; 183(16):4806-4813. 29. Pym AS, Domenech P, Honore N et al. Regulation of catalase-peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis. Mol Microbiol 2001; 40(4):879-889. 30. Gold B, Rodriguez GM, Marras SA et al. The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages. Mol Microbiol 2001; 42(3):851-865. 31. Rodriguez GM, Voskuil MI, Gold B et al. ideR, An essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect Immun 2002; 70(7):3371-3381. 32. Hill PJ, Cockayne A, Landers P et al. SirR, a novel iron-dependent repressor in Staphylococcus epidermidis. Infect Immun 1998; 66(9):4123-4129. 33. Cockayne A, Hill PJ, Powell NB et al. Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter. Infect Immun 1998; 66(8):3767-3774. 34. Edwards KM, Cynamon MH, Voladri RK et al. Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis. Am J Respir Crit Care Med 2001; 164(12):2213-2219. 35. Domenech P, Pym AS, Cellier M et al. Inactivation of the Mycobacterium tuberculosis Nramp orthologue (mntH) does not affect virulence in a mouse model of tuberculosis. FEMS Microbiol Lett 2002; 207(1):81-86. 36. Boechat N, Lagier-Roger B, Petit S et al. Disruption of the gene homologous to mammalian Nramp1 in Mycobacterium tuberculosis does not affect virulence in mice. Infect Immun 2002; 70(8):4124-4131. 37. Govoni G, Vidal S, Cellier M et al. Genomic structure, promoter sequence, and induction of expression of the mouse Nramp1 gene in macrophages. Genomics 1995; 27(1):9-19. 38. Atkinson PG, Blackwell JM, Barton CH. Nramp1 locus encodes a 65 kDa interferon-gammainducible protein in murine macrophages. Biochem J 1997; 325(Pt 3):779-786. 39. Govoni G, Gauthier S, Billia F et al. Cell-specific and inducible Nramp1 gene expression in mouse macrophages in vitro and in vivo. J Leukoc Biol 1997; 62(2):277-286. 40. McMurray DN, Collins FM, Dannenberg AM et al. Pathogenesis of experimental tuberculosis in animal models. Curr Top Microbiol Immunol 1996; 215:157-179. 41. Dannenberg AM, Collins FM. Progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products. Tuberculosis (Edinb) 2001; 81(3):229-242. 42. Rhoades ER, Frank AA, Orme IM. Progression of chronic pulmonary tuberculosis in mice aerogenically infected with virulent Mycobacterium tuberculosis. Tuber Lung Dis 1997; 78(1):57-66.

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CHAPTER 16

Molecular Evolutionary Analysis of the Nramp Family Etienne Richer, Pascal Courville and Mathieu Cellier

Abstract

T

he natural resistance-associated macrophage protein (Nramp) belongs to a family of ion permeases highly conserved in eukaryotes that originated in prokaryotes. Three phylogenetic groups of homologs were identified in Bacteria based on functional genomic approaches. Proteins of group A were characterized as H+-dependent MaNganese Transporter (MntH A) representing functional homologs of eukaryotic Nramp. Heterologous expression in E. coli of MntH B and C proteins modulated bacterial sensitivity to cobalt and cadmium, suggesting conserved function among MntH groups. Genome sequencing of model eukaryotes revealed two groups of eukaryotic Nramp. Conservation in invertebrates of a ‘prototype’ suggests early duplication of an ancestral Nramp gene. Family trees inferred from phylogenetic analyses using various approaches indicate successive divergence of MntH B, MntH A, eukaryotic Nramp and MntH C. MntH C shows high level sequence identity with ‘prototype’ Nramp, heterogeneous variation of a.a. replacement rate among sites, and predicted divergence from ‘archetype’ Nramp. The data suggest a relatively recent origin for MntH C, derived from an eukaryote ‘prototype’ Nramp gene. This proposition is discussed in the context of bacterial infection.

Overview Structural Nramp homologs were characterized in both eukaryotic and prokaryotic species and perform similar metal ion transport function. Amino acid sequence conservation between Nramp homologs corresponds to the preservation of a hydrophobic core of ten predicted transmembrane domains (TMD) and a consensus transmembrane topology prediction with the N-terminus cytoplasmic followed by 11 to 12 predicted transmembrane α-helices. Many of the first ten TMDs display amphiphilic character, as they contain conserved negative charges embedded within the membrane, and may be involved in key structures including proton motive force and divalent cation transport pathways. Phylogenomic studies indicate Nramp family and function originated in Bacteria life domain and highlight the complex evolution of bacterial Nramp homologs. These studies include compiled phylogenetic data using various approaches (distance matrix, maximum parsimony and likelihood, and statistical bootstrapping) and either full-length sequences or a set of conserved, informative positions to infer an extensive Nramp family tree with distance estimates, and verify congruency with the species phylogeny (based on 16S RNA). Additional statistical analyses of a.a. replacement rate variations were done for each group. Comparative studies of sequenced genomic loci were carried out to document vertical or horizontal ancestry. Functional studies were executed by expression in E. coli mntH A strains.

The Nramp Family, edited by Mathieu Cellier and Philippe Gros. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers.

Molecular Evolutionary Analysis of Nramp Family

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Nramp family is subdivided in several branches, most of them congruent with the phylogeny of corresponding organisms, suggesting they represent groups of orthologs that diverged with speciation. Parologs resulting from ‘prototype’ Nramp gene duplication early in eukaryote evolution were found (outparologs); various evidences demonstrate bacterial MntH xenologs, resulting from horizontal gene transfer. The bacterial clade the most distant from eukaryotic Nramp, MntH B, is a monophyletic group of sequences showing moderately tight grouping that were found in distant anaerobic bacteria. MntH A group is closer to eukaryotic Nramp and includes members from both high- and low-GC Gram positive Bacteria and Gram negative Bacteria. MntH A possibly contained the precursor of eukaryotic Nramp. MntH C is subdivided in three subgroups (Cα, Cβ, Cγ) in which horizontal gene transfer is prevalent. MntH Cα sequences belong to distant species but are closely related and remarkably similar to ‘prototype’ Nramp suggesting gene transfer from Protist to Bacteria.

Early Gene Duplication in Eukaryotes Gave the Outparologs ‘Prototype’ and ‘Archetype’ Nramp Nramp protein family was defined using highly similar sequences from mammal, invertebrate, plant and yeast which also demonstrated remarkable conservation in hydrophobicity and predicted amphiphilic helical structures susceptible to form a hydrophilic transmembrane ion transport pathway.1 Similar transport functions were demonstrated using Nramp proteins from distant eukaryotic species and inter-specific genetic complementation.2-6 These data indicate that the role of eukaryotic Nramp proteins is to facilitate cellular chemiosmotic uptake of divalent metal ions (detailed in Chapter 1, Jabado et al, Chapter 7, Mackenzie & Hediger, and Chapter 12, Cohen et al). Recently, genome sequencing and detailed phylogenetic analyses showed eukaryotic Nramp protein sequences segregate in two clusters : ‘prototype’ Nramp, including yeast SMF 1-3, and ‘archetype’ Nramp found mostly in multicellular organisms (including the social amoeba7). Data from the model species Dictyostelium discoideum (Mycetozoa, DdNR1, 2, (Fig. 1) and Anopheles gambiae (Arthropoda, Insecta, AgNR1, 2) -which diverged from the eukaryotic root before the separation of plants and vertebrates, respectively- revealed the coexistence of two divergent types of eukaryotic Nramp genes: ‘prototype’ Nramp encode proteins that are close to each other and to microbial homologs; ‘archetype’ Nramp code for proteins more similar to animal Nramp. The phylogenetic distance between these parologous ‘proto-’ and ‘archetype’ Nramp sequences implies strong divergence after gene duplication (Fig. 1). The divergence of ‘archetype’ Nramp was accompanied by the insertion of at least one spliceosomal intron marking the beginning of Nramp hydrophobic core, before predicted TMD 1. The position of this intron is conserved in Dictyostelium, Drosophila and human genes, including canonical intron boundaries (5'-GT…3'-AG; Fig. 2B). These data raise the possibility that ‘prototype’ and ‘archetype’ Nramp are outparologs that resulted from a gene duplication prior to the separation of Myceotozoa and animal phyla. Yeast SMF proteins are closer to ‘prototype’ Nramp and contain 11 predicted TMD like most other microbial sequences. Yeast genes are intronless and were duplicated twice before the divergence of Saccharomyces cerevisiae and Candida albicans (Fig. 1); S. cerevisiae SMF proteins differ both in cellular location and metallo-regulation (detailed in Chapter 11, Luk et al.). Another recent duplication of SMF1 gene occurred in the pathogen C. albicans ; whether it affects its virulence is not known. A putative SMF1 homolog was found in the distant fungus, Neurospora crassa (Pezizomycotina, Sordariaceae), suggesting SMF1 may be close to yeast ancestral sequence. Aside from the ancestral gene duplication yielding the outparologs ‘prototype’ and ‘archetype’ Nramp, several phyla-specific gene duplications also occurred (inparologs), notably in plants (presented in Chapter 10, Thomine & Schroeder), and worm, fish, and mammals, presumably due to species-specific adaptations.

