Chapter 7

Energy Metabolism in Trypanosoma cruzi Juan Jose Cazzulo

1. INTRODUCTION The American trypanosomiasis, Chagas' disease, affects about 20 million people in Central and South America. The causative agent is a parasitic flagellate, Trypanosoma cruzi, which has a complex life cycle, involving a replicative form, the amastigote, and a nonreplicative form, the bloodstream trypomas-

tigote, in the mammalian host. There is also a replicative form, the epimastigote, and a nonreplicative form, the infective metaCYclic trypomastigote, in the insect vector (Brener, 1973). Although at present all these developmental stages can be obtained in axenic culture (Contreras et al., 1985) or in mammalian cell culture (Andrews and Colli, 1982), or from infected mammals (Gutteridge et al., 1978), most metabolic and enzymological studies so far have been performed with cultured epimastigotes, presumably identical to those naturally present in the insect vector. Comparative studies using epimastigotes, amastigotes, and bloodstream trypomastigotes (Gutteridge and Rogerson, 1979) suggest that, at variance with African trypanosomes (Opperdoes, 1987), the different developmental stages of T. cruzi have qualitatively similar metabolic characteristics. Juan Jose Cazzulo

Instituto de Investigaciones Bioquimicas "Luis F. Leloir," Fundaci6n Campomar-CONICET-Falcultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1405 Buenos Aires, Argentina.

23S

J. L. Avila et al. (eds.), Intracellular Parasites © Springer Science+Business Media New York 1992

Juan JosE Cazzulo

This chapter deals with the energy metabolism in T. cruz; and covers two main, closely related, aspects: carbohydrate catabolism, in particular the aerobic fermentation of glucose, and protein and amino acid catabolism. The relative weight of both metabolic pathways in fulfilling the energetic requirements of the parasite seems to vary both with the parasite developmental stage and with the nutrient availability in the medium. Since some fairly recent reviews have dealt with these subjects (Gutteridge and Rogerson, 1979; Cannata and Cazzulo, 1984a; Cazzulo, 1984), emphasis here will be put on the more recent findings and concepts and on the possible metabolic interrelationships.

2.

TRANSPORT AND UTILIZATION OF CARBOHYDRATE, PROTEINS, AND AMINO ACIDS

It has been known for many years that epimastigotes of T. cruz; are able to take up and consume both carbohydrates and amino acids, excreting them into the medium as some reduced glucose catabolites, chiefly succinate, and NH3 , respectively (von Brand, 1979). Nevertheless, there has been some controversy over which is the most important and natural nutrient for the parasite (Urbina and Azavache, 1984). Some fairly well-established facts at present are: (1) epimastigotes are able to consume both nutrients, although glucose is preferred over amino acids and consumed first when both are present (Cannata and Cazzulo, 1984a; Cazzulo et 01., 1985). This happens despite the fact that the natural environment of the epimastigote, the triatomine's gut, is likely to be rich in proteins and amino acids and poor in carbohydrate. (2) Despite some claims, no culture medium completely devoid of glucose seems to have been successfully used for growth of the parasite, at least for more than a few doublings. (3) Different developmental stages of the parasite seem to prefer one type of nutrient or the other; thus, axenic-cultured amastigotelike forms have an exclusively glycolytic catabolism (Engel et 01., 1987), whereas epimastigotes are able to use both glucose (which is the preferred substrate during the exponential phase of growth) and amino acids (which are better consumed near the stationary phase) (Cazzulo et 01., 1985; Engel et 01., 1987). Metacyclic trypomastigotes have recently been reported as having a nonglycolytic catabolism, deriving their energy exclusively from proteins and amino acids (Urbina et 01., 1989). Both glucose and amino acids are actively taken up from the culture medium by the epimastigotes (see Cannata and Cazzulo, 1984a; Cazzulo, 1984, for review); proteins are incoIporated into the parasites by pinocytosis, which seems to take place mostly at the level of the flagellar pocket (DeSouza, 1984), and they seem to be able to cover the energy requirements of the parasite (Avila et oZ., 1979a, 1983) (see also Chapter 6). Trypanosoma cruzi, like the other Trypanosomatids, does not seem to be able to synthesize and store any reserve

Energy Metabolism in TryJHlllOsoma crud

237

polysaccharide, and therefore carbohydrate needs to be t~en up continuouslytfor utilization (see Cannata and Cazzulo, 1984a, for review). The presence of fructose 1,6-bis-phosphatase in the parasite, recently reported (Adroher et al., 1987), suggests the possibility of the operation of a gluconeogenic pathway. Protein and amino acids, on the other hand, are likely to be an energy reserve for the parasite, which at least under some circumstances, such as the epimastigote-metacyclic trypomastigote differentiation, seems to be able to use as fuel a significant proportion of its own protein (Rodriguez et al., 1990). There is a large amino acid pool, almost half of which is L-alanine (Williamson and Desowitz, 1961), which can be oxidized by the parasite; indeed, it is necessary to starve epimastigotes for 24 hr in order to detect a clear stimulation of respiration by glucose or amino acids (Sylvester and Krassner, 1976). Glucose is consumed at a fairly high rate [0.7 IJ.mollmin - 1 per g (wet weight) - 1] by the epimastigotes; this rate is nevertheless about fivefold lower than that shown by other Trypanosomatids, such as Crithidia jasciculata or Trypanosoma brucei (Cazzulo et al., 1988a).

