Mitchondrial targeting by the vacuolating cytotoxin of Helicobacter Pylori

幽门螺杆菌空泡细胞毒素的线粒体靶向

• 批准号: 9263698
• 负责人: Robin L. Holland
• 金额: $ 3.8万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9263698
• 关键词: Address; Bacteria; Bacterial Toxins; Binding; Biochemical; Carrier Proteins; Cell physiology; Cell surface; Cells; Cellular biology; Complex; Cytosol; Cytotoxin; Development; Disease; Doctor of Philosophy; Endosomes; Epithelial Cells; Exotoxins; Experimental Designs; Fractionation; Gene Deletion; Genes; Health; Helicobacter pylori; Homeostasis; Human; Hybrids; Illinois; Infection; Innate Immune Response; Integration Host Factors; Intoxication; Knock-out; Knowledge; Label; Laboratories; Life; Magnetism; Mammalian Cell; Mediating; Membrane; Metabolic Control; Mitochondria; Mitochondrial Proteins; Modeling; Nuclear; Pathogenesis; Pathogenicity; Pathway interactions; Physiologic pulse; Physiological; Protein Import; Proteins; Proteome; Proteomics; RNA Interference; Reporting; Research; Risk; Stomach; Surface; System; Targeted Toxins; Testing; Time; Toxin; Transport Vesicles; Universities; Vesicle; Vesicle Transport Pathway; Viral; Virulence Factors; base; cell injury; clinically relevant; ferrite; ferromagnetism; insight; interest; knock-down; malignant stomach neoplasm; mitochondrial dysfunction; nanoparticle; novel strategies; novel therapeutics; pathogen; pathogenic bacteria; predictive modeling; protein transport; public health relevance; therapy development; trafficking; uptake

 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.

 描述（应用程序提供）：本应用提案研究以确定幽门螺杆菌吸尘细胞毒素（VACA）在宿主上皮细胞中被输送到线粒体的机制，毒素会诱导线粒体损伤。 VACA与宿主细胞的表面结合并内化为内溶性系统，但是该提案中要解决的知识的主要差距是毒素靶标并定位到线粒体的机制。我们和其他人未能检测到宿主细胞细胞质内的VACA，这表明这种膜相互作用，孔形成毒素不会使用从细胞质溶胶中进口的内源性蛋白使用的现有途径将其用于线粒体。相反，我们当前的模型预测VACA通过囊泡运输将VACA从细胞表面转移到线粒体。鉴于先前尚未确定从细胞表面到线粒体的囊泡运输途径，该应用建议研究以评估含Vaca的蔬菜（VCV）的假说，从早期 - 粘体样品隔间动态地重新建模为有能力用于目标靶向线粒体的运输蔬菜。我将在伊利诺伊大学乌尔巴纳 - 冠军分校的史蒂文·布兰克（Steven Blanke）博士的实验室工作，我将使用提出的一组研究中提出的一组研究来检验这一假设的预测：1）在目标1中提出的研究，提出了一项研究，提议测试以下预测，如果VCV会在vcv上进行动态的蛋白质变化，以至于vcv会变化，以使其成为vcv的蛋白质，以至于会变成一定的蛋白质。和/或囊泡贩运。为了支持拟议的研究，我开发了并优化了新的方法，用于将富含铁电磁纳米粒子标记的VACA的磁分离VCV。 2）在AIM 2中，我将测试以下预测：如果VCV逐渐富含细胞蛋白 线粒体靶向，然后通过RNA干扰或基因缺失对这些细胞蛋白的敲低或敲除，将导致VACA定位降低或阻断对线粒体的定位，而VACA介导的线粒体功能的破坏。拟议研究的完成将解决我们对VACA细胞中毒的理解的主要差距，Vaca是幽门螺杆菌的主要病毒因素，鉴于人类感染会增加胃癌发展的风险。这些研究还将首次揭示出一种细胞内运输机制，通过该机制，蛋白质从宿主细胞的表面转移到线粒体，这可能与临床相关的致病细菌产生的线粒体作用，孔形成的孔形成毒素。识别细菌毒素介导的宿主细胞调节的机制和重要性是开发疗法以阻断有助于发病机理的毒素活性的重要一步。