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Bakker et al. (2001), Glycolysis in Trypanosoma

November 2008, model of the month by Lukas Endler
Original model: BIOMD0000000071

Trypanosomes are a species of kinetoplastids, flagellated unicellular protozoa, that has been of high interest to research as it includes a number human pathogen parasites. Two subspecies of Trypanosoma brucei cause African Trypanosomiasis (or sleeping sickness) in humans - T. b. gambiense and rhodesiense. Another subspecies, T. b. brucei, has been indicated to lead to serious forms of Nagana, the animal counterpart of sleeping sickness, especially in horses, camels and goats. The parasites have a complex life cycle in two different groups of hosts, insects and mammals (figures 1 and 2). Transmission of the disease to mammals mainly occurs through bites of the tsetse fly (genus Glossinia) (figure 3).

Life cycle of Trypanosoma brucei

Figure 1: Life cycle of trypanosoma brucei (figure taken from [1])

blood smear of a patient with trypanosomiasis

Figure 2: Blood smear of a patient with trypanosomiasis (figure taken from [1])

Tsetse fly

Figure 3: Tsetse fly (figure taken from [2])

Kinetoplastids have a peculiar metabolism in that the first seven steps of their glycolytic pathway are separated into an organelle called the glycosome, a property unique to this group of eukaryotes. These organelles seem to be related to the peroxisomes and glyoxyosomes of other eukaryotes [3, 4]. Trypanosomes in the mammalian bloodstream, unlike the forms in their insect hosts, are dependent on glucose from their host since glycolysis is their sole source of ATP. As they neither possess a functional citric acid cycle nor display oxidative phosphorylation, this makes glycolysis a very attractive target for the design of drugs against trypanosomes [5].

In the article discussed here [6], Helfert et al. try to elucidate the role and importance of triose phosphate isomerase (TPI) in the glycolysis of T. brucei in the mammalian bloodstream. As mentioned above, most of the glycolytic reactions in T. brucei are located in the glycosome, whose membrane is assumed to be impermeable to both ATP/ADP and NAD+/NADH. The essential role of the glycosome has been indicated in prior computational studies by some of the authors [7] to prevent hexose kinase (HK) and phospho-fructose kinase (PFK) from being exposed to the high ATP concentrations of the cytosol, as these enzymes seem to be hardly regulated by allosteric effectors as commonly is the case in other eukaryotes. In their mammalian hosts, under anaerobic conditions, the trypanosomes would have a yield of only one ATP per glucose consumed, as they need to regenerate NAD+, which they manage by reducing dihydroxy acetone phosphate (DHAP) to glycerol-3-phosphate (Gly3P) and - after regeneration of ATP - producing glycerol.

Under aerobic conditions reduction equivalents are exported from the glycosome in form of Gly3P, which then is oxidised to DHAP under consumption of O2 by a mitochondrial glycerol phosphate oxidase (GPO). The DHAP produced is then reimported into the glycosome and transformed into glyceraldehyde 3-phosphate (GA-3-P) by triose phosphate isomerase (TPI). Subsequent transformation via the main branch of glycolysis to pyruvate (Pyr) doubles the ATP yield per equivalent glucose compared to anaerobic conditions.

The study was triggered by the failure to knock out the TPI gene in bloodstream derived trypanosomes. Subsequent experiments with tet-regulated TPI showed that TPI depletion was limiting cell growth. To explain why TPI depletion had that harsh an effect on growth rate, a computational model of glycolysis was developed (see figure 4 for an overview).

schematic depiction of the glycolytic pathway in trypanosomes.

Figure 4: Schematic depiction of the glycolytic pathway in trypanosomes. The dotted arrows indicate possible reactions in cell expressing S. cerevisiae TPI. Abbreviations: metabolites: Fru-1,6-P (fructose 1,6-bisphosphate), GA-3-P (glyceraldehyde 3-phosphate), DHAP (dihydroxyacetone phosphate), BPGA (bisphosphoglycerate), 3-PGA (3-phosphoglycerate), 2-PGA (2-phosphoglycerate), PEP (phosphoenol-pyruvate), Gly-3-P (glycerol 3-phosphate); enzymes: ALD (aldolase), HXK (hexokinase), GAPDH1/2 (glyceraldehyde-3-phosphate dehydrogenase), GDH (glycerol-3-phosphate dehydrogenase), GLYK (glycerol kinase), GPO (glycerol-3-phosphate oxidase), PGI (glucose-6-phosphate isomerase), PGK (phosphoglycerate kinase), PYK (pyruvate kinase), PFK (phosphofructokinase), TPI (triosephosphate isomerase). Figure taken from [6].

