Characterization of Accessory Factors in Bacterial Transition Metal Import

细菌过渡金属输入中辅助因素的表征

• 批准号: 8742100
• 负责人: Erik T Yukl
• 金额: $ 12.21万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8742100
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Affinity; Animal Model; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Attenuated; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biological Assay; Biological Models; Biological Process; Calorimetry; Cell membrane; Code; Complex; Crystallization; Cytoplasm; Data; Docking; Drug Targeting; Enterobacter aerogenes bacterium; Environment; Genes; Genetic; Goals; Gram-Negative Bacteria; Growth; Health; Human; In Vitro; Individual; Infection; Klebsiella pneumonia bacterium; Knock-out; Lead; Membrane; Metal Binding Site; Metals; Methods; Modeling; Molecular Chaperones; Mus; Names; Nosocomial Infections; Nutrient; Organism; Paracoccus bacteria; Paracoccus denitrificans; Pathogenesis; Periplasmic Proteins; Phase; Phenotype; Plasmids; Pneumonia; Prevalence; Process; Production; Property; Proteins; Research; Roentgen Rays; Role; Salmonella enterica; Specificity; Streptococcus pneumoniae; Structure; Surface; System; Techniques; Testing; Titrations; Transition Elements; Virulence; Work; X-Ray Crystallography; absorption; base; design; improved; in vivo; inhibitor/antagonist; insight; novel; pathogen; pathogenic bacteria; periplasm; permease; protein complex; protein function; protein protein interaction; public health relevance; simulation; solute; stoichiometry; uptake

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.

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点。一个特别有前途的目标是细菌ATP结合盒（ABC）转运蛋白的超家族，它耦合的ATP水解的各种溶质跨细胞膜的运输。细菌ABC转运蛋白与特异性结合底物并将其递送至转运蛋白的高亲和力溶质结合蛋白（SBP）协同工作。在肠道沙门氏菌和肺炎链球菌等中，破坏编码ABC转运蛋白和锌或锰特异性SBPs的基因显著减弱了动物模型中的毒力，突出了这些系统作为有效的药物靶标。在一些细菌中，第四个，未表征的基因位于邻近的锌或锰ABC转运系统。据推测，这种蛋白质是金属的最佳输入所必需的， 潜在地通过充当ABC转运蛋白和溶质结合蛋白之间的金属伴侣或接头。该项目旨在表征这种ABC转运蛋白系统及其推定的辅助蛋白在Paracoccus dentrificans作为高度同源系统在人类病原体肺炎克雷伯氏菌和产气肠杆菌的模型。这些微生物与广谱抗菌素耐药性和潜在致命的医院感染的病原体有关。辅助蛋白（Pden 1598）以及推定的溶质结合蛋白（Pden 1597）将在体外表征其金属结合和转移特性。将使用ICP-MS评估结合金属的化学计量和同一性，并使用光谱测定法或等温滴定量热法（ITC）测定相关金属的结合亲和力。蛋白质-蛋白质相互作用和金属转移 通过孵育全蛋白和脱辅基蛋白，然后进行色谱分离和金属含量测定，对蛋白质之间的差异进行评估。或者，可以使用光谱技术，例如扩展X射线吸收精细结构（EXAFS）。Pden 1598、Pden 1597以及它们之间形成的任何稳定络合物的晶体结构将被解析，从而提供关于金属结合环境以及金属结合和转移机制的结构信息。最后，两种蛋白质在体内金属输入中的作用将通过在嗜热假单胞菌中产生敲除并评估Zn和Mn限制条件下的细菌生长来确定。这些结构和功能信息将产生新的见解过渡金属进口的机制在细菌和潜在的金属摄取抑制剂作为抗生素的合理设计提供了基础。