Acyl carrier protein is essential for apicoplast biogenesis in malaria parasites independent of fatty acid synthesis
- Department of Biochemistry, University of Utah School of Medicine, United States
- Department of Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, United States
- Department of Biological Chemistry, University of California, Los Angeles, United States
Figures
Figure 1
Schematic depiction of apicoplast acyl carrier protein (ACP) and the type II fatty acid biosynthesis (FASII) pathway in P. falciparum.
CoA = coenzyme A, ACPS = holo-ACP synthase, FabD = malonyl-CoA:ACP S-malonyltransferase, 4-PP = 4-phosphopantetheine. The ACP structural models are based on the X-ray structures of P. falciparum holo-aACP (PDB 3GZM) (Gallagher and Prigge, 2010) and Escherichia coli acyl-ACP (PDB 5USR) (Cory et al., 2017).
Figure 2 with 5 supplements
Knockout or knockdown of acyl carrier protein (ACP) expression blocks parasite growth and apicoplast biogenesis.
(A) Synchronous growth assay of ∆ACP PfMev parasites cultured ±50 µM mevalonate (Mev). Parasitemia values are the average ± SD from biological triplicate samples. (B) Genomic PCR analysis and live parasite imaging show selective loss of the apicoplast genome and disrupted apicoplast morphology based on fluorescence of the ACPL-GFP marker protein in PfMev parasites. Nuc.=nuclear gene (LDH, Pf3D7_1324900), Api.=apicoplast gene (SufB, Pf3D7_API04700), Mito.=mitochondrial gene (CoxI, Pf3D7_MIT02100). (C) Synchronous growth assay of apicoplast ACP-aptamer/TetR-DOZI Dd2 parasites cultured ±1 µM aTc and ±200 µM isopentenyl pyrophosphate (IPP). Parasitemia values are the average ± SD from biological triplicates. Inset: western blot analysis of parasites harvested after 1, 3, or 5 days at the indicated growth conditions and probed with anti-EF1α (cytosolic loading control) or anti-HA (ACP) antibodies. (D) Quantitative PCR analysis of the apicoplast:nuclear genome ratio for parasites in panel C cultured 84 hr in the indicated conditions, based on amplification of apicoplast (SufB, ClpM: Pf3D7_API03600, TufA: Pf3D7_API02900) relative to nuclear (STL: Pf3D7_0717700, I5P: Pf3D7_0802500, ADSL: Pf3D7_0206700) genes. Indicated quantitative PCR (qPCR) ratios were normalized to +aTc and are the average ± SD of biological triplicates. Significance was analyzed by unpaired Student’s t-test to determine the indicated p-values. (E) Immunofluorescence microscopy of parasites in panel C cultured for 5 days (120 hr) in the indicated conditions and stained with anti-apicoplast ACP or DAPI (nucleus). Below: population analysis of apicoplast morphology scored for disrupted (dispersed), punctate, or elongated GFP signal in 50 total parasites from biological triplicates. Scale bars ~1 µm.
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Figure 2—source data 1
PDF file containing uncropped gel images for PCR analysis in Figure 2B and western blot analysis in Figure 2C.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for PCR gel analysis in Figure 2B and western blot analysis in Figure 2C.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig2-data2-v1.zip
Figure 2—figure supplement 1
Integration PCRs for deletion or modification of the apicoplast acyl carrier protein (ACP) gene.
(A) Genotyping PCR for successful deletion of the apicoplast ACP gene (Pf3D7_0208500) in polyclonal PfMev parasites showing selective amplification of sequence at the 5’ and 3’ ends of the drug resistance marker (∆5’ and ∆3’) in knockout parasites but not in parental PfMev parasites and exclusive amplification of sequence at the 5’ and 3’ ends of the ACP CDS in wild-type (WT) but not ∆ACP parasites. Genotype PCR analysis confirming on-target integration of the 3’ aptamer/TetR-DOZI cassette at the apicoplast ACP locus in polyclonal PfMev (B) or clonal Dd2 parasites (C) and the absence of the unmodified WT locus, with parental parasites serving as negative control for integration.
