Profiling of myristoylation in Toxoplasma gondii reveals an N-myristoylated protein important for host cell penetration
- Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, United Kingdom
- Institute for Advanced Biosciences, Team Membrane Dynamics of Parasite-Host Cell Interactions, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, France
- Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, White City Campus, United Kingdom
- The Peptide Chemistry STP, The Francis Crick Institute, United Kingdom
- Department of Life Sciences, Imperial College London, South Kensington, United Kingdom
Figures
Figure 1 with 1 supplement
Metabolic labelling allows for enrichment and visualisation of myristoylated and GPI-anchored proteins in T. gondii.
(A) Metabolic labelling workflow. (B) In gel fluorescence visualisation of YnMyr-dependent enrichment without and with the base treatment (top) and western blot with α-SFP1 (TGGT1_289540) showing …
Figure 1—figure supplement 1
Metabolic labelling optimisation.
(A) In gel fluorescence imaging of protein labelling with increasing concentrations of YnMyr over a 16 hr period in RH parasites. (B) In gel fluorescence visualisation of protein labelling with …
Figure 2 with 1 supplement
Identification of the YnMyr-enriched proteome in T. gondii.
(A) Schematic representation of the MS workflow using non-cleavable and cleavable capture reagents. (B) Venn diagram illustrating the overlap between significantly YnMyr-enriched proteins identified …
Figure 2—figure supplement 1
Identification of the YnMyr-enriched proteome in T. gondii.
(A) Structures of capture reagents used in this study with key functional components highlighted: biotin and azide moieties in blue and bold, respectively, cleavable linkers in grey with the …
Figure 3 with 1 supplement
Chemical inhibition of TgNMT and substrate response.
(A) Prediction of IMP-1002 interaction with TgNMT based on the PvNMT crystal structure. Crystal structure of the PvNMT (PDB: 6MB1, Schlott et al., 2019) active site (orange, red text) with IMP-1002 …
Figure 3—figure supplement 1
Chemical inhibition of TgNMT.
Label free quantification of change in total protein abundance between YnMyr- and NMTi-treated samples. See also Supplementary file 3.
Figure 4 with 1 supplement
The myristoylated proteome of Toxoplasma gondii.
(A) Distribution of the subcellular localisation across our substrate list. Analysis performed using ToxoDB and the build in LOPIT predictor. (B) Substrate orthology within selected Apicomplexans. …
Figure 4—figure supplement 1
The myristoylated proteome of Toxoplasma gondii.
(A) Sequence logo illustrating the amino acid distribution within the 20 N-terminal residues of all targets. Amino acids at each position (1-20) are ordered by the frequency of occurrence. Sequence …
Figure 5 with 1 supplement
MIC7 is myristoylated and is important for T. gondii lytic cycle.
(A) MS-based quantification of MIC7 and MAG1 abundance in tachyzoites [T] and bradyzoites [B] of T. gondii. Significance calculated using two-tailed Student’s t-test, ***p=0.0002, N = 3, error bars …
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Figure 5—source data 1
Numerical data of the graph presented in Figure 5A.
- https://cdn.elifesciences.org/articles/57861/elife-57861-fig5-data1-v2.zip
Figure 5—figure supplement 1
Inducible knock-out of MIC7.
(A) PCR analysis confirming correct integration of the floxed and recodonized version of Mic7 in the iKO line. Primers are indicated by arrows. Red hexagon represents STOP codon. bp – base pairs. (B)…
Figure 6 with 1 supplement
Myristoylation of MIC7 plays a role in the invasion of host cells.
(A) Complementation strategy used to evaluate the functional importance of MIC7 myristoylation. The orientation of cWT and cMut is reversed in the Uprt locus with the Ty1 tag at the C-terminus. Red …
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Figure 6—source data 1
Numerical data of the graphs presented in Figure 6F and G.
- https://cdn.elifesciences.org/articles/57861/elife-57861-fig6-data1-v2.zip
Figure 6—figure supplement 1
Complementation of the MIC7 iKO line.
(A) PCR analysis confirming correct integration of the complementation constructs encoding the WT and myristoylation mutant (Mut) copies of Mic7 at the Uprt locus of the iKO line. Primers are …
Figure 7 with 6 supplements
Functional analysis of MIC7 and its myristoylation in double tagged MIC7 lines.
(A) Schematic representation of MIC7 domain structure with evaluated Myc tag positions indicated by arrows. (B) Western blot analysis showing co-immunoprecipitation of MyccWT and MyccMut (α-Myc) …
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Figure 7—source data 1
Numerical data of the graph presented in Figure 7E.
- https://cdn.elifesciences.org/articles/57861/elife-57861-fig7-data1-v2.zip
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Figure 7—source data 2
Numerical data of the graph presented in Figure 7F.
- https://cdn.elifesciences.org/articles/57861/elife-57861-fig7-data2-v2.zip
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Figure 7—source data 3
Numerical data of the graph presented in Figure 7J.
