Glycolipid Translocation in Mycobacteria

项目总结： 细菌细胞表面糖脂在细菌适应环境和环境的过程中起着关键作用 发病机制。由于它们在细胞外的位置和功能的重要性，已经进行了大量的工作 致力于描述细菌糖脂的生物发生(生物合成和运输)。在取得重大进展的同时 已经在识别转运蛋白方面取得了进展，特别是在革兰氏阴性细菌中，这些过程 在属于放线菌目的特殊革兰氏阳性生物中仍然是完全不透明的。 真菌类群的放线菌(即含有霉菌酸的物种)，它们含有臭名昭著的人类 病原体，包括结核分枝杆菌和白喉棒状杆菌，以细胞为特征 独特的成分和结构暗示高度专门化的生物合成和移位 机械制造。一些研究最多的几种多糖在真菌的细胞膜中起着重要的作用 它们在生理和发病机制中发挥作用的是甘露糖脂和脂多糖，称为 磷脂酰肌醇甘露糖苷(PIMs)及其多糖基化产物脂甘露聚糖(LM)和 脂阿拉伯甘露聚糖(Lipoarabinomanan，LAM)。虽然PIM、LM和LAM生物合成途径中的许多酶已经被 发现，负责PIM中间体跨质膜转运的转运体(S)， 而PIM、LM和LAM对外膜(菌膜)和细胞表面的影响尚不清楚。这 应用研究计划首次深入了解PIM、LM和LAM的分子机制 在分枝菌群细菌的细胞膜的不同层之间的易位。当前的障碍是 这些难以捉摸的转运蛋白的发现是因为缺乏生化工具来从拓扑上和特定地标记 皮姆鞋。为了解决这一不足，我设计了一种涵盖化学生物学的多学科方法， 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 在质膜上发生移位的PIM[目标1]。这些工具与 传统的遗传、生化和蛋白质-蛋白质相互作用方法，以及一种新开发的细胞分选- 基于筛选转座子突变体文库的检测将使特异性PIM和LAM的鉴定成为可能 运输商[目标2]。 这些研究的成功将推进长期存在的关于(糖基)脂类转运的问题 放线菌，特别是分枝杆菌的PIM、LM和LAM的生物发生。新的知识产生了 其中，由AIM 1产生的移位分析可能会进一步在开发创新的 治疗棒状杆菌感染的治疗策略。

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.