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Figure 2—figure supplement 1—source data 1
PDF containing uncropped gel images for integration PCR analyses shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original files for PCR gel analyses in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
Additional microscopy images of ∆ACP PfMev NF54 parasites.
Phase = phase contrast images, api-SFG=ACPL-superfolder GFP, DAPI = nuclear DNA stain. Scale bars ~1 µm.
Figure 2—figure supplement 3
Synchronous growth assays (A), fluorescence microscopy (B), and genomic quantitative PCR (qPCR) (C) of acyl carrier protein (ACP) knockdown in PfMev NF54 parasites.
For the growth assay in panel A, cultures were split on day 6. Samples for genomic qPCR were cultured as indicated for ≥8 days. Gene copy numbers were based on amplification of apicoplast (SufB: Pf3D7_API04700, ClpM: Pf3D7_API03600, TufA: Pf3D7_API02900) or nuclear (ADSL: Pf3D7_0206700) relative to nuclear (STL: Pf3D7_0717700, I5P: Pf3D7_0802500) genes. Indicated qPCR ratios were normalized to +aTc and are the average ± SD of biological triplicates. Scale bars ~1 µm.
Figure 2—figure supplement 4
Additional immunofluorescence assay (IFA) images for apicoplast morphology upon acyl carrier protein (ACP) knockdown in Dd2 parasites in +aTc (A) or –aTc/+isopentenyl pyrophosphate (IPP) (B) conditions.
BF = bright field. Scale bars ~1 µm.
Figure 2—figure supplement 5
Validation of custom anti-apicoplast acyl carrier protein (aACP) antibody.
Indicated amount of purified aACP antigen was loaded and fractionated on SDS-PAGE gel, transferred to membrane, blocked, and probed with 1:1000 dilution of rabbit pre-bleed serum or final serum, washed, and probed with donkey anti-rabbit IRDye680 secondary antibody.
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Figure 2—figure supplement 5—source data 1
PDF containing the uncropped gel image and labels for Figure 2—figure supplement 5.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig2-figsupp5-data1-v1.zip
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Figure 2—figure supplement 5—source data 2
Original file for western blot analysis in Figure 2—figure supplement 5.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig2-figsupp5-data2-v1.zip
Figure 3 with 3 supplements
Holo-acyl carrier protein (ACP) synthase (ACPS) but not FabD is essential for blood-stage parasites and apicoplast biogenesis.
(A) Synchronous growth assay of ∆ACPS PfMev parasites cultured ±50 µM mevalonate. (B) Genomic PCR analysis and (C) live parasite imaging show selective loss of the apicoplast genome and disrupted apicoplast morphology based on fluorescence of the ACPL-GFP marker protein in PfMev parasites. (D) Synchronous growth assay of ∆FabD PfMev parasites cultured ±50 µM mevalonate. (E) Genomic PCR analysis and (F) live parasite imaging indicate retention of the apicoplast genome and normal apicoplast morphology based on ACPL-GFP fluorescence in ∆FabD parasites. Parasitemia values are the average ± SD from biological triplicates. Nuc.=nuclear gene (LDH), Api.=apicoplast gene (SufB), Mito.=mitochondrial gene (CoxI), DIC = differential interference contrast. Scale bars ~1 µm.
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Figure 3—source data 1
PDF containing uncropped gel images for apicoplast status PCRs for Figure 3B and E.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for PCR gel analyses in Figure 3B and E.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig3-data2-v1.zip
Figure 3—figure supplement 1
Integration PCRs for deletion of acyl carrier protein synthase (ACPS) (A) or FabD (B) in PfMev parasites.
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Figure 3—figure supplement 1—source data 1
PDF containing uncropped gel images for integration PCRs for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original files for integration PCR gel analyses in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Additional microscopy images of apicoplast morphology in ∆ACPS (A) and ∆FabD (B) PfMev parasites.
