article: 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.
  Malaria remains a major public health problem with more than 214 million new cases each year and 438,000 deaths world-wide. Significant progress has been made in reducing the number of cases of malaria over the last 15 years, largely as a result of the introduction of different control interventions, including artemisinin-based combination therapies [1]. However, increasing resistance to artemisinin threatens to undermine existing malaria control programs [2] and there remains an ongoing need to develop new therapeutics with a particular focus on drugs that target different parasite developmental stages responsible for pathogenesis and transmission via the mosquito vector. All of the symptoms and pathology associated with malaria are linked to the repeated cycles of infection and lysis of host red blood cells (RBC) completed by asexual blood stages of Plasmodium. Glucose consumption by Plasmodium-infected RBC increases 10-fold and these stages rely primarily on glycolysis for energy generation [3–7]. Notwithstanding their dependence on glycolysis, asexual blood stages maintain a single, poorly cristate mitochondrion [8] and are dependent on electron transport chain (ETC) activity for the re-oxidation of inner membrane dehydrogenases and pyrimidine biosynthesis [9]. The maintenance of the mitochondrial ETC is sustained in part, by the oxidation of pyruvate (diverted from glycolysis) and the uptake and catabolism of glutamine [7,10,11]. Pyruvate can enter the TCA cycle via two pathways; through anaplerotic reactions involving the CO2-fixing enzyme, phosphoenolpyruvate carboxylase (PEPC), or through the activity of a repurposed branched chain α-keto acid dehydrogenase (BCKDH) complex, which substitutes for the activity of the missing mitochondrial pyruvate dehydrogenase in Plasmodium and other apicomplexan parasites [12,13]. Despite the essentiality of the mitochondrion, operation of the TCA cycle is not required for intra-erythrocytic growth of P. falciparum [10,11,14,15]. Plasmodium spp. lack key enzymes involved in gluconeogenesis and all developmental stages are predicted to be dependent on the uptake of sugars. However, in contrast to the asexual blood stages, there is increasing evidence that the mosquito-infective stages of Plasmodium exhibit an increased dependence on the TCA cycle and mitochondrial metabolism [11,14,16,17]. In particular, transcriptomic and proteomic analyses [5,18–20] suggest that enzymes involved in TCA metabolism are elevated in Plasmodium mosquito stages. Specifically, Plasmodium gametocytes develop more complex tubular mitochondrial cristae suggestive of increased mitochondrial function [21]. Metabolomic analyses have confirmed increased TCA metabolism in P. falciparum gametocytes and demonstrated that this is essential for gametocyte maturation [10]. Recent genetic studies have also shown that the TCA cycle is essential for the development of P. falciparum mosquito stages [11], consistent with earlier work in P. berghei demonstrating that the TCA cycle and the electron transport chain are required for ookinete development and oocyst formation [14]. Interestingly, levels of sugars and glutamate/glutamine in the mosquito haemolymph are thought to be comparable to the levels in the blood [22–25] suggesting that these stage-specific shifts in parasite metabolism may be driven by factors other than the availability of specific carbon sources in the mosquito niches. Little is known about the metabolic flexibility of different Plasmodium mosquito stages and the extent to which parasite metabolism impacts on the development of these stages in the mosquito vector. In this study we have utilized a combination of metabolomic and reverse genetic approaches to investigate the metabolic changes that occur in key insect stages of the experimentally tractable species P. berghei and the potential impact of these changes on parasite infection in the mosquito. We find that these stages are highly sensitive to disruptions in multiple pathways in central carbon metabolism including the TCA cycle, the utilisation of glutamine as a carbon source, intermediary carbon metabolism and coenzyme A (CoA) synthesis. The strict dependency of these stages on multiple pathways in carbon metabolism opens new avenues for targeting parasite transmission.
