LOOP DELETION MUTAGENESIS OF FEPA

描述（改编自申请人摘要）：研究者将 研究铁肠杆菌素的转运机制 螯合剂（铁载体，通过其外膜（GM）受体蛋白， FepA。研究计划将标定和表征FepA表面 通过评估其缺失对两个生化指标的影响， FepA生理学的阶段：铁载体在外部 GM的表面，并通过GM内化到周质。 他们将分析FepA功能的以下方面：1。具体 通透性：铁肠杆菌素结合和转运。2.非 比渗透性：FepA通道对抗生素的可及性 和低聚糖。3.构象：抗体和大肠杆菌素的结合 到表面环，以及三聚体结构的维持。4.表面 大肠杆菌、沙门氏菌和 假单胞菌这些实验将使用定点诱变， 铁载体结合和转运研究，流式细胞术测量 表面表位、固相免疫测定、微生物检测， 电子自旋共振光谱学 铁的获取对哺乳动物体内细菌的生长至关重要 hosts.研究人员将研究铁的功能， 肠杆菌素受体蛋白来自埃希氏菌属、沙门氏菌属和 假单胞菌铁载体运输系统在生物体内起着重要的作用。 细菌的传染性，因为它们浓缩铁， 损害GM的阻隔性能，从而使 细菌病原体在哺乳动物宿主中的存活。拟议 研究将阐明铁运输的基本原则， 革兰氏阴性转基因，从而推进对感染性 细菌性疾病 铁肠杆菌素通过FepA的转运依赖于TonB。他们的 实验将识别FepA的表面环， 铁载体，和/或促进其通过GM。这一目标 尚未完成任何TonB依赖性受体蛋白在任何 有机体由于革兰氏阴性细菌病原体获得铁与 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。除了 关于铁肠杆菌素通过FepA的知识， 这项研究将为其他类似的研究提供方法。 细菌转基因蛋白和其他生物。

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.