Visceral leishmaniasis caused by Leishmania are pathogenic protozoans that primarily attack mammalian macrophages, dwells within it as intracellular parasite requiring iron (Fe) from host. Host iron acquisition by Leishmania spp. is crucial for DNA replication, mitochondrial function, cellular iron homeostasis, and redox equilibrium. So, we have evaluated growth and viability of Leishmania promastigote parasites in iron supplemented (ferrous sulphate heptahydrate, 0 µM-4000 µM) and for depleted media conditions (iron chelator deferoxamine, 0 µM-50 µM). Iron supplementation upto 1000 µM was found to be promoting parasite cell growth and differentiation while in iron depleted condition, parasite cell was found to be decreasing with increasing concentration of Fe chelator. The uptake of iron also effects the morphological parameters like cell shape and size both in iron sufficient and deficient conditions. Acquisition of excess or very less iron leads to cytotoxicity and interferes metabolic activity of the parasite.
We have also quantified reactive oxygen species (ROS) using 2,7,-dichlorodihydrofluorescein diacetate (H2DCFDA) and estimated intracellular Thiol using thiol-dithionitrobenzoic acid (DTNB) in iron stress condition to elucidate oxidative stress. We observed the same pattern of ROS and Thiol in both iron conditions where decreasing ROS level and increasing Thiol level suggest Thiol pathway proteins are responsible to scavenge the elevated ROS in these parasites.
Further, we observed LD proteins (TryS, TryR, CS, SAT & Ctxn) involved in the thiol pathway and those involved in Fe-S cluster biosynthesis (NBP, Frataxin & IScU) in iron sufficient and deficient condition confirmed by immunoblotting. As a result, we found higher expression of Thiol and Fe-S protein with increasing concentration of iron which correlates well with the increased ROS level in parasites.
Furthermore, we have successfully established infection in THP-1 macrophages by iron treated and depleted promastigote parasite. Interestingly, we found increasing iron concentration somehow elevates the parasite pathogenesis and increases the virulence of parasites. Lesser infectivity in THP-1 cell line was observed in iron limited condition. Therefore, our present study found iron as micronutrient plays a significant role in infectivity and pathogenicity in host-parasite interaction.