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• W2137062868 abstract "•Six of the eight TCA cycle enzymes were knocked out without affecting asexual growth•Metabolic labeling was analyzed in nine TCA KOs via 13C-labeling and mass spectrometry•The TCA cycle is adaptable, and the effect of a disrupted TCA cycle is stage specific 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 life-cycle 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. 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 life-cycle 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. Malaria is a major global parasitic disease that is responsible for ∼300 million infections and ∼600,000 deaths per year (WHO, 2013WHO (2013). World Malaria Report. http://www.who.int/malaria/publications/world_malaria_report_2013/report/en.Google Scholar). Although there are a number of effective antimalarial drugs available, the continued emergence of drug-resistant parasites (Ariey et al., 2014Ariey F. Witkowski B. Amaratunga C. Beghain J. Langlois A.C. Khim N. Kim S. Duru V. Bouchier C. Ma L. et al.A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.Nature. 2014; 505: 50-55Crossref PubMed Scopus (1227) Google Scholar) 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, 1992Fry M. Pudney M. Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4′-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80).Biochem. Pharmacol. 1992; 43: 1545-1553Crossref PubMed Scopus (394) Google Scholar, Nilsen et al., 2013Nilsen A. LaCrue A.N. White K.L. Forquer I.P. Cross R.M. Marfurt J. Mather M.W. Delves M.J. Shackleford D.M. Saenz F.E. et al.Quinolone-3-diarylethers: a new class of antimalarial drug.Sci. Transl. Med. 2013; 5: 77ra37Crossref Scopus (158) Google Scholar, Phillips et al., 2008Phillips M.A. Gujjar R. Malmquist N.A. White J. El Mazouni F. Baldwin J. Rathod P.K. Triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors with potent and selective activity against the malaria parasite Plasmodium falciparum.J. Med. Chem. 2008; 51: 3649-3653Crossref PubMed Scopus (168) Google Scholar). The parasite’s mitochondrion is highly divergent from its human counterpart (Vaidya and Mather, 2009Vaidya A.B. Mather M.W. Mitochondrial evolution and functions in malaria parasites.Annu. Rev. Microbiol. 2009; 63: 249-267Crossref PubMed Scopus (172) Google Scholar), 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., 2002Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. et al.Genome sequence of the human malaria parasite Plasmodium falciparum.Nature. 2002; 419: 498-511Crossref PubMed Scopus (3375) Google Scholar), which are expressed during the asexual stages (Bozdech et al., 2003Bozdech Z. Llinás M. Pulliam B.L. Wong E.D. Zhu J. DeRisi J.L. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum.PLoS Biol. 2003; 1: E5Crossref PubMed Scopus (1239) Google Scholar). The eight TCA enzymes have been localized to the mitochondrion (Günther et al., 2005Günther S. McMillan P.J. Wallace L.J. Müller S. Plasmodium falciparum possesses organelle-specific alpha-keto acid dehydrogenase complexes and lipoylation pathways.Biochem. Soc. Trans. 2005; 33: 977-980Crossref PubMed Scopus (29) Google Scholar, Hodges et al., 2005Hodges M. Yikilmaz E. Patterson G. Kasvosve I. Rouault T.A. Gordeuk V.R. Loyevsky M. An iron regulatory-like protein expressed in Plasmodium falciparum displays aconitase activity.Mol. Biochem. Parasitol. 2005; 143: 29-38Crossref PubMed Scopus (27) Google Scholar, Takeo et al., 2000Takeo S. Kokaze A. Ng C.S. Mizuchi D. Watanabe J.I. Tanabe K. Kojima S. Kita K. Succinate dehydrogenase in Plasmodium falciparum mitochondria: molecular characterization of the SDHA and SDHB genes for the catalytic subunits, the flavoprotein (Fp) and iron-sulfur (Ip) subunits.Mol. Biochem. Parasitol. 2000; 107: 191-205Crossref PubMed Scopus (35) Google Scholar, Tonkin et al., 2004Tonkin C.J. van Dooren G.G. Spurck T.P. Struck N.S. Good R.T. Handman E. Cowman A.F. McFadden G.I. Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method.Mol. Biochem. Parasitol. 2004; 137: 13-21Crossref PubMed Scopus (344) Google Scholar; H.K., J.M.M., M.W.M., and A.B.V., unpublished data), and TCA cycle intermediates are actively synthesized (Olszewski et al., 2009Olszewski K.L. Morrisey J.M. Wilinski D. Burns J.M. Vaidya A.B. Rabinowitz J.D. Llinás M. Host-parasite interactions revealed by Plasmodium falciparum metabolomics.Cell Host Microbe. 