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. Author manuscript; available in PMC: 2016 Apr 7.
Published in final edited form as: Cell Rep. 2015 Apr 2;11(1):164–174. doi: 10.1016/j.celrep.2015.03.011

Genetic Investigation of Tricarboxylic Acid Metabolism During the Plasmodium falciparum Lifecycle

Hangjun Ke 1,8, Ian A Lewis 2,6,8, Joanne M Morrisey 1, Kyle J McLean 3, Suresh M Ganesan 1,5, Heather J Painter 2,7, Michael W Mather 1, Marcelo Jacobs-Lorena 3, Manuel Llinás 2,4,7, Akhil B Vaidya 1,*
PMCID: PMC4394047  NIHMSID: NIHMS670701  PMID: 25843709

SUMMARY

New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development.

INTRODUCTION

Malaria is a major global parasitic disease that is responsible for ~300 million infections and ~600,000 deaths per year (WHO, 2013). Although there are a number of effective antimalarial drugs available, the continued emergence of drug resistant parasites (Ariey et al., 2014) has made finding new treatments a global health priority. Some existing drugs and promising lead compounds target the parasite’s mitochondrial functions (Fry and Pudney, 1992; Nilsen et al., 2013; Phillips et al., 2008). The parasite’s mitochondrion is highly divergent from its human counterpart (Vaidya and Mather, 2009), which provides a basis for selective toxicity of antimalarial drugs. However, the tricarboxylic acid (TCA) cycle, a fundamental metabolic pathway within the parasite mitochondrion, has not been fully explored as a potential drug target.

Several lines of evidence support the existence of TCA reactions in the human malaria parasite, Plasmodium falciparum. The parasite’s genome encodes all of the TCA cycle enzymes (Gardner et al., 2002), which are expressed during the asexual stages (Bozdech et al., 2003). The eight TCA enzymes have been localized to the mitochondrion (Gunther et al., 2005; Hodges et al., 2005; Takeo et al., 2000; Tonkin et al., 2004; and unpublished data from the Vaidya laboratory), and TCA cycle intermediates are actively synthesized (Olszewski et al., 2009). More recently, isotopic labeling studies have demonstrated an active canonical oxidative TCA cycle. Glutamine and glucose are the main carbon sources for the TCA reactions in P. falciparum (Cobbold et al., 2013; MacRae et al., 2013). Glutamine carbon enters the cycle via α-ketoglutarate, while glucose appears to provide acetyl-CoA (Cobbold et al., 2013; MacRae et al., 2013), as well as some oxaloacetate (Storm et al., 2014), for entry at the citrate synthase (CS) step. The mitochondrial acetyl-CoA is produced from pyruvate by a branched chain ketoacid dehydrogenase (BCKDH) (Oppenheim et al., 2014).

Although recent studies have investigated metabolic flow through the TCA cycle in Plasmodium parasites (Cobbold et al., 2013; MacRae et al., 2013; Oppenheim et al., 2014; Storm et al., 2014), a broad analysis of TCA metabolism using genetic disruptions in P. falciparum has not been conducted until now. Previously, succinate dehydrogenase (SDH) was knocked out in the rodent parasite P. berghei (Hino et al., 2012), and knocked down in the human parasite P. falciparum (Tanaka et al., 2012), without associated metabolomic analyses. MacRae et al. conducted a metabolomic study of TCA and associated intermediates in P. falciparum combined with chemical inhibition of the single TCA enzyme aconitase (MacRae et al., 2013). Disruption of BCKDH in P. berghei forced the parasite to grow in reticulocytes (Oppenheim et al., 2014); consequently, reticulocyte metabolites might influence metabolomic analysis of this KO line. Storm et al. investigated the role of phosphoenolpyruvate carboxylase (PEPC) in P. falciparum but did not directly follow the TCA cycle enzymes (Storm et al., 2014). Therefore, we undertook a study to look at the essentiality, redundancy, and functions of the TCA cycle in P. falciparum. Here, we generated 11 KO lines, disrupting 6 of the 8 TCA cycle enzymes in P. falciparum, and analyzed phenotypic and metabolomic features of these KO lines in different lifecycle stages. The availability of these KO lines also provides a resource for further detailed metabolic studies.

