Characterization of Accessory Factors in Bacterial Transition Metal Import

描述(由申请人提供)：病原菌中抗生素耐药性的流行已成为一个主要的健康问题，并促使人们寻找新的抗生素靶标。一个特别有希望的目标是细菌三磷酸腺苷结合盒(ABC)转运蛋白超家族，它将三磷酸腺苷的水解与多种溶质的跨细胞膜运输联系起来。细菌ABC转运蛋白与高亲和力溶质结合蛋白(SBP)一起工作，SBP专门结合底物并将其输送到TE转运蛋白。在肠沙门氏菌和肺炎链球菌等中，编码ABC转运蛋白和针对锌或锰的SBPs的基因的破坏显著地降低了动物模型的毒力，突显了这些系统作为有效的药物靶点。在许多细菌中，第四个未鉴定的基因与锌或锰ABC转运蛋白系统的基因相邻。假设该蛋白质是最佳金属进口所必需的， 潜在地作为ABC转运蛋白和溶质结合蛋白之间的金属伴侣或接头。该项目旨在将这种ABC转运蛋白系统及其可能的辅助蛋白表征为人类病原体肺炎克雷伯氏菌和产气肠杆菌的高度同源系统的模型。这些细菌与广谱抗菌素耐药性和潜在致命医院感染的病原体有关。辅助蛋白(Pden1598)以及可能的溶质结合蛋白(Pden1597)将在体外对其金属结合和转移特性进行表征。结合金属的化学计量和特性将使用电感耦合等离子体质谱评估，相关金属的结合亲和力将使用光谱分析或等温滴定热法(ITC)确定。蛋白质-蛋白质相互作用与金属转移 将通过孵育全蛋白和载脂蛋白，然后进行层析分离和测定金属含量来评估蛋白质之间的相互作用。或者，可以使用诸如扩展X射线吸收精细结构(EXAFS)的光谱技术。将解决Pden1598、Pden1597和它们之间形成的任何稳定络合物的晶体结构，提供关于金属结合环境和金属结合和转移机制的结构信息。最后，这两种蛋白在体内金属输入中的作用将通过在脱氮假单胞菌中产生敲除基因并评估细菌在锌和锰限制条件下的生长来确定。这些结构和功能信息将为了解过渡金属在细菌中的输入机制提供新的见解，并可能为合理设计金属吸收抑制剂作为抗生素提供基础。

DESCRIPTION (provided by applicant): The prevalence of antibiotic resistance among pathogenic bacteria has become a major health concern and has spurred the search for novel antibiotic targets. A particularly promising target is the superfamily of bacterial ATP-binding cassette (ABC) transporters, which couple the hydrolysis of ATP to the transport of a wide variety of solutes across the cell membrane. Bacterial ABC transporters work in conjunction with a high affinity solute binding protein (SBP) that specifically binds substrate and delivers it to te transporter. In Salmonella enterica and Streptococcus pneumoniae among others, disruption of genes encoding ABC transporters and SBPs specific for Zn or Mn dramatically attenuates virulence in animal models, highlighting these systems as potent drug targets. In a number of bacteria, a fourth, uncharacterized gene is located adjacent to those of Zn or Mn ABC transporter systems. It is hypothesized that this protein is required for optimal import of metals, potentially by acting as a metal chaperone or adaptor between the ABC transporter and the solute binding protein. This project seeks to characterize such an ABC transporter system and its putative accessory protein in Paracoccus dentrificans as a model for highly homologous systems in the human pathogens Klebsiella pneumoniae and enterobacter aerogenes. These organisms are associated with broad-spectrum antimicrobial resistance and the causative agents of potentially deadly nosocomial infections. The accessory protein (Pden1598) as well as the putative solute binding protein (Pden1597) will be characterized in vitro for their metal binding and transfer properties. The stoichiometry and identity of bound metal will be assessed using ICP-MS and binding affinities for relevant metals will be determined using a spectroscopic assay or by isothermal titration calorimetry (ITC). Protein-protein interactions and metal transfer between proteins will be assessed through incubation of holo- and apo-proteins followed by chromatographic separation and determination of metal content. Alternatively, spectroscopic techniques such as extended X-ray absorption fine structure (EXAFS) may be used. Crystal structures of Pden1598, Pden1597 and any stable complex that forms between them will be solved, providing structural information on the metal binding environment and mechanisms of metal binding and transfer. Finally, the role of both proteins in metal import in vivo will be determined by generating knockouts in P. denitrificans and assessing bacterial growth in Zn- and Mn-limiting conditions. Such structural and functional information will yield new insight into the mechanisms of transition metal import in bacteria and potentially provide a basis for the rational design of metal uptake inhibitors as antibiotics.