Trypanosome Glycolysis Research Articles

The death and life of the resurrection drug.

In his own words, it was the most dramatic moment of his scientific career. At the back of an auditorium in Nairobi, Kenya, Cyrus Bacchi met Simon Van Nieuwenhove. Bacchi, at the time, was essentially a biochemist whose interest was the African trypanosome. Van Nieuwenhove was a clinician who had worked for many years in the field in Africa. The topic of their conversation was eflornithine—a small and simple compound that would eventually make a big impact. Bacchi had shown that the compound had an effect on the parasite that causes Human African Trypanosomiasis. Van Nieuwenhove was dosing patients with it. The story of eflornithine did not start with eflornithine.It started because no one knew how to purify an enzyme. Bacchi was working on his thesis, which centred on the α-glycerophosphate shuttle in hemoflagellates. The shuttle acts as a way to transport reducing equivalents from the cytosol to the mitochondrion. α-glycerophosphate dehydrogenase was the enzyme he spent much of his time trying to purify. The reason he could not purify it was it was hidden within another compartment of the trypanosome. It would not be until several years later, and published in 1977, that Fred Opperdoes discovered the glycosome [1]: the organelle that encapsulates glycolysis in trypanosomes, and the organelle that was hiding the enzyme. At every step of the purification process Bacchi lost activity in his enzyme extracts. Magnesium chloride, surprisingly, managed to boost activity. Bacchi looked into other nonmetallic compounds that would act as cations and eventually chose the polyamines: naturally occurring, nitrogen-containing cations whose concentration is closely controlled by the cell. Spermidine and spermine were found to be the best replacements for magnesium, yielding higher activities. With evidence that the enzyme he was trying to purify was most likely locked within the glycosome, Bacchi moved his attention to how the polyamines were made within the trypanosome. This was an area that had amassed a lot of knowledge everywhere apart from in trypanosomes. It was in 1677 that Anton van Leeuwenhoek first observed spermatozoa in humans, dogs, and a host of other organisms, and he later discovered crystals of spermine phosphate in human semen. Modern research had identified the biologically active polyamines—spermidine and spermine—in plants and many types of mammalian cells. The biochemical pathways that make and degrade the polyamines, along with some of their enzymes, had also been identified. At the time, nothing was known about polyamines in protozoa, and in trypanosomes in particular. Did these parasites contain polyamines? Could polyamine metabolism be a useful chemotherapeutic target?

A domino effect in drug action: from metabolic assault towards parasite differentiation

Awareness is growing that drug target validation should involve systems analysis of cellular networks. There is less appreciation, though, that the composition of networks may change in response to drugs. If the response is homeostatic (e.g. through upregulation of the target protein), this may neutralize the inhibitory effect. In this scenario the effect on cell growth and survival would be less than anticipated based on affinity of the drug for its target. Glycolysis is the sole free-energy source for the deadly parasite Trypanosoma brucei and is therefore a possible target pathway for anti-trypanosomal drugs. Plasma-membrane glucose transport exerts high control over trypanosome glycolysis and hence the transporter is a promising drug target. Here we show that at high inhibitor concentrations, inhibition of trypanosome glucose transport causes cell death. Most interestingly, sublethal concentrations initiate a domino effect in which network adaptations enhance inhibition. This happens via (i) metabolic control exerted by the target protein, (ii) decreases in mRNAs encoding the target protein and other proteins in the same pathway, and (iii) partial differentiation of the cells leading to (low) expression of immunogenic insect-stage coat proteins. We discuss how these 'anti-homeostatic' responses together may facilitate killing of parasites at an acceptable drug dosage.

Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes

ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation.

Experimental and in Silico Analyses of Glycolytic Flux Control in Bloodstream Form Trypanosoma brucei

A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.

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.

The trypanocidal Cape buffalo serum protein is xanthine oxidase.

