Receptor-triggered toxin delivery mediated by FhaB of Bordetella

项目摘要 丝状血凝素(FHA)是百日咳杆菌的重要毒力因子，是百日咳杆菌的致病因子。 百日咳，被认为是一种粘附素，促进细菌与宿主细胞的附着。FHA 是其前体FhaB的加工形式，并且它缺乏在FhaB中发现的大的C-末端区域。我们最近 结果使我们假设这个C-末端区域作为毒素/效应器递送装置发挥作用 调节哺乳动物宿主细胞的功能。该提案的目标是通过检查 FhaB与靶哺乳动物细胞结合前后的拓扑结构。为了实现这一目标，我们将利用新的 我们最近开发的技术。这些方法包括一种异源的功能性大肠杆菌FhaB 表达系统，将使我们能够评估FhaB依赖的结合和细胞毒性，以及 基于荧光染料的方法，允许跟踪细菌结合前后的工程FhaB蛋白 以哺乳动物细胞为靶标。我们还将标记假定的毒素结构域，以确定它们是否进入目标细胞，并且我们 将使用显微镜和生化方法追踪它们，以跟踪它们的定位和命运。因为 在波尔德氏菌中的其他因素。可能会导致哺乳动物细胞中毒，我们将补充我们的 利用野生型和工程化波尔德氏菌的正交法分析。在第二个目标中，我们将评估 FhaB片段对宿主细胞生理的影响，扩展了我们的初步研究，表明C- FhaB末端片段诱导人肺细胞变形和丧失活力。我们会表示 FhaB片段在多种病原体相关细胞中的表达及其对细胞生理和细胞功能的影响 生存能力。本方案所产生的信息对改进FHA具有直接的潜在应用价值 通过包括部分FhaB C-末端毒素递送系统来构成疫苗的组成部分。此外，FhaB 将是第一个可以将毒素输送到真核细胞的单一细菌蛋白的例子，这可能 有可能被利用于治疗应用。

Project Summary Filamentous hemagglutinin (FHA) is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and is thought to function as an adhesin that facilitates bacterial attachment to host cells. FHA is a processed form of its precursor, FhaB, and it lacks a large C-terminal region found in FhaB. Our recent results lead us to hypothesize that this C-terminal region functions as a toxin/effector delivery device to modulate mammalian host cell function. The goal of this proposal is to test this hypothesis by examining the topology of FhaB before and after binding to target mammalian cells. Towards this goal, we will leverage new technologies recently developed by us. These methods include a heterologous and functional E. coli FhaB expression system that will allow us to assess FhaB-dependent binding and cellular intoxication and a fluorescent dye-based method that allows tracking of engineered FhaB proteins before and after bacterial binding to target mammalian cells. We will also tag putative toxin domains to determine if they enter target cells, and we will track them using microscopy and biochemical methods to follow their localization and fate. Because additional factors in Bordetella spp. could contribute to mammalian cell intoxication, we will complement our analyses with orthogonal approaches using wild-type and engineered Bordetella. In a second aim, we will assess the effects of FhaB fragments on host cell physiology, extending our preliminary studies that indicate that C- terminal fragments of FhaB induce human lung cells to change shape and lose viability. We will express fragments of FhaB in multiple pathogen-relevant cell types and study their effects on cellular physiology and cell viability. The information generated in this proposal has direct potential application to improving the FHA component of the vaccine by including portions of the FhaB C-terminal toxin delivery system. Moreover, FhaB would be the first example of a single bacterial protein that can deliver toxins into eukaryotic cells, which might be potentially harnessed for therapeutic applications.