Molecular mechanisms of antibiotic resistance

细菌感染对人类健康构成严重威胁。 此外，病毒感染，如流感，经常伴随着细菌感染，这往往是一个致命的组合。由于耐药性的发展，治疗感染的选择大大减少。这种耐药性在很大程度上是由于细菌表达降解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对临床可用的头孢他啶抗生素的耐药性的分子基础，以及β-内酰胺酶中的某些突变如何赋予对这种抗生素的耐药性，以及其他突变如何赋予对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部的反应中间体，并为实现我们的特定目标提供独特的优势：特定目标1：为了验证SHV-1抗性变体在位置M69处引起活性位点变化，其影响涉及3种抑制剂的中间体形成的水平和稳定性，从而允许它们克服3种抑制剂的抗性。抑制作用具体目标二：验证超广谱β-内酰胺酶SHV-2变体（G238 S）和SHV-5变体（G238 S & E240 K）通过拓宽活性位点并与第三代头孢菌素头孢他啶形成新型相互作用而进化为水解该化合物的假设。具体目标3：为了检验以下假设：SHV-6、-8和-24中存在的D179位的变化已通过移动Ω环而进化为水解头孢他啶，从而延伸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。

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.