LOOP DELETION MUTAGENESIS OF FEPA

FEPA 环缺失诱变

• 批准号: 2900868
• 负责人: PHILLIP E KLEBBA
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900868
• 关键词: Escherichia; Pseudomonas; Salmonella; antibiotics; bacterial proteins; bacterial toxins; cell sorting; colicines; conformation; electron spin resonance spectroscopy; epitope mapping; gene deletion mutation; immunologic assay /test; membrane permeability; membrane proteins; membrane transport proteins; mutant; oligosaccharides; pore forming protein; receptor binding; siderophores; site directed mutagenesis

DESCRIPTION (Adapted from applicant's abstract): The investigators will study the transport mechanism of ferric enterobactin, a bacterial iron chelator (siderophore, through its outer membrane (GM) receptor protein, FepA. The research program will demarcate and characterize FepA surface loops by assessing the effects of their deletion on two biochemical stages of FepA physiology: binding of the siderophore on the external surface of the GM, and internalization through the GM to the periplasm. They will assay the following aspects of FepA function: 1. Specific permeability: ferric enterobactin binding and transport. 2. Non- specific permeability: accessibility of the FepA channel to antibiotics and oligosaccharides. 3. Conformation: binding of antibodies and colicins to surface loops, and maintenance of trimeric structure. 4. Surface loop exchange among the FepA proteins of Escherichia, Salmonella, and Pseudomonas. These experiments will use site-directed mutagenesis, siderophore binding and transport studies, flow cytometric measurement of surface epitopes, solid phase immunoassays, microbiological tests, and electron spin resonance spectroscopy. Iron acquisition is essential to the growth of bacteria in mammalian hosts. The investigators will study the functions of the ferric enterobactin receptor proteins from Escherichia, Salmonella, and Pseudomonas. Siderophore transport systems play an important role in the infectivity of bacteria because they concentrate iron without compromising the barrier properties of the GM, thereby enabling the survival of bacterial pathogens in mammalian hosts. The proposed research will clarify basic principles of iron transport through the gram-negative GM, and thereby advance the understanding of infectious bacterial disease. Ferric enterobactin transport through FepA is TonB-dependent. Their experiments will identify surface loops of FepA that recognize siderophores, and/or facilitate their passage through the GM. This goal has not been accomplished for any TonB-dependent receptor protein in any organism. Since gram-negative bacterial pathogens obtain iron with transport systems that are functionally similar at least in their dependence on TonB, the understanding of the FepA transport mechanism is generally relevant to efforts against bacterial pathogenesis. Besides the knowledge gained about ferric enterobactin passage through FepA, this study will generate methods for comparable investigations of other bacterial GM proteins and other organisms.

描述（根据申请人的摘要改编）：调查人员将 研究铁肠霉素的运输机制，一种细菌铁 螯合剂（铁载体，通过其外膜（GM）受体蛋白， fepa。研究计划将划分和表征FEPA表面 通过评估其缺失对两种生化的影响循环 FEPA生理学的阶段：铁载体对外部的结合 GM的表面，并通过GM内部化至周质。 他们将测定FEPA函数的以下方面：1。 渗透性：铁肠霉素的结合和转运。 2。非 - 特定渗透性：FEPA通道对抗生素的可及性 和寡糖。 3。构象：抗体和结肠蛋白的结合 表面环和三聚体结构的维护。 4。表面 大Escherichia，沙门氏菌和 假单胞菌。这些实验将使用位置定向的诱变， 铁载体结合和运输研究，流式细胞仪测量 表面表位，固相免疫测定，微生物学测试和 电子自旋共振光谱。 铁的获取对于哺乳动物细菌的生长至关重要 主持人。研究人员将研究铁的功能 来自大肠菌，沙门氏菌和 假单胞菌。铁载体运输系统在 细菌的感染性，因为它们将铁浓缩而没有 损害通用汽车的屏障特性，从而使 哺乳动物宿主中细菌病原体的存活。提议 研究将阐明铁运输的基本原理 革兰氏阴性总经理，从而提高了对传染性的理解 细菌疾病。 铁通过FEPA的肠乳素转运是tonb依赖性的。他们的 实验将确定识别FEPA的表面环路 铁载体和/或促进他们通过GM的通过。这个目标 尚未针对任何TONB依赖性受体蛋白完成 生物。由于革兰氏阴性细菌病原体与 至少在其功能上相似的运输系统 对tonb的依赖，对FEPA传输机制的理解 通常与针对细菌发病机理的努力有关。除了 关于通过FEPA的肠乳素经验获得的知识， 这项研究将生成用于对其他研究的可比研究的方法 细菌GM蛋白和其他生物。