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• W2805302114 abstract "The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches. The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches. Malaria is a major global health problem, affecting more than 40% of the world's population. This disease is responsible for 196 million to 263 million annual cases, resulting in about 445,000 deaths, most of them occurring in children under the age of 5 in sub-Saharan Africa (1.World Health Organnization.World Malaria Report. 2017; Google Scholar). Malaria is caused by parasites of the genus Plasmodium, and the most lethal human malaria parasite is Plasmodium falciparum. The parasite has a complex life cycle developing in mosquitos and humans, but all clinical features are caused during the repeated invasion of human red blood cells (RBCs). No efficient vaccine is currently available, and resistance to all available treatments has now been observed, making it urgent to identify novel pharmacological targets (1.World Health Organnization.World Malaria Report. 2017; Google Scholar, 2.Cowman A.F. Healer J. Marapana D. Marsh K. Malaria: biology and disease.Cell. 2016; 167: 610-624Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 3.Wells T.N. Hooft van Huijsduijnen R. Van Voorhis W.C. Malaria medicines: a glass half full?.Nat. Rev. Drug Discov. 2015; 14: 424-442Crossref PubMed Scopus (324) Google Scholar). The mature RBC is a very particular host cell for an intracellular parasite. RBCs do not contain organelles and are therefore capable of only few metabolic functions. After invasion, the parasite resides and develops within a parasitophorous vacuole and produces an extensive tubular membrane network in the erythrocyte cytoplasm. During its development, the parasite internalizes and digests the host cell hemoglobin (Hb) in a particular proteolytic compartment called the food vacuole (FV) (4.Goldberg D.E. Hemoglobin degradation.Curr. Top. Microbiol. Immunol. 2005; 295: 275-291Crossref PubMed Scopus (173) Google Scholar). All other nutrients and metabolic precursors that are essential for parasite survival must either be imported from the serum or be synthesized de novo by the parasite. During the intraerythrocytic phase, P. falciparum synthesizes considerable amounts of membranes for its growth within the host cell and for the subsequent formation of up to 32 daughter cells every 48 h. This is associated with a 6-fold increase in the phospholipid (PL) content of the erythrocyte infected with the metabolically highly active late stages of the parasite (trophozoite and schizont) (5.Tran P.N. Brown S.H. Rug M. Ridgway M.C. Mitchell T.W. Maier A.G. Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum.Malar. J. 2016; 15: 73Crossref PubMed Scopus (44) Google Scholar, 6.Vial H.J. Ancelin M.L. Malarial lipids. An overview.Subcell. Biochem. 1992; 18: 259-306Crossref PubMed Scopus (94) Google Scholar). Indeed, Plasmodium membranes are mainly composed of PLs and contain only little cholesterol (7.Déchamps S. Shastri S. Wengelnik K. Vial H.J. Glycerophospholipid acquisition in Plasmodium–a puzzling assembly of biosynthetic pathways.Int. J. Parasitol. 2010; 40: 1347-1365Crossref PubMed Scopus (58) Google Scholar, 8.Maguire P.A. Sherman I.W. Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells.Mol. Biochem. Parasitol. 1990; 38: 105-112Crossref PubMed Scopus (68) Google Scholar). In P. falciparum-infected RBCs (iRBCs), PLs constitute 60% of the total lipid content versus 38% in uninfected RBCs (uRBCs) (5.Tran P.N. Brown S.H. Rug M. Ridgway M.C. Mitchell T.W. Maier A.G. Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum.Malar. J. 2016; 15: 73Crossref PubMed Scopus (44) Google Scholar). In uRBCs, the main PLs are phosphatidylcholine (PC) (30–40%), phosphatidylethanolamine (PE) (25–35%), SM (15%), and phosphatidylserine (PS) (10–20%) (9.Leidl K. Liebisch G. Richter D. Schmitz G. Mass spectrometric analysis of lipid species of human circulating blood cells.Biochim. Biophys. Acta. 2008; 1781: 655-664Crossref PubMed Scopus (122) Google Scholar, 10.Van Deenen L.L. De Gier J. Lipids of the red cell membrane.The red blood cell. 1975; : 147-211Google Scholar). After infection by P. falciparum, an increase of PC and PE and a decrease of PS and SM are observed. In P. falciparum-iRBCs as well as in purified parasites, the main PLs are PC (45–55%), PE (20–35%), PS (4–8%), phosphatidylinositol (∼5%), and SM (3–20%) (5.Tran P.N. Brown S.H. Rug M. Ridgway M.C. Mitchell T.W. Maier A.G. Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum.Malar. J. 2016; 15: 73Crossref PubMed Scopus (44) Google Scholar, 11.Botté 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. et al.Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites.Proc. Natl. Acad. Sci. USA. 2013; 110: 7506-7511Crossref PubMed Scopus (85) Google Scholar, 12.Gulati S. Ekland E.H. Ruggles K.V. Chan R.B. Jayabalasingham B. Zhou B. Mantel P.Y. Lee M.C. Spottiswoode N. Coburn-Flynn O. et al.Profiling the essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum.Cell Host Microbe. 2015; 18: 371-381Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). De novo synthesis of lipids is absent in uRBCs (10.Van Deenen L.L. De Gier J. Lipids of the red cell membrane.The red blood cell. 1975; : 147-211Google Scholar), but Plasmodium possesses its own machinery to produce PLs (7.Déchamps S. Shastri S. Wengelnik K. Vial H.J. Glycerophospholipid acquisition in Plasmodium–a puzzling assembly of biosynthetic pathways.Int. J. Parasitol. 2010; 40: 1347-1365Crossref PubMed Scopus (58) Google Scholar, 13.Ben Mamoun C. Prigge S.T. Vial H. Targeting the lipid metabolic pathways for the treatment of malaria.Drug Dev. Res. 2010; 71: 44-55PubMed Google Scholar). Previous studies have shown that PL synthesis is essential for the viability of P. falciparum at the blood stage (14.Ancelin M.L. Calas M. Vidal-Sailhan V. Herbute S. Ringwald P. Vial H.J. Potent inhibitors of Plasmodium phospholipid metabolism with a broad spectrum of in vitro antimalarial activities.Antimicrob. Agents Chemother. 2003; 47: 2590-2597Crossref PubMed Scopus (77) Google Scholar, 15.Wein S. Maynadier M. Bordat Y. Perez J. Maheshwari S. Bette-Bobillo P. Tran Van Ba C. Penarete-Vargas D. Fraisse L. Cerdan R. et al.Transport and pharmacodynamics of albitiazolium, an antimalarial drug candidate.Br. J. Pharmacol. 2012; 166: 2263-2276Crossref PubMed Scopus (36) Google Scholar). Potent antimalarial activity has been observed with choline (Cho) analogs that target PC synthesis. The lead compound, albitiazolium, was successfully evaluated during human phase I and II clinical trials in adult patients. However, phase II clinical trials in a pediatric population revealed a lower efficiency due to higher drug clearance as compared with adults and led to an arrest of the clinical development (15.Wein S. Maynadier M. Bordat Y. Perez J. Maheshwari S. Bette-Bobillo P. Tran Van Ba C. Penarete-Vargas D. Fraisse L. Cerdan R. et al.Transport and pharmacodynamics of albitiazolium, an antimalarial drug candidate.Br. J. Pharmacol. 2012; 166: 2263-2276Crossref PubMed Scopus (36) Google Scholar, 16.Wein S. Taudon N. Maynadier M. Tran Van Ba C. Margout D. Bordat Y. Fraisse L. Wengelnik K. Cerdan R. Bressolle-Gomeni F. et al.High accumulation and in vivo recycling of the new antimalarial albitiazolium lead to rapid parasite death.Antimicrob. Agents Chemother. 