STRUCTURAL DETERMINATION OF THE LPS FROM RHIZOBIUM

根瘤菌 LPS 的结构测定

• 批准号: 2179935
• 负责人: RUSSELL W CARLSON
• 金额: $ 12.57万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179935
• 关键词: Leguminoseae; Rhizobiaceae; carbohydrate structure; enzyme linked immunosorbent assay; epitope mapping; lipopolysaccharides; mass spectrometry; methylation; monoclonal antibody; mutant; nuclear magnetic resonance spectroscopy; oligosaccharides; symbiosis

The objectives of this grant are to characterize the unique structure of the LPS from R. leguminosarum biovar phaseoli (recently reclassified Rhizobium etli) strain CE3 and to elucidate the role of this molecule in the nitrogen-fixing symbiotic infection of its legume host it is known that Rhizobium LPS are required for a successful infection of the legume host. Gross changes in the LPS, e.g. loss of the O-chain polysaccharide, result in aborted infections. Monoclonal antibodies have shown that during infection there are subtle structural changes that occur in the LPS during infection. By using defined mutants in the LPS biosynthetic pathway (provided by Dr. Dale Noel of Marquette University), the complete structure of the LPS will be determined, as well as those epitope changes that occur during symbiotic infection. Analysis of LPSs from mutants at various stages of the infection process will (a) facilitate the identification of genes in the LPS biosynthetic pathway, and (b) enable us to determine how the O-chain, core oligosaccharides and lipid A linked in the complete LPS molecule. The LPS from these mutants, as well as from the parent strain (CE3) will be used to isolate those LPS fragments that carry the epitopes important in symbiotic infection. These epitopes will be identified by ELISA inhibition assays. The monoclonal antibodies (i.e. JIM26, JIM27, JIM28 and JIM29) have been provided by Dr. Nick Brewin of the John Innes Institute in the UK. Additionally, a graduate student will visit Dr. Noel's laboratory to isolate mutants that are unable to undergo those epitope changes that occur during symbiotic infection. These mutants will be analyzed to determine their structural (by us) and symbiotic (by Dr. Noel) defects. Structural determination of the various LPSs and LPS fragments will be carried out by glycosyl composition and methylation analyses, NMR, and high resolution mass spectrometry. We currently know the structures of the O-chain repeating unit, two core oligosaccharides, and the lipid A from the R. leg. bv. phaseoli CE3 LPS. The lipid A of Rhizobium is unique in that it does not have phosphate, and its sugar backbone consists of galacturonic acid, glucosamine and 2-aminogluconic acid. We have shown that Rhizobium extracts have all the enzymes necessary for the synthesis of a lipid A precursor common to enteric lipid As, Kdo2IVa. Thus, in Rhizobium this common precursor is probably processed to form the unique rhizobial lipid A. One such enzyme, a specific Kdo2IVa 4'- phosphatase has already been identified. The last objective of this proposal is to characterize the other enzymes that process Kdo2lVa into the unique rhizobial lipid A. Dr. Chris Raetz (Duke University) has agreed to collaborate with us in this aspect of the project.

该补助金的目标是表征独特的结构， R.豆科生物变种菜豆（最近重新分类 为了阐明该分子在根瘤菌（Rhizobium etli）菌株CE 3中的作用， 已知其豆科宿主的固氮共生感染 根瘤菌LPS是成功感染豆科植物所必需的 主持人LPS的总体变化，例如O-链多糖的损失， 导致感染中止。 单克隆抗体显示， 在感染过程中，LPS发生了微妙的结构变化， 在感染期间。通过在LPS生物合成途径中使用确定的突变体 （由马奎特大学的戴尔诺埃尔博士提供）， 将确定LPS的结构以及那些表位变化 发生在共生感染期间。来自突变体的LPS的分析， 感染过程的各个阶段将（a）促进 鉴定LPS生物合成途径中的基因，和（B）使我们能够 以确定O-链、核心寡糖和脂质A如何连接， 完整的LPS分子来自这些突变体的LPS，以及来自 亲本菌株（CE 3）将用于分离那些携带 在共生感染中起重要作用的表位。这些表位将是 通过ELISA抑制测定鉴定。单克隆抗体（即， JIM 26、JIM 27、JIM 28和JIM 29）由 英国的约翰·英尼斯研究所此外，研究生将 参观诺埃尔博士的实验室，分离出无法经受 这些表位的变化发生在共生感染。这些突变体 将进行分析，以确定其结构（由我们）和共生（由 博士诺埃尔）缺陷。各种LPS和LPS的结构测定 片段将进行糖基化组成和甲基化 分析、NMR和高分辨率质谱。我们目前所知 O-链重复单元，两个核心寡糖， 和来自R.法例BV.菜豆CE 3 LPS。脂质A 根瘤菌的独特之处在于它不含磷酸盐， 主链由半乳糖醛酸、葡萄糖胺和2-氨基葡萄糖酸组成 酸我们已经证明根瘤菌提取物具有所有必需的酶 为了合成肠脂质A共有的脂质A前体， Kdo2IVa。因此，在根瘤菌中，这种共同的前体可能被加工成 形成独特的根瘤菌脂质A。一种这样的酶，一种特定的Kdo 2 IVa 4 '- 磷酸酶已被鉴定。最后一个目标是 建议是表征将Kdo 2 IVa加工成 独特的根瘤菌脂质A。克里斯·雷茨博士（杜克大学）已经同意 在这个项目上与我们合作。