To survive in its sand fly vector, the trypanosomatid protozoan parasite Leishmania first attaches to the midgut to avoid excretion, but eventually it must detach for transmission by the next bite. In Leishmania major strain Friedlin, this is controlled by modifications of the stage-specific adhesin lipophosphoglycan (LPG). During differentiation to infective metacyclics, d-arabinopyranose (d-Arap) 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 L. major genes (FKP40 and AFKP80) encoding bifunctional proteins with kinase/pyrophosphorylase activities required for salvage and conversion of l-fucose and/or d-Arap into the nucleotide-sugar substrates required by glycosyltransferases. Whereas only AFKP80 yielded GDP-d-Arap from exogenous d-Arap, both proteins were able to salvage l-fucose to GDP-fucose. We now show that Δafkp80− null mutants ablated d-Arap modifications of LPG as predicted, whereas Δfkp40− null mutants resembled wild type (WT). Fucoconjugates had not been reported previously in L. major, but unexpectedly, we were unable to generate fkp40−/afkp80− double mutants, unless one of the A/FKPs was expressed ectopically. To test whether GDP-fucose itself was essential for Leishmania viability, we employed “genetic metabolite complementation.” First, the trypanosome de novo pathway enzymes GDP-mannose dehydratase (GMD) and GDP-fucose synthetase (GMER) were expressed ectopically; from these cells, the Δfkp40−/Δafkp80− double mutant was now readily obtained. As expected, the Δfkp40−/Δafkp80−/+TbGMD-GMER line lacked the capacity to generate GDP-Arap, while synthesizing abundant GDP-fucose. These results establish a requirement for GDP-fucose for L. major viability and predict the existence of an essential fucoconjugate(s).
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This work was supported by a Berg postdoctoral fellowship from the Department of Molecular Microbiology, Washington University School of Medicine (to H. G.), National Institutes of Health Grant R01 AI031078 (to S. J. T. and S. M. B.), and Wellcome Trust Senior Investigator Award 10182 (to M. A. J. F.).
This article contains supplemental Table S1 and Figs. S1–S3.
Guo, Hongjie; Novozhilova, Natalia M.; Bandini, Giulia; Turco, Salvatore J.; Ferguson, Michael A. L.; and Beverley, Stephen M., "Genetic Metabolic Complementation Establishes a Requirement for GDP-Fucose in Leishmania" (2017). Molecular and Cellular Biochemistry Faculty Publications. 129.