Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments

Abstract

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design.

Fig 1. U-¹³C-glucose and U-¹³C¹⁵N-glutamine labelling of glycolytic and TCA cycle intermediates in P. berghei asexual stages.

(A) P. berghei infected RBC (iRBC) and uninfected RBC (uRBC) were metabolically labelled with U-¹³C-glucose and U-¹³C¹⁵N-glutamine for indicated times (0, 6, 12, 18, 24 h post-invasion) and percent labelling of indicated metabolites (mol. % containing one or more ¹³C carbons after correction for natural abundance) determined by GC/MS. (B) Schematic representation of entry of glucose and glutamine as carbon sources and their anticipated fate through glycolysis and TCA cycle. (C) Mass isotopologue distributions of TCA-cycle metabolites at the 24 h time point (schizont stage) in iRBC) and uRBC cultured in the presence of U-¹³C-glucose and U-¹³C¹⁵N-glutamine. The x-axis indicates the number of ¹³C atoms in each metabolite (the ion used to analyse aspartate contains 3 of the 4 carbons as the 4-carbon fragment was below the limit of quantification). Due to the presence of a labelled nitrogen atom when labelling with U-¹³C¹⁵N-glutamine, the isotopologue analyses of nitrogen-containing metabolites include an isotope 1 dalton higher than the U-¹³C-glucose equivalent; i.e. aspartate (Asp, +4), glutamine/glutamate (Glx, +6) and ɣ-aminobutyric acid (GABA, +5). Error bars indicate SD of n = 3 biological replicates. Abbreviations used; glucose (Glc), glucose 6-phosphate (Glc-6-P), 3-phosphoglycerate (3-PGA), phosphoenolpyruvate (PEP), citrate (Cit), succinate (Suc), fumarate (Fum), malate (Mal), aspartate (Asp), glutamine/glutamate (Glx) and ɣ-aminobutyric acid (GABA), Gln, glutamine; Co-A, coenzyme-A; Pyr, pyruvate; OAA, oxaloacetate; Aco, aconitate; k-glu, α-ketoglutarate.

Fig 2. Genetic dissection of central carbon metabolism in asexual blood stages.

(A) Schematic representation of central carbon metabolism in P. berghei showing genes encoding metabolic enzymes disrupted or discussed in this study. Abbreviations as for Fig 1 and: PanK1, pantothenate kinase 1; PanK2, pantothenate kinase 2; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; ACO, aconitase; OAT, ornithine amino transferase; TRP, putative GABA transporter; GDH1, glutamate dehydrogenase 1; GDH3, glutamate dehydrogenase 3; LDC, lysine decarboxylase/ GAD, glutamate decarboxylase; GluS, glutamate synthase. Disrupted genes coloured in blue have no effect on asexual or mosquito stage development, those coloured in red have no effect on asexual stage development but fail to make ookinetes and genes coloured in yellow have no effect on asexual stage development but show a strong phenotype in ookinete to oocyst transition with a block in transmission. See Fig 8 and S3 Table for summary. (B) In vivo growth assay of P. berghei mutants in mixed competition infections with wild type (wt) parasites over 7 generations. Coloured lines represent the non-linear fit of percentage of mutant parasites in the total parasite population (mixed with wt 50–50). Data representative of n = 3 independent biological replicates. P-value ***p < 0.0001, **p < 0.001, *p < 0.01 Two way ANOVA- Dunnett's multiple comparison test with wt control compared to mutant P. berghei parasites per generation. Also see S3 and S7 Figs.

Fig 3. U-¹³C-glucose and U-¹³C¹⁵N-glutamine labelling of glycolytic and TCA cycle intermediates in P. berghei gametocytes during activation.

(A) Gametocytes were activated and then metabolically labelled with U-¹³C-glucose and U-¹³C¹⁵N-glutamine for indicated times during activation (0, 10, 20, 30 min post activation). Percentage labelling (mol% containing one or more ¹³C carbons after correction for natural abundance) in indicated metabolites was determined by GC-MS. (B) Fractional labelling of TCA-cycle intermediates in unactivated gametocytes (0 min) and activated gametes (30 min post activation) cultured in the presence of U-¹³C-glucose and U-¹³C¹⁵N-glutamine. The x-axis indicates the number of ¹³C atoms in each metabolite (the ion used to analyse aspartate contains 3 of the 4 carbons as the 4-carbon fragment was below the limit of quantification). Due to the presence of a labelled nitrogen atom when labelling with U-¹³C¹⁵N-glutamine, the isotopologue analyses of nitrogen-containing metabolites include an isotope 1 dalton higher than the U-¹³C-glucose equivalent; i.e. aspartate (Asp, +4), glutamine/glutamate (Glx, +6) and ɣ-aminobutyric acid (GABA, +5). Error bars indicate SD of n = 3 biological replicates. Abbreviations are same as in Fig 1.

Fig 4. Gametocyte conversions and exflagellation in P. berghei mutant parasites.

(A) Gametocyte conversion was observed during blood stage development in mutant P. berghei parasites over 5 days post infection by either using a wt parent line which expresses GFP in male gametocytes and RFP in female gametocytes (RMgm-164) with P. berghei mutants generated in the same genetic background and analysed using FACS determining the number of gametocytes in infected blood or by observing mature gametocytes in Giemsa stained smears. No significant difference was seen between wt and mutants parasites. Error bars indicate SD of n = 2 biological replicates. (B) Exflagellation (male gamete formation) in mutant P. berghei parasites normalised to wt in an in vitro activation assay. Error bars indicate SD of n = 3 biological replicates. P-values **p < 0.005, *p < 0.05 unpaired two tailed t-test compared to wt.

