Proline Metabolism in Procyclic Trypanosoma brucei Is Down-regulated in the Presence of Glucose
2005; Elsevier BV; Volume: 280; Issue: 12 Linguagem: Inglês
10.1074/jbc.m414274200
ISSN1083-351X
AutoresNadia F. Lamour, Loïc Rivière, Virginie Coustou, Graham H. Coombs, Michael P. Barrett, Frédéric Bringaud,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoProline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-d-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose. Proline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-d-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose. African trypanosomes of the brucei subgroup are responsible for a number of important diseases in man and animals (1Barrett M.P. Burchmore R.J. Stich A. Lazzari J.O. Frasch A.C. Cazzulo J.J. Krishna S. Lancet. 2003; 362: 1469-1480Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). The metabolism of these organisms has been the subject of considerable interest. Bloodstream form trypanosomes are entirely dependent upon glycolytic substrate level phosphorylation for the generation of energy (2Opperdoes F.R. Annu. Rev. Microbiol. 1987; 41: 127-151Crossref PubMed Scopus (429) Google Scholar, 3Michels P.A. Hannaert V. Bringaud F. Parasitol. Today. 2000; 16: 482-489Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The first seven steps of the glycolytic pathway are localized to an unusual organelle, the glycosome (4Opperdoes F.R. Borst P. Spits H. Eur. J. Biochem. 1977; 76: 21-28Crossref PubMed Scopus (75) Google Scholar). A glycerol 3-phosphate:dihydroxyacetone-phosphate shuttle operates between the glycosome and the mitochondrion where a plant like ubiquinone-linked alternative oxidase acts to transfer electrons to oxygen (5Tielens A.G.M. Van Hellemond J.J. Parasitol. Today. 1998; 14: 265-271Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This shuttle is critical in maintaining the NAD+/NADH balance within the glycosome. No net ATP synthesis occurs in the glycosome but ATP is produced cytosolically by the pyruvate kinase reaction (6Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar). Pyruvate is excreted from these cells with no further metabolism and a mere two moles of ATP are synthesized per mole of glucose used. Bloodstream form trypanosomes can sustain this profligate metabolism as they are exposed to a steady supply of high glucose in mammalian blood. They cannot, however, use non-carbohydrate substrates for the generation of energy. Glucose metabolism is thus perceived as an excellent target for therapeutic intervention in bloodstream form trypanosomes (7Verlinde C.L. Hannaert V. Blonski C. Willson M. Perie J.J. Fothergill-Gilmore L.A. Opperdoes F.R. Gelb M.H. Hol W.G. Michels P.A. Drug. Resist. Updat. 2001; 4: 50-65Crossref PubMed Scopus (194) Google Scholar). It appears that the Trypanosoma brucei bloodstream energy metabolism is very simple and contrasts with the more elaborate version present in all the other trypanosomatids analyzed so far, including the insect stages of T. brucei (3Michels P.A. Hannaert V. Bringaud F. Parasitol. Today. 2000; 16: 482-489Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 8Hannaert V. Bringaud F. Opperdoes F.R. Michels P.A. Kinetoplastid Biol. Dis. 2003; 2: 1-30Crossref PubMed Scopus (139) Google Scholar). The life cycle of brucei group trypanosomes is complex. Multiple distinct stages exist in the tsetse fly vector that carries the parasite between mammalian hosts (9Vickerman K. Br. Med. Bull. 1985; 41: 105-114Crossref PubMed Scopus (481) Google Scholar). The metabolism of the parasites, as they proliferate in the midgut of their tsetse fly vector, is markedly different from that used by the bloodstream form. In the tsetse midgut, glucose is scarce but may become transiently abundant following insect blood meals. Proline is a key energy source within the tsetse fly (10Balogun R.A. Comp. Biochem. Physiol. A. 1974; 49: 215-222Crossref PubMed Scopus (24) Google Scholar, 11Bursell E. Billing K.J. Hargrove J.W. McCabe C.T. Slack E. Trans. R. Soc. Trop. Med. Hyg. 1973; 67: 296Abstract Full Text PDF PubMed Scopus (9) Google Scholar), and it has been speculated that this is a main energy source for the procyclic form trypanosomes too (12Ford W.C. Bowman I.B. Trans. R. Soc. Trop. Med. Hyg. 1973; 67: 257Abstract Full Text PDF PubMed Scopus (9) Google Scholar, 13Evans D.A. Brown R.C. J. Protozool. 