Glycolipid Translocation in Mycobacteria

分枝杆菌中的糖脂易位

基本信息

批准号：
10065430
负责人：
heather L hodges
金额：
$ 6.86万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2022-04-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10065430
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

项目摘要

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环境和适应环境方面发挥着关键作用。发病。由于它们的细胞外位置和功能重要性，人们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）。虽然取得了重大进展已经在转运蛋白的鉴定中进行了，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体中保持完全不透明。分枝菌群的放线菌目（即含有分枝菌酸的物种），其中含有臭名昭著的人类病原体，包括结核分枝杆菌和白喉棒杆菌，其特征在于细胞独特的组成和结构的包膜表明高度专业化的生物合成和易位机械。分枝菌属细胞膜中一些研究最多的聚糖具有重要作用它们在生理学和发病机制中发挥作用的是甘露糖化糖脂和脂聚糖，称为磷脂酰肌醇甘露糖苷 (PIM) 及其多糖基化对应物、脂甘露聚糖 (LM) 和脂阿拉伯甘露聚糖（LAM）。虽然 PIM、LM 和 LAM 生物合成途径中的许多酶已被发现负责 PIM 中间体跨质膜易位的转运蛋白， PIM、LM 和 LAM 对外膜（菌膜）和细胞表面的影响尚不清楚。这该应用旨在首次深入了解控制 PIM、LM 和 LAM 的分子机制分枝菌群细菌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由于缺乏生化工具来拓扑和特异性标记 PIM。为了解决这个缺陷，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。首先我会开发一套可以直接标注的工具已发生跨质膜易位的 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，以及新开发的细胞分选- 基于转座子突变体库筛选的测定将能够识别特定的 PIM 和 LAM 运输商[目标 2]。这些研究的成功将推进有关（糖）脂转运的长期存在的问题放线菌，尤其是分枝杆菌中的 PIM、LM 和 LAM 生物发生。产生的新知识其中和目标 1 产生的易位测定可能会进一步在创新药物的开发中找到应用。治疗棒状杆菌感染的治疗策略。