Procyclic Trypanosoma brucei Do Not Use Krebs Cycle Activity for Energy Generation

Abstract

The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitrodifferentiation from wild type and mutant (Δaco::NEO/Δaco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO2. Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei. The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitrodifferentiation from wild type and mutant (Δaco::NEO/Δaco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO2. Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei. acetaat:succinaat CoA-transferase phosphoenolpyruvate salicylhydroxamic acid 4-morpholinoethanesulfonic acid high performance liquid chromatography Trypanosoma brucei, one of the causative agents of African trypanosomiasis, is a unicellular eukaryote, which during its life cycle alternates between the bloodstream of its mammalian host and the blood-feeding insect vector, the tsetse fly (Glossinaspp.) (1Vickerman K. Br. Med. Bull. 1985; 41: 105-114Crossref PubMed Scopus (478) Google Scholar). In the mammalian bloodstream, the long slender form ofT. brucei proliferates, until at the peak of the parasitaemia nonproliferative short stumpy form cells accumulate that are prepared to differentiate to insect stage (procyclic form) parasites. In vivo this takes place in the tsetse midgut shortly after the insect blood meal. In culture this differentiation process (also referred to as transformation) can be induced by a temperature shift and the addition of millimolar concentrations of citrate or cis-aconitate (2Brun R. Schönenberger M. Z. Parasitenk. 1981; 66: 17-24Crossref PubMed Scopus (126) Google Scholar), and in fully differentiation-competent trypanosome strains (termed pleomorphic) transformation is very rapid and perfectly synchronous (3Ziegelbauer K. Quinten M. Schwarz H. Pearson T.W. Overath P. Eur. J. Biochem. 1990; 192: 373-378Crossref PubMed Scopus (118) Google Scholar, 4Rolin S. Paindavoine P. Hanocq-Quertier J. Hanocq F. Claes Y. Le Ray D. Overath P. Pays E. Mol. Biochem. Parasitol. 1993; 61: 115-125Crossref PubMed Scopus (66) Google Scholar, 5Matthews K.R. Gull K. J. Cell Biol. 1994; 125: 1147-1156Crossref PubMed Scopus (100) Google Scholar). Differentiation of T. brucei is accompanied by several profound structural and biochemical changes, including the glucose metabolism. The long slender bloodstream form depends entirely on glycolysis for energy generation and excretes pyruvate as the major end product of carbohydrate metabolism (6Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar, 7Opperdoes F.R. Annu. Rev. Microbiol. 1987; 41: 127-151Crossref PubMed Scopus (429) Google Scholar, 8Clayton C. Michels P. Parasitol. Today. 1996; 12: 465-471Abstract Full Text PDF PubMed Scopus (106) Google Scholar). In the procyclic stage, the end product of glycolysis, pyruvate, is not excreted but further metabolized inside the mitochondrion. Pyruvate is first oxidatively decarboxylated by pyruvate dehydrogenase to acetyl-CoA, which is supposed to be further degraded to carbon dioxide by the Krebs cycle. This presumed flux through the Krebs cycle is, however, only poorly supported by direct experimental data and is mainly based on the detected presence of all the enzyme activities involved (6Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar, 7Opperdoes F.R. Annu. Rev. Microbiol. 1987; 41: 127-151Crossref PubMed Scopus (429) Google Scholar, 9Evans D.E. Brown R.C. J. Protozool. 1972; 19: 686-690Crossref PubMed Scopus (80) Google Scholar, 10Durieux P.O. Schütz P. Brun R. Köhler P. Mol. Biochem. Parasitol. 1991; 45: 19-28Crossref PubMed Scopus (56) Google Scholar). As a result of this incomplete data set, many published metabolic schemes of procyclic T. brucei do not represent main catabolic fluxes but rather present a survey of all possible pathways of carbohydrate breakdown, including a complete Krebs cycle (6Fairlamb A.H. Opperdoes F.R. Morgan M.J. 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In this is by a mitochondrial CoA-transferase and a J.J. Opperdoes F.R. S. PubMed Scopus Google Scholar). is end product of glucose and is excreted by procyclic PubMed Scopus Google Scholar). was that this is inside the S. M. T. P. F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In to bloodstream form T. brucei, procyclic form cells can use for the generation of energy D.E. Brown R.C. J. Protozool. 1972; 19: 686-690Crossref PubMed Scopus (80) Google Scholar). the of the Krebs cycle to carbon fluxes in the energy metabolism of procyclic the of glucose and proline by a and biochemical that was by analysis of a targeted deletion of the aconitase gene of T. brucei K. M. D. Biochem. J. PubMed Google Scholar, J. K. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We that procyclic of aconitase activity were and to the Krebs cycle to energy in the procyclic form a complete but of a enzyme be of by but from a of and metabolic gene deletion of a on the to of targeted the aconitase gene in the bloodstream stage of the parasite where the enzyme is only expressed and is for growth K. M. D. Biochem. J. PubMed Google Scholar, J. K. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). be to rapid and synchronous differentiation to the procyclic stage, aconitase in a pleomorphic T. brucei for which a gene and E. M. Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar). strains of procyclic form with and of aconitase activity. that deletion of the aconitase gene no on differentiation and growth in culture of procyclic T. brucei. analysis of the metabolism of glucose and proline by procyclic to that under the conditions substrates were not degraded by Krebs cycle activity. In no metabolic or growth differences be between wild type and knockout which that Krebs cycle activity is not for the generation of energy in procyclic T. brucei cells. The pleomorphic of T. brucei brucei was in this E. M. Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar). form were on E. M. Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar, 1993; PubMed Scopus Google or in with and E. R. M. C. M. M. Mol. Microbiol. 41: PubMed Scopus Google Scholar). The cells were with a In vitrodifferentiation to the procyclic stage was as E. M. Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar). were in with (2Brun R. Schönenberger M. Z. Parasitenk. 1981; 66: 17-24Crossref PubMed Scopus (126) Google Scholar). was with a of the aconitase in the pleomorphic T. and in by K. M. D. Biochem. J. PubMed Google were were from the blood of after at of and was as by E. R. M. C. M. M. Mol. Microbiol. 41: PubMed Scopus Google Scholar). were with of using a Cell at and the cells were to of and for at The were and of the were The were in the of and to of and for at The culture was to of the and were with or a of and the was strictly cells were by was from as 1993; PubMed Scopus Google Scholar). analysis was R. K. K. in New Scholar). of was on a a and in for at to the with and a and a The were under high conditions for at The were with using the for analysis were to on a and a R. K. K. in New Scholar). The was with J. K. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google in and The was with the mitochondrial from in and The were by Krebs cycle enzyme activities in procyclic T. brucei to change with and culture conditions P.O. Schütz P. Brun R. Köhler P. Mol. Biochem. Parasitol. 1991; 45: 19-28Crossref PubMed Scopus (56) Google Scholar). all and of wild type and are based on of and after in for the of activity were prepared as by Overath P. J. C. Eur. J. Biochem. PubMed Scopus Google Scholar). cells were with or trypanosome in of and by of The was by several through a and with of and and The was for at and the Biochem. PubMed Scopus Google was for the of activity. of the was at with by to the of aconitase activity. activity was with a at to the J. Biol. Full Text PDF PubMed Google Scholar). The was with as The was with and the in at was for The activity was as the of product using of J. Biol. Full Text PDF PubMed Google Scholar). dehydrogenase activity was by the rate of at The and The was by the and dehydrogenase activity was by the rate of at The and The was by and dehydrogenase activity was by the rate of at The and The was by the and activity was not detected in the and and and was not procyclic form cells in growth phase were and in trypanosome The were in liquid and at were using a and and and C. T. D. Biochem. PubMed Scopus Google Scholar). the were in of using a with liquid the was for at in a at the were using a and in of with a of or through the was the after The were to that was from the to Biochem. PubMed Scopus Google Scholar). were for at in of The were after the addition of of or of This that all of the were in complete both glucose and proline with only one of radioactively labeled at a were by addition of of to the to of was through the and the were on after the were with for at In this all carbon dioxide is whereas in the The carbon dioxide was in a of with of and of 1990; Scopus Google Scholar). The in this was in to the by Biochem. PubMed Scopus Google Scholar). of carbon the was from the cells by for at and by the addition of of of the labeled end products by chromatography on a in form P. 1981; Scholar). The was with of of and of The were and with of in a of the were for end products were by The of the major end products in the was by NMR that both wild type and were in the as except all of the glucose present in was NMR of the were using a phase for and a R. P. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar). of was using a of and data after a of were with to of at to at NMR of were using the of the of was using a E. F. H. Scholar), and the was with the using from with and as D. Biochem. PubMed Scopus Google Scholar). to the metabolic of the Krebs cycle in insect stage T. brucei targeted deletion of the gene aconitase J. K. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), which to be in the bloodstream stage of the parasite K. M. D. Biochem. J. PubMed Google Scholar). differentiation of the procyclic stage in to aconitase was for growth of procyclic differentiation of the a and did not with mutant procyclic long for biochemical analysis of this deletion of was in bloodstream form T. brucei of the pleomorphic that can be to differentiate to the procyclic form in with high and and deletion of both of in are in and and wild type were to a differentiation E. M. Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar). Surprisingly, deletion of did not at all change the and of and M. a and did not change the growth rate of the procyclic of aconitase in procyclic was by and activity aconitase activity was the of the in of several and data not In for activities of and dehydrogenase did not difference between wild type procyclic and of procyclic of aconitase activity. The cells were using a and to to in the phase of growth and The represent the of wild type activities in procyclic T. activities are in of The represent the of and wild not not dehydrogenase activity was not in the in a activities are in of The represent the of and wild not not dehydrogenase activity was not in the of the of wild type and was to the intracellular levels of Krebs cycle intermediates succinate, and of glycolytic intermediates glucose and and of and The citrate was in the aconitase knockout confirming a complete of the aconitase In the concentrations of all Krebs cycle and glycolytic intermediates were not in wild type procyclic cells and concentrations in procyclic T. of of represent the of and wild in a The of of represent the of and wild the of the Krebs cycle in the glucose metabolism of procyclic T. brucei, using the end product of glucose The result of and for both the wild type and a very of the glucose was to labeled CO2. which only in labeled pyruvate is further degraded via the Krebs cycle but not during the of acetate, this that Krebs cycle activity is the very of labeled carbon dioxide detected with wild type cells was to the detected in the with the aconitase knockout cells, which Krebs cycle activity of the of This that this of carbon dioxide was not by complete Krebs cycle activity for by a or by the complete of glucose via a end products of by procyclic T. glucose glucose was from the of end end product of was using the of at the end of the of the represent the of wild aconitase glucose was from the of end product of was using the of at the end of the in a of the represent the of wild aconitase only labeled end products are in be that of were detected by The of of the of and succinate. from the We NMR to the of and as major end products of glucose in studies using that from and as major end was as a major end from the NMR be that the of was with that of The of not be in studies with radioactively labeled and glucose are not in the chromatography analysis to and succinate. of from glucose by procyclic T. brucei is in with studies (6Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar). The from the labeled of The of that a of the was labeled at carbon and with a of only in and peak at the of can only be a of at all in the of is that and result in NMR very to the after of both wild type as as aconitase knockout with with labeled labeled in and can only be by a where phosphoenolpyruvate which be is to form by carbon The is via and This is to in procyclic T. brucei F.R. Annu. Rev. Microbiol. 1987; 41: 127-151Crossref PubMed Scopus (429) Google Scholar, 8Clayton C. Michels P. Parasitol. Today. 1996; 12: 465-471Abstract Full Text PDF PubMed Scopus (106) Google Scholar, S. M. T. P. F. J. Biol. Full Text Full Text PDF PubMed Scopus Google of labeled from by procyclic T. brucei. The labeled only in the can be by via the after the of by carbon the is via and decarboxylated to of the via the of this be labeled only in and this is by carbon dioxide via the labeled only in and be The presence of can only be by of carbon dioxide in which is by the be that the of and be via of by Krebs cycle via of labeled from the labeled glucose nor via the use of labeled as for a of Krebs cycle in succinate. the that the of in the of wild type and aconitase knockout procyclic T. brucei was the of a functional Krebs cycle the that procyclic T. brucei use proline as a major energy D.E. Brown R.C. J. Protozool. 1972; 19: 686-690Crossref PubMed Scopus (80) Google Scholar), the of proline was investigated and with that of were in the complete for the except that a of proline was of a of labeled of the end products in the presence of labeled proline that the major end product of proline was succinate, with the of of carbon dioxide No differences in end products be detected between the wild type and aconitase knockout end products of by procyclic T. proline proline was from the of end end product of was using the of at the end of the of the represent the of wild aconitase proline was from the of end product of was using the of at the end of the in a of the represent the of wild aconitase the importance of the of the electron transport both strains in the presence of of the mammalian type respiratory or of the The addition of or in of the of both strains of the mammalian type respiratory chain in a growth of the no differences be detected between the wild type and aconitase knockout procyclic cells The presence of and in of both the wild type and aconitase knockout procyclic In the complete of from the and as was P. 1981; in rapid of the procyclic T. brucei cells are dependent on the use of by the respiratory chain for energy generation and In this investigated the importance of the Krebs cycle for energy generation in the procyclic stage of T. brucei. studies a wild type pleomorphic and several derived mutant in which the aconitase gene was by gene No aconitase activity be detected in confirming that T. brucei not of enzyme to the aconitase the as no differences in the activities of and dehydrogenase were found between wild type and aconitase knockout In to on K. M. D. Biochem. J. PubMed Google Scholar), the aconitase deletion in a fully differentiation tsetse fly to of long culture of the procyclic form parasites. form of aconitase were fully as K. M. D. Biochem. J. PubMed Google and were the procyclic stage with The growth rate of the wild type and procyclic T. brucei very and the energy metabolism of the procyclic T. brucei is not which is from the perfectly ATP/ADP ratio No differences were detected in intracellular levels of and Krebs cycle intermediates between both except for citrate which were up to 90-fold in the knockout of a of aconitase activity. is citrate accumulated in the or inside the aconitase is in both in T. brucei J. K. