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 UDPglucose 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.UDP-Gal ͉ galE ͉ epimerase T he tsetse fly-transmitted protozoan parasite Trypanosoma brucei causes human African sleeping sickness and the related cattle disease Nagana. There are 300,000-500,000 cases of the human disease per year in sub-Saharan Africa (1). The bloodstream form of the parasite divides by binary fission in the blood, lymph, and interstitial fluids of the mammalian host, and causes cachexia and anaemia in cattle and neurological disturbances in man. These conditions are fatal if not treated, and existing chemotherapies are toxic and difficult to administer. The need for new, less-toxic therapeutics is widely acknowledged (1).The bloodstream form of T. brucei is rich in galactosecontaining glycoproteins, most notably the variant surface glycoprotein (VSG) that, at 5 ϫ 10 6 homodimers per cell, forms a dense cell-surface coat. The VSG coat is a macromolecular diffusion barrier that protects the parasite from the innate immune system and also enables the parasite to undergo antigenic variation. Thus, although an individual parasite only expresses one VSG gene at a time, the parasite population can stay ahead of the host's specific immune response to expressed VSGs when a few parasites switch expression to one of several hundred genes encoding immunologically ...
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 UDPGlc 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.
The protozoan parasites Trypanosoma brucei and 7: cruzi are the causative agents of African Sleeping Sickness and Chagas' Disease, respectively. Their surface molecules play a crucial role in protection and infectivity. Galactose residues are thought to play an important role in parasite survival: 7: brucei bloodstream forms are covered by a layer of variant surface glycoprotein (VSG) attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor which is modified by a branched a-Gal side chain. In 7: cruzi p-galactopyranoside residues of m u c k are the acceptor sites for the trans-sialylation reaction which is essential for parasite survival. The oxidoreductase UDP-glucose 4' epimerase interconverts glucose and galactose. In mammals this enzyme is important for converting Gal to Glc because excess Gal can be toxic. In trypanosomes, the reverse reaction (Glc to Gal) is probably more important since Glc is freely available from blood whereas they cannot transport Gal. We have cloned the epimerase from both 7: brucei and 7: cruzi using partial sequences found by database searches. The parasite epimerase genes were cloned into pUCl8; these plasmids successfully rescued an E. coliepimerase mutant (galE-) for growth on McConkey agar with galactose as the sole carbon source. Rescued cells had the epimerase gene in reading frame +1 and grew as large red colonies, whereas their mutant counterparts formed smaller, paler colonies. We have also expressed the parasite epimerases from pET15b and pQE-30. Overexpressed protein will be used for enzyme assays and crystallisation trials. We will also attempt to make epimerase knockouts in I: brucei and 7: cruzi to assess the role of Gal in these parasites.
MFIS: a high throughput technology for studying the ligandreceptor interactionThe Multi-parameter Fluorescence Immunosensor System (MFIS) is designed to study the specificity and cross-reactivity of the ligand-receptor interaction on a large scale. Using a micro-spotting device, many thousands of ligands or receptors can be patterned on a micro-glass slide. A solution containing specific ligands or receptors with distinct fluorescent tags can then be applied on the slide and their binding monitored by fluorescence signals. To investigate the feasibility of this method, we have taken advantage of a wellestablished antigen-antibody system, the dextrans and anti-dextran antibodies. A panel of purified dextran preparations with different linkage compositions and ratios of terminal/internal epitopes were immobilized on a surface-treated glass slide and than incubated with anti-dextran antibodies of defined specificities, either cavitytype or groove-type. The former is specific for the terminal nonreducing end structure of alpha(l,6)dextran; the latter recognizes the internal linear chain of the polysaccharide. When a cavity-type mAb, 16.4.12E, was applied on the glass slide, it bound to the immobilized alpha( 1,6)dextran preparations having branches but not those with an internal linear chain structure. By contrast, a...
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