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Glucose-induced Remodeling of Intermediary and Energy Metabolism in Procyclic Trypanosoma brucei

2008; Elsevier BV; Volume: 283; Issue: 24 Linguagem: Inglês

10.1074/jbc.m709592200

ISSN

1083-351X

Autores

Virginie Coustou, Marc Biran, Marc Breton, Fabien Guegan, Loïc Rivière, Nicolas Plazolles, Derek P. Nolan, Michael P. Barrett, Jean‐Michel Franconi, Frédéric Bringaud,

Tópico(s)

Biochemical and Molecular Research

Resumo

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of 13C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F0/F1-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F0/F1-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability. The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of 13C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F0/F1-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F0/F1-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability. Trypanosomatids are parasitic protozoa, among which several species cause serious diseases in humans such as sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania spp.). These pathogenic trypanosomatids have developed a digenetic lifestyle with one or several vertebrate hosts (including humans) and a hematophagous insect vector that allows their transmission between vertebrate hosts. Recently, the genome sequencing projects of T. brucei (TREU927 strain) (1Berriman M. Ghedin E. Hertz-Fowler C. Blandin G. Renauld H. Bartholomeu D.C. Lennard N.J. Caler E. Hamlin N.E. Haas B. Bohme U. Hannick L. Aslett M.A. Shallom J. Marcello L. Hou L. Wickstead B. Alsmark U.C. Arrowsmith C. Atkin R.J. Barron A.J. Bringaud F. Brooks K. Carrington M. Cherevach I. Chillingworth T.J. Churcher C. Clark L.N. Corton C.H. Cronin A. Davies R.M. Doggett J. Djikeng A. Feldblyum T. Field M.C. Fraser A. Goodhead I. Hance Z. Harper D. Harris B.R. Hauser H. Hostetler J. Ivens A. Jagels K. Johnson D. Johnson J. Jones K. Kerhornou A.X. Koo H. Larke N. Landfear S. Larkin C. Leech V. Line A. Lord A. Macleod A. Mooney P.J. Moule S. Martin D.M. Morgan G.W. Mungall K. Norbertczak H. Ormond D. Pai G. Peacock C.S. Peterson J. Quail M.A. Rabbinowitsch E. Rajandream M.A. Reitter C. Salzberg S.L. Sanders M. Schobel S. Sharp S. Simmonds M. Simpson A.J. Tallon L. Turner C.M. Tait A. Tivey A.R. Van Aken S. Walker D. Wanless D. Wang S. White B. White O. Whitehead S. Woodward J. Wortman J. Adams M.D. Embley T.M. Gull K. Ullu E. Barry J.D. Fairlamb A.H. Opperdoes F. Barrell B.G. Donelson J.E. Hall N. Fraser C.M. Melville S.E. El-Sayed N.M. Science. 2005; 309: 416-422Crossref PubMed Scopus (1320) Google Scholar), T. cruzi (CL Brener strain) (2El-Sayed N.M. Myler P.J. Bartholomeu D.C. Nilsson D. Aggarwal G. Tran A.N. Ghedin E. Worthey E.A. Delcher A.L. Blandin G. Westenberger S.J. Caler E. Cerqueira G.C. Branche C. Haas B. Anupama A. Arner E. Aslund L. Attipoe P. Bontempi E. Bringaud F. Burton P. Cadag E. Campbell D.A. Carrington M. Crabtree J. Darban H. da Silveira J.F. de Jong P. Edwards K. Englund P.T. Fazelina G. Feldblyum T. Ferella M. Frasch A.C. Gull K. Horn D. Hou L. Huang Y. Kindlund E. Klingbeil M. Kluge S. Koo H. Lacerda D. Levin M.J. Lorenzi H. Louie T. Machado C.R. McCulloch R. McKenna A. Mizuno Y. Mottram J.C. Nelson S. Ochaya S. Osoegawa K. Pai G. Parsons M. Pentony M. Pettersson U. Pop M. Ramirez J.L. Rinta J. Robertson L. Salzberg S.L. Sanchez D.O. Seyler A. Sharma R. Shetty J. Simpson A.J. Sisk E. Tammi M.T. Tarleton R. Teixeira S. Van Aken S. Vogt C. Ward P.N. Wickstead B. Wortman J. White O. Fraser C.M. Stuart K.D. Andersson B. Science. 