title: Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and -susceptible Leishmania braziliensis and Leishmania infantum lines.
abstract: BACKGROUND
  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.
  
  
  METHODS
  In 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).
  
  
  RESULTS
  Northern 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.
  
  
  CONCLUSIONS
  Our functional analysis revealed that cTXNPx is involved in the antimony-resistance phenotype in L. braziliensis.
authors.forename: Juvana M
authors.initials: JM
authors.lastname: Andrade
authors.affiliation: Centro de Pesquisas René Rachou/FIOCRUZ, Avenida Augusto de Lima 1715, Belo Horizonte 30190-002, MG, Brazil. silvane@cpqrr.fiocruz.br.
authors.forename: Silvane M F
authors.initials: SM
authors.lastname: Murta
mesh_terms: D000965:Antimony / Q000494:pharmacology*; D000981:Antiprotozoal Agents / Q000494:pharmacology; D004351:Drug Resistance; D015971:Gene Expression Regulation, Enzymologic; D023281:Genomics; D006861:Hydrogen Peroxide / Q000378:metabolism; D007892:Leishmania braziliensis / Q000187:drug effects* / Q000201:enzymology*; D018314:Leishmania infantum / Q000187:drug effects* / Q000201:enzymology*; D018384:Oxidative Stress; D010544:Peroxidases / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D000981:Antiprotozoal Agents; D015800:Protozoan Proteins; D012333:RNA, Messenger; D000965:Antimony; D006861:Hydrogen Peroxide; D010544:Peroxidases; C110959:tryparedoxin peroxidase