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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

Disruption of the P. falciparum CPO gene (PF3D7_1142400) by single-crossover homologous recombination.(A) Schematic depiction of the CPO gene locus before and after incorporation of the donor plasmid via single-crossover recombination. Red arrows indicate the primers used to selectively PCR-amplify either the intact WT or disrupted ∆CPO locus. XhoI and KpnI were used to digest the donor plasmid and gDNA of WT and ∆CPO parasites to give the expected fragment sizes indicated in parentheses for hybridization to a CPO bp 610–1080 oligonucleotide probe. (B) PCR analysis of gDNA from WT and ∆CPO clones using the primers indicated in (A) to selectively amplify the 1.6 kb WT gene or the 1.1 kb truncated gene. (C) Southern blot analysis of gDNA from 3D7 WT and ∆CPO clonal parasites after digestion with XhoI and KpnI, hybridization with a CPO bp 610–1080 oligonucleotide probe, and detection using the Amersham AlkPhos Labeling and CDP-Star chemiluminescent reagents. (D) Homology model of P. falciparum CPO (human CPO template structure 2AEX) to indicate the portion of the protein retained (blue) or lost (green) by single-crossover truncation. The deleted sequence comprises half of the active site binding pocket, identified by the citrate molecule shown in red.DOI:http://dx.doi.org/10.7554/eLife.09143.016
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fig2s4: Disruption of the P. falciparum CPO gene (PF3D7_1142400) by single-crossover homologous recombination.(A) Schematic depiction of the CPO gene locus before and after incorporation of the donor plasmid via single-crossover recombination. Red arrows indicate the primers used to selectively PCR-amplify either the intact WT or disrupted ∆CPO locus. XhoI and KpnI were used to digest the donor plasmid and gDNA of WT and ∆CPO parasites to give the expected fragment sizes indicated in parentheses for hybridization to a CPO bp 610–1080 oligonucleotide probe. (B) PCR analysis of gDNA from WT and ∆CPO clones using the primers indicated in (A) to selectively amplify the 1.6 kb WT gene or the 1.1 kb truncated gene. (C) Southern blot analysis of gDNA from 3D7 WT and ∆CPO clonal parasites after digestion with XhoI and KpnI, hybridization with a CPO bp 610–1080 oligonucleotide probe, and detection using the Amersham AlkPhos Labeling and CDP-Star chemiluminescent reagents. (D) Homology model of P. falciparum CPO (human CPO template structure 2AEX) to indicate the portion of the protein retained (blue) or lost (green) by single-crossover truncation. The deleted sequence comprises half of the active site binding pocket, identified by the citrate molecule shown in red.DOI:http://dx.doi.org/10.7554/eLife.09143.016


Deconvoluting heme biosynthesis to target blood-stage malaria parasites.

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

Disruption of the P. falciparum CPO gene (PF3D7_1142400) by single-crossover homologous recombination.(A) Schematic depiction of the CPO gene locus before and after incorporation of the donor plasmid via single-crossover recombination. Red arrows indicate the primers used to selectively PCR-amplify either the intact WT or disrupted ∆CPO locus. XhoI and KpnI were used to digest the donor plasmid and gDNA of WT and ∆CPO parasites to give the expected fragment sizes indicated in parentheses for hybridization to a CPO bp 610–1080 oligonucleotide probe. (B) PCR analysis of gDNA from WT and ∆CPO clones using the primers indicated in (A) to selectively amplify the 1.6 kb WT gene or the 1.1 kb truncated gene. (C) Southern blot analysis of gDNA from 3D7 WT and ∆CPO clonal parasites after digestion with XhoI and KpnI, hybridization with a CPO bp 610–1080 oligonucleotide probe, and detection using the Amersham AlkPhos Labeling and CDP-Star chemiluminescent reagents. (D) Homology model of P. falciparum CPO (human CPO template structure 2AEX) to indicate the portion of the protein retained (blue) or lost (green) by single-crossover truncation. The deleted sequence comprises half of the active site binding pocket, identified by the citrate molecule shown in red.DOI:http://dx.doi.org/10.7554/eLife.09143.016
© Copyright Policy
Related In: Results  -  Collection

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

fig2s4: Disruption of the P. falciparum CPO gene (PF3D7_1142400) by single-crossover homologous recombination.(A) Schematic depiction of the CPO gene locus before and after incorporation of the donor plasmid via single-crossover recombination. Red arrows indicate the primers used to selectively PCR-amplify either the intact WT or disrupted ∆CPO locus. XhoI and KpnI were used to digest the donor plasmid and gDNA of WT and ∆CPO parasites to give the expected fragment sizes indicated in parentheses for hybridization to a CPO bp 610–1080 oligonucleotide probe. (B) PCR analysis of gDNA from WT and ∆CPO clones using the primers indicated in (A) to selectively amplify the 1.6 kb WT gene or the 1.1 kb truncated gene. (C) Southern blot analysis of gDNA from 3D7 WT and ∆CPO clonal parasites after digestion with XhoI and KpnI, hybridization with a CPO bp 610–1080 oligonucleotide probe, and detection using the Amersham AlkPhos Labeling and CDP-Star chemiluminescent reagents. (D) Homology model of P. falciparum CPO (human CPO template structure 2AEX) to indicate the portion of the protein retained (blue) or lost (green) by single-crossover truncation. The deleted sequence comprises half of the active site binding pocket, identified by the citrate molecule shown in red.DOI:http://dx.doi.org/10.7554/eLife.09143.016
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