Ornithine decarboxylase or gamma-glutamylcysteine synthetase overexpression protects Leishmania (Vianna) guyanensis against antimony.

Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (SbIII)-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in SbIII-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of SbIII in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to SbIII than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to SbIII, and confirms a role of these genes in the SbIII-resistance phenotype.

Molecular characterization of the MRPA transporter and antimony uptake in four New World Leishmania spp. susceptible and resistant to antimony.

ATP-binding cassette (ABC) transporters have been associated with drug resistance in various diseases. The MRPA gene, a transporter of ABCC subfamily, is involved in the resistance by sequestering metal-thiol conjugates in intracellular vesicles of Leishmania parasite. In this study, we performed the molecular characterization of the MRPA transporter, analysis of P-glycoprotein (Pgp) and aquaglyceroporin-1 (AQP1) expression, and determination of antimony level in antimony-susceptible and -resistant lines of L. (V.) guyanensis, L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum. PFGE analysis revealed an association of chromosomal amplification of MRPA gene with the drug resistance phenotype in all SbIII-resistant Leishmania lines analyzed. Levels of mRNA from MRPA gene determined by real-time quantitative RT-PCR showed an increased expression of two fold in SbIII-resistant lines of Leishmania guyanensis, Leishmania amazonensis and Leishmania braziliensis. Western blot analysis revealed that Pgp is increased in the SbIII-resistant L. guyanensis and L. amazonensis lines. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed a reduction in the accumulation of this element in SbIII-resistant L. guyanensis, L. amazonensis and L. braziliensis lines when compared to their susceptible counterparts. Interestingly, a down-regulation of AQP1 protein was observed in the SbIII-resistant L. guyanensis and L. amazonensis lines, contributing for decreasing of SbIII entry in these lines. In addition, efflux experiments revealed that the rates of SbIII efflux are higher in the SbIII-resistant lines of L. guyanensis and L. braziliensis, that may explain also the low SbIII concentration within of these parasites. The BSO, an inhibitor of γ-glutamylcysteine synthetase enzyme, reversed the SbIII-resistance phenotype of L. braziliensis and caused an increasing in the Sb intracellular level in the LbSbR line. Our data indicate that the mechanisms of antimony-resistance are different among species of Leishmania analyzed in this study.