Abstract
BackgroundTryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules. Reports suggest that drug-resistant parasites may increase the levels of TXNPx and other enzymes, thereby protecting them against oxidative stress.MethodsIn this study, the gene encoding cytosolic TXNPx (cTXNPx) was characterized in lines of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum that are susceptible and resistant to potassium antimony tartrate (Sb(III)). We investigated the levels of mRNA and genomic organization of the cTXNPx gene. In addition, we transfected the Leishmania lines with the cTXNPx gene and analysed the susceptibility of transfected parasites to Sb(III) and to hydrogen peroxide (H2O2).ResultsNorthern blot and real-time reverse transcriptase polymerase chain reaction analyses revealed that the level of TXNPx mRNA was approximately 2.5-fold higher in the Sb(III)-resistant L. braziliensis line than in the parental line. In contrast, no significant difference in cTXNPx mRNA levels between the L. infantum lines was observed. Southern blot analyses revealed that the cTXNPx gene is not amplified in the genome of the Sb(III)-resistant Leishmania lines analysed. Functional analysis of cTXNPx was performed to determine whether overexpression of the enzyme in L. braziliensis and L. infantum lines would change their susceptibility to Sb(III). Western blotting analysis showed that the level of cTXNPx was 2 to 4-fold higher in transfected clones compared to non-transfected cells. Antimony susceptibility test (EC50 assay) revealed that L. braziliensis lines overexpressing cTXNPx had a 2-fold increase in resistance to Sb(III) when compared to the untransfected parental line. In addition, these clones are more tolerant to exogenous H2O2 than the untransfected parental line. In contrast, no difference in Sb(III) susceptibility and a moderate index of resistance to H2O2 was observed in L. infantum clones overexpressing cTXNPx.ConclusionOur functional analysis revealed that cTXNPx is involved in the antimony-resistance phenotype in L. braziliensis.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-3305-7-406) contains supplementary material, which is available to authorized users.
Highlights
Tryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules
Genomic organization of the cTXNPX gene Genomic organization of the cytosolic TXNPx (cTXNPx) gene in Sb(III)resistant and -susceptible lines of L. braziliensis and L. infantum was determined by Southern blot analysis of parasite DNA digested with an endonuclease (EcoRI or BamHI)
Hybridization of the blots with an LbcTXNPx gene specific probe revealed that EcoRI-digested DNA gave a major band of 14.0 kb for both L. braziliensis lines, and bands of 0.6, 0.8, 4.0, and 14.0 kb for L. infantum lines (Additional file 1: Figure S1)
Summary
Tryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules. Reports suggest that drug-resistant parasites may increase the levels of TXNPx and other enzymes, thereby protecting them against oxidative stress. Leishmaniasis refers to a spectrum of diseases caused by different species of protozoan parasites belonging to the genus Leishmania. The mechanism of action of antimony has not been fully elucidated. Studies suggest that Sb(V) inhibits macromolecular biosynthesis in amastigotes, possibly altering energy metabolism by inhibiting glycolysis and the oxidative pathway of fatty acids [5,6]. Sb(III) is purported to generate disturbances in the thiol redox potential of the parasite by inducing the efflux of intracellular thiols and by inhibiting trypanothione reductase, resulting in cell death by oxidative stress [7]
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