Manganese Homeostasis and Salmonella

锰稳态和沙门氏菌

• 批准号: 6622052
• 负责人: MICHAEL E MAGUIRE
• 金额: $ 27.2万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/6622052
• 关键词: MDCK cell; Salmonella infections; Salmonella typhimurium; Xenopus oocyte; acid base balance; bacterial cytopathogenic effect; divalent cations; electrodes; electrophysiology; enzyme activity; gene mutation; homeostasis; ion transport; laboratory mouse; manganese; membrane transport proteins; pathologic process; phosphoglycerate mutase; phosphoprotein phosphatase; polymerase chain reaction; radiotracer; site directed mutagenesis; superoxide dismutase; virulence; western blottings

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（自然抗性相关的巨噬细胞蛋白）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室内的分布。 NRAMP1在巨噬细胞中表达，是小鼠中备受的众所周知的ITY/LSH/BC​​G基因座，可对多种病原体（如鼠伤寒链球菌，Leishmania donovania和becobacterial物种）提供抗性。人NRAMP1中的多态性使承载者更容易受到结核分枝杆菌的感染。 NRAMP蛋白主要在真核细胞中表征。然而，许多革兰氏+和革兰氏阴性物种具有紧密的同源物（40％序列身份），表明功能保存。大肠杆菌和鼠伤寒链球菌携带一个单一的NRAMP基因，我们已经克隆并表征了。我们的数据表明，a）细菌NRAMP蛋白是高度选择性的H+刺激的MN2+转运系统（MNTH），其表达受到过氧化物和二价阳离子的调节。 b）MNTH是在NRAMP1+的伤寒链霉菌侵袭而不是NRAMP1巨噬细胞的高度诱导的，c）MNTH的毒力在NRAMP1+小鼠中减弱，但不是NRAMP1-小鼠，而不是NRAMP1-小鼠，d）Puthative Iron Transporters sitabcd是一种物理上相关的MN2+ STABCS。鼠伤寒。如果MN2+摄取的废除导致毒力丧失，则在发病机理的某个时候需要比MN2+本身。在AIM 1中，我们将询问MN2+在哪里，何时以及为什么对发病机理很重要。通过研究MNTH（和SITABCD）突变对NRAMP1+和NRAMP1-巨噬细胞中的鼠伤伤口链球菌的侵袭，存活和增殖的影响，将在何处和何时确定。毒力将在先天性NRAMP1+和NRAMP1-BALB/C小鼠中确定。为什么通过检查很少的MN2+依赖性酶的子集：超氧化物抑制酶（苏打），蛋白质磷酸酶1和2（PRPA/B）和Phoshoglyceromutatse（GPMM）。这些基因座的突变的影响将测量对培养细胞的侵袭和小鼠的毒力。我们还将研究Fur，Oxyr和新的DTXR同源物MNTR对MNTH表达的调节。在AIM 2中，我们将确定MNTH的拓扑结构，进一步检查其传输性能在Xenopus卵母细胞中表达后的54MN2+作为示踪剂以及电生理学，并研究了通过位置的杂物化阳离子中阳离子中保守和带电的阳离子内膜内残基在阳离子中的作用。