The de Novo Synthesis of GDP-fucose Is Essential for Flagellar Adhesion and Cell Growth in Trypanosoma brucei

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. 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. Typanosomatid parasites are protozoan parasites that impose a major health burden on many countries in the developing world, causing a wide range of diseases over three continents. Trypanosoma brucei is the causative agent of African sleeping sickness in humans and nagana in cattle and is spread between mammalian hosts by the bite of the tsetse fly (Glossina sp.). T. brucei transmission occurs when the bloodstream form of the parasite is ingested by a tsetse fly during feeding; the parasite then differentiates into the procyclic form in order to colonize the tsetse fly mid gut. The parasite undergoes further differentiation and migration to the salivary gland of the fly in order to infect a new mammalian host upon a subsequent blood meal. Research in several laboratories has focused on the glycobiology of these organisms to uncover potential targets for therapeutic intervention (1Ferguson M.A. Proc. Natl. Acad. Sci. U. S. 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Parasitol. 2006; 147: 126-136Crossref PubMed Scopus (50) Google Scholar). Sugar nucleotides are activated forms of sugars that are used as the ultimate source of sugar for the majority of glycosylation reactions. Sugar nucleotides are formed in two main ways: by a salvage pathway involving “activation” of the sugar using a kinase and a pryophosphorylase or by a “de novo” pathway involving the bioconversion of an existing sugar/sugar nucleotide. In most cases, sugar nucleotides are synthesized in the cytoplasm and then transported through specific transporters into the lumen of the Golgi apparatus and/or endoplasmic reticulum, where they are used by glycosyltransferases as donor substrates in glycosylation reactions (18Gerardy-Schahn R. Oelmann S. Bakker H. Biochimie (Paris). 2001; 83: 775-782Crossref PubMed Scopus (86) Google Scholar, 19Berninsone P.M. Hirschberg C.B. Curr. Opin. Struct. Biol. 2000; 10: 542-547Crossref PubMed Scopus (115) Google Scholar). 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Parasitol. 1999; 99: PubMed Scopus Google we that of and and and be using this to the conditional null in bloodstream form T. of the of the conditional null is shown in from was by using a TbGMD which a of TbGMD and of the in both life cycle of from the conditional for in of TbGMD in both the bloodstream form and procyclic form as by that we a conditional null and to the TbGMD by the in the absence of in the absence of that was of a that was in reactions of TbGMD in of the of TbGMD on sugar nucleotide we sugar nucleotides several the of from the conditional null In the bloodstream in the of to of GDP-fucose and for in to of GDP-fucose and In the procyclic form of the growth in the of in a in the of GDP-fucose when and that of growth required GDP-fucose to of in the procyclic form TbGMD conditional null In both life cycle a was detected in upon we that this is the that was observed in the in TbGMD nucleotide of bloodstream and procyclic form TbGMD conditional null mutants under and nonpermissive form form in a new of TbGMD the of T. potential growth in conditional null cell under nonpermissive conditions, we in the and absence of and growth by The bloodstream form mutants the for and on the growth and to These growth and to the as the from that these the TbGMD not of from of gene is a common of conditional null mutants of essential genes in bloodstream form T. brucei T. 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