Molecular mechanism of cephalosporin resistance of N. gonorrhoeae conferred by mutated PBP2

项目摘要 淋病奈瑟氏菌是性传播感染淋病的病原体 每年在美国造成超过80万例感染，在全球范围内造成7800万例感染。未经治疗或 无法治愈的感染可导致不孕不育、女性盆腔炎、淋球菌性关节炎 在两性中，感染和传播艾滋病毒的风险都增加了。在过去几十年里， 这种生物体对多种抗生素的抗药性的不可避免地增加已经严重地限制了 淋球菌感染的治疗选择。最令人担忧的是，对扩展频谱的抵制 头孢菌素(ESC)头孢曲松对公众健康构成严重威胁。这种情况需要一个 在分子水平上了解抗生素耐药性，以便能够设计新的抗菌药。 淋球菌对ESC的耐药性是由青霉素结合蛋白2(PBP2)的突变形式引起的。 在这一应用中，我们建议阐明抗性的分子机制，最重要的是 假设PBP2的突变限制了蛋白质的分子动力学。它建立在我们最近的基础上 理解野生型PBP2与ESCs结合时的相互作用及其构象 在ESCR菌株的PBP2中，与结合和酰化相关的变化似乎受到限制。这个 研究内容包括三个方面：一是对野生型PBP2进行结构功能分析 研究当PBP2结合和酰化时形成的特定相互作用的重要性 头孢菌素类。在特定的目标2中，我们将阐明来自ESCR毒株的PBP2中的关键突变是如何存在的 淋球菌减少头孢菌素的失活，同时保留足够的生物学功能来支持 有机体的生长。最后，特定目标3将检查PBP2变体在解决方案中的行为 确定突变是否会阻碍蛋白质的动力学。通过揭示分子机制如何 PbP2的突变克服了β-内酰胺类药物的致死作用，这些研究将使新的策略成为可能 替代抗淋病药物的开发。

Project Summary Neisseria gonorrhoeae, the causative agent for the sexually transmitted infection gonorrhea, is responsible for over 800,000 infections annually in the U.S. and 78 million cases worldwide. Untreated or untreatable infections can lead to infertility, pelvic inflammatory disease (PID) in females, gonococcal arthritis in both sexes, and an increased risk of both contracting and transmitting HIV. Over the past several decades, the inexorable increase of resistance in this organism toward multiple classes of antibiotics has severely limited treatment options for gonococcal infections. Most alarmingly, resistance against the extended-spectrum cephalosporin (ESC) ceftriaxone poses a serious threat to public health. This situation requires an understanding of antibiotic resistance at the molecular level in order to enable design of new antimicrobials. ESC resistance of N. gonorrhoeae is conferred by mutated forms of penicillin-binding protein 2 (PBP2). In this application, we propose to elucidate the molecular mechanism of resistance, with the overarching hypothesis that mutations in PBP2 restrict the molecular dynamics of the protein. It builds upon our recent understanding of the interactions made by wild-type PBP2 when bound by ESCs and how conformational changes associated with binding and acylation appear restricted in PBP2 derived from ESCR strains. The investigation comprises three aims: Specific Aim 1 is a structure-function analysis of wild-type PBP2 to investigate the importance of specific interactions formed when PBP2 is bound and acylated by cephalosporins. In Specific Aim 2, we will elucidate how key mutations present in PBP2 from ESCR strains of N. gonorrhoeae reduce inactivation by cephalosporins while retaining sufficient biological function to support growth of the organism. Finally, Specific Aim 3 will examine the behavior of PBP2 variants in solution to determine whether mutations hinder protein dynamics. By revealing the molecular mechanisms of how mutations in PBP2 overcome the lethal action of β-lactams, these investigations will enable new strategies for the development of replacement anti-gonococcal agents.