2009; 5: 191-199Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). 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., 2013Cobbold S.A. Vaughan A.M. Lewis I.A. Painter H.J. Camargo N. Perlman D.H. Fishbaugher M. Healer J. Cowman A.F. Kappe S.H. Llinás M. Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.J. Biol. Chem. 2013; 288: 36338-36350Crossref PubMed Scopus (59) Google Scholar, MacRae et al., 2013MacRae J.I. Dixon M.W. Dearnley M.K. Chua H.H. Chambers J.M. Kenny S. Bottova I. Tilley L. McConville M.J. Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum.BMC Biol. 2013; 11: 67Crossref PubMed Scopus (152) Google Scholar). Glutamine carbon enters the cycle via α-ketoglutarate, whereas glucose appears to provide acetyl-CoA (Cobbold et al., 2013Cobbold S.A. Vaughan A.M. Lewis I.A. Painter H.J. Camargo N. Perlman D.H. Fishbaugher M. Healer J. Cowman A.F. Kappe S.H. Llinás M. Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.J. Biol. Chem. 2013; 288: 36338-36350Crossref PubMed Scopus (59) Google Scholar, MacRae et al., 2013MacRae J.I. Dixon M.W. Dearnley M.K. Chua H.H. Chambers J.M. Kenny S. Bottova I. Tilley L. McConville M.J. Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum.BMC Biol. 2013; 11: 67Crossref PubMed Scopus (152) Google Scholar), as well as some oxaloacetate (Storm et al., 2014Storm J. Sethia S. Blackburn G.J. Chokkathukalam A. Watson D.G. Breitling R. Coombs G.H. Müller S. Phosphoenolpyruvate carboxylase identified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism.PLoS Pathog. 2014; 10: e1003876Crossref PubMed Scopus (31) Google Scholar), for entry at the citrate synthase (CS) step. The mitochondrial acetyl-CoA is produced from pyruvate by a branched-chain keto acid dehydrogenase (BCKDH) (Oppenheim et al., 2014Oppenheim R.D. Creek D.J. Macrae J.I. Modrzynska K.K. Pino P. Limenitakis J. Polonais V. Seeber F. Barrett M.P. Billker O. et al.BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei.PLoS Pathog. 2014; 10: e1004263Crossref PubMed Scopus (84) Google Scholar). Although recent studies have investigated metabolic flow through the TCA cycle in Plasmodium parasites (Cobbold et al., 2013Cobbold S.A. Vaughan A.M. Lewis I.A. Painter H.J. Camargo N. Perlman D.H. Fishbaugher M. Healer J. Cowman A.F. Kappe S.H. Llinás M. Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.J. Biol. Chem. 2013; 288: 36338-36350Crossref PubMed Scopus (59) Google Scholar, MacRae et al., 2013MacRae J.I. Dixon M.W. Dearnley M.K. Chua H.H. Chambers J.M. Kenny S. Bottova I. Tilley L. McConville M.J. Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum.BMC Biol. 2013; 11: 67Crossref PubMed Scopus (152) Google Scholar, Oppenheim et al., 2014Oppenheim R.D. Creek D.J. Macrae J.I. Modrzynska K.K. Pino P. Limenitakis J. Polonais V. Seeber F. Barrett M.P. Billker O. et al.BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei.PLoS Pathog. 2014; 10: e1004263Crossref PubMed Scopus (84) Google Scholar, Storm et al., 2014Storm J. Sethia S. Blackburn G.J. Chokkathukalam A. Watson D.G. Breitling R. Coombs G.H. Müller S. Phosphoenolpyruvate carboxylase identified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism.PLoS Pathog. 2014; 10: e1003876Crossref PubMed Scopus (31) Google Scholar), 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., 2012Hino A. Hirai M. Tanaka T.Q. Watanabe Y. Matsuoka H. Kita K. Critical roles of the mitochondrial complex II in oocyst formation of rodent malaria parasite Plasmodium berghei.J. Biochem. 2012; 152: 259-268Crossref PubMed Scopus (54) Google Scholar), and knocked down in the human parasite P. falciparum (Tanaka et al., 2012Tanaka T.Q. Hirai M. Watanabe Y. Kita K. Toward understanding the role of mitochondrial complex II in the intraerythrocytic stages of Plasmodium falciparum: gene targeting of the Fp subunit.Parasitol. Int. 2012; 61: 726-728Crossref PubMed Scopus (9) Google Scholar), without associated metabolomic analyses. MacRae et al., 2013MacRae J.I. Dixon M.W. Dearnley M.K. Chua H.H. Chambers J.M. Kenny S. Bottova I. Tilley L. McConville M.J. Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum.BMC Biol. 2013; 11: 67Crossref PubMed Scopus (152) Google Scholar conducted a metabolomic study of TCA and associated intermediates in P. falciparum combined with chemical inhibition of the single TCA enzyme aconitase. Disruption of BCKDH in P. berghei forced the parasite to grow in reticulocytes (Oppenheim et al., 2014Oppenheim R.D. Creek D.J. Macrae J.I. Modrzynska K.K. Pino P. Limenitakis J. Polonais V. Seeber F. Barrett M.P. Billker O. et al.BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei.PLoS Pathog. 2014; 10: e1004263Crossref PubMed Scopus (84) Google Scholar); consequently, reticulocyte metabolites might influence metabolomic analysis of this KO line. Storm et al., 2014Storm J. Sethia S. Blackburn G.J. Chokkathukalam A. Watson D.G. Breitling R. Coombs G.H. Müller S. Phosphoenolpyruvate carboxylase identified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism.PLoS Pathog. 2014; 10: e1003876Crossref PubMed Scopus (31) Google Scholar investigated the role of phosphoenolpyruvate carboxylase (PEPC) in P. falciparum but did not directly follow the TCA cycle enzymes. Therefore, we undertook a study to look at the essentiality, redundancy, and functions of the TCA cycle enzymes in P. falciparum. Here, we generated 11 KO lines, disrupting six of the eight TCA cycle enzymes in P. falciparum, and analyzed phenotypic and metabolomic features of these KO lines in different life cycle stages. The availability of these KO lines also provides a resource for further detailed metabolic studies. To establish the baseline metabolic architecture of wild-type (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 hr 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 hr. In agreement with a previous report (Ellinger et al., 2011Ellinger J.J. Lewis I.A. Markley J.L. Role of aminotransferases in glutamate metabolism of human erythrocytes.J. Biomol. NMR. 2011; 49: 221-229Crossref PubMed Scopus (36) Google Scholar), RBCs converted U-13C glutamine into glutamate and α-ketoglutarate but no other TCA cycle intermediates (Table S2). Similarly, RBCs did not convert U-13C glucose into TCA cycle intermediates during 4 hr incubations (Table S2). In contrast, WT parasites readily converted U-13C glutamine into malate (Figure 1). 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 turning 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, 2005Shen J. In vivo carbon-13 magnetization transfer effect. Detection of aspartate aminotransferase reaction.Magn. Reson. Med. 2005; 54: 1321-1326Crossref PubMed Scopus (25) Google Scholar; Figure 1). The presence of +2 isotopomers in these samples is consistent with glucose-derived acetyl-CoA entering the TCA cycle. 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., 2013Cobbold S.A. Vaughan A.M. Lewis I.A. Painter H.J. Camargo N. Perlman D.H. Fishbaugher M. Healer J. Cowman A.F. Kappe S.H. Llinás M. Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.J. Biol. Chem. 2013; 288: 36338-36350Crossref PubMed Scopus (59) Google Scholar, MacRae et al., 2013MacRae J.I. Dixon M.W. Dearnley M.K. Chua H.H. Chambers J.M. Kenny S. Bottova I. Tilley L. McConville M.J. Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum.BMC Biol. 2013; 11: 67Crossref PubMed Scopus (152) Google Scholar, Storm et al., 2014Storm J. Sethia S. Blackburn G.J. Chokkathukalam A. Watson D.G. Breitling R. Coombs G.H. Müller S. Phosphoenolpyruvate carboxylase identified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism.PLoS Pathog. 2014; 10: e1003876Crossref PubMed Scopus (31) Google Scholar) showing that blood-stage P. falciparum parasites carry out an oxidative TCA metabolism. To determine whether TCA cycle enzymes are essential for parasite survival, we attempted to knock out all eight TCA cycle enzymes through double crossover homologous recombination in D10 parasite line. 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 and 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 and 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 nine KOs, we measured parasitemia over four or five generations (192–240 hr) 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 hr over a 48 hr period. There were only 37 genes that had a statistically significant change at every time point over the 48 hr 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 genes that could directly compensate for the genetic ablations of KDH and IDH. 