The Role of Lipoarabinomannan in Mycobacterium tuberculosis

描述(申请人提供)：结核分枝杆菌是一种主要的人类病原体，在世界范围内造成巨大的人类痛苦。作为单一细菌感染的主要死亡原因，了解该生物体的生物学、生理学和致病性是非常必要的。迫切需要改进诊断、治疗和预防措施。结核分枝杆菌表现出有助于其致病性和潜在感染的某些特征。其中，富含脂质的细胞壁在与宿主的相互作用中起着关键作用，是抵御免疫攻击的第一道防线。这个探索性项目的主要目的是确定关键的主要细胞壁成分之一的脂阿拉伯甘露聚糖(LAM)在结核杆菌中的生理作用。以前不可能在整个细胞的背景下研究LAM，因为LAM阴性突变株还没有被分离出来，我们最近已经证明这是由于它对细菌生存的重要性。虽然不能构建突变体，但我们已经设计出一株结核分枝杆菌，它表达LAM生物合成所需的关键酶之一EMBC，与野生型细胞相比，其水平有所降低，结果是产生了较小尺寸的LAM。我们将确定LAM产量的变化对多种化合物的抗性有何影响，包括乙胺丁醇、DNA损伤剂以及活性氧和氮物种。在结核分枝杆菌中，缺乏EMBC是致命的，但表达减少是可以容忍的。我们将通过构建一株从可控、可滴定的启动子中表达EMBC的菌株，来确定在细菌生存受到影响之前，EMBC的低表达可以降低到什么程度。随后，我们将进行表型分析，以确定EMBC耗尽的生理后果。将特别注意合成的LAM的尺寸和结构，以及对破坏剂和应力的抵抗力。产生的数据将提供LAM在细菌细胞中的生理作用的图像，并为进一步研究致病性提供平台。公共卫生相关性：结核分枝杆菌是人类结核病的病原体，这是一种毁灭性的传染病，每年导致近200万人死亡，800多万人感染。对新的治疗剂和对疾病过程的更多了解存在着迫切的未得到满足的需求。这一建议旨在增加我们对致病的关键细菌效应之一的了解，并确定其在细菌生理学中的作用。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is a major human pathogen causing significant human suffering worldwide. As the leading cause of death from a single bacterial infection, it is imperative to understand the biology, physiology and pathogenicity of the organism. Improved diagnostics, therapeutics and prophylactics are urgently required. M. tuberculosis exhibits certain features which contribute to its pathogenicity and potential to establish latent infections. Among these, the lipid-rich cell wall plays a critical part in interaction with the host, being the first line of defence against immune attack. The primary aim of this exploratory project is to define the physiological role of one of the critical, major cell wall components, lipoarabinomannan (LAM) in the tubercle bacillus. It has not been possible to study LAM in the context of the whole cell previously, since LAM negative mutants have not been isolated and we have recently shown that this is due to its essentiality for bacterial survival. Although, mutants cannot be constructed, we have engineered a strain of M. tuberculosis which expresses EmbC, one of the key enzymes required for LAM biosynthesis, at a reduced level compared to wild-type cells, the consequence of which is that LAM of a smaller size is produced. We will determine what effect changes in LAM production have on resistance to a wide range of compounds including ethambutol, DNA damaging agents and reactive oxygen and nitrogen species. A lack of EmbC is lethal in M. tuberculosis, but reduced expression is tolerated. We will determine how low EmbC expression can be reduced before bacterial survival is compromised by constructing a strain expressing embC from a controllable, titratable promoter. Subsequently, we will conduct phenotypic assays to determine the physiological consequences of EmbC depletion. Particular attention will be paid to the size and structure of LAM synthesized and resistance to damaging agents and stresses. The data generated will provide a picture of the physiological role of LAM in the bacterial cell and provide a platform for further work investigating pathogenicity. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis is the causative agent of human tuberculosis, a devastating infectious disease, which kills nearly 2 million and infects more than 8 million people each year. There is an urgent unmet need for new therapeutic agents and an increased understanding of the disease process. This proposal aims to increase our understanding of one of the key bacterial effectors of pathogenesis and define its role in bacterial physiology.