1972; 19: 686-690Crossref PubMed Scopus (80) Google Scholar, 14ter Kuile B.H. Opperdoes F.R. J. Bacteriol. 1992; 174: 2929-2934Crossref PubMed Google Scholar). However, procyclics make efficient use of glucose, which is the preferred carbon source in the glucose-rich medium commonly used to grow these parasites (15Cross G.A. Klein R.A. Linstead D.J. Parasitology. 1975; 71: 311-326Crossref PubMed Scopus (79) Google Scholar, 16Brun R. Schonenberger M. Acta Trop. 1979; 36: 289-292PubMed Google Scholar). Until recently, it was widely accepted that procyclic trypanosomes produce ATP primarily by the mitochondrial F0/F1-ATP synthase (oxidative phosphorylation) exploiting the proton gradient across the mitochondrial inner membrane generated by the respiratory chain (5Tielens A.G.M. Van Hellemond J.J. Parasitol. Today. 1998; 14: 265-271Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In addition, the respiratory chain was considered to be fed chiefly by NADH produced by the tricarboxylic acid cycle (6Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar). However, recent publications questioned these conclusions and a new model has been proposed (17Besteiro S. Barrett M.P. Rivière L. Bringaud F. Trends Parasitol. 2005; (in press)PubMed Google Scholar) (Fig. 1). A functional Krebs cycle is not essential for energy metabolism and aconitase-defective cells thrive using glucose as an energy source (18Van Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Moreover, blocking the mitochondrial F0/F1-ATP synthase has little impact on growth and does not affect ATP production in glucose-rich medium (19Coustou 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 (86) Google Scholar), while substrate level phosphorylation does appear to be essential to growth (19Coustou 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 (86) Google Scholar, 20Bochud-Allemann N. Schneider A. J. Biol. Chem. 2002; 277: 32849-32854Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Essential sites of ATP production by substrate level phosphorylation include the cytosol (phosphoglycerate kinase and pyruvate kinase) and the mitochondrion (succinyl-CoA synthetase). Interestingly, the succinyl-CoA synthetase, shown to be essential to procyclic trypanosomes, catalyzes the last step of both glucose and proline degradation to produce the excreted end products acetate and succinate, respectively (Fig. 1) (18Van Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 21Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). To date, no in vitro studies using cells grown in glucose-depleted medium, conditions to mimic the midgut environment of tsetse flies between blood meals, appear to have been conducted. We therefore set out to investigate the ability of procyclic trypanosomes to catabolize proline and to determine whether the availability of glucose influences this. To address this question, we adapted two T. brucei procyclic strains (EATRO1125 and 427) to glucose-depleted medium and studied their carbon source comsumption and sensitivity to oligomycin (the most specific inihibitor of the F0/F1-ATP synthase). In addition, we have generated and analyzed a mutant cell line inhibited for the second step of the proline metabolism (proline dehydrogenase (PRODH) 1The abbreviations used are: PRODH, proline dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; FCS, fetal calf serum; INT, iodonitrophenyl tetrazolium; DCPIP, dichlorophenolindophenol; RNAi, RNA interference.). This analysis shows that glucose exerts a negative control on proline metabolism by down-regulating PRODH and affecting proline uptake (Fig. 1). Trypanosomes and Cell Culture—T. brucei strain 427 and EATRO1125 procyclic forms were cultivated at 25 °C in SDM79 medium (16Brun R. Schonenberger M. Acta Trop. 1979; 36: 289-292PubMed Google Scholar) or a glucose-depleted medium derived from SDM79, called SDM80 (1 mm NaH2PO4, 116 mm NaCl, 0.8 mm MgSO4, 5.4 mm KCl, 1.8 mm CaCl2, 26.2 mm NaHCO3, 30.7 mm Hepes, 23.9 mm MOPS, 4 mm pyruvate, 1% (v/v) vitamin mix 100× (Invitrogen, catalog number 010144), 5.2 mm proline (or no proline), 5.9 mm threonine, 1.1 mm