Biochemical and Functional Analysis of Trypanin

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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.