3.

CARBOHYDRATE CATABOLISM: THE AEROBIC FERMENTATION OF GLUCOSE

3.1. End Products of Glucose Catabolism From the early studies of Chang (1948) and Ryley (1956), it is known that T. cruz; epimastigotes and bloodstream trypomastigotes produce organic acids during glucose catabolism. These studies, as well as those of Bowman et al.

(1963) and Caceres and Fernandes (1976), consistently indicated succinate as a major catabolite, although they differed to some extent with reference to other minor products. This question has been reexamined recently using two different approaches: (1) specific enzymatic assay of catabolites in culture medium and cell suspension supernatants; (2) the noninvasive technique of l3C-Iabeled nuclear magnetic resonance (NMR), which allows one to follow glucose catabolism by living cells. Both techniques indicate that succinate is the major catabolite, Lalanine being the second quantitatively important product (Cazzulo et al., 1985; Engel et al., 1987; Frydman et al., 1990); only a little of the enzymatically detected acetate (Cazzulo et al., 1985; Engel et al., 1987) actually originates from glucose (Frydman et al., 1990), most of it probably arising from lipid and amino acid catabolism. The NMR evidence suggests that there are two different alanine pools with different access to deuterium from the medium; one of them is excreted, whereas the other is retained within the cells (Frydman et al., 1990). These experiments, performed with the Thlahuen strain (Thl 0 stock) of the parasite, have given no indication of other catabolites reported by earlier authors,

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Juan Jose CazzuIo

such as lactate, pyruvate, and malate (see Cannata and Cazzulo, 1984a, for review); this discrepancy might be attributed either to the use, in the earlier work, of less specific assay methods or simply to strain differences. Glycerol and ethanol, which are produced by other Trypanosomatids, are not produced at all by T. cruzi epimastigotes (Cazzulo et al., 1988a). Comparison of two developmental forms of the parasite indicated that axenic cultured amastigotelike forms and epimastigotes of the same stock presented only quantitative differences in the end products excreted (Engel et al., 1987).

3.2. Enzyme Pathways for Glucose Catabolism Evidence obtained in several laboratories indicates that both glycolysis and the pentose phosphate shunt are operative in epimastigotes of T. cruzi (see Cannata and Cazzulo, 1984a, for review); the balance between the two pathways seems to depend on the strain of the parasite (Mancilla and Naquira, 1964). Glycolysis in T. cruzi presents two distinctive characters compared with the mammalian host: (1) most of the glycolytic enzymes are placed in a microbody, which for this reason was named "glycosome" by Opperdoes and Borst (1977), who discovered it in T. brucei; and (2) hexokinase and phosphofructokinase, key regulatory enzymes in most glycolytic systems, from bacteria to mammals, are little or not affected by most common effectors (Urbina and Crespo, 1984; Racagni and Machado de Domenech, 1983; Aguilar and Urbina, 1986; Taylor and Gutteridge, 1986b). The only control found so far for a glycolytic enzyme in Trypanosomatids is the strong activation of pyruvate kinase by very low concentrations of fructose 2,6-bis-phosphate (van Schaftingen et al., 1985). The pyruvate kinase from T. cruzi epimastigotes (Juan et al., 1976) is strongly activated by fructose 2,6-bis-phosphate, which is able to counteract (at micromolar concentrations) the inhibitory effects of millimolar concentrations of PI and ATP (Cazzulo et al., 1986b). Millimolar concentrations of fructose 1,6-bisphosphate are also able to active pyruvinate kinase (Juan et al., 1976; Cazzulo et al., 1989b). As a result of the almost complete lack of inhibitory controls, the operation of glycolysis is not influenced appreciably by the presence or absence of oxygen; this is seen as a complete lack of the Pasteur effect (Cazzulo et al., 1988a). The early demonstration of the aerobic fermentation of glucose posed the question of whether the tricarboxylic acid cycle and the respiratory chain were operative in T. cruzi. The present evidence (reviewed in Cannata and Cazzulo, 1984a) indicates that both pathways are actually functional, and all the cycle enzymes, with the only exception of a-oxoglutarate dehydrogenase, have been detected in cell-free extracts. Citrate synthase is a typically eukaryotic enzyme, with the usual response to effectors (Juan et al., 1977). Figure 1 summarizes our present knowledge of these pathways, together with some enzymes involved in catabolite production.

Energy Metabolism In TryptlllOsoma cruzJ GLUCOSE NADP NADPH 111 , Glu-6-P 'i24( I 6-phosphogluconate

121

,

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.

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~

1191

FIGURE 1. Enzyme pathways for glucose catabolism in Trypanosoma cruzi. Abbreviations: Glu-6P, glucose-6-phosphate; Fru-6-P, fructose-6-phosphate; Fru-l,6-diP, fructose l,6-bis-phosphate;

GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphale; G-l,3-diP, 1,3-diphosphoglycerale; 3-PGA, 3-phosphoglycerale; 2-PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvale; Pyr, pyruvale; OA, oxaloacetate; L-mal, L-malate; Fum, fumarate; Suc-CoA, succinyl-CoA; a-OG, a-oxoglutarate; Isocit, isocitrale; Cit, citrale; L-glu, L-glulamale. The enzymes involved are the following: (1) hexokinase; (2) hexose phosphale isomerase; (3) phosphofructokinase; (4) aldolase; (5) triosephosphale isomerase; (6) glyceraldehyde-3-phosphale dehydrogenase; (7) phosphoglycerokinase; (8) phosphoglyceromutase; (9) enolase; (10) pyruvate kinase; (11) a-hydroxyacid dehydrogenase; (12) phosphoenolpyruvale carboxykinase; (13) malic enzynme; (14) pyruvale dehydrogenase; (15) citrale synthase; (16) aconitase; (17) isocitrate dehydrogenase; (18) a-oxoglutarale dehydrogenase; (19) succinale thiokinase; (20) succinale dehydrogenase; (21) fumarale reductase; (22) fumarase; (23) malate dehydrogenase; (24) glucose-6-phosphale dehydrogenase; (25) acetylCoA hydrolase; (26) alanine aminotransferase. Enzyme 18 has not been detected yet in T. cruzi.

Recent studies have focused on the variation of the levels of some glycolytic and Krebs cycle enzymes during differentiation of axenic amastigotelike fonns to epimastigotes (Engel et ai., 1987) and of epimastigotes to metacyclic trypomastigotes (Adroher et al' 1988, 1990). The decreasing levels of glycolytic enzymes and increasing levels of key cycle enzymes during these transitions J

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Juan JoR Cazzulo

support the idea that the amastigotelike forms are exclusively glycolytic, but the epimastigotes have a mixed metabolism and the metacyclic trypomastigotes are almost exclusively oxidative, conducting little glycolysis if at all.

3.3. Systems for Reoxidation of Glycolytic NADH In order to remain functional, all glycolytic systems must have an efficient system for the reoxidation of the NADH generated in the reaction of glyceraldehyde-3-phosphate dehydrogenase. This system is usually the respiratory chain under aerobic conditions and several possible fermentative pathways under anaerobic conditions; among the latter, lactate production by skeletal muscle or ethanol production by yeast are the best known. Trypanosoma cruzi has several possible ways for NADH reoxidation, all of which seem to be operative under aerobic conditions. These are, in addition to reoxidation in the respiratory chain, production of succinate and probably also of L-alanine. Production of lactate, reported by some early workers (Ryley, 1956; Caceres and Fernandes, 1976), and probably mediated by a low lactate dehydrogenase activity (Ryley, 1956; Raw, 1959; Gutteridge and Rogerson, 1979; Taylor et al., 1980), which may be due to one of the isoenzymes of a-hydroxyacid dehydrogenase (Coronel et al., 1981), seems to be of little or no importance, even under anaerobic conditions (Frydman et ai., 1990). Ethanol or other alcohols have not been found as products of glucose catabolism by T. cruzi (Frydman et al., 1990; Cazzulo et al., 1988a). The "alcohol dehydrogenase," acting best with n-propanol as substrate and NADP as coenzyme and proposed as a marker enzyme for taxonomic purposes (Miles et al., 1980), has been recently shown to be an NADP-linked aldehyde reductase, able to use a number of aldehydes as substrates, which is not likely to be involved in the reoxidation of reduced coenzymes (Arauzo and Cazzulo, 1989). This leaves us with two, and perhaps three, main pathways for NADH reoxidation in T. cru~ which are analyzed below.

3.3.1. Succinate Production The production of succinate was shown by Bowman et al. (1963) to depend on CO2 fIxation. Using 14(:02 , both under aerobic and anaerobic conditions, radioactivity appeared in the carboxyl groups of succinate, whereas radioactivity from [l4C]glucose appeared, in anaerobisis, exclusively in the methylene groups. These experiments are consistent with CO2 fIxation on a C3 monocarboxylic acid arising from glycolysis, either phosphoenolpyruvate (PEP) or pyruvate, to give a C4 dicarboxylic acid (Bowman et al., 1963). The primary CO2 fIxation product was initially supposed to be either oxaloacetate or L-malate; the former might be produced by several enzymes, among them pyruvate carboxylase and PEP-carboxykinase (PEPCK), and the latter by malic enzyme.

Energy Metabolism in Trypanosoma cruD

241

CrithidiaJasciculata contains all three enzymes (Cannata et al., 1980; Cazzulo et al., 1980), whereas T. cruzi contains only PEPCK and malic enzyme (Cataldi de Flombaum et al., 1977). Recent [l3C]-NMR evidence (De los Santos et al., 1985) has shown that the CO2-fixing enzyme in C. Jasciculata is unmistakably PEPCK, despite the presence of a low pyruvate carboxylase activity. The labeling pattern in similar experiments performed with T. cruzi is also compatible with this possibility (Frydman et al., 1990) as is the fact that in both Trypanosomatids the relative proportion of succinate and the other major product (ethanol for C. Jasciculata, L-alanine for T. cruzz) changes with the addition of bicarbonate to the incubation medium (De los Santos et al., 1985; Frydman et al., 1990). Moreover, recently Urbina et al. (1989) have shown in vivo, in [l3C]NMR experiments, that addition to the cells of an inhibitor of PEPCK, 3mercaptopicolinic acid, results in a decrease in succinate production with a concomitant increase in L-alanine production. The PEPCK was shown to be ADP-linked, as those from yeast and bacteria, and at variance with the PEPCK of higher animals, which is specific for GDP or lOP (Cataldi de Flombaum et al., 1977), and has been recently purified and studied by Urbina (1987), who showed a strong inhibition of oxaloacetate decarboxylation by ATP, which might be relevant for metabolic regulation in vivo. For succinate production, the oxaloacetate synthesized by the PEPCK reaction would be reduced to L-malate by malate dehydrogenase, L-malate would be dehydrated to fumarate by fumarase (Baernstein, 1953), and fumarate would be finally reduced to succinate by a fumarate reductase (Boveris et al., 1986). Reduction to L-malate would be enough for the reoxidation of all glycolytic NADH, if both PEP molecules were converted into succinate, with no energy loss, since one ATP is synthesized by both pyruvate kinase and PEPCK. The

reason for succinate production and excretion is not clear yet, but has been consistently found by all research workers studying this subject, whereas Lmalate itself has been only seldom reported (Caceres and Fernandes, 1976).

