The suppression of galactose metabolism in Trypanosoma cruzi epimastigotes causes changes in cell surface molecular architecture and cell morphology.

The cell surface of the epimastigote form of Trypanosoma cruzi is covered by glycoconjugates rich in galactose. The parasite cannot take up galactose through its hexose transporter, suggesting that the epimerisation of UDP-glucose to UDP-galactose may be the parasite's only route to this sugar. The T. cruzi UDP-glucose 4'-epimerase is encoded by the TcGALE gene. We were unable to make a CL-Brener strain T. cruzi epimastigote TcGALE-/- null mutant, suggesting that the gene is essential. Two TcGALE+/- single-allele knockout clones displayed aberrant morphology and haploid deficiency with respect to galactose metabolism. The morphological phenotypes included shortened flagella, increased incidence of spheromastigotes, agglutination and a novel walnut-like appearance. The reduced supply of UDP-galactose was manifest in the two clones as a six- or nine-fold reduction in the expression of galactopyranose-containing cell surface mucins and negligible or two-fold reduction in the expression of galactofuranose-containing glycoinositolphospholipids. The major loss of mucins as opposed to glycoinositolphospholipids may indicate that the latter are more important for basic parasite survival in culture. The apparent haploid deficiency suggests that epimerase levels are close to limiting, at least in the epimastigote form, and might be exploited as a potential drug target.

Engineered gene over-expression as a method of drug target identification.

The proposed target of aminobisphosphonate (aBP) bone resorption inhibitors, both in mammalian osteoclasts and in Dictyostelium, is the enzyme farnesyl diphosphate synthase (FDP synthase). The genetic evidence, obtained with Dictyostelium, derives from variant strains that over-express FDP synthase and that are relatively resistant to aBPs. We show that forced FDP synthase over-expression also leads to aBP resistance; by placing FDP synthase under control of a semi-constitutive promoter, transforming it into Dictyostelium cells and selecting with the aBP alendronate. This combination of drug and dominant selectable marker provides a novel selection system for transformation. We further show that, when a population of Dictyostelium cells expressing an entire growth stage cDNA library is placed under alendronate selection, FDP synthase is the only cDNA insert that confers drug resistance. This confirms FDP synthase as the primary target of aBPs and suggests a general method of drug target identification based upon engineered gene over-expression.

The suppression of galactose metabolism in procylic form Trypanosoma brucei causes cessation of cell growth and alters procyclin glycoprotein structure and copy number.

Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4'-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDP-Glc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins., were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.

Cloning and characterisation of the UDP-glucose 4'-epimerase of Trypanosoma cruzi.

Trypanosoma cruzi incorporates galactose into many of its cell-surface glycoconjugates but it is unable to transport this sugar through its hexose transporter. Epimerisation of UDP-glucose to UDP-galactose by UDP-glucose 4'-epimerase may be the only way that the parasites can obtain galactose. Here, we describe cloning the T. cruzi UDP-Glc 4'-epimerase (TcGALE) gene and show that it is functional by complementing an Escherichia coli epimerase-deficient strain. The T. cruzi GALE gene encodes a 42.4 kDa protein and the recombinant protein expressed in E. coli is a homodimer in solution with a specific activity of 3.8 U mg(-1) and K(m) for UDP-Gal of 114 microM. Unlike the human epimerase, T. cruzi UDP-Glc 4'-epimerase is unable to inter-convert UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. This may explain why T. cruzi initiates O-glycosylation of its abundant GPI-anchored surface mucins via GlcNAcalpha1-O-Thr/Ser rather than the GalNAcalpha1-O-Thr/Ser linkage that is common for mucins from many other eukaryotes.

High-resolution crystal structure of Trypanosoma brucei UDP-galactose 4'-epimerase: a potential target for structure-based development of novel trypanocides.

The crystal structure of UDP-galactose 4'-epimerase from the protozoan parasite Trypanosoma brucei in complex with the cofactor NAD(+) and a fragment of the substrates, UDP, has been determined at 2.0 A resolution (1 A = 0.1 nm). This enzyme, recently proven to be essential for this pathogenic parasite, shares 33% sequence identity with the corresponding enzyme in the human host. Structural comparisons indicate that many of the protein-ligand interactions are conserved between the two enzymes. However, in the UDP-binding pocket there is a non-conservative substitution from Gly237 in the human enzyme to Cys266 in the T. brucei enzyme. Such a significant difference could be exploited by the structure-based design of selective inhibitors using the structure of the trypanosomatid enzyme as a template.

Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei.

The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana. The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor. These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose. The parasite is unable to take up galactose, suggesting that it may depend on the action of UDP-glucose 4'-epimerase for the conversion of UDP-Glc to UDP-Gal and subsequent incorporation of galactose into glycoconjugates via UDP-Gal-dependent galactosyltransferases. In this paper, we describe the cloning of T. brucei galE, encoding T. brucei UDP-Glc-4'-epimerase, and functional characterization by complementation of a galE-deficient Escherichia coli mutant and enzymatic assay of recombinant protein. A tetracycline-inducible conditional galE null mutant of T. brucei was created using a transgenic parasite expressing the TETR tetracycline repressor protein gene. Withdrawal of tetracycline led to a cessation of cell division and substantial cell death, demonstrating that galactose metabolism in T. brucei proceeds via UDP-Glc-4'-epimerase and is essential for parasite growth. After several days without tetracycline, cultures spontaneously recovered. These cells were shown to have undergone a genetic rearrangement that deleted the TETR gene. The results show that enzymes and transporters involved in galactose metabolism may be considered as potential therapeutic targets against African trypanosomiasis.