Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum
- James I MacRae†1,
- Matthew WA Dixon†1, 2,
- Megan K Dearnley1, 2,
- Hwa H Chua1,
- Jennifer M Chambers1,
- Shannon Kenny1, 2,
- Iveta Bottova1, 2,
- Leann Tilley†1, 2 and
- Malcolm J McConville†1Email author
© MacRae et al.; licensee BioMed Central Ltd. 2013
Received: 3 May 2013
Accepted: 10 June 2013
Published: 13 June 2013
The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved.
We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13C-glucose and 13C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death.
Our metabolomics approach has allowed us to revise current models of P. falciparum carbon metabolism. In particular, we found that both asexual and sexual blood stages utilize a conventional TCA cycle to catabolize glucose and glutamine. Gametocyte differentiation is associated with a programmed remodeling of central carbon metabolism that may be required for parasite survival either before or after uptake by the mosquito vector. The increased sensitivity of gametocyte stages to TCA-cycle inhibitors provides a potential target for transmission-blocking drugs.
KeywordsMalaria Central carbon metabolism TCA cycle Metabolomics Gametocyte
The human parasite Plasmodium falciparum is the major cause of disease and death from malaria . Disease is associated with the development of asexual parasite stages that undergo repeated cycles of invasion and replication in red blood cells (RBCs). Following establishment of infection, a small proportion of parasites (<1%) differentiate to gametocytes [2, 3]. Gametocytogenesis is essential for subsequent transmission because the mature gametocyte is the only stage that can undergo sexual development in the mosquito vector, which is a prerequisite for the spread of disease. Both the asexual RBC stages and gametocytes are thought to be primarily dependent on glucose uptake and glycolysis for ATP synthesis and survival. Glucose uptake in infected RBCs increases more than 75-fold compared with uninfected RBCs , and the resultant increased lactate production contributes to lactic acidosis, a major cause of morbidity and death during severe malaria . Despite their reliance on glycolysis, the asexual stages of P. falciparum retain a single mitochondrion that is essential for parasite growth. Maintenance of the mitochondrial respiratory chain appears to be required for both the transport of proteins and metabolites into the mitochondrion and for the reoxidation of inner-membrane dehydrogenases, such as the dihydroorotate dehydrogenase involved in de novo pyrimidine biosynthesis . As a result, the asexual stages and gametocytes are sensitive to electron transport chain inhibitors, including the antimalarial atovaquone [6–9].
Mitochondrial dehydrogenases require a source of reducing equivalents which could, in principal, be generated in the mitochondrion or in the cytoplasm. P. falciparum encodes all of the enzymes needed for a complete TCA cycle, but the genes encoding a mitochondrial pyruvate dehydrogenase (PDH) complex are missing , and it is generally assumed that a TCA cycle utilizing glycolytic pyruvate does not operate in the blood stages [11–16]. The possibility that an unusual branched TCA cycle may operate in the asexual stages of P. falciparum, fuelled by the catabolism of glutamine via both the oxidative and reductive arms of the TCA cycle, was recently proposed , but subsequently retracted . More recent genetic studies have also suggested that operation of a complete mitochondrial TCA cycle is not required for the development of the asexual stages in the related murine parasite, Plasmodium berghei[18, 19]. The extent to which a conventional or unconventional TCA cycle operates in P. falciparum RBC stages therefore remains unresolved.
Despite lacking a recognizable mitochondrial PDH in P. falciparum, there is increasing evidence that a conventional TCA cycle can operate in the insect stages of these parasites [18, 20] and of other apicomplexan parasites. In particular, we have recently shown that the TCA cycle is essential for the growth of intracellular stages of Toxoplasma gondii.
In this study, we therefore reinvestigated the potential role of mitochondrial metabolism in P. falciparum asexual stages, and the possibility that the TCA cycle is important for the development of gametocytes.
