The Role of Protein Interactions in Microbial Copper/Silver Resistance

描述(申请人提供)：微生物中的抗生素耐药系统对人类健康构成威胁。银和铜等金属在各种环境中用作抗菌剂，特别是在烧伤创面治疗中用作银，因为必须适当地保持细胞内金属水平才能使生物体存活。然而，致病微生物和非致病微生物都有在高环境金属水平条件下生存的抗性机制。在革兰氏阴性细菌中，质子-底物逆向转运体系统，类似于多种药物出口系统，将铜和银输送到细胞外空间。这些金属转运体与多药系统的不同之处在于，它们有第四种成分，位于周质中。这项建议的目的是表征这第四个周质成分的结构和功能，以了解其在金属动态平衡中的作用。这个成分，CusF在CUS系统中来自大肠杆菌，在它与外排复合体的其余部分CusCBA的相互作用中，预计将发挥金属配位蛋白功能或金属依赖的调节功能。为了验证CusF作为金属配位体功能的假设，我们将在体外确定CusF是否将金属转移到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。CusF与CusCBA的比例将被确定，因为CusF与复合体其余部分的比例过剩可能表明其作为金属配位伴侣的作用，而较低的化学计量比可能表明调节功能。此外，还将确定CusF在体内与之相互作用的蛋白质。通过结构和生物化学实验确定了CusF和CusB的金属和蛋白质相互作用的原子级细节。CusF在金属结合态的结构将被确定。最近已经获得了CusB的晶体，我们预计结构细节将会公布。CusF/CusB络合物的结构信息以及该络合物对金属的配位作用将通过核磁共振和EXAFS实验来确定。CusF和CusB的亲和力以及选定的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测量。有关微生物对金属抗生素耐药性的基本信息将从拟议的实验中获得。在未来广谱抗生素的开发和使用中，了解微生物产生抗生素耐药性的机制将对公众健康产生影响。

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.