Interaction of frataxin, an iron binding protein, with IscU of Fe-S clusters biogenesis pathway and its upregulation in AmpB resistant Leishmania donovani.

Abstract Leishmania donovani is a unicellular protozoon parasite that causes visceral leishmaniasis (VL), which is a fatal disease if left untreated. Certain Fe–S proteins of the TCA cycle and respiratory chain have been found in the Leishmania parasite but the precise mechanisms for their biogenesis and the maturation of Fe–S clusters remains unknown. Fe–S clusters are ubiquitous cofactors of proteins that perform critical cellular functions. The clusters are biosynthesized by the mitochondrial I ron– S ulphur C luster (ISC) machinery with core protein components that include the catalytic cysteine desulphurase IscS, the scaffold proteins IscU and IscA, and frataxin as an iron carrier/donor. However, no information regarding frataxin, its regulation, or its role in drug resistance is available for the Leishmania parasite. In this study, we characterized Ld-frataxin to investigate its role in the ISC machinery of L. donovani . We expressed and purified the recombinant Ld-frataxin protein and observed its interaction with Ld-IscU by co-purification and pull-down assay. Furthermore, we observed that the cysteine desulphurase activity of the purified Ld-IscS protein was stimulated in the presence of Ld-frataxin and Ld-IscU, particularly in the presence of iron; neither Ld-frataxin nor Ld-IscU alone had significant effects on Ld-IscS activity. Interestingly, RT-PCR and western blotting showed that Ld-frataxin is upregulated in AmpB-resistant isolates compared to sensitive strains, which may support higher Fe–S protein activity in AmpB-resistant L. donovani . Additionally, Ld-frataxin was localized in the mitochondria, as revealed by digitonin fractionation and indirect immunofluorescence. Thus, our results suggest the role of Ld-frataxin as an iron binding/carrier protein for Fe–S cluster biogenesis that physically interacts with other core components of the ISC machinery within the mitochondria.