The operation of a conventional TCA cycle in the P. falciparum asexual stages was confirmed by complementary 13C-glutamine labeling experiments. Incubation of infected RBC with 13C-glutamine resulted in labeling of all detectable TCA-cycle intermediates, with the highest 13C-enrichment seen in α-ketoglutarate and C4 dicarboxylic acids (Figure 1A). No labeling of TCA-cycle intermediates was detected in uninfected RBCs (Figure 1A). The predominant isotopomers of succinate, malate, and fumarate in 13C-glutamine-labeled parasites were fully labeled, indicating that most of the carbon skeletons that enter the TCA cycle via glutamate are not continuously cycled through the TCA reactions, and are presumably exported from the mitochondrion. This was supported by the low level of labeling of citrate compared with the C4 dicarboxylic acids and the predominant presence of the +4 citrate isotopomer. However, all intermediates contained readily detectable levels of +2 isotopomers, consistent with cycling of a sub-pool of C4 dicarboxylic acids around a canonical oxidative TCA cycle. Importantly, and in contrast to a previous report , citrate isotopomers containing +5 labeled carbons were not detected, indicating minimal catabolism of α-ketoglutarate via the reductive arm of the TCA cycle (Figure 1B). Collectively, these analyzes show that the P. falciparum asexual stages catabolize both pyruvate and glutamate in a conventional TCA cycle, and argue against the operation of a bifurcated TCA cycle, as previously proposed [13, 17]. However, these results do support a degree of compartmentalization within this cycle, with glutamate sustaining a major flux from α-ketoglutarate to malate/oxaloacetate, and with glucose-derived pyruvate and oxaloacetate contributing to a minor flux towards citrate synthesis.
To further define the metabolic state of gametocytes, Stage III gametocytes were cultured in the presence of 13C-glucose or 13C-glutamine, and enrichment in intracellular intermediates was determined by GC-MS. Marked differences were seen in the relative level of labeling of the TCA-cycle intermediates in gametocyte stages compared with the asexual stage parasites (Figure 1A). Specifically, TCA-cycle intermediates in gametocytes were strongly labeled with 13C-glucose, whereas the levels of enrichment in 13C-glutamine-fed gametocytes were low. The rapid labeling of citrate isotopomers with +2, +4, and eventually +6 labeled carbons after 2–4 hours demonstrates the operation of an active canonical TCA cycle (Figure 2B). Similarly, the rapid labeling of +3 isotopomers of oxaloacetate (indicated by aspartate labeling) and malate and the +5 isotopomer of citrate indicated that phosphoenolpyruvate may feed directly into the TCA cycle via PEP carboxylase or PEP carboxykinase (Figure 2B). Although labeling of gametocyte TCA-cycle intermediates with 13C-glutamine was relatively low compared with that in 13C-glucose-fed gametocytes, isotopomer analysis again confirmed conventional TCA-cycle operation. Specifically, a major +4 isotopomer was detected in all TCA intermediates, and this reached a maximum after 4 hours (Figure 2B). In contrast, +2 isotopomers were generated with slower kinetics, indicative of loss of labeled carbon with repeated cycles around the TCA cycle. As for the asexual stages, the absence of a predominant +5 isotopomer in citrate confirmed that the TCA cycle operates primarily in the oxidative direction in P. falciparum gametocytes (Figure 2B). GABA was labeled with both 13C-U-glucose and 13C-U-glutamine to the same maximal labeling level as other metabolites, although with slower kinetics (Figure 2B). Together, these findings suggest that there is substantial remodeling of mitochondrial metabolism in gametocytes, with glucose providing most of the carbon skeletons for operation of a complete TCA cycle (Figure 1A). Quantification of the rates of glucose consumption and lactate production provided further support for increased flux of glucose-derived pyruvate into the TCA cycle of gametocytes. Specifically, whereas more than 93% of the glucose internalized by the asexual stages was secreted as lactate, this was reduced to 80% in gametocytes, reflecting increased mitochondrial catabolism (Figure 2A).