2017; 61: e00352-17Crossref PubMed Scopus (6) Google Scholar). Targeting PC synthesis in P. falciparum is thus a proven strategy. It is therefore even more important to decipher the PL metabolism in order to identify additional pharmacological targets. The PL metabolism of P. falciparum is unique in its intensity and in the multiplicity of its pathways. Its metabolism combines pathways generally found in prokaryotes and eukaryotes. The three main PLs are PC, PE, and PS. PC can be synthesized from Cho via the three enzymatic steps of the CDP-Cho-dependent Kennedy pathway (17.Ancelin M.L. Vial H.J. Several lines of evidence demonstrating that Plasmodium falciparum a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine.FEBS Lett. 1986; 202: 217-223Crossref PubMed Scopus (30) Google Scholar, 18.Ancelin M.L. Vial H.J. Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes.Biochim. Biophys. Acta. 1989; 1001: 82-89Crossref PubMed Scopus (51) Google Scholar, 19.Vial H.J. Thuet M.J. Philippot J.R. Cholinephos­photransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers.Biochim. Biophys. Acta. 1984; 795: 372-383Crossref PubMed Scopus (35) Google Scholar) (Fig. 1). However, PC can also be synthesized from ethanolamine (Etn) that enters the same pathway after the triple methylation of phosphoethanolamine (P-Etn) to phosphocholine (P-Cho) by the P-Etn methyltransferase (PMT), an enzyme generally found in plants (20.Pessi G. Kociubinski G. Mamoun C.B. A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation.Proc. Natl. Acad. Sci. USA. 2004; 101: 6206-6211Crossref PubMed Scopus (133) Google Scholar). On the other hand, triple methylation of PE to PC, as present in mammalian cells, is absent in P. falciparum (21.Witola W.H. El Bissati K. Pessi G. Xie C. Roepe P.D. Mamoun C.B. Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects.J. Biol. Chem. 2008; 283: 27636-27643Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). PE can be synthesized via the CDP-Etn-dependent Kennedy pathway from Etn (17.Ancelin M.L. Vial H.J. Several lines of evidence demonstrating that Plasmodium falciparum a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine.FEBS Lett. 1986; 202: 217-223Crossref PubMed Scopus (30) Google Scholar, 19.Vial H.J. Thuet M.J. Philippot J.R. Cholinephos­photransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers.Biochim. Biophys. Acta. 1984; 795: 372-383Crossref PubMed Scopus (35) Google Scholar). For PC and PE synthesis, Etn can also be provided by the decarboxylation of serine (Ser) by the Ser decarboxylase (22.Elabbadi N. Ancelin M.L. Vial H.J. Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation.Biochem. J. 1997; 324: 435-445Crossref PubMed Scopus (60) Google Scholar), an enzyme generally found in plants (23.Rontein D. Nishida I. Tashiro G. Yoshioka K. Wu W.I. Voelker D.R. Basset G. Hanson A.D. Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme.J. Biol. Chem. 2001; 276: 35523-35529Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The activity of this enzyme has been detected in the parasite (22.Elabbadi N. Ancelin M.L. Vial H.J. Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation.Biochem. J. 1997; 324: 435-445Crossref PubMed Scopus (60) Google Scholar), but its corresponding gene has still not been identified. PE can also be generated by decarboxylation of PS through the PS decarboxylase (PSD) (24.Baunaure F. Eldin P. Cathiard A.M. Vial H. Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum.Mol. Microbiol. 