Structure of LAM in Relation to Biology and Biosynthesis

描述(申请人提供)：由于缺乏有效的化疗药物和开发新的抗生素，结核病再次成为一个公共卫生问题变得更加复杂。已知其病原体结核分枝杆菌的细胞壁是一些最有效的抗分枝杆菌药物的靶点，其中包括乙胺丁醇，已知的是抑制细胞壁本身和相关的脂阿拉伯甘露糖(LAM)的阿拉伯糖的生物合成。十多年来的各种生物学研究共同提供了令人信服的证据，表明LAM是宿主与病原体相互作用的关键表面分子。由于天然和实验室诱导的对乙胺丁醇的耐药性而导致的未成熟LAM或截短LAM的发现，以及EMBC基因敲除事件，为旨在确定LAM在发病机制中的相关性的结构和功能研究提供了宝贵的模型化合物。 具体地说，随着新酶的推出，先进的化学、红外光谱和质谱分析工具可以结合在一起，以获得阿拉伯聚糖组装的精细细节以及阿拉伯聚糖与甘露聚糖核心的连接方式和位置(S)。与临床分离株特定生物学属性正相关的酶修饰结构阿拉伯糖基序将从LAM中衍生出来，并将产生新的阿拉伯脂脂用于功能研究。将努力解决LAM的异质性，并将其与生物学联系起来。CD1限制的T细胞对LAM的识别将在结核的细胞免疫及其伴随的细胞因子分泌诱导的背景下进行检验。最后，将利用基因敲除突变体、无细胞分析和人工合成的阿拉伯呋喃糖基受体来确定与LAM的阿拉伯聚糖组装有关的代谢事件，几乎对此一无所知，随后将鉴定涉及的蛋白质(转移酶)。 因此，这项研究建议的统一主题是LAM的结构分析和操纵，辅之以遗传和生物合成研究，从而更好地了解其生物学和生物合成。

DESCRIPTION (provided by applicant): The re-emergence of tuberculosis as a public health problem has been complicated by the lack of effective chemotherapeutic agents and the development of new antibiotics. The cell wall of its causative agent, Mycobacterium tuberculosis, is known to be a target of some of the most effective antimycobacterial drugs including ethambutol which has been known to inhibit the biosynthesis of arabinan of the cell wall proper and associated lipoarabinomannan (LAM). A diverse range of biological studies over a decade has collectively provided compelling evidence implicating LAM as a key surface molecule in host-pathogen interactions. The finding of immature LAM or truncated LAM as a consequence of natural and laboratory induced resistance to ethambutol, and embC gene knockout events provide invaluable model compounds for both structural and functional studies aiming at defining the relevance of LAM in pathogenesis. Specifically, with the availability of new enzymes, advanced chemical, NrvIR and mass spectrometric tools can be combined to yield the fine details of the arabinan assembly and the mode and site(s) of arabinan attachment to the mannan core. Enzymatically modified structural arabinan motifs positively correlating with particular biological attributes of clinical isolates will be derived from LAM, and neoarabinolipids will be generated for functional studies. Efforts will be given in resolving the heterogeneity in LAM and relate it to biology. CD1 restricted recognition of LAM by T cells will be examined in the context of cell mediated immunity in tuberculosis and its concomitant induction of cytokine secretion. Finally, gene knock-out mutants, cell-free assays and synthetic arabinofuranosyl acceptors will be utilized to establish the metabolic events involved in the arabinan assembly of LAM about which almost nothing is known, followed by identification of proteins (transferases) involved. Thus, the unifying theme of this Research Proposal is the structural analysis and manipulation of LAM, supplemented by genetic and biosynthetic studies leading to a better understanding of its biology and biosynthesis.