Lipids of mycobacteria

描述（由申请人提供）：分枝杆菌是一种复杂的细菌属，其独特的细胞壁富含复杂的脂质、碳水化合物和多酮。该属最具临床意义的成员是结核分枝杆菌（TB），这是一种致命的专性人类病原体，具有巨大的全球负担。尽管针对这种病原体进行了数十年的研究，但结核病仍然感染着世界三分之一的人口。为了开发新的和创新的治疗策略，需要更好地了解结核病的生理学和发病机制。近年来，基因组学和蛋白质组学技术的出现极大地促进了我们对结核病的研究。然而，缺乏合适的互补、快速的技术来研究脂质、聚酮和碳水化合物（有机）库，这些已知在结核病和其他致病性分枝杆菌的生理和毒力中起重要作用。为此，我们一直在开发新的快速分析技术来研究分枝杆菌脂类、聚酮类和碳水化合物。我们开始开发一种简单的补充技术，通过二维1H-13C HSQC核磁共振分析，通过最小操作分析富含13C的粗脂提取物，快速查看结核杆菌的脂质和聚酮谱。在这些复杂的二维HSQC图谱中，有可能找到可用于识别和量化关键脂质物种存在的不同信号。然后我们发现这些脂质谱可以用于观察由于药物治疗、基因突变和生理环境变化、物种特征和毒力因子表达的变化而引起的细胞壁变化。重要的是，与基于质谱的脂质组学方法不同，该分析技术的响应在脂质种之间是等摩尔的。这允许在同一样品中对几种不同的脂质物种进行快速比较分析。然而，在该方法真正用于全面定量的脂质组学研究之前，还需要进一步完善和验证该方法。这种基于R21应用程序的方法开发解决了最终确定该技术所需的研究，以便它可以用于分枝杆菌的系统生物学研究，与其他脂质组学、基因组学、蛋白质组学和糖组学方法相补充，并被结核病研究界作为一个整体。本提案的目的是：(i)开发和验证一种新的基于核磁共振的快速结核脂质分析方法；（ii）进行2项试点研究，旨在评估该方法的稳健性和将该方法应用于高临床相关性研究时产生的数据质量。公共卫生相关性：结核分枝杆菌是结核病的病原体。结核分枝杆菌富含脂质和有机物质，这是其生存和致病能力所必需的。在本提案中，计划开发新的快速方法来同时研究所有这些材料，以便可以推断出它们在疾病过程中的作用。

DESCRIPTION (provided by applicant): Mycobacteria are a complex bacterial genus characterized by a unique cell wall rich in complex lipids, carbohydrates and polyketides. The most clinically significant member of this genus is Mycobacterium tuberculosis (TB) a deadly obligate human pathogen with an immense global burden. Despite decades of research effort against this pathogen, TB still infects one third of the world population. In order to develop new and innovative treatment strategies against TB, a better understanding of the physiology and pathogenesis of TB is needed. The advent of genomics and proteomics techniques has significantly boosted how we study TB in recent years. However, there is a lack of suitable complementary, rapid techniques to study the lipid, polyketide and carbohydrate (organic) pools that are known to play important roles in the physiology and virulence of TB and other pathogenic mycobacteria. Towards these ends, we have been developing new rapid analysis techniques to study mycobacterial lipids, polyketides and carbohydrates. We initiated the development of a simple complementary technique to quickly view the lipid and polyketide profiles of the TB bacilli using 13C enriched crude lipid extracts analyzed with minimal manipulation by 2D 1H-13C HSQC NMR analysis. Within these complex 2D HSQC maps it was possible to find distinct signals that could be used to identify and quantify the presence of key lipid species. We then showed that these lipid profiles could be used for observing changes in the cell wall due to drug treatment, gene mutation and changes in the physiological environment, species characterization and virulence factor expression. Importantly, the response of this analysis technique is equimolar between lipid species unlike mass spectrometric based lipidomic methods. This allows for rapid comparative analysis of several different lipid species within the same sample. However, before this method can be truly used for comprehensive and quantitative lipidomics studies, work is required to further refine and validate the method. This method development based R21 application addresses the studies required to finalize this technique so that it can be used in systems biology studies of mycobacteria, complementary to other lipidomic, genomic, proteomic and glycomic methods, and by the TB research community as a whole. The aims of this proposal are (i) Development and validation of a new rapid NMR based TB lipid profiling method; (ii) to perform 2 pilot studies designed to evaluate the robustness of the method and the quality of the data produced when the method is applied to studies of high clinical relevance. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis. M. tuberculosis is rich in lipids and organic materials that are essential for its survival and disease causing abilities. In this proposal it is planned to develop new rapid methods to study all these material simultaneously so that a new understanding of their role in the disease process can be deduced.