Mitchondrial targeting by the vacuolating cytotoxin of Helicobacter Pylori

 描述（由申请人提供）：本申请提出了研究，以确定幽门螺杆菌空泡细胞毒素（VacA）被运输至宿主上皮细胞中的线粒体的机制，其中毒素诱导线粒体损伤。VacA结合到宿主细胞的表面，并被内化到内溶酶体系统中，但在该提案中要解决的知识的主要差距是毒素靶向和定位于线粒体的机制。我们和其他人未能在宿主细胞胞质溶胶中检测到VacA，这表明这种膜相互作用，孔形成毒素不被线粒体利用从胞质溶胶输入的内源性蛋白质所使用的现有途径。相反，我们目前的模型预测，VacA是从细胞表面运输到线粒体的囊泡运输。考虑到先前尚未鉴定出从细胞表面到线粒体的囊泡运输途径，本申请提出了研究以评估以下假设：含VacA的囊泡（VCV）从早期内体样区室动态地重新建模为能够靶向线粒体的运输囊泡。在伊利诺伊大学厄巴纳-香槟分校史蒂文·布兰克博士的实验室工作，我将使用两个特定目标中提出的一组研究来测试这一假设的预测：1）在目标1中，提出了研究来测试预测，如果VCV被动态重构，VCV的蛋白质组将作为时间的函数而改变，以变得富含介导线粒体靶向和/或囊泡运输的蛋白质。为了支持所提出的研究，我开发并优化了磁性隔离富含VacA的VCV的新方法，这些VCV用铁磁纳米颗粒标记。2)在目标2中，我将测试这样的预测，即如果VCV逐渐富含细胞蛋白质， 如果线粒体靶向所需的蛋白质是VacA，则通过RNA干扰或基因缺失敲低或敲除这些细胞蛋白质将导致VacA定位于线粒体的减少或阻断，以及VacA介导的线粒体功能破坏。完成拟议的研究将解决一个主要的差距，我们的理解细胞中毒的VacA，这是一个主要的毒力因子H。幽门螺杆菌是一种引起人类严重健康问题的病原体，它会增加胃癌发生的风险。这些研究还将首次揭示蛋白质从宿主细胞表面移动到线粒体的细胞内运输机制，这可能与临床相关病原菌产生的更广泛的肠道作用，孔形成毒素有关。确定细菌毒素介导的宿主细胞调节的机制和重要性是开发用于阻断有助于发病机制的毒素活性的疗法的重要一步。

 DESCRIPTION (provided by applicant): This application proposes studies to identify the mechanism by which the Helicobacter pylori vacuolating cytotoxin (VacA) is trafficked to mitochondria in host epithelial cells, where the toxin induces mitochondrial damage. VacA binds to the surface of host cells and is internalized into the endolysosomal system, but a major gap in knowledge to be addressed in this proposal is the mechanism by which the toxin targets and localizes to mitochondria. We and others have failed to detect VacA within the host cell cytosol, suggesting that this membrane-interacting, pore-forming toxin is not taken up into mitochondria using existing pathways used by endogenous proteins that are imported from the cytosol. Rather, our current model predicts that VacA is transported from the cell surface to mitochondria by vesicular trafficking. Given that vesicular trafficking pathways from the cell-surface to mitochondria have not previously been identified, this application proposes studies to evaluate the hypothesis that VacA-containing vesicles (VCVs) are dynamically re-modeled from early- endosomal-like compartments to trafficking vesicles that are competent for targeting mitochondria. Working in the laboratory of Dr. Steven Blanke at the University of Illinois at Urbana-Champaign, I will test the prediction of this hypothesis using a set of studies proposed within 2 Specific Aims: 1) In Aim 1, studies are proposed to test the prediction that, if VCVs are dynamically remodeled, the proteome of VCVs will change as a function of time to become enriched with proteins that mediate mitochondrial targeting and/or vesicular trafficking. In support of the proposed studies, I have developed and optimized new approaches for magnetically isolating VCVs enriched in VacA labeled with ferromagnetic nano-particles. 2) In Aim 2, I will test the prediction that if VCVs become progressively enriched with cellular proteins required for mitochondrial targeting, then knockdown or knockout of these cellular proteins by RNA interference or gene deletion will result in decreased or blocked VacA localization to mitochondria, and, VacA-mediated disruption of mitochondrial function. Completion of the proposed studies will address a major gap in our understanding of cellular intoxication by VacA, which is a major virulence factor of H. pylori, a pathogen of significant health concern given human infection increases the risk for the development of gastric cancer. These studies will also reveal, potentially for the first time, an intracellular trafficking mechanism by which proteins move from the surface of host cells to the mitochondria, which may be relevant to the broader class of mitochondrial-acting, pore-forming toxins generated by clinically relevant pathogenic bacteria. Identifying the mechanisms and importance of bacterial toxin-mediated modulation of host cells is an important step towards developing therapies for blocking toxin activities that contribute to pathogenesis.