Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.


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Figure 1 -Figure supplement 2. Phylogenic tree of mammalian, plant, algal, and hematozoan HOs.Nodes are annotated with bootstrap values for each branch.Ortholog groups independently predicted by OrthoMCL 3 are annotated for each colored section.Only select Plasmodium HOs are included for clarity, but all other known proteins of OG6_156412 are displayed.PfHO is marked in red.

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Figure 1 -Figure supplement 3. X-ray crystallographic data collection and structure refinement statistics for PfHO.Statistical values given in parentheses refer to the highest resolution bin.

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Figure 1 -Figure supplement 4. Sequence and structural alignment of PfHO and plant HOs.A) Sequence alignment of PfHO with Arabidopsis thaliana (O48782) and Glycine max (C6THA4) HO1s (Uniprot ID).Heme-coordinating histidine and distal helix glycines in plant HOs are highlighted in red.B) Sequence identity of PfHO with proteins identified in our sequence homology analysis showing N-terminal targeting sequence and HO-domain separately.C) Structural alignment of the 2.8 Å-resolution PfHO crystal structure (blue, PDB: 8ZLD) with X-ray structures of A. thaliana HO1 (green, PDB: 7EQH) and G. max HO1 (yellow, PDB: 7CKA).

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Figure 3 -Figure supplement 1. Schemes for modification of the PfHO genomic locus to integrate Cterminal GFP-DHFRDD or HA2-glmS tags.A) Schematic of single-crossover strategy for tagging PfHO gene locus with C-terminal GFP-DHFRDD.1kb sequence at 3' of PfHO coding region (green line) was used as a probe to test for integration by Southern blot, and enzyme digestion sites with expected sizes are indicated.B) Southern blot of digested parasite DNA harvested from wildtype, polyclonal PfHO-GFP-DHFRDD, and select clonal PfHO-GFP-DHFRDD cultures.C) Schematic of single-crossover integration strategy for tagging PfHO gene locus with C-terminal HA2-glmS.Primer sites used to probe integration by genome PCR are indicated.D) Genome PCR of parasite DNA harvested from wildtype parental and PfHO-HA2-glmS cultures.

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Figure 3 -Figure supplement 2. Widefield immunofluorescence microscopy of fixed 3D7 parasites endogenously expressing PfHO-GFP-DHFRDD and stained with anti-GFP and anti-apicoplast acyl carrier protein (ACP) antibodies, and DAPI.Pearson correlation coefficient (rp) of red and green channels is shown in merged images in yellow.

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Figure 3 -Figure supplement 4. Scheme for modification of the PfHO genomic locus to integrate the aptamer/TetR-DOZI system.A) Schematic of double-crossover integration strategy for replacing PfHO gene with cDNA encoding PfHO (Toxoplasma gondii codon bias) and RNA aptamers at the 5' and 3' ends of the gene.580 bp sequence in 3' UTR of PfHO (green line) was used as a probe to test for integration by Southern blot, and enzyme digestion sites with expected sizes marked on locus and plasmid.Primers used to probe integration by genome PCR are marked on wildtype and tagged loci.B) Genome PCR of parasite DNA harvested from wildtype parental, polyclonal PfHO-aptamer/TetR-DOZI, and select PfHOaptamer/TetR-DOZI clonal cultures.C) Southern blot of digested parasite DNA harvested from wildtype, polyclonal PfHO-aptamer/TetR-DOZI, and select clonal PfHO-aptamer/TetR-DOZI cultures.D) Complete PfHO cDNA sequence used to replace the endogenous PfHO gene (identical encoded protein sequence).

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Figure 3 -Figure supplement 5. Validation of custom PfHO antibody specificity.Western blot specificity tests of custom PfHO rabbit antibody in parasite lysates.A) Lysates from asynchronous or synchronous parasite cultures stained with 1:1,000 dilution of rabbit serum prior to inoculation with PfHO protein antigen.B) Lysates from E. coli expressing PfHO 84-305 and from equal numbers of synchronous 3D7 parasites stained with 1:1,000 dilution of crude serum from the final bleed of a rabbit inoculated with PfHO protein antigen.Both blots were stained with an anti-rabbit HRP secondary antibody and visualized by chemiluminescence.

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Figure 3 -Source data 1.Uncropped western blots of parasites endogenously expressing PfHO-GFP-DHFRDD that were untreated or Dox/IPP-treated for 5 days.Membrane was stained with goat anti-GFP and custom rabbit anti-PfHO primary antibodies then anti-goat-IRDye800 and anti-rabbit IRDye680 secondary antibodies.Stained membrane was visualized on a Licor CLx imager.

