How do you build a parasite?

描述(由申请人提供)：弓形虫是一种普遍存在的病原体，估计感染了三分之一的美国人口和全球10%-90%的个人(取决于国家、各种社会和行为因素等)。这种寄生虫在广泛的细胞类型中进行细胞内复制，并可以潜伏(组织)囊状形式持续多年。除了弓形虫，尖端原生动物门还包括许多其他具有临床和/或兽医重要性的寄生虫。尽管这些生物引起的疾病在性质上有很大的不同(例如，将疟疾与弓形虫病或球虫病进行比较)，但所有顶复合体寄生虫的致病影响最终归因于增殖，这使得了解寄生虫复制成为一个重要目标。所有的顶端复合体都通过一个独特的过程进行复制，在这个过程中，多个子代同时在母细胞内聚集(称为分裂生殖)。这项申请建议从分子方面表征细胞骨架细胞器的结构和组成，这些细胞器是启动子体组装的焦点。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，使得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)目前可用于弓形虫的广泛的细胞生物学和分子遗传学工具。特别是，荧光蛋白记者现在允许几乎所有已知的亚细胞结构在活体寄生虫中可视化，瞬时转基因的效率允许快速评估重组质粒功能(即使是致命的转基因)。成像技术可以利用定量时间推移视频显微镜和图像去卷积、激光扫描共聚焦显微镜、荧光光漂白和恢复以及激光消融来分析不同亚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有针对性的方法对基本上任何寄生虫基因进行突变，并识别负责的病变。我们的目标是确定需要什么蛋白质，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.