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：
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
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/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

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）：致病细菌中抗生素耐药性的患病率已成为主要的健康问题，并促使人们寻求新的抗生素靶标。一个特别有希望的目标是细菌ATP结合盒（ABC）转运蛋白的超家族，它们将ATP的水解与整个细胞膜的各种溶质的运输息息相关。细菌ABC转运蛋白与高亲和力溶质结合蛋白（SBP）结合起来，该蛋白（SBP）专门结合底物并将其传递给TE转运蛋白。在肠沙门氏菌和肺炎链球菌等中，编码ABC转运蛋白的基因和针对Zn或MN的SBP的破坏会大大减弱动物模型中的毒力，从而强调这些系统作为有效的药物靶标。在许多细菌中，第四个未表征的基因位于Zn或Mn ABC转运蛋白系统的基因附近。假设该蛋白是最佳进口金属所必需的，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适配器。该项目旨在将这种ABC转运蛋白系统及其在dentrificans中的假定辅助蛋白描述为人类病原体高度同源系统的模型kleblebsiella肺炎和肠杆菌的模型。这些生物与广谱抗菌素耐药性和潜在致命医院感染的病因有关。辅助蛋白（PDEN1598）以及推定的溶质结合蛋白（PDEN1597）的金属结合和转移特性将在体外表征。将使用ICP-MS评估结合金属的化学计量和相关金属的结合亲和力，将使用光谱测定法或通过等温滴定量热法（ITC）确定。蛋白质 - 蛋白质相互作用和金属转移蛋白质之间将通过温育全蛋白和apo蛋白进行评估，然后进行色谱分离和金属含量的测定。或者，可以使用光谱技术，例如扩展的X射线吸收细胞（EXAF）。 PDEN1598，PDEN1597及其之间形成的任何稳定复合物的晶体结构将得到解决，从而提供有关金属结合环境和金属结合和转移机制的结构信息。最后，两种蛋白质在体内的金属进口中的作用将通过在丹尼替夫人菌中产生敲除并评估Zn-和Mn限速条件下的细菌生长来确定。这种结构和功能信息将为过渡金属在细菌中进口的机理提供新的见解，并有可能为金属摄取抑制剂作为抗生素的合理设计提供基础。