LOOP DELETION MUTAGENESIS OF FEPA

描述(改编自申请人的摘要)：调查人员将 细菌铁--肠杆菌素转运机制的研究 螯合剂(铁载体，通过其外膜(GM)受体蛋白， 费帕。研究计划将对Fepa表面进行标定和表征 通过评估它们的缺失对两个生化指标的影响 FEPA生理学的不同阶段：铁载体与外部的结合 GM的表面，并通过GM内化到周质。 他们将分析FepA功能的以下几个方面：1.特异性 通透性：三价铁结合和转运。2.非 比通透性：FepA通道对抗生素的可及性 和低聚糖。3.构象：抗体与结肠素的结合 到表面环，以及三聚体结构的维护。4.地面 大肠杆菌、沙门氏菌和沙门氏菌FepA蛋白之间的环交换 假单胞菌。这些实验将使用定点突变， 铁载体结合和运输研究，流式细胞术测量 表面表位、固相免疫分析、微生物测试和 电子自旋共振谱。 铁的获取对哺乳动物体内细菌的生长是必不可少的 主持人。调查人员将研究铁的功能。 来自大肠杆菌、沙门氏菌和沙门氏菌的肠杆菌素受体蛋白 假单胞菌。铁载体运输系统在 细菌的传染性，因为它们浓缩了铁而不 损害GM的阻挡特性，从而使 细菌病原体在哺乳动物宿主中的生存。建议数 研究将阐明铁通过管道运输的基本原理 革兰氏阴性杆菌，从而提高对感染性疾病的认识 细菌性疾病。 铁通过FepA转运是TonB依赖的。他们的 实验将识别Fepa的表面环，识别 铁载体，和/或协助它们通过全球机制。这个目标 对于任何依赖于TonB的受体蛋白在任何 有机体。由于革兰氏阴性细菌通过 至少在功能上相似的运输系统 对TonB的依赖，对FEPA转运机制的理解 通常与对抗细菌致病的努力有关。此外 通过Fepa获得的关于铁的肠肌蛋白通道的知识， 这项研究将为其他可比调查提供方法 细菌转基因蛋白质和其他生物体。

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.