  Uninfected RBC (uRBC) and synchronised P. berghei ring stage infected RBC populations (iRBC, 10% parasitaemia) containing ~35% reticulocytes were generated as described previously [26]. uRBC and iRBC were metabolically labelled with U-13C-glucose and U-13C15N-glutamine and harvested at various time points (0,6, 12,18 and 24 h) over the intra-erythrocytic developmental cycle. Asexual parasites from a recently generated gametocyte non-producer line (GNPm9) [27] were used to avoid any low level contamination of RBC asexual stages with gametocytes. U-13C-glucose and U-13C15N-glutamine labelling studies were performed on P. berghei gametocytes generated from the gametocyte producer parent line, RMgm-164 [27]. Mature gametocytes were obtained from P. berghei infected mice, magnetically purified and incubated for 2 h at 37°C in minimal media containing U-13C-glucose or U-13C15N-glutamine prior to activation. To form gametes, gametocytes were then switched to minimal media containing the gametocyte activation factor, xanthurenic acid [29] and labelled carbon sources at 21°C (mimicking mosquito midgut conditions). Cells were harvested at 0,10,20 and 30 min time points and metabolism was rapidly quenched prior to metabolite extraction and quantification of 13C enrichment by GC-MS. Gametocytes from the gametocyte producer parent line, RMgm-164 [27] as described above and RMgm-15 (Pb137, p48-/45-), which produces viable female gametes but non-viable male gametes [36], were activated and cultured for 21 h at 21°C. RMgm-164 produces fertilised female gametes which mature into ookinetes; therefore RMgm-15 was used to discriminate between activated, unfertilised and fertilised female gametes at the metabolic level. Gametes were labelled with U-13C-glucose or U-13C15N-glutamine; samples were collected after 10 h (retort stage) and 21 h (mature ookinetes) and magnetically purified at 21°C before rapidly quenching metabolism, metabolite extraction and quantification of 13C enrichment by GC-MS.
  Comprehensive 13C-labeling experiments confirmed that all P. berghei stages tested, including asexual RBC stages, gametocytes, gametes and ookinetes, exhibit high rates of glucose utilization which is required to drive anaerobic glycolysis (for ATP synthesis) and the pentose phosphate pathway, as well as pathways of sugar nucleotide and glycoconjugate biosynthesis. This dependence on glucose uptake and catabolism, which was confirmed in the case of microgametogenesis using the inhibitor 2-deoxyglucose, is not surprising as Plasmodium spp. lack the key gluconeogenic enzyme fructose 1,6-bisphosphatase and are therefore completely dependent on glucose uptake for de novo synthesis of hexose phosphates. Against this background of constitutive glucose utilization, we observed a progressive increase in the extent to which pyruvate was further catabolized in the TCA cycle following the differentiation of P. berghei asexual RBC stages to gametocytes and subsequently to mosquito stages (gametes and ookinetes) (Figs 1,3 and 6). Consistent with recent studies in P. falciparum [10,11,17] and P. berghei [13], we showed that pyruvate flux into the TCA is very low in P. berghei asexual RBC stages and that the low but detectable flux through the TCA cycle in these stages is largely maintained by the uptake and catabolism of glutamine (glutaminolysis) and a partial GABA shunt. The low overall flux of pyruvate and glutamine into the TCA cycle of P. berghei asexual stages suggests that this plays a minor role in ATP synthesis and may be required to maintain redox balance and membrane potential of the mitochondria. A proportion of asexual RBC stages differentiate to gametocytes, which are required for transmission and production of gametes in the mosquito midgut. Mature circulating gametocytes are cell cycle-arrested and sexually dimorphic, eventually producing male and female forms that have different biological activities upon transfer to the mosquito midgut environment. Labelling studies indicated that mature P. berghei gametocytes exhibit elevated levels of TCA cycle activity sustained by increased catabolism of pyruvate and glutaminolysis (Fig 3), as has also been observed in P. falciparum gametocytes [10]. The elevated TCA cycle flux in the gametocyte stages of both murine and human malaria parasites is associated with enlargement of the mitochondrion and increased cristae formation in both male and female gametocytes [37,38]. Following transmission to the mosquito vector, male and female gametocytes initiate quite distinct developmental programs. Most strikingly, female gametocytes retain a mitochondrion, while this organelle is largely degenerate in the mature male gametocyte [20,39–41]. Upon activation, the male gametocyte initiates three rounds of energy intensive rapid nuclear replication and packages the resulting nuclei into eight male gametes [42,43]. Our labelling experiments demonstrated that these motile parasite stages exhibit high rates of glycolysis