Molecular mechanisms of antibiotic resistance

抗生素耐药性的分子机制

• 批准号: 7064635
• 负责人: FOCCO VAN DEN AKKER
• 金额: $ 27.04万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7064635
• 关键词: Raman spectrometry; X ray crystallography; active sites; antibiotics; bacteria; bacterial genetics; bacterial proteins; beta lactam antibiotic; beta lactamase; drug resistance; enzyme inhibitors; enzyme mechanism; gene mutation; hydrolysis; protein structure; structural biology

Bacterial infections pose serious threats to human health. Furthermore, viral infections such as the flu are frequently accompanied by bacterial infections which is often a deadly combination. Due to the development of resistance, the options for treating infections have dwindled substantially. This resistance is in large part due to the bacterial expression of beta-lactamases that degrade beta-lactam antibiotics. Our main goal is to structurally understand the molecular basis of resistance against the clinically available ceftazidime antibiotic and how certain mutations in beta-lactamases confer resistance to this antibiotic and other mutations confer resistance to the beta-lactamase inhibitors tazobactam, sulbactam, and clavulanic acid. Our structure-function studies involve a novel synergy between X-ray and time-resolved Raman crystallography. Our innovative inter-disciplinary approach allows us to identify and track reaction intermediates inside crystals prior to X-ray analysis and provides a unique advantage to accomplish our Specific Aims: Specific Aim 1: To test the hypothesis that SHV-1 resistance variants at position M69 cause active site changes that affect the level and stability of the intermediate formation involving the 3 inhibitors thus allowing them to overcome the inhibition. Specific Aim 2: To test the hypothesis that the extended-spectrum beta-lactamase SHV-2 variant (G238S) and SHV-5 variant (G238S & E240K) have evolved to hydrolyze the third-generation cephalosporin ceftazidime by widening the active site and forming novel interactions with this compound. Specific Aim 3: To test the hypothesis that the changes at position D179 which are present in SHV-6, -8, and -24 have evolved to hydrolyze ceftazidime by shifting the omega loop thereby extending the active site to accommodate ceftazidime. The structural knowledge gained from the proposed research could aid in the design of new antibiotic drugs.

细菌感染对人类健康构成严重威胁。 此外，流感等病毒感染经常伴有细菌感染，这往往是致命的组合。由于耐药性的发展，治疗感染的选择已大大减少。这种耐药性在很大程度上是由于细菌表达了可降解β-内酰胺抗生素的β-内酰胺酶。我们的主要目标是从结构上了解临床上可用的头孢他啶抗生素耐药性的分子基础，以及β-内酰胺酶的某些突变如何导致对该抗生素的耐药性以及其他突变如何导致对β-内酰胺酶抑制剂他唑巴坦、舒巴坦和克拉维酸的耐药性。我们的结构功能研究涉及 X 射线和时间分辨拉曼晶体学之间的新颖协同作用。我们创新的跨学科方法使我们能够在 X 射线分析之前识别和跟踪晶体内部的反应中间体，并为实现我们的具体目标提供独特的优势： 具体目标 1：测试 M69 位置处的 SHV-1 抗性变异的假设引起活性位点的变化，影响涉及 3 种抑制剂的中间体形成的水平和稳定性，从而使它们克服抑制作用。具体目标 2：检验超广谱 β-内酰胺酶 SHV-2 变体 (G238S) 和 SHV-5 变体 (G238S & E240K) 已进化为通过加宽活性位点并形成水解第三代头孢菌素头孢他啶的假设与该化合物的新相互作用。具体目标 3：检验以下假设：SHV-6、-8 和 -24 中存在的 D179 位点的变化已进化为通过移动 omega 环来水解头孢他啶，从而扩展活性位点以容纳头孢他啶。从拟议的研究中获得的结构知识可以帮助设计新的抗生素药物。