Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection - PubMed (original) (raw)
Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection
Viswanathan Arun Nagaraj et al. PLoS Pathog. 2013.
Abstract
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-(14)C] 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.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. De novo heme-biosynthetic pathway of P. falciparum.
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 P. berghei for the first (ALAS) and last (FC) enzymes of this pathway.
Figure 2. Strategy for the generation and characterization of _Pb_ALASKO and _Pb_FCKO.
(A) Double crossover recombination strategy to generate _Pb_ALAS and _Pb_FC KOs. (B,C) Genomic DNA-PCR analysis indicating the targeted deletion of ALAS and FC sequences in the KOs. (D,E) RT-PCR analysis indicating absence of mRNAs for ALAS and FC in the KOs. (F,G) Southern analysis of DNA from _Pb_WT, _Pb_ALAS and _Pb_FC KOs. For _Pb_ALASKO confirmation, respective genomic DNA and transgenic plasmid (TP) were digested with BglII and hybridized with 3′UTR specific probe. For _Pb_FCKO, digestion was carried out with SphI and BspDI. Transgenic plasmids were included to rule out the presence of episomes. (H,I) Northern analysis indicating the absence of mRNAs for ALAS and FC in the KOs. (J) Northern analysis for PBGD in the _Pb_ALAS and _Pb_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 _Pb_WT and _Pb_KOs giving positive signal (control).
Figure 3. Growth curves for intraerythrocytic stages of P. berghei WT and KO parasites in mice.
Mice were injected intraperitoneally with 105 P. berghei infected-RBCs/reticulocytes and the parasite growth was routinely monitored as described in Materials and Methods. Multiple fields were used to quantify the parasite infected cells. The data provided represent the mean ± S.D. obtained from 6 animals.
Figure 4. Acquisition of radiolabeled heme by P.berghei and P.falciparum in short-term cultures.
_P.berghei_-infected reticulocytes were isolated from mice infected with WT and KO parasites. Infected reticulocytes were also isolated after CQ treatment. Radiolabeling of P. berghei and P. falciparum with [4-14C]ALA in short-term cultures was carried out as described in Materials and Methods. 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 _Pb_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 P.falciparum. Pb, P. berghei; Pf, P. falciparum.
Figure 5. Ookinete formation in the midgut of _P.berghei_-infected (WT and KOs) mosquitoes.
(A) Quantification of ookinetes formed in vitro using gametocyte cultures. The data represent three independent experiments; P>0.05. (B) Ookinetes formed in vitro and stained with Giemsa reagent. Scale bar: 5 µm. (C) Quantification of ookinetes formed in vivo. (D) Ookinetes formed in vivo and stained with Giemsa reagent. Scale bar: 5 µm. The in vivo data are from 30 mosquitoes from 3 different batches; P>0.05.
Figure 6. Oocyst and sporozoite formation in _P.berghei_-infected (WT and KOs) mosquitoes.
(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 _Pb_ALASKO and _Pb_FCKO with respect to WT are <0.02. P value for _Pb_ALASKO(Mq+ALA) with respect to _Pb_ALASKO is <0.01 and _Pb_FCKO(Mq+Blood) with respect to _Pb_FCKO is >0.05. The data represent 90 mosquitoes from 3 different batches. (D) Quantification of sporozoites. P values for _Pb_ALASKO, _Pb_FCKO, _Pb_ALASKO(Mq+ALA) and _Pb_FCKO(Mq+Blood) with respect to WT are <0.01. The data represent 90 mosquitoes from 3 different batches. UI, uninfected; Mq, mosquitoes; _Pb_ALASKO(Mq+ALA) and _Pb_FCKO(Mq+Blood), P. berghei KO parasites from mosquitoes supplemented with ALA and blood feeding, respectively.
Figure 7. Ability of P.berghei sporozoites (WT and KOs) to infect mice with and without ALA supplement to the animals.
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; _Pb_ALASKO(Mq+ALAMi+ALA), _Pb_ALASKO supplemented with ALA in mosquitoes and mice; _Pb_ALASKO(Mq+ALAMi−ALA), _Pb_ALASKO supplemented with ALA in mosquitoes but not in mice; _Pb_FCKO(Mq+Blood), _Pb_FCKO supplemented with blood feeding in mosquitoes.
Figure 8. Model depicting the possible routes of heme transport from hemoglobin and biosynthetic heme in the intraerythrocytic stages of malaria parasite.
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.
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