3.3.2. L-Alanine Production We have recently proposed (Cazzulo et al., 1988a) that the production of Lalanine might be linked to the reoxidation of glycolytic NADH. The reduced coenzyme can be reoxidized in the reaction of the NAD-linked glutamate dehydrogenase, leading to L-glutamate synthesis; the latter will then react with the pyruvate produced by glycolysis, by the transamination reaction catalyzed by alanine aminotransferase present in the parasite (Barros and Caldas, 1983). LGlutamate and a-oxoglutarate would be required in catalytic amounts, whereas NH3 would be consumed. It is noteworthy that excretion of NH3 into the medium is low or negligible during glucose consumption (Cazzulo et al., 1985, 1988a; Engel et al., 1987); this may be due to actual lack of production or to preferential

Juan JIIR Cazzulo

242

GLUCOSE Oxaloacetate L-glutamate

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I I I I I

I I

,

0< -oxoglutarate

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oc-oxoglutarate

,-....:==:e Pyruvate _ _ _CD,"",- -... IL-ALANINEI------

FIGURE 2. Possible participation of the glutamate dehydrogenases in the reoxidation of glycolytic NADH in Trypanosoma cruzi. The enzymes indicated are: I, alanine aminotransferase; 2, NADlinked glutamate dehydrogenase; 3, NADP-linked glutamate dehydrogenase; 4, malic enzyme; 5, malate dehydrogenase.

use for transamination. Production of L-alanine or succinate would be equivalent in tenns of NADH oxidation. The subcellular localization of the enzymes required for this pathway has not been determined yet; in the case of alanine aminotransferase, it is known that there are, in most T. cruzi stocks, at least two isoenzymes (Miles et ai., 1978). The existence of cytosolic and mitochondrial isoenzymes is therefore possible, and the results of Taylor and Gutteridge (1986a), although not conclusive, support this possibility. L-Alanine might also be obtained in the cytosol from L-malate (synthesized by the reduction of oxaloacetate inside the glycosome) through the concerted action of malic enzyme, the NADP-linked glutamate dehydrogenase and alanine aminotransferase; this pathway (Figure 2) would convert L-malate and NH3 into L-alanine and CO2 and would have similar requirements as the one involving the NAD-linked glutamate dehydrogenase, plus catalytic amounts of NADP. The observed decrease in L-alanine production in the presence of added NaHC0 3 might be due to product inhibition of the decarboxylation reaction catalyzed by malic enzyme. This latter pathway has the advantage that it does not require the existence of a cytosolic pool of NADH arising from glycolysis, even if the

Energy MetaboUsm in Trypanosoma cruzi

243

pathway occurs in the soluble fraction; malic enzyme IT and the NADP-linked glutamate dehydrogenase are known to be cytosolic (Cannata et ai., 1980; Cannata and Cazzulo, 1984a,b). The pathway involving the NADP-linked glutamate dehydrogenase may be essential in the case of C. /asciculata, which has a very low pyruvate kinase activity (Cazzulo et ai., 1989b) and must nevertheless obtain pyruvate to convert it into ethanol, with concomitant NADH oxidation (Cazzulo et al., 1985, 1988a).

3.3.3. Respiratory Chain At least part of the glycolytic NADH in T. cruz; is reoxidized through a respiratory chain consisting of cytochromes b, CSS8 ' and two alternative terminal oxidases, cytochrome oxidase (a + a3 ) and cytochrome 0 (Stoppani et al., 1980; Gutteridge, 1981). Cytochrome d seems also to be present (Affranchino et al., 1986). Evidence obtained by different groups (Carneiro and Caldas, 1982; Affranchino et al., 1986) suggests the presence of at least three different terminal oxidases in T. cruz; epimastigotes, namely, one sensitive to cyanide and azide, a second sensitive to cyanide but insensitive to azide, and a third insensitive to both inhibitors. The fIrst, cytochrome oxidase, seems to be the most important terminal oxidase in the parasite; the second would be cytochrome 0 and the third might be cytochrome d (Affranchino et al., 1986). Recent studies indicate the existence of wide variations in the cytochrome contents of different parasite stocks (Schwartz de Tarlovsky et al., 1985), and also in different developmental stages; Engel et al. (1987) showed that the extracellular amastigotelike fonns completely lacked spectrophotometrically detectable cytochromes, whereas these were acquired on differentiation to epimastigotes. Intracellular (tissue) amastigotes, as well as bloodstream trypomastigotes, on the other hand, contain a full complement of cytochromes (Gutteridge and Rogerson, 1979). The amounts of cytochromes b and a + a3 present in epimastigotes are signifIcantly lower than those found in mammalian mitochondria (Boiso et al., 1979a), and the resulting relative inefficiency of the respiratory chain might be the reason for the existence of the aerobic fennentation, as a complementary pathway for NADH oxidation. Mitochondrial particles obtained from culture epimastigotes and bloodstream trypomastigotes and tissue amastigotes are able to oxidize NADH, succinate, and glycerol 3-phosphate (Gutteridge and Rogerson, 1979). Cyanide and antimycin A are inhibitors of both the endogenous respiration of epimastigotes (Stoppani et al., 1980) and the consumption of oxygen in the presence of glucose (Gutteridge and Rogerson, 1979). Antimycin A inhibited growth of the epimastigotes and considerably increased production of succinate from labeled glucose or CO2 , with a decrease in alanine production (Boiso et al., 1979b). These fIndings suggest the importance of the respiratory chain for both NADH oxidation and ATP synthesis, through coupled oxidative phosphorylation (see below).

