The Role of Protein Interactions in Microbial Copper/Silver Resistance

微生物中的抗生素耐药系统对人类健康构成威胁。金属，如银和 铜在各种环境中用作抗菌剂，尤其是烧伤创面中的银 治疗。因为为了有机体的生存，必须适当地维持细胞内的金属水平。然而， 致病和非致病微生物都有抵抗机制，可以在以下条件下生存 高环境金属水平。在革兰氏阴性细菌中，质子-底物逆向转运蛋白系统，类似于 多药物输出器系统将铜和银输送到细胞外空间。这些金属 转运蛋白与多药系统的不同之处在于，它们有第四个组成部分，位于 周质。 这项提议的目标是描述第四个周质的结构和功能 了解其在金属动态平衡中的作用。CUS系统中的这个组件，来自E. Coli中，被认为具有金属配位蛋白功能或金属依赖的调节功能 与外排复合体的其余部分相互作用，CusCBA。 为了检验CusF作为金属配位体的功能这一假设，我们将确定CusF 在体外将金属转移到CusB。为了确定CusF是否发挥了监管作用，我们将调查金属 CusF在体内的转移是其发挥作用的关键。CusF与CusCBA的比例将确定， 因为过量的CusF与络合物的其余部分成比例可以指示作为金属配位络合物的作用， 而较低的化学计量比可能表示一种调节功能。此外，CusF与之结合的蛋白质 活体内的相互作用将被确定。建议进行结构和生物化学实验来确定 CusF和CusB金属和蛋白质-蛋白质相互作用的原子水平细节。中的CusF结构 金属结合状态将被确定。最近已经获得了CuB的晶体，我们预计 结构细节将会公布。CusF/CusB复合体的结构信息及其配位 将通过核磁共振和EXAFS实验来确定该络合物对金属的影响。CusF和CusF的亲和力 CusB和选定的金属和蛋白质的突变体将通过等温滴定进行测量 量热法。 与微生物对金属类抗生素的耐药性有关的基本信息将从 拟议中的实验。对微生物产生抗生素的机制的理解 在未来广谱抗生素的开发和使用中，耐药性将对公众健康产生影响。

Antibiotic resistance systems in microorganisms pose a threat to human health. Metals such as silver and copper are used as antimicrobial agents in a variety of settings, notably for silver in burn wound treatment.since intracellular metals levels must be properly maintained for an organism's survival. However, both pathogenic and non-pathogenic microbes have resistance mechanisms to survive under conditions of high environmental metal levels. In gram-negative bacteria, proton-substrate antiporter systems, similar to the multidrug exporter systems, transport copper and silver to the extracellular space. These metal transporters differ from the multidrug systems in that they have a fourth component which is located in the periplasm. The goals of this proposal are to characterize the structure and function of this fourth periplasmic component to understand its role in metal homeostasis. This component, CusF in the Cus system from E. coli, is expected to serve a metallochaperone function or a metal-dependent regulatory function in its interactions with the rest of the efflux complex, CusCBA. To test the hypothesis that CusF functions as a metallochaperone, we will determine whether CusF transfers metal to CusB in vitro. To establish whether CusF play a regulatory role, we will investigate if metal transfer from CusF in vivo is crucial to its function. The proportions of CusF to CusCBA will be determined, as an excess of CusF in proportion to the rest of the complex may indicate a role as a metallochaperone, while a lower stoichiometry may indicate a regulatory function. Additionally, the proteins with which CusF interact in vivo will be identified. Structural and biochemical experiments are proposed to determine the atomic level details of metal and protein-protein interactions of CusF and CusB. The structure of CusF in the metal-bound state will be determined. Crystals of CusB have been recently obtained, and we anticipate that structural details will be forthcoming. Structural information for the CusF/CusB complex and the coordination of metal by this complex will be determined by NMR and EXAFS experiments. The affinities of CusF and CusB and selected mutants for metals and for the protein partner will be measured by isothermal titration calorimetry. Fundamental information pertinent to microbial resistance to metal-based antibiotics will be obtained from the proposed experiments. An understanding of the mechanisms by which microorganisms confer antibiotic resistance will have impact on public health in the future development and use of broad spectrum antibiotics.