Mechanisms of Copper and Silver Resistance in Microbial Systems

描述（由申请人提供）：金属广泛用于抑制微生物种群的生长。特别地，银通常在医院环境中用作对人类具有低毒性的有效广谱杀生物剂。然而，蛋白质系统在高金属浓度条件下增强微生物的存活并降低金属作为广谱杀生物剂的功效。其中一个系统是E.大肠杆菌中的一种蛋白质，负责对环境中高水平的银（I）和铜（I）进行感知和响应。尽管其对公共卫生的重要性，但Cus系统处理铜和银的机制尚未得到很好的理解。我们建议，铜和银处理的机制的理解将提供一个目标，为未来的药物开发，并帮助了解金属基杀生物剂的局限性。在我们以前的工作中，我们一直专注于使用Cus系统作为一个大类的微生物金属抗性系统的模型系统。我们的生物化学和结构特征的两个组件，CusF和CusB，跨膜金属外排系统（CusCFBA），积极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相结合。在目标1中，我们将研究Cus系统的金属转运机制，并检验Cus系统对周质的解毒至关重要的假设，即通过金属从Cus周质蛋白（CusB和CusF）转移到膜结合的Cus蛋白（CusA和CusC）。在目标2中，我们将测试CusS通过金属结合到周质结构域而被激活的假设，导致最终导致Cu（I）/Ag（I）抗性系统激活的构象变化。在这些研究的结论，我们将有一个详细的了解微生物反击金属污染的环境。我们将确定一种特定的金属是如何通过感觉和反应系统，运输过程的机制，以及运输发生后的进一步解毒策略与其他金属区分开来的。这些研究将为我们提供工具，以了解微生物对金属杀虫剂的反应，并提供有效使用金属杀虫剂控制人类细菌感染的策略。

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.