2005; 309: 409-415Crossref PubMed Scopus (1136) Google Scholar), and Leishmania major (Friedlin strain) (3Ivens A.C. Peacock C.S. Worthey E.A. Murphy L. Aggarwal G. Berriman M. Sisk E. Rajandream M.A. Adlem E. Aert R. Anupama A. Apostolou Z. Attipoe P. Bason N. Bauser C. Beck A. Beverley S.M. Bianchettin G. Borzym K. Bothe G. Bruschi C.V. Collins M. Cadag E. Ciarloni L. Clayton C. Coulson R.M. Cronin A. Cruz A.K. Davies R.M. De Gaudenzi J. Dobson D.E. Duesterhoeft A. Fazelina G. Fosker N. Frasch A.C. Fraser A. Fuchs M. Gabel C. Goble A. Goffeau A. Harris D. Hertz-Fowler C. Hilbert H. Horn D. Huang Y. Klages S. Knights A. Kube M. Larke N. Litvin L. Lord A. Louie T. Marra M. Masuy D. Matthews K. Michaeli S. Mottram J.C. Muller-Auer S. Munden H. Nelson S. Norbertczak H. Oliver K. O'Neil S. Pentony M. Pohl T.M. Price C. Purnelle B. Quail M.A. Rabbinowitsch E. Reinhardt R. Rieger M. Rinta J. Robben J. Robertson L. Ruiz J.C. Rutter S. Saunders D. Schafer M. Schein J. Schwartz D.C. Seeger K. Seyler A. Sharp S. Shin H. Sivam D. Squares R. Squares S. Tosato V. Vogt C. Volckaert G. Wambutt R. Warren T. Wedler H. Woodward J. Zhou S. Zimmermann W. Smith D.F. Blackwell J.M. Stuart K.D. Barrell B. Myler P.J. Science. 2005; 309: 436-442Crossref PubMed Scopus (1134) Google Scholar) have been completed, providing wonderful tools to determine their metabolic complexities (1Berriman M. Ghedin E. Hertz-Fowler C. Blandin G. Renauld H. Bartholomeu D.C. Lennard N.J. Caler E. Hamlin N.E. Haas B. Bohme U. Hannick L. Aslett M.A. Shallom J. Marcello L. Hou L. Wickstead B. Alsmark U.C. Arrowsmith C. Atkin R.J. Barron A.J. Bringaud F. Brooks K. Carrington M. Cherevach I. Chillingworth T.J. Churcher C. Clark L.N. Corton C.H. Cronin A. Davies R.M. Doggett J. Djikeng A. Feldblyum T. Field M.C. Fraser A. Goodhead I. Hance Z. Harper D. Harris B.R. Hauser H. Hostetler J. Ivens A. Jagels K. Johnson D. Johnson J. Jones K. Kerhornou A.X. Koo H. Larke N. Landfear S. Larkin C. Leech V. Line A. Lord A. Macleod A. Mooney P.J. Moule S. Martin D.M. Morgan G.W. Mungall K. Norbertczak H. Ormond D. Pai G. Peacock C.S. Peterson J. Quail M.A. Rabbinowitsch E. Rajandream M.A. Reitter C. Salzberg S.L. Sanders M. Schobel S. Sharp S. Simmonds M. Simpson A.J. Tallon L. Turner C.M. Tait A. Tivey A.R. Van Aken S. Walker D. Wanless D. Wang S. White B. White O. Whitehead S. Woodward J. Wortman J. Adams M.D. Embley T.M. Gull K. Ullu E. Barry J.D. Fairlamb A.H. Opperdoes F. Barrell B.G. Donelson J.E. Hall N. Fraser C.M. Melville S.E. El-Sayed N.M. Science. 2005; 309: 416-422Crossref PubMed Scopus (1320) Google Scholar). Trypanosomatids depend on the carbon sources present in their hosts for their energy metabolism (4Bringaud F. Rivière L. Coustou V. Mol. Biochem. Parasitol. 2006; 149: 1-9Crossref PubMed Scopus (314) Google Scholar). For example, the trypomastigote forms of T. brucei and T. cruzi (bloodstream forms) use d-glucose, which is abundant in the fluids of their vertebrate host(s) (5Cannata J.J. Cazzulo J.J. Comp. Biochem. Physiol. B. 1984; 79: 297-308Crossref PubMed Scopus (51) Google Scholar, 6Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp, New York1986Google Scholar). In contrast, the insect vectors obtain their energy from l -proline and/or l -glutamine, the prominent constituent of their hemolymph and tissue fluids (7Bursell E. The Role of Proline in Energy Metabolism. Plenum Publishing Corp., New York1981: 183-224Google Scholar). Consequently, the insect stages of T. brucei and T. cruzi rely on amino acid catabolism, with a preference for l -proline. However, these parasites prefer d-glucose when grown in medium rich in this sugar. Because glucose-rich media are routinely used to grow these parasites, d-glucose metabolism has received the most attention, and relatively little is known about their amino acid metabolism. Recent advances in understanding about trypanosomatid catabolism have focused on procyclic T. brucei, which has significant overlap with other species regarding its metabolism, but for which RNA interference (RNAi) 2The abbreviations used are: RNAi, RNA interference; FRDg, glycosomal fumarate reductase; SDH, succinate dehydrogenase; PDH, pyruvate dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; MEc, cytosolic malic enzyme; MEm, mitochondrial malic enzyme; ATPϵ-F1β, F1 complex β-subunit; PBS, phosphate-buffered saline. 