Kehres D G, Zaharik M L, Finlay B B, Maguire M E
Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4965, USA.
Mol Microbiol. 2000 Jun;36(5):1085-100. 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+ ( 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.
天然抗性相关巨噬细胞蛋白（NRAMPs）在哺乳动物中被鉴定为参与铁代谢和宿主对某些病原体抗性的二价过渡金属转运蛋白。病原体抗性机制被认为涉及Fe2+和Mn2+的隔离，这两种离子是原核生物和真核生物过氧化氢酶及超氧化物歧化酶的辅因子，不仅用于保护巨噬细胞免受自身产生的活性氧的伤害，还能阻止阳离子被病原体用于合成其保护酶。细菌中也存在NRAMP同源物。我们报道了大肠杆菌和鼠伤寒沙门氏菌中单个NRAMP基因的克隆与特性分析，以及从铜绿假单胞菌克隆出的两个不同的NRAMP基因和洋葱伯克霍尔德菌中一个NRAMP基因的内部片段。这些基因被命名为mntH，因为两种肠杆菌的NRAMPs编码受H+刺激的、高度选择性的锰（II）转运系统，在所测试条件下占每个物种所有Mn2+摄取量。对于鼠伤寒沙门氏菌的MntH，54Mn2+的Km（约0.1微摩尔）与pH无关，但最大摄取量随pH降低而增加。单价阳离子、渗透压、Mg2+和Ca2+不抑制54Mn2+摄取。Ni2+、Cu2+和Zn2+抑制摄取，其Ki大于100微摩尔，Co2+的Ki为20微摩尔，Fe2+的Ki从pH 7.6时的100微摩尔降至pH 5.5时的10微摩尔。Fe3+和Pb2+抑制作用较弱，Ki为50微摩尔，而Cd2+是强效抑制剂，Ki约为1微摩尔。大肠杆菌的MntH有类似的抑制谱，只是Ki高3至10倍。鼠伤寒沙门氏菌和大肠杆菌的MntH也都转运55Fe2+，然而，其Km等同于Fe2+抑制Mn2+摄取的Ki，因此过高而无生理相关性。在鼠伤寒沙门氏菌和大肠杆菌中，mntH:lacZ构建体受过氧化氢强烈诱导，受EDTA弱诱导，对百草枯无反应，这与启动子中存在Fur和OxyR结合位点一致。过表达mntH的菌株比野生型更易受Mn2+和Cd2+抑制生长，而缺乏功能性mntH基因的菌株更易被过氧化氢杀死。在鼠伤寒沙门氏菌SL1344菌株中，mntH突变体在侵袭培养的HeLa或RAW264.7巨噬细胞或在其中存活方面无缺陷；然而，巨噬细胞侵袭3小时后，mntH:lacZ的表达被诱导增加数倍。鼠伤寒沙门氏菌mntH突变体在BALB/c小鼠中仅表现出轻微的毒力减弱。因此，NRAMP Mn2+转运蛋白MntH和Mn2+在细菌对活性氧的反应中起作用，并且可能在发病机制中起作用。
