Biochemical and Functional Analysis of Trypanin

描述(申请人提供)：非洲锥虫(布氏锥虫)是一种原生动物寄生虫，在人类中引起一种称为非洲昏睡病的致命疾病，在动物中引起相关疾病。这些疾病具有破坏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。细胞运动被认为在疾病的发病机制和寄生虫的发展中起着关键作用，但以前还不可能直接研究这种关系。我们已经分离出一个锥虫突变体，它不能定向细胞运动，但令人惊讶的是，它没有瘫痪。该突变体缺乏一种名为“Trypanin”的蛋白质，该提议的广泛、长期目标是利用Trypanin(-)突变体和重组Trypanin蛋白质作为工具，以促进我们对布氏毛虫鞭毛细胞运动的机制和生物学的理解。在哺乳动物宿主和寄生虫发育以及采采蝇体内，也将使用胰酶(-)突变体来检测细胞运动在疾病发病机制中的作用。前两个具体目标是确定胰酶和胰酶相关蛋白(Trp1)的生化性质，并确定这些蛋白是否相互作用。Trypanin是布鲁氏毛滴虫细胞运动所需的独特鞭毛附着复合体的一部分，下一个具体目标是使用生物化学和遗传学相结合的方法来分离和鉴定该复合体的其他成分。最后，由于Trypanin(-)突变体具有活跃的鞭毛而不是瘫痪的，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失去胰酶是否改变了哺乳动物宿主的感染过程和/或阻止了采采蝇载体内寄生虫的发展。 布氏锥虫鞭毛附着复合体为研究细胞骨架-膜相互作用的基本方面提供了独特的机会。因此，除了提供与寄生虫病相关的信息外，这些研究有望与真核细胞生物学具有广泛的相关性。

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.