This model is based on an earlier one from Bakker et al.[8], in which a detailed computational reconstruction of the entire glycolytic pathway of trypanosomes in mammals had been created. It was enhanced to incorporate more detailed kinetics with measured parameters for glucose-6-phosphate isomerase (PGI) and TPI. Variation of the maximal velocity of TPI indicated that, as was to be expected for this enzyme, TPI had a large overcapacity, with little changes to the ATP levels or glucose fluxes over a wide range of activities (figure 5B). At low relative activities--below 15 percent of the measured maximal velocity of the wild type--the flux through pyruvate started to decrease and that through the glycerol branch increased as expected. In contrast to the anaerobic scheme, however, no regime with equimolar glycerol and pyruvate production was reached, but all glycolytic steady state fluxes decreased to zero (figure 5C). This was found to stem from the activity of the mitochondrial GPO, which unlike under anaerobic conditions, still oxidises Gly-3-P exported from the glycosome to DHAP. The export of reduction equivalents leads to an increase of the NAD+/NADH ratio and therefore to a possible accumulation of DHAP, as it cannot be reduced to Gly-3-P anymore. This accumulation in turn could lead to inhibition of ALD and a reduction of glycolytic flux. To confirm these predictions, measurements of Pyr production, intracellular ATP and DHAP levels were performed (see Table 1). Qualitatively these measured values fitted the predictions quite well, while there were still some quantitative disparities. Use of an inhibitor of GPO, salicylhydroxamic acid, indicated that GPO activity might be essential. A dsRNA construct of the alternative oxidase part, TAO, of GPO was created to reduce TAO mRNA and consequently GPO activity. This resulted in a reduction of oxygen consumption to 22% and a reduction of the growth rate. Oxygen consumption of 22% of the wild type would translate to a maximal velocity reduction to 4.5% in the computational model, which corresponded with the decrease of the measured mRNA levels of TAO. The model predicts a reduction of ATP production to 44% at this GPO activity,which also fits the reduced growth rate (figures 6A and 6B).

Computational predictions of steady states under varying TPI activities.

Figure 5: Computational predictions of steady states under varying TPI activities. The percentages indicate relative TPI activity. A) cytosolic and glyoxysomal ATP/ADP ratios and glycosomal DHAP, B) and C) pyruvate and glycerol production as well as glucose consumption. Figure taken from [6]

Effects of TAO dsRNA under a tetracycline inhibitor.

Figure 6: A) Effects of TAO dsRNA under a tetracycline inhibitor on TAO mRNA levels. Addition of tetracycline downregulates TAO mRNA. B) Growth rates of strains carrying the tetracycline regulated TAOdsRNA under different conditions: -tet, -gly: no tetracycline/glycerol; +tet: 100ng/ml tetracycline; + gly: 1mM glycerol. The strains were subsequently diluted to 2.5e5 cells/ml every day. Figure taken from [6].

Computational results and measurements of pyruvate production and total DHAP/ATP

Table 1: Computational results and averages of at least 3 independent measurements of pyruvate production and total DHAP/ATP. Table taken from [6]

In this study experimental and computational methods were combined to elucidate the roles of key enzymes of trypanosomal glycolysis. The use of the model gave explanations for the reduced viability of strains having reduced TPI or GPO activity, both of which might be good targets for the development of new drugs against sleeping sickness and other trypanosome caused diseases.

Bibliographic References

  3. C.E. Clayton, P. Michels. Metabolic compartmentation in African trypanosomes. Parasitol Today 12:465-471, 1996. [SRS@EBI]
  4. P.A. Michels, F. Bringaud, M. Herman, V. Hannaert. Metabolic functions of glycosomes in trypanosomatids. Biochim Biophys Acta 1763:1463-1477, 2006. [SRS@EBI]
  5. C.L. Verlinde, V. Hannaert, C. Blonski, M. Willson, J.J. Périé, L.A. Fothergill-Gilmore, F.R. Opperdoes, M.H. Gelb, W.G. Hol, P.A. Michels. Glycolysis as a target for the design of new anti-trypanosome drugs. Drug Resist Updat 4:50-65, 2001. [SRS@EBI]
  6. S. Helfert, A.M. Estévez, B. Bakker, P. Michels, C. Clayton. Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei. Biochem J 357:117-125, 2001. [SRS@EBI]
  7. B.M. Bakker, F.I. Mensonides, B. Teusink, P. van Hoek, P.A. Michels, H.V. Westerhoff. Compartmentation protects trypanosomes from the dangerous design of glycolysis. PNAS 97:2087-2092, 2000.[SRS@EBI]
  8. B.M. Bakker, P.A. Michels, F.R. Opperdoes, H.V. Westerhoff. Glycolysis in bloodstream form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes. J Biol Chem 272:3207-3215, 1997. [SRS@EBI]