Plasma and serum from Cape buffalo (Syncerus caffer) kill bloodstream stages of all species of African trypanosomes in vitro. The trypanocidal serum component was isolated by sequential chromatography on hydroxylapatite, protein A-G, Mono Q, and Superose 12. The purified trypanocidal protein had a molecular mass of 150 kDa, and activity correlated with the presence of a 146-kDa polypeptide detected upon reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences of three peptide fragments of the 146-kDa reduced polypeptide, ligand affinity and immunoaffinity chromatography of the native protein, and sensitivity to pharmacological inhibitors, identified the trypanocidal material as xanthine oxidase (EC 1.1.3.22). Trypanocidal activity resulted in the inhibition of trypanosome glycolysis and was due to H2O2 produced during catabolism of extracellular xanthine and hypoxanthine by the purine catabolic enzyme.

Regulation and control of compartmentalized glycolysis in bloodstream formTrypanosoma brucei

Unlike other eukaryotic cells, trypanosomes possess a compartmentalized glycolytic pathway. The conversion of glucose into 3-phosphoglycerate takes place in specialized peroxisomes, called glycosomes. Further conversion of this intermediate into pyruvate occurs in the cytosol. Due to this compartmentation, many regulatory mechanisms operating in other cell types cannot work in trypanosomes. This is reflected by the insensitivity of the glycosomal enzymes to compounds that act as activity regulators in other cell types. Several speculations have been raised about the function of compartmentation of glycolysis in trypanosomes. We calculate that even in a noncompartmentalized trypanosome the flux through glycolysis should not be limited by diffusion. Therefore, the sequestration of glycolytic enzymes in an organelle may not serve to overcome a diffusion limitation. We also search the available data for a possible relation between compartmentation and the distribution of control of the glycolytic flux among the glycolytic enzymes. Under physiological conditions, the rate of glycolytic ATP production in the bloodstream form of the parasite is possibly controlled by the oxygen tension, but not by the glucose concentration. Within the framework of Metabolic Control Analysis, we discuss evidence that glucose transport, although it does not qualify as the sole rate-limiting step, does have a high flux control coefficient. This, however, does not distinguish trypanosomes from other eukaryotic cell types without glycosomes.

Anti-trypanosomal compounds. I. In vivo and in vitro activity of inhibitors of glycolysis in trypanosomes

Carboxylated and phosphorylated esters of glycerol, possible inhibitors of the atypical glycolysis in trypanosomes have been synthesised and tested on Trypanosoma equiperdum in vitro and Trypanosoma brucei, on mice, and some of them also on glycerol phosphate dehydrogenase (GDH). Two of these compounds which showed no in vitro activity behaved as good competitive inhibitors of GDH; 3 other compounds were shown to have a good activity on cultures of trypanosomes, but owing to their weak solubility, tests on the enzyme could not be conclusive. Indications for the design of other compounds have been given.

Compartmentation of glycolysis in trypanosomes: a potential target for new trypanocidal drugs.

In African trypanosomes most enzymes of the glycolytic pathway are found in a microbody-like organelle, called the glycosome. The analysis of their structural and functional properties has shown that these glycosomal enzymes possess some specific features which are absent from the cytosolic proteins of trypanosomes and from the glycolytic enzymes of other organisms, where glycolysis is not compartmentalized within an organelle. The specific properties of the glycosomal enzymes may be responsible for the routing of the proteins from their site of synthesis, the cytosol, into the glycosome, or they may be involved in the proper functioning of the enzymes within the organelle. Whatever the role of the unique features, they are potential targets for compounds that could specifically interfere with glycolysis in trypanosomes. Therefore, a detailed study of the glycolytic enzymes of trypanosomes may lead to the development of therapeutically useful drugs against these harmful parasites.

Subcellular compartmentation of glycolytic intermediates in Trypanosoma brucei.

