Antibiotic Recognition and Signaling in Vancomycin-Resistant Enterococci (VRE)

项目摘要 医院获得性肠球菌病原体抗生素耐药性的上升构成了严重的临床 威胁特别是，肠球菌现在极有可能显示出对万古霉素的耐药性， 在历史上，抗生素是这些病原体的最后手段， 治疗选择万古霉素耐药在这些生物体中总是涉及获得 在移动的DNA元件上编码的抗性操纵子。这些操纵子（最终来源于 产磷土壤微生物）编码重塑细菌细胞壁的酶，从而赋予 对抗生素的抗药性。重要的是，这些重塑酶的表达是由两个- 组分系统VanS/VanR，其感测抗生素的存在并通过以下方式响应该信号： 增加抗性基因的转录。很少有人知道VanS，传感器组氨酸激酶 这种双组分系统，检测万古霉素;事实上，基本细节仍然模糊，包括 VanS是否直接结合万古霉素，或者它是否检测到一些下游的结果， 万古霉素作用。本提案主要关注两种主要形式的万古霉素耐药肠球菌（VRE） 在患者中发现，即A型和B型耐药。结合使用生物化学、生物物理和 微生物的方法，拟议的实验将解决潜在的分子机制 这两种类型的VRE中的抗生素识别。该提案基于初步观察结果， A和B耐药表型似乎依赖于根本不同的抗生素机制， 感应因此，一个目标将集中于来自B型肠球菌（VanSB）的VanS蛋白，其显示为 直接与万古霉素结合。这种相互作用的细节将得到阐明， 抗生素对VanSB酶活性的影响。第二个目标是VanSA，它不 直接对抗生素产生反应。因此，将探测激活信号的精确身份。一 第三个目标将提供有关VanS和VanR蛋白的结构信息，以告知和指导 的功能性努力。总的来说，拟议的工作将产生基本的见解的运作， 免疫传感途径;为合理重新设计治疗方法提供潜在的切入点， 使它们能够逃避病原体的检测;并为抗生素佐剂的开发提供信息， 克服细菌监测系统，恢复万古霉素等经过充分测试的药物的疗效。

PROJECT SUMMARY The rise of antibiotic resistance in hospital-acquired enterococcal pathogens constitutes a serious clinical threat. In particular, enterococci are now extremely likely to display resistance to vancomycin, which has historically functioned as the antibiotic of last resort for these pathogens, leaving the clinician with few viable therapeutic options. Vancomycin resistance in these organisms invariably involves the acquisition of resistance operons encoded on mobile DNA elements. These operons (which are ultimately derived from antibiotic-producing soil microbes) encode enzymes that remodel the bacterial cell wall, and thereby confer resistance to the antibiotic. Importantly, expression of these remodeling enzymes is controlled by a two- component system, VanS/VanR, that senses the presence of the antibiotic and responds to this signal by increasing transcription of the resistance genes. Little is known about how VanS, the sensor histidine kinase of this two-component system, detects vancomycin; indeed, fundamental details remain obscure, including whether VanS directly binds vancomycin, or whether it instead detects some downstream consequence of vancomycin action. This proposal focuses on the two main forms of vancomycin-resistant enterococci (VRE) found in patients, namely A- and B-type resistance. Using a combination of biochemical, biophysical, and microbiological approaches, the proposed experiments will address the molecular mechanisms underlying antibiotic recognition in these two types of VRE. The proposal builds upon preliminary observations revealing that the A and B resistance phenotypes appear to rely on fundamentally different mechanisms of antibiotic sensing. Hence, one Aim will focus on the VanS protein from B-type enterococci (VanSB), which is shown to bind directly to vancomycin. The details underlying this interaction will be elucidated, as will the effects of antibiotic action upon the enzymatic activities of VanSB. A second Aim focuses on VanSA, which does not respond directly to the antibiotic. Accordingly, the precise identity of the activating signal will be probed. A third Aim will provide structural information about the VanS and VanR proteins, in order to inform and direct the functional efforts. Overall, the proposed work will yield fundamental insights into the functioning of an antibiotic-sensing pathway; provide potential entry points for the rational redesign of therapeutics that will enable them to evade detection by pathogens; and inform the development of antibiotic adjuvants that can overcome bacterial surveillance systems and restore efficacy to well-tested drugs such as vancomycin.