CELL WALL MUTANTS OF MYCOBACTERIUM TUBERCULOSIS

结核分枝杆菌细胞壁突变体

• 批准号: 2070721
• 负责人: GURDYAL S BESRA
• 金额: $ 13.97万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2070721
• 关键词: Mycobacterium tuberculosis; SDS polyacrylamide gel electrophoresis; arabinose; bacterial genetics; carbohydrate structure; cell wall; cytokine; galactans; gas chromatography mass spectrometry; gene complementation; glycolipids; high performance liquid chromatography; host organism interaction; laboratory mouse; leukocyte activation /transformation; lipopolysaccharides; mannose; molecular cloning; mutant; peptidoglycan; phosphatidylinositols; recombinant DNA; transposon /insertion element; trehalose; virulence

In this proposal, we return to the "ugly" aspect of Mycobacterium tuberculosis, to the "armor coat," the "waxy layer," i.e., the mycolates, the mycocerosates, the sulfatides, the cord factors, and the arabinosides of arabinogalactan and lipoarabinomannan, that lend the organism and the disease such uniqueness among prokaryotes and infections. We have assembled a unique consortium that can address from a modern perspective the dominant surface components of M. tuberculosis that underlie mechanisms of pathogenesis, immune-evasion, virulence and persistence. The development of novel cloning vectors for introducing recombinant DNA into Mycobacterium spp., our comprehensive understanding of the finite biochemical structure of the molecules that comprise the cellular envelope, and our ability to isolate and characterize well defined cell wall mutants now allow a novel approach to understanding disease function. Thus the central theme of this proposal is to isolate mutants deficient in major cell wall structures and to use them in definition of biosynthetic genes and biological functions. Specifically, cell wall mutants will be generated by chemical or transposon mutagenesis. Also, spontaneous mutants will be selected by screening of clinical isolates, and the formation of spontaneous mutants will be "forced" by growth of M. tuberculosis under stringent nutrient conditions. Mutants will be selected by a variety of protocols such as antibiotic sensitivity, colony morphology, radiolabeling, and probing with monoclonal antibodies and lectins. Once specific defects are chemically characterized, the genes responsible for the synthesis of the deficient entities will be defined by complementation with genes from virulent M. tuberculosis, and stable well-defined mutants will be constructed using gene replacement methodologies. Finally, these specific cell wall defined mutants will provide the means by which to measure the contribution of major cell wall components to phagocytosis, stimulation of macrophage, release of cytokines, and activation of the host cell to kill the bacterium in well established animal models. The combined abilities of this group now provide the means to solve the long- time need for the generation, characterization and evaluation of cell-wall deficient variants of M. tuberculosis.

在这个提议中，我们回到分枝杆菌“丑陋”的一面 结核病，到“盔甲外套”，“蜡层”，即，霉菌， 蜡酸菌、硫苷脂、脐带因子和阿拉伯糖苷 阿拉伯半乳聚糖和脂阿拉伯甘露聚糖， 这种疾病在原核生物和感染中的独特性。我们有 组建了一个独特的联盟，可以从现代的角度解决 M.结核病是导致 致病机制、免疫逃避、毒力和持久性。 新型重组DNA导入载体的研究进展 分枝杆菌属，我们对有限的 组成细胞的分子的生化结构 包膜，以及我们分离和表征定义明确的细胞的能力 壁突变体现在允许一种新的方法来理解疾病的功能。 因此，该建议的中心主题是分离缺乏以下基因的突变体： 主要的细胞壁结构，并将其用于生物合成的定义 基因和生物功能。具体来说，细胞壁突变体将 通过化学或转座子诱变产生。还有，自发突变体 将通过筛选临床分离株进行选择，并形成 自发突变体将被M.肺结核 严格的营养条件。 突变体将由各种 例如抗生素敏感性，菌落形态， 放射性标记和用单克隆抗体和凝集素探测。一旦 特定的缺陷是化学特征，基因负责 缺陷实体的合成将通过互补来定义 带有致命的M.结核病和稳定的明确的突变体 将使用基因替换方法构建。最后这些 特定的细胞壁限定的突变体将提供手段， 测量主要细胞壁组分对吞噬作用的贡献， 刺激巨噬细胞，释放细胞因子，激活 宿主细胞在良好建立的动物模型中杀死细菌。的 这一群体的综合能力现在提供了解决长期- 细胞壁生成、表征和评价所需的时间 缺陷型M.结核