Final Activity Report Summary - T. BRUCEI GTS (Functional genomics relating to the biosynthesis of trypanosome glycoconjugates) The African trypanosome Trypanosoma brucei is the causative agent of African sleeping sickness and the cattle disease Nagana. This protozoan parasite is very dependent on glycoconjugates for both its survival and its infectivity and it is known that galactose and N-acetylglucosamine metabolism is essential for T. brucei. The main aim of this project is to identify and characterise the genes involved in the biosynthesis of trypanosomal glycoconjugates. By applying a post-genomic/bioinformatic approach we managed to identify 21 putative glycosyltransferase (GT) genes in the recently completed T. brucei genome. We showed the developmental regulation of some of these putative GTs in the T. brucei life cycle by analysing the mRNA levels of these genes in the bloodstream and procyclic form of the parasite.Several examples of these putative GTs have been experimentally analysed in our laboratory by generating gene null mutants in the bloodstream and procyclic forms of the parasite and characterising their phenotypes. The Tb10.389.1450 gene, for example, presents higher mRNA levels in the procyclic form of the parasite. This GT, probably a GlcNAc-transferase, plays and important role in the biosynthesis of the glycosylphosphatidylinositol (GPI) membrane anchors that attach the procyclins to the surface of the parasite. Procyclins is the main glycoprotein in the procyclic, or insect form, of the parasite. Tb10.389.1450 is concretely involved in the formation of the poly-N-acetyl-lactosamine side chains of the GPI anchors. It has been previously suggested that these side chains form a glycocalyx that protects the parasite surface from proteolisis in the Tsetse fly midgut, and it is interesting the fact that the Tb10.389.1450 null mutants show problems to colonise their Tsetse fly vector when compared to the wild type cells.Tb927.2.3370 is a very similar case to the previous example. Its mRNA levels are higher in the procyclic form of the parasite and the analysis of the null mutant phenotype showed that the gene is as well involved in the biosynthesis of the poly-N-acetyl-lactosamine side chains of the GPI anchors. Preliminary experiments suggest that this GT is, as well, a GlcNAc transferase.Despite of their important function in the biosynthesis of the GPI anchors in the procyclic form of the parasite, these two GTs play as well a role in the formation of the giant N-glycans of glycoproteins present in the flagellar pocket of the bloodstream form of the parasite. Therefore, this N-glycans, that contain an average of 54 N-acetyllactosamine repeats, are synthesised by GTs involved as well in the biosynthesis of procyclic GPI-anchors.