which are likely to be the major source of ATP for male microgamete motility [30,31] and exflagellation. Inhibition of glycolysis with 2DG prevented exflagellation and microgametogenesis, but did not affect female gamete maturation. While female gametes may be able to switch to using glutamine as an alternative (short term) carbon source for ATP synthesis under these conditions, the absence of a functional mitochondrion and TCA cycle in male gametes prevent such a switch. The absence of an active TCA cycle metabolism in male gametes may also make these stages more dependent on enzymes involved in the interconversion of exogenously scavenged glutamine and glutamate (which is normally synthesized from TCA cycle intermediates in other stages) consistent with reduced male gamete production in the P. berghei mutants lacking genes involved in GABA and glutamine catabolism (described below) (Fig 4B). In the mosquito mid-gut, male and female gametes fuse to form an apolar zygote which further develops into a motile polar ookinete. Ookinetes must be able to sustain high motility in order to transit across the gut epithelial layer and escape from the hostile environment of the blood meal and the mosquito digestive tract. Our data demonstrate that maturing ookinetes are metabolically active and can co-catabolize glucose and glutamine via the TCA cycle. Interestingly, glutamine was the predominant carbon source utilized in the TCA cycle indicating a preference for glutaminolysis for ATP synthesis. This may indicate metabolic compartmentalization of glucose utilization and preferential direction of hexose phosphates into essential pathways such as the pentose phosphate pathway and nucleotide synthesis. Unfertilized activated female gametes also exhibited a similar metabolic programme to fertilized ookinetes, suggesting that fertilization is not a prerequisite for these metabolic changes. Even though glycolysis was confirmed to be present in uRBC, U-13C-glucose was found be consumed in iRBC much more rapidly than in uRBC (Fig 1A). This is in line with previous studies [44] and pointed towards the rapid turnover of glycolytic end products in iRBC to downstream metabolites via the TCA cycle and/or intermediary carbon metabolism or for rapid biomass generation to help in proliferative schizogony [45]. Although pre-existing pools of glucose in the host cell can contribute to glycolysis in the parasite, the direct role of the host cell glycolytic pathway in development of intracellular parasites is unclear as the parasite glycolytic machinery is well established [46] and is supported by increased internalisation of glucose into the infected cell owing to expression of glucose transporters on the surface of the infected cell [32]. However, the ability of the host erythrocyte to maintain its own ATP pools via glycolysis ensures its viability and supports the survival of the resident parasite. Moreover, the parasite can also supply the host cell with ATP in order to improve this process [47]. It is notable that host reticulocytes also retain residual mitochondrial metabolism and TCA cycle flux is fed by glutaminolysis, although this cycle appears to function primarily in reductive mode, with conversion of α-ketoglutarate (derived from glutamine) to citrate. Reductive carboxylation has been observed in cells with functionally compromised mitochondria [48] such as occurs in maturing reticulocytes [49]. In other systems, reductive metabolism facilitated by glutaminolysis has been shown to generate lipogenic acetyl-CoA [50,51]. The function of a reductive TCA cycle in P. berghei-infected reticulocytes and its impact on the intracellular parasite is unknown at this stage and it would be interesting to investigate whether host cell generated two-carbon units from this pathway contribute to parasite metabolism. In order to determine which, if any, of these stage-specific changes in P. berghei central carbon metabolism were important for parasite development and mosquito transmission, mutant lines lacking aconitase (ACO), a key enzyme required for catabolism of pyruvate in the TCA cycle and several enzymes involved in glutamate/GABA metabolism (GluS, GDH 1 and 3, LDC/GAD, OAT) were generated (S3 Table). Interestingly, a delayed growth phenotype was seen in aco- mutants in the asexual RBC stages which have the least active mitochondrial TCA cycle of all the stages and little effect was seen on the development of P. berghei gametocytes and gametes. Any further development of these mutants was blocked in the mosquito. These findings suggest that any loss of glycolytic pyruvate catabolism in the P. berghei TCA cycle is compensated for by either a much larger steady-state of pre-existing TCA cycle metabolites or increased glutaminolysis. The slow growth phenotype seen in P. berghei aco- mutant parasites could be caused by an imbalanced redox state in the mitochondria due to decrease in NADPH production. Furthermore, species-specific differences may