2004; 51: 33-46Crossref PubMed Scopus (27) Google Scholar). Finally, biosynthesis of PS from Ser has been demonstrated biochemically in P. falciparum (25.Déchamps S. Maynadier M. Wein S. Gannoun-Zaki L. Marechal E. Vial H.J. Rodent and non-rodent malaria parasites differ in their phospholipid metabolic pathways.J. Lipid Res. 2010; 51: 81-96Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The only PS synthase (PSS) present in P. falciparum (PF3D7_1366800) is similar to the mammalian PS synthase 2 (PSS2), which catalyzes the base-exchange reaction between PE and Ser (Fig. 1). A previously predicted CDP-diacylglycerol (DAG)-dependent PSS enzyme (7.Déchamps S. Shastri S. Wengelnik K. Vial H.J. Glycerophospholipid acquisition in Plasmodium–a puzzling assembly of biosynthetic pathways.Int. J. Parasitol. 2010; 40: 1347-1365Crossref PubMed Scopus (58) Google Scholar) has now been reannotated as a phosphatidylglycerolphosphate synthase (PF3D7_0820200) in the Plasmodium database (www.plasmodb.org). In the present study, we determined the contribution of the different pathways to the biosynthesis of PC, PE, and PS, and we analyzed their regulation. To this purpose, we undertook a lipidomic analysis of P. falciparum-iRBCs using deuterium-labeled Cho, Etn, and Ser as tracers to follow the different PL pathways (supplemental Fig. S1). We identified the reaction intermediates and the final PL products of each pathway by LC/MS/MS and quantified the relative contribution of each pathway to the biosynthesis of the three main PLs. When we started our study, Cho from serum (10–15 µM) (Human Metabolome Data Base, http://www.hmdb.ca) was considered to be the only source of Cho for the parasite. Our results suggest that the parasite uses almost exclusively lysophosphatidylcholine (LysoPC) from serum to synthesize PC through the Kennedy pathway. In accordance with our data, Brancucci et al. showed recently that the majority of free Cho in the parasite derives from LysoPC (26.Brancucci N.M.B. Gerdt J.P. Wang C. De Niz M. Philip N. Adapa S.R. Zhang M. Hitz E. Niederwieser I. Boltryk S.D. et al.Lysophosphatidylcholine regulates sexual stage differentiation in the human malaria parasite Plasmodium falciparum.Cell. 2017; 171: 1532-1544.e15Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). PE can be provided by both Etn and Ser by three different routes. We also show that Ser used for PS synthesis is predominantly provided by Hb degradation. Comparative lipidomics with a transgenic parasite line in which the gene encoding PMT activity is deleted clarified the interplay between the different pathways showing that P. falciparum is able to compensate for the lack of Cho by using Etn or Ser. Metabolites LysoPC and SM from egg yolk and internal standards were obtained from Sigma-Aldrich, with the exception of cytidine-5′-diphosphate, which was purchased from MP Biomedicals Inc. (Illkirch, France) and myo-inositol-1-phosphate from Interchim (Montluc, France). LysoPC contains primarily palmitic and stearic acids. SM contains primarily palmitic acid. All the deuterated compounds were purchased from CDN Isotopes (Québec, Canada). RPMI 1640 medium and RPMI 1640 without Cho, Ser, methionine, inositol, and folic acid were obtained from Gibco. P. falciparum (3D7 and pfpmtΔ strains) cell cultures were performed as previously described (27.Trager W. Jensen J.B. Human malaria parasites in continuous culture.Science. 