RESULTS

TCA architecture in wildtype P. falciparum

To establish the baseline metabolic architecture of wildtype (WT) parasites, we incubated infected red blood cells (RBCs) (D10 strain, ~90% parasitemia at the late trophozoite/schizont stages) in a culture medium containing either uniformly 13C-labeled (U-13C) glutamine or U-13C glucose for 4 h and monitored the appearance of 13C in TCA intermediates by high performance liquid chromatography-mass spectrometry (HPLC-MS). As controls, uninfected RBCs were labeled with U-13C glutamine or U-13C glucose for 4 h. In agreement with a previous report (Ellinger et al., 2011), RBCs converted U-13C glutamine into glutamate and α-ketoglutarate, but no other TCA cycle intermediates (Table S2). RBCs did not convert U-13C glucose into TCA cycle intermediates during 4 h incubations (Table S2). In contrast, WT parasites readily converted U-13C glutamine into malate (Figure 1), in agreement with recent findings (Cobbold et al., 2013; MacRae et al., 2013). The abundant +4 isotopomers (normal mass plus four atomic mass units) of succinate, fumarate and malate observed indicated that TCA metabolism progressed through canonical oxidative reactions with the majority of carbon entering the cycle as α-ketoglutarate and leaving the cycle as malate (Figure 1). The presence of +4 citrate in these samples indicated that a small fraction of +4 malate was oxidatively converted into citrate. Oxidative t urning of the TCA cycle was further confirmed by analyzing metabolites extracted from parasites incubated in U-13C glucose. Mass spectrometry analysis showed low, but significant, isotopic enrichment in +2 citrate, +2 α-ketoglutarate, +2 succinate, +2 malate and +2 aspartate (a proxy for oxaloacetate, as it is normally in equilibrium with aspartate via transamination (Shen, 2005)) (Figure 1). The presence of +2 isotopomers in these samples is consistent with glucose-derived acetyl-CoA entering the TCA cycle (Cobbold et al., 2013; MacRae et al., 2013, Oppenheim et al., 2014). The abundance of glucose-derived carbon in TCA cycle intermediates was much lower than glutamine-derived carbon, indicating that glucose is a minor contributor to TCA flux in asexual blood stages (Figure 1). Similarly, the low intensity of the +5 citrate signal in U-13C glucose labeled samples indicated that anaplerotic carbon input from glucose (i.e. oxaloacetate from cytosolic PEPC reaction) was small. Our results are in general agreement with the recent publications (Cobbold et al., 2013; MacRae et al., 2013; Storm et al., 2014) showing that blood stage P. falciparum parasites carry out an oxidative TCA metabolism.

Figure 1. TCA architecture in the asexual blood stages of WT P. falciparum.

Figure 1

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Bar graphs show the percent isotopic enrichment (y-axes) for 13C isotopomers (x-axes) of TCA metabolites extracted from D10 WT parasites incubated for 4 h with either U-13C glucose (blue bars) or U-13C glutamine (orange bars). Please note different scales for distinct metabolites. These data are the average of three biological replicates, each carried out in triplicate. The molecular structures corresponding to the most abundant glucose and glutamine-derived isotopomers are shown (*). Abbreviations: KDH, α-ketoglutarate dehydrogenase; SCS, succinyl-CoA synthase; SDH, succinate dehydrogenase; FH, fumarate hydratase; MQO, malate quinone oxidoreductase; CS, citrate synthase; ACO, aconitase; IDH, isocitrate dehydrogenase. Cofactors: Q, ubiquinone; QH2, ubiquinol; NAD+, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate.

Most TCA cycle enzymes are dispensable in asexual blood stages

To determine if TCA cycle enzymes are essential for parasite survival, we attempted to knock out all eight TCA cycle enzymes through double crossover homologous recombination. We successfully knocked out six TCA enzymes, including genes encoding α-ketoglutarate dehydrogenase E1 subunit (ΔKDH, PF3D7_0820700), succinyl-CoA synthetase α subunit (ΔSCS, PF3D7_1108500), SDH flavoprotein subunit (ΔSDH, PF3D7_1034400), CS (ΔCS, PF3D7_1022500), aconitase (ΔAco, PF3D7_1342100) and isocitrate dehydrogenase (ΔIDH, PF3D7_1345700) (Figures S1, S2). In addition, we also produced three double KO lines: 1) ΔKDH/ΔSCS, which should prevent glutamine-derived carbon from entering the canonical oxidative TCA cycle and block all of the biosynthetic routes to succinyl-CoA; 2) ΔKDH/ΔIDH, which should prevent utilization of glutamine derived carbon in the TCA cycle; and 3) ΔSCS/ΔSDH, which should block oxidative turning of the cycle and substrate-level ATP generation in the mitochondrion (Figures S1, S2). In contrast, we were unable to disrupt the genes encoding fumarate hydratase (FH, PF3D7_0927300) and malate quinone oxidoreductase (MQO, PF3D7_0616800), despite multiple trials using a variety of approaches (data not shown), suggesting that these two enzymes may be essential in the asexual blood stages.

To test the growth phenotypes of all 9 KOs, we measured parasitemia over 4 to 5 generations (192–240 h) relative to the WT (D10) parasites. Surprisingly, no significant growth defects were detected in any of the KO parasite lines when parasites were grown in complete RPMI-1640 medium (data not shown). To assess the possibility that growth defects may become apparent under nutritionally restrictive conditions, we also examined the growth phenotypes of the ΔKDH/ΔIDH and ΔAco lines under various nutritional stresses (e.g., glucose, glutamine, and aspartate starvation) but found no differences between the KO and WT parasites (data not shown). These results show that TCA metabolism is not essential in asexual blood stages in vitro.

We also examined possible transcriptional alterations that may accompany the disruption of the TCA cycle during the intra-erythrocytic development cycle (IDC) in the ΔKDH/ΔIDH double KO line. A whole genome expression profile was determined through microarray analysis of RNA extracted from tightly synchronized parasite cultures sampled every 6 h over a 48 h period. There were only 37 genes that had a statistically significant change at every time point over the 48 h IDC (overall p across time < 0.002) (Table S3). Although these variations were statistically significant, there were no clear coordinated changes in expression of TCA cycle or mitochondrial electron transport chain (mtETC) genes that could directly compensate for the genetic ablations of KDH and IDH. All microarray data are deposited at Gene Expression Omnibus database (GEO accession number GSE59015).

