Abstract
Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules. In the present study, the APX gene was overexpressed in Leishmania braziliensis to investigate its contribution to the trivalent antimony (SbIII)-resistance phenotype. Western blot results demonstrated that APX-overexpressing parasites had higher APX protein levels in comparison with the wild-type line (LbWTS). APX-overexpressing clones showed an 8-fold increase in the antimony-resistance index over the parental line. In addition, our results indicated that these clones were approximately 1.8-fold more tolerant to H2O2 than the LbWTS line, suggesting that the APX enzyme plays an important role in the defence against oxidative stress. Susceptibility tests revealed that APX-overexpressing L. braziliensis lines were more resistant to isoniazid, an antibacterial agent that interacts with APX. Interestingly, this compound enhanced the anti-leishmanial SbIII effect, indicating that this combination represents a good strategy for leishmaniasis chemotherapy. Our data demonstrate that APX enzyme is involved in the development of L. braziliensis antimony-resistance phenotype and may be an attractive therapeutic target in the design of new strategies for leishmaniasis treatment.
Highlights
Leishmaniasis is an important neglected tropical disease caused by different species of unicellular protozoan parasites belonging to the Leishmania genus
Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules, regulating oxidative stress in Leishmania and avoiding damage to the parasite cells.[10]. Previous studies demonstrated that Trypanosoma cruzi extracts contained ascorbate-dependent peroxidase activity.[11,12,13] Nogueira et al[14] showed that the APX level was increased in benznidazole-resistant T. cruzi populations
Since APX is absent in humans and it presents an important role in the antioxidant defence of the trypanosomatids, this enzyme may be considered an excellent drug target for chemotherapy of these parasites.[17]
Summary
Leishmaniasis is an important neglected tropical disease caused by different species of unicellular protozoan parasites belonging to the Leishmania genus. Normalisation of the results with the monoclonal anti-α-tubulin antibody (1:15,000) (Sigma, St. Louis, USA) revealed that the APX protein level was 5-fold higher in transfected clones 4 and 13 from L. braziliensis than in the wild-type or transfected with empty vector (controls) (Fig. 1A). APX-overexpressing L. braziliensis clones were subjected to susceptibility assays with H2O2 to analyse their tolerance to oxidative stress produced by several concentrations (100-400 μM) of this compound during a 48 h incubation.
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