Zn(II) metallochaperones in E. coli

大肠杆菌中的 Zn(II) 金属伴侣

• 批准号: 7230202
• 负责人: MICHAEL W CROWDER
• 金额: $ 16.99万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230202
• 关键词: Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biological Assay; Bioterrorism; Categories; Cell Line; Cells; Class; Clinical; Complex; DNA Microarray Chip; DNA Microarray format; Escherichia coli; Gel; Goals; Ions; Knock-out; Membrane; Metals; Preparation; Protein Binding; Proteins; Ribosomes; Stress; Structure; Thinking; Transcript; Western Blotting; design; gel electrophoresis; mutant; novel; polyclonal antibody; porin; research study; trafficking

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand Zn(ll) trafficking in cells. The main goal of this study is to identify Zn(ll)-metallochaperones in E. coli involved specifically with Zn(ll) import and transport. Specifically, we propose to identify the putative, soluble Zn(ll)-metallochaperones that are thought to deliver Zn(ll) from outer membrane-bound channels/porins to the ribosomes and/or proteins. We propose to utilize the following strategy to achieve our goals. DNA microarrays were used to identify five transcripts (of Zn(ll)-metallochaperone candidates) that were up-regulated in E. coli cells stressed with Zn(ll) deficiency. (1) These five candidate proteins will be over-expressed, purified, and characterized for structure and Zn(ll) binding. (2) All candidates that bind Zn(ll) will be used in pulldown experiments in an effort to identify any proteins that form complexes with the candidates. In addition, E. coli cells lacking the Zn(ll) binding candidates (knockout lines) will be obtained. (3) All proteins identified in the pulldown experiments will be over-expressed, purified, and characterized for Zn(ll) binding and structure. Polyclonal antibodies against these proteins will be generated. (4) Wild-type and mutant E. coli cells will be cultured in the presence of 65Zn, and the resulting soluble proteins will be subjected to native gel electrophoresis. The gels will be analyzed for differential amounts of 65Zn, and western blots of the gels will be analyzed using the aforementioned polyclonal antibodies. The knockout lines will also be assayed for Zn(ll) transport by using a recently developed assay in the lab. Since Zn(ll) is an essential metal ion for bacteria and metallochaperones deliver metal ions to specific proteins, we believe that understanding Zn(ll)-transport in E. coli will present novel protein targets for the design and preparation of a new class of antibiotics. The need for new classes of antibiotics is particularly important currently to combat the clinical crisis of antibiotic resistance and to combat the Category A and B bacterial strains on the CDC's bioterrorism watch list.

描述（由申请人提供）：该项目的长期目标是了解细胞中的 Zn(II) 运输。本研究的主要目标是鉴定大肠杆菌中专门参与 Zn(II) 输入和运输的 Zn(II)-金属伴侣。具体来说，我们建议鉴定假定的可溶性 Zn(II)-金属伴侣，其被认为将 Zn(II) 从外膜结合通道/孔蛋白递送至核糖体和/或蛋白质。我们建议利用以下策略来实现我们的目标。 DNA 微阵列用于鉴定在 Zn(II) 缺乏胁迫的大肠杆菌细胞中上调的 5 个转录本（Zn(II)-金属伴侣候选物）。 (1) 这五种候选蛋白将被过表达、纯化并表征其结构和 Zn(II) 结合。 (2) 所有结合 Zn(II) 的候选蛋白都将用于下拉实验，以鉴定与候选蛋白形成复合物的任何蛋白质。此外，将获得缺乏 Zn(II) 结合候选物（敲除系）的大肠杆菌细胞。 (3) 在下拉实验中鉴定的所有蛋白质都将被过表达、纯化，并表征 Zn(II) 结合和结构。将产生针对这些蛋白质的多克隆抗体。 (4)将野生型和突变型大肠杆菌细胞在65Zn存在下培养，所得可溶性蛋白将进行非变性凝胶电泳。将分析凝胶中 65Zn 的差异量，并使用上述多克隆抗体分析凝胶的蛋白质印迹。还将使用实验室最近开发的检测方法对敲除株系的 Zn(II) 转运进行检测。由于 Zn(II) 是细菌必需的金属离子，并且金属伴侣将金属离子传递给特定蛋白质，因此我们相信，了解大肠杆菌中 Zn(II) 的转运将为设计和制备新型抗生素提供新的蛋白质靶标。目前，为了应对抗生素耐药性的临床危机以及对抗 CDC 生物恐怖主义观察名单上的 A 类和 B 类细菌菌株，对新型抗生素的需求尤为重要。

项目成果

Absence of ZnuABC-mediated zinc uptake affects virulence-associated phenotypes of uropathogenic Escherichia coli CFT073 under Zn(II)-depleted conditions.

• DOI: 10.1111/j.1574-6968.2009.01762.x
• 发表时间: 2009-11
• 期刊: FEMS microbiology letters
• 影响因子: 2.1
• 作者: Gunasekera TS;Herre AH;Crowder MW
• 通讯作者: Crowder MW