Abstract
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).
Highlights
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 bite
We show that ⌬afkp80؊ null mutants ablated D-Arap modifications of LPG as predicted, whereas ⌬fkp40؊ null mutants resembled wild type (WT)
To dissect the potentially overlapping roles of AFKP80 and FKP40 in vivo and the relative contributions of D-Arap and fucose to Leishmania biology, we focused on the role of A/FKPs through the generation of null mutants
Summary
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 bite. 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. Of Molecular and Cell Biology, Boston University These results establish a requirement for GDP-fucose for L. major viability and predict the existence of an essential fucoconjugate(s). These PCRs establish the presence or loss of either or both of the A/FKP genes. These PCRs establish the presence or loss of A/FKP genes as expected. Rescue was obtained following expression of the GDP-fucose de novo pathway genes GMD and GMER, thereby establishing that GDP-fucose is required for the survival of L. major
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