1976; 193: 673-675Crossref PubMed Scopus (6181) Google Scholar) in human A+ erythrocytes and complete medium (RPMI 1640 medium supplemented with 25 mM HEPES) and 10% AB+ human serum or 0.5% Albumax I (Gibco), at 37°C under a gaseous mixture of 5% CO2, 5% O2, and 90% N2. Parasites were synchronized using a VarioMACS magnetic cell separator (CS column, Miltenyi Biotec, Paris, France) followed by a 5% sorbitol treatment (28.Ahn S.Y. Shin M.Y. Kim Y.A. Yoo J.A. Kwak D.H. Jung Y.J. Jun G. Ryu S.H. Yeom J.S. Ahn J.Y. et al.Magnetic separation: a highly effective method for synchronization of cultured erythrocytic Plasmodium falciparum.Parasitol. Res. 2008; 102: 1195-1200Crossref PubMed Scopus (20) Google Scholar, 29.Lambros C. Vanderberg J.P. Synchronization of Plasmodium falciparum erythrocytic stages in culture.J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2839) Google Scholar). Free parasites were prepared from mature infected erythrocytes by adding 10 vols of 0.02% (wt/vol) saponin in cold PBS (116 mM NaCl, 8.3 mM Na2HPO4, and 3.2 mM KH2PO4, pH 7.4) for 1 min, followed by two washes by centrifugation at 1,260 g for 5 min at 4°C and resuspension in PBS. Uninfected human blood and serum were provided by the local blood bank (Etablissement Français du Sang) under the approval number 21PLER2016-0103. For metabolic labeling, RBCs and iRBCs at late stages were first washed three times in modified RPMI 1640 and put back into culture in the appropriate medium. Culture medium was composed of modified RPMI 1640 supplemented with 20 mM HEPES buffer (Gibco, pH 7.4), glutamine (0.3 g/l), hypoxanthine (15 µg/ml), inositol (0.035 g/l), gentamycin (40 µg/ml), deuterated precursors (4–1,000 µM Cho-d9, 2–500 µM Etn-d4, and 20–1,000 µM Ser-d3), and 10% human serum AB+ or 0.5% Albumax I. Serum-free culture medium was prepared according to Mitamura et al. (30.Mitamura T. Hanada K. Ko-Mitamura E.P. Nishijima M. Horii T. Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum.Parasitol. Int. 2000; 49: 219-229Crossref PubMed Scopus (61) Google Scholar) as follows. Lipid-free BSA stock solution [600 µM in RPMI 1640 without Cho, Ser, and inositol (Gibco) (modified RPMI)] was added to dried lipid precipitate (oleic acid and palmitic acid, 30 µM final), and the mixture was sonicated for 1 min to allow FAs to bind to BSA. The final concentration of lipid-free BSA was 30 µM. This solution was supplemented with 20 mM HEPES buffer (Gibco, pH 7.4), glutamine (0.3 g/l), hypoxanthine (15 µg/ml), inositol (0.035 g/l), 140 µM Ser, 10 µM Etn, and gentamycin (40 µg/ml). Then, serum-free culture medium was supplemented or not with 20 or 400 µM Cho, or 20 µM LysoPC or 20 µM SM. For supplementation with 20 µM LysoPC or SM, lipid-free BSA stock solution was added to dried lipid precipitate containing oleic acid, palmitic acid, and LysoPC or SM and then sonicated. FAs, LysoPC, and SM were thus bound to BSA, mimicking serum conditions and avoiding micelle formation. In this case, the final concentration of lipid-free BSA was 40 µM. When labeling was done in serum-free culture medium, Etn and Ser were replaced by 10 µM Etn-d4 and 140 µM Ser-d3 and supplemented with 20 µM Cho-d9. For labeling experiments, the parasite cultures were washed in modified RPMI and then maintained in culture medium containing deuterated precursors for 48 or 96 h at 37°C, under an atmosphere of 5% CO2, 5% O2, and 90% N2. Medium was changed every 24 h. Lipidomic analyses were generally performed on iRBCs (and not on isolated parasites) because these preparations contained all membrane compartments, within the parasite and exported by the parasite to the host erythrocyte. For this, mature iRBCs were purified using VarioMACS. Occasionally, parasites were isolated from mature iRBCs using 0.01% saponin for 1 min at 4°C in cold PBS buffer. Cells were washed with cold PBS buffer, resuspended in 100 µl of water, and stored at −80°C until extraction of water-soluble metabolites. Pellets for extraction of the PLs were directly stored at −80°C. Water-soluble metabolites were extracted according to the method of Storm et al. (31.Storm J. Sethia S. Blackburn G.J. Chokkathukalam A. Watson D.G. Breitling R. Coombs G.H. Muller S. Phosphoenolpyruvate carboxylase identified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism.PLoS Pathog. 