Altered M. tuberculosis Mannosylation and the Macrophage

改变结核分枝杆菌甘露糖化和巨噬细胞

基本信息

批准号：
7750035
负责人：
Larry S. Schlesinger
金额：
$ 35.19万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-15 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7750035
关键词：
Aerosols Alginates Alveolar Macrophages Anabolism Anti-Inflammatory Agents Anti-inflammatory Apoptosis Arts Bacteria Behavior Binding Biochemical Biology C-Type Lectins Carbohydrates Cell Wall Cells Cellular biology Chromosomes Cosmids Dendritic Cells Environment Enzymes Escherichia coli Event Gene Targeting Genes Genome Genus Mycobacterium Goals Guanosine Diphosphate Mannose Human Human Genetics Immune response Immunobiology Infection Inflammatory Response Intercellular Adhesion Molecules Knock-out Knowledge Laboratories Lectin Libraries Ligands Lipids Lysosomes Mannans Mannose Mannose-1-phosphate guanylyltransferase Mannose-6-Phosphate Isomerase Mannosides Mediating Metabolism Modeling Molecular Molecular Mimicry Morbidity - disease rate Mus Mycobacterium Infections Mycobacterium tuberculosis Nature Nucleotides Open Reading Frames Organism Pathogenesis Pathway interactions Pattern recognition receptor Phagocytes Phagocytosis Phagosomes Phenotype Phosphatidylinositols Phosphomannomutase Pilum Population Predisposition Procedures Process Production Pseudomonas aeruginosa Pulmonary Surfactant-Associated Protein D Reaction Receptor Cell Regulation Research Research Personnel Role Salmonella Screening procedure Surface System Techniques Tuberculosis Virulent cell envelope comparative genomics cytokine fructose-6-phosphate hexokinase lipoarabinomannan lipomannan macrophage mannoproteins mannose 1-phosphate mannose 6 phosphate mannose receptor microbial host microbicide monocyte mortality mutant mycobacterial new therapeutic target overexpression phosphatidylinositol mannoside programs receptor receptor binding scavenger receptor stable cell line trafficking two-dimensional

项目摘要

Tuberculosis continues to cause tremendous morbidity and mortality throughout the world's population. Critical in establishment of a M. tuberculosis (M.tb) infection are entry and survival in the macrophage. The M.tb cell envelope is heavily mannosylated with the abundant lipoglycans lipoarabinomannan (ManLAM), lipomannan (LM), and phosphatidyl inositol mannosides (PIMs). These lipoglycans bind to C-type lectins expressed on macrophages and dendritic cells (DC). The mannose receptor (MR) is a C-type lectin that mediates phagocytosis of virulent strains of M.tb by human macrophages. DC-SIGN is expressed abundantly on DCs and is also expressed on alternatively activated macrophages such as alveolar macrophages. ManLAM is a major ligand for the MR and DC-SIGN. We hypothesize that the nature of surface mannosylation of M.tb has a major impact on the ability of the bacterium to interact with C-type lectins (the MR and DC-SIGN) and thereby modulate macrophage function and host responses. The cytosolic nucleotide mannose carrier GPD-mannose is an essential donor either directly or indirectly in the biosynthesis of LAM, LM, and PIMs as well as cell wall mannoproteins. This proposal focuses on the core enzymes that are required to produce GPD-mannose in mycobacteria. Genes encoding these enzymes will be over-expressed or knocked out inM. smegmatis and M.tb and mutant bacteria with altered surface mannosylation will then be studied in human macrophages and in the mouse aerosol model of TB pathogenesis. One such gene is M.tb manB which we have found encodes a functional phosphomannomutase. Overexpression of manB in M. smegmatis leads to a significant increase in LAM, LM, and PIMs produced as well as a 13-fold increase in bacterial association with macrophages. In addition to the targeted gene approach, we will screen M.tb libraries for clones that are altered in surface mannosylation. Specific aims: Aim 1: Clone, over-express and knock out a defined set of M.tb genes involved in GDP-mannose biosynthesis, Aim 2: Determine whether mycobacteria mutants are altered in surface mannosylation, Aim 3: Determine how genes involved in mannose biosynthesis impact M.tb interactions with the MR and DC-SIGN, the biology of bacteria in human macrophages, and M.tb pathogenesis in the mouse, Aim 4: Screen a M.tb transposon library and a M. smegmatis library over-expressing M.tb genes using Salmonella mannose-binding (type 1)pili and surfactant protein D to identify mutants and clones altered in surface mannosylation. The overall goal of this proposal is to combine molecular, biochemical, and cell biology techniques to identify key enzymes for GDP-mannose biosynthesis in M.tb and, by altering then- level of expression, determine their impact on 1)M.tb cell wall mannosylation and 2) the biology of bacterial behavior in human macrophages and the mouse. Information gained from these studies should enhance our knowledge of TB pathogenesis and also potentially identify new therapeutic targets since mannose metabolism has been shown to be essential for the survival of mycobacteria.

