Altered Mycobacterium tuberculosis Mannosylation and the Macrophage

改变结核分枝杆菌甘露糖基化和巨噬细胞

• 批准号: 7169641
• 负责人: Larry S. Schlesinger
• 金额: $ 36.23万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169641
• 关键词: Adhesions; Aerosols; Alginates; Alveolar Macrophages; Anabolism; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Arts; Bacteria; Binding; Biochemical; Biochemistry; Biological Assay; Biology; C-Type Lectins; Carbohydrates; Cell Adhesion; Cell Communication; Cell Wall; Cells; Cellular biology; Cessation of life; Chromosomes; Complement; Cosmids; Dendritic Cells; Environment; Enzymes; Escherichia coli; Event; Genes; Genetic; Genetic Transcription; Genome; Genomics; Genus Mycobacterium; Glycoconjugates; Goals; Guanosine Diphosphate Mannose; Homologous Gene; Human; Human Genetics; Immune response; Immunobiology; Infection; Inflammatory Response; Inositol; Intercellular Adhesion Molecules; Knock-out; Knowledge; Laboratories; Lectin; Libraries; Ligands; Lipids; Lysosomes; Mannans; Mannose; Mannose-1-phosphate guanylyltransferase; Mannose-6-Phosphate Isomerase; Mediating; Metabolism; Microspheres; Modeling; Molecular; Molecular Mimicry; Morbidity - disease rate; Mus; Mutation; Mycobacterium Infections; Mycobacterium tuberculosis; Nature; Nucleotides; Open Reading Frames; Organism; Pathogenesis; Pathway interactions; Pattern recognition receptor; Phagocytes; Phagocytosis; Phagosomes; Phenotype; Phosphomannomutase; Pilum; Polymerase Chain Reaction; Population; Predisposition; Procedures; Process; Production; Pseudomonas aeruginosa; Pulmonary Surfactant-Associated Protein D; Rate; Reaction; Receptor Cell; Regulation; Research; Research Personnel; Role; Salmonella; Screening procedure; Solid; Structure; Surface; System; Techniques; Time; Transferase; Tuberculosis; Virulent; cell envelope; comparative; cytokine; fructose-6-phosphate; hexokinase; lipoarabinomannan; lipomannan; macrophage; mannoproteins; mannose 1-phosphate; mannose 6 phosphate; mannose receptor; microbial host; microbicide; monocyte; mortality; mutant; mycobacterial; novel; novel therapeutics; phosphatidylinositol mannoside; programs; receptor; receptor binding; scavenger receptor; stable cell line; therapeutic target; trafficking; two-dimensional

DESCRIPTION (provided by applicant): Tuberculosis continues to cause tremendous morbidity and mortality throughout the world's population. Critical in establishment of a M. tuberculosis (M.tb) infection within its human host are entry and survival in the macrophage. The macrophage mannose receptor (MR) participates in the phagocytosis of virulent strains of M.tb. Components of the M.tb cell wall serve as ligands for host cell receptors and can modulate host microbicidal and inflammatory responses. The M.tb cell envelope is heavily mannosylated containing lipoglycans such as lipoarabinomannan (LAM) which serves as a ligand for the MR. We hypothesize that the nature of surface mannosylation of M.tb has a major impact on the ability of M.tb to interact with the MR as well as to modulate macrophage function and consequently host responses, enabling the establishment of infection. PimB was recently described as M.tb phosphatidyl myo-inositol monomannoside transferase (pimB). We have used allelic-exchange to inactivate pimB in M.tb strain Erdman. Macrophages display marked cellular adhesion following infection with wild-type M.tb. In contrast, macrophages infected with the pimB mutant display minimal cellular adhesion and a significant increase in the rate of macrophage death. We have developed an assay in which Salmonella mannose-specific binding pili agglutinate M.tb LAM coated microspheres that we will develop as a screen for alterations in M.tb surface mannosylation. We propose to further characterize the role of pimB and other selected enzymes potentially involved in mannosylation of M.tb surface molecules in the biology of the M.tb-host interaction, to develop a novel screening strategy for M.tb clones altered in surface mannosylation, and to evaluate these bacterial clones for anomalous host cell interaction. Our specific aims are to: 1A. Determine the mechanism for reduced homotypic adhesion and increased rate of macrophage death following infection with the pimB mutant of M.tb: 1 B. Determine the biochemical nature of the pimB mutation. Analyze the structure of LAM and other mannosylated cell wall glycoconjugates from wild type, the pimB mutant, and pimB overproducing M.tb strains. 2. Perform transcription and genetic studies of genes encoding the biosynthetic enzymes of LAM and mannose glycoconjugates: 2A. Quantify the level of transcription of pimB and its homologues in M.tb grown in broth, solid medium and within human macrophages using the AbI 7700 (TaqMan) "real-time" quantitative PCR system; 2B. Construct and analyze genetically defined M.tb strains with alterations in the mannose biosynthetic genes. 3). Utilize Salmonella mannose-binding (type 1) pili to screen for M.tb mutants and clones respectively altered in surface mannosylation from an M.tb transposon library and M. smegmatis library complemented with M.tb genes to characterize the effect of alterations in bacterial mannosylation on macrophage interaction. The assembled investigators will combine techniques in genetics, biochemistry, and cell biology to accomplish the goals of this proposal.