Metabolic consequences of TCA cycle disruptions in asexual blood stage parasites

One possible explanation for the surprising absence of a growth phenotype in the KO parasite lines could be the presence of unannotated enzymes with redundant functions. To test this, we conducted a series of isotope labeling experiments and used the diagnostic pattern of isotopomers to determine the metabolic capacity of the parasites using our 9 KO lines. These experiments were conducted with U-13C glutamine, since this amino acid is the main carbon source for the TCA cycle in WT parasites (Figure 1, and (Cobbold et al., 2013; MacRae et al., 2013)). In general (but with some exceptions, see below), transgenic parasites incubated in U-13C glutamine showed a consistent phenotype across the panel of TCA KO lines: metabolites upstream of the disrupted enzyme show ed significant isotopic enrichment, whereas downstream metabolites showed significantly diminished levels of enrichment (Figure 2). The ΔSDH parasites, for example, accumulated +4 succinate (p<0.01) but showed no appreciable production of +4 fumarate (p<0.001) or +4 malate (p<0.001). Similarly, the ΔKDH line, which interferes with the first committed step in TCA-related glutamine utilization, resulted in no detectable downstream labeling (p<0.001 for all comparisons). These data show that P. falciparum does not contain redundant enzymes to bypass the deleted TCA enzymatic steps.

Figure 2. Metabolic consequences of TCA cycle disruptions in the asexual blood stages.

Figure 2

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(Top) A linearized depiction of oxidative TCA metabolism showing each of the expected isotopomers produced from U-13C glutamine labeling. Among them, aspartate (*) undergoes rapid exchange with oxaloacetate (oxaloacetate cannot be stably measured by the methods used in this work); +2 succinate (far right) is derived from a second round of the TCA cycle. Levels of isotopically enriched metabolites observed in extracts from uninfected RBCs, D10 WT and 9 different TCA KO lines are shown. For each line, data are averaged from at least three biological replicates, which were carried out in duplicate or triplicate. Each row shows a complete profile of the TCA cycle metabolites. Each column corresponds to the ratio of isotopomer in each KO line relative to the D10 WT. Orange circles show the positions of carbons labeled by U-13C glutamine. Red triangles represent the ratio of each individual measurement relative to the D10 WT. Grey triangles indicate metabolites that are below the threshold of detection. Blue Xs indicate the enzymatic steps that were disrupted in the KO lines. Abbreviations: Glu, glutamate; αKG, α-ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Asp, aspartate; Cit, citrate. For genetic and phenotypic analyses of these KOs, see Figures S1, S2 and Table S3.

Although the majority of the parasite lines showed the anticipated metabolic accumulation upstream of the deleted enzymes, the ΔSCS and ΔIDH lines showed deviations from the overall pattern. In the case of the ΔSCS line, a reduced level of isotope labeling was observed in metabolites downstream of succinyl-CoA (Figure 2). Metabolic flux past the deleted enzyme could be attributable to the spontaneous conversion of succinyl-CoA to succinate (Simon and Shemin, 1953). In the ΔIDH line, parasites showed unexpectedly diminished levels of labeling in metabolites upstream of IDH (Figure 2), while the upstream flux in ΔCS and ΔAco lines was not affected (Figure 2). The mechanisms behind the diminished levels of TCA intermediates in ΔIDH line are unclear at this point and need further investigation.

Mixing of glucose- and glutamine-derived carbon in the mitochondrion

Citrate is a diagnostic metabolite of TCA metabolism that is only generated in the parasite mitochondrion. Our ΔAco line is a convenient tool in this context because it accumulates citrate (Figure 2) and thus amplifies the mitochondrial signal. As shown in Figure S3, we incubated WT and ΔAco parasites in medium containing 2-13C glucose (only 1 carbon at position 2 is labeled) plus U-13C glutamine and analyzed the isotopomer pattern of citrate. Infected cells incubated in the dual glucose/glutamine labeled medium showed significant accumulation of +5 citrate (p<0.001), which arises when glutamine-derived +4 oxaloacetate condenses with glucose-derived +1 acetyl-CoA (Figure S3). Importantly, these data also suggest that the two carbon substrate, acetyl-CoA, is only derived from glucose (not from glutamine), most likely via the BCKDH reaction (Oppenheim et al., 2014).

Plasticity of TCA metabolism in P. falciparum

Our glutamine labeling data showed that enzyme redundancy does not play a role in the survival of the TCA KO parasites (Figure 2). Another strategy that organisms can use to compensate for metabolic deficiencies is to increase the flow of carbon through alternative pathways. To test this possibility, we incubated parasites in U-13C glucose and examined the isotope labeling of TCA-related metabolites. Glucose-derived carbon enters the TCA cycle via two classical mechanisms: 1) as two carbon acetyl-CoA units, which balance the two CO2 molecules lost on each turn of the cycle and 2) via anaplerotic reactions (i.e. PEPC reaction) that contribute four-carbon oxaloacetate or malate to the cycle (Cobbold et al., 2013; MacRae et al., 2013; Oppenheim et al., 2014; Storm et al., 2014).

