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: 7590317
• 负责人: MEGAN M MCEVOY
• 金额: $ 29.05万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7590317
• 关键词: Affinity; Antibiotic Resistance; Antibiotics; Bacterial Proteins; Binding; Biochemical; Burn injury; Calorimetry; Cells; Complex; Copper; Development; Environment; Escherichia coli; 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

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.

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。金属（例如银和铜）用作各种环境中的抗菌剂，尤其是用于烧伤伤口治疗中的银，因为必须适当地维护细胞内金属水平以进行生存。但是，病原和非致病微生物都具有在高环境金属水平的条件下生存的抗性机制。在革兰氏阴性细菌中，质子 - 基底抗植物系统，类似于多药出口系统，传输铜和银色到细胞外空间。这些金属转运蛋白不同于多药系统，因为它们具有位于周质中的第四个成分。该提案的目标是表征第四个周围成分的结构和功能，以了解其在金属稳态中的作用。该组件是来自大肠杆菌的CUS系统中的CUSF，预计将在其与外排配合物的其余部分CUSCBA相互作用中提供金属伴侣函数或金属依赖性调节函数。为了检验CUSF充当金属伴侣的假设，我们将确定CUSF是否会在体外转移金属转移到CUSB。为了确定CUSF是否发挥调节作用，我们将研究金属从CUSF在体内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，CUSF过量可能表明是金属伴侣的作用，而较低的化学计量法可能表明调节功能。另外，将鉴定出与体内相互作用的蛋白质。提出了结构和生化实验，以确定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中的CUSF的结构。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复合物的结构信息以及该复合物对金属的协调将由NMR和EXAFS实验确定。 CUSF和CUSB的亲和力以及金属和蛋白质伴侣的选定突变体将通过等温滴定量热法测量。与金属基抗生素有关的基本信息将从拟议的实验中获得。对微生物赋予抗生素耐药性的机制的理解将对未来的开发和使用广谱抗生素的使用产生影响。