The Role of Protein Interactions in Microbial Copper/Silver Resistance

描述（由申请人提供）：微生物中的抗生素抗性系统对人类健康构成威胁。银和铜等金属在多种环境中用作抗菌剂，特别是烧伤伤口治疗中的银，因为必须适当维持细胞内金属水平才能保证生物体的生存。然而，致病微生物和非致病微生物都具有在高环境金属水平条件下生存的抵抗机制。在革兰氏阴性细菌中，质子底物反向转运系统与多药物输出系统类似，将铜和银转运到细胞外空间。这些金属转运蛋白与多药物系统的不同之处在于它们具有位于周质中的第四种成分。该提案的目标是表征第四种周质成分的结构和功能，以了解其在金属稳态中的作用。该成分（来自大肠杆菌的 Cus 系统中的 CusF）预计在与外排复合物的其余部分 CusCBA 相互作用时发挥金属伴侣功能或金属依赖性调节功能。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，我们将研究体内 CusF 的金属转移是否对其功能至关重要。将确定 CusF 与 CusCBA 的比例，因为与复合物的其余部分相比，过量的 CusF 可能表明其作为金属伴侣的作用，而较低的化学计量可能表明具有调节功能。此外，还将鉴定与 CusF 在体内相互作用的蛋白质。建议通过结构和生化实验来确定 CusF 和 CusB 的金属和蛋白质-蛋白质相互作用的原子水平细节。将确定金属结合状态下 CusF 的结构。 CusB 晶体最近已获得，我们预计结构细节即将公布。 CusF/CusB 配合物的结构信息以及该配合物与金属的配位将通过 NMR 和 EXAFS 实验确定。 CusF 和 CusB 以及所选突变体对金属和蛋白质伴侣的亲和力将通过等温滴定量热法进行测量。与微生物对金属抗生素耐药性相关的基本信息将从拟议的实验中获得。了解微生物赋予抗生素耐药性的机制将对未来广谱抗生素的开发和使用中的公众健康产生影响。

DESCRIPTION (provided by applicant): 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.