Intracellular metabolites extracted from parasites grown in U-13C glucose showed that TCA KO parasites (ΔAco, ΔKDH, ΔIDH, ΔKDH/ΔIDH, and ΔSDH) accumulated the intracellular products of PEPC (i.e. +3 aspartate and +3 malate) to levels 1.7–5.3 times higher than those seen in the WT (Table S4; p< 0.02 for all pairwise comparisons to WT; p = 0.06 for ΔAco). In addition, an analysis of metabolites excreted into the growth medium indicated that TCA KO parasites committed significantly more of their glucose-derived carbon to mitochondrial reactions (p< 0.05, Figure 3). As illustrated in Figure 3A, PEPC-derived metabolites can be divided into pre- and post-mitochondrial species, which can be differentiated on the basis of their isotopomer patterns. Pre-mitochondrial metabolites include +3 malate and +3 aspartate (as surrogate for +3 oxaloacetate), whereas post-mitochondrial metabolites include +5 citrate, +4/+5 α-ketoglutarate, and +4/+5 glutamate. The concentrations of these pre- and post-mitochondrial metabolites in the medium excreted by the D10 WT, ΔKDH and ΔKDH/ΔIDH lines are shown in Figure 3B. In WT parasites, 89% of the excreted PEPC-derived carbon pool was pre-mitochondrial (+3 malate and +3 aspartate) (Figure 3C). Thus, the majority of this potentially anaplerotic carbon pool was excreted without having been committed to mitochondrial reactions. In contrast, the KO lines committed significantly more of this glucose-derived carbon to mitochondrial reactions. As shown in Figure 3C, the percentages of post-mitochondrial metabolites in ΔKDH and ΔKDH/ΔIDH lines increased up to 3 fold in comparison to the WT. In ΔKDH parasites, post-mitochondrial PEPC-derived carbon was excreted primarily as +4/+5 glutamate whereas ΔKDH/ΔIDH primarily excreted +5 citrate (Figure 3B). This excretion pattern is consistent with the intracellular labeling patterns of these KO lines (Figure S4). These data showed that 1) parasites can draw on either glucose or glutamine as significant carbon sources for the TCA cycle, 2) parasites can secrete a variety of mitochondrial metabolites into the medium, and 3) KO parasites with impaired glutamine utilization commit a significantly higher proportion of their glucose-derived carbon to mitochondrial TCA reactions.

Figure 3. Anaplerotic compensation for impaired glutamine metabolism.

Figure 3

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(A) A schematic representation of the cytosolic and mitochondrial pathways used by PEPC-derived oxaloacetate. Blue arrows depict glucose utilization without mitochondrial participation (pre-mitochondrial flux) and red arrows indicate glucose utilization involving mitochondrial processes (post-mitochondrial flux). (B) Extracellular concentrations of individual metabolites in D10 WT, ΔKDH, ΔKDH/ΔIDH lines. (C) Pre- and post-mitochondrial metabolites excreted into the medium by the D10 WT and various KO lines. The total excretion of glucose-derived carbon through PEPC is the sum of the concentrations of +3 malate and +3 aspartate (pre-mitochondrial, blue bars), and +5 citrate, +4/+5 α-ketoglutarate and +4/+5 glutamate (post-mitochondrial, red bars). Data are derived from three biological replicates. The glucose labeling patterns in other TCA KO lines from the parasite pellet samples are shown in Figure S4 and Table S4.

Mitochondrial electron transport chain inhibition blocks flux through the TCA cycle

The mitochondrial electron transport chain (mtETC) is an established target of antimalarial drugs. To assess the connection between the TCA cycle and mtETC in P. falciparum, we conducted metabolic analyses in parasites under conditions where the mtETC was inhibited at Complex III by atovaquone (Fry and Pudney, 1992; Srivastava et al., 1997). Atovaquone-treated parasites are unable to recycle ubiquinol to ubiquinone and thus become functional KOs for all ubiquinone-requiring enzymes including SDH, MQO, and dihydroorotate dehydrogenase (DHOD). Since atovaquone is toxic to WT parasites, these experiments were conducted with an mtETC independent transgenic line that expresses the cytosolic ubiquinone-independent Saccharomyces cerevisiae DHOD (yDHOD) (Ke et al., 2011; Painter et al., 2007). These parasites have a functional mtETC that can be inhibited by atovaquone, but are able to grow in the presence of the drug because they generate pyrimidines via yDHOD. WT and yDHOD-transgenic parasite lines were treated with or without 100 nM atovaquone (~100 times EC50) in medium containing U-13C glucose for 4 h. Labeling data showed that atovaquone-treated parasites did not assimilate glucose-derived carbon into TCA intermediates (Figure 4). The characteristic +2 isotopomers of TCA metabolites typically observed in control parasites were completely eliminated in atovaquone-treated parasites (Figure 4, Figure S5). Therefore, our data indicate that mtETC inhibitors prevent flux through the TCA cycle, as well as block the electron transport chain.

Figure 4. mtETC inhibition blocks the TCA flux.

