Valdiation of Trypanosoma brucei trypanothione synthetase as drug target

Abstract

In trypanosomes, the parasite-specific thiol trypanothione [T(SH) 2] fulfills various functions, the best established being detoxification of H 2O 2 and organic hydroperoxides and ribonucleotide reduction. Recently, a trypanothione synthetase ( Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-TryS catalyzed the entire synthesis of T(SH) 2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ function of the complex Tb-TryS activities and to evaluate the importance of T(SH) 2 metabolism in T. brucei, TryS suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH) 2 and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H 2O 2 and tert.-butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidine-conjugated thiols (Gsp and T(SH) 2) and further confirms the significance of trypanothione in the defense against oxidative stress and the maintenance of viability and proliferation in unstressed parasites.