Bacterial Cell Wall Composition and the Influence of Antibiotics

细菌细胞壁的组成和抗生素的影响

• 批准号: 9319788
• 负责人: Lynette S Cegelski
• 金额: $ 28.31万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9319788
• 关键词: Antibiotic Resistance; Antibiotics; Architecture; Biochemical; Biological Assay; Carbon; Cell Survival; Cell Wall; Cells; Communicable Diseases; Complement; Coupled; Data; Defensins; Development; Dissection; Electron Microscopy; Goals; Gram-Positive Bacteria; High Pressure Liquid Chromatography; Human; Impairment; Infection; Interruption; Isotopes; Label; Measurement; Measures; Metabolic; Metabolism; Methods; Molecular; Multi-Drug Resistance; Mycobacterium tuberculosis; Nitrogen; Organism; Peptidoglycan; Pharmaceutical Preparations; Physiologic pulse; Polymers; Protein Synthesis Inhibitors; Protocols documentation; Reporting; Resistance; Sampling; Site; Staphylococcus aureus; System; Teichoic Acids; Testing; Thick; Vancomycin; Virulence Factors; Work; analog; base; cell growth; cell preparation; clinical development; crosslink; design; drug development; drug discovery; experimental study; inhibitor/antagonist; insight; next generation; novel strategies; pathogen; solid state nuclear magnetic resonance; tool

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.

项目概要 这个时代可能会被人们铭记为传染病肆虐的时代。 由于抗生素耐药性上升和抗生素数量有限，该病在全球范围内卷土重来 在药物开发管道中。需要新的抗生素，并且耐药性的出现 几乎所有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，这对细胞至关重要 生存，是抗生素的主要目标。量化细胞壁组成的变化是 对于评估药物作用方式至关重要，特别是对于目前正在流行的人类病原体而言 对金黄色葡萄球菌等多种抗生素具有耐药性。然而，作为异质不溶物 聚合物基质、细胞壁对成分和成分的非微扰分析提出了挑战 建筑学。固态核磁共振已成为测量成分参数的强大工具 和完整细胞壁和整个细胞背景下的结构，并被用来定义 万古霉素类似物的作用方式，包括奥利万星 (OrbactivTM)，特别侧重于 标记的样品和两个关键的肽聚糖位点（交联和桥联）。分析未标记或 统一标记的样品更广泛地报告细胞中碳和氮的总体组成 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 标签扰乱和稀释，并可以识别不同类别抗生素引起的变化。 在这个项目中，我们将引入新的协议，将它们与现有的方法集成，并且 构建并描述有效的固态 NMR 工具包来定义和量化细菌细胞壁 完整细胞制剂中的成分，包括肽聚糖和磷壁酸成分 壁和整个细胞。这些方法将整合不同的标签策略以及 适当的 NMR 脉冲序列。这些方法的结果补充了广泛的 新旧抗生素的生化数据通常已经可用，并且将在以下领域发挥独特作用： 他们能够识别和定量比较完整样品中的细胞壁成分，而无需 降解水解和扰动限制了基于溶液的定量比较 测定，对于细胞壁完全抵抗的革兰氏阳性生物体尤其重要 溶解。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型抗生素的作用方式。