l-arginine, 0.58 mm l-methionine, 0.68 mm l-phenylalanine, 0.75 mm l-tyrosine, 0.1 mm l-cystine, 0.2 mm l-histidine, 0.4 mm l-isoleucine, 0.76 mm l-leucine, 0.4 mm l-lysine, 0.05 mm l-tryptophan, 0.4 mm l-valine, 2.25 mm l-alanine, 0.1 mm l-asparagine, 0.1 mm l-aspartic acid, 0.09 mm l-glutamic acid, 0.49 mm l-serine, 0.1 mm glycine, 1.28 mm taurine, 0.46 mm glutamine, 0.2 mm β-mercaptoethanol, 0.1 mm hypoxanthine, 0.017 mm thymidine, 0.1 mm kanamycin, 0.008 mm hemin). Fetal calf serum (FCS) dialyzed by ultrafiltration against 0.15 m NaCl (molecular mass cutoff: 10,000 Da) and heat inactivated (Sigma: catalog number F0392, glucose concentration: 1 mm) was added to 9% (v/v) and heat-inactivated FCS (glucose concentration: 5 mm) to 1% (v/v). The glucose concentration in the SDM80 medium is 0.15 mm as compared with 6 mm in the SDM79 medium. The T. brucei cell line 29-13 (derived from strain 427) used in RNA interference experiments was grown in SDM79 medium containing 15 μg·ml-1 G418 and 25 μg·ml-1 hygromycin B. Determination of Metabolite Consumption and Excretion—To determine the concentration of metabolites consumed or excreted by the EATRO1125 procyclic trypanosomes, cells (inoculated at 106 cells·ml-1) were grown in SDM80 medium (with 1.6 mm l-glutamine and 8 mm l-threonine) either containing or lacking 10 mm d-glucose, until stationary phase was reached. Aliquots of the growth medium were collected twice a day for analysis. The quantity of d-glucose present in the medium was determined using the "glucose trinder kit" (Sigma). Pyruvate concentration was determined enzymatically as described previously (22Czok R. Lamprecht W. Bergmeyer H.U. Methods of Enzymatic Analysis. 3. Verlag Chemie/Academic Press, New York1974: 1585-1589Google Scholar). The concentration of each of the 20 amino acids present in the medium was determined by chromatography on an automatic amino acid analyzer coupled to a computing integrator (Beckman), after deproteinization of the samples by perchloric acid treatment. Additionally, a colorimetric assay (23Bates L.S. Waldren R.P. Teare I.D. Plant Soil. 1973; 39: 205-207Crossref Scopus (13286) Google Scholar) was used to determine the proline concentration. Determination of Oligomycin Sensitivity—The EATRO1125 and 427 procyclic cells (2 × 106 cells·ml-1) were incubated in SDM80 either containing or lacking 6 mm glucose, in the presence of decreasing quantities of oligomycin (from 50 μg·ml-1 to 0.01 ng·ml-1). The assay was performed in 96-well microtitre plates with 100 μl of cell suspension per well, and the cell viability was determined optically. The oligomycin concentration required to kill all of the cells (lethal dose 100: LD100) was determined 24, 48, 72, and 96 h after drug addition. Cloning of the Proline Dehydrogenase Gene and DNA/RNA Analysis—T. brucei genomic DNA was made using a mini-prep method (24Medina-Acosta E. Cross G.A. Mol. Biochem. Parasitol. 1993; 59: 327-329Crossref PubMed Scopus (236) Google Scholar). Total RNA was produced using TRIzol® reagent (Invitrogen), and Pfu DNA polymerase (Promega) was used for all PCR reactions using a GeneAmp PCR system 2400 (PerkinElmer Life Sciences). For reverse transcriptase PCR experiments, the enzyme SUPERScript II, RNase H- reverse transcriptase (Invitrogen) was used. The 5′ end of the proline dehydrogenase gene was obtained using a specific internal primer (RTTb1 5′-ACATGCCTGTCACCTTAACAGC) and spliced leader primers (SL1 5′-TAACGCTATATAAGTATCAGTTTC and SL2 5′-AGTATCAGTTTCTGTACTTTATTG). Southern and Northern blotting (using 5 μg of DNA or 4 μg of RNA per lane, respectively) were as described (25Sambrook J. Fritsch E.F. Maniatis T. 2nd Ed. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.37-16.68Google Scholar). 32P-Labeled probes were made using the Prime It® II kit (Stratagene). Vector NTI (v 6.0) was used to analyze DNA and protein sequences (searching for open reading frames and restriction sites and construction of cloning and expression plasmids). SignalP (26Nielsen H. Engelbrecht J. von Heijne G. Brunak S. Proteins. 1996; 24: 165-177Crossref PubMed Scopus (72) Google Scholar), Predotar (Institut National de Recherche Agronomique), and TargetP (27Emanuelsson O. Nielsen H. Brunak S. von Heijne G. J. Mol. Biol. 2000; 300: 1005-1016Crossref PubMed Scopus (3638) Google Scholar), MitoProt (28Claros M.G. Vincens P. Eur. J. Biochem. 