In cell-fractionation experiments most of the glycolytic enzymes in bloodstream forms of Trypanosoma brucei are recovered in a microbody, called the glycosome [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360-364]. To see whether this compartmentation of glycolytic enzymes is accompanied by compartmentation of metabolites we have pulse-labelled intact T. brucei with [U-14C]glucose and followed the incorporation of radioactivity into glycolytic intermediates separated by anion-exchange chromatography. The kinetics of incorporation provide direct evidence for the existence of two pools of glycolytic intermediates. One pool is completely labelled within 15 s and represents 20-30% of total cellular metabolites. Radioactively labelled pyruvate is already produced after 15 s. Since this pool is directly involved in the glycolytic flux, we conclude that it is present in the glycosome. The second pool which represents 70-80% of the total appears not to be directly involved in glycolysis. Its content equilibrates relatively slowly with the glycosomal pool. It probably represents the cytosol. Incorporation of radioactivity into the total glycerol 3-phosphate pool is more rapid than for the other metabolites studied. This indicates rapid mixing of the glycosomal and cell-sap pools of glycerol 3-phosphate, as required for the extra-glycosomal oxidation of glycerol 3-phosphate by the mitochondrial oxidase. In the presence of 1 mM salicylhydroxamic acid, which mimics anaerobiosis, the trypanosome produces equimolar amounts of pyruvate and glycerol. Labelling of glycerol and glycerol 3-phosphate proceeds at identical rates but at all times the specific activity of glycerol is less than that of glycerol 3-phosphate. This is compatible with our earlier proposal that glycerol is made from glycerol 3-phosphate by glycerol kinase. We conclude that glycolysis in trypanosomes takes place in the glycosome and that the membrane of this organelle is poorly permeable to most glycolytic intermediates.

DNA rearrangements involving the genes for variant antigens in Trypanosoma brucei.

Trypanosomes are unicellular, flagellate protozoa responsible for some of the most widespread parasitic diseases of humans and domestic animals (Hoare 1972). In itself, this would explain the increasing popularity of trypanosomes as research objects. However, trypanosomes also have a number of intriguing attributes that are of scientific interest: (1) The mitochondrial DNA of trypanosomes is the most bizarre DNA in nature; it consists of a large network of catenated circles, usually called kinetoplast DNA (see Borst and Hoeijmakers 1979). (2) Trypanosome glycolysis is unique since it is organized in a separate organelle, the glycosome (see Opperdoes and Borst 1977). (3) Trypanosomes can completely suppress mitochondrial biogenesis (Hoare 1972), a property they share only with yeast. (4) Although trypanosomes are primitive eukaryotes with much less genetic complexity than that of Escherichia coli, they share some of the typical aspects of eukaryotic genome organization, i.e., simple sequence satellites and a large fraction...

The potential use of inhibitors of glycerol-3-phosphate oxidase for chemotherapy of African trypanosomiasis

The bloodstream forms of African trypanosomes, responsible for sleeping sickness in man, are completely dependent on glycolysis for their energy supply, because the biogenesis of a mitochondrial respiratory chain is suppressed in the vertebrate host [l-4] . The present ideas on trypanosome glycolysis are presented in a simplified form in fig.1. Grant and Fulton [5] showed that the flow of carbon is consistent with the view that the Embden-Meyerhof scheme is the only pathway of glucose utilization. Pyruvate is not further metabolized because of the absence of a functional Krebs cycle [6,7] and the lack of lactate dehydrogenase [8] and is excreted into the host blood. The NAD’ reduced in the glyceraldehyde phosphate dehydrogenase step is reoxidized by a Lglycerol-3-phosphate oxidase system [9] , unique to trypanosomes (boxed in fig. 1). According to fig. 1 this oxidase system is essential for survival of bloodstream trypanosomes. Since it is absent in the mammalian host, it should provide an ideal target for a selective chemotherapeutic agent. This paper deals with the effect of salicylhydroxamic acid (SHAM), a powerful inhibitor of the glycerolphosphate oxidase system in trypanosomes, on glycolysis and on the energy production of isolated, intact trypanosomes. Although the oxidase is completely blocked by 1 mM SHAM, we have found that motility and ATP production are not drastically