- https://cdn.elifesciences.org/articles/57861/elife-57861-fig7-data3-v2.zip
Figure 7—figure supplement 1
Generation of doubly-tagged MIC7 lines.
(A) Plaque assay confirming that introduction of Myc tag between EGF5 and TM domains of MIC7 yields non-functional protein. (B) Co-localisation of ectopically expressed Myc tagged MIC7 versions …
Figure 7—figure supplement 2
Functional analysis of MIC7 and its myristoylation.
(A) Time lapses of bright-field (BF) and green fluorescent channel (GFP-GPI) showing successful/failed invasions of tachyzoites in U2OS cells that express a GFP-GPI marker. The marker allows …
Figure 7—video 1
Related to Figure 7F.
MIC7HA tachyzoite enters a GFP-GPI U2OS cell. Invasion proceeds with the typical invagination of the host cell plasma membrane that initiates the folding of the nascent parasitophorous vacuole while …
Figure 7—video 2
Related to Figure 7F.
Early abortive invasion of a GFP-GPI U2OS cell by a MIC7HA-treated tachyzoite (+RAPA). The tachyzoite apex starts to engage into the host cell membrane promoting a narrow and limited invagination of …
Figure 7—video 3
Related to Figure 7F.
MyccWT-treated tachyzoite (+RAPA) enters a GFP-GPI U2OS cell. Invasion proceeds with the same features and kinetics as for the MIC7HA tachyzoite. The parasite passes though the constricted …
Figure 7—video 4
Related to Figure 7F.
Early abortive invasion of a GFP-GPI U2OS cell by a MyccMut-treated tachyzoite (+RAPA). The tachyzoite engages the conoid into the cell membrane, which locally invaginates, but does not proceed …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Synthetic Gene (Toxoplasma gondii) | Microneme protein 7 (MIC7) | GeneArt, Life Technologies | TGGT1_261780 (http://toxodb.org) | Floxed and HA tagged sequence |
| Cell line (Homo sapiens) | Human foreskin fibroblasts (HFFs) | ATCC | Cat# SCRC-1041, RRID:CVCL_3285 | The cell line is available from the American Type Culture Collection (ATCC) |
| Cell line (Toxoplasma gondii) | RH ∆ku80 ∆hxgprt | Huynh and Carruthers, 2009 | Used in all mass spectrometry experiments | |
| Cell line (Toxoplasma gondii) | RH DiCre ∆ku80 ∆hxgprt | Hunt et al., 2019 | The second-generation DiCre-expressing cell line in Toxoplasma gondii | |
| Cell line (Toxoplasma gondii) | iKO MIC7; RH DiCre ∆ku80 ∆hxgprt_LoxMic7_HA | This paper | The endogenous Mic7 gene was replaced with a floxed and HA-tagged Mic7 gene | |
| Cell line (Toxoplasma gondii) | cWT MIC7 | This paper | As described for the iKO MIC7 line, however a Mic7-Ty1 expressing construct was integrated into the UPRT locus. | |
| Cell line (Toxoplasma gondii) | cMut MIC7 | This paper | As described for the iKO MIC7 line, however a Mic7(G2K/G3A)-Ty1 expressing construct was integrated into the UPRT locus. | |
| Cell line (Toxoplasma gondii) | Myc cWT MIC7 | This paper | As described for the cWT MIC7 line, however a Myc-Mic7-Ty1 expressing construct was integrated into the UPRT locus. | |
| Cell line (Toxoplasma gondii) | Myc cMut MIC7 | This paper | As described for the cMut MIC7 line, however a Myc-Mic7(G2K/G3A)-Ty1 expressing construct was integrated into the UPRT locus. | |
| Antibody | Rat anti-HA monoclonal clone 3F10 | Roche | Cat# 11867423001 RRID:AB_390919 | WB (1:1000) IFA (1:1000) |
| Antibody | Mouse anti-Myc monoclonal clone 4A6 | Millipore | Cat# 05–724 RRID:AB_11211891 | WB (1:1000) IFA (1:1000) |
| Antibody | Mouse anti-Ty1 monoclonal clone BB2 | Thermo Fisher | Cat# MA5-23513 RRID:AB_2610644 | WB (1:2000) IFA (1:500) |
| Antibody | Mouse anti-Toxoplasma monoclonal clone TP3 | Abcam | Cat# ab8313 RRID:AB_306466 | WB (1:1000) |
| Antibody | Mouse anti-MIC2 monoclonal clone 6D10 | other | Provided by Vernon Carruthers Lab WB (1:1000) | |
| Antibody | Rabbit anti-MIC2 polyclonal | other | Provided by Vernon Carruthers Lab WB (1:500) IFA (1:5000) | |
| Antibody | Rabbit anti-TgCAP polyclonal | Hunt et al., 2019 | WB (1:2000) | |
| Antibody | Rabbit anti-Gra29 polyclonal | Young et al., 2020 | WB (1:1000) | |
| Antibody | Rabbit anti-SFP1 polyclonal | Young et al., 2020 | WB (1:1000) | |
| Antibody | Mouse anti-CDPK1 polyclonal | other | Provided by Matthew Child and Matt Bogyo WB (1:3000) | |
| Antibody | Rabbit anti-SAG1 monoclonal | other | Provided by John Boothroyd Lab WB (1:10,000) | |
| Antibody | Rabbit anti-GAP45 polyclonal | other | Provided by Peter Bradley Lab WB (1:1000) | |
| Antibody | Rabbit anti-phospho-Stat6 polyclonal | Cell Signaling | Cat# 9361 RRID:AB_331595 | IFA (1:600) |
