Novel intracellular SbV reducing activity correlates with antimony susceptibility in Leishmania donovani

The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (Sb-V). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-RIS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. Amastigotes (the intracellular form) and promastigotes (the extracellular form) accumulate Sb-V and Sb-III via separate transport systems. Stage-specific intracellular Sb-V reducing activity was apparent in amastigotes, which reduced the negligibly toxic Sb-V to highly toxic Sb-III. This amastigote-specific reducing activity was deficient in the Pentostam-resistant mutant L. donovani Ld1S.20. These data indicate that parasite susceptibility to Sb-V correlates with its level of Sb-V reducing activity. Also, in promastigotes of both wild-type L. donovani and the Pentostam-resistant mutant L. donovani Ld1S.20, Sb-V inhibited the toxicity of Sb-III but not of Asm. Both Sb-V and Sb-III were toxic to wild-type amastigotes. However, as observed in promastigotes, in mutant amastigotes Sb-V inhibits Sb-III but not As-III activity, Anion exchange chromatography showed that intracellular antimony metabolism occurred in both promastigotes and amastigotes. These data demonstrate that the interaction between the two antimony oxidation states occurs intracellularly, within the parasite. The results also indicate that Sb-V anti-leishmanial activity is dependent on its reduction to Sb-III. The mechanism of this novel intracellular Sb-V reduction has yet to be identified, and it may or may not be enzymatic, This is the first description of intracellular Sb-V reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell.