ABSTRACT
Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.
Subject(s)
Antioxidants/metabolism , Glutamate-Cysteine Ligase/genetics , Glutathione/analogs & derivatives , Protozoan Proteins/genetics , Spermidine/analogs & derivatives , Thioredoxins/genetics , Trypanosoma cruzi/drug effects , Amide Synthases/genetics , Amide Synthases/metabolism , Buthionine Sulfoximine/pharmacology , Cell Line , Drug Combinations , Drug Resistance/genetics , Fibroblasts/parasitology , Gene Expression Regulation , Glutamate-Cysteine Ligase/metabolism , Glutathione/antagonists & inhibitors , Glutathione/biosynthesis , Humans , Hydrogen Peroxide/pharmacology , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Nitroimidazoles/pharmacology , Oxidation-Reduction , Oxidative Stress , Peroxidases/genetics , Peroxidases/metabolism , Protozoan Proteins/metabolism , Signal Transduction , Spermidine/antagonists & inhibitors , Spermidine/biosynthesis , Thioredoxins/metabolism , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/geneticsABSTRACT
Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.