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Figure 3 -Source data 2. Uncropped western blots of Dd2 parasites tagged with PfHO-Aptamer/TetR-DOZI grown in +aTC or -aTC/IPP conditions for 7 days, stained with rabbit anti-Ef1α and custom rabbit anti-PfHO primary antibodies and anti-rabbit-IRDye800 secondary antibody.Stained membrane was visualized on a Licor CLx imager.

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Figure 3 -Source data 7. Uncropped western blot of lysates from asynchronous or synchronous 3D7 parasites stained with 1:1,000 dilution of rabbit serum prior to inoculation with PfHO protein antigen.

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Figure 3 -Source data 8. Uncropped western blot of lysates from E. coli expressing PfHO 84-305 and from equal numbers of synchronous 3D7 parasites stained with 1:1,000 dilution of crude serum from the final bleed of a rabbit inoculated with PfHO protein antigen.

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Figure 4 -Figure supplement 1. Peptide coverage of PfHO sequence detected by mass spectrometry.A) Red residues correspond to sequence detected by tryptic digest and tandem mass spectrometry after PfHO isolation from parasites.B) List of individual peptides detected within PfHO N-term and HO-domains.

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Figure 4 -Source data 1.Uncropped western blots of parasites endogenously expressing PfHO-HA2 and lysates from E. coli expressing PfHO HO-like domain (PfHO 84-305 -HA2) stained with rat anti-HA and custom rabbit anti-PfHO primary antibodies and anti-rabbit-IRDye680 and anti-rat-IRDye800 secondary antibodies then visualized with LICOR CLx imager.Indicated molecular masses were estimated using LICOR Image Studio software based on migration of the protein standards.

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Figure 5 -Figure supplement 1. Spectral counts for proteins that co-purified with PfHO and not with either mitochondrial control in both IP/MS experiments.PfHO is marked in red, and proteins predicted to be apicoplast localized based on prior IP/MS studies are marked in green.The full list of proteins is provided in Figure 5 -source data 1.

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Figure 5 -Figure supplement 2. List of apicoplast-localized proteins co-purified with PfHO in two IP/MS experiments ordered by average of spectral counts for each experiment.

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Figure 5 -Figure supplement 3. Functional pathway predictions for the 65 apicoplast-localized proteins that co-purified with PfHO in two IP/MS experiments.

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Figure 5 -Figure supplement 4. Quantification of parasite DNA fragment size by Agilent Bioanalyzer DNA analysis after pulse-sonication shearing of parasite lysates.Our DNA shearing method produced two populations of DNA fragment sizes, 1100-1600 bp and 150-300 bp.

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Figure 5 -Figure supplement 6.Additional ChIP experiments in parasites with GFP-tagged PfHO constructs.(A) Relative abundance of apicoplast-encoded genes in DNA co-purified with the indicated GFP-tagged PfHO constructs by ChIP normalized to PfHO-GFP.B) Distribution and orientation of target genes on 35 kb apicoplast genome.Orange lines mark the location of the ~100bp qPCR amplicon for each gene.C) Relative abundance of apicoplast-encoded genes in DNA co-purified with PfHO-GFP by ChIP normalized to input.D) Steady-state PCR amplification of nuclear-encoded (aACP: Pf3D7_0208500, GGPPS: Pf3D7_1128400) and apicoplast-encoded (SufB: Pf3D7_API04700, ClpM: Pf3D7_API03600) genes from DNA co-purified with full-length PfHO-GFP by ChIP ±PFA crosslinking.

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Figure 5 -Figure supplement 7. RT-qPCR data for additional apicoplast genes.Transcript levels of apicoplast, nuclear, and mitochondrial genes were assessed in PfHO-Aptamer/TetR-DOZI parasites grown in ±aTC with 200 µM IPP for 3 days (84 hours post-synchronization).Each transcript is normalized to average of two nuclear transcripts -I5P (Pf3D7_0802500) and ADSL (Pf3D7_0206700).As an additional control, mitochondrial-encoded CytB (Pf3D7_MIT02300) transcript abundance is also measured relative to nuclear controls.Error bars represent average ±SD of independent biological triplicates.

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Figure 5 -Figure supplement 8. Representative time-course showing ClpM: nuclear transcript levels at indicated times during first and second cycle of parasite growth ±aTC in the presence of IPP.Large error bars between independent replicate experiments are due to large variance in total ClpM expression.Within each experiment, ClpM transcripts are >10-fold less abundant in -aTC parasites at cycle 2 timepoints 72, 84, and 90 hours.Error bars represent average ±SD of independent biological triplicates.

Figure 5 -Figure 5 -
Figure 5 -Source data 1.Table of proteins identified in PfHO IP/MS experiments Excel file

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Figure 5 -Source data 3. Uncropped PCR gel of nuclear-encoded and apicoplast-encoded genes from DNA co-purified with GFP-tagged PfHO constructs ±crosslinking. SeMet a Calculated for equivalent reflections (within I + or I -).