Glycolipid Translocation in Mycobacteria

分枝杆菌中的糖脂易位

• 批准号: 9911276
• 负责人: heather L hodges
• 金额: $ 6.53万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911276
• 关键词: Actinobacteria class; Actinomycetales; Address; Anabolism; Area; Bacteria; Biochemical; Biogenesis; Biological Assay; Biology; Cell Separation; Cell membrane; Cell surface; Chemicals; Collaborations; Corynebacterium diphtheriae; Cytoplasm; Development; Environment; Enzymes; Genetic; Genus Mycobacterium; Glycobiology; Glycolipids; Gram-Negative Bacteria; Immune system; Infection; Inositol; Knowledge; Label; Libraries; Lipids; Literature; Location; Mannose; Membrane; Metabolic; Modification; Molecular; Monosaccharides; Mutate; Mycobacterium tuberculosis; Mycolic Acid; Organism; Pathogenesis; Pathway interactions; Penetration; Pharmaceutical Preparations; Physiology; Play; Polysaccharides; Process; Proteins; Reaction; Recombinants; Research; Role; Side; Structure; Suggestion; Surface; Techniques; Therapeutic; Vesicle; Work; analog; base; biochemical tools; cell envelope; comparative genomics; design; extracellular; human pathogen; innovation; insight; interdisciplinary approach; lipid transport; lipoarabinomannan; lipomannan; mutant; mycobacterial; overexpression; periplasm; phosphatidylinositol mannoside; professor; protein protein interaction; reconstitution; success; tool; translocase; virtual

Project Summary: Bacterial cell surface glycolipids function in critical roles in the adaption of bacteria to their environment and pathogenesis. Owing to their extracellular location and functional importance, considerable work has been devoted to delineating bacterial glycolipid biogenesis (biosynthesis and transport). While significant advances have been made in the identification of transporters in particular in Gram-negative bacteria, these processes remain wholly opaque in the specialized Gram-positive organisms belonging to the order Actinomycetales . Actinomycetales of the mycolata group (i.e., mycolic acid containing species), which contain notorious human pathogens, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, are characterized by a cell envelope of unique composition and structure suggestive of highly specialized biosynthetic and translocation machineries. Some of the most studied glycans in the cell envelope of mycolata species for the important roles they play in physiology and pathogenesis are mannosylated glycolipids and lipoglycans known as phosphatidylinositol mannosides (PIMs) and their multi-glycosylated counterparts, lipomannan (LM) and lipoarabinomannan (LAM). While many enzymes in the PIM, LM, and LAM biosynthetic pathway have been discovered, the transporter(s) responsible for translocation of PIM intermediates across the plasma membrane, and of PIM, LM and LAM to the outer membrane (mycomembrane) and cell surface are not known. This application proposes to gain the first insights into the molecular mechanisms governing PIM, LM and LAM translocation across the different layers of the cell envelope of mycolata group bacteria. A current obstacle to the discovery of these elusive transporters is the lack of biochemical tools to topologically and specifically label PIMs. To address this deficiency, I have devised a multidisciplinary approach encompassing chemical biology, glycobiology, genetics and protein-protein interactions. First, I will develop a set of tools that can directly label PIMs that have undergone translocation across the plasma membrane [Aim 1]. These tools, in combination with traditional genetic, biochemical and protein-protein interaction approaches, and a newly developed cell sorting- based assay to screen of a transposon mutant library will enable the identification of specific PIM and LAM transporters [Aim 2]. Success in these studies would advance long-standing questions regarding (glyco)lipid transport in Actinomycetes, and PIM, LM and LAM biogenesis in mycobacteria in particular. The new knowledge generated therein and translocation assays arising from Aim 1 may further find applications in the development of innovative therapeutic strategies to treat Corynebacterineae infections.

项目摘要： 细菌细胞表面糖脂在细菌适应其环境中的关键作用和 发病。由于它们的细胞外位置和功能重要性，大量工作已经 致力于描述细菌性糖脂生物发生（生物合成和转运）。虽然取得了重大进展 这些过程尤其是在革兰氏阴性细菌中识别转运蛋白的鉴定 在属于放线菌的专业革兰氏阳性生物中保持完全不透明。 菌根组的放线菌（即含有霉菌酸的物种），其中包含臭名昭著的人类 病原体，包括结核分枝杆菌和甲状腺杆菌，以细胞为特征 独特的组成和结构的包络，暗示了高度专业的生物合成和易位 机械。 Mycolata物种细胞包膜中研究最多的Glycans的一些重要角色 他们在生理和发病机理中发挥作用是甘露糖基化的糖脂和脂肪聚糖 磷脂酰肌醇甘露糖苷（PIMS）及其多糖基化对应物，Lipomannan（LM）和 脂肪动物群（LAM）。虽然PIM，LM和LAM生物合成途径中的许多酶已经 发现，负责PIM中间体在质膜上易位的转运蛋白， PIM，LM和LAM的外膜（MyComembrane）和细胞表面尚不清楚。这 应用建议获得有关PIM，LM和LAM的分子机制的首次见解 Mycolata组细菌的细胞膜不同层的易位。当前的障碍 这些难以捉摸的转运蛋白的发现是缺乏拓扑和特殊标签的生化工具 PIMS。为了解决这种缺陷，我设计了一种跨学科方法，包括化学生物学， 糖生物学，遗传学和蛋白质 - 蛋白质相互作用。首先，我将开发一组可以直接标记的工具 在整个质膜上经历了易位的PIM [AIM 1]。这些工具，结合 传统的遗传，生化和蛋白质 - 蛋白质相互作用方法，以及新开发的细胞分选 基于转座子突变库屏幕的基于测定将使特定的PIM和LAM识别 转运蛋白[AIM 2]。 在这些研究中的成功将提出有关（Glyco）脂质转运的长期问题 尤其是分枝杆菌中的放线菌和PIM，LM和LAM生物发生。产生的新知识 在其中和AIM 1产生的易位测定法可能会进一步找到创新的应用 治疗Corynebacterineae感染的治疗策略。