P. falciparum and other apicomplexan parasites lack a mitochondrial isoform of PDH, and the enzyme involved in converting pyruvate to acetyl-CoA remains to be characterized. A possible candidate for this function is the mitochondrion-located branched chain α-keto acid dehydrogenase (BCKDH) complex . This complex is retained in all apicomplexa (except Cryptosporidium spp.), as well as more distantly related protists that lack a mitochondrial PDH , and has been shown to utilize pyruvate in some organisms [33, 34]. The transcriptional upregulation of key subunits in the BCKDH complex (PF3D7_1312600) in P. falciparum gametocytes , may underlie the increased flux of pyruvate into the TCA cycle seen in this stage. However, entry of glutamine/glutamate into the P. falciparum TCA cycle could be regulated by either the glutamate dehydrogenase or by amino acid transaminases [36, 37]. Because the P. falciparum glutamate dehydrogenase is not required for growth of asexual stages , it is likely that amino acid transaminases alone can fulfill this role. We have previously shown that P. falciparum encodes a putative glutamate/GABA transaminase (PF3D7_0608800), as well as the proteins needed for synthesis of GABA (PF3D7_0405700) and import of this metabolite into the mitochondrion (PF3D7_0209600) . The finding that GABA is synthesized in both asexual and sexual RBC stages, and that pyruvate is converted to alanine is consistent with both metabolites contributing to transamination reactions in the mitochondrion, which convert glutamate to α-ketoglutarate (Figure 4) . It is notable that P. falciparum lacks an obvious homolog for succinate semi-aldehyde dehydrogenase, which is required for the conversion of GABA to the TCA-cycle intermediate succinate . This differs from the situation in the related apicomplexan parasite, Toxoplasma gondii, which is able to utilize GABA as a short-term energy reserve , highlighting further specializations in the carbon metabolism of the Apicomplexa.
Despite entering a non-proliferative state, gametocytes exhibit increased levels of glucose utilization, glycolytic flux, and TCA catabolism of pyruvate. This switch to a more efficient method of energy generation may be needed to sustain gametocyte development under conditions of hypoglycemia, which is common in patients with severe malaria [39, 40]. The upregulation of TCA function in gametocytes might also reflect increased energy demands in female gametes preparing for the post-fertilization stages, during which access to glucose in the mosquito hemolymph may be limited . Indeed, there is accumulating evidence that a functional TCA cycle is essential for the development of the ookinete . Interestingly, deletion of the TCA-cycle enzyme succinate dehydrogenase had no observable effect on the production of P. berghei gametocytes . By contrast, we found that partial inhibition of the TCA cycle with NaFAc inhibits gametocyte development, and high concentrations of NaFAc are lethal to this stage. These apparent differences could reflect species-specific differences in their dependence on the TCA for energy or maintenance of the respiratory chain, or the possibility that inhibition of the aconitase reaction results in a stronger metabolic phenotype, including reduced yield of NADH and/or the accumulation of intermediates such as citrate to toxic levels. Further gene knockout studies are required to definitively validate these possibilities.
The metabolic switch to increased TCA-cycle catabolism of glucose in gametocytes could also reflect changes in carbon-source availability in the infected RBC. Glutamine uptake in trophozoite-infected RBC is mediated by a specific RBC amino acid transporter  and novel permeation pathways (NPPs) induced by the parasite . There is evidence that the NPPs are downregulated in developing gametocytes , resulting in reduced glutamine uptake. The reduced availability of glutamine in gametocytes might explain the switch to glucose as the preferred carbon source for TCA metabolism.
Recent transcriptional studies have suggested the existence of at least two physiological states of blood stage parasites in the human host, including a glycolysis-dependent asexual state and a ‘weakly gametocyte/sporozoite-like’ state that seems to have upregulated non-glycolytic energy metabolism, including the TCA cycle and oxidative phosphorylation [40, 45, 46]. Our results add to these findings, and suggest that the transcriptional upregulation of TCA cycle enzymes in response to glucose limitation or other metabolic stresses possibly represents a switch to a more energy-efficient metabolism.
Our findings reveal major changes in metabolic fluxes in P. falciparum bloodstream stages, which are not reflected in transcriptional-profiling studies, highlighting the importance of post-transcriptional regulatory mechanisms in controlling parasite metabolism. Stage-specific changes in metabolic fluxes in core metabolic pathways may be exploited in drug development. In particular, our results suggest that inhibitors of the mitochondrial enzyme responsible for converting pyruvate to acetyl-CoA could lead to a defect in gametocytogenesis. Field studies and mathematical modeling show that such transmission-blocking inhibitors will be needed to achieve the long-term goal of malaria elimination [47, 48].
