How do you build a parasite?

如何构建寄生虫？

• 批准号: 6999369
• 负责人: JOHN M. MURRAY
• 金额: $ 37.41万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-15 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6999369
• 关键词: Toxoplasma gondii; confocal scanning microscopy; cryoelectron microscopy; fluorescence microscopy; gene targeting; green fluorescent proteins; intracellular parasitism; microorganism reproduction; protein protein interaction; protein structure function; protozoal genetics; tubulin; video microscopy

DESCRIPTION (provided by the applicant): Toxoplasma gondii is a ubiquitous pathogen infecting an estimated one-third of the U.S. population and 10-90% of individuals worldwide (depending on the country, various sociological and behavioral factors, etc). This parasite replicates intracellularly in a wide range of cell types and can persist for years in latent (tissue) cyst form. In addition to Toxoplasma, the protozoan phylum Apicomplexa includes many other parasites of clinical and/or veterinary importance. Although the diseases caused by these organisms differ greatly in nature (compare malaria, for example, with toxoplasmosis, or coccidiosis), the pathogenic impact of all apicomplexan parasites is ultimately attributable to proliferation, which makes understanding parasite replication an important goal. All Apicomplexans replicate by a distinctive process in which multiple daughters assemble simultaneously within the mother cell (termed 'schizogony'). This application proposes to characterize in molecular terms the structure and composition of the cytoskeletal organelles that serve as the focal point for initiation of daughter assembly. We use Toxoplasma for these studies because (1) T. gondii normally forms only two parasites at a time, making studies on the morphology of replication much more tractable than in Plasmodium or Eimeria species, and (2) a wide range of cell biological and molecular genetic tools are now available for T. gondii. In particular, fluorescent protein reporters now permit virtually all known subcellular structures to be visualized in living parasites, and the efficiency of transient transfection permits rapid assessment of recombinant plasmid function (even for lethal transgenes). Imaging techniques permit the analysis of relationships between various subcellular organelles over time, using quantitative time-lapse video microscopy and image de-convolution, laser scanning confocal microscopy, fluorescence photo-bleaching and recovery, and laser ablation. Molecular genetic approaches permit the mutation of essentially any parasite gene by either random or targeted methods, and identification of the lesions responsible. We aim to determine what proteins are needed, the function of each protein, and how they are arranged in 3D to provide the scaffold for building a parasite.

描述（由申请人提供）：弓形虫Gondii是一种无处不在的病原体，感染了估计的美国人口的三分之一，全球10-90％的人（取决于国家，各种社会学和行为因素等）。该寄生虫在各种细胞类型中重复细胞内复制，并且可以以潜在（组织）囊肿形式持续多年。除弓形虫外，原生动物门神经膜还包括许多其他临床和/或兽医重要性的寄生虫。尽管这些生物引起的疾病在自然界中（例如，将疟疾与弓形虫病或球虫病进行比较），但所有Apicomplexan寄生虫的致病影响最终归因于增殖，这使得理解寄生虫复制成为重要的目标。所有apicomplexans都通过一个独特的过程复制，其中多个女儿在母细胞中同时组装（称为“精神分裂症”）。该应用建议用分子角度表征细胞骨架细胞器的结构和组成，这些细胞器是启动子组装的焦点的焦点。我们在这些研究中使用弓形虫，因为（1）T。gondii通常一次仅形成两个寄生虫，使对复制的形态进行研究比疟原虫或eimeria属物种更容易触犯，并且（2）现在可用于T. gondii。特别是，荧光蛋白报道现在允许在活寄生虫中可视化几乎所有已知的亚细胞结构，并且瞬时转染的效率允许快速评估重组质粒功能（即使对于致命转基因）。成像技术允许使用定量的延时视频显微镜和图像去卷积，激光扫描共聚焦显微镜，荧光照相和恢复以及激光消融来分析各种亚细胞细胞器之间的关系。分子遗传学方法允许通过随机或靶向方法基本上任何寄生虫基因的突变，并鉴定出负责的病变。我们旨在确定需要哪种蛋白质，每种蛋白质的功能以及如何在3D中排列以提供建造寄生虫的支架。