Genetic metabolic complementation establishes a requirement for GDP-fucose in .

To survive in its sand fly vector, the trypanosomatid protozoan parasite first attaches to the midgut to avoid excretion, but eventually it must detach for transmission by the next bite. In strain Friedlin, this is controlled by modifications of the stage-specific adhesin lipophosphoglycan (LPG). During differentiation to infective metacyclics, d-arabinopyranose (d-Ara) caps the LPG side-chain galactose residues, blocking interaction with the midgut lectin PpGalec, thereby leading to parasite detachment and transmission. Previously, we characterized two closely related genes ( and ) encoding bifunctional proteins with kinase/pyrophosphorylase activities required for salvage and conversion of l-fucose and/or d-Ara into the nucleotide-sugar substrates required by glycosyltransferases. Whereas only AFKP80 yielded GDP-d-Ara from exogenous d-Ara, both proteins were able to salvage l-fucose to GDP-fucose. We now show that Δ null mutants ablated d-Ara modifications of LPG as predicted, whereas Δ null mutants resembled wild type (WT). Fucoconjugates had not been reported previously in , but unexpectedly, we were unable to generate / double mutants, unless one of the A/FKPs was expressed ectopically. To test whether GDP-fucose itself was essential for viability, we employed "genetic metabolite complementation." First, the trypanosome pathway enzymes GDP-mannose dehydratase (GMD) and GDP-fucose synthetase (GMER) were expressed ectopically; from these cells, the Δ/Δ double mutant was now readily obtained. As expected, the Δ/Δ/+ line lacked the capacity to generate GDP-Ara, while synthesizing abundant GDP-fucose. These results establish a requirement for GDP-fucose for viability and predict the existence of an essential fucoconjugate(s).