Intracellular trafficking of the mitochondrial targeting toxin VacA from Helicobacter pylori

 描述(由申请人提供)：这项探索性的R21申请提出了一些研究，以确定和表征空泡细胞毒素(VacA)的机制，空泡细胞毒素(VacA)是由人类胃部病原体幽门螺杆菌产生的一种重要毒力因子，它针对中毒宿主细胞内的线粒体。VacA是一种膜通道形成毒素，与宿主细胞表面结合后，从细胞膜被摄取到含有VacA的囊泡(VCV)中，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，我们提出了一些研究，挑战细胞内作用的细菌毒素中毒宿主细胞的典型模型，这些模型通常需要在毒素介导的细胞内靶标修饰之前，将酶活性片段从内溶体系统或内质网转移到胞浆。相反，在内化之后，我们认为VacA在VCV内运输到线粒体，然后毒素直接转移到线粒体。然而，Vcv蛋白对VacA靶向/运输到线粒体的重要性的识别很难预测，部分原因是直到最近，线粒体还没有被普遍认为从内溶酶体系统接收蛋白质货物，因此，几乎不知道前往线粒体的细胞内运输间隔。为了解决这一认识上的差距，我们将评估一种假设，即以VacA为靶向的线粒体需要包含VacA的囊泡(VCV)从早期的内体样室重塑为具有靶向功能并与线粒体融合的囊泡。利用基于磁分离的方法从中毒不同阶段的细胞中分离富含铁的VCV进行蛋白质组分析，我们将识别VCV相关蛋白的变化，并表征它们在VacA靶向线粒体和线粒体功能障碍中的功能重要性。这些研究将首次详细描述宿主细胞机制，这对线粒体靶向非常重要，并有助于我们理解VacA调控宿主细胞的基本生物学基础。

 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.