ACPS = acyl carrier protein synthase, Phase = phase contrast, api-SFG = apicoplast-targeted superfolder GFP. Scale bars ~1 µm.
Figure 3—figure supplement 3
Growth of ∆ACP (A) and ∆FabD (B) PfMev parasites in low-lipid conditions with daily media changes.
ACP = acyl carrier protein.
Figure 4 with 3 supplements
Apicoplast acyl carrier protein (ACP) associates with apicoplast pyruvate kinase II.
(A) Proximity biotinylation study of Dd2 parasites episomally expressing apicoplast ACP tagged with C-miniTurbo, showing log2 enrichment ratio of known apicoplast-targeted proteins compared to parental Dd2 parasites, based on spectral intensity of proteins detected by tandem mass spectrometry. (B) Immunoprecipitation-mass spectrometry (IP-MS) study of apicoplast ACP interacting proteins, based on anti-HA-tag IP of apicoplast versus mitochondrial ACP (mACP)-HA2 in Dd2 parasites lysed in Triton X-100 or RIPA buffer. Axes display the log2 enrichment ratio for detection of each protein in IP samples of aACP versus mACP. Dashed diagonal line has a slope of one. (C) IP-MS analysis based on anti-HA-tag IP of lysates from Dd2 parasites episomally expressing wild-type (WT) or the S95A mutant of apicoplast ACP-HA2 and lysed in digitonin or RIPA buffer. Axes display the log2 enrichment ratio for detection of each protein in IP samples of WT versus S95A ACP. Dashed diagonal line has a slope of one. The identity of numbered proteins in panels A–C is shown in Supplementary file 1. A list of all proteins detected by mass spectrometry is shown in Figure 4—source data 3. (D) Affinity pull-down and western blot analysis of lysates from E. coli bacteria heterologously expressing parasite His6-tagged PKII or apicoplast ACP-HA2 (WT or S95A mutant). PKII was affinity isolated from bacterial lysates using nickel-nitrilotriacetic acid (Ni-NTA) resin, eluted with free imidazole, and analyzed by western blot for co-purification with aACP. Membranes were probed with anti-His6 and anti-HA-tag antibodies. The signal intensity for WT or S95A ACP relative to PKII in pull-down samples was quantified by densitometry in biological triplicate samples and plotted as the average ± SD, with significance analyzed by Student’s t-test to determine the indicated p-value.
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Figure 4—source data 1
PDF containing uncropped gels for western blot analyses in Figure 4D.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot gel images in Figure 4D.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig4-data2-v1.zip
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Figure 4—source data 3
Excel file of all proteins detected in proximity biotinylation and immunoprecipitation studies of apicoplast acyl carrier protein (ACP).
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Western blot detection of biotinylated proteins in lysates from parental, apicoplast acyl carrier protein (aACP)-miniTurbo, or aACP-BioID2 Dd2 parasites.
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Figure 4—figure supplement 1—source data 1
PDF containing uncropped gel image for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for western blot gel analysis in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
Enriched protein interactors of apicoplast acyl carrier protein (ACP) detected by proximity biotinylation or immunoprecipitation (IP).
(A) Apicoplast ACP interactors identified by miniTurbo versus BioID2. (B) Interacting proteins of ACP enriched in IP samples of apicoplast compared to mitochondrial ACP in Triton X-100 or RIPA buffer. (C) Interacting proteins of ACP enriched in IP samples of wild-type (WT) compared to S95A ACP in RIPA or digitonin buffer. All axes display the log2 enrichment ratio.
Figure 4—figure supplement 3
Replicate western blot images for Ni-NTA pull-down of pyruvate kinase II (PKII) association with wild-type (WT) (A, B) or Ser95Ala (C, D) acyl carrier protein (ACP) in E. coli.
