Mitchondrial targeting by the vacuolating cytotoxin of Helicobacter Pylori

 描述(申请人提供)：本申请提出研究，以确定幽门螺杆菌空泡毒素(VacA)运输到宿主上皮细胞线粒体的机制，在那里该毒素导致线粒体损伤。VacA结合在宿主细胞表面，并内化到内溶酶体系统中，但这一提议中需要解决的一个主要知识空白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浆中检测到VacA，这表明这种与膜相互作用、形成孔的毒素没有通过从胞浆进口的内源蛋白所使用的现有途径被吸收到线粒体。相反，我们目前的模型预测VacA是通过囊泡运输从细胞表面运输到线粒体的。鉴于囊泡从细胞表面到线粒体的运输途径以前还没有被确定，本应用提出了评估含有VacA的囊泡(VCV)从早期内体样室动态地重新建模为运输有能力靶向线粒体的囊泡的假设的研究。在伊利诺伊大学香槟分校Steven Blanke博士的实验室工作，我将使用两个特定目标内提出的一组研究来检验这一假说的预测：1)在目标1中，建议进行研究以测试预测，如果VCV被动态重塑，VCV的蛋白质组将随着时间的变化而变化，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，我开发和优化了新的方法，用于磁隔离富含用铁磁性纳米颗粒标记的VacA的VCV。2)在目标2中，我将测试这样的预测：如果VCV逐渐富含细胞蛋白质 通过RNA干扰或基因缺失来敲除或敲除这些细胞蛋白，将导致VacA在线粒体上的定位减少或受阻，并导致VacA介导的线粒体功能中断。拟议研究的完成将解决我们对VacA细胞中毒的了解中的一个主要空白，VacA是H.Pylori的一个主要毒力因素，鉴于人类感染增加了患胃癌的风险，Hp是一种严重影响健康的病原体。这些研究还可能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类别的线粒体作用、形成孔洞的毒素有关。确定细菌毒素介导的宿主细胞调控的机制和重要性是开发阻断毒素活性的治疗方法的重要一步。

 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.