The de Novo Synthesis of GDP-fucose Is Essential for Flagellar Adhesion and Cell Growth in Trypanosoma brucei*
- Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD15EH, United Kingdom
- 1 To whom correspondence should be addressed: MSI/WTB complex, Dow St., Dundee DD15EH, United Kingdom. Tel.: 44-1382-384219; Fax: 44-1382-348896; E-mail: m.a.j.ferguson{at}dundee.ac.uk.
Abstract
The protozoan parasite Trypanosoma brucei causes human African sleeping sickness in sub-Saharan Africa. The parasite makes several essential glycoproteins, which has led to the investigation of the sugar nucleotides and glycosyltransferases required to synthesize these structures. Fucose is a common sugar in glycoconjugates from many organisms; however, the sugar nucleotide donor GDP-fucose was only recently detected in T. brucei, and the importance of fucose metabolism in this organism is not known. In this paper, we identified the genes encoding functional GDP-fucose biosynthesis enzymes in T. brucei and created conditional null mutants of TbGMD, the gene encoding the first enzyme in the pathway from GDP-mannose to GDP-fucose, in both bloodstream form and procyclic form parasites. Under nonpermissive conditions, both life cycle forms of the parasite became depleted in GDP-fucose and suffered growth arrest, demonstrating that fucose metabolism is essential to both life cycle stages. In procyclic form parasites, flagellar detachment from the cell body was also observed under nonpermissive conditions, suggesting that fucose plays a significant role in flagellar adhesion. Fluorescence microscopy of epitope-tagged TbGMD revealed that this enzyme is localized in glycosomes, despite the absence of PTS-1 or PTS-2 target sequences.
Footnotes
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↵2 The abbreviations used are: GMD, GDP-mannose 4,6-dehydratase; GMER, GDP-4-keto-6-deoxymannose epimerase reductase; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; LC-MS/MS, high performance liquid chromatography tandem mass spectrometry; MOPS, 3-(n-morpholino)propanesulfonic acid; GST, glutathione S-transferase; ORF, open reading frame; HA, hemagglutinin; UTR, untranslated region; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRITC, tetramethylrhodamine isothiocyanate.
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↵* This work was supported by a Wellcome Trust studentship (to D. C. T.), a Marie Curie postdoctoral fellowship (to L. I.), and Wellcome Trust Programme Grant 071463 (to M. A. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received June 8, 2007.
- Revision received July 19, 2007.
