The characterization of Plasmodium falciparum iron regulatory-like protein (PfIRPa) in asexual stage Plasmodium falciparum parasites

Iron is an indispensable nutrient for the erythrocytic forms of Plasmodium falciparum, but the metabolic pathways of iron homeostasis in Plasmodium falciparum are poorly understood. An iron regulatory-like protein expressed by Plasmodium falciparum (PfIRPa) is a plausible candidate for having a role in iron homeostasis of asexual Plasmodium falciparum parasites. This protein has homology to mammalian iron regulatory proteins/aconitases, and is capable of binding RNA iron response elements (IREs). Western blot analysis of digitonin-permeabilized trophozoites suggests that the localization of PfIRPa is predominantly in membranous compartments of the parasite, such as the mitochondrion. However, immunofluorescence analysis shows that PfIRPa is localized in the mitochondria and the parasitic cytosol. Under conditions favoring iron-sulfur cluster formation, recombinant PfIRPa (rPfIRPa) displays aconitase activity, as detected by a colorimetric NADPH-MTT (3-[4.5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) assay. Spectrophotometric measurements of cis-aconitate hydration at 240 nm show that rPfIRPa had an overall catalytic efficiency (Kcat/Km) that was similar in magnitude to human cytosolic IRP1/aconitase and human mitochondrial aconitase. PflRPa immunoprecipitated from parasite lysates also has aconitase activity, as assessed by an MTT-based assay. PfIRPa binds specifically to an IRE-like stem loop structure found in the 3' untranslated region of malarial gene PF13_0080. The location of this IRE is consistent with the possibility that PfIRPa could protect PF13_0080 mRNA from endonucleolytic cleavage by binding to this IRE-like structure. To test this hypothesis, two small (25-mer) RNA-mimicking oligonucleotides were designed: one identical and one complementary to the stem-loop structure of PF13_0080 IRE. Contrary to expectations, the addition of either oligonucleotide to erythrocytic Plasmodium falciparum led to increased levels of PF13_0080 mRNA, decreased levels of PfIRPa mRNA, and sustained malaria growth despite substrate limitations caused by high parasitemia culturing conditions. The results presented in this work provide evidence that PfIRPa localizes in the mitochondrion and in the cytosol and is able to demonstrate aconitase activity. The results also suggest that PfIRPa might act as a post-transcriptional repressor of PF13_0080 mRNA. Further understanding of the role of PfIRPa/aconitase in the regulation of Plasmodium falciparum iron homeostasis may contribute towards the development of novel antimalarial strategies against plasmodial species.