Biochemical and Functional Analysis of Trypanin

描述（由申请人提供）：非洲锥虫（布鲁氏锥虫）是一种原生动物寄生虫，可导致一种致命疾病，即人类的非洲昏睡病和动物的相关疾病。这些疾病具有毁灭性的健康和经济后果。这些寄生虫通过采采蝇媒介的叮咬传播到哺乳动物宿主的血液中。细胞运动被怀疑在疾病发病和寄生虫发育中起关键作用，但以前不可能直接研究这种关系。我们已经分离出一种不能定向细胞运动的锥虫突变体，但令人惊讶的是，它并没有瘫痪。该突变体缺乏一种名为“锥虫蛋白”的蛋白质，本研究的长远目标是利用锥虫蛋白（-）突变体和重组锥虫蛋白作为工具，促进我们对布鲁氏T.鞭毛细胞运动的机制和生物学的理解。锥虫蛋白（-）突变体也将用于研究细胞运动在哺乳动物宿主和寄生虫发育以及采采蝇中疾病发病机制中的作用。前两个具体目标是确定锥虫蛋白和锥虫蛋白相关蛋白（TRP 1）的生化特性，并确定这些蛋白是否相互作用。锥虫氨酸是一种独特的鞭毛附着复合体的一部分，这种复合体是布鲁氏虾细胞运动所必需的，下一个具体目标是采用生化和遗传相结合的方法分离和鉴定这种复合体的其他成分。最后，由于锥虫蛋白（-）突变体有一个主动跳动的鞭毛，并且没有瘫痪，它们提供了一个独特的机会来专门研究定向细胞运动在哺乳动物和昆虫宿主中的作用。因此，第四个具体目标是确定锥虫蛋白的缺失是否会改变哺乳动物宿主的感染过程和/或阻止采采蝇媒介内的寄生虫发育。

DESCRIPTION (provided by the applicant): African trypanosomes (Trypanosoma brucei) are protozoan parasites that cause a fatal disease known as African sleeping sickness in humans and related diseases in animals. These diseases have devastating health and economic consequences. These parasites are transmitted to the bloodstream of a mammalian host by the bite of a tsetse fly vector. Cell motility is suspected to play a pivotal role in disease pathogenesis and parasite development, but it has not previously been possible to investigate this relationship directly. We have isolated a trypanosome mutant that is incapable of directional cell motility but, surprisingly, is not paralyzed. This mutant lacks a protein called "trypanin" and the broad, long-term objectives of this proposal are to exploit trypanin (-) mutants and recombinant trypanin proteins as tools to advance our understanding of the mechanism and biology of flagellum based cell motility in T. brucei. Trypanin (-) mutants will also be used to examine the role of cell motility in disease pathogenesis in the mammalian host and parasite development and within the tsetse fly. The first two specific aims are to determine the biochemical properties of trypanin and a trypanin-related protein (TRP 1) and to determine whether these proteins interact with one another. Trypanin is part of a unique flagellum attachment complex that is required for T. brucei cell motility and the next specific aim is to employ a combined biochemical and genetic approach to isolate and identify additional components of this complex. Finally, because trypanin (-) mutants have an actively beating flagellum and are not paralyzed, they provide a unique opportunity to specifically examine the role of directional cell motility in the mammalian and insect hosts. Thus, the fourth specific aim is to determine whether loss of trypanin alters the course of infection in the mammalian host and/or blocks parasite development within the tsetse fly vector. T. brucei flagellum attachment complexes offer unique opportunities to investigate fundamental aspects of cytoskeleton-membrane interactions. Thus, in addition to providing information relevant to parasitic disease, these studies are expected to have broad relevance to eukaryotic cell biology.