title: Biophysical and Pharmacological Characterization of Energy-Dependent Efflux of Sb in Laboratory-Selected Resistant Strains ofLeishmania(Viannia) Subgenus.
abstract: The growing resistance of leishmaniasis to first-line drugs like antimonials in some regions limits the control of this parasitic disease. The precise mechanisms involved inLeishmaniaantimony resistance are still subject to debate. The reduction of intracellular SbIIIaccumulation is a common change observed in both laboratory-selected and field isolated resistantLeishmaniastrains, but the exact transport pathways involved in antimony resistance have not yet been elucidated. In order to functionally characterize the antimony transport routes responsible for resistance, we performed systematic transport studies of SbIIIin wild-type and resistant strains ofL. (Viannia)guyanensisandL. (V.) braziliensis. Those include influx and efflux assays and the influence of ABC transporters and metabolism inhibitors: prochlorperazine, probenecid, verapamil, BSO, and sodium azide. The mRNA levels of genes associated with antimony resistance (MRPA, GSH1, ODC, AQP1, ABCI4, andARM58) were also investigated in addition to intracellular thiol levels. A strong reduction of Sb influx was observed inL. guyanensisresistant mutant (LgSbR), but not inL. braziliensis(LbSbR). Both mutants showed increased energy-dependent efflux of SbIII, when compared to their respective parental strains. In LgSbR, BSO and prochlorperazine inhibited antimony efflux and resistance was associated with increasedMRPAandGSH1mRNA levels, while in LbSbR antimony efflux was inhibited by probenicid and prochlorperazine in absence of resistance-associated gene modulation. Intracellular thiol levels were increased in both Sb-resistant mutants. An energy-dependent SbIIIefflux pathway sensitive to prochlorperazine was clearly evidenced in both Sb-resistant mutants. In conclusion, the present study allowed the biophysical and pharmacological characterization of energy-dependent Sb efflux pathway apparently independent of MRPA, ABCI4, and ARM58 upregulation, inLeishmania(Vianna) mutant selectedin vitrofor resistance to SbIII. Prochlorperazine has also been identified as an effective chemosensitizer in both Sb resistant mutants, which acts through inhibition of the active efflux of Sb.
authors.affiliation: Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas GeraisBelo Horizonte, Brazil; Departamento de Farmácia/Ensino e Pesquisa, Hospital João XXIII - Fundação Hospitalar do Estado de Minas GeraisBelo Horizonte, Brazil.
authors.forename: Priscila G
authors.initials: PG
authors.lastname: Dos Reis
authors.affiliation: Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou - CPqRR/FIOCRUZ Belo Horizonte, Brazil.
authors.forename: Rubens L
authors.initials: RL
authors.lastname: do Monte-Neto
authors.affiliation: Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais Belo Horizonte, Brazil.
authors.forename: Maria N
authors.initials: MN
authors.lastname: Melo
authors.affiliation: Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais Belo Horizonte, Brazil.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Frézard
keywords: ABC transporter; Leishmania; Viannia; antimony; chemosensitizer; efflux; influx; resistance