Manganese Homeostasis and Salmonella

NRAMP蛋白（天然耐药相关巨噬细胞蛋白）是H+刺激的二价阳离子转运蛋白。哺乳动物NRAMP2/DCT1介导Fe2+和Mn2+的肠道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，是小鼠中众所周知的Ity/Lsh/Bcg位点，可抵抗多种病原体，如鼠伤寒沙门氏菌、多诺瓦利什曼原虫和分枝杆菌。人类NRAMP1基因的多态性使其携带者更容易感染结核分枝杆菌。NRAMP蛋白主要在真核细胞中被表征；然而，许多革兰氏+和革兰氏菌种类具有相近的同源物（40%的序列同一性），表明功能守恒。大肠杆菌和鼠伤寒沙门氏菌携带一个单一的NRAMP基因，我们已经克隆并鉴定了该基因。我们的数据表明，a)细菌NRAMP蛋白是一个高度选择性的H+刺激的Mn2+运输系统（mntH），其表达受过氧化物和二价阳离子的调节。b) MntH可高度诱导鼠伤寒沙门氏菌侵袭NRAMP1+而非NRAMP1-巨噬细胞，c) MntH在NRAMP1+小鼠而非NRAMP1-小鼠中的毒力减弱，d)假定的铁转运体SitABCD是生理上相关的Mn2+转运体，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，那么在发病过程的某个时刻需要Mn2+本身。在目标1中，我们将询问Mn2+在何时、何地以及为何对发病机制起重要作用。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，将确定何时何地。将在基因NRAMP1+和NRAMP1- BALB/c小鼠中测定毒力。为什么将通过检查极少数依赖于Mn2+的酶的一个亚群来研究：超氧化物歧化酶（sodA）、蛋白磷酸酶1和2 （prpA/B）和甘油变磷酸酶（gpmM）。这些基因位点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表达的调控。在Aim 2中，我们将确定MntH的拓扑结构，使用54Mn2+作为示踪剂进一步研究其在爪蟾卵母细胞中表达后的转运特性，并通过定点诱变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.