Rupak Datta
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Researcher at Indian Institute of Science Education and Research (IISER) Kolkata
Natural resistance associated macrophage protein 1 (Nramp1) was discovered as a genetic determinant of resistance against multiple intracellular pathogens, including Leishmania. It encodes a transmembrane protein of the phago-endosomal vesicles, where it functions as an iron transporter. But how Nramp1 expression is regulated in an infected macrophage is unknown. Its role in controlling iron availability to the intracellular pathogens and in determining the final outcome of an infection also remains to be fully deciphered. Here we report that Nramp1 protein abundance undergoes temporal changes in Leishmania major infected macrophages. At 12 hours post infection, there was drastic lowering of Nramp1 level accompanied by increased phagolysosomal iron availability and enhanced parasite growth. Leishmania infection-induced downregulation of Nramp1 was found to be caused by ubiquitin-proteasome degradation pathway. In fact, blocking of Nramp1 degradation with proteasome inhibitor resulted in depletion of phagolysosomal iron pool with significant reduction in the number of intracellular parasites. Further, we uncovered that this degradation process is mediated by the iron regulatory peptide hormone hepcidin that binds to Nramp1. Interestingly, Nramp1 protein level was restored to normalcy after 30 hours of infection with a concomitant drop in the phagolysosomal iron level, which is suggestive of a host counter defense strategy to deprive the pathogen of this essential micronutrient. Taken together, our study implicates Nramp1 as a central player in the host-pathogen battle for iron. It also unravels Nramp1 as a novel partner for hepcidin. The hitherto unidentified 'hepcidin-Nramp1 axis' may have a broader role in regulating macrophage iron homeostasis.
Copper is essential for all life forms; however in excess it is extremely toxic. Toxic properties of copper are utilized by hosts against various pathogenic invasions. Leishmania, in its both free-living and intracellular forms was found to exhibit appreciable tolerance towards copper-stress. To determine the mechanism of copper-stress evasion employed by Leishmania we identified and characterized the hitherto unknown Copper-ATPase in Leishmania major and determined its role in parasites survival in host macrophage cells. L. major Cu-ATPase, LmATP7, exhibits high homology with its orthologues at multiple conserved motifs. In promastigotes, LmATP7 localized to the plasma membrane with a fraction in intracellular puncta. Upon copper treatment, LmATP7 expression increases few folds. LmATP7 is capable of complementing copper transport in Cu-ATPase-{Delta} yeast strain. Promastigotes overexpressing LmATP7 exhibits higher survival upon copper stress indicating efficacious copper export compared to wild type parasites. We explored macrophage-Leishmanial interaction with respect to copper stress subjected by the host upon parasite and the parasites reciprocating response thereon to evade the stress. The Copper-P-type-ATPases ATP7A/7B serves as major copper exporter in eukaryotes that maintain cellular copper levels. We found that Leishmania infection, triggers ATP7A upregulation in macrophages. Additionally, as part of host response, ATP7A traffics from trans-Golgi network and transports copper to the endosomal and endolysosomal compartments harbouring the Leishmania amastigotes. Finally, we show LmATP7 overexpression in this parasite, increased amastigote survivability within infected macrophages, establishing its role in combating host-induced copper stress.