Unraveling the mechanism by which Leishmania secretory factor GP63 targets the phagolysosomal iron transporter Nramp1 for efficient iron acquisition

Samanta, Suman (2025) Unraveling the mechanism by which Leishmania secretory factor GP63 targets the phagolysosomal iron transporter Nramp1 for efficient iron acquisition. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Suman Samanta (19RS083)) 19RS083.pdf - Submitted Version Restricted to Repository staff only Download (4MB)

Abstract

Nramp1 (Natural Resistance-Associated Macrophage Protein 1) is a critical phagolysosomal iron exporter in macrophages and plays a pivotal role in host defense against various intracellular pathogens, including Leishmania. Despite its central importance in host-pathogen interactions, the mechanisms by which Nramp1 levels are modulated during Leishmania infection remained unclear. Our previous studies demonstrated that L. major infection leads to significant depletion of Nramp1, thereby increasing phagolysosomal iron availability and enhancing parasite survival. We further showed that this depletion is mediated by ubiquitin-proteasome-dependent degradation, triggered by the iron-regulatory hormone hepcidin, whose expression is markedly upregulated upon infection. However, the mechanism by which Leishmania induces hepcidin expression and targets Nramp1 for degradation remained unresolved. Taking cue from an intriguing observation that Nramp1 depletion was observed even in bystander uninfected cells in close proximity to Leishmania infected macrophages, we hypothesized that a parasite-secreted factor might be driving this process. To test this hypothesis, we treated macrophages with Leishmania conditioned media (Lm-CM), which induced proteasomal degradation of Nramp1 to a similar extent as direct infection and also increased the endo/lysosomal iron content in the Lm-CM-treated macrophages compared to the untreated macrophages. The effect was abolished when Lm-CM was treated with heat and trypsin, indicating the involvement of a proteinaceous component. To identify the responsible factor, we isolated extracellular vesicles (EVs) from Lm-CM and observed that both EVs and the post-EV supernatant fraction induced Nramp1 depletion to a similar extent as Lm-CM treatment. Pharmacological inhibition of GP63 activity using EDTA and 1,10-phen. abrogated Lm-CM induced Nramp1 depletion. To further confirm it, we generated a GP63-deficient strain (LmGP63⁻/⁻), and LmGP63⁻/⁻ -CM failed to suppress Nramp1 level. Further analysis revealed that GP63 facilitates ubiquitin-proteasome-mediated degradation of Nramp1 by upregulating the iron regulatory protein hepcidin. We also found that GP63 modulates hepcidin mRNA levels by altering pre-miRNA-122 levels through the depletion of DICER1, a key enzyme in miRNA processing. In vivo studies in BALB/c mice validated these findings: wild-type L. major infection reduced Nramp1 and DICER1 while elevating hepcidin, whereas these effects were absent in LmGP63⁻/⁻ infections. Collectively, our findings establish GP63 as the key secretory factor responsible for Nramp1 depletion, promoting its ubiquitin-proteasome-mediated degradation by upregulating hepcidin through DICER1 depletion, which alters pre-miRNA-122 expression, creating a more favorable environment for Leishmania.

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