exist to the extent that glutaminolysis compensates for loss of glucose catabolism in the TCA cycle. Specifically, disruption of the gene encoding aconitase [11] or chemical inhibition of aconitase [10] in P. falciparum inhibited gametocyte maturation. While this apparent requirement for aconitase for P. falciparum but not P. berghei gametocytogenesis may reflect species-specific differences in the metabolic networks of these parasites, it may also result from the different host cell environments occupied by the gametocytes of each species. Indeed we have previously shown that P. berghei is more permissive to other metabolic gene knock-outs than P. falciparum due to differences in their tropism for different host cells [26]. Moreover, it has been reported [14] that TCA cycle disruption by deletion of a gene of flavoprotein (Fp) subunit Pbsdha (PBANKA_051820), one of the four components of complex II, a catalytic subunit for succinate dehydrogenase activity also does not affect gametocyte maturation in P. berghei and the point of disruption (whether it is before the entry of alpha-ketoglutarate or after) does not seem to be crucial. As suggested in the P. falciparum aconitase disruption study [11], accumulation of citrate to toxic levels which seems to hinder the process of gametocytogenesis in P. falciparum over 7–10 days in aco- mutants does not happen in a 48 hour asexual life cycle of P. falciparum. As P. berghei has even shorter blood stage developmental stages (24 hour asexual life cycle, 30 hours for gametocyte maturation), cytotoxic effects of accumulating citrate may well be avoided. Gametocytes thus formed seem to be able to activate and form gametes as glycolysis is unaffected in these mutants but failed to develop further in the mosquito. Sexual crosses revealed that this defect was not sex specific and both male and female gametes were affected. The increased reliance of extra-cellular stages on a more efficient process of TCA cycle and oxidative phosphorylation for energy generation in the mosquito in the absence of the glucose rich environment of the mammalian host, halts its growth due to disruption of TCA metabolism in the aco- mutant as seen in other studies [11,13,14]. Disruption of glutamate synthase (GluS), the enzyme up-stream of glutamate dehydrogenases (GDHs) responsible for the reversible interconversion of glutamate and glutamine (with concomitant utilization/production of α-ketoglutarate and NAD/NADPH) also had no effect on the growth of asexual RBC stages, gametocyte development and ookinete motility, but resulted in a defect in exflagellation, reduced oocyst production and an effective block in sporozoite production and mosquito transmission. We were unable to generate P. berghei mutant clones lacking GDH2, an apicoplast-located enzyme responsible for conversion of glutamine to α-ketoglutarate. This isoform of GDH may contribute to mitochondrial metabolism through transfer of intermediates between closely linked apicoplast and mitochondrion [52,53]. Another essential role of GDH2 could be the production of NADPH for type II fatty acid synthesis, isoprenoid synthesis or ferredoxin reduction for maintaining the redox balance in the absence of a pentose phosphate pathway or a photosystem in this plant-derived organelle [54]. The deletion of the other two cytosolic GDHs (GDH1 and GDH3), individually or in combination, had no major impact on parasite development and is consistent with the findings in P. falciparum [55]. These results highlight the importance of glutamine catabolism, either for glutamate synthesis and/or catabolism in the TCA cycle in these latter stages. Consistent with this conclusion, previous studies have shown that defects in the TCA cycle enzymes downstream of the entry of glutamate carbon skeletons (i. e. succinate dehydrogenase in P. berghei [14] and α-ketoglutarate dehydrogenase in P. falciparum [11]) were also associated with impaired development of ookinetes and ookinete to oocyst transition. The absence of a distinct phenotype in other stages of P. berghei glus- mutant may reflect the extent to which glutamine-dependent transamindases can bypass the need for glutamate synthase and/or the availability of high glucose levels in blood, which may not be accessible in the mosquito vector. We have previously shown that glutamate can be converted to GABA via a glutamate decarboxylase (GAD) (annotated as a lysine decarboxylase (LDC) in Plasmodium [10]) in the related apicomplexan parasite, Toxoplasma gondii [28]. In Plasmodium spp. , GABA may be further converted to succinate semialdehyde by mitochondrial transaminases, providing a mechanism for balancing the levels of pyruvate and alanine. However Plasmodium spp. , unlike T. gondii, appear to lack the next enzyme in a canonical GABA shunt that converts succinate semialdehyde to succinate which would be required for catabolism of GABA in the TCA cycle) [28,46]. As such, it is unlikely that GABA catabolism acts as a shunt to