P. falciparum asexual and gametocyte cultures were prepared as described previously . Briefly, asexual stage P. falciparum 3D7 parasite-infected RBCs were cultured in O-positive RBCs (Australian Red Cross Blood Service (ARCBS), Carlton, VIC, Australia) at 4 to 5% hematocrit, in a commercial medium (RPMI-GlutaMAX(™)-HEPES; Invitrogen) supplemented with 5% v/v human serum (ARCBS), and 0.25% w/v lipid-rich bovine serum albumin (AlbuMAX II; Invitrogen). Sorbitol synchronizations were used to obtain ring-stage cultures. Gametocyte cultures were initiated at 2% trophozoites (5% hematocrit) and grown until they reached 8 to 10% trophozoites. Volumes of parasite cultures were expanded four times, resulting in a 2% trophozoite stage parasite culture (day 0). Gametocytes were grown in the presence of 62.5 mmol/l N-acetylglucosamine from day 1 of culture. Development of the cultures was monitored by Giemsa-stained thin smears. Stage III (day 5) and Stage V (day 11) gametocytes were enriched by magnetic separation as previously described . For inhibitor studies, the culture medium was supplemented with either 1or 10 mmol NaFAc or NaAc. The concentration of these supplements was maintained during subsequent medium changes. All cell lines used in this study were certified to be free of Mycoplasma contamination by PCR of culture-derived DNA using Mycoplasma-specific primers (WEHI Antibody Facility, Bundoora, Victoria, Australia).
Stable isotope labeling and metabolite extraction of P. falciparum-infected and uninfected RBCs
Stable isotope labeling and metabolite extraction was adapted from that previously described . Synchronized P. falciparum-infected cultures (at 8 to 10% parasitemia) and uninfected RBC were pelleted (800 × g, 10 min, 4°C) and the medium replaced with glucose-free RPMI medium (Sigma-Aldrich, St Louis, MO, USA) or glutamine-free RPMI media (Invitrogen), supplemented as above with an additional 8 mmol/l final concentration of 13C-U-glucose or 13C-U-glutamine (Spectra Stable Isotopes, Columbia, MD, USA), as indicated. At required time points, cultures were rapidly transferred to a 50 ml centrifuge tube, and cellular metabolism was quenched by immersing the tube in a dry ice/ethanol slurry to chill the suspension to 0°C . Schizont-infected RBCs were purified from uninfected and ring-infected RBCs by passage through a size LD column and magnetic unit apparatus (varioMACS; Miltenyi Biotec, Bergisch Gladbach, Germany) , at 4°C. Mature trophozoite-infected and schizont-infected RBCs were eluted with ice-cold PBS at one-fifth of the culture volume. Gametocyte cultures were magnet-purified at the desired stage of development 1 day prior to commencement of the labeling experiments. Infected and uninfected RBCs were pelleted by centrifugation (800 × g for 10 minutes at 4°C), and washed three times with ice-cold PBS. Aliquots of 108 cell equivalents were extracted with chloroform:methanol (2:1 v/v) for 1 hour at 4°C with periodic sonication. The samples were separated by centrifugation (18,000 × g for 10 minutes at 4°C), the supernatant retained, and the pellet re-extracted with methanol:water (2:1 v/v containing 1 nmol scyllo-inositol as internal standard) for 1 hour, as above. After centrifugation, the supernatants were pooled and dried under nitrogen. Polar and apolar metabolites were separated by phase partitioning (chloroform:methanol:water, 1:3:3 v/v). Polar metabolite extracts were dried in a rotary evaporator, washed twice with methanol, derivitized by methoximation and trimethylsilylation, and analyzed by GC-MS . The level of labeling of individual metabolites was estimated as the percentage of the metabolite pool containing one or more 13C atoms after correction for natural abundance. The mass isotopomer distributions of individual metabolites were corrected for the occurrence of natural isotopes in both the metabolite and the derivitization reagent . To ensure that the starting medium was consistent between experiments, aliquots (10 μl) were washed, derivitized, and analyzed by GC-MS (as above) with each experiment.
