The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

Abstract

Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito.

Figure 1. PfGMD transfection construct and integration events.

(A) Schematic representation of the transfection plasmid (pHH1-PfGMD) used to target and disrupt GMD gene in P. falciparum 3D7 parasites (3D7) and the expected recombination event (3D7∆GMD). The small white box in front of F1 fragment represents the absence of the first two ‘AT’ nucleotides in the start codon. The black boxes represent upstream and downstream DNA sequence flanking the PfGMD gene locus. The position of HindIII (H) and EcoRI (E) restriction sites, the position of GMD probe (thick black line) and the predicted length of the restriction fragments (thin black lines) are shown. (B) Southern Blot analysis of EcoRI and HindIII digested genomic DNA from 3D7 and 3D7ΔGMD parasites. Hybridisation of GMD probe to digested DNA from 3D7ΔGMD revealed restriction fragment sizes consistent with the disruption of PfGMD by the single recombination of the plasmid. *Matches with the expected size of the episomic plasmid copy. The panel shows a cropped blot and full-length blot is shown in Supplementary data (Supplementary Fig. S3).

Figure 2. P. falciparum 3D7ΔGMD grow at comparable rates to 3D7 control cell lines.

(A) Synchronous ring-stage 3D7ΔGMD and 3D7 growth was monitored over two complete life cycles (96h) by flow cytometry. (B) Intraerythrocytic development (x1000 magnification) of wild-type (3D7) and PfGMD (ΔGMD) null mutants of P. falciparum 3D7 strain. Time indicates hours post-invasion of tightly (5 hours window) synchronised in vitro cultures by means of a combination of Percoll and sorbitol treatments. (C) Inhibition of 3D7 (dark grey bar) and 3D7ΔGMD (light grey bar) growth by a 3-h heat-shock at 41.5 °C. Values are the average of three independent replicas, with standard deviation, and represent percentage of growth relative to identical cultures not subjected to heat-shock. Heat-shock was performed when parasites were at the trophozoite stage and parasitemia was measured by FACS at the next generation. Statistical analysis, performed by applying one-way analysis of variance Tukey’s post-test, showed no significant differences.

Figure 3. A pool of GDP-Fuc is detected in 3D7ΔGMD mutants.

(A) The levels of GDP-Fuc and other sugar nucleotides are quantified in wild type (3D7; white bars) and 3D7ΔGMD mutants (grey bars). Values are shown as relative amounts to wild type sugar nucleotide levels and indicate the average variation ± S.D. of five different analyses. Analyses of every single experiment were always performed in triplicate. (B) Picomols of free ³H-fucose (light grey bars) and GDP-[³H]Fuc (dark grey bars) incorporated by wild type (3D7) and 3D7ΔGMD mutant parasites in culture (top) or after saponin treatment to release parasites (bottom). ³H-L-glucose (white bars) is included for comparison purposes, as it is incorporated through equilibrative (not active) transport mechanisms. A control of ³H-glucosamine, incorporated by parasites, was also used in every experiment (not shown).

Figure 4. UEA-I labelling is abrogated in 3D7ΔGMD mutants.

(A) Deconvolving micrographs of P. falciparum infected RBCs show a specific UEA-I labelling (green) on the surface of wild type parasites and host RBCs. GSL-II also binds to the surface of parasites (B) UEA-I labelling disappears in 3D7ΔGMD mutant surface (bottom panels) whereas GSL-II binding is not altered.