Biochemical and Functional Analysis of Trypanin

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ABSTRACT African trypanosomes (e.g. Trypanosoma brucei) are protozoan parasites that cause African sleeping sickness, a fatal disease with devastating health and economic impact. These parasites are digenetic organisms, spending part of their life cycle in a mammalian host and part in an insect vector (the tsetse fly). Trypanosomes are highly motile in both life cycle stages and motility is central to parasite development and disease pathogenesis. Motility is mediated by a single flagellum that is an essential and multifunctional organelle with critical roles in cell motility, host-parasite interaction, cell morphogenesis and cell division. Surprisingly, we know very little about the flagellar apparatus at the molecular level. In particular, we lack an understanding of how flagellar proteins are assembled into supramolecular structures within the axoneme and how they function individually and collectively to drive cell motility and other flagellum functions. The long-term goal of the proposed research is to advance our understanding of the trypanosome flagellum and to exploit trypanosomes as a model to investigate the eukaryotic cilium. This will be done using a combination of functional and structural approaches. RNAi, site-directed mutagenesis and ultrastructural analyses will be used to investigate the function of trypanin and other components of the dynein regulatory complex (DRC). The DRC is part of a signal transduction pathway that regulates flagellar motility and is essential in bloodstream-form trypanosomes, making it a candidate drug target. The trypanosome flagellum is analogous to cilia and flagella in other eukaryotes, including humans. Flagella are required for motility of several human pathogens and are present on most tissues of the human body. They perform motility, transport and sensory functions. Infectious diseases caused by pathogens that require cilia include African sleeping sickness and Malaria. Together, these diseases are responsible for mortality and morbidity in approximately 0.4 billion people world-wide. Heritable human diseases caused by cilia defects include: hydrocephalus, infertility, epilepsy, left-right axis defects, eye disorders, polycystic kidney disease and obesity. Therefore, in addition to addressing fundamental questions in cell biology, this research directly impacts efforts to understand and treat infectious diseases and genetic diseases in humans.