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Figure 1. Phylogenetic analysis of the Nramp / MntH super-family. An unrooted tree obtained by Maximum Likelihood phylogenetic analysis using 292 parsimonious positions and the model of a.a. substitution BLOSUM 62 is presented. Statistical confidence is indicated in percent at the right of each node. The scale of 0.1 a.a. replacement per site is indicated. Systematic names for proteins are composed of the initial of genus name in capital, two to four letters from the species names followed by NR for eukaryotic Nramp proteins or A, B, or Cα, Cβ, and Cγ for prokaryotic MntH proteins; numbers have been added in multigenic organisms. The taxonomic distribution of the species possessing Bacterial MntH and corresponding phylogenetic grouping is presented in Table1. Eukarya: Ag= Anopheles gambiae; At= Arabidopsis thaliana; Bt= Bos taurus; Ca= Candida albicans; Ce= Caenorhabditis elegans; Cre= Chlamydomonas reinhardtii; Dd= Dictyostelium discoideum; Dm= Drosophila melanogaster; Gg= Gallus gallus; Hs= Homo sapiens; Ncr= Neurospora crassa; Om= Oncorhynchus mykiss; Os= Oryza sativa; Mm= Mus musculus; Sc= Saccharomyces cerevisiae.


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Figure 2. Duplication of Nramp gene in eukaryote yielded ‘prototype’ and ‘archetype’ Nramp. A) Condensed tree obtained by the minimal evolution method, using distance-based calculation and the least square method to infer the phylogenetic tree; 3000 replica of statistical bootstraping were performed to evaluate the significance of each node (here, above 50%); branch length is not proportional to sequence relationship. B) Nramp protein sequence alignment and corresponding gene sequences showing conservation of the position of the first intron found in the ‘archetype’ Nramp genes of Dictyostelium discoideum, Drosophila melanogaster and Homo sapiens.

Two distinct types of Nramp are found in plants (plant Nramp I and II, (Fig. 1, 2A). Plant I appears intermediate between ‘prototype’ and ‘archetype’ Nramp clusters, and Plant II group is part of ‘archetype’ Nramp. Plant I and II divergence predated the speciation of eudicot (A. thaliana) and monocot (O. sativa). In the protochordate C. intestinalis and all vertebrates, only ‘archetype’ Nramp was conserved (animal Nramp). The gene duplication that produced mammalian Nramp1 and 2 probably occurred in vertebrates (Fig. 2B), which would be consistent with their different tissue and cell specific expression (presented in details in Chapter 8, EH Morgan, and Chapter 9, Canonne-Hergaux & Gros). Whereas Nramp2 is expressed in many tissues and cell types and is required for iron homeostasis, Nramp1 gene is expressed in mature professional phagocytes contributing mainly to innate immunity (e.g., see ref. 8).

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Current data obtained with yeast and animal Nramp suggest ‘prototype’ and ‘archetype’ Nramp are functionally conserved but distinct (see Chapter 12, Cohen et al). Study of Dictyostelium ‘archetype’ and ‘prototype’ Nramp cellular function and location will enable to compare the properties of Nramp outparologs in a model organism. D. discoideum feeds on bacteria that are ingested and degraded within a phagosome ; it will be of interest to determine –if and- which Dictyostelium Nramp is involved in antimicrobial defence.

Characterization of Bacterial Nramp Orthologs: MntH A and B The existence of bacterial Nramp homologs -denominated MntH- suggests this family existed prior to the emergence of eukaryotes. The polyphyletic origins of mntH genes question the ancestry of eukaryotic Nramp and hypothetical role of mntH genes in bacterial virulence.

MntH A and Bacterial Virulence

Several MntH A proteins were studied once the role of yeast SMF1 protein in Mn2+ uptake2 and the existence of a M. leprae Nramp candidate homolog were established.1 One aim was to determine whether bacterial MntH could perform a similar transport function and would compete with host Nramp1 for metal acquisition within the phagosome during intracellular infection. Enteric E. coli MntH A was characterized by gene knockout and add-back experiments as a proton-dependent Mn2+ transporter facilitating the uptake of other divalent metal ions, in whole cell transport assays, and by complementation of a E. coli metal-dependent mutant.9 As E. coli does not normally survive in macrophages, targeted mutations were also generated in Mycobacteria and Salmonella to address the possible role of mntH A homologs in virulence. Salmonella enterica serovar Typhimurium (S. typhimurium) has two Mn2+ transporters : a pH-dependent Mn2+ permease MntH A and an ABC-type transporter SitABCD (‘Salmonella iron transporter’10-13). The mntH A gene is carried by a chromosome fragment conserved in E. coli (~120 kb) and Yersinia spp. (~ 22 kb), whereas sitABCD is encoded by Salmonella pathogenicity island 1,14 and also found close to mobile DNA in E. coli CFT 073 and Y. pestis pathogens. Salmonella MntH A is required for resistance to hydrogen peroxide (H2O2) and regulated transcriptionally by the metallo-repressors Fur and MntR and the H2O2 responsive activator OxyR (detailed in Chapter 13, Helmann JD, and in Chapter 14, and Papp et al.). SitABCD mediates both Mn2+ and Fe2+ uptake at alkaline pH. It is a virulence factor in the systemic phase of the murine typhoid that is expressed intracellularly and in response to iron privation,15 suggesting regulatory mechanisms similar to mntH A gene. The role of S. typhimurium mntH A in virulence was tested, compared to the role of sitABCD and feoB, encoding the GTP-dependent ferrous iron transporter, by gene knockout experiments and systemic infections in Nramp1+ or Nramp1-/- mice.10 The mortality and morbidity induced by infection demonstrated that both sitABCD and feoB were required for full virulence in Nramp1-/- animals, whereas all Nramp1+ animals survived the intravenous challenge (103 bacteria). In vitro, Nramp1 expression by Raw macrophages inhibited the growth of all Salmonella strains tested; the double mutant mntH A sitABCD, lacking Mn2+ uptake, was most affected. Infection of Nramp1-/- macrophages required the addition of a cell permeant Fe2+ chelator to observe diminished intracellular growth of S. typhimurium mutants; sitABCD mutant was also affected. The results are consistent with the observation that Salmonella do not experience iron shortage during in vitro infection of Nramp1-/- macrophages.16 In vivo, the acquisition of divalent metals is important for Salmonella virulence in Nramp1-/- animals and the primary transporters of Mn2+ and Fe2+ are more important than MntH A. High dose infections will be required to study Salmonella survival in presence of Nramp1 and to address the possible competition between host and bacterial Nramp homologs. MntH A proteins were also studied in both low- and high-GC Gram positive species (Table 1). B. subtilis mntH A expression was found MntR- and Mn-dependent, and required for growth in Mn-limited medium.17 The role of M. tuberculosis mntH A in virulence in mice was


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tested using mntH A knockout mutant and both high- and low-dose intravenous infections. The results presented in Chapter 15 (Domenech and Cole) appear consistent with the Salmonella study, indicating that although mntH A may contribute to resistance to metal starvation and/or oxidative stress, primary transporters of divalent metals (feo, sit) may be more important during the systemic phase of infection.

