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Deconvoluting heme biosynthesis to target blood-stage malaria parasites.

Sigala PA, Crowley JR, Henderson JP, Goldberg DE - Elife (2015)

Bottom Line: Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX).We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen.This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St Louis, United States.

ABSTRACT
Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.

No MeSH data available.


Related in: MedlinePlus

Analysis of heme biosynthesis activity in parasite-infected erythrocytes after saponin permeabilization and culture in 13C-labelled ALA.(A) Parasite-infected erythrocytes were permeabilized in 0.02% saponin, washed to remove the erythrocyte cytoplasm, and placed back into culture medium containing 200 µM 5-[13C4]-ALA for 12 hr prior to DMSO extraction and analysis by LC-MS/MS. Bright field and fluorescence image of live (B) asexual trophozoite and (C) stage IV sexual gametocyte treated with 0.02% saponin and stained with 20 nM MitoTracker Red. (D) LC-MS/MS quantification of 13C-labelled heme, PPIX, and CPP in DMSO extracts of intact WT 3D7 asexual parasites, saponin-released asexual parasites, and saponin-released gametocytes cultured overnight in 200 µM 5-[13C4]-ALA.DOI:http://dx.doi.org/10.7554/eLife.09143.024
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fig5: Analysis of heme biosynthesis activity in parasite-infected erythrocytes after saponin permeabilization and culture in 13C-labelled ALA.(A) Parasite-infected erythrocytes were permeabilized in 0.02% saponin, washed to remove the erythrocyte cytoplasm, and placed back into culture medium containing 200 µM 5-[13C4]-ALA for 12 hr prior to DMSO extraction and analysis by LC-MS/MS. Bright field and fluorescence image of live (B) asexual trophozoite and (C) stage IV sexual gametocyte treated with 0.02% saponin and stained with 20 nM MitoTracker Red. (D) LC-MS/MS quantification of 13C-labelled heme, PPIX, and CPP in DMSO extracts of intact WT 3D7 asexual parasites, saponin-released asexual parasites, and saponin-released gametocytes cultured overnight in 200 µM 5-[13C4]-ALA.DOI:http://dx.doi.org/10.7554/eLife.09143.024

Mentions: Our observation that disruption of the parasite-encoded PBGD and CPO does not affect biosynthetic production of heme and PPIX from ALA suggests that host enzyme activity in the erythrocyte cytoplasm provides the dominant contribution to PPIX biosynthesis in ALA-treated intraerythrocytic parasites. To dissect these parallel pathways and test whether the parasite pathway alone, in the absence of host enzymes, can support heme biosynthesis from ALA, we fractionated parasite-infected erythrocytes using saponin to selectively permeabilize host cell membranes while leaving parasite membranes intact (Figure 5A). Under these conditions, soluble erythrocyte proteins can be washed away to leave the intact live parasite natively embedded within the resulting erythrocyte ghost (Figure 5B,C). Parasites treated in this fashion remain metabolically active for 5–6 hr or longer, retain a membrane potential, accumulate fluorescent dyes such as MitoTracker Red (Figure 5B,C), and carry out DNA synthesis (Izumo et al., 1987; Cobbold et al., 2011). After saponin treatment and washout, we placed parasites back into culture medium containing 13C-ALA and incubated them overnight before extracting them for analysis by LC-MS/MS. We failed to detect biosynthesis of heme, PPIX, or CPP in fractionated asexual and sexual blood-stage parasites (Figure 5D), suggesting that the apicoplast-localized portion of the parasite heme biosynthesis pathway is largely or completely inactive during blood-stage infection.10.7554/eLife.09143.024Figure 5.Analysis of heme biosynthesis activity in parasite-infected erythrocytes after saponin permeabilization and culture in 13C-labelled ALA.


Deconvoluting heme biosynthesis to target blood-stage malaria parasites.

Sigala PA, Crowley JR, Henderson JP, Goldberg DE - Elife (2015)

Analysis of heme biosynthesis activity in parasite-infected erythrocytes after saponin permeabilization and culture in 13C-labelled ALA.(A) Parasite-infected erythrocytes were permeabilized in 0.02% saponin, washed to remove the erythrocyte cytoplasm, and placed back into culture medium containing 200 µM 5-[13C4]-ALA for 12 hr prior to DMSO extraction and analysis by LC-MS/MS. Bright field and fluorescence image of live (B) asexual trophozoite and (C) stage IV sexual gametocyte treated with 0.02% saponin and stained with 20 nM MitoTracker Red. (D) LC-MS/MS quantification of 13C-labelled heme, PPIX, and CPP in DMSO extracts of intact WT 3D7 asexual parasites, saponin-released asexual parasites, and saponin-released gametocytes cultured overnight in 200 µM 5-[13C4]-ALA.DOI:http://dx.doi.org/10.7554/eLife.09143.024
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4532139&req=5

fig5: Analysis of heme biosynthesis activity in parasite-infected erythrocytes after saponin permeabilization and culture in 13C-labelled ALA.(A) Parasite-infected erythrocytes were permeabilized in 0.02% saponin, washed to remove the erythrocyte cytoplasm, and placed back into culture medium containing 200 µM 5-[13C4]-ALA for 12 hr prior to DMSO extraction and analysis by LC-MS/MS. Bright field and fluorescence image of live (B) asexual trophozoite and (C) stage IV sexual gametocyte treated with 0.02% saponin and stained with 20 nM MitoTracker Red. (D) LC-MS/MS quantification of 13C-labelled heme, PPIX, and CPP in DMSO extracts of intact WT 3D7 asexual parasites, saponin-released asexual parasites, and saponin-released gametocytes cultured overnight in 200 µM 5-[13C4]-ALA.DOI:http://dx.doi.org/10.7554/eLife.09143.024
Mentions: Our observation that disruption of the parasite-encoded PBGD and CPO does not affect biosynthetic production of heme and PPIX from ALA suggests that host enzyme activity in the erythrocyte cytoplasm provides the dominant contribution to PPIX biosynthesis in ALA-treated intraerythrocytic parasites. To dissect these parallel pathways and test whether the parasite pathway alone, in the absence of host enzymes, can support heme biosynthesis from ALA, we fractionated parasite-infected erythrocytes using saponin to selectively permeabilize host cell membranes while leaving parasite membranes intact (Figure 5A). Under these conditions, soluble erythrocyte proteins can be washed away to leave the intact live parasite natively embedded within the resulting erythrocyte ghost (Figure 5B,C). Parasites treated in this fashion remain metabolically active for 5–6 hr or longer, retain a membrane potential, accumulate fluorescent dyes such as MitoTracker Red (Figure 5B,C), and carry out DNA synthesis (Izumo et al., 1987; Cobbold et al., 2011). After saponin treatment and washout, we placed parasites back into culture medium containing 13C-ALA and incubated them overnight before extracting them for analysis by LC-MS/MS. We failed to detect biosynthesis of heme, PPIX, or CPP in fractionated asexual and sexual blood-stage parasites (Figure 5D), suggesting that the apicoplast-localized portion of the parasite heme biosynthesis pathway is largely or completely inactive during blood-stage infection.10.7554/eLife.09143.024Figure 5.Analysis of heme biosynthesis activity in parasite-infected erythrocytes after saponin permeabilization and culture in 13C-labelled ALA.

Bottom Line: Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX).We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen.This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St Louis, United States.

ABSTRACT
Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.

No MeSH data available.


Related in: MedlinePlus