Receptor-triggered toxin delivery mediated by FhaB of Bordetella

博德特氏菌 FhaB 介导的受体触发毒素递送

基本信息

批准号：
10308546
负责人：
Diego Acosta-Alvear
金额：
$ 22.27万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-11-27 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10308546
关键词：
Acellular Vaccines Affect Analysis of Variance Animals Architecture Bacteria Bacterial Adhesins Bacterial Attachment Site Bacterial Proteins Binding Biochemical Biochemistry Biogenesis Bioinformatics Bordetella Bordetella Virulence Factors Bordetella bronchiseptica Bordetella pertussis C-terminal Cell Surface Proteins Cell Survival Cell physiology Cell surface Cells Cellular Morphology Complement Data Devices Dyes Engineering Ensure Escherichia coli Eukaryotic Cell Fluorescent Dyes Foundations Future Genetic Goals Growth Hemagglutinin Human Immune response Immune system Intoxication Knowledge Label Laboratories Lead Lung Mammalian Cell Maps Measures Mediating Membrane Methods Microscopy Modeling Molecular Morphology Pathogenesis Pathway interactions Pertussis Physiological Physiology Play Process Proteins Proteolytic Processing Publishing Reproducibility Role Shapes Students Surface System Targeted Toxins Testing Therapeutic Toxic effect Toxin Vaccines Work airway epithelium alpha Toxin antimicrobial base cell type design experimental study improved medical schools mutant new technology pathogen pathogenic bacteria prevent receptor

项目摘要

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.

项目概要丝状血凝素 (FHA) 是百日咳博德特氏菌的重要毒力因子，百日咳博德特氏菌是百日咳博德特氏菌的病原体。百日咳，被认为具有促进细菌附着到宿主细胞的粘附素的功能。联邦住房管理局是其前体 FhaB 的加工形式，并且缺少 FhaB 中的大 C 末端区域。我们最近的结果使我们假设该 C 末端区域充当毒素/效应物传递装置调节哺乳动物宿主细胞功能。该提案的目标是通过检查 FhaB 与目标哺乳动物细胞结合之前和之后的拓扑结构。为了实现这一目标，我们将利用新的我们最近开发的技术。这些方法包括异源和功能性大肠杆菌 FhaB 表达系统将使我们能够评估 FhaB 依赖性结合和细胞中毒以及基于荧光染料的方法，可追踪细菌结合前后的工程 FhaB 蛋白以哺乳动物细胞为目标。我们还将标记假定的毒素结构域以确定它们是否进入靶细胞，并且我们将使用显微镜和生化方法追踪它们，以追踪它们的定位和命运。因为博德特氏菌属中的其他因素。可能会导致哺乳动物细胞中毒，我们将补充我们的使用野生型和工程化博德特氏菌采用正交方法进行分析。在第二个目标中，我们将评估 FhaB 片段对宿主细胞生理学的影响，扩展了我们的初步研究表明 C- FhaB 的末端片段会诱导人肺细胞改变形状并失去活力。我们将表达多种病原体相关细胞类型中的 FhaB 片段，并研究它们对细胞生理和细胞的影响生存能力。本提案中生成的信息可直接用于改善联邦住房管理局 (FHA) 通过包含 FhaB C 端毒素递送系统的部分，成为疫苗的组成部分。此外，FhaB 这将是第一个可以将毒素输送到真核细胞中的单一细菌蛋白的例子，这可能可能被用于治疗应用。