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

项目总结 医院获得性肠球菌对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对万古霉素表现出耐药性，万古霉素已经 历史上作为这些病原体的最后手段的抗生素，给临床医生留下了几个可行的 治疗选择。在这些生物中，万古霉素耐药性总是涉及到获得 编码在移动DNA元件上的抗性操纵子。这些操纵子(它们最终源自 产生抗生素的土壤微生物)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，这些重塑酶的表达受两个- 组件系统，VANS/VanR，感知抗生素的存在并通过以下方式对该信号做出反应 增加抗性基因的转录。关于VANS是如何感受组氨酸激酶的，人们知之甚少 在这个双组分系统中，检测到万古霉素；实际上，基本细节仍然不清楚，包括 VANS是否直接结合万古霉素，或者它是否检测到某些下游后果 万古霉素作用。本提案重点介绍两种主要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。使用生化、生物物理和 微生物学方法，拟议的实验将解决潜在的分子机制 抗生素在这两类VRE中的识别。该提案建立在初步观察的基础上，揭示了 A和B耐药表型似乎依赖于根本不同的抗生素机制 感官。因此，一个目标将集中在来自B型肠球菌的VANS蛋白(VanSB)，它被证明是 直接与万古霉素结合。这种相互作用背后的细节将被阐明，以及 抗生素对VanSB酶活性的影响。第二个目标是Vansa，它没有 对抗生素有直接反应。相应地，将探索激活信号的精确同一性。一个 第三个目标将提供有关VANS和VanR蛋白的结构信息，以便提供信息和指导 功能上的努力。总体而言，拟议的工作将产生对 抗生素感应途径；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良好测试的药物的疗效。

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.