Characterization of the ferrous iron transporter Feo

腹泻疾病霍乱造成巨大的健康、经济和政治负担。 全世界。霍乱的病原体霍乱弧菌是水生栖息地的地方病，但它 能够感染人类的小肠，引起大规模的、危及生命的腹泻 回应。霍乱弧菌要在宿主上定居并致病，必须获得必要的营养， 包括铁，在宿主环境中。铁的主要形式存在于小的 肠道是铁质的。因此，了解亚铁摄取的机制是至关重要的。 在霍乱弧菌中。霍乱弧菌主要的铁离子转运体是FeO。FEO系统是 广泛分布于所有细菌物种中，在致病力中具有重要作用 几种致病物种；然而，人们对其结构和机制知之甚少 交通工具。在霍乱弧菌中，FeO转运蛋白由三种蛋白组成，FeoA，FeoB， 和FeoC。FeoB的膜包埋的C-末端结构域可能形成孔 铁的转运，有趣的是，它的N-末端结构域与真核小G有同源性 蛋白质，表明了一种新的运输机制。FeoA和FeoC的作用是 未知。我们最近证明了这三种FeO蛋白结合在一起形成了更高的 体内有序复合体。这是第一次对成熟的膜进行结构分析- 体内嵌入的活性FeO络合物。这项提案的目标是确定总体上的 这个大型复合体的结构，以便建立和测试铁的机制模型 运输。在我们的第一个具体目标中，我们将确定原生生物的质量和化学计量比 Feo情结。这将为建立活性FeO转运体的结构模型奠定基础。 在目标2中，我们将通过描述 建筑群的成员。然后，我们将测试这些交互的功能重要性 复杂的形成和铁的运输活动。在目标3中，我们将确定能源的来源 通过Feo的运输。这些研究将极大地提高我们对 这一重要而独特的铁运输车的结构和功能。值得注意的是，我们所有的研究 将使用活性的、膜相关的FeO络合物在体内进行，给出我们的结果 这与目前主导FeO领域的体外研究有不可否认的相关性。作为我们的 前人的工作表明，霍乱弧菌是研究FeO的理想模式菌。我们有 已经组装了所需的大部分菌株和试剂，而我们和我们的合作者， 拥有进行拟议实验所需的专业知识。

The diarrheal disease cholera poses enormous health, economic, and political burdens worldwide. The causative agent of cholera, Vibrio cholerae, is endemic in aquatic habitats, but it is capable of infecting the human small intestine, causing a massive, life-threatening diarrheal response. To colonize the host and cause disease, V. cholerae must acquire essential nutrients, including iron, in the host environment. The predominant form of iron present in the small intestine is ferrous iron. Thus, it is critical to understand the mechanisms of ferrous iron uptake in V. cholerae. The major ferrous iron transporter in V. cholerae is Feo. The Feo system is widely distributed among all bacterial species, and has important functions in the virulence of several pathogenic species; nevertheless, very little is known about its structure and mechanism of transport. In V. cholerae, the Feo transporter is composed of three proteins, FeoA, FeoB, and FeoC. The membrane-embedded C-terminal domain of FeoB is likely to form the pore for iron transport, and, interestingly, its N-terminal domain has homology to small eukaryotic G proteins, suggesting a novel mechanism of transport. The roles of FeoA and FeoC are unknown. We recently demonstrated that the three Feo proteins associate to form a higher order complex in vivo. This represents the first structural analysis of the mature, membrane- embedded, active Feo complex in vivo. The goal of this proposal is to determine the overall structure of this large complex in order to build and test models for the mechanism of iron transport. In our first specific aim, we will determine the mass and stoichiometry of the native Feo complex. This will lay the groundwork for a structural model of the active Feo transporter. In aim 2, we will refine this model by delineating the sites of interaction within and between the members of the complex. We will then test the functional significance of these interactions for complex formation and iron transport activity. In aim 3, we will determine the source of energy for transport through Feo. These studies will significantly advance our knowledge of the structure and function of this important and unique iron transporter. Significantly, all our studies will be carried out using active, membrane-associated Feo complexes in vivo, giving our results an undeniable relevance over the in vitro studies that currently dominate the Feo field. As our previous work shows, V. cholerae is an ideal model organism for the study of Feo. We have already assembled most of the strains and reagents needed, and we, and our collaborators, have the expertise required to carry out the proposed experiments.