Abstract
In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action.
Highlights
The expansion of malaria intervention has had a tremendous impact on malaria incidence and mortality worldwide; the lack of a commercial vaccine and the increase in drug-resistant strains highlight the importance of identifying new mechanisms to combat the disease
To investigate the host BV in Plasmodium culture, the amounts of BV in the supernatant of uninfected and P. falciparum-infected red blood cell (RBC) cultures were measured by high-performance liquid chromatography (HPLC-UV-Vis and HPLC-mass spectrometry (MS); Fig. 1)
BV quantification, which was performed by the 4 used for PCR of 5′ and 3′ fragments for knockout construct are indicated. (B) Schematic representation of the gene-tagging construct used for tagging of the endogenous pbho locus in P. berghei by single homologous recombination with gfp
Summary
The expansion of malaria intervention has had a tremendous impact on malaria incidence and mortality worldwide; the lack of a commercial vaccine and the increase in drug-resistant strains highlight the importance of identifying new mechanisms to combat the disease. The host haemoglobin is degraded and used as a source of amino acids[3] This process generates haem, a toxic molecule that is scavenged by the malaria parasite through the formation of haemozoin polymer[4]. In Plasmodium, three eIF2α kinases were identified: PfeIK1, PfeIK2 and PfPK418. A.P. Han et al 200120 identified the presence of an eIF2α kinase in reticulocytes whose action is modulated by haem. We investigated changes in the phosphorylation pattern of Plasmodium eIF2α factor upon the addition of BV and haem into P. falciparum-infected RBCs. To the best of our knowledge, this work is the first to investigate the effect of BV on intraerythrocytic development and its potential targets in P. falciparum. Our data indicate BV as a non-competitive inhibitor at the low micromolar range, and molecular modelling suggests a BV-binding mode for P. falciparum enolase
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