Biogenesis of lipoarabinomannan in mycobacteria

性状（由申请人提供）：磷脂酰肌醇甘露糖苷（PIM）及其多糖基化对应物脂甘露聚糖（LM）和脂阿拉伯甘露聚糖（LAM）是在所有分枝杆菌种属的包膜中发现的复合糖脂/脂聚糖。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。虽然在过去的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进展，但对它们的生物起源知之甚少。阐明其生物合成途径，除了提供有关结核分枝杆菌生物化学的基础知识外，还将导致发现可能代表新型抗结核药物关键靶点的必需酶。定义的M的可用性。结核和M.在PIM/LM和LAM合成的某些方面有缺陷的牛BCG突变体也将能够精确测量这些分子对结核病免疫发病机制的贡献，并提出针对这种疾病的新疫苗策略。 我们建议利用放线菌产生的LAM样分子的结构的最新知识，以及越来越多的脂多糖产生放线菌的基因组序列的可用性，进行基因组比较研究，确定参与PIM，LM和LAM的生物合成的糖基转移酶，并构建分枝杆菌的脂多糖缺陷型突变体。利用CS-35抗LAM单克隆抗体和我们研制的其他抗体，我们还计划筛选M. tuberculosis，M. bovis BCG和M.对于LAM合成的某些方面有缺陷的突变体，将使用常规方法和新型分析工具对突变体进行生物化学表征，将为新鉴定的生物合成酶开发无细胞测定法，并确定这些蛋白质的三维结构。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.