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• W2079968363 abstract "The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a “PDH-like” enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage. The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a “PDH-like” enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage. Over the course of its 48-h intraerythrocytic developmental cycle the human malaria parasite, Plasmodium falciparum, matures and replicates. This rapid development necessitates a constant supply of exogenous nutrients, with the parasite primarily dependent upon glucose to sustain its energy demands (for review, see Refs. 1Olszewski K.L. Llinás M. Central carbon metabolism of Plasmodium parasites.Mol. Biochem. Parasitol. 2011; 175: 95-103Crossref PubMed Scopus (62) Google Scholar, 2Polonais V. Soldati-Favre D. Versatility in the acquisition of energy and carbon sources by the Apicomplexa.Biol. Cell. 2010; 102: 435-445Crossref PubMed Scopus (35) Google Scholar, 3Mogi T. Kita K. Diversity in mitochondrial metabolic pathways in parasitic protists PlasmodiumCryptosporidium.Parasitol. Int. 2010; 59: 305-312Crossref PubMed Scopus (73) Google Scholar). Parasite-infected erythrocytes consume up to 100-fold more glucose than uninfected erythrocytes via glycolysis (4Mehta M. Sonawat H.M. Sharma S. Glycolysis in Plasmodium falciparum results in modulation of host enzyme activities.J. Vector Borne Dis. 2006; 43: 95-103PubMed Google Scholar, 5Roth Jr., E.F. Raventos-Suarez C. Perkins M. Nagel R.L. Glutathione stability and oxidative stress in P. falciparum infection in vitro. Responses of normal and G6PD-deficient cells.Biochem. Biophys. Res. Commun. 1982; 109: 355-362Crossref PubMed Scopus (67) Google Scholar, 6Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. The accumulation of lactic acid and its influence on the growth of Plasmodium falciparum in synchronized cultures.In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar). Most of this glucose is metabolized to pyruvate that is subsequently reduced to lactate and excreted (7Cranmer S.L. Conant A.R. Gutteridge W.E. Halestrap A.P. Characterization of the enhanced transport of l- and d-lactate into human red blood cells infected with Plasmodium falciparum suggests the presence of a novel saturable lactate proton cotransporter.J. Biol. Chem. 1995; 270: 15045-15052Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8Elliott J.L. Saliba K.J. Kirk K. Transport of lactate and pyruvate in the intraerythrocytic malaria parasite, Plasmodium falciparum.Biochem. J. 2001; 355: 733-739Crossref PubMed Scopus (67) Google Scholar, 9Kanaani J. Ginsburg H. Transport of lactate in Plasmodium falciparum-infected human erythrocytes.J. Cell Physiol. 1991; 149: 469-476Crossref PubMed Scopus (51) Google Scholar, 10Pfaller M.A. Krogstad D.J. Parquette A.R. Nguyen-Dinh P. Plasmodium falciparum. Stage-specific lactate production in synchronized cultures.Exp. Parasitol. 1982; 54: 391-396Crossref PubMed Scopus (75) Google Scholar, 11Vander Jagt D.L. Hunsaker L.A. Campos N.M. Baack B.R. d-Lactate production in erythrocytes infected with Plasmodium falciparum.Mol. Biochem. Parasitol. 1990; 42: 277-284Crossref PubMed Scopus (108) Google Scholar). However, P. falciparum possess several metabolic pathways that are typically supplied by pyruvate-derived acetyl-CoA including the mitochondrial tricarboxylic acid cycle (TCA), cytosolic fatty acid elongation, and nuclear histone acetylation (12Botté C.Y. Yamaryo-Botté Y. Rupasinghe T.W. Mullin K.A. MacRae J.I. Spurck T.P. Kalanon M. Shears M.J. Coppel R.L. Crellin P.K. Maréchal E. McConville M.J. McFadden G.I. Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 7506-7511Crossref PubMed Scopus (85) Google Scholar, 13Cui L. Miao J. Chromatin-mediated epigenetic regulation in the malaria parasite Plasmodium falciparum.Eukaryot. Cell. 2010; 9: 1138-1149Crossref PubMed Scopus (86) Google Scholar, 14MacRae 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 (159) Google Scholar, 15Fritzler J.M. Millership J.J. Zhu G. Cryptosporidium parvum long-chain fatty acid elongase.Eukaryot. Cell. 2007; 6: 2018-2028Crossref PubMed Scopus (28) Google Scholar, 16Miao J. Lawrence M. Jeffers V. Zhao F. Parker D. Ge Y. Sullivan Jr., W.J. Cui L. Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development.Mol. Microbiol. 