Glycolipid Translocation in Mycobacteria

分枝杆菌中的糖脂易位

• 批准号: 10387912
• 负责人: heather L hodges
• 金额: $ 0.25万
• 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 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387912
• 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.

项目概要： 细菌细胞表面糖脂在细菌适应其环境中起关键作用， 发病机制由于它们的细胞外位置和功能重要性， 致力于描述细菌糖脂的生物合成（生物合成和运输）。虽然重大进展 在鉴定转运蛋白，特别是革兰氏阴性细菌中，这些方法 在属于放线菌目的特化革兰氏阳性生物体中保持完全不透明。 放线菌目的分枝菌组（即，含有分枝菌酸的物种），其中含有臭名昭著的人类 包括结核分枝杆菌和白喉棒状杆菌在内的病原体的特征在于细胞 一种独特的组成和结构的包膜，提示高度专门化的生物合成和转运 机械。一些研究最多的聚糖在真菌物种的细胞被膜中起着重要作用 在生理学和发病机制中起作用的是甘露糖基化糖脂和脂聚糖， 磷脂酰肌醇甘露糖苷（PIM）和它们的多糖基化对应物脂甘露聚糖（LM）， 脂阿拉伯甘露聚糖（LAM）。虽然PIM、LM和LAM生物合成途径中的许多酶已经被发现， 发现，负责PIM中间体跨质膜转运的转运蛋白， 以及PIM、LM和LAM对外膜（菌膜）和细胞表面的影响尚不清楚。这 应用程序提出，以获得第一次深入了解的分子机制，管理PIM，LM和LAM 在分枝菌群细菌的细胞被膜的不同层之间的易位。目前的障碍， 这些难以捉摸的转运蛋白的发现是缺乏拓扑学和特异性标记的生化工具 PIM。为了解决这个问题，我设计了一个多学科的方法，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，我将开发一套工具，可以直接标记 已经发生跨质膜易位的PIM [目的1]。这些工具与 传统的遗传、生物化学和蛋白质-蛋白质相互作用方法，以及新开发的细胞分选- 筛选转座子突变体文库的基于测定的方法将能够鉴定特异性PIM和LAM 运输工具[目标2]。 这些研究的成功将推进长期存在的关于（糖）脂转运的问题， 放线菌，特别是分枝杆菌中的PIM、LM和LAM生物发生。产生的新知识 其中，由Aim 1产生的易位测定可以进一步应用于开发创新的 治疗棒状杆菌属感染的治疗策略。