Molecular mechanisms of antibiotic resistance

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随细菌感染，而细菌感染往往是致命的组合。由于耐药性的发展，治疗感染的选择大幅减少。这种耐药性在很大程度上是由于细菌表达的能降解β-内酰胺类抗生素的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的分子基础，以及β-内酰胺酶的某些突变如何导致对这种抗生素的耐药性，以及其他突变如何导致对他唑巴坦、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结晶学之间的一种新的协同作用。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的反应中间体，并为实现我们的特定目标提供了独特的优势：特定目标1：测试假设，即位于M69位的SHV-1抗药性变异会导致活性部位变化，影响涉及3种抑制剂的中间形成的水平和稳定性，从而使它们能够克服抑制。具体目的2：验证超广谱β-内酰胺酶SHV-2变异体(G238S)和SHV-5变异体(G238S和E240K)通过拓宽活性部位和与第三代头孢他啶形成新的相互作用而进化为第三代头孢菌素头孢他啶水解酶的假说。具体目标3：验证SHV-6、-8和-24中存在的D179位的变化已进化为通过移动omega环从而扩展活性部位以适应头孢他啶的假设。从拟议的研究中获得的结构知识可能有助于设计新的抗生素药物。

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.