2013; 89: 660-675Crossref PubMed Scopus (68) Google Scholar). In most organisms acetyl-CoA is predominantly synthesized through the action of the pyruvate dehydrogenase (PDH) 3The abbreviations used are: PDHpyruvate dehydrogenaseBCKDHbranched-chain keto acid dehydrogenase complexKDHketoglutarate dehydrogenaseACSacetyl-CoA synthetaseIDCintraerythrocytic developmental cycleHPIhours post invasionPEPCphosphoenolpyruvate carboxylasePEPCKphosphoenolpyruvate carboxykinase. complex. In Plasmodium, however, the PDH is localized within the apicoplast, a plastid-like organelle enclosed by four membranes (17Foth B.J. Stimmler L.M. Handman E. Crabb B.S. Hodder A.N. McFadden G.I. The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast.Mol. Microbiol. 2005; 55: 39-53Crossref PubMed Scopus (143) Google Scholar, 18Ralph S.A. Strange organelles. Plasmodium mitochondria lack a pyruvate dehydrogenase complex.Mol. Microbiol. 2005; 55: 1-4Crossref PubMed Scopus (25) Google Scholar). Although it is clear that P. falciparum uses glucose-derived acetyl-CoA to supply the mitochondrial TCA cycle (14MacRae 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 (159) Google Scholar), it remains unclear to what extent PDH-derived acetyl-CoA is utilized by the parasite. In particular, transport of acetyl-CoA out of the apicoplast (via an unknown transport mechanism) would be required, which is highly unlikely. pyruvate dehydrogenase branched-chain keto acid dehydrogenase complex ketoglutarate dehydrogenase acetyl-CoA synthetase intraerythrocytic developmental cycle hours post invasion phosphoenolpyruvate carboxylase phosphoenolpyruvate carboxykinase. PDH is a large multienzyme complex that converts pyruvate and co-enzyme A into acetyl-CoA using thiamine, NAD, and lipoic acid as cofactors. PDH is comprised of the E1 pyruvate dehydrogenase (which exists as a heteromer of E1α and E1β), E2 dihydrolipoamide acetyltransferase, and E3 dihydrolipoamide dehydrogenase subunits (19Zhou Z.H. McCarthy D.B. O'Connor C.M. Reed L.J. Stoops J.K. The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 14802-14807Crossref PubMed Scopus (171) Google Scholar). The E1 subunit mediates the covalent attachment of pyruvate to thiamine pyrophosphate, decarboxylating pyruvate to an acetyl group, and then transfers the acetyl group to lipoic acid. The acetyl group is then transferred to co-enzyme A (CoA) via the E2 subunit, and dihydrolipoic acid is oxidized back to lipoic acid by the E3 subunit. Because of its localization to the apicoplast in malaria parasites, the E3 subunit is not shared between other dehydrogenase complexes (17Foth B.J. Stimmler L.M. Handman E. Crabb B.S. Hodder A.N. McFadden G.I. The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast.Mol. Microbiol. 2005; 55: 39-53Crossref PubMed Scopus (143) Google Scholar). In the rodent malaria Plasmodium yoelii, disrupting the E1α or E3 subunits of PDH has no effect on blood-stage development but prevents parasites from developing into liver-stage exo-erythrocytic merozoites (20Pei Y. Tarun A.S. Vaughan A.M. Herman R.W. Soliman J.M. Erickson-Wayman A. Kappe S.H. Plasmodium pyruvate dehydrogenase activity is only essential for the parasite's progression from liver infection to blood infection.Mol. Microbiol. 2010; 75: 957-971Crossref PubMed Scopus (75) Google Scholar). Because this phenotype is consistent with the fatty acid synthase II knock-out parasites, PDH is thought to be involved in de novo fatty acid synthesis during liver-stage development (17Foth B.J. Stimmler L.M. Handman E. Crabb B.S. Hodder A.N. McFadden G.I. The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast.Mol. Microbiol. 2005; 55: 39-53Crossref PubMed Scopus (143) Google Scholar, 21Vaughan A.M. O'Neill M.T. Tarun A.S. Camargo N. Phuong T.M. Aly A.S. Cowman A.F. Kappe S.H. Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.Cell Microbiol. 2009; 11: 506-520Crossref PubMed Scopus (303) Google Scholar, 22Yu M. Kumar T.R. Nkrumah L.J. Coppi A. Retzlaff S. Li C.D. Kelly B.J. Moura P.A. Lakshmanan V. Freundlich J.S. Valderramos J.C. Vilcheze C. Siedner M. Tsai J.H. Falkard B. Sidhu A.B. Purcell L.A. Gratraud P. Kremer L. Waters A.P. Schiehser G. Jacobus D.P. Janse C.J. Ager A. Jacobs Jr., W.R. Sacchettini J.C. Heussler V. Sinnis P. Fidock D.A. The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites.Cell Host Microbe. 