Juan Jose Cazzulo

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The transport of glycolytic NADH from the glycosome, where it is produced, to the mitochondrion for oxidation in the respiratory chain may be mediated by L-malate, synthesized inside the glycosome, and oxidized in the mitochondrion (see Section 3.4). Mitochondrial oxidation ofNADH produced in the cytoplasm might be accomplished through the shuttle system a-oxoisocaproate-a-hydroxyisocaproate; isoenzyme II of a-hydroxyacid dehydrogenase and leucine aminotransferase, essential to obtain a-oxoisocaproate from L-Ieucine, have been reported as present both in the cytosol and in the mitochondrion (Montamat et al., 1987). The importance of such a cytosol-mitochondrion shuttle system in Trypanosomatids is still a matter of speculation; moreover, Taylor and Gutteridge (1986a) have reported that a-hydroxyacid dehydrogenase is present in the glycosome and in the cytosol of T. cruzi epimastigotes, whereas the transaminases are mostly cytosolic, with a small particulate, presumably mitochondrial, component. The possible action of a glycerol 3-phosphate shuttle system, like the one present in mammalian cells and also present in C. fasciculata (Bacchi et al., 1968), seems to be ruled out by the absence in T. cruzi of an NAD-linked glycerol 3-phosphate dehydrogenase (Cazzulo et al., 1988a), present in the glycosome of T. brucei and Crithidia luciliae (Opperdoes et al., 1977).

3.4. SubceUular Compartmentation and Regulation of Glucose Catabolism

As described above, inhibitory controls usually present in most organisms and responsible for the Pasteur effect, are lacking in Trypanosomatids. The organisms seem to be adapted to consume as much glucose as is able to enter the cell, and the only apparently important control point is the activation of the cytosolic pyruvate kinase by fructose 2, 6- (or 1,6-) bis-phosphate (Cazzulo et al., 1989b). Even if lack of controls is useful for the parasite, which has no obvious reason to save its host's glucose, some kind of regulatory mechanism is necessary to prevent futile cycles, which might interfere with the aerobic fermentation process. In this sense, the reaction catalyzed by malic enzyme must be controlled to avoid recycling of C4 dicarboxylic acid, synthesized by PEPCK from the C3 PEP, back to the C3 pyruvate. The present evidence suggests that subcellular compartmentation is the major factor involved in the regulation of glycolysis in Trypanosomatids. First, the high rate of glucose consumption seems to be due to the very high concentration of both glycolytic enzymes and intermediates inside the glycosome; this has been particularly well studied in T. brucei (Opperdoes, 1987). Glycosomes are FIGURE 3. Compartrnentation of enzymes involved in glucose catabolism in Trypanosoma cruzi. Abbreviations are the same as in Figure 1, with the addition of ALA for L-alanine; some of the reactions, shown in Figure I, have been omitted here for the sake of clarity.

G

OSOME

[LACTAtt]