1996; 241: 779-786Crossref PubMed Scopus (1386) Google Scholar), and Tmpred (29Hofmann K. Stoffel W. Biol. Chem. 1993; 374: 166Google Scholar) were used to search for secretory, mitochondrial, or other targeting signals and transmembrane domains, respectively. Production of Anti-PRODH Antibodies and Protein Analysis—Recombinant PRODH lacking the N-terminal 72 amino acids that included the mitochondrial targeting sequence and a putative transmembrane domain (preliminary experiments showed that the presence of these domains precluded soluble expression) was created after amplification using primers TbPRODH72 (5′-AAACATATGAAGCGTGCTGAGGCAATTTTT) and Tbrev (5′-CCCAAGCTTCATCCAAAAGACGCG). The product was ultimately cloned into pET21a+ and expressed in Escherichia coli strain BL21(DE3). Protein was produced in soluble form after expression at 16 °C in the presence of 1 mm isopropyl β-d-thiogalactopyranoside. The purification was performed on the Biocad 700E work station using Ni2+ chelate chromatography (Novagen) with the elution buffer recommended by the manufacturer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 500 mm imidazole). Antiserum was raised in rabbits (Diagnostics Scotland), with an initial injection of 0.4 ml of protein solution at 1 mg·ml-1 followed by three more 0.2-ml injections at 1-month intervals. Antibodies were purified using an AminoLink® (Pierce) kit carrying immobilized purified PRODH on a solid support. Purified antibodies were aliquoted and stored at -20 °C until use. Western blotting of SDS-PAGE separated proteins on nitrocellulose membranes involved blocking for 3 h at room temperature with 1× TBS (0.02 m Tris-HCl, 13.7 mm NaCl) containing 5% (w/v) powdered milk and 0.2% (v/v) Tween 20. The primary antibody, antihistidine tag (diluted 1:2000) or specific PRODH antibody (diluted 1:500 to 1:5000), was made up in 1× TBS, 1% (w/v) powdered milk, and 0.1% (v/v) Tween 20, before being added to the membrane and shaken overnight at 4 °C. The SuperSignal chemiluminescent substrate (Pierce) protocol with 1:2000 diluted anti-mouse IgG conjugated to peroxidase or with 1:500 to 1:5000 anti-rabbit horseradish peroxidase-coupled IgG (Sigma) allowed the detection of the proteins according to manufacturer's specifications. Proline Dehydrogenase Assay—PRODH activity was measured following the reduction of the electron-accepting dye iodonitrophenyl tetrazolium (INT), at 520 nm (30Menzel R. Roth J. J. Mol. Biol. 1981; 148: 21-44Crossref PubMed Scopus (52) Google Scholar), or dichlorophenolindophenol (DCPIP) (31Johnson A.B. Strecter H.J. J. Biol. Chem. 1962; 237: 1876-1882Abstract Full Text PDF PubMed Google Scholar), at 600 nm (32Brown E.D. Wood J.M. J. Biol. Chem. 1993; 268: 8972-8979Abstract Full Text PDF PubMed Google Scholar). Log phase procyclic cells were harvested and washed twice with phosphate-buffered saline, pH 7.9, before being resuspended in 500 μl of TSE buffer (25 mm Tris-HCl, pH 8, 1 mm EDTA, 0.25 m sucrose) and lysed by sonication before testing for PRODH activity (33Obungu V.H. Kiaira J.K. Njogu R.M. Olembo N.K. Comp. Biochem. Physiol. B. 1999; 123: 59-65Crossref PubMed Scopus (11) Google Scholar, 34Besteiro 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). The INT reaction mixture contained variable proline concentrations, 16% (v/v) ethylene glycol, 0.4% (v/v) Tween 20; 0.16 m Tris-HCl, pH 8.5, 0.04 mg of gelatin, and 0.5 mm INT. The DCPIP reaction mixture contained 11 mm MOPS, 11 mm MgCl2, 11% (v/v) glycerol, 0.28 mm phenazine methosulfate, and 56 μm of DCPIP, pH 7.5. Variable proline concentrations were added to 900-950 μl of the stock assay mix, and the reaction was started by adding the enzyme (1-50 μl). Activity was monitored spectrophotometrically in cuvettes with a 1-cm light path at 600 nm. RNA Interference (RNAi)—A 606-bp, XbaI-flanked, fragment of the PRODH gene was amplified by PCR from T. brucei gDNA using specific primers each containing an XbaI restriction site (5′-ATTTTCTAGACTCGGACCCATCCATATTTCG and 5′-CCCTCTAGAATCACAACATTACACTCCTCC). This PCR product was cloned into the plasmid p2T7 (35LaCount D.J. Bruse S. Hill K.L. Donelson J.E. Mol. Biochem. Parasitol. 