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.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 (1-5). These targets include enzymes of glycosylphosphatidylinositol biosynthesis (1, 6 -9) and enzymes of sugar nucleotide biosynthesis. For example, GDP-mannose biosynthesis has been shown to be essential for the infectivity of Leishmania mexicana (10 -13) and UDPglucose 4Ј-epimerase, the only source of UDP-galactose in T.

Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

“…This sugar has been found previously in gp72 of T. cruzi (8,12), a homologue of the T. brucei Fla-1 and Fla-2 flagellar adhesion zone glycoproteins (17, 31), but has not been previously reported in any T. brucei glycoconjugate. However, the sugar nucleotide GDP-fucose was recently identified in bloodstream and procyclic-form T. brucei and shown to be essential for both life cycle stages (52,53).…”

Structural Basis for the Broad Substrate Range of the UDP-Sugar Pyrophosphorylase from Leishmania major

“…2.7.7.9) is the largest UDP-sugar pool in the cell and is used to produce UDP-Galp by epimerization. 5,6 UDP-Galp can be converted into UDP-galactofuranose and is thus essential for the biosynthesis of lipophosphoglycan, proteophosphoglycans, and glycoinositolphospholipids. [7][8][9] Besides its involvement in the biosynthesis of the surface glycocalyx, UDP-Glc is presumably involved in the synthesis of β-D-glucosylhydroxymethyluracil, a hypermodified DNA base found in telomeres and known as base J.…”

“…4 A small amount of GDP-fucose was also detected in L. major extract, although no fucosylated structures have been described in this organism to date. 5 The pool of UDP-Glc synthesized from the abundant metabolites UTP and glucose-1-phosphate (Glc-1-P) by a UDP-glucose pyrophosphorylase (UGP; E.C. 2.7.7.9) is the largest UDP-sugar pool in the cell and is used to produce UDP-Galp by epimerization.…”

A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly

“…The primer overhangs then target the tagging construct to the correct locus which inserts using homologous recombination after direct transfection of the PCR product. Alternatively, the tagging construct can be cloned into a plasmid that contains a larger (~500 bp) targeting sequence, which is then digested to release the completed construct prior to transfection [13,38,39]. The primer approach benefits from speed of assembly; only successful PCR is necessary to generate a construct that can then be directly introduced into cells.…”