2014; 10: e1003876Crossref PubMed Scopus (31) Google Scholar). Briefly, after rapid thawing, 400 µl of cold chloroform/methanol (1/3, vol/vol) was added to cell lysate or culture medium aliquot. Samples were then vigorously mixed, sonicated at 4°C using a sonicating water bath, and incubated at 4°C under shaking conditions during 1 h. After centrifugation at 16,000 g at 4°C, the supernatant was stored at −80°C until LC/MS/MS analysis. The cellular lipids were extracted according to the procedure of Folch et al. (32.Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), as modified by Rock (33.Rock R.C. Incorporation of 14C-labelled fatty acids into lipids of rhesus erythrocytes and Plasmodium knowlesi in vitro.Comp. Biochem. Physiol. B. 1971; 40: 893-906Crossref PubMed Scopus (24) Google Scholar). Briefly, after rapid thawing, 2.1 ml of methanol was added to 350 µl of cell pellet and shaken vigorously. After 15 min at 4°C, 1.05 ml of chloroform was added, and the sample was incubated during 30 min at room temperature, then centrifuged 10 min at 450 g. The supernatant was collected, and the pellet was resuspended in 1.68 ml of chloroform/methanol/water (37.5/50/12.5: vol/vol/vol). After 10 min of centrifugation at 1,800 g, the supernatant was collected and pooled, and then 4.2 ml of chloroform and 1.75 ml of water were added. After 10 min under shaking conditions, the sample was centrifuged 10 min at 1,800 g, and the aqueous phase was removed. The organic phase was dried with nitrogen and resuspended in 100 µl of chloroform/methanol (2:1), transferred into vials under nitrogen, and stored at −80°C until LC/MS/MS analysis. Reverse-phase LC (RPLC) was carried out at 40°C on a Luna 5u C8 (2.Cowman A.F. Healer J. Marapana D. Marsh K. Malaria: biology and disease.Cell. 2016; 167: 610-624Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) 100 Å, 150 × 1 mm, 5 µm particles (Phenomenex, Le Pecq, France). The gradient elution program was a combination of eluent A (isopropanol/methanol/water: 50/10/40: vol/vol/vol) with 0.2% formic acid and 0.028% ammonium hydroxide and eluent B (isopropanol with 0.2% formic acid and 0.028% ammonium hydroxide) with 30% B (0 min), 50% B (0–5 min), 80% B (5–30 min), and 30% B (30–35 min). The flow rate was set at 40 µl × min−1, and 3 µl sample volumes were injected. RPLC/MS/MS analyses were performed with a 5500 QTRAP (AB Sciex) instrument coupled to an LC system (Ultimate 3000, Dionex). Five microliters of PL extracts dissolved in isopropanol/methanol/water (50/10/40: vol/vol/vol) (3.75 × 105 cells/µl) was added to 5 µl of internal standards (PE and PC at 5 µM and PS at 10 µM) and dissolved in 40 µl of isopropanol/methanol/water (50/10/40: vol/vol/vol). Analyses were carried out in the negative (PE and PS) and positive (PC) modes with fast polarity switching (50 ms). RPLC/MS/MS method in multiple reaction monitoring (MRM) mode was adapted for MRM transitions from Buré et al. (34.Buré C. Ayciriex S. Testet E. Schmitter J.M. A single run LC-MS/MS method for phospholipidomics.Anal. Bioanal. Chem. 2013; 405: 203-213Crossref PubMed Scopus (44) Google Scholar). Nitrogen was used as curtain gas (set to 20), gas1 (set to 25), and gas2 (set to 0). Needle voltage was at −4,500 or + 5,500 V without needle heating; declustering potential was between −180 and −85 V or set at +40 V. The collision gas was nitrogen; collision energy varied from 47 to 62 eV on a compound-dependent