Involvement of nucleoside diphosphate kinase b and elongation factor 2 in Leishmania braziliensis antimony resistance phenotype

BACKGROUND: Nucleoside diphosphate kinase b (NDKb) is responsible for nucleoside triphosphates synthesis and it has key role in the purine metabolism in trypanosomatid protozoans. Elongation factor 2 (EF2) is an important factor for protein synthesis. Recently, our phosphoproteomic analysis demonstrated that NDKb and EF2 proteins were phosphorylated and dephosphorylated in antimony (Sb(III))-resistant L. braziliensis line compared to its Sb(III)-susceptible pair, respectively. METHODS: In this study, the overexpression of NDKb and EF2 genes in L. braziliensis and L. infantum was performed to investigate the contribution of these proteins in the Sb(III)-resistance phenotype. Furthermore, we examined the role of lamivudine on Sb(III) susceptibility in clones that overexpress the NDKb gene, and the effect of EF2 kinase (EF2K) inhibitor on the growth of EF2-overexpressing parasites. RESULTS: Western blot analysis demonstrated that NDKb and EF2 proteins are more and less expressed, respectively, in Sb(III)-resistant line of L. braziliensis than its wild-type (WTS) counterpart, corroborating our previous phosphoproteomic data. NDKb or EF2-overexpressing L. braziliensis lines were 1.6 to 2.1-fold more resistant to Sb(III) than the untransfected WTS line. In contrast, no difference in Sb(III) susceptibility was observed in L. infantum parasites overexpressing NDKb or EF2. Susceptibility assays showed that NDKb-overexpressing L. braziliensis lines presented elevated resistance to lamivudine, an antiviral agent, but it did not alter the leishmanicidal activity in association with Sb(III). EF2-overexpressing L. braziliensis clone was slightly more resistant to EF2K inhibitor than the WTS line. Surprisingly, this inhibitor increased the antileishmanial effect of Sb(III), suggesting that this association might be a valuable strategy for leishmaniasis chemotherapy. CONCLUSION: Our findings represent the first study of NDKb and EF2 genes overexpression that demonstrates an increase of Sb(III) resistance in L. braziliensis which can contribute to develop new strategies for leishmaniasis treatment.