Understanding the Role of Natural Resistance Associated Macrophage Protein 1 (Nramp1) in Determining the Outcome of Leishmania major Infection

Banerjee, Sourav (2022) Understanding the Role of Natural Resistance Associated Macrophage Protein 1 (Nramp1) in Determining the Outcome of Leishmania major Infection. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sourav Banerjee (15RS046)) 15RS046.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Natural Resistance Associated Macrophage Protein 1 (Nramp1) is a phago-endosomal iron transporter, originally discovered because of its association in regulating different pathogenic infections including Leishmania sp. Human population based studies have shown that mutation in this gene results into susceptibility towards this parasitic infection. However, how the status of this protein is modulated during Leishmania infection in order to cause susceptibility is unknown. In this background, using the murine macrophage- L. major infection model, we have found that Leishmania infection significantly downregulates Nramp1 protein expression which in turn increases phagolysosomal iron content thereby supporting intracellular parasite replication. In order to determine the mechanism behind Nramp1 downregulation, we found that Nramp1 follows ubiquitin- proteasomal degradation pathway during infection. Importantly, treatment of Leishmania infected macrophage cells with heparin, an agonist of hormone hepcidin, blocked degradation of Nramp1 that resulted into depletion of phagolysosomal iron pool which in turn significantly reduced intracellular parasite burden. Further, co-immunoprecipitation studies confirmed the physical interaction between Nramp1 and hepcidin, establishing Nramp1 to be a novel target of this hormone. Importantly, taeoverexpression of Nramp1 drastically reduced phagolysosomal iron level which in turn significantly inhibited parasite replication within infected macrophage cells. Together, these findings provided a detailed mechanism of how L. major manipulates macrophage Nramp1 protein in order establish infection. However, these intriguing observations further prompted us to check the status of Nramp1 under in vivo infection condition. For this investigation, we infected Balb/c mice with L. major promastigotes in left- hind footpad. We found that L. major infection significantly upregulated transferrin receptor expression whereas downregulates Nramp1 protein at the site of infection. Perturbation in these two essential iron recycling proteins resulted into increased accumulation of iron that in turn supported amastigote proliferation. In order to find the source of this increased iron to the site of infection we observed that serum iron level in the infected animals is significantly increased as a result of depleted iron storage in liver. Although splenic iron content was unchanged in those infected animals, splenomegaly was observed. Importantly, histopathological studies revealed increased accumulation of megakaryocytes and significant upregulation of erythroid genes in infected mouse spleen. Additionally, hemoglobin level as well as bone marrow iron content in the infected animals dropped significantly with the progression of disease which accompanied development of morphologically altered erythrocytes suggesting onset of iron deficiency anemia upon L. major infection. Therefore, our observations for the first time describes how L. major infection manipulates Nramp1 protein both in vitro and in vivo and its connection with altered systemic iron homeostasis during cutaneous leishmaniasis.

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