Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity

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 (gamma ECS), 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 gamma ECS and TryS control T(SH)(2) synthesis by 60-74% and 15-31%, respectively. gamma ECS 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. gamma ECS 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 gamma ECS and TXN1 in the T(SH)(2) antioxidant pathway may compromise the parasite's viability and infectivity.