描述（由申请人提供）：结核病继续在全世界人口中造成巨大的发病率和死亡率。 在人类宿主体内建立结核分枝杆菌（M.tb）感染的关键是巨噬细胞的进入和存活。巨噬细胞甘露糖受体 (MR) 参与结核分枝杆菌强毒株的吞噬作用。 M.tb 细胞壁的成分作为宿主细胞受体的配体，可以调节宿主的杀菌和炎症反应。 M.tb 细胞包膜被严重甘露糖基化，含有脂聚糖，例如脂阿拉伯甘露聚糖 (LAM)，它充当 MR 的配体。我们假设 M.tb 表面甘露糖基化的性质对 M.tb 与 MR 相互作用以及调节巨噬细胞功能和宿主反应从而实现感染建立的能力有重大影响。 PimB 最近被描述为 M.tb 磷脂酰肌醇单甘露糖苷转移酶 (pimB)。我们使用等位基因交换来灭活 M.tb 菌株 Erdman 中的 pimB。感染野生型结核分枝杆菌后，巨噬细胞表现出明显的细胞粘附。相比之下，感染 pimB 突变体的巨噬细胞表现出最小的细胞粘附和巨噬细胞死亡率的显着增加。我们开发了一种检测方法，其中沙门氏菌甘露糖特异性结合菌毛凝集 M.tb LAM 包被的微球，我们将开发该方法作为 M.tb 表面甘露糖基化变化的筛选。我们建议进一步表征 pimB 和其他可能参与 M.tb 表面分子甘露糖基化的酶在 M.tb-宿主相互作用的生物学中的作用，以开发一种新的筛选策略，用于表面甘露糖基化改变的 M.tb 克隆，并评估这些细菌克隆的异常宿主细胞相互作用。我们的具体目标是： 1A．确定 M.tb 的 pimB 突变体感染后同型粘附减少和巨噬细胞死亡率增加的机制：1 B. 确定 pimB 突变的生化性质。分析来自野生型、pimB 突变体和 pimB 过量生产 M.tb 菌株的 LAM 和其他甘露糖基化细胞壁糖缀合物的结构。 2. 对编码 LAM 和甘露糖复合糖生物合成酶的基因进行转录和遗传学研究：2A。使用 AbI 7700 (TaqMan)“实时”定量 PCR 系统量化肉汤、固体培养基和人巨噬细胞内生长的 M.tb 中 pimB 及其同源物的转录水平； 2B。构建并分析甘露糖生物合成基因发生改变的基因定义的 M.tb 菌株。 3）。利用沙门氏菌甘露糖结合（1 型）菌毛从 M.tb 转座子文库和 M.smegmatis 文库中筛选表面甘露糖基化发生改变的 M.tb 突变体和克隆，并补充 M.tb 基因，以表征细菌中改变的影响甘露糖基化对巨噬细胞相互作用的影响。聚集的研究人员将结合遗传学、生物化学和细胞生物学技术来实现该提案的目标。