Identification of MntH B in Anaerobes Group B sequences were revealed by genome sequencing of anaerobe species ; phylogenetic analyses showed they represent a separate group.18 Heterologous expression of C. tepidum and Clst. acetobutylicum MntH B in E. coli conferred increased sensitivity to divalent cations,7 modulating the Fe-dependent temperature sensitive growth of E. coli hflB1 Ts mutant. The results suggest that divalent metals including Fe2+ are substrates for MntH B proteins, implying conserved function with MntH A despite relatively low sequence similarity. Group B sequences show relatively low level identity and loose phylogenetic grouping (Fig. 3), consistent with sizeable phylogenetic distances between the corresponding species (based on 16S RNA, Green sulfur bacteria, Cytophaga-Flexibacter-Bacteroides group, and low-GC Gram positive Clostridiaceae and lactic Bacteria). A high value of the shape parameter alpha (1.3) was obtained with relatively few MntH B protein sequences, indicating a uniform gamma distribution of the a. a. replacement rate among sites in this group. The data seem consistent with an early origin of the ancestor of MntH B group.7 MntH B site-specific a.a. substitution rate (coefficient theta) was found more similar to the value obtained for MntH A than for any other group, suggesting limited functional (type 1) divergence between MntH A and B. However, MntH A group also showed low level site-to-site difference in a.a. replacement (alpha shape parameter, 1.22), due partly to the phylogenetic distance between species composing MntH A group and to the number of sequences which increases diversity. The significance of predicted functional divergence from MntH B was increased after considering less diverse MntH A subsets, notably low- vs. high-GC Gram positive Bacteria, but not in the case of the subgroup MntH A* that comprises proteins from α- and β-Proteobacteria possessing additional mntH Cγ genes. Two low-GC Gram positive Clostridiaceae spp. possess both one mntH A and one mntH B gene. Clostridiaceae MntH A sequences form a sister clade of Bacillaceae MntH A, presumably due to the presence of the additional mntH B gene in their genomes (Fig. 1). The potential type I functional divergence predicted between clostridial MntH B and A proteins might suggest mntH A and B genes diverged after an ancestral gene duplication.7

α, Cβ β, Cγγ Study of MntH Xenologs: MntH Cα A third group of bacterial Nramp homologs was found (MntH C) that displayed higher similarity to ‘prototype’ Nramp than to MntH A and B groups, being phylogenetically closer to eukaryotic homologs. mntH C genes are present in distant phyla including α, β- and γ-Proteobacteriaceae, low-GC Gram positive Bacteria and Cyanobacteria, due to prevalent horizontal transmission.

MntH Cα This clade shows the shortest distances between members despite they derive from distant Bacteria (based on 16S RNA phylogeny, Table 1). MntH Cα also contains the closest sequences to the protist Chlamydomonas sequence and to other ‘prototype’ Nramp (Fig. 1). Site-to-site a.a. replacement rate in MntH Cα group follows a nonuniform gamma distribution that indicates preservation of a pattern of a.a. conservation/variability. This result contrasts with those obtained for MntH A and B, and may be explained by a more recent origin of MntH Cα. Consistently, the gamma distributions calculated for pairwise combinations of MntH Cα, Cβ or Cγ subgroups remained heterogeneous, indicating conservation of the a. a. substitution pattern across these subgroups and suggesting they derive from a common ancestor.7

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Figure 3. Horizontal gene transfer of mntH Cβ1 in group B Streptococcus (GBS). The sequence of mntH loci was compared using the complete genomes of three strains of S. agalactiae serotypes Ia (A909), III (NEM316) and V (2603 V/R) and one strain of S. mutans. Strains A909 and NEM316 were similar in gene content and order and possess only one mntH Cβ2 gene, conserved in sequence and genomic context with S. mutans mntH Cβ2 gene (A). The strain 2603 V/R was unique, exhibiting an additional mntH Cβ1 gene within a presumably mobile DNA element (B).

Prediction of potential divergence of function for MntH Cα was significant with ‘archetype’ Nramp, MntH A and B, but minimal between MntH Cα, Cβ and Cγ. Preferential divergence from ‘archetype’ Nramp was also observed for MntH Cβ, MntH Cγ and yeast Nramp, suggesting it may reflect in part the divergence between ‘prototype’ and ‘archetype’ Nramp. This would support the proposition that MntH C ancestor belonged to the ‘prototype’ Nramp group. Several α-Proteobacteria possess a mntH Cα gene. This phylum also includes the genus Rickettsia that is the closest known to the ancestor of mitochondria, raising the possibility that


Table 1. Taxonomic distribution of bacterial homologs of MntH groups B, A, A*, Cα, Cβ, Cγ Taxonomic Group

Species

Abbreviation

Phylogenetic MntH Group

Archaea Euryarchaeota

Halobacteriales

Natromonas pharaonis

CFB/Green Sulfur Bacteria

Cytophagales/ Flavobacteria/ Bacteroidetes

Bacteroides fragilis B. thetaiotaomicron

Green sulfur bacteria

Chlorobium tepidum

Ctepi

B

Low GC Gram positive

Clostridiales

Clostridium acetobutylicum Carboxydothermus hydrogenoformans

Cacet Chydr

B

A A

Thermoanaerobiales Bacillales

Thermoanaerobacter tengcongensis Bacillus subtilis B. cereus B. anthracis Listeria hydrogenoformans L. inocua Staphylococcus aureus S. epidermidis

Tteng Bsubt Bcere Banth Lmono Lino Saur Sepi

B

A A A A

Enterococcus faecalis E. faecium Lactobacillus brevis L. plantarum Oenococcus oeni Leuconostoc mesenteroides Streptococcaceae agalactiae S. cricetus S. gordonii S. mitis S. mutans Lactococcus lactis

Efa Efcm Lbre Lpl Ooe Lmes Sag Scri Sgor Smit Smut Llact

Nphar

Outgroup

Bfrag Btheta

B B

Bacteria

Lactobacillales

Cα Cα Cβ Cβ

185

B^

Cβ1,2 Cβ Cβ Cβ1,2,3 Cβ1,2 Cβ Cβ1+,2 Cβ# Cβ Cβ1,2 Cβ Cβ continued on next page

186

Table 1. Continued Taxonomic Group

Species

Abbreviation


Bacteria High GC Gram positive

Actinomycetales

Mycobacterium tuberculosis M. leprae M. avium M. marinum M. smegmatis Clavivacter michiganensis Corynebacterium diphteriae

Mtube Mlpr Maviu Mmari Msmeg Cmich Cdiph

A A A A A A A#

Bifidobacteriales

Bifidobacterium longum

Blong

A A

Thermus/Deinococcus

Deinococcales

Deinococcus radiodurans

Dradi

Cyanobacteria

Nostocales

Nostoc sp. PCC 7120 N. punctiforme

Nost Npun

Cα Cα

Rhizobiales

Rhodopseudomonas palustris Bradyrhizobium japonicum Brucella suis Mesorhizobium loti Agrobacterium tumefaciens Rhizobium leguminosarum

Rpal Bjap Bsui Mlot Atum Rleg

Cα Cα Cα Cα Cα Cα

Proteobacteria

Alpha Subdivision

Rhodobacter capsulatus

Rcap

Magnetospirillum magnetotacticum

Mmag

Burkholderiales

Burkholderia mallei B. pseudomallei B. cepacia B. fungorum Ralstonia solanacearums

Bma Bps Bce Bfu Rsol

A*1 Cα A*2 A*2 A*1,2

Cγ1^,2 Cγ1 Cγ2 Cγ1,2 Cγ2

continued on next page

The Nramp Family

Beta Subdivision

Rhodobacterales Rhodospirillales


Table 1. Continued Taxonomic Group

Species

Abbreviation


Bacteria Gamma Subdivision

Enterobacteriales

Wigglesworthia brevipalpis Klebsiella pneumoniae Escherichia coli Shigella dysenteriae Sh. flexeneri Salmonella typhi S. typhimurium S. bongori Yersinia pestis Y. enterocolitica Erwinia chrysanthemi E. carotovora