~

I

LACTATE

,

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Juan J_ CazzuIo

certainly present in T. cruzi (Taylor et al., 1980; Cannata et al., 1982) and have recently been purified and characterized by Taylor and Gutteridge (1987), who showed that hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, and glyceraldehyde 3-phosphate dehydrogenase are glycosomal enzymes, whereas phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase are cytosolic. Second, the possible futile cycle between PEPCK and malic enzyme seems to be prevented by the fact that the former is glycosomal (Cannata et al., 1982), whereas the latter has two isoenzymes, one mitochondrial and the other cytosolic (Cazzulo et al., 1980; Cannata et al., 1982). Malate dehydrogenase also has two different isoenzymes, one glycosomal and one mitochondrial (Cannata and Cazzulo, 1984b). The subcellular distribution of PEPCK and the malate dehydrogenases is identical to that demonstrated for the procyclic trypomastigotes ofT. brucei (Opperdoes, 1987). Figure 3 summarizes the data on subcellular compartmentation of enzymes in T. cruzi, as well as the interconnections between glycolysis and the tricarboxylic acid cycle. Phosphoenol pyruvate is produced in the cytosol, since Taylor et al. (1980) showed this location for enolase, and it can then follow two different pathways: (1) it can be taken up by the glycosome and carboxylated to oxaloacetate by PEPCK; or (2) it can be converted into pyruvate by the cytosolic pyruvate kinase. Glycosomal oxalocetate will be converted into L-malate by the glycosomal malate dehydrogenase, whereas cytosolic pyruvate will be transaminated to L-alanine. Pyruvate can also be obtained in the cytosol by the reaction of malic enzyme II or in the mitochondrion by the reaction of malic enzyme I; this raises the possibility of two different pools of pyruvate, which might explain the presence of two different Lalanine pools, recently detected by [13C]-NMR (Frydman et al., 1990). Production of L-malate inside the glycosome will leave this organelle in redox balance, if both molecules of PEP are taken up by the glycosome and converted into oxaloacetate and afterward into L-malate. L-Malate will probably be transferred then to the mitochondrion via cytosol; part of it may be decarboxylated to pyruvate by malic enzyme II, producing NADPH for cytosolic biosynthetic processes. When L-malate reaches the mitochondrial matrix, its fate will depend on whether the Krebs cycle is actively operating or not. Under anaerobic conditions, or iffor some reason the operation of the cycle is low, some oxaloacetate obtained by the malate dehydrogenase reaction will accumulate and it will be able to inhibit the highly sensitive (Ki for oxaloacetate 9~M) malic enzyme I (Cannata et al., 1979), thus blocking L-malate decarboxylation and diverting most of the L-malate to succinate via fumarate. On the other hand, if the cycle is working actively, the oxaloacetate concentration will be kept low by the citrate synthase reaction, malic enzyme I will be active, and part of the L-malate will be decarboxylated to pyruvate and enter the cycle as acetyl-CoA to be oxidized. Activity of malic enzyme I during operation of the tricarboxylic acid cycle can be inferred from the results of Stoppani et al. (1980), who showed that during oxidation of [l4C]-acetate by the epimastigotes, a

Energy Metabolism in Trypanosoma cruzi

247

substantial amount of labeled L-alanine is fonned. Labeled L-alanine can be obtained in these circumstances only from pyruvate arising from a Krebs cycle intermediate, like L-malate. The present evidence suggests a central role for L-malate in the aerobic fennentation process and a key position of malic enzyme in the regulation of the pathway.

4. OXIDATIVE PHOSPHORYLATION 4.1.

Phosphorylation in Vivo and in Submitochondrial Particles

Oxidation of substrates in the respiratory chain of T. crLlzi is coupled to phosphorylation, as was first shown by Stoppani and Boiso (1973), though the evidence was indirect. In fact, these authors showed that the intracellular ATP content was decreased by about 50% in vivo by the addition of several wellknown inhibitors of oxidative phosphorylation (N,N' -dicyclohexylcarbodiimide, or DCCD) and the respiratory chain (cyanide) and also by uncouplers (carbonyl cyanide m-chlorophenylhydrazone, or CCP). Only recently direct evidence of oxidative phosphorylation in this parasite has been obtained (Affranchino et al., 1985). Submitochondrial particles obtained by differential centrifugation after parasite disruption by grinding with glass powder in a mortar, were shown to be able to oxidize substrates such as L-malate, glycerol-3-phosphate, succinate and ascorbate plus tetramethyl-p-phenylendiamine (TMPD). Evidence of respiratory control, with values in the range of 2.8-2.0, was found and suggested the operation of coupling sites II and III; respiration and phosphorylation were uncoupled by CCP or sonication. NADH oxidation was not controlled by ADP nor inhibited by antimycin or cyanide, thus suggesting the possible presence of a special NADH dehydrogenase in the outer mitochondrial membrane, as found in other organisms (Affranchino et al., 1985). The apparent absence of phosphorylation site I might be a common property of Trypanosomatids, since it was previously reported for two species of Crithidia (Kusel and Storey, 1972; Toner and Weber, 1972; Opperdoes et al., 1976).

4.2. Mitochondrial ATPase As in all other systems known, either prokaryotic or eukaryotic, oxidative phosphorylation is mediated in T. cruzi by a mitochondrial oligomycin-sensitive Mg2+-ATPase. The enzyme was detected in epimastigotes (De Sastre and Stoppani, 1973; Frasch et al., 1978b), solubilized from the mitochondrial membranes (Frasch et al., 1978a), and purified to electrophoretic homogeneity (Cataldi de Flombaum et al., 1979). The ATPase is made up of subunits similar to those

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found in other systems and is inhibited by oligomycin (in its membrane-bound form) or efrapeptin, aurovertin, and citreoviridin (both membrane-bound and soluble forms) (Cataldi de Flombaum and Stoppani, 1981), and also by organomercurial compounds, in particular by fluorescein mercuric acetate (Cataldi de Flombaum et al., 1979).

5. PROTEIN AND AMINO ACID CATABOLISM 5.1. Production of NH3 Proteins and amino acids are consumed by epimastigotes in culture, with production of ammonia (von Brand, 1979), which is liberated into the medium, raising again the pH lowered by the excretion of organic acids (Caceres and Fernandes, 1976). Trypanosoma cruz;, at variance with other Trypanosomatids, is strictly ariunonotelic (Yoshida and Camargo, 1978) and lacks all the enzymes of the urea cycle (Camargo et al., 1978).

