Manganese Homeostasis and Salmonella

锰稳态和沙门氏菌

基本信息

批准号：
6438468
负责人：
MICHAEL E MAGUIRE
金额：
$ 28.96万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-01-11 至 2005-12-31
项目状态：
已结题

项目摘要

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.

NRAMP 蛋白（自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 NRAMP2/DCT1 介导肠道对 Fe2+ 和 Mn2+ 的摄取以及这些阳离子在细胞室内的分布。 NRAMP1 在巨噬细胞中表达，是小鼠中长期已知的 Ity/Lsh/Bcg 位点，可对多种病原体（如鼠伤寒沙门氏菌、杜氏利什曼原虫和分枝杆菌）产生抵抗力。人类 NRAMP1 的多态性使携带者更容易受到结核分枝杆菌感染。 NRAMP 蛋白主要在真核细胞中进行表征；然而，许多革兰氏阳性细菌和革兰氏阴性细菌具有密切的同源性（40% 序列同一性），表明功能保守。大肠杆菌和鼠伤寒沙门氏菌携带单一 NRAMP 基因，我们已克隆并鉴定了该基因。我们的数据表明，a) 细菌 NRAMP 蛋白是一种高度选择性的 H+- 刺激的 Mn2+ 转运系统 (mntH)，其表达受过氧化物和二价阳离子调节。 b) MntH 在鼠伤寒沙门氏菌侵入 NRAMP1+ 而非 NRAMP1-巨噬细胞时被高度诱导，c) mntH 的毒力在 NRAMP1+ 小鼠中减弱，但在 NRAMP1- 小鼠中不减弱，d) 假定的铁转运蛋白 SitABCD 是一种生理相关的 Mn2+ 转运蛋白，并且 3 ) mntH 和itABCD 的突变使得鼠伤寒沙门氏菌无毒力。如果消除 Mn2+ 摄取会导致毒力丧失，则在发病机制的某个时刻需要 Mn2+ 本身。在目标 1 中，我们将询问 Mn2+ 在何处、何时以及为何对发病机制如此重要。何时何地将通过研究mntH（和sitABCD）突变对NRAMP1+和NRAMP1-巨噬细胞以及上皮细胞系中鼠伤寒沙门氏菌的侵袭、存活和增殖的影响来确定。将在同源 NRAMP1+ 和 NRAMP1- BALB/c 小鼠中测定毒力。我们将通过检查极少数 Mn2+ 依赖性酶的子集来研究原因：超氧化物歧化酶 (sodA)、蛋白磷酸酶 1 和 2 (prpA/B) 以及磷酸甘油变位酶 (gpmM)。将测量这些位点突变对培养细胞的侵袭和小鼠毒力的影响。我们还将研究 Fur、OxyR 和新的 DtxR 同源物 MntR 对 mntH 表达的调节。在目标 2 中，我们将确定 MntH 的拓扑结构，使用 54Mn2+ 作为示踪剂进一步检查其转运特性，并在非洲爪蟾卵母细胞中表达后进行电生理学检查，并通过定点诱变研究保守且带电的膜内残基在阳离子通量中的作用。