basis. The dwell time was set to 3 ms. MS/MS experiments were performed by 73 positive MRM scans and 300 negative MRM scans according to the PL identifications obtained by shotgun MS. The area of LC peaks was determined using MultiQuant software (version 3.0, AB Sciex). Only labeled and unlabeled PC, PE, and PS were searched for and quantified in the samples. Shotgun-mass spectrometry analysis by ESI was carried out according to the protocol previously described (34.Buré C. Ayciriex S. Testet E. Schmitter J.M. A single run LC-MS/MS method for phospholipidomics.Anal. Bioanal. Chem. 2013; 405: 203-213Crossref PubMed Scopus (44) Google Scholar). Dried PL extracts were dissolved in 500 µl of a mixture of chloroform/methanol (2/1: vol/vol) containing 7.5 mM ammonium acetate. Each sample was infused into the TurboV electrospray source of a QTRAP 5500 mass spectrometer (AB Sciex, Concord, Canada) at a flow rate of 7 µl·min−1, with fast polarity switching (50 ms) at a scan rate of 200 Da/s. ESI-MS/MS experiments in precursor ion scan were performed in negative ion mode to screen for PE and PS and led to 53 precursor ion scans. In positive ion mode, one precursor ion scan allowed screening for PC. PL species were identified using Lipid View software (version 1.2, AB Sciex). The analysis of selected reaction intermediates was performed by the method of Vo Duy et al. (35.Vo Duy S. Besteiro S. Berry L. Perigaud C. Bressolle F. Vial H.J. Lefebvre-Tournier I. A quantitative liquid chromatography tandem mass spectrometry method for metabolomic analysis of Plasmodium falciparum lipid related metabolites.Anal. Chim. Acta. 2012; 739: 47-55Crossref PubMed Scopus (24) Google Scholar). LC instrumentation included an ACQUITYTM Ultra Performance Liquid Chromatography integrated system from Waters (Milford, MA). Mass spectrometry experiments were performed using a triple quadrupole mass spectrometer TSQ Quantum UltraTM (Thermo Fisher Scientific Inc., Waltham, MA). Metabolites were separated using an Acquity UPLC BEH Shield RP18 column (150 mm × 2.1 mm, 1.7 µm; Waters, Saint-Quentin en Yvelines, France) in the positive ionization mode. Heptafluorobutyric acid was added to the mobile phase as an ion pair reagent. Quantitation was performed using one internal standard. Each metabolite was quantified against a calibration curve prepared by spiking RBC cell pellets (3 × 106 cells) with appropriate working solutions of all metabolites and the internal standard. Using the method Vo Duy et al. (35.Vo Duy S. Besteiro S. Berry L. Perigaud C. Bressolle F. Vial H.J. Lefebvre-Tournier I. A quantitative liquid chromatography tandem mass spectrometry method for metabolomic analysis of Plasmodium falciparum lipid related metabolites.Anal. Chim. Acta. 2012; 739: 47-55Crossref PubMed Scopus (24) Google Scholar) the quantities of P-Cho were overestimated because of a lack of specificity. The introduction of a second product ion for P-Cho allowed us to increase the specificity of the method and to obtain correct values. For the water-soluble metabolites and the final PLs, the area of peaks were determined and reported as percent of the total contents of the corresponding metabolite or PL. PE can be provided by the Kennedy pathway or by the PSD pathway. To distinguish between the two origins, we assume that the relative amounts of CDP-Etn are conserved in the relative amounts of PE. In other words, the 56.8% of CDP-Etn from Etn-d4 gave the 34.7% of PE from Etn-d4. Conserving this ratio, we calculated that 6.4% of CDP-Etn from Ser-d3 should correspond to 3.9% of PE from Ser-d3, and 36.8% of CDP-Etn from unlabeled" @default.
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