Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

<div><p>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 <i>de novo</i>, 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 <i>Plasmodium berghei</i> (<i>Pb</i>). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out <i>in vitro</i> radiolabeling of heme in <i>Pb</i>-infected mouse reticulocytes and <i>Plasmodium falciparum</i>-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 <i>de novo</i> pathway is absolutely essential for parasite development in the mosquito and liver stages. <i>Pb</i>KO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in <i>Pb</i>ALASKO parasites recovered when mosquitoes received an ALA supplement. <i>Pb</i>ALASKO 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.</p></div

Oocyst and sporozoite formation in <i>P.berghei</i>-infected (WT and KOs) mosquitoes.

<p>(A) Mercurochrome staining of oocysts in the midgut preparations. Arrows indicate oocysts and the magnified images of oocysts are provided in insets. Scale bar: 100 µm. (B) Sporozoites in the salivary glands. Magnified images of sporozoites are provided in insets. Scale bar: 50 µm. (C) Quantification of oocysts. P values for <i>Pb</i>ALASKO and <i>Pb</i>FCKO with respect to WT are <0.02. P value for <i>Pb</i>ALASKO(Mq^+ALA) with respect to <i>Pb</i>ALASKO is <0.01 and <i>Pb</i>FCKO(Mq^+Blood) with respect to <i>Pb</i>FCKO is >0.05. The data represent 90 mosquitoes from 3 different batches. (D) Quantification of sporozoites. P values for <i>Pb</i>ALASKO, <i>Pb</i>FCKO, <i>Pb</i>ALASKO(Mq^+ALA) and <i>Pb</i>FCKO(Mq^+Blood) with respect to WT are <0.01. The data represent 90 mosquitoes from 3 different batches. UI, uninfected; Mq, mosquitoes; <i>Pb</i>ALASKO(Mq^+ALA) and <i>Pb</i>FCKO(Mq^+Blood), <i>P. berghei</i> KO parasites from mosquitoes supplemented with ALA and blood feeding, respectively.</p

Ability of <i>P.berghei</i> sporozoites (WT and KOs) to infect mice with and without ALA supplement to the animals.

<p>Mosquitoes were allowed to feed on mice (30 mosquitoes/mouse) and parasitemia in blood and mortality of the animals were assessed. The data represent 9 mice each from three different batches. Mq, mosquito; Mi, mice; <i>Pb</i>ALASKO(Mq^+ALAMi^+ALA), <i>Pb</i>ALASKO supplemented with ALA in mosquitoes and mice; <i>Pb</i>ALASKO(Mq^+ALAMi^−ALA), <i>Pb</i>ALASKO supplemented with ALA in mosquitoes but not in mice; <i>Pb</i>FCKO(Mq^+Blood), <i>Pb</i>FCKO supplemented with blood feeding in mosquitoes.</p

Growth curves for intraerythrocytic stages of <i>P. berghei</i> WT and KO parasites in mice.

<p>Mice were injected intraperitoneally with 10⁵<i>P. berghei</i> infected-RBCs/reticulocytes and the parasite growth was routinely monitored as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003522#s4" target="_blank">Materials and Methods</a>. Multiple fields were used to quantify the parasite infected cells. The data provided represent the mean ± S.D. obtained from 6 animals.</p

Acquisition of radiolabeled heme by <i>P.berghei</i> and <i>P.falciparum</i> in short-term cultures.

<p><i>P.berghei</i>-infected reticulocytes were isolated from mice infected with WT and KO parasites. Infected reticulocytes were also isolated after CQ treatment. Radiolabeling of <i>P. berghei</i> and <i>P. falciparum</i> with [4-¹⁴C]ALA in short-term cultures was carried out as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003522#s4" target="_blank">Materials and Methods</a>. Radiolabeling of total parasite heme, hemozoin and mitochondrial cytochrome complex were assessed with (+) and without (−) succinyl acetone (SA) treatment. (A) Radiolabeling of total parasite heme. (B) Radiolabeling of hemozoin-heme. (C–E) Radiolabeling of parasite mitochondrial cytochrome complex. (F,G) Radiolabeling of hemozoin-heme and mitochondrial cytochrome complex after chloroquine (CQ) treatment. Equal numbers of infected reticulocytes were used to perform the radiolabeling of <i>Pb</i>FCKO parasites and the data obtained for CQ treatment were compared with untreated control. (H–J) Radiolabeling of total heme, hemozoin-heme and mitochondrial cytochrome complex in <i>P.falciparum</i>. <i>Pb</i>, <i>P. berghei</i>; <i>Pf</i>, <i>P. falciparum.</i></p

Strategy for the generation and characterization of <i>Pb</i>ALASKO and <i>Pb</i>FCKO.

<p>(A) Double crossover recombination strategy to generate <i>Pb</i>ALAS and <i>Pb</i>FC KOs. (B,C) Genomic DNA-PCR analysis indicating the targeted deletion of <i>ALAS</i> and <i>FC</i> sequences in the KOs. (D,E) RT-PCR analysis indicating absence of mRNAs for <i>ALAS</i> and <i>FC</i> in the KOs. (F,G) Southern analysis of DNA from <i>Pb</i>WT, <i>Pb</i>ALAS and <i>Pb</i>FC KOs. For <i>Pb</i>ALASKO confirmation, respective genomic DNA and transgenic plasmid (TP) were digested with <i>BglII</i> and hybridized with 3′UTR specific probe. For <i>Pb</i>FCKO, digestion was carried out with <i>SphI</i> and <i>BspDI</i>. Transgenic plasmids were included to rule out the presence of episomes. (H,I) Northern analysis indicating the absence of mRNAs for <i>ALAS</i> and <i>FC</i> in the KOs. (J) Northern analysis for <i>PBGD</i> in the <i>Pb</i>ALAS and <i>Pb</i>FC KOs giving positive signals (control). (K,L) Western analysis indicating the absence of ALAS and FC proteins in the KOs. (M) Western analysis for hsp60 in the <i>Pb</i>WT and <i>Pb</i>KOs giving positive signal (control).</p

<i>De novo</i> heme-biosynthetic pathway of <i>P. falciparum</i>.

<p>The enzymes are localized in three different cellular compartments - mitochondrion, apicoplast and cytosol. The transporters involved in the shuttling of intermediates are yet to be identified. Red bars represent the knockouts generated in <i>P. berghei</i> for the first (ALAS) and last (FC) enzymes of this pathway.</p

Model depicting the possible routes of heme transport from hemoglobin and biosynthetic heme in the intraerythrocytic stages of malaria parasite.

<p>H, heme; Hb, hemoglobin; FV, food vacuole; M, mitochondrion; Ap, apicoplast; Gly, glycine; SCoA, succinyl CoA; PBG, porphobilinogen; UROG, uroporphyrinogen III; COPROG, coproporphyrinogen III; PROTOG, protoporphyrinogen IX; PROTO, protoporphyrin IX.</p

Ookinete formation in the midgut of <i>P.berghei</i>-infected (WT and KOs) mosquitoes.

<p>(A) Quantification of ookinetes formed <i>in vitro</i> using gametocyte cultures. The data represent three independent experiments; P>0.05. (B) Ookinetes formed <i>in vitro</i> and stained with Giemsa reagent. Scale bar: 5 µm. (C) Quantification of ookinetes formed <i>in vivo</i>. (D) Ookinetes formed <i>in vivo</i> and stained with Giemsa reagent. Scale bar: 5 µm. The <i>in vivo</i> data are from 30 mosquitoes from 3 different batches; P>0.05.</p