Bacterial Cell Wall Composition and the Influence of Antibiotics

项目总结 这个时代可能会被人们记住，因为在这个时代里，传染病 由于抗生素耐药性的上升和抗生素数量的有限，全球范围内的复苏 在药物开发流水线中。需要新的抗生素，并出现对 几乎任何一种抗生素都强调需要了解抗生素模式的分子细节。 帮助指导新抗生素开发的行动。 革兰氏阳性细菌周围有一层厚厚的细胞壁，这是细胞必不可少的 存活，是抗生素的主要靶点。细胞壁成分的量化变化是 对评估药物作用模式至关重要，尤其对现在的人类病原体 对多种抗生素耐药，如金黄色葡萄球菌。然而，作为一种异质的不可溶物质 聚合物基质，细胞壁对组成和组成的非微扰分析提出了挑战 建筑。固体核磁共振已成为测量组成参数的有力工具 以及在完整的细胞壁和整个细胞的背景下的体系结构，并被用来定义 包括oritvancin(Orbactive TM)在内的万古霉素类似物的作用方式，重点是 标记的样品和两个关键的肽聚糖位点(交联链和桥胶链接)。对未贴标签的OR的分析 统一标记的样本更广泛地报告了细胞中的总体碳和氮组成 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 标签扰乱和稀释，并可以识别由于不同类别的抗生素造成的变化。 在这个项目中，我们将引入新的协议，将这些协议与现有方法集成，并 建立并描述用于定义和量化细菌细胞壁的有效固态核磁共振工具包 完整细胞制剂中的成分，包括肽聚糖和磷壁酸成分 墙壁和整个牢房。这些方法将整合不同的标签策略 适当的核磁共振脉冲序列。这些方法的结果补充了广泛的 生化数据通常已经可以用于新旧抗生素，并将在 它们识别和定量比较完整样品中细胞壁成分的能力 限制基于溶液的定量比较的降解性水解和扰动 对于细胞壁完全抵抗的革兰氏阳性生物来说，化验尤其重要 解散。这些方法将用一组既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.