2The abbreviations used are: RNAi, RNA interference; FRDg, glycosomal fumarate reductase; SDH, succinate dehydrogenase; PDH, pyruvate dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; MEc, cytosolic malic enzyme; MEm, mitochondrial malic enzyme; ATPϵ-F1β, F1 complex β-subunit; PBS, phosphate-buffered saline. has been extensively developed. RNAi is not functional in T. cruzi or in most Leishmania subspecies (8Ullu E. Tschudi C. Chakraborty T. Cell. Microbiol. 2004; 6: 509-519Crossref PubMed Scopus (217) Google Scholar), although it appears that Leishmania braziliensis does have the machinery required for RNAi (9Peacock C.S. Seeger K. Harris D. Murphy L. Ruiz J.C. Quail M.A. Peters N. Adlem E. Tivey A. Aslett M. Kerhornou A. Ivens A. Fraser A. Rajandream M.A. Carver T. Norbertczak H. Chillingworth T. Hance Z. Jagels K. Moule S. Ormond D. Rutter S. Squares R. Whitehead S. Rabbinowitsch E. Arrowsmith C. White B. Thurston S. Bringaud F. Baldauf S.L. Faulconbridge A. Jeffares D. Depledge D.P. Oyola S.O. Hilley J.D. Brito L.O. Tosi L.R. Barrell B. Cruz A.K. Mottram J.C. Smith D.F. Berriman M. Nat. Genet. 2007; 39: 839-847Crossref PubMed Scopus (586) Google Scholar). Procyclic trypanosomes grown in rich media primarily convert d-glucose into succinate and acetate, with smaller amounts of lactate and CO2 (10Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 11van Weelden S.W.H. Fast B. Vogt A. van der Meer P. Saas J. van Hellemond J.J. Tielens A.G.M. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Succinate represents ∼70% of the excreted end products of d-glucose metabolism. It is produced in the glycosomes (peroxisome-like organelles specialized in d-glucose metabolism in trypanosomatids) and the mitochondrion by two NADH-dependent fumarate reductase isoforms, FRDg and FRDm1 (mitochondrial NADH-dependent fumarate reductase), respectively (10Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 12Coustou V. Besteiro S. Rivière L. Biran M. Biteau N. Franconi J.-M. Boshart M. Baltz T. Bringaud F. J. Biol. Chem. 2005; 280: 16559-16570Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 13Coustou V. Biran M. Besteiro S. Rivière L. Baltz T. Franconi J.-M. Bringaud F. J. Biol. Chem. 2006; 281: 26832-26846Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Acetyl-CoA produced in the mitochondrion is not metabolized to CO2 through the tricarboxylic acid cycle (11van Weelden S.W.H. Fast B. Vogt A. van der Meer P. Saas J. van Hellemond J.J. Tielens A.G.M. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), but is converted into acetate by the mitochondrial acetate:succinate CoA-transferase and another as yet unknown enzymatic activity (14van Hellemond J.J. Opperdoes F.R. Tielens A.G.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (111) Google Scholar, 15Rivière L. van Weelden S.W.H. Glass P. Vegh P. Coustou V. Biran M. van Hellemond J.J. Bringaud F. Tielens A.G.M. Boshart M. J. Biol. Chem. 2004; 279: 45337-45346Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Procyclic forms of several T. brucei strains were successfully adapted to glucose-depleted medium, with no significant effect on growth rate (16Furuya T. Kessler P. Jardim A. Schnaufer A. Crudder C. Parsons M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14177-14182Crossref PubMed Scopus (106) Google Scholar, 17Morris J.C. Wang Z. Drew M.E. Englund P.T. EMBO J. 2002; 21: 