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.

项目摘要 这个时代可能被人们铭记为传染病引起戏剧性的一个时代 由于抗生素耐药性的兴起和抗生素数量有限，全球复兴 在药物开发管道中。需要新的抗生素，并且对 几乎所有抗生素都强调了了解抗生素模式的分子细节 帮助指导新抗生素的开发。 革兰氏阳性细菌用厚的细胞壁包围，这对细胞必不可少 生存，是抗生素的主要靶标。量化细胞壁组成的改变是 对于评估药物的作用模式至关重要，对于现在的人类病原体尤其重要 对多种抗生素（例如金黄色葡萄球菌）抗性。然而，作为一个异质的不溶性 聚合物矩阵，细胞壁对组成和非扰动分析构成了挑战 建筑学。固态NMR已成为测量组成参数的强大工具 和在完整的细胞壁和全细胞的背景下进行体系结构，并被用于定义 万古霉素类似物的作用方式，包括oritvancin（orbactivtm），专门针对 标记的样品和两个关键的肽聚糖位点（交联和bridgelinks）。分析未标记或 统一标签的样品报告了细胞中总体碳和氮组成的更广泛报告 墙壁和全细胞可以很容易地扩展到其他生物，不需要考虑 标签和稀释标签，可以识别由于不同类别的抗生素而引起的变化。 在这个项目中，我们将介绍新协议，将它们与现有方法集成在一起，然后 构建和描述有效的固态NMR工具包，以定义和量化细菌细胞壁 组成，包括肽聚糖和Teichoic酸成分，完整的细胞制剂 墙壁和全细胞。这些方法将整合不同的标签策略与 适当的NMR脉冲序列。这些方法的结果补充了广泛的 生化数据通常已用于新旧抗生素，并且将独特地启用 他们识别和定量比较完整样品中细胞壁成分的能力 降解水解和扰动限制基于溶液的定量比较 测定法，对于革兰氏阴性生物尤其重要 解散。该方法将通过一组已建立的抗生素进行测试，并将用于 研究不同的细菌菌株和四种新抗生素的作用模式。