| Chemical compound | Myristic acid (Myr) | Tokyo Chemical Industry | Cat# M0476 | |
| Chemical compound | Alkyne-myristic acid (YnMyr) | Iris Biotech | Cat# RL-2055 | |
| Chemical compound | Azide-PEG3-biotin (N3-biotin) | Sigma-Aldrich | Cat# 762024 | Capture reagent 1 |
| Chemical compound | Trypsin cleavable reagent | Broncel et al., 2015 | The reagent used here was synthesised in-house by the Peptide Chemistry science technology platform, The Francis Crick Institute | |
| Chemical compound | TEV cleavable reagent | Speers and Cravatt, 2005 | The reagent used here was synthesised in-house by the Peptide Chemistry science technology platform, The Francis Crick Institute | |
| Chemical compound | IMP-1002 | Schlott et al., 2019 | The reagent used here was synthesised by the Tate Laboratory, Imperial College London | |
| Chemical compound | Rapamycin | Sigma-Aldrich | Cat# R8781 | |
| Chemical compound | 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) | Howard et al., 2015 | The reagent used here was synthesised in-house by the Peptide Chemistry science technology platform, The Francis Crick Institute | |
| Software, algorithm | MaxQuant (version 1.5.0.25 and 1.5.2.8) | Cox and Mann, 2008 | RRID:SCR_014485 | Free software for searching of mass spectrometry acquisition files |
| Software, algorithm | Perseus (version 1.5.0.9) | Tyanova et al., 2016 | RRID:SCR_015753 | Free software for processing of MaxQuant output files |
| Software, algorithm | PyMOL (version 1.3r1) | Schrodinger LLC | RRID:SCR_000305 | Commercial software for molecular visualisation |
| Software, algorithm | Prism 8 (version 8.1.1) | GraphPad Software, Inc | RRID:SCR_002798 | Commercial software for statistical analysis |
Additional files
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Supplementary file 1
related to Figure 2.
Identification of base-dependent YnMyr enrichment in T. gondii. Sheet 1: Toxoplasma proteins with YnMyr intensities quantified irrespective of base treatment. Sheet 2: Proteins with base-sensitive enrichment. Sheet 3: MG proteins insensitive to base treatment and robustly enriched in a YnMyr-dependent manner with N3-biotin reagent (1). Sheet 4: Analysis of proteomes (supernatants post enrichment).
- https://cdn.elifesciences.org/articles/57861/elife-57861-supp1-v2.xlsx
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Supplementary file 2
related to Figure 2.
Identification of myristoylated proteins and myristoylated peptides in T. gondii. Sheet 1: Toxoplasma proteins bearing the MG motif. Sheet 2: Substrates significantly enriched with Trypsin reagent (2). Sheet 3: Substrates selected based on fold change in YnMyr/Myr enrichment with TEV reagent (3). Sheet 4: Myristoylated peptides found with Trypsin reagent (2). Sheet 5: Myristoylated peptides found with TEV reagent (3). Sheet 6: Human proteins bearing the MG motif. Sheet 7: Human substrates significantly enriched with Trypsin and TEV reagents.
- https://cdn.elifesciences.org/articles/57861/elife-57861-supp2-v2.xlsx
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Supplementary file 3
related to Figure 3.
Chemical inhibition of TgNMT. Sheet 1: Response of YnMyr enriched Toxoplasma proteins to NMTi. Sheet 2: NMTi does not significantly affect Toxoplasma proteome. Sheet 3: Response of base-sensitive Toxoplasma proteins to NMTi. Sheet 4: Response of YnMyr enriched Human proteins to NMTi. Sheet 5: NMTi does not significantly affect Human proteome.
- https://cdn.elifesciences.org/articles/57861/elife-57861-supp3-v2.xlsx
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Supplementary file 4
related to Figure 4.
Myristoylated proteome of T. gondii. Sheet 1: Substrate list and annotation. Sheet 2: Myristoylated proteins in P. falciparum and their orthologues in Toxoplasma. Sheets 3–9: Substrate orthologues in selected Apicomplexans.
- https://cdn.elifesciences.org/articles/57861/elife-57861-supp4-v2.xlsx
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Supplementary file 5
related to Figure 5.
MIC7 expression in tachyzoites and bradyzoites.
- https://cdn.elifesciences.org/articles/57861/elife-57861-supp5-v2.xlsx
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Supplementary file 6
Primers used for plasmid and parasite lines generation.
- https://cdn.elifesciences.org/articles/57861/elife-57861-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57861/elife-57861-transrepform-v2.docx
