Fred R. Opperdoes

Localization of nine glycolytic enzymes in a microbody‐like organelle in Trypanosoma brucei: The glycosome

are completely dependent on glycolysis for energy production in the bloodstream of the vertebrate host, because the biosynthesis of respiratory chain and Krebs cycle enzymes is repressed (see ref. [l] ). The glycolytic pathway is modified in two respects: glucose is converted into pyruvate rather than lactate and the NADH produced in glycol- ysis is reoxidized indirectly by O2 via the coupled action of NAD’-linked glycerol-3-phosphate dehydro- genase and glycerol-3-phosphate oxidase [2] . We have shown previously that in

Glycolysis in bloodstream form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes.

In trypanosomes the first part of glycolysis takes place in specialized microbodies, the glycosomes. Most glycolytic enzymes of Trypanosoma brucei have been purified and characterized kinetically. In this paper a mathematical model of glycolysis in the bloodstream form of this organism is developed on the basis of all available kinetic data. The fluxes and the cytosolic metabolite concentrations as predicted by the model were in accordance with available data as measured in non-growing trypanosomes, both under aerobic and under anaerobic conditions. The model also reproduced the inhibition of anaerobic glycolysis by glycerol, although the amount of glycerol needed to inhibit glycolysis completely was lower than experimentally determined. At low extracellular glucose concentrations the intracellular glucose concentration remained very low, and only at 5 mM of extracellular glucose, free glucose started to accumulate intracellularly, in close agreement with experimental observations. This biphasic relation could be related to the large difference between the affinities of the glucose transporter and hexokinase for intracellular glucose. The calculated intraglycosomal metabolite concentrations demonstrated that enzymes that have been shown to be near-equilibrium in the cytosol must work far from equilibrium in the glycosome in order to maintain the high glycolytic flux in the latter.

Plant-like traits associated with metabolism of Trypanosoma parasites.

Trypanosomatid parasites cause serious diseases among humans, livestock, and plants. They belong to the order of the Kinetoplastida and form, together with the Euglenida, the phylum Euglenozoa. Euglenoid algae possess plastids capable of photosynthesis, but plastids are unknown in trypanosomatids. Here we present molecular evidence that trypanosomatids possessed a plastid at some point in their evolutionary history. Extant trypanosomatid parasites, such as Trypanosoma and Leishmania, contain several “plant-like” genes encoding homologs of proteins found in either chloroplasts or the cytosol of plants and algae. The data suggest that kinetoplastids and euglenoids acquired plastids by endosymbiosis before their divergence and that the former lineage subsequently lost the organelle but retained numerous genes. Several of the proteins encoded by these genes are now, in the parasites, found inside highly specialized peroxisomes, called glycosomes, absent from all other eukaryotes, including euglenoids.

What Controls Glycolysis in Bloodstream Form Trypanosoma brucei

On the basis of the experimentally determined kinetic properties of the trypanosomal enzymes, the question is addressed of which step limits the glycolytic flux in bloodstream formTrypanosoma brucei. There appeared to be no single answer; in the physiological range, control shifted between the glucose transporter on the one hand and aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glycerol-3-phosphate dehydrogenase (GDH) on the other hand. The other kinases, which are often thought to control glycolysis, exerted little control; so did the utilization of ATP. We identified potential targets for anti-trypanosomal drugs by calculating which steps need the least inhibition to achieve a certain inhibition of the glycolytic flux in these parasites. The glucose transporter appeared to be the most promising target, followed by ALD, GDH, GAPDH, and PGK. By contrast, in erythrocytes more than 95% deficiencies of PGK, GAPDH, or ALD did not cause any clinical symptoms (Schuster, R. and Holzhütter, H.-G. (1995) Eur. J. Biochem. 229, 403–418). Therefore, the selectivity of drugs inhibiting these enzymes may be much higher than expected from their molecular effects alone. Quite unexpectedly, trypanosomes seem to possess a substantial overcapacity of hexokinase, phosphofructokinase, and pyruvate kinase, making these “irreversible” enzymes mediocre drug targets.

Ether--lipid (alkyl-phospholipid) metabolism and the mechanism of action of ether--lipid analogues in Leishmania.