Fig 5. 2-Deoxyglucose (2DG) specifically inhibits male gametocyte activation.

(A) Plot showing the number of activation centres observed in cultures containing 2DG compared to control in ookinete media alone. Error bars indicate SD of n = 6 biological replicates. (B) Microscopy images of male gametocytes after activation in the presence of 2DG. GFP expressing male gametocytes of the RMgm-164 line exhibited a characteristic shape (arrowed in left panel) and were positive for the red blood cell marker Ter119 (arrowed in right panel) indicating that they had not emerged from the host red blood cell. Scale bar = 5 μm. (C) Left Panel: Female gametocytes emerged in the presence of 2DG as counted under fluorescent microscope after counterstaining with a fluorescent conjugated red blood cell marker Ter119. Error bars indicate SD of n = 6 biological replicates. Right panel fluorescent microscope images showing the red fluorescent female parasites of the RMgm-164 line with the green FITC-Ter119 red blood cell membrane marker (top panel), DIC image (lower panel) for unactivated (arrowed, left) and activated emerged females (right). (D) Plot showing the percentage of male gametes activated in cultures containing 25 mM 2DG (+25 mM 2DG) is reduced significantly compared to the control (-2DG) and this can be rescued by addition of an equimolar amount of glucose (+25 mM Glc). Error bars indicate SD of n = 4 biological replicates. P-value ***p < 0.0005, **p < 0.005, *p < 0.05, paired two tailed t-test compared to -2DG. (E) Conversion to ookinetes after 2DG treatment is inhibited. Parasites were activated then 2DG (25 mM) was added 30 min post activation (post). Alternatively parasites were incubated in BSA enriched PBS containing 25 mM 2DG for 30 min before activation in media also containing 2DG (pre). Conversion rates were calculated after 21 h post activation. Error bars indicate SD of n = 2 biological replicates. P-value ***p < 0.0005, **p < 0.005, *p < 0.05, paired two tailed t-test compared to Pre-.

Fig 6. U-¹³C-glucose and U-¹³C¹⁵N-glutamine labelling of glycolytic and TCA cycle intermediates in P. berghei ookinetes and unfertilised (UF) female gametes.

(A) P. berghei ookinetes and unfertilized gametes were metabolically labelled with U-¹³C-glucose and U-¹³C¹⁵N-glutamine for 10 hr and 21 hr post activation, and percentage labelling (mol. % containing one or more ¹³C carbons after correction for natural abundance) was determined by GC-MS. (B) Absolute abundance of GABA in uninfected erythrocytes (uRBC) and P. berghei infected erythrocytes (iRBC) at ring, trophozoite, schizont, gametocyte and ookinete stages in nmol per 6 × 10⁵ cells (normalised to magnetically purified parasite numbers). (C) Fraction labelling of TCA-cycle isotopologues in mature ookinetes and unfertilised (UF) female gametes at 21 h post activation cultured in the presence of U-¹³C-glucose and U-¹³C¹⁵N-glutamine. The x-axis indicates the number of ¹³C atoms in each metabolite (the ion used to analyse aspartate contains 3 of the 4 carbons as the 4-carbon fragment was below the limit of quantification in our GC-MS data). Due to the presence of a labelled nitrogen atom when labelling with U-¹³C¹⁵N-glutamine, the isotopologue analyses of nitrogen-containing metabolites include an isotope 1 dalton higher than the U-¹³C-glucose equivalent; i.e. aspartate (Asp, +4), glutamine/glutamate (Glx, +6) and ɣ-aminobutyric acid (GABA, +5). Error bars indicate SD of n = 3 biological replicates. Abbreviations are same as in Fig 1.

Fig 7. Mosquito stage development of P. berghei mutant parasites.

(A) In vitro ookinete conversion of mutant P. berghei parasites as compared to wt. The error is given as the SD of n = 3 independent biological replicates. P-value **p < 0.005 unpaired two tailed t-test compared to wt. (B) Number of mature oocysts at 7–12 days post-P. berghei mutant parasite-infected blood feed in mosquito mid guts. n = 40 mosquitoes cumulative of two independent biological replicates. P-values ****p < 0.00005, ***p < 0.0005, **p < 0.005, *p < 0.05 unpaired two tailed t-test compared to wt. (C) in vitro ookinete conversion assay to measure fertility of aco^- P. berghei gametocytes. Fertility of aco^- P. berghei gametocytes was analysed by their capacity to form ookinetes by crossing gametes with RMgm-348 (Pb270, p47^-) which produces viable male gametes but non-viable female gametes and RMgm-15 (Pb137, p48/45^-) which produces viable female gametes but non-viable male gametes. p47^- and p48/45^- self crosses serve as negative controls and p47^- x p48/45^- cross is the positive control. aco^- cross with either p47^- or p48/45^- did not produce any ookinetes. The error is given as the S.D. of n = 2 independent biological replicates. P values ***p<0.0005, **p<0.005 unpaired two tailed t-test compared to cross p47—x p48/45 –. (D) Ookinete motility assay. Mature ookinetes were embedded in matrigel and tracks were constructed on Image J. Displacement in 10.5 min was calculated for ookinetes moving in a straight line and represented as speed of motility in μm/min. (n = mean 40 ookinetes).

Fig 8. A schematic representation of the life cycle of P. berghei and the effect of disrupting metabolic genes in this study.