2008; 4: 567-578Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The catalytically active domain of the E2 subunit was demonstrated to have acetyl-CoA synthetic activity (17Foth B.J. Stimmler L.M. Handman E. Crabb B.S. Hodder A.N. McFadden G.I. The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast.Mol. Microbiol. 2005; 55: 39-53Crossref PubMed Scopus (143) Google Scholar), but it has not been determined whether the PDH complex significantly contributes to acetyl-CoA metabolism or if it is essential for blood stages of the human malaria parasite P. falciparum. Herein, we have applied a rapid stable-isotope labeling technique in combination with high resolution mass spectrometry to study blood-stage acetyl-CoA metabolism and determine the contribution of the PDH to central carbon metabolism. In a comparative analysis of wild type versus PDH-deficient (pdh e1α-) parasites, we come to the surprising conclusion that the PDH does not appreciably contribute to the acetyl-CoA pool or metabolic pathways downstream of this central precursor. Moreover, we observe significant fluxes through acetyl-CoA-dependent pathways despite the absence of a functional PDH. We propose that acetyl-CoA is predominantly synthesized through the mitochondrial branched-chain keto acid dehydrogenase complex (BCKDH), an enzyme complex typically associated with amino acid degradation. We show that this PDH-like metabolism can account for the majority of acetyl-CoA synthesis and that direct synthesis of acetyl-CoA from acetate can also contribute to the acetyl-CoA pool when parasites are supplied with an abundant acetate source. These findings demonstrate that blood-stage parasites use a PDH-like enzyme and acetate fixation for supplying acetyl-CoA pools. To further investigate central carbon metabolism of P. falciparum, we measured the relative contribution of glucose- and glutamine-derived carbon to the TCA cycle throughout the intraerythrocytic developmental cycle. We demonstrate that as the parasite matures, it reduces the incorporation of anapleurotic carbon from glucose and increases incorporation via glutamine. This finding highlights the parasite's ability to restructure metabolism to meet its developmental requirements. P. falciparum NF54 and pdh e1α-G2/B9 clones were cultured and synchronized by standard methods (23Lambros C. Vanderberg J.P. Synchronization of Plasmodium falciparum erythrocytic stages in culture.J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2832) Google Scholar, 24Trager W. Jensen J.B. Human malaria parasites in continuous culture.Science. 1976; 193: 673-675Crossref PubMed Scopus (6161) Google Scholar) with the modification that culture flasks were maintained at 37 °C in an atmospherically controlled incubator set at 5% CO2, 6% O2. Parasitemia and synchronicity was monitored using microscopy and Giemsa-stained thin-blood smear slides. Uninfected erythrocytes (from the same donor) were cultured for 48-h in parallel before any experimentation. Mycoplasma testing was performed routinely to ensure contamination-free cultures. Cell counts were taken for all experiments with a Neubauer hemocytometer. Anopheles stephensi mosquitoes (originating from the Walter Reed Army Institute of Research) were maintained at 27 °C and 75% humidity on a 12-h light/dark cycle. Larval stages were reared after standard protocols as described in the MR4 manual with larval stages maintained on finely ground Tetramin fish food and adult mosquitoes maintained on 8% dextrose in 0.05% para-aminobenzoic acid water. In vitro P. falciparum NF54 blood stage cultures were maintained in RPMI 1640 (25 mm HEPES, 2 mm l-glutamine) supplemented with 50 μm hypoxanthine and 10% A+ human serum in an atmosphere of 5% CO2, 5% O2, and 90% N2. Cells were subcultured into O+ erythrocytes. Gametocyte cultures were initiated at 5% hematocrit and 0.8–1% parasitemia (mixed stages) and maintained for up to 17 days with daily media changes. Non blood-fed adult female mosquitoes (3–7 days post-emergence) were fed on gametocyte cultures. Gametocyte cultures were quickly spun down, and the pelleted infected erythrocytes were diluted to a 40% hematocrit with fresh A+ human serum and O+ erythrocytes. Mosquitoes were allowed to feed through Parafilm for up to 20 min. After blood feeding, mosquitoes were maintained for up to 19 days at 27 °C, 75% humidity and provided with 8% dextrose solution in PABA water. Infection prevalence was checked at days 7–10 by examining dissected midguts under light microscopy for the presence of oocysts with salivary gland dissections performed at days 14–19. Parasite cultures were enriched using a modified magnetic enrichment method. A custom-built magnetic-separation apparatus was designed using Google Sketch and produced by Pokono via high density plastic three-dimensional printing. 