4429-4438Crossref PubMed Scopus (127) Google Scholar, 18Lamour N. Rivière L. Coustou V. Coombs G.H. Barrett M.P. Bringaud F. J. Biol. Chem. 2005; 280: 11902-11910Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The rate of l -proline consumption in these growth conditions is increased by ∼6-fold (18Lamour N. Rivière L. Coustou V. Coombs G.H. Barrett M.P. Bringaud F. J. Biol. Chem. 2005; 280: 11902-11910Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). It has been proposed that the procyclic form of T. brucei, as well as other trypanosomatids, adapts its energy metabolism to available carbon sources (4Bringaud F. Rivière L. Coustou V. Mol. Biochem. Parasitol. 2006; 149: 1-9Crossref PubMed Scopus (314) Google Scholar); however, this view has been recently questioned (19van Weelden S.W.H. van Hellemond J.J. Opperdoes F.R. Tielens A.G.M. J. Biol. Chem. 2005; 280: 12451-12460Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 20van Hellemond J.J. Hoek A. Schreur P.W. Chupin V. Ozdirekcan S. Geysen D. van Grinsven K.W. Koets A.P. Van den Bossche P. Geerts S. Tielens A.G.M. Eukaryot. Cell. 2007; 6: 1693-1696Crossref PubMed Scopus (7) Google Scholar). Here, we illustrate the metabolic flexibility of procyclic trypanosomes by comparing their l -proline metabolism when grown in glucose-rich and glucose-depleted media. In glucose-rich conditions, the l -proline degradation pathway is truncated, and complexes II and IV of the respiratory chain and the mitochondrial F0/F1-ATP synthase become of greatly reduced significance to the energy pathways of the cell. We conclude that d-glucose availability leads to down-regulation of the proline degradation pathway, along with proton flow through the mitochondrial F0/F1-ATP synthase and electron flow through the respiratory chain (in addition to negative regulation of the rate of l -proline consumption). Trypanosome and Cell Cultures—The procyclic form of T. brucei EATRO1125 was cultured at 27 °C in SDM79 medium containing 10% (v/v) heat-inactivated fetal calf serum and 3.5 mg/ml hemin (21Brun R. Schonenberger M. Acta Trop. 1979; 36: 289-292PubMed Google Scholar) or in a glucose-depleted medium derived from SDM79, called SDM80 (18Lamour N. Rivière L. Coustou V. Coombs G.H. Barrett M.P. Bringaud F. J. Biol. Chem. 2005; 280: 11902-11910Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). SDM80 medium is supplemented with 9% (v/v) heat-inactivated fetal calf serum dialyzed by ultrafiltration against 0.15 m NaCl (molecular mass cutoff of 10,000 Da; Sigma F0392; d-glucose concentration of 1 mm) and 1% (v/v) heat-inactivated fetal calf serum (d-glucose concentration of 5 mm). The d-glucose concentration in SDM80 medium is 0.15 mm compared with 6 mm in SDM79 medium and 6.15 mm in SDM80 medium supplemented with d-glucose (SDM80glu). Inhibition of Gene Expression by RNAi—The inhibition by RNAi of gene expression in the procyclic forms (22Ngo H. Tschudi C. Gull K. Ullu E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14687-14692Crossref PubMed Scopus (600) Google Scholar) was performed by expression of stem-loop "sense/antisense" RNA molecules of the targeted sequences (23Bringaud F. Robinson D.R. Barradeau S. Biteau N. Baltz D. Baltz T. Mol. Biochem. Parasitol. 2000; 111: 283-297Crossref PubMed Scopus (47) Google Scholar) introduced into the pLew100 or pLew79 expression vector (kindly provided by E. Wirtz and G. Cross) (24Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1110) Google Scholar) or the p2T7Ti-177 vector (kindly provided by B. Wickstead and K. Gull) (25Wickstead B. Ersfeld K. Gull K. Mol. Biochem. Parasitol. 2002; 125: 211-216Crossref PubMed Scopus (204) Google Scholar) as described previously (10Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 12Coustou V. Besteiro S. Rivière L. Biran M. Biteau N. Franconi J.