RESUMEN
Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.
Asunto(s)
Antimonio/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos , Leishmania infantum/efectos de los fármacos , Animales , Línea Celular , Enfermedades Endémicas , Humanos , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Leishmaniasis Visceral/parasitología , Estadios del Ciclo de Vida , Pruebas de Sensibilidad Parasitaria , FenotipoRESUMEN
The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmania species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantum cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC50s], about 1.8 mg/ml and 22 microg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus m-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC50s, of 0.54 and 1.32 microg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the Leishmania amastigote stage.