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

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 H 2 O 2 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.

Motility patterns of Trypanosoma cruzi trypomastigotes correlate with the efficiency of parasite invasion in vitro

“…After 13 days of interaction, almost all the cells have been infected. From these results, we concluded that the fraction of infected cells after 18 h of the initial cell-parasite interaction can be used as an estimate for the parasite invasion efficiency, in agreement with former results 12,[18][19][20] .…”

Computational Drug Repositioning for Chagas Disease Using Protein-Ligand Interaction Profiling

“…Finally, GBD had an IC 50 = 66 ± 12 μM. For comparison, under similar exposure protocols, the IC 50 for benznidazole was 12 ± 2 μM [ 35 ] whereas for NFX the value was 3 ± 0.6 μM (Aketzalli Silva Carmona & Emma Saavedra, unpublished results). In addition, the compounds were evaluated on human foreskin fibroblasts (HFF1) to measure cytotoxicity; the effects on these cells are summarized in Table 4 .…”

ATrypanosoma bruceiORFeome-based Gain-of-Function Library identifies genes that promote survival during melarsoprol treatment

“…Despite the established relationship between melarsoprol and trypanothione, the specific mode of melarsoprol cell killing remains unknown (24). Because the biosynthetic and redox utilization pathways contain enzymes unique to Trypanosomatids, they have been broadly explored as drug targets against American trypanosomes and Leishmania species (15,(28)(29)(30)(31)(32).…”

“…6). The essential gene encoding gglutamylcysteine synthetase (GSH1, Tb927.10.12370)(41), which is the rate-limiting step of trypanothione biosynthesis(28,32,33,42), was 191-fold overrepresented in melarsoprol survivors(TABLE 2). Overexpression of GSH1 in T. brucei and other Trypanosomatids increases the concentration of intracellular trypanothione and has been shown to promote melarsoprol resistance under laboratory conditions(33).…”