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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.

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Artemisinin and curcumin show an additive interaction in killing Plasmodium falciparum in culture. In vivo, 3 oral doses of curcumin following a single injection of ␣,␤-arteether to Plasmodium berghei-infected mice are able to prevent recrudescence due to ␣,␤-arteether monotherapy and ensure almost 100% survival of the animals.Artemisinin derivative-based combination therapy (ACT) has been advocated as the therapy of choice to handle widespread drug resistance in Plasmodium falciparum malaria, at the same time preventing recrudescence due to artemisinin monotherapy. However, most of the combinations are less than ideal because of side effects, pharmacokinetic mismatch, and cost (8,15). Studies in this laboratory have shown that curcumin isolated from the roots of Curcuma longa (turmeric) has antimalarial activity in P. falciparum culture and in Plasmodium berghei-infected mice (10). In this study, we propose a novel artemisinin-curcumin therapy to treat malaria.Artemisinin and curcumin (98% curcuminoid content) were purchased from Sigma Chemicals, Bangalore, India. ␣,␤-Arteether (EMAL, a synthetic derivative of artemisinin) developed by Central Drug Research Institute, Lucknow, India, was a kind gift from IPCA Laboratories Ltd., Mumbai, India. [ 3 H]hypoxanthine was purchased from PerkinElmer, Singapore.The P. falciparum FCK strain, a local chloroquine-resistant isolate, was cultured in human O-positive washed erythrocytes using standard protocols (14). Parasites were synchronized using 5% (wt/vol) D-sorbitol, and the cultures were pooled and the parasites released from the erythrocyte with 0.15% (wt/vol) saponin (3) for further processing. The 50% inhibitory concentrations (IC 50 s) for artemisinin and curcumin were determined in P. falciparum cultures by measuring [3 H]hypoxanthine uptake into the parasite as a measure of viability (2). Fractional inhibitory concentrations (FIC) were calculated using the formulawhere A C and B C are the concentrations of A and B in the combination associated with a particular level of effect, e.g., IC 60 , and A E and B E are the concentrations of A and B when used singly to produce the same level of effect, on the basis that a simple additive interaction should lead to a value of 1 (7). FIC were derived from graphs drawn using GraphPad Prism 4.Means Ϯ standard deviations and Student's t test were analyzed using Microsoft Excel. For in vivo studies, Swiss mice (25 to 28 g) were injected intraperitoneally with P. berghei-infected mouse blood (60 to 70% parasitemia) on day 0, such that the animals developed high parasitemia and died in about 5 to 8 days. The artemisinin derivative ␣,␤-arteether was injected intramuscularly at different doses on day 1. This was followed by oral feeding of curcumin in dimethyl sulfoxide on days 1, 2, and 3 (100 mg/kg of body weight). The mice were observed for external symptoms and mortality when the drugs were given alone or in combination. Blood from the tail vein was analyzed on different days for parasitemia using Giemsa to stain the slides.De...

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