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微生物剂，特别是在烧伤伤口中用作银 因为细胞内的金属水平必须适当地维持生物体的生存。然而，在这方面， 致病性和非致病性微生物都具有抵抗机制， 环境金属含量高。在革兰氏阴性菌中，质子底物反向转运系统，类似于 多药物输出系统将铜和银转运到细胞外空间。这些金属 转运蛋白与多药系统的不同之处在于它们具有第四组分，该组分位于 周质 这个提议的目标是描述第四个周质的结构和功能 了解其在金属稳态中的作用。该成分为E. 大肠杆菌，预计在其代谢过程中发挥金属伴侣功能或金属依赖性调节功能。 与外排复合物CusCBA的其余部分相互作用。 为了检验CusF作为金属伴侣的假设，我们将确定CusF是否 在体外将金属转移到CusB。为了确定CusF是否发挥调节作用，我们将研究金属是否 在体内从CusF转移对其功能至关重要。将确定CusF与CusCBA的比例， 由于与复合物的其余部分成比例的过量的CusF可指示作为金属伴侣的作用， 而较低的化学计量可以指示调节功能。此外，与CusF结合的蛋白质 将鉴定体内相互作用。结构和生物化学实验提出了确定 CusF和CusB的金属和蛋白质-蛋白质相互作用的原子水平细节。CusF的结构 将确定金属结合状态。最近已经获得了CusB的晶体，我们预计， 结构细节即将公布。CusF/CusB复合物的结构信息和配位 通过NMR和EXAFS实验确定了配合物对金属的吸附量。CusF和 将通过等温滴定法测量CusB和金属和蛋白质伴侣的选定突变体 量热法 有关微生物对金属基抗生素耐药性的基本信息将从 提出的实验。了解微生物赋予抗生素的机制 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.