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Figure 4—figure supplement 3—source data 1
PDF containing uncropped gel image for Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
Original files for western blot gel analysis in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig4-figsupp3-data2-v1.zip
Figure 5 with 1 supplement
Acyl carrier protein (ACP) knockdown destabilizes pyruvate kinase II (PKII) and impairs apicoplast DNA and RNA levels.
(A) Western blot analysis of apicoplast ACP-aptamer/TetR-DOZI Dd2 parasites episomally expressing PKII C-Myc3 and cultured for 3 days±1 µM aTc with 200 µM isopentenyl pyrophosphate (IPP). The blot was probed with α-Myc (PKII), α-HA (ACP), and α-ΕF1β (cytosolic loading control) antibodies and stained with Ponceau S for total protein. (B) Densitometry quantification of PKII and aACP protein levels normalized to ΕF1β based on western blot analysis of three biological replicates. Graph depicts the average ± SD, normalized to +aTc conditions. (C) Quantitative PCR analysis of DNA isolated from biological triplicate samples of apicoplast ACP-aptamer/TetR-DOZI parasites cultured for 36 or 84 hr±1 µM aTc with 200 µM IPP, with normalization of Ct values averaged from three apicoplast genes (SufB, TufA, ClpM) to Ct values averaged from three nuclear genes (STL, I5P, ADSL). Graphs depict the average ± SD of each timepoint, normalized to +aTc conditions. (D) Quantitative RT-PCR analysis of RNA isolated from biological triplicate samples obtained from parasites cultured under identical conditions as for panel C to determine normalized levels of apicoplast (SufB, TufA, ClpM) relative to nuclear (STL, I5P, ADSL) transcripts. Indicated p-values were determined by Student’s t-test analysis.
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Figure 5—source data 1
PDF containing the uncropped western blot images for Figure 5A.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig5-data1-v1.zip
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Figure 5—source data 2
Original image files for western blot analysis in Figure 5A.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig5-data2-v1.zip
Figure 5—figure supplement 1
Replicate western blot images for acyl carrier protein (ACP) knockdown and pyruvate kinase II (PKII) levels.
Indicated masses in panels A and B are kDa.
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Figure 5—figure supplement 1—source data 1
PDF containing uncropped gel images for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for western blot gel analysis in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/111494/elife-111494-fig5-figsupp1-data2-v1.zip
Figure 6 with 1 supplement
Scheme of acyl carrier protein (ACP) interaction with pyruvate kinase II (PKII) and dysfunctions upon ACP knockdown.
Loss of ACP synthase (ACPS) is expected to phenocopy loss of ACP. PEP = phosphoenolpyruvate, (d)NTPs = nucleoside triphosphates and deoxynucleoside triphosphates. The aACP structural model is based on PDB 3GZM and 5USR. The structural model of aACP bound to PKII was generated using AlphaFold 3.
Figure 6—figure supplement 1
AlphaFold 3 model of apicoplast acyl carrier protein (ACP) bound to pyruvate kinase II (PKII), rendered in PyMOL.
The unstructured N-terminus of parasite PKII, which is expected to function as the apicoplast-targeting sequence and be cleaved upon organelle import, was not included in modeling. The presence of the 4-phosphopantetheine group (4-PP) group was modeled by superposition with ACP in the PDB file 5USR.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Plasmodium falciparum) | Dd2 | PMID:1970614 | BEI Resources MRA-156 | |
| Cell line (P. falciparum) | NF54 PfMev | PMID:32059044 | Can be obtained from Prigge lab | |
| Cell line (P. falciparum) | NF54 PfMev aACP-HA-FLAG aptamer/TetR-DOZI | Made for this study | Can be obtained from Sigala lab | |
| Cell line (P. falciparum) | NF54 PfMev aACP knockout | Made for this study | Can be obtained from Prigge lab | |
| Cell line (P. falciparum) | NF54 PfMev ACPS knockout | Made for this study | Can be obtained from Prigge lab | |
| Cell line (P. falciparum) | NF54 PfMev FabD knockout | Made for this study | Can be obtained from Prigge lab | |
| Cell line (P. falciparum) | Dd2 mACP-HA2 (pTEOE) | PMID:34612205 | Can be obtained from Sigala lab | |
| Cell line (P. falciparum) | Dd2 aACP-HA2 (pTEOE) | PMID:34612205 | Can be obtained from Sigala lab | |
| Cell line (P. falciparum) | Dd2 S95A aACP-HA2 (pTEOE) | Made for this study | Can be obtained from Sigala lab | |
| Cell line (P. falciparum) | Dd2 aACP-BioID2-HA5 (pTEOE) | Made for this study | Can be obtained from Sigala lab | |
| Cell line (P. falciparum) | Dd2 aACP-MiniTurbo-HA5 (pTEOE) | Made for this study | Can be obtained from Sigala lab | |
| Cell line (P. falciparum) | Dd2 aACP-HA-FLAG aptamer/TetR-DOZI | Made for this study | Can be obtained from Sigala lab | |
| Cell line (P. falciparum) | Dd2 PKII-Myc3 (pTEOE) aACP-HA-FLAG aptamer/TetR-DOZI | Made for this study | Can be obtained from Sigala lab | |
| Cell line (Escherichia coli) | BL21/DE3 MBP-PKII-HIS6 (pMAL) aACP-HA2 (pET28a) | Made for this study | Can be obtained from Sigala lab | |
| Cell line (E. coli) | BL21/DE3 MBP-PKII-HIS6 (pMAL) S95A-aACP-HA2 (pET28a) | Made for this study | Can be obtained from Sigala lab | |
| Antibody | Anti-HA (3F10) (rat, monoclonal) | Sigma (Roche) | Cat. No. 11867423001 | (1:1000) |
| Antibody | Anti-aACP (rabbit, polyclonal) | Made for this study | (1:1000) Can be obtained from Sigala lab | |
| Antibody | Anti-His6 DyLight680 (mouse, monoclonal) | Thermo Fisher | Cat. No. MA121315D680, RRID:AB_2536987 | (1:1000) |
| Antibody | Anti-EF1α (rabbit, polyclonal) | PMID:11251817 | (1:1000) | |
| Antibody | Anti-Myc (mouse, monoclonal) | Invitrogen | Cat. No. MA1-21316 | (1:1000) |
Additional files
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Supplementary file 1
Supplementary tables 1–5.
Supplementary table 1. Table of apicoplast-targeted proteins identified in parasites expressing acyl carrier protein (ACP)-miniTurbo or ACP-BioID2. Supplementary table 2. Table of apicoplast-targeted proteins identified by immunoprecipitation/tandem mass spectrometry (IP/MS) of apicoplast acyl carrier protein (ACP)-HA2. Supplementary table 3. Table of apicoplast-targeted proteins identified by immunoprecipitation/tandem mass spectrometry (IP/MS) of wild-type (WT) or Ser95Ala acyl carrier protein (ACP)-HA2. Supplementary table 4. Table of apicoplast-targeted protein interactors of apicoplast ACP (aACP) identified by both miniTurbo and immunoprecipitation/tandem mass spectrometry (IP/MS). Supplementary table 5. List of PCR primers.
- https://cdn.elifesciences.org/articles/111494/elife-111494-supp1-v1.pdf
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Supplementary file 2
Scheme and primers for genomic PCR analyses to confirm gene disruptions.
- https://cdn.elifesciences.org/articles/111494/elife-111494-supp2-v1.pdf
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MDAR checklist
- https://cdn.elifesciences.org/articles/111494/elife-111494-mdarchecklist1-v1.pdf
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Acyl carrier protein is essential for apicoplast biogenesis in malaria parasites independent of fatty acid synthesis
eLife 15:RP111494.
https://doi.org/10.7554/eLife.111494.3