结核病继续给全世界人口带来巨大的发病率和死亡率。建立的关键 a 结核分枝杆菌（M.tb）感染进入并在巨噬细胞中存活。 M.tb 细胞包膜被严重甘露糖基化丰富的脂聚糖脂阿拉伯甘露聚糖 (ManLAM)、脂甘露聚糖 (LM) 和磷脂酰肌醇甘露糖苷 (PIM)。这些脂聚糖与巨噬细胞和树突细胞 (DC) 上表达的 C 型凝集素结合。甘露糖受体 (MR) 是 C 型凝集素介导人类巨噬细胞对结核分枝杆菌强毒株的吞噬作用。 DC-SIGN 在 DC 上大量表达并且也在替代激活的巨噬细胞上表达，例如肺泡巨噬细胞。 ManLAM 是 MR 的主要配体和 DC-SIGN。我们假设 M.tb 表面甘露糖基化的性质对 M.tb 的能力有重大影响。细菌与 C 型凝集素（MR 和 DC-SIGN）相互作用，从而调节巨噬细胞功能和宿主回应。胞质核苷酸甘露糖载体 GPD-甘露糖是直接或间接的重要供体。 LAM、LM 和 PIM 以及细胞壁甘露糖蛋白的生物合成。该提案的重点是核心酶分枝杆菌中产生 GPD-甘露糖所需的。编码这些酶的基因将在 M 中过度表达或被敲除。然后将在人类巨噬细胞和结核病发病机制的小鼠气溶胶模型。其中一个基因是 M.tb manB，我们发现它编码一个功能性的磷酸甘露糖变位酶。 manB 在耻垢分枝杆菌中的过度表达导致产生的 LAM、LM 和 PIM 显着增加以及与巨噬细胞的细菌关联增加 13 倍。除了靶向基因方法外，我们还会筛选用于表面甘露糖基化发生改变的克隆的 M.tb 文库。具体目标：目标 1：克隆、过度表达并敲除定义一组参与 GDP-甘露糖生物合成的 M.tb 基因，目标 2：确定分枝杆菌突变体是否在表面甘露糖基化，目标 3：确定参与甘露糖生物合成的基因如何影响 M.tb 与 MR 和 DC-SIGN、人类巨噬细胞中细菌的生物学以及小鼠 M.tb 发病机制，目标 4：筛选 M.tb 转座子使用沙门氏菌甘露糖结合（1 型）菌毛和表面活性剂过表达 M.tb 基因的耻垢分枝杆菌文库和耻垢分枝杆菌文库蛋白 D 用于鉴定表面甘露糖基化发生改变的突变体和克隆。该提案的总体目标是结合分子、生化和细胞生物学技术来识别关键酶 M.tb 中的 GDP-甘露糖生物合成，并通过改变 then- 表达水平，确定其对 1)M.tb 细胞壁的影响甘露糖基化和 2) 人类巨噬细胞和小鼠中细菌行为的生物学。从这些获得的信息研究应增强我们对结核病发病机制的了解，并有可能确定自甘露糖以来的新治疗靶点新陈代谢已被证明对于分枝杆菌的生存至关重要。