Ether-lipid (alkyl-phospholipid) analogues such as Miltefosine possess potent in vitro and in vivo anti-leishmanial activity and these compounds are currently undergoing clinical trials in humans. These analogues are also effective against Trypanosoma cruzi and Trypanosoma brucei subspecies but their mode of action is not known. Leishmania have high levels of ether-lipids and these are mainly found in the glycosylphosphatidylinositol-anchored glycolipids and glycoproteins present on the surface of the parasites. In Leishmania mexicana promastigotes we have studied both the initiating steps for the biosynthesis of ether-lipids, and key remodelling steps. The effect of Miltefosine and Edelfosine, on key enzymes involved in the metabolism of ether-lipids has been studied. The enzymes include dihydroxyacetonephosphate acyltransferase, sn-l-acyl-2-lyso-glycero-3-phosphocholine and sn-l-alkyl-2-lyso-glycero-3-phosphocholine acyltransferases. We confirm that the initiating steps in ether-lipid metabolism in Leishmania are present in glycosomes, and that Miltefosine or Edelfosine did not perturb these enzymes. The metabolism of the latter phosphatidylcholine base intermediates, which may be involved in the remodelling of acyl- and alkyl-glycerophospholipids, was also seemingly associated with glycosomes. Both Miltefosine and Edelfosine inhibited this microbody (glycosomal) located alkyl-specific-acyl-CoA acyltransferase in a dose-dependent manner with an inhibitory concentration of 50 microM. It is suggested therefore that a perturbation of ether-lipid remodelling could be responsible for the anti-leishmanial action of these drugs.

The occurrence of glycosomes (microbodies) in the promastigote stage of four major Leishmania species.

Evidence is presented for the occurrence of glycosomes (organelles resembling peroxisomes) in four major species of Leishmania (viz. L. major, L.m. mexicana, L. b. braziliensis and L. donovani), based on latency as well as differential and isopycnic centrifugation studies. The enzymes involved in glycolysis; (hexokinase, phosphoglucose isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase); glycerol metabolism (sn-glycerol-3-phosphate dehydrogenase and glycerol kinase); carbon dioxide fixation (phosphoenolpyruvate carboxykinase and possibly malate dehydrogenase); together with an enzyme involved in the beta-oxidation of fatty acids (3-beta-hydroxybutyryl coenzyme A dehydrogenase); a key enzyme in the synthesis of ether lipids (dihydroxyacetone phosphate acyltransferase) as well as the ADP utilising enzyme adenylate kinase, were all found associated, at least in part, with a subcellular organelle which had a buoyant density in sucrose gradients of 1.21 to 1.24 g cm-3. Little variance in enzyme composition was found between the different species of Leishmania or in comparison with other members of the Trypanosomatidae, supporting the unifying principle that glycosomes are a unique characteristic of this family. The occurrence of important catabolic, anabolic and anaplerotic pathways in the glycosomes of Leishmania renders them prime targets for chemotherapy.

Carbohydrate Metabolism in African Trypanosomes, with Special Reference to the Glycosome

Over the last decade, our knowledge of the biochemistry and molecular biology of trypanosomes has expanded so much that the African trypanosome, Trypanosoma brucei, is now the equivalent of E. coli to the biochemical parasitologist. Trypanosomes are of interest to scientists not only because of their medical and veterinary importance, but also because of several unique features of their biochemistry and molecular biology. Two such features have been reviewed recently: the mitochondrial DNA network (the kinetoplast) and its role in the life cycle (Hajduk, 1978; Borst and Hoeijmakers, 1979; Barker, 1980; Englund et al., 1982), and the variant surface glycoprotein and its role in evading the immune response of the host (Englund et al., 1982; Turner, 1982).

Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias.

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.

Metabolic control analysis of glycolysis in trypanosomes as an approach to improve selectivity and effectiveness of drugs.

Glycolysis is the only ATP-generating process in bloodstream form trypanosomes and is therefore a promising drug target. Inhibitors which decrease significantly the glycolytic flux will kill the parasites. Both computer simulation and experimental studies of glycolysis in bloodstream form Trypanosoma brucei indicated that the control of the glycolytic flux is shared by several steps in the pathway. The results of these analyses provide quantitative information about the prospects of decreasing the flux by inhibition of any individual enzyme. The plasma membrane glucose transporter appears the most promising target from this perspective, followed by aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and glycerol-3-phosphate dehydrogenase. Non-competitive or irreversible inhibitors would be most effective, but it is argued that potent competitive inhibitors can be suitable, provided that the concentration of the competing substrate cannot increase unrestrictedly. Such is the case for inhibitors that compete with coenzymes or with blood glucose.

Effects of antimycin, glucose deprivation, and serum on cultures of neurons, astrocytes, and neuroblastoma cells.

The resistance of cultured mouse neuroblastoma cells, primary cultures of rat cerebellar neurons, and rat brain astrocytes to a block of aerobic metabolism was studied. Parameters such as lactate production and ATP content were measured in the presence of antimycin A and under various conditions of glucose, oxygen, and serum supply. The following conclusions can be drawn: (1) All cell types studied were characterized by an active production of lactate; (2) Incubation of the various cell types in the absence of glucose at normal oxygen tension did not affect ATP levels; (3) Respiration blocked by antimycin led to a Pasteur effect; (4) Neuroblastoma cells, but not the other cell types, were fully resistant to inhibition of respiration provided that sufficient glucose was supplied; (5) In the absence of glucose no stores of energy or utilizable substrate were present in the cell types studied when respiration was blocked; (6) In the presence of fetal calf serum anoxic neurons showed irreversible signs of degeneration.