Authors

Discussion

The importance of N- and/or O-linked glycosylation in the Plasmodium parasite is far from certain. Glycosylphosphatidylinositol (GPI) anchored proteins are however abundant in these parasite, as they are present in all extracellular life cycle stages and anchor proteins essential for both egress and host cell invasion. Thus, the enzymes involved in the synthesis of activated mannose, which is crucial for GPI-anchor biosynthesis are potential novel drug targets. Here we to shed further light on the abundance of mannose derived glycosylation in P. falciparum and assess several enzymes involved in this process as candidate drug targets. Sub-cellular localisation of GDP-Man pyrophosphorylase (GDP-Man PP) and dolichol-phosphate-mannose synthase (DPMS) has been determined. Additionally, various genetically altered cell lines (including knock out and over expression) for these genes have been produced to elucidate their function and essentiality. Biochemical analysis of these cell lines demonstrate that the regulation of the two enzymes are intricately linked in a mono-directional manner. Focused metabolomics by multi-reactant monitoring of the overexpression lines suggests that the fucosylation pathway may have a novel function within the parasite, possibly acting as a dynamic store for activated fucose/mannose. In order to determine the cellular concentration of key metabolites within the parasite, the volume of the intra-erythrocytic stages have been determined. This shows that the concentration of metabolites in the ring stage parasites is substantially higher than previously thought. The recombinant expression of DPMS in E. coli allowed activity assays and active site residues to be investigated, as well as establishing a platform for inhibitor screening. Finally, inhibitors of the T. brucei DPMS enzyme have been screened against the P. falciparum parasites in culture.