Intracellular pathogen Leishmania intervenes in iron loading into ferritin by cleaving chaperones in host macrophages as an iron acquisition strategy.

Iron (Fe) sequestration is one of the most important strategies of the host to control the growth and survival of invading pathogens. Ferritin (Ft) plays a pivotal role in the sequestration mechanism of mammalian hosts by storing Fe. Recent evidence suggests that poly(rC)-binding proteins (PCBPs) act as chaperones for loading Fe into Ft. Incidentally, modulation of host PCBPs in respect to storing Fe in Ft during any infection remains unexplored. Among PCBPs, PCBP1 and PCBP2 are present in every cell type and involved in interacting with Ft for Fe loading. Leishmania donovani (LD) resides within macrophages during the mammalian stage of infection, causing life-threatening visceral leishmaniasis. Here, we reveal the ability of LD to cleave PCBP1 and PCBP2 in host monocytes/macrophages. LD cleaves PCBP1-FLAG into two fragments and PCBP2-FLAG into multiple fragments, thus affecting their interactions with Ft and resulting in decreased Fe loading into Ft. LD-derived culture supernatant or exosome-enriched fractions are also able to cleave PCBPs, suggesting involvement of a secreted protease of the parasite. Using an immune-depletion experiment and transgenic mutants, we confirmed the involvement of zinc-metalloprotease GP63 in cleaving PCBPs. We further revealed that by cleaving host PCBPs, Leishmania could inhibit Fe loading into Ft to accumulate available Fe for higher intracellular growth. This is the first report of a novel strategy adopted by a mammalian pathogen to interfere with Fe sequestration into Ft by cleaving chaperones for its survival advantage within the host.

Leishmania donovani inhibits ferroportin translation by modulating FBXL5-IRP2 axis for its growth within host macrophages.

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn-FLAG in LD infected J774 macrophage confirms that Fpn down-regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by 35 S-methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5'UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F-box and leucine-rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD-infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2-FBXL5 axis to inhibit host Fpn expression.