5.2. Proteolytic Enzymes Epimastigotes of T. cruzi have been known for many years to have a considerable intracellular amino acid pool, about half of which is made up by

L-

alanine (Williamson and Desowitz, 1961); proline, glycine, and glutamic acid are also abundant (O'Daly et al., 1983). These free amino acids, which can be used for the synthesis of proteins or some derived substances or for energy production, are likely to be produced by proteolysis of exogenous or endogenous proteins or to be taken up from the culture medium (see also Chapter 6). In the case of L-alanine, a substantial proportion can originate in glucose catabolism, as discussed above. A number of proteolytic activities have been described in T. cruzi epimastigotes, starting with the work of Itow and Camargo (1977) and Avila et al. (l979b); it is difficult to tell whether all the activities described belong to different enzymes, since these early studies were performed with crude extracts (see Cazzulo, 1984, for review). Three proteinases, all belonging to the group of the cysteine proteinases, have been purified to homogeneity from epimastigotes. One of them has a high molecular weight (about 200 kDa) and hydrolyzes efficiently benzoyl-arginine-p-nitroanilide (BAPA) (Bongertz and Hungerer, 1978). This proteinase might be the same enzyme recently reported and studied in further detail by Ashall (1990); the other two have molecular weight values of about 60 kDa (Rangel et al., 1981a,b; Bontempi et at., 1984) and might be the same enzyme, except for some differences in substrate and inhibitor specificity (see Cazzulo, 1984, for review). The study of the proteinases in trypanosomes

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and leishmanias as well as in other protozoal parasites, such as plasmodia, and in helminths, such as the schistosomes, has received considerable interest in recent years, since, in addition to their obvious participation in the nutrition of the parasite at the expense of the host, they may be relevant to other aspects of the host-parasite relationship. In the case of T. cruz;, proteinases might be involved in penetration of the trypomastigote into the host cell (Piras et al., 1985), as well as in evasion from the immune response of the host (Krettli et al., 1980; Teixeira and Santana, 1989). The 6O-kDa cysteine proteinase has been studied in detail recently; it is a high-mannose-type glycoprotein (Cazzulo et al., 1989a, 1990a) placed in the lysosomes (Bontempi et al., 1989), which presents in the sequenced parts of the molecule high homology with cathepsin L and papain (Cazzulo et al., 1989a; Eakin et al., 1990), as well as with the cysteine proteinase recently cloned and sequenced in T. brucei (Mottram et al., 1989). The enzyme, for which the name "cruzipain" has been proposed (Cazzulo et al., 1990b), seems to be the major cysteine proteinase of the parasite, present not only in epimastigotes, but also in cell-culture trypomastigotes and amastigotes (Campetella et al., 1990). The levels of the proteinase, as detected both by enzyme activity and immunoreactivity in Western blots, are considerably lower in amastigotes and trypomastigotes than in epimastigotes, which suggests that the expression of the enzyme is developmentally regulated (Campetella et al., 1990). Recent self-proteolysis experiments (U. Hellman, C. Wernstedt, and J. J. Cazzulo, unpublished data) indicate that the enzyme possesses a 25-kDa proteolysis-resistant, glycosylated C-terminal extension, which may have some similarity with that recently described for the T. brucei enzyme (Mottram et aI., 1989). In addition to cysteine proteinases, which are by far the best studied in this parasite, recent evidence has been presented (by DNA sequencing) for the presence of a serine proteinase (Sakanari et al., 1989); there also is a recent report of a metalloproteinase, which might have some resemblance to the Leishmania gp63 (Greig and Ashall, 1990). There is no evidence for the possible presence of aspartic proteinases in T. cruzi, and high-molecular-weight multicatalytic proteinases have not been reported yet in the parasite, although their presence seems likely, considering their universal distribution (Dahlmann et al., 1989).

5.3. Amino Acid Catabolism Trypanosoma cruz; epimastigotes are able to oxidize a number of amino acids, presumably through the tricarboxylic acid cycle (Mancilla et al., 1967; Sylvester and Krassner, 1976). The catabolic pathways for amino acids such as leucine and proline are probably similar to those present in other organisms, but so. far as I know, they have not been examined in detail and none of the enzymes involved has been demonstrated. The only exception would be serine hy-

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droxymethyltransferase, which is involved in the serine-glycine interconversion, which has been characterized by Nosei and Avila (1985). On the other hand, the enzymes responsible for the exchange and disposal of amino nitrogen have been studied in much greater detail. The enzymes involved in the liberation of the amino group of the different amino acids as ammonia are the transaminases, many of which transfer the amino group to a-oxoglutarate to give L-glutamate, and the glutamate dehydrogenases, which liberate the - NH2 group as NH3 . The contribution of amino acid oxidases to the production of ammonia is in general of little importance, and none of these enzymes has been reported so far in Trypanosomatids. Since the early studies of Bash-Lewinson and Grossowicz (1957) and Zeled6n (1960), it is known that T. cruzi contains transaminase activities. A number of amino acids were shown to be able to transaminate with a-oxoglutarate or pyruvate to yield Lglutamate or L-alanine, respectively (Zeled6n, 1960). The enzymes responsible, aspartate aminotransferase (Cazzulo et al., 1977) and alanine aminotransferase (Barros and Caldas, 1983) have been partially purified from epimastigotes and characterized; they have molecular weights in the vicinity of 100 kDa and seem not to differ significantly from their mammalian counterparts. Both enzymes have at least two isoenzymes in most T. cruzi isolates and are among the six enzyme systems proposed by Miles et al. (1978, 1980) for the classification of isolates of the parasite into zymodemes. As noted above, leucine aminotransferase has also been detected in T. cruzi epimastigotes (Montamat et al., 1987). Trypanosoma cruzi epimastigotes contain two different glutamate dehydrogenases, one NADP-linked and one NAD-linked (Cazzulo et al., 1979; Walter and Ebert, 1979). The parasite therefore resembles bacteria, fungi, and plants in this respect and clearly differs from higher animals, which possess only one glutamate dehydrogenase able to act both with NAD and NADP. Both enzymes have been purified to homogeneity (Juan et al., 1978; Walter and Ebert, 1979) and their properties studied in some detail (see Cazzulo, 1984, for review). The regulatory properties of the NAD-linked enzyme have been studied by Urbina and Azavache (1984). Recently, the NADP-linked enzyme has been shown to be very similar to the enzyme from Escherichia coli in terms of amino acid composition and of N-terminal sequence; 33 amino acids at the N-terminus presented 65% identity with the E. coli enzyme, including a 14-amino-acid N-terminal extension, which is not present in the enzyme from Neurospora crassa (Cazzulo et al., 1988b). The role both enzymes play in T. cruzi metabolism is still doubtful; there is some evidence suggesting that the NADP-linked glutamate dehydrogenase might be biosynthetic (Carneiro and Caldas, 1983), whereas the NADlinked enzyme might have a catabolic role, since its activity increases in the late exponential phase of growth, when amino acids are certainly being consumed and NH3 is produced (Cazzulo et at., 1985) and is strongly inhibited by ATP