-M. Boshart M. Baltz T. Bringaud F. J. Biol. Chem. 2005; 280: 16559-16570Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 13Coustou V. Biran M. Besteiro S. Rivière L. Baltz T. Franconi J.-M. Bringaud F. J. Biol. Chem. 2006; 281: 26832-26846Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Rivière L. van Weelden S.W.H. Glass P. Vegh P. Coustou V. Biran M. van Hellemond J.J. Bringaud F. Tielens A.G.M. Boshart M. J. Biol. Chem. 2004; 279: 45337-45346Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 23Bringaud F. Robinson D.R. Barradeau S. Biteau N. Baltz D. Baltz T. Mol. Biochem. Parasitol. 2000; 111: 283-297Crossref PubMed Scopus (47) Google Scholar, 26Coustou V. Besteiro S. Biran M. Diolez P. Bouchaud V. Voisin P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: 49625-49635Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). These three vectors contain the phleomycin resistance gene. Construction of the pLew-ASCT-SAS, pLew-FRDm1-SAS, pLew-FRDg-SAS, pLew-FRDg/m1-SAS, pLew-FHm-SAS, pLew-FHc-SAS, and p2T7Ti-177-FHc/mSAS plasmids, used to produce the RNAiASCT-hp4, RNAiFRDm1-G7, RNAiFRDg-C2, RNAiFRDg/m1-B5, RNAiFHmA1, RNAiFHc-A5 and RNAiFHc/m-F10 cell lines, respectively, has been described (12Coustou V. Besteiro S. Rivière L. Biran M. Biteau N. Franconi J.-M. Boshart M. Baltz T. Bringaud F. J. Biol. Chem. 2005; 280: 16559-16570Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 13Coustou V. Biran M. Besteiro S. Rivière L. Baltz T. Franconi J.-M. Bringaud F. J. Biol. Chem. 2006; 281: 26832-26846Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Rivière L. van Weelden S.W.H. Glass P. Vegh P. Coustou V. Biran M. van Hellemond J.J. Bringaud F. Tielens A.G.M. Boshart M. J. Biol. Chem. 2004; 279: 45337-45346Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) (the RNAiXXX cell lines were previously called ΔXXX). The pLew-SDH-SAS construct targets a fragment (bp 12–452) of the succinate dehydrogenase (SDH) gene (Tb09.160.4380). Briefly, a PCR-amplified 524-bp fragment containing the antisense SDH sequence (440 bp of targeted sequence plus 96 bp used as a spacer to form the loop between the annealing sense and antisense sequences, respectively) with restriction sites added to the primers was inserted into the HindIII and BamHI restriction sites of the pLew100 plasmid. Then, a PCR-amplified 474-bp fragment containing the sense SDH sequence was inserted upstream of the antisense sequence using HindIII and XhoI restriction sites (XhoI was introduced at the 3′-extremity of the antisense PCR fragment). The resulting plasmid (pLew-SDH-SAS) contains a sense and antisense version of the targeted gene fragment, separated by a 96-bp fragment, under the control of the PARP promoter linked to a prokaryotic tetracycline operator. The pLew-PDH.E2-SAS plasmid (designed to inhibit, by RNAi, the expression of the E2 subunit of the pyruvate dehydrogenase (PDH) complex) was generated in the pLew79 vector with the same strategy described above, employing the same restriction sites. The targeting cassette is composed of a sense and antisense version of the Tb10.6k15.3080 gene (bp 69–632), separated by an 84-bp fragment. To target inhibition of expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene (Tb927.2.4210), the 3′-end of the coding sequence and the 3′-untranslated region were selected. The targeted sequences correspond to the last 130 bp (bp 1448–1578 of the PEPCK gene) of the coding sequence followed by 197 bp of the 3′-untranslated region. The sense and antisense fragments, separated by 40 bp, were cloned into the pLew100 vector. To simultaneously target both malic enzyme genes, which encode the cytosolic and mitochondrial isoforms (MEc (Tb11.02.3120) and MEm (Tb11.02.3130), respectively), we selected