The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen

• Published in: Molecular microbiology 2000-06, Vol.36 (5), p.1085-1100
• Main Authors: Kehres, David G., Zaharik, Michelle L., Finlay, B. Brett, Maguire, Michael E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Burkholderia cepacia - genetics; Burkholderia cepacia - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cation Transport Proteins; Cloning, Molecular; DNA, Bacterial; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Female; Gene Expression Regulation, Bacterial; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Iron-Binding Proteins; Manganese; Manganese - metabolism; manganese transporter; Membrane Proteins - genetics; Membrane Proteins - metabolism; Metals - metabolism; Mice; Mice, Inbred BALB C; Molecular Sequence Data; natural resistance-associated macrophage proteins; NRAMP protein; Phenotype; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - metabolism; Reactive Oxygen Species - metabolism; Salmonella typhimurium; Salmonella typhimurium - genetics; Salmonella typhimurium - metabolism; Transcription, Genetic
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.2000.01922.x

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+ (≈ 0.1 µM) 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 µM, Co2+ with a Ki of 20 µM and Fe2+ with a Ki that decreased from 100 µM at pH 7.6 to 10 µM at pH 5.5. Fe3+ and Pb2+ inhibited weakly, exhibiting Kis of 50 µM, while Cd2+ was a potent inhibitor with a Ki of about 1 µM. 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 i