Wbre Kpne Ecoli Sdys Sflex Styph Stypm Sbong Ypest Yente Echry Ecaro

Pseudomonadales

Pseudomonas aeruginosa P. syringae P. fluorescens

Pae Psyr Pflu

Cα

Xanthomonadales

Xanthomonas axonopodis X. campestris Xylella fastidiosa

Xaxo Xcam Xfas

Cα Cα Cα

Cβ A A A A A A A A A A A Cγ2 Cγ2 Cγ2

^= pseudogene; += strain polymorphism; #= small plasmid

187

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The Nramp Family

closeness between α-Proteobacterial MntH Cα and ‘prototype’ Nramp reflects horizontal gene transfer (HGT) from prokaryote to the eukaryotic nucleus. However, mntH genes in α-Proteobacteria are diverse, including one mntH A* in R. capsulatus, and two types of mntH Cα. One comprises sequences from closely related α-Proteobacteria (Rleg Cα, Atum Cα, Rpal Cα, Bsui Cα) ; the other type includes sequences from α- and γ-Proteobacteria (Mlot Cα, Mmag Cα, Xcam Cα, Xaxo Cα, Xfas Cα) that are encoded by a conserved divergent operon formed by mntR and mntH genes.7 It was localised in a region of Xanthomonas genome rich in transposable elements and strain specific genes, likely involved in host colonization and pathogenesis.19 Together these data indicate a recent origin for MntH Cα and raise the possibility that recent HGT in Bacteria resulted from an initial transfer from eukaryote to prokaryote.

MntH Cβ mntH Cβ genes are mainly restricted to lactic Bacteria and other low-GC Gram positive spp. such as Staphylococcaceae. Many species possess two mntH Cβ genes and several examples of likely recent HGT were documented. Most intriguing is the horizontal transfer of a mntH Cβ gene from a low-GC Gram positive spp. to the γ-Proteobacterium Wigglesworthia brevipalpis. Wigglesworthia is a primary endosymbiont of the tsetse fly possessing one of the smallest known bacterial genome with the lowest known GC content.20 Wbre Cβ is the single Wigglesworthia protein clearly more related to Staphylococcus or Streptococcus than to Enterics homologs. Wbre Cβ mntH gene is linked to an Asn-tRNA gene, which may facilitate recombination and gene transfer (e.g., Yersinia high pathogenicity island7). It will be of interest to establish whether mntH Cβ gene transfer occurred prior to or after establishment of endosymbiosis and how it contributes to Wigglesworthia fitness in the mutualistic interaction with its host. In Streptococacceae, several examples of mntH Cβ genes linked to potentially mobile insertion sequences (IS) also suggested horizontal transfer of mntH Cβ genes.7 An example of recent HGT was revealed by the genomic sequencing of several serotypes of the group B Streptococcus (GBS) S. agalactiae, a commensal of the human gastrointestinal and urogenital tracts that can cause life-threatening infections sepsis and meningitis in neonates and infants.21,22 Strains of GBS serotypes Ia (A909), III (NEM316) and V (2603 V/R) share together with S. mutans, another commensal but cariogenic species, a genomic DNA fragment carrying genes encoding (5' to 3' orientation) the peptidase U32, MntH Cβ2, and the lysyl-t-RNA synthetase. Connecting DNA segments at both extremities of mntH Cβ2 are different between S. mutans and S. agalactiae species, compatible with species-specific genetic plasticity (Fig. 3A). The GBS strain 2603 V/R possesses an additional gene (Fig. 3B), encoding a MntH Cβ1 protein highly similar to those of Enterococcus faecalis, Oenococcus oenii, Lactococcus lactis and Lactobacillus plantarum (Fig. 1, Table 1). S. agalactiae gene is localized at the 3' end of a DNA element presumed mobile, as its presence does not correlate with capsular serotypes; it is related to S. thermophilus integrative and conjugative element (ICESt1) that is suspected to facilitate HGT between lactic Bacteria spp. It is not known at present if and how this additional MntH Cβ1 protein increases GBS fitness. MntH Cβ1 proteins display 12 predicted transmembrane domains (TMD), a number normally found in eukaryotic Nramp proteins (except yeast proteins). Both proteins EfaCβ1 (12 TMD) and EfaCβ2 (11 TMD) were functionally expressed in E. coli, conferring increased sensitivity to Co and Cd.7

MntHCγ The subgroup Cγ is peculiar, displaying a relatively homogeneous gamma distribution of a.a. replacement rate among sites (alpha=1.1) despite fewer sequences. The subset MntH Cγ2 shows less uniform gamma distribution (alpha=0.65) and high sequence relationship to MntH Cα, whereas MntH Cγ1 is more related to MntH A* -a subgroup comprising species with both mntH A and Cγ genes. The low level of functional divergence predicted between MntH Cγ or Cγ2 groups and MntH A* vs. other MntH A subsets (e.g., Enterics, Gram positive Bacteria), argues against the suggestion that mntH A* and Cγ genes diverged after gene duplication in a


189

single species. In addition, the limited sequence divergence of MntH A* and Cγ2 from other groups, ‘archetype’ Nramp in particular, may be interpreted as an indication of their recent origin. One possibility is that species possessing both mntH A* and Cγ genes already had a mntH A gene before acquiring a novel mntH C gene by HGT, subsequently maintaining both functions. Consequently, both MntH Cγ and MntH A* sequences would appear specifically different. If eukaryotic ‘prototype’ Nramp genes were ancestors of mntH C genes, the presence of three subgroups Cα, Cβ and Cγ would suggest repeated opportunities for gene transfer. Each subgroup contains species living intracellularly, indicating that close association with eukaryotic host may have favoured HGT of a ‘prototype’ Nramp gene.7

Proposed Evolutionary Pathway of Bacterial Nramp Genes Several possible steps in the evolution of Nramp family are proposed (Fig. 4): (i) early origin in Bacteria, probably an anaerobe fermenting species, of MntH B -a proton-dependent divalent metal cations symporter (ferrous iron?); (ii) emergence of MntH A, before the divergence of Gram positive and negative clusters and Deinococcus group, with a preference for Mn2+; (iii) horizontal transfer of a mntH A gene to the eukaryotic nucleus after an endosymbiotic event giving rise to ‘prototype’ Nramp; (iv) gene duplication in eukaryotes, predating the separation of Mycetozoa, animals and plants enabling divergence of ‘archetype’ Nramp; (v) horizontal transfer of eukaryotic ‘prototype’ Nramp gene to Bacteria, creating MntH C and further spread by horizontal transfer. MntH B deep branching in Nramp tree implies early emergence (Fig. 1). Bacteroides / Chlorobia group diverged early in the evolution of Bacteria24-26 and C. tepidum performs anoxygenic photosynthesis using an iron-based reaction center (RC I), a likely precursor of cyanobacterial Mn-dependent RC II required for oxygenic photosynthesis.27,28 Thermoanaerobacter spp. fermentate sugars using thiosulfate and sulphur, but not sulfate or sulfite, as electron acceptor.29 These anaerobic Bacteria have thus growth requirements compatible with early earth atmosphere (anaerobiosis, reduced sulfur, soluble ferrous iron, high temperature, light) i.e., prior to the onset of aerobiosis (ca 2.4 gyrs, estimated by iron oxidation and sulfur isotope variation records.28,30 MntH A is an evolutionary intermediate between MntH B and C. Similarly to MntH B, but contrary to MntH C, MntH A sequence relationships (except MntH A*) appear congruent with the phylogeny and gene content of corresponding species, and are supported by analyses of a.a. replacement rate variations. These properties indicate a likely origin of MntH A prior to the separation of Gram positive and negative clusters, and Deinococcus group. The demonstration of MntH A-dependent high affinity Mn2+ uptake in D. radiodurans, whose thermo-resistance and DNA repair capacity is affected by Mn31 would support this proposition. It is a possibility that MntH A diverged from MntH B after gene duplication; also, the sequence of another low-GC Gram positive species, Carboxydothermus hydrogenoformans, was found either branching between MntH B and A groups or diverging before MntH B. On the other hand, the closer relationship of MntH A (vs. MntH B) with eukaryotic Nramp suggests a mntH A gene as likely endosymbiotic ancestor of eukaryotic Nramp. Together these observations support an early emergence of MntH B prior to MntH A.18 That eukaryotic ‘prototype’ Nramp derived from prokaryotic MntH implies adaptation to novel cellular compartments, membrane composition and mechanisms of regulation for efficient divalent metal uptake (possibly in competition with MntH expressing Bacteria). Mycetozoa and Arthropoda share the closely related ‘prototype’ homologs (DdNR1 and AgNR1, Fig. 1), indicating a common eukaryotic ancestry rather than separate horizontal acquisitions of bacterial MntH, given the actual MntH diversity (Fig. 1). After the gene duplication that yielded ‘archetype’ Nramp, some protists and invertebrates maintained ‘prototype’ Nramp, whereas others and the vertebrates lost it. Fungi possess several ‘prototype’ but no ‘archetype’ Nramp ; this suggests fungi either diverged before the ancestral

