Abstract
The Trypanosoma brucei flagellum is a multifunctional organelle with critical roles in motility, cellular morphogenesis, and cell division. Although motility is thought to be important throughout the trypanosome lifecycle, most studies of flagellum structure and function have been restricted to the procyclic lifecycle stage, and our knowledge of the bloodstream form flagellum is limited. We have previously shown that trypanin functions as part of a flagellar dynein regulatory system that transmits regulatory signals from the central pair apparatus and radial spokes to axonemal dyneins. Here we investigate the requirement for this dynein regulatory system in bloodstream form trypanosomes. We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes, in a pattern identical to that seen in procyclic cells. Surprisingly, trypanin RNA interference is lethal in the bloodstream form. These knockdown mutants fail to initiate cytokinesis, but undergo multiple rounds of organelle replication, accumulating multiple flagella, nuclei, kinetoplasts, mitochondria, and flagellum attachment zone structures. These findings suggest that normal flagellar beat is essential in bloodstream form trypanosomes and underscore the emerging concept that there is a dichotomy between trypanosome lifecycle stages with respect to factors that contribute to cell division and cell morphogenesis. This is the first time that a defined dynein regulatory complex has been shown to be essential in any organism and implicates the dynein regulatory complex and other enzymatic regulators of flagellar motility as candidate drug targets for the treatment of African sleeping sickness.
Highlights
African trypanosomes, e.g. Trypanosoma brucei and related subspecies, are protozoan parasites that cause African trypanosomiasis in humans and animals
T. brucei is the causative agent of human African trypanosomiasis, a fatal disease that is commonly referred to as ‘‘African sleeping sickness.’’ In the last three decades there has been a dramatic rise in the incidence of human sleeping sickness and it is estimated that 500,000 new infections occur annually [1,2]
We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes with a pattern that is indistinguishable from that seen in procyclic cells
Summary
E.g. Trypanosoma brucei and related subspecies, are protozoan parasites that cause African trypanosomiasis in humans and animals. T. brucei is transmitted via a tsetse fly vector and alternates between bloodstream form and insect form (procyclic) lifecycle stages, which are adapted to survive in mammalian hosts and tsetse flies, respectively. These parasites are highly motile in both lifecycle stages and motility is suspected to be important for parasite development in the tsetse fly, as well as pathogenesis in the mammalian host [3]. Motility in trypanosomes is mediated by a single flagellum that emerges from the flagellar pocket at the posterior end of the cell. Recent work using RNA interference (RNAi) in procyclic cells has demonstrated functional conservation for a few axonemal proteins and confirmed that both the axoneme and PFR are critical for normal cell motility [9,10,11,12]
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