1.5 in3 magnets (pull force 220 lbs) were purchased, and metal inserts cut on-site. The apparatus allowed the simultaneous use of four CS VarioMacs columns (Miltenyi Biotech) and the design can be downloaded from 3dwarehouse. Enrichment was performed by resuspending 1 ml of packed cell culture (10% parasitemia) in 15 ml (∼8% hematocrit) and passed through a single CS column with constant addition of complete RPMI until the eluent was transparent. Infected erythrocytes were then eluted separate from the magnetic apparatus, yielding 85–98% parasitemia. Separate strains were enriched simultaneously to maintain consistency between cell lines and allowed to recover at 37 °C for 1 h before experimentation. For [13C]glucose labeling experiments, complete RPMI was made using glucose-free RPMI powder (Sigma), and an appropriate amount of [6-13C]glucose (all stable-isotope compounds were acquired from Cambridge Isotopes) was added to give a final concentration of 11 mm. For thiamine-free/oxythiamine experiments, RPMI was reconstituted using 50× RPMI amino acid solution (Sigma) and individual vitamins, salts, and oxythiamine purchased from Sigma and Fischer. Fully reconstituted RPMI was always made in parallel and used as a control condition to ensure there was no variation in parasite growth between batches. The IC50 of oxythiamine was determined, and a low dose long-term incubation strategy was required to allow conversion of oxythiamine to oxythiamine pyrophosphate. [5-13C]Glutamine was added to glutamine-free RPMI at a final concentration of 2 mm for [5-13C]glutamine labeling experiments. [6-13C,15N]leucine labeling experiments were performed in complete custom-made RPMI without leucine and supplemented with [6-13C,15N]leucine at the RPMI concentration. [2-13C]Acetate and [1-13C]pyruvate (label in the 2-C position) labeling experiments were performed using complete RPMI with an appropriate amount of each compound added to give a concentration of 2 and 10 mm, respectively. Media were pH-titrated and added as a 1:1 ratio to cell suspensions containing label-free RPMI, giving a final concentration of 1 mm (acetate) and 5 mm (pyruvate). 11 mm unlabeled glucose was present in all experiments. Four methods were employed to extract metabolites. Steady-state incubations were extracted using the methods described previously (25Olszewski K.L. Llinás M. Extraction of hydrophilic metabolites from Plasmodium falciparum-infected erythrocytes for metabolomic analysis.Methods Mol. Biol. 2013; 923: 259-266Crossref PubMed Google Scholar) with slight modifications. Briefly, infected erythrocytes were transferred to microcentrifuge tubes and spun at 14,000 × g for 30 s. The supernatant was rapidly aspirated, and the cell pellet was extracted with 1 ml of 90% ice-cold methanol (containing the internal standard [4-13C,15N]aspartate). Samples were dried under nitrogen flow and stored at −80 °C until analysis. Isolated parasites were extracted from 10% parasitemia cultures by saponin lysis (0.08%) in Eppendorf tubes. Samples were rapidly washed 3 times with ice-cold PBS, and the parasite pellet was extracted with 90% methanol. The rapid-labeling and extraction method was adapted from previously described radiolabeled flux techniques (26Cobbold S.A. Martin R.E. Kirk K. Methionine transport in the malaria parasite Plasmodium falciparum.Int. J. Parasitol. 2011; 41: 125-135Crossref PubMed Scopus (21) Google Scholar). 