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The Nramp Family

Figure 4. Hypothetical evolutionary time scale of Nramp evolution (in billions years ago, bya). A) Estimated molecular time for the divergence of Archaea and Bacteria (~ 3.5 bya), origin of Eukarya (~ 2.7 bya), apparition of Cyanobacteria (~ 2.6 bya), the rise of atmospheric O2 from below 1% to above 15% (~2.3 bya), origin of mitochondria (~2.0 bya), the divergence between animals, plants and fungi (~1.6 bya), and between invertebrates and vertebrates (~ 1.0 bya), the divergence of birds (~0.3 bya) and between Eudicot and Monocot plants as well as the radiation of mammals (~0.1 bya) are from Ref.26,28,43-45 B) Prokaryotic MntH B could date back to the apparition of anoxygenic photosynthetic organisms which may have been able to colonize meso-thermophile habitats (e.g., shallow water setting); MntH A may have evolved in more oxygenic conditions and could have been selected in aerobic organisms. Eukaryotic Nramp likely derive from a mntH A gene transferred to the eukaryotic nucleus after an endosymbiotic event (between 2.7 and 2.0 bya) and was duplicated in protist (before 1.6 bya) yielding the proto- and archetype Nramp outparologs. MntH C probably appeared afterward following horizontal transfer of a eukaryotic prototype Nramp gene.

Nramp gene duplication or selectively retained ‘prototype’ and lost ‘archetype’ Nramp. As fungi are generally viewed close to metazoans, and given the presence of ‘archetype’ Nramp in protozoans, believed to have diverged early in eukaryotic evolution (e.g., Plasmodia), selective maintenance of ‘prototype’ Nramp in fungi may seem more likely. MntH C possibly results from the transfer of a eukaryotic ‘prototype’ Nramp gene to a Bacterium and further propagation by HGT. MntH C grouping may reflect shared ecological niches by the respective species, e.g., gastro-intestinal tract for MntH Cβ or intracellular milieu for MntH Cα, aiding to create (contact or gain-of–function) opportunities for mntH gene transfers. Minimal functional divergence was predicted between MntH Cα and Cβ suggesting their main differences could be the type of gene that was acquired, e.g., ‘prototype’ Nramp or mntH C, and presence or absence of other mntH genes in the recipient Bacteria. Common MntH C properties would include deriving from a ‘prototype’ Nramp ancestor and improving bacterial divalent metals acquisition at the interface with eukaryotic cells.


191

Significance and Implications in the Context of Bacterial Infection Presence and Diversity of MntH in Bacteria No sequence was yet demonstrated to encode an Archaeal homolog. Biological membranes are more permeable to proton at high temperatures and hyperthermophilic Bacteria and Archaea use the less permeable ion sodium to maintain a motive force;32 they also use preferably ATPor GTP- driven systems for carbon and energy sources uptake (e.g., MntA or FeoB homologs, respectively33). In addition, Archaea have fewer transporters than most Bacteria.32 Hence biology and lifestyle may explain the absence of proteins similar to MntH in Archaea. In absence of Archaeal homolog, eukaryotic Nramp likely derived from a Bacterial ancestor. MntH A group sequence properties support an origin predating eukaryotic Nramp; MntH B group appeared even more ancient whereas MntH C clearly had a more recent origin. Sequence differences between MntH groups B and A may thus reflect their antiquity, relating to divalent metals uptake by Bacteria using the proton-motive force. In contrast, MntH C sequence diversity would relate to prevalent HGT and eukaryotic ‘prototype’ Nramp origin of mntH C genes.

MntH C and Bacterial Fitness A logical correlate to the horizontal transfer of mntH C genes is a gain of function for recipient Bacteria, as acquisition of genomic islands or islets is a common evolutionary mechanism used by Bacteria to adapt to their environment.34 HGT concerns genes subjected to positive selection for increasing bacterial fitness in various conditions (ecological, saprophytic, symbiotic or pathogenic fitness). A single function, e.g., iron uptake, may favour the growth of related Bacteria either pathogen (Yersinia), saprophyte (Escherichia) or environmental (Klebsiella 34). Considering MntH membrane transport a simple operational function, it is expected to find examples of mntH HGT. However, the high prevalence of HGT in MntH C group suggests distinctive properties, useful in different conditions given the diversity in bacterial species in this group. Novel membrane transport properties could have resulted from the adaptation of ‘prototype’ Nramp in eukaryotic cells and might explain subsequent spreading of mntH C xenologs among various Bacteria spp. This could be tested by comparing the role of MntH C and A in bacterial infection of eukaryotic hosts. One possibility is that MntH C proteins could confer increased resistance to metal starvation and oxidative stress conditions encountered during host infection, or to other conditions unfavourable for microbial growth (e.g., food preservation). Intracellular accumulation of Mn can increase bacterial growth and fitness, providing elemental protection against reactive oxygen species.35 Several virulent bacteria use this strategy during infection whereas host defences aim at depleting microbial environment of metal ions.36,37 Bacterial pathogens, including Enterics, rely primarily on ABC-type transporters related to SitABCD to maintain full virulence in vivo.15,38 However, inactivation of both mntH Cβ and mntABC was required to observe reduced growth of Staphylococcus aureus in a murine abscess model.39 Future studies will aim at establishing how MntH C vs. MntH A-dependent transport can increase bacterial fitness and if it is important for infection. Mn improves resistance to various stresses as it is a cofactor of many detoxifying enzymes.37 For example, Mn- and O2-dependent decarboxylases create a “proton-motive metabolic cycle’’ used to take solute up for intracellular decarboxylation and efflux of the product, in turn producing ATP and protecting cells against acidification.40 The lactic bacterium O. oeni uses the malolactic fermentation cycle to cope with external pH ~ 3.5 in the presence of ethanol, which increases proton membrane permeability, and is used to balance wine.41 Similar Mn-dependent activity may be required to resist hoop bitter compound in beer; identification of Lactobacillus brevis mntH Cβ -the closest known relative of O. oeni mntH Cβ2- as a putative genetic marker of strains growing in beer, could indicate a role of MntH Cβ proteins in bacterial stress resistance.42 MntH C proteins could thus increase resistance to harsh environmental conditions,

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including metal starvation, acid and oxidative stresses, and favour bacterial growth in an infectious setting.

