Manganese Homeostasis and Salmonella

NRAMP蛋白（Natural Resistance Associated Macrophage Protein，天然抗性相关巨噬细胞蛋白）是H+刺激的二价阳离子转运蛋白。哺乳动物NRAMP 2/DCT 1介导Fe 2+和Mn 2+的肠摄取以及这些阳离子在细胞隔室中的分布。NRAMP 1在巨噬细胞中表达，并且是小鼠中长期已知的Ity/Lsh/Bcg基因座，其提供对多种病原体如沙门氏菌的抗性。鼠伤寒、杜氏利什曼原虫和分枝杆菌属。人NRAMP 1中的多态性使得携带者对M.肺结核感染。NRAMP蛋白主要在真核细胞中表征;然而，许多革兰氏阳性和革兰氏阴性细菌物种具有密切同源物（40%序列同一性），表明功能保守。E. coli和革兰氏阳性菌S.鼠伤寒杆菌携带我们已经克隆和表征单个NRAMP基因。我们的数据表明：a）细菌NRAMP蛋白是一种高选择性的H+刺激的Mn 2+转运系统（mntH），其表达受过氧化物和二价阳离子的调节。B）MntH对S的诱导作用强。鼠伤寒沙门氏菌侵入NRAMP 1+巨噬细胞而非NRAMP 1-巨噬细胞，c）mntH的毒力在NRAMP 1+小鼠中减弱而非NRAMP 1-小鼠中减弱，d）推定的铁转运蛋白SitABCD是生理学相关的Mn 2+转运蛋白，以及3）mntH和sitABCD的突变使S.鼠伤寒无毒性。如果Mn 2+摄取的消除导致毒力丧失，那么在发病的某些点上需要Mn 2+本身。在目的1中，我们将询问Mn 2+在何处、何时以及为何对发病机制重要。通过研究mntH（和sitABCD）突变对S. NRAMP 1+和NRAMP 1-巨噬细胞以及上皮细胞系中的鼠伤寒沙门氏菌。将在同源性NRAMP 1+和NRAMP 1- BALB/c小鼠中测定毒力。原因将通过检查极少数Mn 2+依赖性酶的子集来研究：超氧化物歧化酶（sodA）、蛋白质磷酸酶1和2（prpA/B）和磷酸甘油变位酶（gpmM）。将测量这些基因座突变对培养细胞侵袭力和小鼠毒力的影响。我们还将研究Fur、OxyR和一种新DtxR同系物MntR对mntH表达的调节。在第二个目标中，我们将确定MntH的拓扑结构，进一步以54 Mn 2+为示踪剂研究其转运特性，并在非洲爪蟾卵母细胞中表达后进行电生理学研究，并通过定点突变研究膜内保守和带电残基在阳离子通量中的作用。

NRAMP proteins (Natural Resistance Associated MacroPhage Protein) are H+-stimulated divalent cation transporters. Mammalian NRAMP2/DCT1 mediates intestinal uptake of Fe2+ and Mn2+ and distribution of these cations within cellular compartments. NRAMP1 is expressed in macrophages and is the long known Ity/Lsh/Bcg locus in the mouse that provides resistance to diverse pathogens such as S. typhimurium, Leishmania donovania and Mycobacterial species. Polymorphisms in human NRAMP1 render the bearer more susceptible to M. tuberculosis infection. NRAMP proteins have been characterized primarily in eukaryotic cells; however, many Gram+ and Gram-bacterial species have close homologs (40% sequence identity), suggesting conservation of function. E. coli and S. typhimurium carry a single NRAMP gene which we have cloned and characterized. Our data indicate that a) the bacterial NRAMP protein is a highly selective H+- stimulated Mn2+ transport system (mntH) whose expression is regulated by peroxide and divalent cation. b) MntH is highly induced upon S. typhimurium invasion of NRAMP1+ but not NRAMP1-macrophages, c) virulence of mntH is attenuated in NRAMP1+ mice but not NRAMP1- mice, d) the putative iron transporter SitABCD is a physiologically relevant Mn2+ transporter and 3) mutation of both mntH and sitABCD renders S. typhimurium avirulent. If abrogation of Mn2+ uptake results in loss of virulence, than Mn2+ itself is needed at some point in pathogenesis. In Aim 1, we will ask where, when and why Mn2+ is important for pathogenesis. Where and when will be determined by investigation of the effect of mutations in mntH (and sitABCD) on invasion, survival and proliferation of S. typhimurium in NRAMP1+ and NRAMP1- macrophages and also epithelial cell lines. Virulence will be determined in congenic NRAMP1+ and NRAMP1- BALB/c mice. Why will be investigated by examining a subset of the very few Mn2+-dependent enzymes: superoxide dismustase (sodA), protein phosphatases 1 and 2 (prpA/B), and phoshoglyceromutatse (gpmM). The effects of mutations at these loci will be measured on invasion of cultured cells and virulence in mice. We will also investigate regulation of mntH expression by Fur, OxyR and a new DtxR homolog MntR. In Aim 2, we will determine the topology of MntH, further examine its transport properties using 54Mn2+ as tracer and also electrophysiologically after expression in Xenopus oocytes, and investigate the role of conserved and charged intramembrane residues in cation flux by site-directed mutagenesis.