2000; 111: 67-76Crossref PubMed Scopus (157) Google Scholar) yielding the plasmid p2T7PRODH. 8 μg of NotI-linearized p2T7PRODH was transfected into the T. brucei cell line 29-13 (strain 427), washed, and resuspended in Zimmerman's post-fusion medium by electroporation in a 0.4-cm cuvette, using a single pulse at a voltage of 1.5 kV and 25-microfarad capacitance with an infinite resistance using a Bio-Rad Gene Pulser. Transfected parasites were transferred into prewarmed SDM79 medium supplemented with 10% (v/v) heat-inactivated FCS, 15 μg·ml-1 G418, and 25 μg·ml-1 hygromycin B at 25 °C. 24 h later 10 μg·ml-1 phleomycin was added to the medium to select stably expressing cell lines. Clones were selected by limiting dilution in SDM79 supplemented with 10% (v/v) FCS and 25% (v/v) conditioned medium collected and filtered after cultivation of wild-type parasites. Cultures of one clone (Δprodh, clone F2) were initiated using 5 × 105 parasites·ml-1 in the absence or the presence of 1 μg·ml-1 tetracycline (tetracycline induces the expression of dsRNA). 10 ml of complete SDM79 medium was used or 10 ml of SDM80 medium supplemented with proline and/or glucose at different concentrations as stated in the text. Parasite density was measured every 24 h with an improved Neubauer hematocytometer (Weber Scientific). Growth was also analyzed using the Alamar® blue assay (36Raz B. Iten M. Grether-Buhler Y. Kaminsky R. Brun R. Acta Trop. 1997; 68: 139-147Crossref PubMed Scopus (641) Google Scholar). Proline Uptake Assay—T. brucei strain 427 grown in SDM80 medium, either containing or lacking 10 mm glucose, or Δprodh clone F2 grown in SDM79 were harvested during the mid-log phase of growth by centrifuging at 3000 × g for 10 min at 4 °C before being washed twice in Carter's balanced saline solution (37Carter N.S. Fairlamb A.H. Nature. 1993; 361: 173-176Crossref PubMed Scopus (311) Google Scholar) at 4 °C. The pellet was resuspended in Carter's balanced saline solution at the density of 108 parasite·ml-1. Radiolabeled l-[2,3,4,5-3H]proline (112 Ci·mmol-1) (PerkinElmer Life Sciences) was used in uptake assays. Uptake involved the oil stop transport assay as described (37Carter N.S. Fairlamb A.H. Nature. 1993; 361: 173-176Crossref PubMed Scopus (311) Google Scholar, 38Hasne M.P. Barrett M.P. Mol. Biochem. Parasitol. 2000; 111: 299-307Crossref PubMed Scopus (18) Google Scholar) with 100 μl of the test solution cushioned above 90 μl of oil mixture (di-n-butylphthalate and mineral oil at a ratio of 7:1). 100 μl of cells (at 108 cells·ml-1) were added to each tube and incubated for times ranging between 3 s and 3 h depending on the experiment. The transport assays were terminated by centrifugation (12,000 × g, 1 min at room temperature). Samples were flash-frozen in liquid nitrogen before removing the pellets using a tube cutter and transferring them to scintillation vials containing 200 μl of 2% (w/v) SDS. 3 ml of scintillation fluid (Ecoscint A, National Diagnostics) was added and tubes left overnight. Radioactivity levels were determined by using an LKB Wallac 1219 Rackbeta liquid scintillation counter. Background levels, which correspond to the non-transported radiolabeled compounds associated with the cells (in the interstitial space), were measured by performing uptake assays on ice for <3 s. Data were analyzed using the PRISM and Grafit 4.0 software (Erithacus). Kinetic constants were determined by non-linear regression analysis using the Michaelis-Menten equation. Increase of Proline Consumption in T. brucei Cultivated in Glucose-depleted Medium—The SDM79 semidefined medium has become the growth medium of choice for procyclic form T. brucei (16Brun R. Schonenberger M. Acta Trop. 1979; 36: 289-292PubMed Google Scholar). This insect stage parasite, grown in SDM79 or equivalent media, primarily consumes glucose as a carbon source and also depletes medium of threonine and to a lesser extent proline, glutamine, and pyruvate (15Cross G.A. Klein R.A. Linstead D.J. Parasitology. 