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

基本信息

批准号：
8911847
负责人：
Erik T Yukl
金额：
$ 14.28万
依托单位：
NEW MEXICO STATE UNIVERSITY LAS CRUCES
依托单位国家：
美国
项目类别：
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/8911847
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 Inductively Coupled Plasma Mass Spectrometry 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 simulation solute stoichiometry uptake

项目摘要

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普遍存在已成为一个主要的健康问题，并刺激了对新抗生素靶点的寻找。一个特别有前途的目标是细菌 ATP 结合盒 (ABC) 转运蛋白超家族，它将 ATP 的水解与多种溶质跨细胞膜的转运结合起来。细菌 ABC 转运蛋白与高亲和力溶质结合蛋白 (SBP) 协同工作，该蛋白特异性结合底物并将其递送至转运蛋白。在肠沙门氏菌和肺炎链球菌等中，编码 ABC 转运蛋白和锌或锰特异性 SBP 的基因的破坏可显着减弱动物模型中的毒力，凸显这些系统作为有效的药物靶点。在许多细菌中，第四个未表征的基因位于 Zn 或 Mn ABC 转运系统的基因附近。据推测，这种蛋白质是金属最佳导入所必需的，可能通过充当 ABC 转运蛋白和溶质结合蛋白之间的金属伴侣或接头。该项目旨在表征牙周球菌中的 ABC 转运蛋白系统及其假定的辅助蛋白，作为人类病原体肺炎克雷伯菌和产气肠杆菌中高度同源系统的模型。这些微生物与广谱抗菌药物耐药性和潜在致命的医院感染的病原体有关。辅助蛋白 (Pden1598) 以及推定的溶质结合蛋白 (Pden1597) 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特性，并使用光谱测定或等温滴定量热法 (ITC) 确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，可以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)之类的光谱技术。 Pden1598、Pden1597 以及它们之间形成的任何稳定复合物的晶体结构将被解析，提供有关金属结合环境以及金属结合和转移机制的结构信息。最后，这两种蛋白质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细菌导入过渡金属的机制提供新的见解，并可能为合理设计抗生素等金属吸收抑制剂提供基础。