Biogenesis of lipoarabinomannan in mycobacteria

描述(申请人提供)：磷脂酰肌醇甘露糖苷(PIM)及其多糖基化的类似物，脂甘露聚糖(LM)和脂阿拉伯甘露聚糖(LAM)是复杂的糖脂/脂多糖，存在于所有分枝杆菌物种的包膜中。它们是结核病和麻风病过程中重要的免疫调节分子，也是促进分枝杆菌进入吞噬细胞和非吞噬细胞的关键配体。尽管在过去的15年里，在破译这些分子的结构和结构-功能关系方面取得了很大的进展，但人们对它们的生物发生知之甚少。阐明它们的生物合成途径，除了提供关于结核分枝杆菌生物化学的基本知识外，还将导致发现关键的酶，这些酶可能是新型抗结核药物的关键靶点。在PIM/LM和LAM合成的某些方面缺乏的确定的结核分枝杆菌和牛分枝杆菌卡介苗突变体的可用性也将使人们能够精确地测量这些分子在结核病免疫发病机制中的贡献，并提出针对这种疾病的新的疫苗策略。 我们建议利用放线菌产生的LAM类分子结构的最新知识，以及越来越多的产生脂多糖的放线菌基因组序列的可用性来进行基因组比较研究，鉴定参与PIM、LM和LAM生物发生的糖基转移酶，并构建脂糖缺乏的分枝杆菌突变体。利用CS-35抗LAM单抗和我们开发的其他抗体，我们还建议筛选结核分枝杆菌、牛分枝杆菌和耻垢分枝杆菌的转座子突变体库，以寻找在LAM合成方面存在缺陷的突变株。突变体将使用传统方法和新的分析工具进行生化表征，新鉴定的生物合成酶的无细胞分析将被开发，这些蛋白质的三维结构将被确定。最后，将研究PIM/LM/LAM突变体与宿主细胞的相互作用及其免疫调节特性。

DESCRIPTION (provided by applicant): Phosphatidylinositol mannosides (PIM) and their multiglycosylated counterparts, lipomannan (LM) and lipoarabinomannan (LAM) are complex glycolipids/ lipoglycans that are found in the envelopes of all mycobacterial species. They are important immunomodulatory molecules in the course of tuberculosis and leprosy as well as key ligands promoting the entry of mycobacteria into phagocytic and non-phagocytic cells. Although much progress has been made over the last 15 years in deciphering the structures and structure-function relationships of these molecules, little is known about their biogenesis. The elucidation of their biosynthetic pathways, in addition to providing fundamental knowledge about the biochemistry of Mycobacterium tuberculosis, would lead to the discovery of essential enzymes that could represent crucial targets for novel anti-tuberculosis drugs. The availability of defined M. tuberculosis and M. bovis BCG mutants deficient in some aspects of PIM/ LM and LAM synthesis would also enable a precise measurement of the contribution of these molecules to the immunopathogenesis of tuberculosis and suggest new vaccine strategies against this disease. We propose to exploit the recent knowledge of the structures of LAM-like molecules produced by actinomycetes together with the availability of a growing number of genome sequences of lipoglycan-producing actinomycetes to perform genome comparison studies, identify glycosyltransferases involved in the biogenesis of PIM, LM and LAM and construct lipoglycan-deficient mutants of mycobacteria. Using the CS- 35 anti-LAM monoclonal antibody and other antibodies that we have developed, we also propose to screen transposon mutant libraries of M. tuberculosis, M. bovis BCG, and M. smegmatis for mutants defective in some aspects of LAM synthesis. Mutants will be biochemically characterized using conventional methods and novel analytical tools, cell-free assays will be developed for the newly identified biosynthetic enzymes and the three-dimensional structure of these proteins will be determined. Finally, the interactions of PIM/ LM/ LAM mutants with host cells and their immunomodulatory properties will be investigated.