Date Published: August 1, 2013
Publisher: Public Library of Science
Author(s): Viswanathan Arun Nagaraj, Balamurugan Sundaram, Nandan Mysore Varadarajan, Pradeep Annamalai Subramani, Devaiah Monnanda Kalappa, Susanta Kumar Ghosh, Govindarajan Padmanaban, Maria M. Mota.
Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-14C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.
Plasmodium falciparum (Pf) and Plasmodium vivax account for more than 95% of human malaria. P. falciparum is widely resistant to the antimalarial drugs chloroquine (CQ) and antifolates. Sporadic resistance is also seen in P. vivax. Emerging resistance to the artemisinin-based combination therapies  and the absence of an effective vaccine highlight an urgent need to develop new drug targets and vaccine candidates , . The de novo heme-biosynthetic pathway of the malaria parasite offers potential drug targets and new vaccine candidates. The malaria parasite is capable of de novo heme biosynthesis despite its ability to acquire heme from red blood cell (RBC) hemoglobin. During the intraerythrocytic stages, the parasite detoxifies hemoglobin-heme by converting it into hemozoin , . The source of the heme used in the parasite mitochondrial cytochromes and the parasite heme requirements during the mosquito and liver stages are yet unknown. Hence, the role of the de novo heme-biosynthetic pathway throughout the entire parasite life cycle is a subject of considerable interest .
In this study, we assessed the role of parasite-synthesized heme in all stages of malaria parasite growth. We generated ALAS and FC KOs in P. berghei. We used the KOs to track parasite-synthesized heme and host hemoglobin-heme during the intraerythrocytic stages of the parasite. The KOs did not affect parasite growth in mice when the parasites were injected intraperitoneally. All infected animals died within 10 to 12 days, when parasitemia reached around 60%. The synthesis of mitochondrial cytochromes is essential for parasite survival, so our results mean that the PbKO parasites used hemoglobin-heme to synthesize cytochromes during the intraerythrocytic stages. We demonstrated this by radiolabeling hemoglobin-heme with [4-14C]ALA in short-term mouse reticulocyte cultures.