Siderophore transport mechanism via membrane receptors

说明(申请人提供)：铁是除乳杆菌和血链球菌以外的所有微生物所必需的重要元素。从几种病原体中获取铁的能力与它们的毒力有关。一些铁运输系统也自然地运输抗生素。因此，铁运输系统可以被阻断或被利用来输送抗生素，以控制病原体在宿主体内的生长。在铁缺乏的条件下，许多革兰氏阴性细菌，包括几种病原体，表达复杂的铁运输系统。这些系统包括： 1.一种被称为铁载体的铁螯合分子， 2.识别铁-铁载体复合体的特异性外膜受体蛋白， 3.周质结合蛋白， 4.依赖于ATP的ABC型转运体和 5.TonB-ExbB-ExbD复合体位于内膜。结合的铁载体络合物以能量依赖的方式通过外膜受体运输。这种能量可能是通过TonB复合体传递的，它利用细胞质膜电位。铁-铁载体复合体的结合已经得到了很好的表征，但结合的铁载体通过受体运输的机制却知之甚少。该项目的长期目标是在分子水平上了解铁的运输机制。这一知识可能有助于通过扰乱铁的运输来控制细菌感染，还可以设计通过这些系统输送的新型抗生素。将使用遗传学、生化和结晶学方法相结合的方法。这个项目的目的是利用大肠杆菌中的受体FepA和FhuA来识别特定参与运输过程而不是结合的氨基酸残基。这将通过以下方式完成： 1.通过19-受体铁载体蛋白的多序列比对确定保守区域。这些残基将首先在FepA中进行突变和分析，以确定结合正常和转运缺陷的突变体； 2.在FhuA中，相应的结合正常和转运缺陷的残基会发生突变； 3.将测试被认为参与TonB相互作用的残基通过甲醛交联与TonB物理相互作用的能力，并使用TonB菌株测试它们对TonB的依赖性； 4.对结合正常、转运有缺陷的突变蛋白，将规范纯化和结晶方法。

DESCRIPTION (provided by applicant): Iron is a vital element required by all microorganisms except Lactobacilli and Streptococcus sanguis. The ability to acquire iron in several pathogens has been related to their virulence. Some iron-transport systems also naturally transport antibiotics. Thus iron-transport systems could either be blocked or exploited to deliver antibiotics to control pathogens' growth inside the body of the host. Under iron-depleted conditions many gram-negative bacteria including several pathogens, express complex iron transport systems. These systems consist of: 1. An iron-chelating molecule called a siderophore, 2. A specific outer membrane receptor protein recognizing iron-siderophore complexes, 3. A periplasmic binding protein, 4. An ATP dependent ABC type transporter and 5. TonB-ExbB-ExbD complexes located in the inner membrane. The bound iron-siderophore complexes are transported through the outer membrane receptors in an energy dependent manner. The energy is presumably transduced by the TonB complex, which utilizes the cytoplasmic membrane potential. The binding of the iron-siderophore complex has been well characterized but the mechanism of the transport of the bound siderophore across the receptor is poorly understood. The long-term goal of this project is to understand the mechanism of iron transport at the molecular level. This knowledge could help to control bacterial infections by disrupting iron transport and also to design novel antibiotics to be delivered through these systems. A combination of genetic, biochemical and crystallographic methods will be used. The aim of this project is to identify the amino acid residues specifically involved in the transport process and not in binding using the receptors FepA and FhuA in Escherichia coli. This will be done by: 1. Identification of the conserved regions through multiple sequence alignment of the 19-receptor proteins transporting ferric siderophores. These residues will be mutated and analyzed first in FepA to identify mutants with normal binding and defective transport; 2. The corresponding residues showing normal binding and defective transport will be mutated in FhuA; 3. The residues believed to be involved in TonB interaction will be tested for their ability to physically interact with TonB by formaldehyde cross-linking and also for their TonB dependence using a TonB strain; 4. For the mutant proteins showing normal binding and defective transport, methods of purification and crystallization will be standardized.