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+ (≈ 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 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.

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…However, the regulation of manganese importers is slightly at odds with this model: the proton-driven manganese importer MntH is induced (by OxyR) when E. coli is exposed to H 2 O 2 (141), not O 2 − -and the ATP-driven importer MntABC is induced in Stapholococcus aureus under the same circumstances by its PerR system (142). Further, mutations that eliminate these systems sensitize a variety of microbes to H 2 O 2 (143) (141).…”

“…Further, mutations that eliminate these systems sensitize a variety of microbes to H 2 O 2 (143) (141). In explaining these data, some workers have speculated that Mn may scavenge H 2 O 2 as well as O 2 − , since carbonate-bound manganese shows a catalase-like activity in vitro (144).…”

Bacterial outer membrane channel for divalent metal ion acquisition

“…Nutritional metals, such as manganese, copper, cobalt, and zinc are available as the divalent cation in aerobic environments, and are thus soluble. Whereas cytoplasmic (inner) membrane transporters of free metal ions are well characterized in bacteria (9)(10)(11)(12), translocation across the outer membrane and into the periplasm has not been described. In principle, outer membrane pores with no or low selectivity should readily accommodate the diffusion of these small, soluble nutrients that are needed only in low quantities (4,5).…”

Overexpression, purification, and site-directed spin labeling of the Nramp metal transporter from Mycobacterium leprae

“…Thus, our finding that divalent metals increase levels of protein in the membrane by 90-120% is significant. Recent studies on prokaryotic Nramp homologues from S. typhimurium and E. coli have shown that transcription of these proteins is diminished when the cells are exposed to divalent transition ions (11,12), presumably caused by promoter elements that recognize divalent metals (12). In our case, we suspect that the metals serve a substrate-protective role, through structural stabilization, which may benefit insertion and͞or shield the protein from proteases.…”

“…Recently, the manganese transport functions of bacterial Nramp homologues (MntH) from Escherichia coli, Salmonella typhimurium, and M. tuberculosis have been demonstrated (11)(12)(13). Thus the resistance of the host against the pathogen, and the pathogenicity of the microorganism, may depend on the performance of the individual Nramp or homologous protein.…”