Figure 4

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(A) A schematic representation of carbon input from glucose into the TCA metabolites. Blue arrows/circles show anaplerotic input through PEPC reaction, while gold arrows/circles indicate flux from acetyl-CoA. Atovaquone (ATV) blocks the mtETC, thereby inhibiting MQO. (B) Enrichment of each metabolite in yDHOD transgenic parasites in the presence or absence of ATV is shown. The +2 and +5 citrate isotopomers are diagnostic of flux through the citrate synthase reaction. Data are the average of three biological replicates. A more comprehensive set of TCA cycle isotopomers is presented in Figure S5.

TCA metabolism is essential for malaria transmission

All KO lines described above were derived from the P. falciparum D10 strain that is defective in sexual stage conversion. Therefore, we knocked out the KDH-E1 and Aco genes, individually, in the gametocyte-producing line NF54 strain. Disruption of KDH prevents glutamine-derived carbon from entering the TCA cycle, while Aco disruption blocks the full utilization of glucose as a TCA carbon source at an early step.

As observed for D10 lines, neither ΔKDH nor ΔAco in the NF54 background exhibited growth defects in asexual stages (Figure S6A), suggesting that TCA metabolism is conserved among different parasite genetic backgrounds. When induced to generate gametocytes, NF54-ΔKDH and NF54-ΔAco parasites behaved differently. NF54-ΔKDH was able to fully complete the gametocytogenesis process, forming mature stage V gametocytes in about 7–10 days (Figure S6B) with gametocytemia indistinguishable from that of WT NF54 (Figure 5A). In addition, there was no significant difference in the exflagellation profile of NF54-ΔKDH compared to the WT parasites (Figure 5B), suggesting that disruption of the TCA cycle did not harm gamete formation. Although the NF54-ΔAco line progressed normally until stage III, it failed to form mature stage V gametocytes (Figure S6C–D). Overall gametocyte production in NF54-ΔAco line was largely diminished as well (Figure 5D). Due to its lack of stage V gametocytes, NF54-ΔAco did not form gametes (Figure 5E). Our observations now confirm the role of Aco in gametocytogenesis, and are consistent with the results observed when parasites were treated with 10 mM sodium fluoroacetate (NaFAc), an Aco inhibitor (MacRae et al., 2013).

Figure 5. TCA cycle is essential to mosquito stage development of P. falciparum.

Figure 5

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(A) Gametocytemia of NF54 WT and NF54-ΔKDH parasites on days 14–20 post induction. (B) Exflagellation percentage in NF54 WT and NF54-ΔKDH lines on days 14–20 post induction. (C) Infectivity of NF54 WT and NF54-ΔKDH parasites as measured by the number of oocysts per mosquito on day 8 after blood feeding. Panels D to F correspond to panels A to C, respectively, showing results from NF54-ΔAco line. In (C) and (F), data are from two independent feeding experiments. Morphological data are shown in Figure S6.

To assess the direct requirement of TCA flux and mtETC for exflagellation (gamete formation), mature WT NF54 parasites were incubated with atovaquone prior to exflagellation induction. As shown in Figure S6E, incubating mature WT gametocytes with 100 nM atovaquone for up to 24 h had no effect on the parasite’s exflagellation rate. These data indicate that mtETC and TCA fluxes are dispensable for male gamete formation by mature gametocytes. The absence of defects in NF54-ΔKDH parasites is also consistent with the conclusion that halting TCA metabolism is not harmful for gametocyte or gamete development. Thus, the defect in NF54-ΔAco gametocytes may be due to reasons other than disruption of the TCA cycle (see Discussion).

To examine the role of TCA metabolism in mosquito stages, female Anopheles gambiae mosquitoes were fed with blood containing mature gametocytes derived from NF54 WT or NF54-ΔKDH parasites. The ability of parasites to mate successfully and develop further in the insect was assessed by counting oocysts in each mosquito 8 days after the blood feed. We found that none of the mosquitoes fed on blood with NF54-ΔKDH gametocytes produced oocysts, while those fed on blood containing WT gametocytes generated normal numbers of oocysts (Figure 5C). As expected, there were no oocysts formed in mosquitoes fed on NF54-ΔAco gametocytes (Figure 5F), which fail to progress to mature gametocytes or gametes (Figure S6D). Since KDH disruption had no effect on gametocyte development and gamete formation (Figure 5A–B), the inability to form oocysts in this KO suggests that a fully functional TCA metabolism is only essential for parasite development in mosquitoes subsequent to gamete formation.

DISCUSSION

By combining genetic manipulation and metabolic analysis in unprecedented detail, this study significantly clarifies our understanding of TCA metabolism in the human malaria parasite, P. falciparum. The derivation of 11 KO lines also provides a resource for future investigations of the biological consequences of TCA cycle disruptions. Our study reveals several key findings, including: 1) the TCA cycle is not essential for parasite survival in asexual blood stages, but is required for parasite transmission; 2) the TCA metabolism can utilize different carbon sources and alter its pathway fluxes when the normal flux is disrupted; and 3) this metabolic plasticity is essential for parasites to meet bioenergetic demands in the multiple stages of their complex lifecycle. A schematic representation of our principle findings is illustrated in Figure 6.

Figure 6. Models for TCA metabolism under various conditions.

