CELL WALL MUTANTS OF MYCOBACTERIUM TUBERCULOSIS

在这项提议中，我们回到分枝杆菌“丑陋”的一面 结核病，到“盔甲”，“蜡质层”，也就是分枝杆菌， 霉菌、硫脂、脐带因子和阿拉伯糖苷 阿拉伯半乳聚糖和脂肪阿拉伯甘露聚糖，它们为生物体和 这种疾病在原核生物和传染病中具有独特性。我们有 组建了一个独特的财团，可以从现代的角度解决 构成结核分枝杆菌的主要表面成分 致病机制、免疫逃避机制、毒力机制和持久性机制。 用于导入重组DNA的新型克隆载体的研究进展 进入分枝杆菌属，我们对有限的 组成细胞的分子的生化结构 包膜，以及我们分离和鉴定定义明确的细胞的能力 壁突变体现在为理解疾病功能提供了一种新的方法。 因此，这项建议的中心主题是分离出缺乏 主要细胞壁结构及其在生物合成定义中的应用 基因和生物功能。具体地说，细胞壁突变体将是 由化学或转座子诱变产生的。此外，自发的突变体 将通过对临床分离株的筛选，形成 自发的突变体将被结核分枝杆菌的生长“强迫” 严格的营养条件。突变体将由各种不同的 抗生素敏感性、菌落形态、 放射性标记，并用单抗和凝集素进行探测。一次 特定的缺陷具有化学特征，这些基因负责 缺陷实体的合成将通过互补来确定 带有来自强毒结核分枝杆菌的基因，以及稳定的明确定义的突变体 将使用基因替换方法构建。最后，这些 特定的细胞壁定义的突变体将提供 测量主要细胞壁成分对吞噬作用的贡献， 刺激巨噬细胞，释放细胞因子，激活巨噬细胞 在已建立的动物模型中，宿主细胞可以杀死细菌。这个 这一群体的综合能力现在提供了解决长期- 细胞壁的产生、表征和评价需要时间 结核分枝杆菌的缺陷变种。

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.