1975; 71: 311-326Crossref PubMed Scopus (79) Google Scholar, 19Coustou 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 (86) Google Scholar) at significant rates. We previously estimated that the rate of glucose consumption of the EATRO1125 strain grown in the SDM79 medium is about 3-fold higher than the rate of proline consumption, during mid-log phase growth (19Coustou 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 (86) Google Scholar). To determine whether the rate of consumption of other potential carbon sources is influenced by the absence of glucose, the EATRO1125 strain was adapted to the glucose-depleted medium SDM80 (0.15 mm glucose), a derivative of SDM79 (6 mm glucose). The 40-fold reduction in glucose concentration does not affect the growth rate of this procyclic cell line nor that of the T. brucei 427 strain (with doubling time being the same in SDM80, SDM80 containing 6 mm glucose, and SDM79 media (Fig. 2, top panels)). For the EATRO1125 line, the quantity of each of the 20 amino acids, and pyruvate, present in the growth medium, was determined for cells grown in the glucose-depleted (0.15 mm) and glucose-rich (6 mm) SDM80 media. For some amino acids, the rate of consumption (threonine, pyruvate, and glutamine) or production (glycine, alanine, and glutamate) was not different regardless of the medium used. The concentrations of other amino acids were not significantly altered in either medium. However, the rate of proline consumption increased severalfold in the glucose-depleted medium (Fig. 2). To quantify the relative proline consumption increase during the mid-log phase, both the EATRO1125 and 427 strains were incubated at a higher cell density (5 × 106), and the proline concentration in the growth medium was determined periodically over a period of 2 days (Fig. 3). The rate of proline consumption is ∼2-fold higher for the 427 cell line than for the EATRO1125 cell line in the presence (0.31 versus 0.15 μmol·h-1·mg protein-1) or the absence (1.8 versus 0.93 μmol·h-1·mg protein-1) of glucose. These differences may be related to the higher growth rate observed for the 427 strain (doubling time: 10.5 versus 13.8 h). However, the rate of proline consumption in the glucose-depleted medium is ∼6-fold increased in both EATRO1125 (6.3-fold) and 427 (5.7-fold) strains. We also attempted to grow the 427 cell line in medium lacking both glucose and proline but containing each of the other 19 commonly used l-amino acids at 10 mm. Proline, but no other amino acid, could support robust growth in glucose-depleted conditions (Fig. 4). This included l-glutamate, a direct product of the proline degradation pathway and glutamine, which is also converted into glutamate. Moreover, l-threonine could not support growth despite the fact that this amino acid is consumed in great quantities by procyclic T. brucei. In other experiments (not shown) proline was also included at low concentration (0.1 m) to ensure that non-energy requirements of this amino acid (e.g. protein biosynthesis) were provided. The presence of proline did not affect the outcome. Oxidative Phosphorylation Is Essential for Growth in Glucose-depleted Medium—We previously observed that in glucose-rich medium, pyruvate kinase, which produces ATP in the cytosol by substrate level phosphorylation, is essential for procyclic trypanosomes. In contrast, oligomycin (the most specific known inihibitor of the mitochondrial F0/F1-ATP synthase) does not affect the steady state amount of intracellular ATP and only moderately affects parasite growth (19Coustou 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 (86) Google Scholar). We proposed that production of ATP by substrate level phosphorylation is essential, while ATP generation by the F0/F1-ATP synthase (oxidative phosphorylation) is available but not essential (19Coustou 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 (86) Google Scholar) for procyclic cells grown in glucose-rich medium. In the absence of glucose, the overall carbon source consumption decreases significantly, although the relative rate of proline consumption increases. We determined the oligomycin concentration required to kill all of the cells (LD100) grown in glucose-rich or glucose-depleted medium (Fig. 5). At day 3 of incubation with oligomycin, the EATRO1125 and 427 strains are 2000 and 5000 times more sensitive to the metabolic effector, respectively, when grown in glucose-depleted medium as compared with glucose-rich
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