2-ml microcentrifuge tubes were loaded with 300 μl of 30% trifluoroacetic acid with 700 μl of an oil mixture (5:4 dibutyl phthalate:dibutyl octanol, density 1.02) layered above. Time courses were initiated with the 1:1 addition of cell suspensions to pre-warmed isotope-labeled media (1–2% final hematocrit) and incubated at 37 °C. At the appropriate time points, 1 ml of cell suspension was layered on top of the oil mixture and immediately centrifuged at 14,000 × g for 30 s. The supernatant and oil layer was removed, and the acid layer was mixed with 700 μl of 90% ice-cold methanol (containing the internal standard [4-13C,15N]aspartate) and centrifuged. The metabolite extracts were transferred to a fresh microcentrifuge tube and immediately dried under nitrogen flow. Extracts were resuspended in 100 μl of H2O pH neutralized with 5–10 μl of 1 m ammonia bicarbonate, transferred to a fresh microcentrifuge tube, dried under nitrogen flow, and stored at −80 °C until analysis. Validation of this method was achieved by comparison to the standard 90% methanol extraction method. Fatty acid extractions were performed as described previously (27Kamphorst J.J. Fan J. Lu W. White E. Rabinowitz J.D. Liquid chromatography-high resolution mass spectrometry analysis of fatty acid metabolism.Anal. Chem. 2011; 83: 9114-9122Crossref PubMed Scopus (70) Google Scholar) with processing blanks extracted in parallel. [16-13C]Palmitate was used as an internal standard and used to normalize all signals. After data acquisition, processing blanks were subtracted from biological samples, providing the total ion counts associated with each condition. For all LC-MS analysis, samples were reconstituted in 100 μl of H2O and analyzed on an Exactive Orbitrap mass spectrometer as previously described (28Xu Y.F. Létisse F. Absalan F. Lu W. Kuznetsova E. Brown G. Caudy A.A. Yakunin A.F. Broach J.R. Rabinowitz J.D. Nucleotide degradation and ribose salvage in yeast.Mol. Syst. Biol. 2013; 9: 665Crossref PubMed Scopus (47) Google Scholar). The data presented in the glutamine panel of Fig. 5C were collected on a Finnigan TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with an electrospray ionization source, operating in positive mode. Uninfected and enriched infected erythrocytes were incubated for 2 h in [6-13C]glucose RPMI and subsequently extracted with 90% methanol. Samples were dried down under N2 gas, then resuspended in 99.9% D2O and titrated to pH 7.40 (±0.01; uncorrected glass electrode reading). Two-dimensional 13C,1H heteronuclear single quantum correlation NMR spectra were collected on a 500-MHz Bruker spectrometer equipped with a 1H-optimized triple resonance cryoprobe. Heteronuclear single quantum correlations were acquired in 4 transients, with 8192 directly acquired points and 1028 increments. Spectra were Fourier transform with shifted sine bell window function, zero-filled, and phased in TopSpin. Spectra were analyzed using rNMR (29Lewis I.A. Schommer S.C. Markley J.L. rNMR. Open source software for identifying and quantifying metabolites in NMR spectra.Magn. Reson. Chem. 2009; 47: S123-S126Crossref PubMed Scopus (146) Google Scholar), and metabolites were identified and quantified using established methods (30Lewis I.A. Schommer S.C. Hodis B. Robb K.A. Tonelli M. Westler W.M. Sussman M.R. Markley J.L. Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra.Anal. Chem. 2007; 79: 9385-9390Crossref PubMed Scopus (226) Google Scholar). Thermo Fisher mass spectrometry RAW files were converted from profile mode into centroid mode using the ReAdW program (31Keller A. Eng J. Zhang N. Li X.J. Aebersold R. A uniform proteomics MS/MS analysis platform utilizing open XML file formats.Mol. Syst. Biol. 2005; 1 (0017)Crossref PubMed Scopus (598) Google Scholar) and loaded into MAVEN, a publicly available analysis program (32Clasquin M.F. Melamud E. Rabinowitz J.D. LC-MS data processing with MAVEN. A metabolomic analysis and visualization engine.Curr. Protoc. Bioinformatics. 2012; (Chapter 14, Unit 14.11)PubMed Google Scholar). Correct assignment was confirmed via the addition of pure standard for all metabolites presented. Isotope-labeled forms were identified using the expected mass shifts given by 13C and 15N. Peaks below an ion count of 1000 were excluded from analysis. Raw signals were normalized to the internal control, corrected for natural abundance where appropriate, and adjusted to ion count/108 cells using cells counts taken for each sample. For quantification of glycolytic intermediates the signal ratio of the 13C-labeled metabolite (complete pool labeling was achieved with [13C]glucose and [13C]acetate for 2 h) to its unlabeled internal standard was used and to convert ion count/108 cells into intracellular concentration as previously described (33Bennett B.D. Yuan J. Kimball E.H. Rabinowitz J.D. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach.Nat. Protoc. 2008; 3: 1299-1311Crossref PubMed Scopus (291) Google Scholar), assuming the intracellular volume of an infected erythrocyte is 75 fl (34Saliba K.J. Horner H.A. Kirk K. Transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite Plasmodium falciparum.J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). For TCA cycle intermediates, pure 13C-labeled standards were added to unlabeled parasite extracts and quantified as described above. Graphical representations and statistical analysis were performed in GraphPad. A paired two-tail Student's t test was performed for pairwise statistical comparisons, and a one-way analysis of variance was performed for multiple statistical comparison. See supplemental Methods for further experimental details. To investigate central carbon metabolism of P. falciparum-infected erythrocytes, we used stable-isotope labeling. Unfortunately, parasite biology is not amenable to existing rapid-labeling techniques utilized in bacterial metabolomics studies, which use filter paper-adhered cells to rapidly transfer samples between labeled media to extraction solution (35Amador-Noguez D. Brasg I.A. Feng X.J. Roquet N. Rabinowitz J.D. Metabolome remodeling during the acidogenic-solventogenic transition in Clostridium acetobutylicum.Appl. Environ. Microbiol. 2011; 77: 7984-7997Crossref PubMed Scopus (101) Google Scholar, 36Xu Y.F. Amador-Noguez D. Reaves M.L. Feng X.J. Rabinowitz J.D. Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase.Nat. Chem. Biol. 2012; 8: 562-568Crossref PubMed Scopus (63) Google Scholar). Therefore, we developed a rapid stable-isotope labeling technique to measure dynamic glycolytic flux in Plasmodium (see “Experimental Procedures”). Magnet-enriched uninfected and infected erythrocytes (trophozoite stage) suspended in [12C]glucose RPMI were supplemented with [6-13C]glucose (final concentration 11 mm at an isotope ratio of 1:1 12C:13C). Turnover of intracellular glycolytic intermediates was monitored over a 1-h period via LC-MS (Fig. 1A and supplemental Fig. S1). The total metabolite pool (presented as intracellular concentration; Fig. 1A, top panel) was unchanged across the time course, indicating that the conditions used did not perturb parasite metabolism. Isotopic labeling kinetics in glycolytic intermediates indicated differential labeling rates between the upper and lower halves of glycolysis; fructose 1,6-bisphosphate was completely labeled in less than 2 min, whereas phosphoenolpyruvate, pyruvate, and lactate required an hour to reach isotopic equilibrium (Fig. 1A). Infected erythrocytes (NF54) showed a significant increase in the total pool of most metabolic intermediates and in the glycolytic flux relative to uninfected erythrocytes (supplemental Table S1; all p < 0.01). Surprisingly, unlike glycolytic intermediates, the acetyl-CoA pool did not completely label in the conditions tested, labeling only 42 ± 9% of the total pool after 1 h and 77 ± 3% after 3 h (Fig. 1B). The E1 subunit of the PDH complex mediates the thiamine pyrophosphate-dependent decarboxylation of pyruvate and is considered critical for normal PDH function. To ensure that P. falciparum produce the E1 subunit and all of the additional subunits of the PDH complex, we first used MS/MS proteomics to identify the subunits using the NF54 strain during the blood stage (supplemental Fig. S2 and Table S2). Knowing that the complex was expressed, we generated a pdh e1α- line by homologous recombination to investigate acetyl-CoA metabolism in the parasite (supplemental Fig. S3). Surprisingly, PDH-disrupted parasites had no signification alterations in glycolytic flux (Fig. 1A). Moreover, PDH disrupted parasites converted [6-13C]glucose into +2 atomic mass unit-shifted acetyl-CoA (consistent with the incorporation of two 13C atoms, referred to henceforth as M+2) and M+2 citrate (the first product of acetyl-CoA metaboli" @default.
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• W2079968363 title "Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum" @default.
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