How do you build a parasite?

如何构建寄生虫？

• 批准号: 6869951
• 负责人: JOHN M. MURRAY
• 金额: $ 37.8万
• 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/6869951
• 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）：弓形虫是一种普遍存在的病原体，估计感染三分之一的美国人口和全世界 10-90% 的个人（取决于国家/地区、各种社会学和行为因素等）。这种寄生虫在多种细胞类型中进行细胞内复制，并且可以以潜伏（组织）囊肿的形式持续存在多年。除了弓形虫之外，原生动物门顶复门还包括许多其他具有临床和/或兽医重要性的寄生虫。尽管这些生物体引起的疾病在本质上差异很大（例如，将疟疾与弓形虫病或球虫病进行比较），但所有顶复门寄生虫的致病影响最终都可归因于增殖，这使得了解寄生虫复制成为一个重要目标。所有顶复门都通过独特的过程进行复制，其中多个子体在母细胞内同时组装（称为“分裂生殖”）。该申请提出以分子术语表征作为子体组装启动焦点的细胞骨架细胞器的结构和组成。我们使用弓形虫进行这些研究，因为 (1) 刚地弓形虫通常一次仅形成两种寄生虫，这使得对复制形态的研究比疟原虫或艾美耳球虫物种更容易处理，以及 (2) 广泛的细胞生物学和现在可用于弓形虫的分子遗传学工具。特别是，荧光蛋白报告基因现在几乎可以在活寄生虫中可视化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（甚至对于致死转基因）。成像技术允许使用定量延时视频显微镜和图像去卷积、激光扫描共焦显微镜、荧光光漂白和恢复以及激光烧蚀来分析各种亚细胞细胞器之间随时间的关系。分子遗传学方法允许通过随机或靶向方法对基本上任何寄生虫基因进行突变，并识别引起的病变。我们的目标是确定需要哪些蛋白质、每种蛋白质的功能以及它们如何在 3D 中排列以提供构建寄生虫的支架。