by-pass key reactions in the TCA cycle down-stream of α-ketoglutarate. Transcriptomic studies have shown that ldc/gad mRNA is elevated in P. berghei gametocytes [56]. Disruption of ldc/gad in P. berghei did not affect asexual RBC stages and gametocyte formation, although exflagellation was reduced. Nevertheless, ldc-/gad- parasites could be transmitted through the mosquito, suggesting that GABA dependent transamination reactions in the mitochondrion are dispensable for P. berghei transmission. Curiously, it was not possible to obtain a clonal population for a P. berghei mutant lacking the putative GABA transporter (PBANKA_030670) [10]. This suggests that this transporter may be involved in transporting other substrates or that loss of transport may lead to toxic build-up of GABA in all stages. The enzymes phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK) play important roles in maintaining the levels of cytoplasmic pools of pyruvate, PEP and C4 dicarboxylic acids [7,12,26]. Transcription of pepck is up-regulated in P. falciparum gametocytes and zygotes [57] and P. berghei gametocytes and ookinetes [56] suggesting a role in mosquito stage development of the parasite. We demonstrated that PEPCK is not essential in blood stages and that pepck- mutants also exflagellated to form wild type-like gametes and gave comparable ookinete conversion rates. Furthermore, pepck- ookinetes were motile, although they failed to develop into mature oocysts and subsequent sporozoites, thus blocking transmission. PEPCK-derived PEP can be converted to pyruvate in the cytosol by the glycolytic enzyme, pyruvate kinase 1 [58] and be further catabolized in the mitochondrion TCA cycle [13] generating the ATP required for multiple rounds of DNA replication in sporogony. Alternatively it can enter the apicoplast where it can be converted to pyruvate and acetyl-CoA by pyruvate kinase 2 [59] and apicoplast-resident pyruvate dehydrogenase [60], respectively (Fig 2A). Apicoplast-derived acetyl-CoA is required for de novo fatty acid synthesis during sporozoite development in the mosquito by the FASII pathway in P. falciparum [61] and an unidentified route in P. berghei [62–64]. Pantothenic acid is the precursor for CoA, and essential co-factor for the oxidation of pyruvate (both in the mitochondrion and the apicoplast) and the substrate for acetyl-CoA synthesis. In P. falciparum it has been shown that structural analogues of pantothenate are growth inhibitors of the asexual blood stages in vitro [65,66]. In P. berghei, deletion of both pantothenate kinase 1 and 2 (pank1 & 2) enzymes (either individually or in tandem) generated mutants that grew similarly to wild type in the asexual developmental stages, but showed a defect in the ookinete to oocyst transition. The synthesis of CoA is not identical across the Plasmodium genus: the avian malaria parasite, P. lophurae, is incapable of de novo CoA synthesis and scavenges CoA from the host erythrocyte whereas both P. falciparum and P. berghei can synthesise CoA de novo [67]. However, P. berghei pank mutants may also scavenge CoA from the host reticulocytes since these cells are known to support the growth of metabolically-compromised P. berghei parasites [26], whilst P. falciparum, with its dependence primarily on mature RBCs in vitro, would lack such exogenous supplies. The CoA synthesis pathway in the parasite terminates in the apicoplast as the last enzyme of this pathway, dephospho-CoA kinase, is localised to this organelle [68]. It is possible that the CoA required for oxidation of pyruvate to power the TCA cycle (during mosquito stage development) or the CoA required for fatty acid synthesis (in oocyst sporogony) is compromised in the pank mutants, thereby arresting the ookinete to oocyst transition. Expression of both pank1 and pank2 is maximal in the ookinete stage in P. berghei [56] supporting the notion that CoA biosynthesis is important during transmission of P. berghei. As a number of inhibitors of this pathway are currently being investigated for asexual inhibition of the malaria parasite [65,66,69–71], these studies should be extended to assess the transmission-blocking capacity of these inhibitors. In summary, we have assessed the importance of central carbon metabolism in different blood and mosquito stages of the rodent malaria parasite P. berghei using 13C-stable isotope labelling, targeted metabolomics and reverse genetics. We show that these parasites rely on glucose metabolism throughout the life cycle, while co-utilized glutamine is the major driver of TCA metabolism and crucial to the development of the extra-erythrocytic ookinete stage and ookinete to oocyst transition. Both intermediary carbon metabolism and CoA synthesis are also important for ookinete to oocyst transition with a possible role in oocyst sporogony. This study adds to the existing knowledge of stage specific metabolism in the malaria parasite and also points at key differences between different Plasmodium species.