Analysis of P. falciparum-infected and uninfected RBC culture medium
Synchronized P. falciparum-infected cultures and uninfected RBCs (2 × 109 cells) were cultured in 10 ml glucose-free medium supplemented as above, with 8 mmol 13C-U-glucose and 8 mmol 12C-U-glutamine. Both early trophozoite-infected and Stage III gametocyte-infected cultures were at 10% infection. At required time points, 2 × 600 μl aliquots were removed and separated by centrifugation (18,000 × g at room temperature for 1 minute) to remove RBCs. The volume of culture remaining at each time point was measured to account for evaporative losses. Culture supernatants (540 μl) were gently pre-mixed with 5 mmol D6-DSS in deuterium oxide (D2O) (60.0 μl, containing 0.2% w/v NaN3) and 21.4 mmol 13C-U-glycerol in D2O (5.00 μl, containing 0.2% w/v NaN3), prior to analysis by NMR. 13C spectra at 200 MHz were obtained using an 800 MHz NMR spectroscope (Avance; Bruker-Biospin, Rheinstetten, Germany) fitted with a cryoprobe. Samples were maintained at 25°C and spun at 20 Hz during sample collection. 13C spectra were acquired using the Avance zgpg pulse program with power-gated 1H decoupling. A pre-scan delay of 80.78 μ seconds, a delay between pulses of 2.0 seconds, and an acquisition time of 0.78 seconds were used. For each sample, four dummy scans were followed by 4000 scans with receiver gain set to 2050. The resulting 13C free induction decays were processed with Bruker TOPSPIN version 2.0 (the exponential function with line broadening = 5.0 Hz was applied in the frequency domain prior to Fourier transformation, baseline correction, and integration). Metabolite abundances were quantified as described previously  by multiplication of the metabolite integration area(s) with a correction factor derived from five T1 relaxation NMR experiments of known metabolite concentrations and normalization to the internal standard (13C-glycerol).
Gametocyte morphology analysis
Purified Stage II to III gametocytes were used to initiate 10 to 15% parasitemia cultures at 1% hematocrit. Treatment groups included complete culture media with or without NaFAc (10 mmol/l) or sodium acetate (10 mmol/l), and were performed in duplicate and on two separate occasions. Culture media and drug were exchanged daily. Development of gametocytes were monitored daily by Giemsa-stained slides, and percentage parasitemia was calculated from the counts of approximately 20 random fields of view (approximately 2000 uninfected RBCs) from each slide, and mean values and standard errors were estimated. Gametocyte morphology was classified as previously described .
Membrane organization within the gametocytes was assessed by staining (BODIPY-TR-ceramide; Invitrogen Corp. Carlsbad, CA, USA). Parasites were incubated overnight in the presence of BODIPY-TR-ceramide in complete culture medium at a final concentration of 0.7 μmol/l, as previously described . Mitochondrial membrane potential was investigated using the membrane potential dye Rho123 (Invitrogen Corp. Carlsbad, CA, USA). Staining was performed essentially as described previously . Briefly, gametocytes were resuspended in 0.1 μg/ml Rho123 in complete culture media, and incubated for 30 minutes at 37°C. The cells were pelleted and resuspended in normal culture medium, and incubated for an additional 30 minutes at 37°C. Cells were imaged using a microscopy system (DeltaVision Elite; Applied Precision, Issaquah, WA, USA). Images were deconvolved using the default settings in the softWoRx acquisition software (version 5.0). Images were further processed using NIH ImageJ (version 1.47c; ).
Branched chain α-keto acid dehydrogenase
Extracted ion chromatogram
Gas chromatography–mass spectrometry
Nuclear magnetic resonance spectroscopy
Novel permeation pathway
Red blood cell
Total ion chromatogram.
This work was supported by grants from the Australian National Health & Medical Research Council (NHMRC) and the Australian Research Council (ARC). MWAD is a NHMRC Early Career Fellow. IB is supported by the Swiss National Science Foundation. MJM is a NHMRC Principal Research Fellow. LT is an ARC Australian Professorial Fellow.
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