Figure 6

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A schematic representation of TCA flux is shown for (A) WT, (B) ΔAco, (C) ΔKDH, and (D) atovaquone-treated parasites. Metabolic fluxes are depicted qualitatively as major (thick blue lines), minor (thin black lines), or zero (dotted lines). Some minor fluxes have been excluded for clarity of presentation. At the bottom of each panel, the progression of the parasite lifecycle is indicated. IDC, intra-erythrocytic development cycle, is comprised of ring (R), trophozoite (T) and schizont (S). Gametocyte development progresses from stage I to stage V. Mosquito stages include gamete (G), zygote (Z), ookinete (OK) and oocyst (OC). Proposed reasons for developmental arrest at each of these s tages are indicated by numbers within red octagons.

Our baseline metabolic data collected on WT parasites are in agreement with the current understanding of TCA cycle function in the parasite (Cobbold et al., 2013; MacRae et al., 2013; Oppenheim et al., 2014; Storm et al., 2014). Malaria parasites possess a canonical oxidative TCA cycle with the majority of TCA flux in asexual blood stages flowing from glutamine-derived α-ketoglutarate to malate (Figures 1, 2, 6). Beyond these observations, we show that the parasite’s mitochondrion can alter metabolic fluxes through alternative pathways when one route of carbon utilization is blocked. This metabolic plasticity was illustrated by our observations of substrate utilization in mutant lines lacking various TCA cycle enzymes (Figures 3, 6, S4). Most TCA KO lines showed significant elevation in their utilization of glycolytically-derived carbon in the remaining TCA reactions. In the extreme case of the KDH deletion, which prevents parasites from using glutamine as a carbon source for TCA reactions, parasites excrete glucose-derived glutamate into the medium (Figures 3). Thus, instead of the flow of carbon proceeding from glutamate to malate as in the WT (Figure 6A), in ΔKDH parasites it proceeds from glucose-derived malate to glutamate, utilizing the MQO to IDH segment of the cycle (Figure 6C). This metabolic plasticity may allow multiple carbon sources to feed the TCA cycle at various times in the parasite’s complicated lifecycle.

A previous study in a related Apicomplexan parasite, Toxoplasma gondii (MacRae et al., 2012), provided evidence for a γ-aminobutyric acid (GABA) shunt, in which succinate is generated directly from α-ketoglutarate via the action of GABA α-ketoglutarate aminotransferase, glutamic acid decarboxylase, and succinic semialdehyde dehydrogenase enzymes. By extension, it has been proposed that a similar GABA shunt might also operate in P. falciparum (McRae et al. 2013), although genes encoding all the requisite enzymes for the shunt cannot be detected in the P. falciparum genome. However, we found that the level of +4 succinate in the ΔKDH parasites labeled with U-13C glutamine was very low (Figure 2), which would not be expected if a robust GABA shunt were to bypass the KDH reaction to feed the downstream TCA reactions. In light of the lack of evidence for GABA shunt enzymes in P. falciparum, we propose that the low level of +4 succinate in the ΔKDH line is likely a product of widely conserved α-ketoglutarate-dependent oxygenases, which generate succinate through iron- and oxygen-dependent decarboxylation of α-ketoglutarate (Schofield and Zhang, 1999). These enzymes carry out various protein hydroxylation and histone demethylation reactions, and genes encoding them are found in the P. falciparum genome (e.g. lysine-specific histone demethylase 1, PF3D7_1211600; and JmjC domain containing protein, PF3D7_0809900).

In asexual blood stages, parasites are remarkably resistant to disruption of TCA metabolism; six of the eight TCA cycle enzymes can be deleted with no detectable growth defects (data not shown). In the absence of redundancy (Figure 2), these data show that a full turning of the TCA cycle is not essential in asexual blood stages. The dispensability of TCA metabolism in blood stages extends to TCA-dependent pathways. For example, provision of succinyl-CoA via the TCA cycle to the heme biosynthetic pathway is not essential for blood stage P. falciparum (Ke et al., 2014) or P. berghei (Nagaraj et al., 2013).

It was surprising to observe that disruption of the TCA cycle at the KDH step had no significant effect on sexual differentiation and gamete formation (Figure 5). Previous studies have shown up-regulation of the TCA enzymes in gametocytes (Young et al., 2005), suggesting functional importance of the TCA cycle in gametocytogenesis. However, our results with the ΔKDH line indicate that a complete turning of the TCA cycle is not essential for sexual differentiation. In contrast, the parasite line lacking aconitase failed to produce mature gametocytes and gametes (Figure 5). Our results now provide genetic evidence to confirm the importance of aconitase in gametocytogenesis shown by MacRae et al. using the inhibitor NaFAc (MacRae et al., 2013). The specific necessity for aconitase for later gametocyte development might be due to the following possible reasons: 1) aconitase is required for the production of mitochondrial NADPH, which is crucial for maintaining the mitochondrial redox balance, defense against oxidative damage and sustaining NADPH-dependent biosynthetic enzymes; 2) aconitase converts citrate to downstream metabolites, preventing the accumulation of citrate to a potentially toxic level. The fact that asexual stages of ΔAco and ΔIDH parasites are not affected may suggest that the 48 h lifecycle is insufficient to produce lethal effects, whereas the 7–10 days required for gametocyte maturation results in accumulation of damage beyond the threshold of tolerability in the ΔAco parasite.