a fragment from the MEc gene (bp 427–824) that showed the highest homology to the MEm gene (80% identity). The sense and antisense fragments, separated by 26 bp, were cloned in the pLew100 vector. To inhibit expression of the mitochondrial F0/F1-ATP synthase, the sense and antisense versions of a fragment (bp 471–934) of the β-subunit gene of the F1 complex (ATPϵ-F1β; Tb927.3.1380), separated by a 58-bp fragment, were cloned into the BamHI and HindIII restriction sites of the p2T7Ti-177 vector. Trypanosome Transfection, Adaptation to SDM80 Medium, and RNAi Induction—The EATRO1125 procyclic form cell line (EATRO1125.T7T), constitutively expressing the T7 RNA polymerase gene and the tetracycline repressor under the control of a T7 RNA polymerase promoter for tetracycline-inducible expression (23Bringaud F. Robinson D.R. Barradeau S. Biteau N. Baltz D. Baltz T. Mol. Biochem. Parasitol. 2000; 111: 283-297Crossref PubMed Scopus (47) Google Scholar), was transformed with each of the plasmids. Trypanosome transfection and selection of phleomycin-resistant clones were performed as reported previously (27Bringaud F. Baltz D. Baltz T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7963-7968Crossref PubMed Scopus (108) Google Scholar). Briefly, all of the RNAi-harboring cell lines were selected in glucose-rich SDM79 medium containing hygromycin B (25 μg/ml), neomycin (10 μg/ml), and phleomycin (5 μg/ml). Aliquots of each were frozen in liquid nitrogen to provide stocks of each line that had not been cultivated long term in medium. The selected cell lines were then adapted to glucose-rich SDM80glu medium and glucose-depleted SDM80 medium containing the same concentration of the three antibiotics. The cell lines were first transferred to SDM80glu medium and maintained for at least 2 weeks before adaptation to SDM80 medium. All analyzed cell lines were maintained for 2 weeks in SDM80 or SDM80glu medium before addition of 1 μg/ml tetracycline for RNAi induction. Enzyme Assays—Sonicated (5 s at 4 °C) crude extracts of trypanosomes resuspended in cold hypotonic buffer (10 mm potassium phosphate, pH 7.8) were tested for pyruvate kinase (EC 2.7.1.40) (28Callens M. Opperdoes F.R. Mol. Biochem. Parasitol. 1992; 50: 235-243Crossref PubMed Scopus (19) Google Scholar), fumarase (EC 4.2.1.2) (13Coustou V. Biran M. Besteiro S. Rivière L. Baltz T. Franconi J.-M. Bringaud F. J. Biol. Chem. 2006; 281: 26832-26846Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), FRD (EC 1.3.1.6) (10Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), PDH (EC 1.2.2.2) (29Else A.J. Clarke J.F. Willis A. Jackman S.A. Hough D.W. Danson M.J. Mol. Biochem. Parasitol. 1994; 64: 233-239Crossref PubMed Scopus (18) Google Scholar), ME (EC 1.1.1.40) (30Klein R.A. Linstead D.J. Wheeler M.V. Parasitology. 1975; 71: 93-107Crossref PubMed Scopus (52) Google Scholar), and SDH (EC 1.3.99.1) (31Brown J.M. Quinton M.S. Yamamoto B.K. J. Neurochem. 2005; 95: 429-436Crossref PubMed Scopus (82) Google Scholar) activities as described. Western Blot Analyses—Total protein extracts of the procyclic form of T. brucei (2 × 106 cells) were separated by SDS-PAGE (10%) and immunoblotted on Immobilon-P filters (Millipore) (32Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). Immunodetection was performed as described (32Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar, 33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) using, as primary antibodies, the rabbit antiserum against the F1 moiety of the mitochondrial F0/F1-ATP synthase isolated from Crithidia fasciculata (a gift from D. Speijer) (34Speijer D. Breek C.K. Muijsers A.O. Hartog A.F. Berden J.A. Albracht S.P. Samyn B. Van Beeumen J. Benne R. Mol. Biochem. Parasitol. 