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 nine KO lines. These experiments were conducted with U-13C glutamine, because this amino acid is the main carbon source for the TCA cycle in WT parasites (Figure 1; Cobbold et al., 2013Cobbold S.A. Vaughan A.M. Lewis I.A. Painter H.J. Camargo N. Perlman D.H. Fishbaugher M. Healer J. Cowman A.F. Kappe S.H. Llinás M. Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.J. Biol. Chem. 2013; 288: 36338-36350Crossref PubMed Scopus (59) Google Scholar, MacRae et al., 2013MacRae J.I. Dixon M.W. Dearnley M.K. Chua H.H. Chambers J.M. Kenny S. Bottova I. Tilley L. McConville M.J. Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum.BMC Biol. 2013; 11: 67Crossref PubMed Scopus (152) Google Scholar). 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 showed 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. 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, 1953Simon E.J. Shemin D. The preparation of S-succinyl coenzyme A.J. Am. Chem. Soc. 1953; 75: 2520Crossref Scopus (536) Google Scholar). In the ΔIDH line, parasites showed unexpectedly diminished levels of labeling in metabolites upstream of IDH (Figure 2), whereas 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. 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 one 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., 2014Oppenheim R.D. Creek D.J. Macrae J.I. Modrzynska K.K. Pino P. Limenitakis J. Polonais V. Seeber F. Barrett M.P. Billker O. et al.BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei.PLoS Pathog. 2014; 10: e1004263Crossref PubMed Scopus (84) Google Scholar). 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., 2013Cobbold S.A. Vaughan A.M. Lewis I.A. Painter H.J. Camargo N. Perlman D.H. Fishbaugher M. Healer J. Cowman A.F. Kappe S.H. Llinás M. Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.J. Biol. Chem. 2013; 288: 36338-36350Crossref PubMed Scopus (59) Google Scholar, MacRae et al., 2013MacRae J.I. Dixon M.W. Dearnley M.K. Chua H.H. Chambers J.M. Kenny S. Bottova I. Tilley L. McConville M.J. Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum.BMC Biol. 2013; 11: 67Crossref PubMed Scopus (152) Google Scholar, Oppenheim et al., 2014Oppenheim R.D. Creek D.J. Macrae J.I. Modrzynska K.K. Pino P. Limenitakis J. Polonais V. Seeber F. Barrett M.P. Billker O. et al.BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei.PLoS Pathog. 2014; 10: e1004263Crossref PubMed Scopus (84) Google Scholar, Storm et al., 2014Storm J. Sethia S. Blackburn G.J. Chokkathukalam A. Watson D.G. Breitling R. Coombs G.H. Müller S. Phosphoenolpyruvate carboxylase identified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism.PLoS Pathog. 2014; 10: e1003876Crossref PubMed Scopus (31) Google Scholar). 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. The mitochondrial electron transport chain is an established target of antimalarial drugs. To assess the connection between the TCA cycle and mitochondrial electron transport chain in P. falciparum, we conducted metabolic analyses in parasites under conditions where the mitochondrial electron transport chain was inhibited at complex III by atovaquone (Fry and Pudney, 1992Fry M. Pudney M. Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4′-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80).Biochem. Pharmacol. 1992; 43: 1545-1553Crossref PubMed Scopus (394) Google Scholar, Srivastava et al., 1997Srivastava I.K. Rottenberg H. Vaidya A.B. Atovaquone, a broad spectrum antiparasitic drug, collapses mitochondrial membrane potential in a malarial parasite.J. Biol. Chem. 1997; 272: 3961-3966Crossref PubMed Scopus (336) Google Scholar). 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). Because atovaquone is toxic to WT parasites, these experiments were conducted with a mitochondrial-electron-transport-chain-independent transgenic line that expresses the cytosolic ubiquinone-independent Saccharomyces cerevisiae DHOD (yDHOD) (Ke et al., 2011Ke H. Morrisey J.M. Ganesan S.M. Painter H.J. Mather M.W. Vaidya A.B. Variation among Plasmodium falcip" @default.
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