In stark contrast to the asexual blood stages, parasite development in mosquitoes was completely inhibited by TCA cycle disruptions (Figure 5C). These results clearly establish the evolutionary necessity for maintenance of the TCA cycle by P. falciparum, since inhibition of transmission through mosquito would render the parasite extinct. This observation also suggests a major switch in mitochondrial functions as the parasite transitions from its vertebrate to invertebrate host. The motile ookinete has to survive outside a host cell for 24 h and invade the mosquito midgut epithelium. Whereas blood stage parasites are able to thrive through substrate-level generation of a mere 2 ATP molecules per glucose via glycolysis, survival in mosquitoes may place a much greater value on more economical energy generation through oxidative phosp horylation powered by the TCA cycle. In the ΔKDH line, input of TCA derived electrons into mtETC would be significantly reduced, thereby decreasing mitochondrial oxidative efficiency, potentially causing parasites to arrest due to energy insufficiency. Observations of defective mosquito stage development by P. berghei with gene KOs of SDH (Hino et al., 2012), NADH dehydrogenase (Boysen and Matuschewski, 2011) and BCKDH (Oppenheim et al., 2014) also lend support to this argument.

Interestingly, we found that two of the TCA cycle enzymes, FH and MQO, could not be genetically ablated even in asexual blood stages. The inability to disrupt the MQO gene was surprising, since we have previously shown that biochemical activity of the enzyme could be functionally disrupted in yDHOD transgenic parasites (Ke et al., 2011; Painter et al., 2007). One possible reason could be an essential non-enzymatic structural function of MQO in mitochondrial biogenesis. This interpretation is supported by the observation that MQO is conserved among all Apicomplexan parasites, including Cryptosporidium species, which have lost all other mtETC proteins (Abrahamsen et al., 2004). The essentiality of FH, on the other hand, could be explained by its role in a fumarate cycle serving the purine salvage pathway, as suggested by Bulusu et al. (Bulusu et al., 2011). Plasmodium parasites are unable to synthesize purines and rely entirely on the purine salvage pathway. Two important enzymes in this pathway are adenylosuccinate synthase and adenylosuccinate lyase. These enzymes utilize aspartate and generate fumarate in the process of converting inosine monophosphate to adenosine monophosphate. Mitochondrial FH could convert fumarate derived from purine salvage into malate in the mitochondrion, which could be transported to the cytosol and converted back to aspartate through successive reactions with malate dehydrogenase and aspartate aminotransferase. The fact that we can completely eliminate TCA-derived malate in several KO lines (Figure 2) suggests that the role of FH in the TCA cycle is dispensable. The essentiality of FH is, therefore, likely related to the requirements of the purine salvage pathway. It is interesting to note that two mitochondrial enzymes critical for parasite survival in blood stages, DHOD and FH, appear to serve pyrimidine biosynthesis and purine salvage, respectively.

Storm et al. found that PEPC could be knocked out, but only when the parasites were provided with malate or fumarate in the medium (Storm et al., 2014). These authors suggested that PEPC is a key enzyme in P. falciparum central carbon metabolism by providing anaplerotic carbon for the TCA cycle. However, our ΔAco, ΔCS, and ΔIDH lines grew normally despite their inability to incorporate anaplerotic carbon from glucose into downstream TCA reactions. Moreover, the survival of ΔIDH parasites indicates that mitochondrial NADPH production may not be essential in asexual blood stages, in contrast to the suggestion made by Storm et al. (Storm et al., 2014). Consequently, we attribute the essentiality of PEPC to factors other than maintenance of the TCA cycle.

Our analysis of atovaquone-treated yDHOD transgenic parasites revealed another unexpected metabolic finding: oxaloacetate is evidently not transported into the mitochondrion, but malate is (Figure 4, S5). This is surprising given that the parasite’s mitochondrial dicarboxylate-tricarboxylate carrier (PfDTC) efficiently transports oxaloacetate in vitro (Nozawa et al., 2011). The absence of +2 and +5 citrate, despite normal levels of aspartate (observed as surrogate for oxaloacetate), argues that PfDTC does not import oxaloacetate into the mitochondrion in vivo (Figures 4, S5). Data from ΔKDH/ΔIDH line treated with atovaquone also support this argument (Figure S5); the residual signals (+2 and +5 citrate) were likely due to the delayed effect of atovaquone, which was added concurrently with 13C-glucose to the culture during the 4 h labeling. This observation also explains the parasite’s propensity for secreting malate when using glutamine as a carbon source for TCA metabolism. Specifically, mitochondrial α-ketoglutarate/malate transport may be inherently linked through the action of an antiporter (presumably PfDTC).

It was interesting to note that upon atovaquone treatment, the level of +3 succinate accumulated to a much higher degree in D10 WT parasites when labeled with U-13C glucose (Figure S5). Since input from glucose into the TCA cycle is inhibited under this condition, one potential source of +3 succinate could be a reverse reaction of SDH that reduces fumarate to succinate. Fumarate reductase activity was proposed by a previous study (Takashima et al., 2001). At this point the nature of the electron donor for this reaction remains unclear, but it could be the high level of ubiquinol that would accumulate upon inhibition of the cytochrome bc1 complex.

