Intracellular trafficking of the mitochondrial targeting toxin VacA from Helicobacter pylori

幽门螺杆菌线粒体靶向毒素 VacA 的细胞内运输

基本信息

批准号：
8873801
负责人：
Steven R. Blanke
金额：
$ 18.93万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-15 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8873801
关键词：
Address Bacteria Bacterial Toxins Bacterial exotoxin Binding Biology Cell Death Cell Surface Proteins Cell Surface Receptors Cell membrane Cell surface Cells Cholera Toxin Chronic Clostridium difficile Complement Factor H Cytosol Cytotoxin Data Diphtheria Toxin Disease Effectiveness Employee Strikes Endoplasmic Reticulum Functional disorder Gastric ulcer Helicobacter pylori Human Infection Inner mitochondrial membrane Intervention Intoxication Ion Channel Ions Iron Knock-out Knowledge Magnetism Malignant Neoplasms Mediating Membrane Membrane Potentials Metabolic Methods Mitochondria Mitochondrial Proteins Modeling Modification Molecular Nature Outer Mitochondrial Membrane Paper Process Property Protein Import Proteins Proteome Salmonella typhimurium Staging Stomach Stomach Diseases Surface System Targeted Toxins Testing Time Toxin Vesicle Virulence Factors Work anthrax toxin base disorder risk enteropathogenic Escherichia coli insight killings knock-down longitudinal analysis mitochondrial dysfunction pathogen pathogenic bacteria public health relevance trafficking uptake

项目摘要

DESCRIPTION (provided by applicant): This exploratory R21 application proposes studies to identify and characterize the mechanism by which the vacuolating cytotoxin (VacA), an important virulence factor produced by the human gastric pathogen Helicobacter pylori, targets mitochondria within intoxicated host cells. VacA is a membrane-channel forming toxin, which subsequent to binding the surface of host cells, is taken up from the plasma membrane into VacA- containing vesicles (VCVs), and subsequently targets the inner membrane of mitochondria, resulting in metabolic dysfunction and cell death. Here, we propose studies that challenge canonical models of host cell intoxication by intracellular-acting bacterial toxins, whic generally require the translocation of enzymatic active fragments from the endolysosomal system or endoplasmic reticulum to the cytosol prior to toxin- mediated modification of intracellular targets. Rather, subsequent to internalization, we propose that VacA is transported within VCVs to mitochondria, where the toxin is then directly transferred to mitochondria. However, the identity of VCV proteins important for VacA targeting/trafficking to mitochondria is difficult to predict, due in part to the fact that until very recently, mitochondria were not generlly thought to receive protein cargo from the endolysosomal system and, as a consequence, almost nothing is known about intracellular trafficking compartments destined for mitochondria. To address this gap in knowledge, we will evaluate the hypothesis that VacA-targeting of mitochondria requires remodeling of VacA-containing vesicles (VCVs) from early endosomal-like compartments to vesicles that are functionally competent for targeting and fusion with mitochondria. Using magnetic-based separation methods to isolate iron-enriched VCVs from cells at different stages of intoxication for proteome analysis, we will identify changes in VCV-associated proteins, and, characterize their functional importance for VacA targeting of mitochondria and mitochondrial dysfunction. These studies will provide the first detailed characterization of the host cell machinery that is important for mitochondrial targeting, and, contribute to our understanding of the fundamental biology underlying the modulation of host cells by VacA.

描述（由申请人提供）：该探索性 R21 申请提出了研究，以鉴定和表征空泡细胞毒素（VacA）（人胃病原体幽门螺杆菌产生的重要毒力因子）针对中毒宿主细胞内的线粒体的机制。一种膜通道形成毒素，随后与宿主细胞表面结合，从质膜吸收到含有 VacA 的细胞中囊泡（VCV），随后靶向线粒体内膜，导致代谢功能障碍和细胞死亡。在这里，我们提出的研究挑战了细胞内作用的细菌毒素引起的宿主细胞中毒的典型模型，该模型通常需要酶活性的易位。在毒素介导的细胞内靶标修饰之前，VacA 片段从内溶酶体系统或内质网进入细胞质，相反，在内化之后，VacA 被转运。然而，对于 VacA 靶向/运输到线粒体很重要的 VCV 蛋白的身份很难预测，部分原因是直到最近，线粒体还不是普遍存在的。人们认为 VacA 是从内溶酶体系统接收蛋白质货物，因此，对于前往线粒体的细胞内运输隔室几乎一无所知。为了解决这一知识空白，我们将评估 VacA 靶向线粒体需要重塑的假设。使用基于磁性的分离方法从处于不同中毒阶段的细胞中分离出富铁的 VCV，以进行蛋白质组分析，我们将鉴定 VCV 相关蛋白的变化，并表征它们对 VacA 靶向线粒体和线粒体功能障碍的功能重要性。这些研究将提供宿主的第一个详细特征。细胞机制对于线粒体靶向非常重要，并且有助于我们了解 VacA 调节宿主细胞的基础生物学。