title: The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen.
abstract: NRAMPs (natural resistance-associated macrophage proteins) have been characterized in mammals as divalent transition metal transporters involved in iron metabolism and host resistance to certain pathogens. The mechanism of pathogen resistance is proposed to involve sequestration of Fe2+ and Mn2+, cofactors of both prokaryotic and eukaryotic catalases and superoxide dismutases, not only to protect the macrophage against its own generation of reactive oxygen species, but to deny the cations to the pathogen for synthesis of its protective enzymes. NRAMP homologues are also present in bacteria. We report the cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp. typhimurium, and the cloning of two distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia. The genes are designated mntH because the two enterobacterial NRAMPs encode H+-stimulated, highly selective manganese(II) transport systems, accounting for all Mn2+ uptake in each species under the conditions tested. For S. typhimurium MntH, the Km for 54Mn2+ ( approximately 0.1 microM) was pH independent, but maximal uptake increased as pH decreased. Monovalent cations, osmotic strength, Mg2+ and Ca2+ did not inhibit 54Mn2+ uptake. Ni2+, Cu2+ and Zn2+ inhibited uptake with Kis greater than 100 microM, Co2+ with a Ki of 20 microM and Fe2+ with a Ki that decreased from 100 microM at pH 7. 6 to 10 microM at pH 5.5. Fe3+ and Pb2+ inhibited weakly, exhibiting Kis of 50 microM, while Cd2+ was a potent inhibitor with a Ki of about 1 microM. E. coli MntH had a similar inhibition profile, except that Kis were three- to 10-fold higher. Both S. typhimurium and E. coli MntH also transport 55Fe2+ however, the Kms are equivalent to the Kis for Fe2+ inhibition of Mn2+ uptake, and are thus too high to be physiologically relevant. In both S. typhimurium and E. coli, mntH:lacZ constructs were strongly induced by hydrogen peroxide, weakly induced by EDTA and unresponsive to paraquat, consistent with the presence of Fur and OxyR binding sites in the promoters. Strains overexpressing mntH were more susceptible to growth inhibition by Mn2+ and Cd2+ than wild type, and strains lacking a functional mntH gene were more susceptible to killing by hydrogen peroxide. In S. typhimurium strain SL1344, mntH mutants showed no defect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cells; however, expression of mntH:lacZ was induced severalfold by 3 h after invasion of the macrophages. S. typhimurium mntH mutants showed only a slight attenuation of virulence in BALB/c mice. Thus, the NRAMP Mn2+ transporter MntH and Mn2+ play a role in bacterial response to reactive oxygen species and possibly have a role in pathogenesis.
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4965, USA. dgk2@po.cwru.edu
authors.forename: D G
authors.initials: DG
authors.lastname: Kehres
authors.forename: M L
authors.initials: ML
authors.lastname: Zaharik
authors.forename: B B
authors.initials: BB
authors.lastname: Finlay
authors.forename: M E
authors.initials: ME
authors.lastname: Maguire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D033862:Iron-Binding Proteins; D008345:Manganese / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010641:Phenotype; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D004269:DNA, Bacterial; D006861:Hydrogen Peroxide; D033862:Iron-Binding Proteins; D008345:Manganese; D008565:Membrane Proteins; D008670:Metals; D017382:Reactive Oxygen Species; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2