Our demonstration that the antimalarial drug atovaquone eliminated anaplerotic carbon input from glucose into TCA metabolism has implications for the potent efficacy of mtETC inhibitors in blocking transmission of parasites to mosquitoes (Fowler et al., 1994; Nilsen et al., 2013). Greater demand for mitochondrial contribution to bioenergetics in insect stages would make mtETC, and the TCA cycle that primes it with reducing equivalents, more critical for parasite survival. Our results show that mtETC inhibitors not only affect parasite respiration but also interfere with the TCA cycle (Figures 4, S5). This assault on mitochondrial functions is the likely reason for the exquisite sensitivity of the mosquito stage development of parasites exposed in the mammalian host to mtETC inhibitors (Nilsen et al., 2013). While our results clearly argue against potential TCA cycle inhibitors as antimalarial drug leads, since they are unlikely to inhibit blood stage parasite growth, such compounds could serve as potent transmission blocking agents. One TCA cycle enzyme with potential to be an attractive drug target is FH, inhibition of which would be effective at both vertebrate and invertebrate stages of the parasite. Selectively toxic compounds could be envisioned since the parasite possesses a type I FeS-dependent FH, which is substantially different from the human type II enzyme (Woods et al., 1988).

In summary, this study describes a comprehensive analysis of TCA cycle function in the human parasite P. falciparum. As shown in Figure 6A, malaria parasites maintain an oxidative TCA cycle with the main flux supplied by glutamine. In ΔAco parasites (Figure 6B), maturation of gametocytes is prevented, perhaps due to an accumulation of damage related to the loss of mitochondrial NADPH production and/or high levels of citrate. In the ΔKDH line (Figure 6C), blood stage parasites metabolize glucose into glutamate, while the insect stage parasites fail to survive. Compounds targeting the mtETC, such as atovaquone, completely block the flux of metabolites through the TCA cycle (Figure 6D). Our study also reveals that the parasites have a flexible carbon metabolism, which may be important for making the transitions between the different environments encountered during the lifecycle. In addition, the availability of 11 different KO lines described here provide an important resource for investigating biological consequences of disrupting the TCA cycle under various conditions, such as nutritional restrictions, in P. falciparum.

EXPERIMENTAL PROCEDURES

Gene KO protocol

WT P. falciparum parasites were transfected with each KO construct and subjected to positive and negative selections. Deletion of the target gene was mediated via double cross-over recombination.

Metabolite labeling and HPLC-MS

Uninfected RBCs from heparinized blood were sedimented through 65% Percoll to remove any contaminated reticulocytes, platelets, and white blood cells prior to parasite culturing. Parasites were tightly synchronized, grown to late trophozoite stage, and infected RBCs were isolated by density centrifugation using a Percoll step gradient (35%, 60%, and 65%). Purified parasites (late trophozoite to schizont stages) were incubated with the labeling medium for 4 h. All of the isotopes used in this study (U-13C glutamine, U-13C glucose, and 2-13C-glucose) were 99% pure and were purchased from Cambridge Isotope Laboratories, Inc.

Methanolic metabolite extracts were dried under a stream of N2 gas and reconstituted in 200 µL (4 times the original extraction volume) of HPLC-grade H2O. High-resolution MS data were collected on a Thermo Scientific Exactive mass spectrometer in negative mode using ion-pairing C18 chromatography following previously published methods (Lu et al., 2010). Metabolite data were analyzed using MAVEN (Melamud et al., 2010) and isotopomers were corrected for naturally-occurring 13C using established methods (Fan et al., 2014). Data from technical replicates were averaged and error bars report error across biological replicates. All p values were calculated by two-tailed t-test. Raw data for all metabolites are provided in Supplementary Table S5 (for U-13C glutamine labeling of the wild type and KO lines) and Supplementary Table S6 (for multiple stable isotope labeling of the wild type and selected KO lines).

For all other procedures, please see Supplementary Experimental Procedures.

Supplementary Material

1
2
3
4

ACKNOWLEDGEMENT

We thank Dr. Praveen Balabaskaran Nina for providing the SDH KO construct and April M. Pershing for assistance with parasite culture. We thank Abhai Tripathi and Chris Kizito of the JHMRI Parasite and Insectary core facilities for their help. We also thank John Miller for help in creating Figures 1 and 6, and Jing Fan and Junyoung Park for their assistance with isotope correction. This project was funded by a grant from NIH (R01 AI028398) to A.B.V and support from the Burroughs Welcome Fund, an NIH Director’s New Innovators award (1DP2OD001315-01), and the Center for Quantitative Biology (P50 GM071508) to M.L.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Author Contributions: This work was designed by A.B.V, H.K., M.W.M., M.L. and I.A.L.; H.K. and J.M.M. generated the KOs and conducted the isotope labeling experiments and other analyses; HPLC-MS and data analyses were done by I.A.L. S.M.G. made MQO KO attempts. K.J.M. and M.J-L conducted mosquito feeding experiments. H.J.P. analyzed the microarray data. H.K., I.A.L, M.W.M, M.L. and A.B.V wrote the manuscript with input from all authors.

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Associated Data

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Supplementary Materials

1
NIHMS670701-supplement-1.docx (45.2KB, docx)
2
NIHMS670701-supplement-2.pdf (5.1MB, pdf)
3
NIHMS670701-supplement-3.xlsx (57.9KB, xlsx)
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