title: The de novo and salvage pathways of GDP-mannose biosynthesis are both sufficient for the growth of bloodstream-form Trypanosoma brucei.
abstract: The sugar nucleotide GDP-mannose is essential for Trypanosoma brucei. Phosphomannose isomerase occupies a key position on the de novo pathway to GDP-mannose from glucose, just before intersection with the salvage pathway from free mannose. We identified the parasite phosphomannose isomerase gene, confirmed that it encodes phosphomannose isomerase activity and localized the endogenous enzyme to the glycosome. We also created a bloodstream-form conditional null mutant of phosphomannose isomerase to assess the relative roles of the de novo and salvage pathways of GDP-mannose biosynthesis. Phosphomannose isomerase was found to be essential for parasite growth. However, supplementation of the medium with low concentrations of mannose, including that found in human plasma, relieved this dependence. Therefore, we do not consider phosphomannose isomerase to be a viable drug target. We further established culture conditions where we can control glucose and mannose concentrations and perform steady-state [U-(13) C]-D-glucose labelling. Analysis of the isotopic sugar composition of the parasites variant surface glycoprotein synthesized in cells incubated in 5 mM [U-(13) C]-D-glucose in the presence and absence of unlabelled mannose showed that, under physiological conditions, about 80% of GDP-mannose synthesis comes from the de novo pathway and 20% from the salvage pathway.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Sabine
authors.initials: S
authors.lastname: Kuettel
authors.forename: Majken C T
authors.initials: MC
authors.lastname: Wadum
authors.forename: Maria Lucia S
authors.initials: ML
authors.lastname: Güther
authors.forename: Karina
authors.initials: K
authors.lastname: Mariño
authors.forename: Carolin
authors.initials: C
authors.lastname: Riemer
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
mesh_terms: D001769:Blood / Q000469:parasitology*; D055786:Gene Knockout Techniques; D006155:Guanosine Diphosphate Mannose / Q000096:biosynthesis*; D006801:Humans; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*; D008830:Microbodies / Q000201:enzymology; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism / Q000472:pathogenicity*
chemical_list: D006155:Guanosine Diphosphate Mannose; D008359:Mannose-6-Phosphate Isomerase