Outlook The Nramp family of divalent metal transporters originated early in the evolution of Bacteria before the divergence of Gram positive and negative clusters; MntH B homologs found in few Bacteria could represent the closest relatives to the ancestor of the family. Despite relatively low sequence identity, MntH B share with MntH A several characteristics (sequence length, hydrophobicity, and relative site-specific divergence rates) suggesting that sequence difference resulted from successive times of emergence and/or adaptation to changing environmental conditions (e.g., aerobiosis). The closer relationship between MntH A and Nramp suggests the endosymbiotic transfer of a prokaryotic mntH A gene towards eukaryotic nucleus. Two types of eukaryotic Nramp, ‘proto-’ and ‘archetype’, are believed to result from an ancient gene duplication in the common ancestor of plant, animal and fungi, followed by extensive sequence divergence. The functional and/or structural correlates of Nramp diversification are presently not known. ‘Archetype’ Nramp are expressed in vertebrates at host/microbe interface (e.g., epithelia and phagocytes) and required for metal homeostasis and resistance to infection. It will be interesting to study the role of these ‘prototype’ and ‘archetype’ Nramp proteins in host resistance to infection in eukaryotic species possessing both types. The close sequence relationship between ‘prototype’ Nramp and prokaryotic MntH C, subjected to prevalent horizontal gene transfer, strongly suggests the possibility of transfer between domains (Eukarya, Bacteria). The importance of horizontal gene transfer in adaptive processes of Bacteria is widely recognised. The potential role of MntH C in bacterial infection deserves further attention given the importance of eukaryotic Nramp for host resistance and the existence of endo-symbiotic or parasitic Bacteria that express MntH C. The use of a phylogenetic model will help understanding the evolution of Nramp mechanism of transport of divalent metals and its role in metal homeostasis.

Acknowledgments This work was supported by a research grant from the Canadian Institutes of Health Research (CIHR). E. R. is supported by the CIHR and Dr. Cellier is a scholar from the Fonds pour la Recherche en Santé du Québec.

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10. Boyer E, Bergevin I, Malo D et al. Acquisition of Mn(II) in addition to Fe(II) is required for full virulence of Salmonella enterica serovar Typhimurium. Infect Immun 2002; 70(11):6032-6042. 11. Kehres DG, Zaharik ML, Finlay BB et al. ME. The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen. Mol Microbiol 2000; 36(5):1085-1100. 12. Kehres DG, Janakiraman A, Slauch JM et al. Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H(2)O(2), Fe(2+), and Mn(2+). 13. Kehres DG, Janakiraman A, Slauch JM et al. SitABCD is the alkaline Mn(2+) transporter of Salmonella enterica serovar Typhimurium. J Bacteriol 2002; 184(12):3159-3166. 14. Zhou D, Hardt WD, Galan JE. Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island. Infect Immun 1999; 67(4):1974-1981. 15. Janakiraman A, Slauch JM. The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of Salmonella typhimurium. Mol Microbiol 2000; 35(5):1146-1155. 16. Eriksson S, Lucchini S, Thompson A et al. Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol 2003; 47(1):103-118. 17. Que Q, Helmann JD. Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol Microbiol 2000; 35(6):1454-1468. 18. Cellier MF, Bergevin I, Boyer E et al. Polyphyletic origins of bacterial Nramp transporters. Trends Genet 2001; 17(7):365-370. 19. da Silva FR, Vettore AL, Kemper EL et al. Fastidian gum: The Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity. FEMS Microbiol Lett 2001; 203(2):165-171. 20. Akman L, Yamashita A, Watanabe H et al. Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia. Nat Genet 2002; 32(3):402-407. 21. Tettelin H, Masignani V, Cieslewicz MJ et al. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc Natl Acad Sci 2002; 99(19):12391-12396. 22. Glaser P, Rusniok C, Buchrieser C et al. Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol Microbiol 2002; 45(6):1499-1513. 23. Burrus V, Pavlovic G, Decaris B et al. The ICESt1 element of Streptococcus thermophilus belongs to a large family of integrative and conjugative elements that exchange modules and change their specificity of integration. Plasmid 2002; 48(2):77-97. 24. Brochier C, Philippe H. Phylogeny: A nonhyperthermophilic ancestor for bacteria. Nature 2002; 417(6886):244. 25. Brochier C, Bapteste E, Moreira D et al. Eubacterial phylogeny based on translational apparatus proteins. Trends Genet 2002; 18(1):1-5. 26. Hedges SB. The origin and evolution of model organisms. Nat Rev Genet 2002; 3(11):838-849. 27. Baymann F, Brugna M, Muhlenhoff U et al. Daddy, where did (PS)I come from? Biochim Biophys Acta 2001; 1507(1-3):291-310. 28. Nisbet EG, Sleep NH. The habitat and nature of early life. Nature 2001; 409(6823):1083-1091. 29. Zhou XJ, Vaziri ND, Pandian D et al. Urinary concentrating defect in experimental hemochromatosis. J Am Soc Nephrol 1996; 7(1):128-134. 30. Wiechert UH. Geology. Earth’s early atmosphere. Science 2002; 298(5602):2341-2342. 31. Levin-Zaidman S, Englander J, Shimoni E et al. Ringlike structure of the Deinococcus radiodurans genome: A key to radioresistance? Science 2003; (5604):254-256. 32. Konings WN, Albers SV, Koning S et al. The cell membrane plays a crucial role in survival of bacteria and archaea in extreme environments. Antonie Van Leeuwenhoek 2002; 81(1-4):61-72. 33. Koning SM, Albers SV, Konings WN et al. Sugar transport in (hyper)thermophilic archaea. Res Microbiol 2002; 153(2):61-67. 34. Hacker J, Carniel E. Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes. EMBO Rep 2001; 2(5):376-381. 35. Horsburgh MJ, Wharton SJ, Karavolos M et al. Manganese: Elemental defence for a life with oxygen. Trends Microbiol 2002; 10(11):496-501. 36. Posey JE, Gherardini FC. Lack of a role for iron in the Lyme disease pathogen. Science 2000; 288(5471):1651-1653. 37. Jakubovics NS, Jenkinson HF. Out of the iron age: New insights into the critical role of manganese homeostasis in bacteria. Microbiology 2001; 147(Pt 7):1709-1718. 38. Claverys JP. A new family of high-affinity ABC manganese and zinc permeases. Res Microbiol 2001; 152(3-4):231-243.

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39. Horsburgh MJ, Wharton SJ, Cox AG et al. MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake. Mol Microbiol 2002; 44(5):1269-1286. 40. Foster JW, Moreno M. Inducible acid tolerance mechanisms in enteric bacteria. Novartis Found Symp 1999; 221:55-69. 41. Graca da Silveira M, Vitoria San Romao M, Loureiro-Dias MC et al. Flow cytometric assessment of membrane integrity of ethanol-stressed Oenococcus oeni cells. Appl Environ Microbiol 2002; 68(12):6087-6093. 42. Hayashi N, Ito M, Horiike S et al. Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillus brevis strains capable of growing in beer. Appl Microbiol Biotechnol 2001; 55(5):596-603. 43. Kasting JF. Earth history. The rise of atmospheric oxygen. Science 2001; 293(5531):819-820. 44. Archibald JM, Keeling PJ. Recycled plastids: A ‘green movement’ in eukaryotic evolution. Trends Genet 2002; 18(11):577-584. 45. Hedges SB, Kumar S. Genomic clocks and evolutionary timescales. Trends Genet 2003; 19(4):200-206.

INDEX

A

C

Anemia 5-7, 38, 39, 48, 66, 68, 69, 73, 74, 76, 77, 79, 82, 83, 88, 90, 100, 101, 103, 108, 155 Arabidopsis 5, 49, 113-121, 129, 158, 159, 181 Aspergillus nidulans 47 Association studies 29, 30, 33, 34 Atopy 37, 38, 53-58, 62 Autoimmune disease 5, 29, 36, 37, 53-59, 61, 62

Cadmium 76, 113, 114, 116, 120, 121, 125, 130, 131, 178 Caenorhaditis elegans 4, 157 Candida albicans 4, 99, 156, 179, 181 Ceruloplasmin 108, 109 Chagas’ disease 37 Chelator 6, 8, 10, 121, 125, 126, 136, 142, 150, 182 Chicken 16-21, 23, 59 Chlamydia 4, 158 Collagen-induced arthritis (CIA) 59-61 Comparative genomics 16, 17 Complementation of yeast 115, 116, 119, 135, 136, 141 CorAD 8 crnA 47 Crohn’s Disease (CD) 36, 53-55

B Bacillus Calmette-Guerin (BCG) 3, 17, 18, 23, 29, 37, 38, 44-46, 48, 53-56, 58, 61, 62, 158, 159, 172-174 Bacillus subtilis 4, 9, 146-151, 156, 159, 163, 174, 182, 184 Bacteria 2, 4, 8, 10, 18, 21, 45, 47, 99, 113, 117, 129, 136-139, 141, 142, 146-148, 151, 154-156, 159, 161, 163, 165-169, 172, 174-176, 178, 179, 182-184, 186-192 Belgrade rat 74, 82-84, 86, 91, 100, 101 Blood-brain barrier (BBB) 83, 91 Bovine 21, 23, 24, 157 Brain 6, 32, 61, 65, 68, 82, 83, 87, 91, 108 Breeding 17, 21, 22, 24 Brucella abortus 3, 21-24 Brucella suis 16, 21, 22, 187 Brucellosis 16, 21-24 BSD2 129 Bsd2 129-131

D Diabetes 37, 53-55, 61 Disease resistance 16, 21, 23, 24, 32 Divalent cation 1, 5-8, 10, 12, 32, 38, 44, 48, 49, 53, 54, 57, 62, 66, 88, 99, 138, 139, 147, 154, 155, 161-163, 167, 168, 173-175, 178, 183 Divalent metal 7, 8, 10, 11, 30, 66, 67, 73, 82, 88, 90, 96, 99, 104, 106, 110, 129, 135-137, 139, 141, 142, 147, 161, 162, 179, 182, 183, 189, 190-192 Divalent metal transporter 66, 82, 96, 99, 192 Divalent metal transporter 1 (DMT1) 4, 65-70, 73-79, 82-93, 96, 99-110, 115, 118, 125, 127, 129 Drosophila melanogaster 4, 69, 117, 142, 155, 157, 180, 181

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196

E

G

EIN2 5, 49, 114-117, 121, 159 ein2 115 Endosymbiosis 188 Enterocytes 67, 69, 77, 78, 82-86, 103, 104, 106 Erythroid cell 77, 82, 83, 88, 90, 96, 103, 106 Erythroid tissue 88 Erythropoiesis 65, 69, 105, 106 Erythropoietic cells 82, 90 Escherichia coli 4, 5, 48, 117, 146, 148-150, 156, 159, 161-163, 168, 174, 178, 182, 183, 186, 188 Eukaryotic Nramp1 167, 172, 173, 175 Experimental autoimmune encephalomyelitis (EAE) 59, 61 Expressed sequence tag (EST) 17, 113-115

Gastrointestinal tract 83, 84 Gene duplication 179, 181, 183, 188-190, 192 Gene expression 4, 19, 24, 32, 45, 48, 97, 99, 118, 119, 129, 130, 146, 147 Glial cells 82, 91 Golgi 98, 124, 127, 129, 131 Granulocyte-macrophage colony stimulating factor (GM-CSF) 47, 97

F Fe2+ 5-8, 11, 12, 44, 48, 49, 66, 67, 73-78, 82, 135-137, 139, 154, 155, 161-166, 168, 174, 182, 183 feoABC 8 feoB 8, 167, 182, 191 fepBCDG 8 Ferric uptake repressor (Fur) 8, 146, 148, 150, 151, 154, 163, 165, 166, 174, 182 Ferritin 39, 58, 61, 104, 106, 108, 109, 119, 138, 174 Fibrinogen 46 Fibroblasts 58, 83 Fibronectin 46 Fluorescence in situ hybridization (FISH) 17, 19 Francisella tularensis 4

H Heme iron 65-67, 69, 70, 77, 83, 84, 86, 87, 91, 99, 105, 107, 108 Hemochromatosis 39, 69, 73, 79, 105, 107, 136 Hepatocytes 65, 68, 82, 83, 87, 88, 102, 103, 107, 109 Hereditary hemochromatosis (HHC) 39, 73, 79, 105, 107 Heterologous expression 115, 121, 135, 139, 178, 183 HLA-DR 44 Horizontal gene transfer 179, 185, 188, 192 Host resistance 1, 2, 16-20, 24, 96, 155, 192 Human immunodeficiency virus (HIV) 1, 34, 35

I Immunoglobulin E (IgE) 37, 38, 54-56, 62 Infection 1-5, 7, 8, 16-24, 29, 32-37, 44-46, 48, 49, 62, 73, 96, 97, 99, 136, 137, 139, 142, 154, 155, 163, 166, 167, 172, 173, 175, 178, 182, 183, 188, 191, 192 Inflammatory bowel disease (IBD) 32, 36, 39, 53-55, 57

Index

Interferon γ (INFγ) 4, 19, 21, 32, 44, 45 Interleukin 1β (IL-1β) 47 Intestinal absorption 39, 48, 65, 73, 77, 83, 87, 99, 105 Intestinal iron absorption 39, 69, 77, 78, 83, 96, 155 Intestine 65, 66, 69, 73, 77, 79, 82-89, 104-106 Intracellular pathogen 3, 7, 16-18, 44, 45, 49, 66, 96, 99, 114, 146, 165, 172, 174 IRE-binding protein 77 Iron 5-8, 10, 36-39, 47-49, 58, 59, 61, 65, 66-70, 73, 75-79, 82-93, 96, 99, 101, 103-110, 113-115, 118, 119, 124-127, 129-131, 135-138, 140, 141, 143, 146-148, 150, 151, 155, 163-165, 167, 168, 174, 175, 181, 182, 189, 191 Iron deficiency 5, 6, 66-69, 82, 85, 88, 90, 99, 101, 107, 126, 129, 155 Iron metabolism 6, 39, 65, 68, 79, 82, 92, 99, 101, 105, 107, 124 Iron regulatory protein (IRP) 39, 48, 86, 99 Iron transport 6, 7, 38, 39, 48, 65-68, 70, 73, 76-78, 82, 83, 88, 90, 91, 106, 107, 114, 119, 127, 131, 140, 164, 167, 182 Iron-deficiency anemia 68 Iron-responsive element (IRE) 23, 32, 38, 68, 77-79, 83-85, 88, 90, 91, 93, 99, 101, 105-107, 119, 146 IRP1 77 Ity/Lsh/Bcg 3, 4, 54, 96

K Kawasaki disease 35, 56 Kidney 65, 68, 70, 78, 82, 83, 86, 90, 92, 102, 104-106, 155 Kinase 4, 47-49

197

L Legionella 4 Leishmania 2, 4, 5, 16-18, 23, 31, 35, 45, 49, 54, 57, 96, 98, 136, 154 Leishmania donovani 2, 3, 16-18, 23, 45, 46, 54, 96, 136, 154 Leprosy 2, 31, 32, 34, 35 Linkage studies 2, 11, 29, 33-35, 39 Lipopolysaccharide (LPS) 4, 22, 32, 46, 55, 56, 58, 97, 155, 175 Listeria 4, 45, 184 Liver 2, 19, 32, 37, 45, 47, 65, 66, 68, 77, 79, 82, 83, 86-88, 105, 107, 108, 155

M Macrophage 1-4, 6-11, 16, 18, 19, 21-24, 30, 32, 35-37, 44-49, 53, 54, 57-59, 61, 62, 65, 66, 68, 70, 73, 77, 78, 85, 96-99, 102, 105, 107, 108, 110, 113, 114, 136-139, 141, 142, 146, 154, 155, 165-168, 172-175, 178, 182 Major histocompatibility class II 44 Manganese 18, 49, 66, 113, 115, 124-126, 127, 129-131, 135, 137, 140, 143, 146-151, 165, 178 Manganese transport regulator 146, 149 Membrane protein 1, 2, 4, 44, 47, 96, 98, 114, 125, 155 Membrane transport 5, 65, 82, 107, 127, 191 Membrane-associated interleukin 1 (MaIL-1) 47 Menkes’ disease 114, 146 Metal homeostasis 114, 115, 118, 119, 121, 146, 192 Metal-ion 73, 135-143 Metal-ion transport 139-141 Mitochondria 82, 83, 106, 109, 115, 124, 125, 127, 129, 131, 184, 191 Mn2+ 5-7, 11, 44, 48, 49, 66, 75, 135-139, 154, 155, 161-168, 173, 174, 182, 189

