The Role of Protein Interactions in Microbial Copper/Silver Resistance

蛋白质相互作用在微生物铜/银抗性中的作用

• 批准号: 8051948
• 负责人: MEGAN M MCEVOY
• 金额: $ 7.63万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8051948
• 关键词: Affinity; Antibiotic Resistance; Antibiotics; Bacterial Proteins; Binding; Biochemical; Burn injury; Calorimetry; Cells; Complex; Copper; Development; Environment; Extracellular Space; Future; Goals; Gram-Negative Bacteria; Health; Heavy Metals; Homeostasis; Human; In Vitro; Measures; Membrane; Metals; Microbe; Modeling; Molecular; Organism; Orthologous Gene; Periplasmic Proteins; Play; Proteins; Protons; Public Health; Resistance; Rest; Role; Silver; Structure; System; Testing; Titrations; Ursidae Family; antimicrobial drug; antiporter; base; in vivo; metal complex; microbial; microorganism; multidrug transport; mutant; periplasm; promoter; protein protein interaction; research study; resistance mechanism; stoichiometry; trafficking; wound

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.

微生物中的抗生素抗性系统对人类健康构成威胁。金属等金属 铜在多种环境中用作抗菌剂，特别是烧伤的银 在细胞内金属水平上必须适当维护生物的生存。然而， 致病性和非致病微生物具有抗药性机制，可在 高环境金属水平。在革兰氏阴性细菌中 多药出口系统，将铜和银传输到细胞外空间。这些金属 转运蛋白不同于多饮用系统，因为它们具有位于 周期。 该提案的目标是表征该第四个周期性的结构和功能 了解其在金属稳态中的作用的组成部分。该组件，来自E的CUS系统中的CUSF。 大肠杆菌有望在其中发挥金属伴侣功能或金属依赖性调节功能 与其他外排配合物库斯巴的相互作用。 为了测试CUSF充当金属伴侣的假设，我们将确定CUSF是否 将金属转移到体外。为了确定CUSF是否发挥调节作用，我们将调查是否金属 体内从CUSF转移对其功能至关重要。 CUSF与CUSCBA的比例将被确定， 作为与络合物其余部分成比例的CUSF的过量，可能表明是金属伴侣的作用， 较低的化学计量法可能表明调节功能。另外，cusf的蛋白质 将识别体内相互作用。提出了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。 CUSF在 将确定金属结合的状态。最近已经获得了CUSB的晶体，我们预计 结构细节将即将到来。 CUSF/CUSB复合物和协调的结构信息 该复合物的金属将由NMR和EXAFS实验确定。 CUSF和 CUSB和选择的金属和蛋白质伴侣的突变体将通过等温滴定测量 量热法。 与金属基抗生素有关的微生物抗性有关的基本信息将从 提出的实验。了解微生物赋予抗生素的机制 抵抗将对未来的开发和广泛抗生素的使用对公共卫生产生影响。