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 PbALASKO and PbFCKO.
(A) Double crossover recombination strategy to generate PbALAS and PbFC 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 PbWT, PbALAS and PbFC KOs. For PbALASKO confirmation, respective genomic DNA and transgenic plasmid (TP) were digested with BglII and hybridized with 3′UTR specific probe. For PbFCKO, 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 PbALAS and PbFC 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 PbWT and PbKOs 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 PbFCKO 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 PbALASKO and PbFCKO with respect to WT are <0.02. P value for PbALASKO(Mq+ALA) with respect to PbALASKO is <0.01 and PbFCKO(Mq+Blood) with respect to PbFCKO is >0.05. The data represent 90 mosquitoes from 3 different batches. (D) Quantification of sporozoites. P values for PbALASKO, PbFCKO, PbALASKO(Mq+ALA) and PbFCKO(Mq+Blood) with respect to WT are <0.01. The data represent 90 mosquitoes from 3 different batches. UI, uninfected; Mq, mosquitoes; PbALASKO(Mq+ALA) and PbFCKO(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; PbALASKO(Mq+ALAMi+ALA), PbALASKO supplemented with ALA in mosquitoes and mice; PbALASKO(Mq+ALAMi−ALA), PbALASKO supplemented with ALA in mosquitoes but not in mice; PbFCKO(Mq+Blood), PbFCKO 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.