Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and FoF1-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and FoF1-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by FoF1-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.