Manganese Homeostasis and Salmonella

NRAMP蛋白是一种受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内的分布。NRAMP1在巨噬细胞中表达，是小鼠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等具有抵抗力的ity/LSH/BCG基因座。人类NRAMP1基因的多态性使携带者更容易感染结核分枝杆菌。NRAMP蛋白主要是在真核细胞中鉴定的；然而，许多革兰氏阳性和革兰氏阴性细菌具有密切的同源性(40%的序列一致性)，这表明功能是保守的。大肠杆菌和鼠伤寒沙门氏菌携带单一的NRAMP基因，我们已经克隆并鉴定了该基因。我们的研究结果表明：a)细菌NRAMP蛋白是一个高选择性的H+刺激的Mn2+转运系统，其表达受过氧化氢和二价阳离子的调节。(B)mnth可被NRAMP1+而不是NRAMP1-巨噬细胞高度诱导入侵；c)mnth对NRAMP1+小鼠的毒力降低，但对NRAMP1-小鼠无影响；d)可能的铁转运蛋白SitABCD是一种与生理相关的Mn2+转运蛋白；3)mnth和SitABCD的突变使鼠伤寒沙门氏菌无毒。如果取消对Mn2+的摄取导致毒力丧失，那么在致病过程中的某个点上需要Mn2+本身。在目标1中，我们将询问Mn2+在哪里、何时以及为什么在发病机制中起重要作用。何时何地将通过研究mnth(和SitABCD)突变对鼠伤寒沙门氏菌在NRAMP1+和NRAMP1-巨噬细胞以及上皮细胞系中的侵袭、存活和增殖的影响而确定。将确定同基因NRAMP1+和NRAMP1-BALB/c小鼠的毒力。为什么将通过检测极少数依赖Mn2+的酶的子集来研究：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(GpmM)。这些基因座的突变对培养细胞的侵袭和小鼠的毒力的影响将被测量。我们还将研究Fur、OxyR和一个新的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，以54Mn2+为示踪剂进一步检测其在非洲爪哇卵母细胞中的转运特性以及表达后的电生理特性，并通过定点突变来研究保守的和带电的膜内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.