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

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).

An essential, kinetoplastid-specific GDP-Fuc: β-D-Gal α-1,2-fucosyltransferase is located in the mitochondrion of Trypanosoma brucei

“…The essentiality and mitochondrial location of the orthologous protein in L. major , LmjFUT1, was reported in (Guo et al 2021). Here we asked whether TbFUT1 could functionally substitute for LmjFUT1, using a plasmid shuffle approach (Guo et al, 2017; Murta et al, 2009). We started with an L. major homozygous fut1 -null mutant expressing LmjFUT1 from the episomal pXNGPHLEO vector which additionally expresses GFP (Guo et al, 2017; Murta et al, 2009) and transfected it with an episomal construct pIR1NEO- TbFUT1 (±HA) expressing WT or HA-tagged TbFUT1 .…”

“…T. brucei utilizes the de novo pathway in which GDP-Fuc is synthesised from GDP-Mannose via GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER) (Guther et al, 2021; Turnock et al, 2007; Turnock and Ferguson, 2007). Conversely, L. major has two related bifunctional D-arabinose/L-fucose kinase/pyrophosphorylase, AFKP80 and FKP40, that synthesise GDP-Fuc from free fucose (Guo et al, 2017). Despite the aforementioned essentialities for GDP-Fuc in T. brucei and L. major , the only structurally-defined fucose-containing oligosaccharide in trypanosomatids is the low-abundance Ser/Thr-phosphodiester-linked glycan on T. cruzi gp72, a glycoprotein that has been implicated in flagellar attachment (Allen et al, 2013; Cooper et al, 1993; Ferguson et al, 1983; Haynes et al, 1996).…”

Proteins involved in the biosynthesis of lipophosphoglycan in Leishmania: a comparative genomic and evolutionary analysis

“…However, for d-Arap to be inserted into this structure, it must first be associated with the GDP molecule, a process that involves piro-and phosphorylation catalyzed by the protein encoded by AFKP80. This complex is then transported by LPG2 to the Golgi complex, where it becomes available to arabinotransferases encoded by SCA1 and SCA2 [59]. A paralog of AFKP80 (termed FKP40) was identified in the genome of L. major Friedlin with a 99.75% identity, differing by only three amino acids in the N-terminal located in the enzymatic domain responsible for pyrophosphorylase activity.…”

“…A paralog of AFKP80 (termed FKP40) was identified in the genome of L. major Friedlin with a 99.75% identity, differing by only three amino acids in the N-terminal located in the enzymatic domain responsible for pyrophosphorylase activity. This minor variation was sufficient to provoke the loss of its pyrophosphorylase action [59]. Due to the high degree of conservation between this gene and its paralog, our search for orthologs of AFKP80 or FKP40 was conservatively defined in order to identify any of their possible orthologs, which is why both genes appear collapsed in Figure 3.…”

Nucleocytoplasmic O-glycosylation in protists

“…Evidence exists for more extensive nucleocytoplasmic glycosylation beyond these two prototype examples. A type of fucosylation associated with mitochondria and the cytoplasm was described in Leishmania, a parasite group that inflicts a range of visceral and cutaneous infections in humans, though dependence on a candidate essential fucosyltransferase has yet to be established [55]. Bioinformatics studies suggest the occurrence of several cytoplasmically localized glycosyltransferases in Dictyostelium and T. gondii that are yet to be assigned [56, 9⦁].…”