The Nramp Family

198

MntH 4, 5, 8, 141, 142, 146-148, 150, 154, 161-166, 168, 172-176, 178, 179, 181-184, 186-192 MntR 146-151, 154, 163-166, 174, 182, 188 Monocyte 4, 22, 32, 44, 97 Multiple sclerosis (MS) 37, 53-55, 58, 59 Mycobacteria 1-3, 6-9, 18, 24, 29, 32, 35, 44, 45, 48, 53-55, 58, 62, 73, 137, 139, 172, 173, 182 Mycobacterium avium 3, 4, 6, 9, 10, 35, 48, 54, 137, 159, 173, 187 Mycobacterium bovis 3, 9, 10, 22, 29, 44, 48, 54, 61, 96, 154, 158, 159, 172-174 Mycobacterium intracellulare 3, 45 Mycobacterium lepraemurium 3, 45 Mycobacterium tuberculosis 1, 3, 5, 9, 24, 32, 34, 44, 46, 54, 55, 141, 146, 147, 151, 158, 159, 172-176, 182, 187

N Neutrophils 4, 8, 18, 58, 97, 99 Nitric oxide (NO) 21, 22, 46, 48, 49, 58, 59, 61 Nramp (NRAMP) 4-8, 12, 29, 31, 38, 40, 44, 46-49, 66, 68, 70, 82, 96, 97, 108, 113-119, 121, 124, 125, 127, 129-131, 135-137, 139, 142, 146-148, 154, 155, 157-159, 161-163, 167, 168, 172, 173, 178-184, 188-192 Nramp1 (NRAMP1) 1-12, 16-21, 23, 24, 29-38, 32, 40, 44, 46-48, 53, 54, 66, 68, 70, 73, 96-99, 108, 110, 113, 114, 129, 136-139, 141-143, 154, 155, 157, 161, 162, 166, 167, 172-175, 181, 182 NRAMP2 5, 29, 31, 38, 39, 99, 115, 118 NRMIT 135, 137, 139, 141, 142

O Oxidative stress 124, 126, 131, 148, 151, 174, 183, 191, 192 OxyR 146, 148, 150, 151, 154, 163, 166, 182

P Pasteurella pneumotropica 3 Pathogenesis 24, 36, 165, 167, 168, 188 PerR 151 pH 1, 5-8, 10, 12, 18, 38, 48, 73-76, 82, 88, 92, 110, 117, 135, 137-141, 143, 161, 163-165, 172-174, 182, 191 Phagocytic cells 1, 12, 48, 108 Phagolysosome 8, 9, 44, 99, 108, 173 Phagosome 1, 2, 4, 6, 7, 9, 10, 12, 47, 48, 77, 78, 98, 99, 102, 108, 110, 136-139, 141, 155, 167, 172-174, 182 Phosphatases 49 Phylogenetic analysis 181 Phylogeny 178, 179, 183, 189 Placenta 70, 82, 83, 93, 109 Placental transfer 93 Plant 113-121, 129, 136, 154, 179, 181, 192 Primary biliary cirrhosis (PBC) 31, 37, 53-55 Pro-inflammatory response 53, 54, 57, 58, 61, 62 Protein kinase C (PKC) 4, 23, 47-49 Pseudomonas 4, 117, 161, 186

R Reactive oxygen species 146, 150, 155, 163, 191 Regulation 7, 8, 19, 21, 23, 24, 36, 44, 47-49, 58, 59, 61, 65, 68, 69, 73, 76-79, 83, 85, 88, 90, 105-107, 118, 119, 121, 124, 129-131, 135, 146-148, 150, 151, 163, 164, 166, 174, 179, 189

Index

199

Restriction fragment length polymorphisms (RFLP) 17 Reticulocyte 7, 39, 77, 88-90, 93, 106, 107 Rheumatoid arthritis (RA) 29, 36, 37, 53-55, 58 Rice 114, 115, 117, 118

SOD2 127, 129 Staphylococcus aureus 4, 148, 149, 151, 156, 159, 161, 163, 184, 191 Superoxide dismutase 8, 11, 127, 129, 163, 174

S

TNF-α 46, 49, 57, 58 Toxoplasma gondii 4, 155 Transferrin 6, 39, 58, 65-67, 70, 73, 77-79, 82, 83, 86-93, 105-107, 110, 173 Transferrin cycle 39, 65-67, 70, 77 Transferrin receptor 58, 65, 77-79, 88-91, 93, 105, 106, 173 Transport 4-8, 11, 12, 19, 23, 30, 32, 38, 39, 44, 47, 48, 65-68, 70, 73-78, 82-84, 88-93, 100, 101, 104, 106-110, 114-119, 121, 124-126, 127, 129-131, 135-142, 146-149, 154, 155, 159, 161-165, 167, 168, 174, 178, 179, 182, 191, 192 Transporter 1, 2, 5, 18, 21, 30, 36, 38, 47, 48, 53, 54, 62, 65, 66, 68, 70, 73, 74, 76, 82, 83, 90, 96, 99, 106-108, 113-119, 121, 124-127, 129-131, 135-139, 141-143, 146-148, 150, 154, 159, 161-164, 167, 168, 172-176, 178, 182, 183, 191, 192 Trypanosoma cruzi 36, 37 Tuberculosis 1-5, 7, 9, 23, 24, 29, 31-35, 44, 46, 54, 55, 57, 58, 141, 146, 147, 151, 158, 159, 172-176, 182, 187

Saccharomyces cerevisiae 4, 5, 117, 119, 124, 125, 127, 129-132, 135, 136, 155, 161, 179, 181 Salmonella 2-4, 8-11, 16-21, 23, 24, 45-47, 54, 96, 99, 117, 136, 142, 146, 148, 150, 154, 159, 165, 174, 182, 183, 186 Salmonella containing vacuoles (SCV) 10, 11, 165, 167, 168 Salmonella enteritidis 18-21 Salmonella gallinarum 18, 19 Salmonella pathogenicity island 2 (SPI2) 8 Salmonella pullorum 16, 18, 19 Salmonellosis 16, 18, 19 Signaling 44, 48, 49, 115, 159 Single nucleotide polymorphism (SNP) 17, 30, 35, 38, 39 sitA-D 8 SLC11A1 30, 53-59, 62 SLC11A2 38, 58, 73, 79, 99 Slip 135, 139-141 Slip phenomenon 139 SMF1 6, 74, 75, 114, 115, 117, 118, 121, 125-127, 136, 179, 182 smf1 5, 48, 115, 116, 125-127, 129, 131, 135, 141, 143 Smf1p 124-127, 129-131, 135-137, 139-141 SMF2 6, 117, 125, 127, 137 smf2 5, 48, 127, 129, 131 Smf2p 124, 125, 127, 129-131 SMF3 6, 117, 124, 127, 131, 137 smf3 5, 127, 129 Smf3p 124, 125, 127, 129, 131

T

U Ulcerative colitis (UC) 36, 53-55 Uptake 6, 7, 39, 45, 47, 65, 66, 68, 69, 73-78, 82-84, 86-90, 92, 93, 104-107, 109, 113-116, 118-121, 124-127, 129, 131, 135-137, 139,

The Nramp Family

200

141, 142, 146-148, 150, 151, 154, 161, 163-165, 174-176, 179, 182, 189, 191

V Vacuole 9-11, 114, 124, 127, 129-131, 165, 172, 173 Vesicle trafficking 32 Virulence 1, 2, 8, 12, 142, 147, 154, 167-169, 174, 175, 179, 182, 191

W Wilson’s disease 114, 146

Y Yeast 5-7, 48, 74, 98, 113-121, 124-127, 129-131, 135-137, 141, 143, 146, 179, 182, 184, 188 Yeast mutants 48, 113-115, 118, 125, 127, 129, 136, 141

Z Zinc 49, 113, 114, 117, 125, 130, 131, 135, 146, 147 Zinc uptake repressor (Zur) 146 Zn2+ 5, 6, 44, 48, 49, 66, 75, 135-137, 140, 154, 155, 161, 163-165


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The Nramp Family

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The Nramp Family

The Family

The Family

The Family

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The Family

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The Nramp Family

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The widow_s family

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The family comes on

Murder In the Family

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The Family Busts Loose

The First Family

Secrets- The Wallace Family

Heart of the Family

Dead in the Family

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