Intracellular trafficking of the mitochondrial targeting toxin VacA from Helicobacter pylori

 描述（由申请人提供）：本探索性R21申请提出了研究，以确定和表征空泡细胞毒素（VacA）（一种由人类胃病原体幽门螺杆菌产生的重要毒力因子）靶向中毒宿主细胞内线粒体的机制。VacA是一种膜通道形成毒素，其在结合宿主细胞表面后，从质膜被摄取到含VacA的囊泡（VCV）中，并随后靶向线粒体的内膜，导致代谢功能障碍和细胞死亡。在这里，我们提出了挑战宿主细胞中毒的典型模型的研究细胞内作用的细菌毒素，这通常需要易位酶活性片段从内溶酶体系统或内质网的胞质溶胶之前，毒素介导的修饰细胞内的目标。相反，在内化之后，我们提出VacA在VCV内转运到线粒体，然后毒素直接转移到线粒体。然而，对于VacA靶向/运输至线粒体重要的VCV蛋白的身份难以预测，部分原因是直到最近，线粒体通常不被认为从内溶酶体系统接收蛋白质货物，因此，几乎不知道用于线粒体的细胞内运输区室。为了解决这一知识差距，我们将评估这样的假设，即线粒体的VacA靶向需要将含有VacA的囊泡（VCV）从早期的内体样隔室重塑为功能上能够靶向并与线粒体融合的囊泡。使用基于磁性的分离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，我们将鉴定VCV相关蛋白的变化，并表征其对VacA靶向线粒体和线粒体功能障碍的功能重要性。这些研究将提供对线粒体靶向重要的宿主细胞机制的第一个详细表征，并有助于我们理解VacA调节宿主细胞的基本生物学。

 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.