Bacterial Cell Wall Composition and the Influence of Antibiotics

项目摘要 这个时代可能会被人们记住，因为在这个时代，传染病对人类的健康产生了巨大的影响。 由于抗生素耐药性的增加和抗生素数量有限， 在药物研发管道中。需要新的抗生素，耐药性的出现， 几乎所有的抗生素都强调需要了解抗生素模式的分子细节， 帮助指导新抗生素的开发。 革兰氏阳性菌的细胞壁很厚， 是抗生素的主要目标。细胞壁组成的定量变化是 这对评估药物作用模式至关重要，对现在被认为是 对多种抗生素耐药，如金黄色葡萄球菌。然而，作为一种异质的不溶性 聚合物基质，细胞壁对组成的非微扰分析提出了挑战， 架构固体核磁共振技术已成为测量化学组成参数的有力工具 和架构的背景下，完整的细胞壁和整个细胞，并被用来定义 万古霉素类似物包括奥利万星（OrbactivTM）的作用模式，特别关注 标记的样品和两个关键的肽聚糖位点（交联和桥链）。未标记或 统一标记的样品更广泛地报告了细胞中的总体碳和氮组成， 细胞壁和整个细胞，可以很容易地扩展到其他生物体，不需要考虑 标签加扰和稀释，并可以识别由于不同类别的抗生素的变化。 在这个项目中，我们将引入新的协议，将它们与现有的方法相结合， 建立和描述一个有效的固态NMR工具包来定义和量化细菌细胞壁 在完整的细胞制备物中的包括肽聚糖和磷壁酸组分的组合物 墙壁和整个细胞。这些方法将整合不同的标记策略， 适当的NMR脉冲序列。这些方法的结果补充了广泛的 生物化学数据通常已经可用于新旧抗生素，并且将在 它们能够识别和定量比较完整样品中的细胞壁组分， 降解水解和扰动，限制了基于溶液的定量比较 检测，特别是对革兰氏阳性微生物与细胞壁抵抗完全 解散这些方法将用一组已确定的抗生素进行测试，并将用于 研究不同的细菌菌株和四种新抗生素的作用方式。

PROJECT SUMMARY This era may come to be remembered as one in which infectious diseases made a dramatic worldwide resurgence, owing to the rise of antibiotic resistance and the limited number of antibiotics in the drug development pipeline. New antibiotics are needed and the emergence of resistance to almost any antibiotic underscores the need to understand the molecular details of antibiotic modes of action to help guide the development of new antibiotics. Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Yet, as a heterogeneous insoluble polymeric matrix, the cell wall poses a challenge to non-perturbative analyses of composition and architecture. Solid-state NMR has emerged as a powerful tool to measure parameters of composition and architecture in the context of intact cell walls and whole cells and was employed to define the mode of action of vancomycin analogs including oritvancin (OrbactivTM), focusing on specifically labeled samples and two key peptidoglycan sites (crosslinks and bridgelinks). Analyses of unlabeled or uniformly-labeled samples report more broadly on overall carbon and nitrogen composition in cell walls and whole cells, can be readily extended to other organisms, do not require considerations of label scrambling and dilution, and can identify alterations due to different classes of antibiotics. In this project, we will introduce new protocols, integrate these with existing approaches, and build and describe an effective solid-state NMR toolkit to define and quantify bacterial cell-wall composition, including peptidoglycan and teichoic acid components, in intact preparations of cell walls and whole cells. These approaches will integrate different labeling strategies coupled with appropriate NMR pulse sequences. The results from these approaches complement extensive biochemical data usually already available for old and new antibiotics and will be uniquely enabling in their ability to identify and quantitatively compare cell-wall components in intact samples without degradative hydrolyses and perturbations that limit quantitative comparisons in solution-based assays, particularly important for Gram-positive organisms with cell walls that resist complete dissolution. The methods will be tested with a panel of established antibiotics and will be used to investigate different bacterial strains and the modes of action of four new antibiotics.