Mechanisms of Copper and Silver Resistance in Microbial Systems

描述(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，银通常用于医院环境中，作为一种对人类低毒的有效广谱杀菌剂。然而，蛋白质系统已经到位，可以提高微生物在高金属浓度条件下的生存能力，并降低金属作为广谱杀生剂的效力。其中一个系统，即大肠杆菌的CUS系统，负责感应和响应环境中银(I)和铜(I)水平的升高。尽管铜和银对公众健康具有重要意义，但铜和银由铜和银系统处理的机制尚不清楚。我们认为，对铜和银处理的机械理解将为未来药物的开发提供一个靶点，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系统作为一大类微生物金属电阻系统的模型系统。我们对跨膜金属外排系统(CusCFBA)的两个成分CusF和CusB进行了生化和结构表征，CusCFBA可以主动地从细胞中去除金属。此外，我们还开发了一种新的、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CUS系统的金属运输机制，并通过将金属从CUS周质蛋白(CusB和CusF)转移到膜结合的CUS蛋白(CusA和CusC)来检验CUS系统对周质解毒起主要作用的假设。在目标2中，我们将验证这样的假设，即金属与周质结构域的结合激活了cuss，导致构象变化，最终导致铜(I)/银(I)抗性系统的激活。在这些研究的结论中，我们将对微生物对金属污染环境的反击有一个详细的了解。我们将确定一种特定的金属如何通过感官和反应系统与其他金属区分开来，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，并为有效利用它们控制人类种群中的细菌感染提供策略。

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.