1997; 85: 171-186Crossref PubMed Scopus (55) Google Scholar) and the rabbit antiserum against glycerol-3-phosphate dehydrogenase (27Bringaud F. Baltz D. Baltz T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7963-7968Crossref PubMed Scopus (108) Google Scholar, 35Denise H. Giroud C. Barrett M.P. Baltz T. Eur. J. Biochem. 1999; 259: 339-346Crossref PubMed Scopus (29) Google Scholar) diluted 1:100. Peroxidase-conjugated goat anti-mouse or anti-rabbit Ig (1:10,000 dilution) was used as a secondary antibody, and revelation was performed using ECL™ Western blotting detection reagents (Amersham Biosciences) as described by the manufacturer. For quantitative analyses, membranes or x-ray films were scanned, and protein bands were quantified using NIH Image software. Determination of Sensitivity to Metabolic Drugs—EATRO1125 procyclic cells (2 × 106 cells/ml) were incubated in SDM80 medium either containing or lacking 6 mm d-glucose in the presence of decreasing quantities of rotenone (from 0.5 mm to 20 nm), salicylhydroxamic acid (from 10 mm to 1.5 μm), potassium cyanide (from 10 mm to 80 nm), malonate (from 10 mm to 80 nm), and oligomycin (from 50 μg/ml to 0.01 ng/ml). The assay was performed in 96-well microtiter plates with 100 μl of cell suspension/well, and the cell viability was determined optically. The drug concentration required to kill all of the cells (100% lethal dose, LD100) was determined 1, 2, 3, 4, and 5 days after drug addition. d-Glucose and l -Proline Measurements—To determine the rate of d-glucose and l -proline consumption, cells (inoculated at 1–1.5 × 107 cells/ml) were grown in 10 ml of SDM80 (0.15 mm d-glucose) or SDM80glu (6.15 mm d-glucose) medium. Aliquots of each growth medium (500 μl) were collected 0, 1, 6, 9, 10, 23, and 24 h after incubation at 27 °C. Cells were lysed by sonication in 500 μl of 5 mm phosphate buffer, pH 7.5. The quantity of d-glucose present in the medium was determined using the Glucose GOD-PAP kit (Biolabo S.A.). l -Proline concentration was determined with a colorimetric assay as described previously (36Bates L.S. Waldren R.P. Teare I.D. Plant Soil. 1973; 39: 205-207Crossref Scopus (12458) Google Scholar) after deproteinization of the samples by perchloric acid treatment. At 0, 9, and 24 h, the cell density and cellular protein concentration were determined to estimate the amount of carbon source consumed per mg of cellular protein. NMR Experiments—6 × 108 T. brucei procyclic cells were collected by centrifugation at 1400 × g for 10 min, washed once with phosphate-buffered saline (PBS), and incubated in 30 ml of incubation buffer (PBS supplemented with 24 mm NaHCO3, pH 7.3). For the analysis of d-glucose metabolism, the cells were incubated for 6 h at 27°C in incubation buffer containing 110 μmol of d-[1-13C]glucose. For the analysis of l -proline metabolism, the cells were incubated in PBS, pH 7.3 (without NaHCO3), containing 20 μmol of l -[4-13C]proline and in the presence or absence of 100 μmol of unenriched d-glucose or 100 μmol of unenriched pyruvate. The d-glucose concentration in the medium was determined with the Glucose GOD-PAP kit. l -Proline concentration was determined with a calorimetric assay as described previously (36Bates L.S. Waldren R.P. Teare I.D. Plant Soil. 1973; 39: 205-207Crossref Scopus (12458) Google Scholar) after deproteinization of the samples by perchloric acid treatment. The integrity of the cells during the incubation was checked by microscopic observation. After centrifugation for 10 min at 1400 × g, the supernatant was lyophilized and redissolved in 485 μl of D2O, and 15 μl of pure dioxane was added as an external reference. 13C NMR spectra were collected at 125.77 MHz with a Bruker DPX500 spectrometer equipped with a 5-mm broadband probe head. Measurements were

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