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(Cazzulo et al., 1979; Walter and Ebert, 1979; Urbina and Azavache, 1984) and acetyl-CoA (Urbina and Azavache, 1984).

6.

RELATIONSIllP BETWEEN CARBOHYDRATE AND AMINO ACID CATABOLISM

As stated in Section 1, there seems to be a delicate balance between the metabolic pathways for energy production from either carbohydrates or amino acids. Oxidation of the carbon chains of the latter compounds requires the function of the tricarboxylic acid cycle (Sylvester and Krassner, 1976). The question of which catabolic pathway is more important to the parasite has been raised and is still a matter of speculation. The oxidation in the cycle of intermediates of the cycle itself, such as oxaloacetate and a-oxoglutarate, arising from amino acid catabolism, requires their previous conversion into acetyl-CoA. This can be accomplished through the action of malic enzyme on L-malate or through the action of an oxaloacetate decarboxylase on oxaloacetate; the latter role can be fulfilled by the concerted action of PEPCK and pyruvate kinase just as well. Urbina and Azavache (1984) and Urbina (1987) suggest that the preferential use of glucose before amino acids by T. cruzi epimastigotes is due to the tight regulation of NAD-linked glutamate dehydrogenase and PEPCK and the lack of regulation of the glycolytic pathway; the former enzymes would remain inhibited during active glycolysis by the high ATP concentration, whereas they would become fully active after glucose is exhausted and the ATP contents decreases. However, the experimental evidence of PEPCK participation in succinate excretion mentioned above indicates that under conditions of active glycolysis the enzyme is acting as an auxiliary to this pathway, despite the inhibition of the carboxylation reaction by ATP reported by Urbina (1987). According to this author, amino acid catabolism would be mediated by transport of L-malate from the mitochondrion to the glycosome, formation of oxaloacetate by malate dehydrogenase, decarboxylation of this intermediate to PEP by PEPCK, transport of PEP to the cytosol, formation of pyruvate by pyruvate kinase, and transport of pyruvate into the mitochondrion where it would be oxidized to acetyl-CoA and incorporated into the cycle through the citrate synthase reaction. In support of this proposal, Urbina et al. (1989) report inhibition of proline oxidation by 3mercaptopicolinic acid. Although this pathway is feasible, since the reaction catalyzed by PEPCK is freely reversible, the possibility that mitochondrial malic enzyme I is involved instead of PEPCK is also likely. In fact, in this way the conversion of Krebs cycle intermediates into acetyl-CoA would take place in only one subcellular compartment, the mitochondrion, instead of requiring three (mitochondrion, glycosome, cytosol, and mitochondrion again), as in Urbina and Azavache's proposal. As pointed out above, the strong inhibition of the malic

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enzyme I by oxaloacetate suggests that it will be active only when the concentration of this intermediate is kept low by citrate synthase and the cycle is fully active. There can be little doubt that PEPCK in T. cruzi and other Trypanosomatids has a catabolic function, as suggested by its lack of the glucose repression characteristic of PEPCKs having a gluconeogenic role (Cazzulo et al., 1985). This applies to any of its proposed roles in the parasite's catabolism. Although more experimental evidence will be required to solve the remaining doubts about the relative relevance of carbohydrate and amino acid catabolism in T. cruzi, it seems reasonable to assume that both have similar importance and that one or the other will be preferred according to substrate availability and also to the enzyme makeup of the different developmental forms of the parasite.

ACKNOWLEDGMENTS. I am indebted to Professor J. J. B. Cannata for helpful discussions. The work performed at the author's laboratory and reviewed here was aided by grants from the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and the Secretaria de Ciencia y Tecnica (SECYT) de la Republica Argentina; the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR); and the Swedish Agency for Research Cooperation with Developing Countries (SAREC). The author is a member of the Carrera del Investigador Cientifico from CONICET.

7.

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Energy metabolism in Trypanosoma cruzi.

Chapter 7 Energy Metabolism in Trypanosoma cruzi Juan Jose Cazzulo 1. INTRODUCTION The American trypanosomiasis, Chagas' disease, affects about 20 m...
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