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

NRAMPs (natural resistance-associated macrophage proteins) have been charac terized in mammals as divalent transition metal transporters involved in ir on metabolism and host resistance to certain pathogens. The mechanism of pa thogen resistance is proposed to involve sequestration of Fe2+ and Mn2+, co factors of both prokaryotic and eukaryotic catalases and superoxide dismuta ses, not only to protect the macrophage against its own generation of react ive oxygen species, but to deny the cations to the pathogen for synthesis o f its protective enzymes. NRAMP homologues are also present in bacteria. We report the cloning and characterization of the single NRAMP genes in Esche richia coli and Salmonella enterica ssp. typhimurium, and the cloning of tw o distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia. The genes are designated mntH be cause the two enterobacterial NRAMPs encode H+-stimulated, highly selective manganese(II) transport systems, accounting for all Mn2+ uptake in each sp ecies under the conditions tested. For S. typhimurium MntH, the K-m for Mn- 54(2+) (approximate to 0.1 mu M) was pH independent, but maximal uptake inc reased as pH decreased. Monovalent cations, osmotic strength, Mg2+ and Ca2 did not inhibit Mn-54(2+) uptake. Ni2+, Cu2+ and Zn2+ inhibited uptake wit h K(i)s greater than 100 mu M, Co2+ with a K-i of 20 mu M and Fe2+ with a K -i that decreased from 100 mu M at pH 7.6 to 10 mu M at pH 5.5. Fe3+ and Pb 2+ inhibited weakly, exhibiting K(i)s of 50 mu M, while Cd2+ was a potent i nhibitor with a K-i of about 1 mu M. E. coli MntH had a similar inhibition profile, except that K(i)s were three- to 10-fold higher. Both S. typhimuri um and E. coli MntH also transport Fe-55(2+) however, the K(m)s are equival ent to the K(i)s for Fe2+ inhibition of Mn2+ uptake, and are thus too high to be physiologically relevant. In both S. typhimurium and E. coli, mntH::l acZ constructs were strongly induced by hydrogen peroxide, weakly induced b y EDTA and unresponsive to paraquat, consistent with the presence of Fur an d OxyR binding sites in the promoters. Strains overexpressing mntH were mor e susceptible to growth inhibition by Mn2+ and Cd2+ than wild type, and str ains lacking a functional mntH gene were more susceptible to killing by hyd rogen peroxide. In S. typhimurium strain SL1344, mntH mutants showed no def ect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cell s; however, expression of mntH::lacZ was induced severalfold by 3 h after i nvasion of the macrophages. S. typhimurium mntH mutants showed only a sligh t 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.