  All animal work was approved by the University of Glasgow’s Animal Welfare and Ethical Review Body and by the UK’s Home Office (PPL 60/4443). The animal care and use protocol complied with the UK Animals (Scientific Procedures) Act 1986 as amended in 2012 and with European Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes. For mouse infections, female Theilers Original (TO) outbred mice of body weight 26–30 g were used. Cryopreserved blood stages were thawed at room temperature and 0. 02–0. 5 mL of the suspension was injected intraperitoneally into a mouse. For infection with blood stages obtained from an infected mouse (mechanical passage), one droplet of tail blood (5 μL) was collected from an infected animal with a parasitaemia of 5–15% in 10 mL PBS and 0. 1 mL of the suspension was injected intraperitoneally into a mouse. On day 4–7 after injection the parasitaemia increased from 0. 1 to 5–20%. On day 4 or 5 after injection the parasitaemia ranged between 0. 5–3%. Unlabelled P. berghei cultures were maintained for one cycle using standard methods. Minimal medium (S1 Table) was used as the growing medium. A comparative analysis of growth of all P. berghei stages in vitro in our minimal media showed growth dynamics similar to RPMI1640 (S6 Fig). Flasks were gassed for 30 sec with a gas mix containing 5% CO2,5% O2, and 90% N2 and incubated overnight at 37°C on a shaker at a minimal speed just to keep the cells in suspension. P. berghei schizonts (from lines ANKA cl15cy1, RMgm-7 which expresses GFP constitutively under the eef1a promoter and from line RMgm-164 which expresses GFP in male gametocytes and RFP in female gametocytes) were transfected with linear DNA constructs containing the yfcu-hdhfr selectable marker flanked by homology arms (generated using primers in S2 Table) corresponding to 5’UTR and 3’UTR of the orf/catalytic domains of the gene of interest respectively for double crossover homologous recombination, injected intravenously in female TO mice and selected by pyrimethamine in drinking water as described in [72]. Resulting transfectants were analysed by PCR for 5’ and 3’ integration (using primers in S2 Table), cloned by limiting dilution and the absence/disruption of open reading frame in the mutants confirmed by PCR. Equal numbers of mutant parasites (106 cells) made in RMgm-7 background expressing GFP constitutively under eef1a promoter were mixed with wild type parasites (106 cells) (RMgm- 86) expressing RFP under the same promoter and the mixture was injected into a mouse. The population of infected erythrocytes (iRBCs) was monitored by Hoechst staining and the proportion of iRBCs expressing GFP and RFP was recorded by FACS analysis over the course of seven generations. Infected blood from the first mouse was sequentially passaged into two to three mice to avoid multiple infections over this period. Mutants made in the RMgm-164 background which expresses GFP in male gametocytes and RFP in female gametocytes along with wild type were grown in mice and peripheral blood was monitored by FACS analysis by checking for infected erythrocytes (iRBCs) by Hoechst staining and the proportion of iRBCs expressing GFP and RFP, indicative of the presence of male and female gametocytes. Mutants made in RMgm-7 background or ANKA cl15cy1 were stained with Giemsa and mature male and female gametocytes were quantified by manual observation and counting a minimum of 100 iRBCs. Mice infected with P. berghei were given sulfadiazine in drinking water which killed all asexual stage parasites in 48 h and circulating gametocytes remain in blood. Mice were bled and infected blood was collected in unlabelled RPMI1640 containing 5 g/L Albumax II and 100 μM xanthurenic acid to activate gametocytes. Cultures were incubated at 21°C for 21 h and Giemsa stained smears were made for counting mature ookinetes and female gametes whose cumulative ratio to female gametes only gave the ookinete conversion rate. Equal numbers of gametocytes from two P. berghei lines obtained from infected TO mice treated with sulfadiazine in drinking water were taken and mixed in activation media. The suspension was then incubated at 21°C for 21 h and Giemsa smears were made for counting mature ookinetes and female gametes. Ookinetes were embedded in Matrigel™ (BD Biosciences) and allowed to set for 1 h at 21°C. Fluorescent ookinetes were observed on a Leica M205 FA Fluorescence Stereomicroscope for 10. 