title: Involvement of nucleoside diphosphate kinase b and elongation factor 2 in Leishmania braziliensis antimony resistance phenotype.
abstract: 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 (SbIII)-resistant L. braziliensis line compared to its SbIII-susceptible pair, respectively.
  
  
  
  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 SbIII-resistance phenotype. Furthermore, we examined the role of lamivudine on SbIIIsusceptibility in clones that overexpress the NDKb gene, and the effect of EF2 kinase (EF2K) inhibitor on the growth of EF2-overexpressing parasites.
  
  
  
  Western blot analysis demonstrated that NDKb and EF2 proteins are more and less expressed, respectively, in SbIII-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 SbIIIthan the untransfected WTS line. In contrast, no difference in SbIIIsusceptibility 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 SbIII. EF2-overexpressing L. braziliensis clone was slightly more resistant to EF2K inhibitor than the WTS line. Surprisingly, this inhibitor increased the antileishmanial effect of SbIII, suggesting that this association might be a valuable strategy for leishmaniasis chemotherapy.
  
  
  
  Our findings represent the first study of NDKb and EF2 genes overexpression that demonstrates an increase of SbIIIresistance in L. braziliensis which can contribute to develop new strategies for leishmaniasis treatment.
authors.affiliation: Centro de Pesquisas René Rachou CPqRR, Fundação Oswaldo Cruz - FIOCRUZ, Avenida Augusto de Lima 1715, Belo Horizonte, MG, Brazil.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Moreira
authors.affiliation: Centro de Pesquisas René Rachou CPqRR, Fundação Oswaldo Cruz - FIOCRUZ, Avenida Augusto de Lima 1715, Belo Horizonte, MG, Brazil. silvane@cpqrr.fiocruz.br.
authors.forename: Silvane M F
authors.initials: SM
authors.lastname: Murta
mesh_terms: D000965:Antimony / Q000633:toxicity*; D015153:Blotting, Western; D004351:Drug Resistance; D020869:Gene Expression Profiling; D007892:Leishmania braziliensis / Q000187:drug effects* / Q000235:genetics*; D018314:Leishmania infantum / Q000187:drug effects / Q000235:genetics; D054778:NM23 Nucleoside Diphosphate Kinases / Q000032:analysis* / Q000235:genetics; D020652:Peptide Elongation Factor 2 / Q000032:analysis* / Q000235:genetics
chemical_list: D054778:NM23 Nucleoside Diphosphate Kinases; D020652:Peptide Elongation Factor 2; D000965:Antimony
keywords: Antimony resistance; Chemotherapy; Elongation factor 2; Leishmania spp.; Nucleoside diphosphate kinase b