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In biochemical the of the Krebs cycle for energy generation in procyclic T. brucei was by studies using labeled the absence of aconitase activity in pathways of energy generation, using the of inside the via via by as as via in the Krebs one the to Krebs cycle activity was by the absence of the knockout cells did not of with the wild type wild type and knockout procyclic T. brucei to and as major end No of be that a Krebs cycle is not involved in energy in wild type as as in knockout cells. demonstrated the absence of of glucose via a complete Krebs cycle in brucei. NMR studies with were to the of and during glucose The of by procyclic T. not a mitochondrial of this the activity of a J. J. Protozool. 1991; PubMed Scopus Google Scholar). however, S. M. T. P. F. J. Biol. Full Text Full Text PDF PubMed Scopus Google that mainly inside the by a and analysis of the of the in can on the by which this was The NMR demonstrated that was involved in the of is to which is via to succinate. of the with that this was by on this via of the or including the of carbon dioxide This the of the carbon dioxide was whereas carbon dioxide is present in the is however, that a of the glucose inside the via the which in the of carbon dioxide Scopus Google Scholar), and was that the major of from glucose inside the S. M. T. P. F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The of pathways the of labeled carbon dioxide in a of the be that of the of Krebs cycle activity in the but are in in with the of by S. M. T. P. F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). studies that the acid from the by the insect stage of T. brucei is a main of energy and carbon D.E. Brown R.C. J. 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This result demonstrated that proline is only degraded to and not further degraded via the Krebs cycle this in a The in which the glucose and proline were in both in the complete the only difference the of This that for both substrates in the presence of the and that the of and of end products of glucose and proline at the of the of end products from in complete that in the presence of glucose and glucose is the the rate of glucose was as high as that of be however, that proline be the in the insect where glucose concentrations are We that procyclic T. brucei, wild type or knockout mutants aconitase are to under of both the mammalian type respiratory chain and the in the survival and growth only one of the was on growth to be to of energy was found of the mammalian type respiratory chain as with of the no differences be detected between the wild type and knockout procyclic T. brucei. that both of the respiratory chain a in the of procyclic cells Mol. Biochem. Parasitol. PubMed Scopus Google Scholar), but the activity of only one of the of the respiratory chain is for survival and growth. The of aconitase is but no aconitase in procyclic is not in the generation of energy via the Krebs cycle. The end products of the of the wild type with labeled glucose and proline demonstrated that Krebs cycle activity not a in the of The that no metabolic differences be detected between procyclic wild type and procyclic aconitase knockout is of the of Krebs cycle activity for energy generation in the procyclic life cycle the the metabolism of procyclic T. brucei is not is as a electron for survival and growth. The absence of a growth of dehydrogenase or dehydrogenase in procyclic T. brucei J. Biol. Full Text Full Text PDF PubMed Scopus Google is in with that the Krebs cycle is not involved in energy generation in this The differences in the of of Krebs cycle enzymes during bloodstream to procyclic differentiation 10Durieux P.O. Schütz P. Brun R. Köhler P. Mol. Biochem. Parasitol. 1991; 45: 19-28Crossref PubMed Scopus (56) Google and there are with a of the Krebs cycle in the procyclic of African are in many including the of the mitochondrial and the during mitochondrial gene the that a is to the and the of and in the life cycle. a of is that of procyclic form T. brucei substrates to mainly and succinate, whereas the of Krebs cycle enzymes and a functional respiratory chain including which is only present in and The presence of a complete with the incomplete of substrates in a in between and the fully for C. J.J. Biochem. Full Text Full Text PDF Scopus Google Scholar). This is by the presence of enzyme in type that Krebs cycle is not for complete of glucose or be that of the Krebs cycle is for the of proline to succinate. The enzyme of this of the Krebs cycle is in the cycle J.J. Opperdoes F.R. S. PubMed Scopus Google and to be essential for in mitochondrial and for growth of procyclic J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). be that the Krebs cycle at the first is in the wild type citrate levels were in the aconitase knockout strain. the the possible of aconitase and Krebs cycle enzymes in procyclic is but no the is, is not to the generation of energy in the procyclic The is that the Krebs cycle a in energy generation in of the life cycle of the parasite in the tsetse fly in the or under for is not all of the are a of and published studies J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. T. P. F. J. Biol. Full Text Full Text PDF PubMed Scopus Google with in which for the first the glucose and proline metabolism of procyclic T. brucei of a pleomorphic under in a model of the main pathways of glucose as in This metabolic the pathways to in of procyclic T. brucei and for the of glucose to acetate, succinate, and alanine, the of proline to via of the Krebs and the essential of the respiratory chain in the of in is for