5 min and snapshots acquired every 10 sec. Tracks were constructed using Fiji software. P. berghei parasites were pre-incubated in BSA enriched PBS with the addition of 2-deoxyglucose (2DG) at indicated concentrations at 37°C for 30 min. Parasites were then activated by replacing the media with activation media at 21°C containing the same concentration of 2DG and activation centres were counted on a haemocytometer after 30 min. Parasites were observed at later time points to confirm that there was no delayed activation. Female gametocytes emerged in the presence of 2-deoxyglucose were counted under fluorescent microscope after counterstaining with a fluorescent conjugated red blood cell marker Ter119. At least 50 female gametocytes were counted per experiment. To observe the effect of 2DG on ookinete development, gametocytes were activated then 2-deoxyglucose (25 mM) was added 30 min post activation (post). Alternatively parasites were incubated in BSA enriched PBS containing 25 mM 2-deoxyglucose for 30 min before activation in media also containing 2-deoxyglucose (pre). Conversion rates were calculated as the number of ookinetes as a percentage of the total ookinetes and unconverted females gametocyte. P. berghei infected mice with a parasitaemia of 5–10% were used to blood-feed a cage of 250 mosquitoes for 10 min. Mature oocysts were counted in mosquito midguts between days 8–14 using a Leica M205 FA Fluorescence Stereomicroscope. Salivary gland sporozoites were checked between days 21–25. Infected mosquitoes were allowed to feed on naïve mice for 10 min between days 21–25 and these mice were observed for parasites by making Giemsa stained blood smears between days 3–14 to check for successful transmission. Asexual and sexual stage parasites were cultured as described above in minimal media prepared based on known nutritional requirement of Plasmodium parasites in in vitro cultures [73–76] (S1 Table) lacking the unlabelled equivalent of the following labelled carbon sources, 13C U-glucose or 13C15N U-glutamine (Cambridge Isotope Laboratories) which were included at 2000 mg/L and 300 mg/L respectively. Briefly, for asexual cultures, parasites from the line 820m9w21dm1cl1 (gametocyte non-Producer) [27] were used to grow a synchronous infection in mice (pre-treated with phenylhydrazine-HCl 5 days before to induce reticulocytosis) with 10% parasitaemia at ring stage. Mice were bled and leucocytes were removed and each replicate containing 108 cells was incubated in 12 mL media containing unlabelled or the indicated labelled carbon source. The flasks were gassed and kept at 37°C with slight shaking at 35 rpm. Each flask was harvested to collect samples for 0 h, 6 h, 12 h, 18 h, and 24 h time points (the first time point was collected after allowing for cells to equilibrate for 2 h). Uninfected mice treated similarly were also bled at the same time and samples were processed exactly as for infected blood. For gametocyte cultures, parasites from line 820em1dcl2TBB (RMgm-164, the parent producer line) [27] were grown in mice and the mice were given sulfadiazine in drinking water (30 mg/L) when the parasitaemia reached ~30% for 48 h which led to killing of all asexual stage parasites and left mature circulating gametocytes. Mice were bled and parasites were harvested as described above. Gametocytes were magnetically purified and incubated for 2 h in labelled media at 37°C before activation to allow for metabolic equilibration and were then transferred to activation media at 21°C containing labelled carbon sources, harvested at 10 min, 20 min and 30 min time points and used for quenching metabolism and metabolite extraction (as the activation process is quick, cells were purified first then activated and quenched). Ookinete cultures were performed in activation media at 21°C containing labelled carbon sources, harvested at 10 h and 21 h, magnetically purified at 21°C and used for quenching metabolism and metabolite extraction. At each time point, flasks were immersed in a dry ice-ethanol bath to rapidly quench metabolism. The temperature of the cell suspension was monitored using a thermometer and the flasks were removed when the temperature decreased to 8°C. The culture suspension (12 mL) was then spun at 1300 × g at 4°C and supernatant was removed. The cells were washed once in 500 μL cold PBS and spun again at 1300 × g at 4°C and supernatant was removed. The pellet was resuspended in 150 μL extraction solvent (chloroform: methanol: water: 1: 3: 1 v/v) with dispersion of the pellet by pipetting and mixed vigorously on shaker in cold room for 1 h. The suspensions were then sonicated for 10 min in ice-cold water bath and then centrifuged for 5 min at 15,300 × g at 4°C. 50 μL of the supernatant was then put into glass vials, dried under nitrogen flow at room temperature and stored at -80°C before analysis. Extracts were reconstituted with extraction solvent containing an internal standard (scyllo-Inositol, SI, 1 nmol) and dried in vacuum. Then 20 μL of 30 mg/mL methoxyamine-HCl in pyridine was added to the vials, mixed and incubated overnight at 30°C. This was followed by addition of 20 μL of BSTFA + 1% TMCS Silylation reagent, mixing and overnight incubation at 37°C. 1 μL of the reconstituted and derivatised sample mix was injected onto an Agilent 7890A-5975C GC-MS instrument, equipped with a VF5-MS column (30 m, 0. 25 mm inner diameter) with helium as the carrier gas. The oven temperature was held at 70°C (1 min), then ramped at 1°C/min to 76°C, then 5°C/min to 325°C and held for 10 min. As a control, a mixture of glycolytic and TCA cycle standards were also derivatized and run with the samples to serve as authentic reference standards for retention time and spectra. Data analysis was performed manually on the Agilent ChemStation platform and metabolites were identified based on their GC retention time and mass spectra compared to authentic reference standards. The level of labelling was estimated as the percent of metabolite pool containing one or more labelled 13C atoms and the distribution of mass isotopologues of individual metabolites was corrected for naturally abundant isotopes in both the metabolite and derivatizing agent as described previously [10,77]. All stable isotope labelling data is available to download as a summary table in MS-Excel and raw MS files from the Figshare repository accessible from https: //figshare. com/s/aabe6ed0eba25acddcc1.
summary: Malaria kills almost half a million people worldwide every year and more than two hundred million people are diagnosed with this deadly disease annually. It is caused by the protozoan parasite Plasmodium spp. , mostly in sub-Saharan Africa and Asia and is transmitted by bites of infected female Anopheles mosquitoes. Due to an increase in resistance to existing drugs and lack of an effective vaccine, new intervention strategies which target development of parasite in human host and transmission through the mosquito vector are urgently needed. In this study, we explored the metabolic capacity of different developmental stages of the malaria parasite to determine carbon source utilization in different host niches and whether any stage-specific switches in metabolism could be exploited in new therapies aimed at eradicating malaria. Using stable isotope labelling and metabolomics, we have identified considerable nutritional adaptability of malaria parasites between the mammalian host and the mosquito vector. Gene disruption in the rodent malaria parasite P. berghei was used to identify the metabolic pathways which are crucial to the survival and development of the parasite. Our data also point at key metabolic differences in different Plasmodium species highlighting the importance of integrating metabolomics analyses with molecular tools and identifies possible transmission blocking candidates for malaria intervention.
keywords: carbohydrate metabolism
  parasite groups
  medicine and health sciences
  body fluids
  chemical compounds
  plasmodium
  metabolic processes
  gametocytes
  parasitic diseases
  organic compounds
  glucose metabolism
  parasitology
  germ cells
  apicomplexa
  acidic amino acids
  amino acids
  molecular biology techniques
  research and analysis methods
  cell labeling
  animal cells
  proteins
  chemistry
  molecular biology
  hematology
  biochemistry
  metabolic labeling
  blood
  cell biology
  organic chemistry
  anatomy
  physiology
  glutamine
  biology and life sciences
  cellular types
  citric acid cycle
  physical sciences
  metabolism
title: Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments