Structure of LAM in Relation to Biology and Biosynthesis

描述（由申请人提供）：由于缺乏有效的化疗药物和耐药菌株的发展，结核病作为一个公共卫生问题的重新出现变得更加复杂。已知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，包括乙胺丁醇，已知其抑制细胞壁阿拉伯半乳聚糖（AG）和相关的脂阿拉伯糖甘露聚糖（LAM）的生物合成。在过去几年中，各种各样的生物学研究共同提供了令人信服的证据，表明LAM是宿主-病原体相互作用的关键表面分子。由于耐药和基因操作导致的截断、突变的LAM变体的可用性为旨在确定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。具体来说，阿拉伯糖组装的精细细节及其与磷脂酰肌醇甘露聚糖核心的附着点将被表征，与临床分离物的特定生物学特性正相关的结构细节将被确定。LAM中微观异质性的结构基础将被定义，化学和/或酶修饰的阿拉伯糖和甘露聚糖将从LAM中提取，用于结构/生物学研究。作为一个主要的分支，最近由于embCAB蛋白的遗传操作而产生的相应细胞壁突变的可用性，以及对基因产物的分析，现在使我们能够合理地剖析LAM/AG阿拉伯蛋白形成的途径。同样，对结核分枝杆菌中LAM突变体的研究以及我们在产生LAM缺失结核分枝杆菌方面的共同努力，将直接有助于解决LAM在生物体存活/感染性中的作用。因此，本研究计划的统一主题包括LAM的结构分析和操作，辅以基因探针来改变其结构并使耻垢分枝杆菌和结核分枝杆菌中的LAM突变，这些都与生物学和生物合成有关。

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 drug-resistant strains. The cell wall of the pathogen Mycobacterium tuberculosis, is known to be the target of some of the most effective anti-mycobacterial drugs including ethambutol which is known to inhibit the biosynthesis of the arabinan of cell wall arabinogalactan (AG) and the associated lipoarabinomannan (LAM). A diverse range of biological studies over the past few years has collectively provided compelling evidence implicating LAM as a key surface molecule in host-pathogen interactions. The availability of truncated, mutated LAM variants as a consequence of drug resistance and genetic manipulation provides invaluable model compounds for both structural and functional studies aiming at defining the relevance of LAM in pathogenesis. Specifically, the fine details of the arabinan assembly and its point(s) of attachment to the phosphatidylinositol mannan core will be characterized and structural niceties positively correlating with particular biological attributes of clinical isolates will be identified. Structural basis of microheterogeneity in LAM will be defined and chemically and/or enzymatically modified arabinan and mannan will be derived from LAM for structural/biological studies. As a major spin off, the recent availability of the consequential cell wall mutants due to genetic manipulation of the embCAB proteins, and analyses of the gene products now allows us rationally to dissect the pathway to the formation of the arabinan of LAM/AG. In the same vein, studies on LAM mutants in M. tuberculosis and our concerted efforts on generating LAM depleted M. tuberculosis will contribute directly into addressing the role of LAM in survival/infectivity of the organism. Thus, the unifying theme of this Research Proposal encompasses structural analysis and manipulation of LAM, supplemented by genetic probes to alter its structure and mutate LAM in M. smegmatis and M. tuberculosis all in relation to biology and biosynthesis.