Mechanisms of Copper and Silver Resistance in Microbial Systems

描述（由申请人提供）：金属被广泛用于抑制微生物种群的生长。特别是，银通常在医院环境中作为一种有效的广谱杀菌剂，对人体毒性低。然而，蛋白质系统的存在提高了微生物在高金属浓度条件下的存活率，并降低了金属作为广谱杀菌剂的功效。其中一个系统，大肠杆菌的Cus系统，负责感知和响应环境中银(I)和铜(I)水平的升高。尽管铜和银在公共卫生方面具有重要意义，但cu系统处理铜和银的机制尚不清楚。我们提出，对铜和银处理的机制理解将为未来的药物开发提供一个目标，并有助于理解金属基杀菌剂的局限性。在我们之前的工作中，我们专注于使用Cus系统作为大型微生物金属抗性系统的模型系统。我们对跨膜金属外排系统（CusCFBA）的两种组分CusF和CusB进行了生物化学和结构表征，CusCFBA能有效地从细胞中去除金属。此外，我们还开发了一种新颖而强大的技术来监测蛋白质之间的金属转移。这项先前工作的自然发展是将这些发现与系统中的其他蛋白质结合起来。在目标1中，我们将研究cu系统的金属运输机制，并验证cu系统对周质解毒主要重要的假设，通过金属从cu周质蛋白（CusB和CusF）转移到膜结合的cu蛋白（CusA和CusC）。在目标2中，我们将验证CusS被金属结合到周围质域激活的假设，导致构象变化，最终导致Cu(I)/Ag(I)抗性系统的激活。在这些研究的结论，我们将有一个详细的了解微生物对金属污染环境的反击。我们将确定一种特定的金属是如何通过感官和反应系统与其他金属区分的，运输过程的机制，以及运输发生后的进一步解毒策略。这些研究将为我们了解微生物对金属杀菌剂的反应提供工具，并为有效使用金属杀菌剂控制人类细菌感染提供策略。

DESCRIPTION (provided by applicant): Metals are in widespread use to inhibit the growth of microbial populations. In particular, silver is commonly used in hospital settings as an effective broad spectrum biocide that has low toxicity to humans. However, protein systems are in place which enhance survival of microbes under conditions of high metal concentrations and reduce the efficacy of metals as broad spectrum biocides. One of these systems, the Cus system of E. coli, is responsible for sensing and responding to elevated levels of silver(I) and copper(I) in th environment. Despite its public health importance, the mechanisms of copper and silver handling by the Cus system are not well understood. We propose that mechanistic understandings of copper and silver handling will provide a target for future development of drugs and aid in understanding the limitations of metal-based biocides. In our previous work, we have focused on using the Cus system as a model system for a large class of microbial metal resistance systems. We have biochemically and structurally characterized two of the components, CusF and CusB, of the membrane spanning metal efflux system (CusCFBA) that actively removes metals from the cell. Additionally we have developed a novel and powerful technique to monitor metal transfer between proteins. The natural development of this previous work is to integrate these findings with the other proteins in the system. In aim 1 we will investigate the mechanism of metal transport by the Cus system and test the hypothesis that the Cus system is primarily important for detoxification of the periplasm, through metal transfer from the Cus periplasmic proteins (CusB and CusF) to the membrane-bound Cus proteins (CusA and CusC). In aim 2, we will test the hypothesis that CusS is activated by metal binding to the periplasmic domain, leading to conformational changes that ultimately result in activation of Cu(I)/Ag(I) resistance systems.. At the conclusion of these studies, we will have a detailed understanding of the microbial counterattack to metal contaminated environments. We will have determined how a specific metal is discriminated from other metals by a sensory and response system, the mechanisms of the transport process, and the further detoxification strategy after transport has occurred. These studies will give us the tools to understand the microbial response to metal biocides and provide strategies for their effective use in controlling bacterial infections in human populations.