Iron transport pathways in the human malaria parasite Plasmodium falciparum revealed by RNA-sequencing

Host iron deficiency is protective against severe malaria as the human malaria parasite Plasmodium falciparum depends on free iron from its host to proliferate. Due to the absence of transferrin, ferritin, ferroportin, and a functional heme oxygenase, the parasite’s essential pathways of iron acquisition, storage, export, and detoxification differ from those in humans and may thus be excellent targets for therapeutic development. However, the P. falciparum proteins involved in these processes remain largely unknown. Here, we show that parasites cultured in erythrocytes from an iron-deficient donor displayed significantly reduced growth rates compared to those grown in red blood cells from healthy controls. Sequencing of parasite RNA revealed diminished expression of genes involved in overall metabolism, hemoglobin digestion, and metabolite transport under low-iron versus control conditions. Supplementation with hepcidin, a specific ferroportin inhibitor, resulted in increased free iron levels in erythrocytes, enhanced parasite replication, and transcriptional upregulation of genes responsible for merozoite motility and host cell invasion. Based on endogenous GFP tagging of differentially expressed putative transporter genes followed by confocal live-cell imaging, proliferation assays with knockout and knockdown lines, and protein structure predictions, we identified six proteins that are likely required for ferrous iron transport in P. falciparum . Pf E140 may be involved in iron uptake into the parasite cytosol across the plasma membrane and Pf MRS3 could mediate import of the metal ion into the mitochondrion. Pf VIT may transport excess iron from the cytosol into cytoplasmic vesicles, and Pf ZIPCO could be implicated in Zn ²⁺ and Fe ²⁺ export from these organelles, while Pf NRAMP and Pf CRT are likely responsible for Fe ²⁺ efflux from the digestive vacuole. Our results provide new insights into the mechanisms of iron transport in P. falciparum and the parasite’s response to iron status alterations in the host. Pf E140 and Pf CRT are particularly promising candidate targets for novel antimalarial drugs, as these are essential to the parasite and lack human orthologs.