How do you build a parasite?

描述（由申请人提供）：刚地弓形虫是一种普遍存在的病原体，感染了大约三分之一的美国人口和全世界10-90%的个体（取决于国家、各种社会和行为因素等）。这种寄生虫在细胞内以多种细胞类型复制，并能以潜伏（组织）囊肿形式持续数年。除弓形虫外，顶复虫门的原生动物还包括许多其他具有临床和/或兽医重要性的寄生虫。虽然这些生物引起的疾病在性质上差别很大（例如，将疟疾与弓形虫病或球虫病进行比较），但所有顶复体寄生虫的致病作用最终可归因于增殖，这使得了解寄生虫的复制成为一个重要目标。所有顶复合体都通过一个独特的过程进行复制，在这个过程中，多个子细胞同时在母细胞内组装（称为“分裂”）。本应用程序提出了表征在分子方面的结构和组成的细胞骨架细胞器，作为启动子组装的焦点。我们使用弓形虫进行这些研究是因为：(1)弓形虫通常一次只形成两种寄生虫，这使得研究弓形虫的复制形态比在疟原虫或艾美耳虫物种中更容易处理；(2)现在有广泛的细胞生物学和分子遗传学工具可用于弓形虫。特别是，荧光蛋白报告器现在允许几乎所有已知的亚细胞结构在活的寄生虫中可视化，并且瞬时转染的效率允许快速评估重组质粒的功能（即使是致命的转基因）。成像技术允许分析各种亚细胞细胞器之间随时间的关系，使用定量延时视频显微镜和图像反卷积，激光扫描共聚焦显微镜，荧光光漂白和恢复，以及激光消融。分子遗传学方法允许通过随机或有针对性的方法对基本上任何寄生虫基因进行突变，并确定负责的病变。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。

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.