Dynamic phosphoproteomics and proteomics uncover Leishmania donovani-driven ferritin hijacking, contributing to the control of iron homeostasis and iron-related oxidative stress

Leishmania donovani, the causative agent of visceral leishmaniasis, survives within the parasitophorous vacuole (PV) of mammalian macrophages by extensively rewiring host cellular pathways. Although transcriptional and proteomic changes in infected macrophages have been characterized, the impact on the host phosphoproteome, a pivotal, reversible regulator of signalling, remains largely unexplored. To address this gap, we combined time-resolved quantitative phosphoproteomics and proteomics to map the dynamic response of murine macrophages to L. donovani infection. Early after infection, the parasite rapidly attenuates the macrophage signaling cascades normally triggered by phagocytosis. Between 24 h and 48 h post-infection we observed a progressive de-phosphorylation of proteins involved in innate immunity, apoptosis and other stress-responsive pathways, consistent with a partial shutdown of multiple host kinases. From 24 h onward, a global decline in protein abundance was also detected, most notably within lysosomal network. Strikingly, only five host proteins were consistently up-regulated, suggesting their importance for parasite survival. Ferritin light chain (Ftl1) displayed the largest increase. Immunofluorescence and transmission-electron microscopy revealed that Ftl1 accumulates within the parasitophorous vacuole, colocalizes with the ferritin receptor Ncoa4, and yet fails to undergo degradation, indicating that Leishmania co-opts ferritin as its own intracellular iron-storage compartment. Ferritin is not confined to the PV; it is also internalized by the parasite. Within L. donovani, ferritin accumulates not only in the cytoplasm but also in the nucleus, where it may function both as an iron-storage depot and as an iron buffer that protects the parasite from oxidative damage. Finally, we demonstrate that Leishmania donovani pre-conditions its host macrophage for the iron-rich environments of the liver and spleen by driving the assembly of ferritin particles enriched in ferritin-light chain. This contrasts with L. amazonensis, which, residing in the iron-poor skin, induces ferritin predominated by ferritin-heavy chain (Fth1). Knock-down of Ftl1 did not decrease parasite survival because macrophages compensated for its loss by up-regulating Hspb1, which encodes a protein that limits lipid peroxidation by inhibiting the Fenton reaction. Although this response protects the host cell, it also diminishes iron import, imposing a metabolic cost on Leishmania. Hspb1 represents only one example; additional, as yet unidentified, compensatory pathways are likely activated by the parasite to mitigate the loss of ferritin-light chain. Collectively, our data uncover a previously unknown strategy whereby L. donovani hijacks host ferritin trafficking to create a protected iron reservoir, thereby preventing ferroptosis. This mechanism sets Leishmania, a eukaryotic intracellular parasite, apart from the canonical iron-acquisition tactics employed by bacteria and fungi.