Parasites and human cell cultures
Table of Contents
Primary HFFs (ATCC CCL-171) were cultured in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 10 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) buffer pH 7.2, 2 mM l-glutamine and 50 μg ml−1 penicillin and streptomycin (Invitrogen). Cells were incubated at 37 °C and 5% CO2. Toxoplasma strains used in this study and listed in Supplementary Table 1 were maintained in vitro by serial passage on monolayers of HFFs. Cultures were free of mycoplasma as determined by qualitative PCR.
Reagents
The following primary antibodies were used in the immunofluorescence, immunoblotting and/or ChIP assays: rabbit anti-TgHDAC3 (Research Resource Identifier (RRID): AB_2713903), rabbit anti-TgGAP45 (gift from Prof. Dominique Soldati), mouse anti-HA tag (Roche, RRID: AB_2314622), rabbit anti-HA Tag (Cell Signaling Technology, RRID: AB_1549585), rabbit anti-mCherry (Cell Signaling Technology, RRID: AB_2799246), rabbit anti-Flag (Cell Signaling Technology, RRID: AB_2798687), mouse anti-MYC clone 9B11 (RRID: AB_2148465), H3K9me3 (Diagenode, RRID: AB_2616044), rabbit anti-acetyl-histone H4, pan (Lys5,8,12) (Millipore, RRID: AB_310270), rat anti-IMC7 and centrin 1 (gift from Prof. M. J. Gubbels), mouse anti-IMC1 (gift from Prof. G. E. Ward), mouse anti-AtRx antibody clone 11G8 (ref. 24) and mouse anti-GRA11b14. We have also raised homemade antibodies to linear peptides in rabbits corresponding to the following proteins: MORC_Peptide2 (C+SGAPIWTGERGSGA); AP2XI-2 (C+HAFKTRRTEAAT); TGME49_273980 or GRA80 (C+RPPWAPGAGPEN); TGME49_243940 or GRA81 (C+QKELAEVAQRALEN); TGME49_277230 or GRA82 (C+SDVNTEGDATVANPE); TGME49_209985 or ROP26 (CQETVQGNGETQL); SRS48 family (CKALIEVKGVPK); SRS59B or K (C+IHVPGTDSTSSGPGS); TGME49_314250 or BRP1 (C+QVKEGTKNNKGLSDK); TGME49_307640 or CK2 kinase (C+IRAQYHAYKGKYSHA); and TGME49_306455 (C+DGRTPVDRVFEE). They were manufactured by Eurogentec and used for immunofluorescence, immunoblotting and/or ChIP. Secondary immunofluorescent antibodies were coupled with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific). Secondary antibodies used in western blotting were conjugated to alkaline phosphatase (Promega).
Auxin-induced degradation
Degradation of AP2XII-1–mAID–HA, AP2XI-2–mAID–HA and AP2XII-1–mAID–HA plus AP2XI-2–mAID–MYC was achieved with IAA (Sigma-Aldrich number 45533). A stock of 500 mM IAA dissolved in 100% ethanol at a ratio of 1:1,000 was used to degrade mAID-tagged proteins to a final concentration of 500 μM. The mock treatment consisted of an equivalent volume of 100% ethanol at a final concentration of 0.0789% (w/v). To monitor the degradation of mAID-tagged proteins, parasites grown in HFF monolayers were treated with auxin or ethanol alone for various time intervals at 37 °C. After treatment, parasites were collected and analysed by immunofluorescence or western blotting.
Immunofluorescence microscopy
Toxoplasma-infected HFF cells grown on coverslips were fixed in 3% formaldehyde for 20 min at room temperature, permeabilized with 0.1% (v/v) Triton X-100 for 15 min, and blocked in phosphate-buffered saline (PBS) containing 3% (w/v) BSA. Cells were then incubated with primary antibodies for 1 h, followed by the addition of secondary antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes). Nuclei were stained with Hoechst 33258 (2 μg ml−1 in PBS) for 10 min at room temperature. After washing four times in PBS, coverslips were mounted on a glass slide with Mowiol mounting medium, and images were acquired with a fluorescence microscope ZEISS ApoTome.2 and processed with ZEN software (Carl Zeiss).
For IFA of in vivo stages in the cat, small intestines of infected kittens from a previous study16 embedded in paraffin were sectioned to 3 μm and dried overnight at 37 °C. Deparaffinization was carried out first three times for 2 min in xylene, and then the sections were washed twice for 1 min in 100% ethanol and finally rehydrated sequentially for 1 min in 96% ethanol, and then 70% ethanol and water. For antigen retrieval, samples were boiled in a pressure cooker for 20 min in citrate buffer at pH 6.1 (Dako Target Retrieval Solution, S2369) and transferred to water. Cells were permeabilized in 0.3% Triton X-100 in PBS and blocked with fetal calf serum (FCS). Staining was carried out overnight at 4 °C using the following combinations: mouse anti-GRA11b14 with rabbit anti-GRA80, anti-IMC1 or anti-GAP45 (the last two being gifts from Prof. Dominique Soldati) or rabbit anti-GRA80 with rat immune serum recognizing merozoite proteins in 20% FCS and 0.3% Triton X-100 in PBS. The samples were then washed and incubated with 1 μg ml−1 DAPI, 20% FCS and 0.3% Triton X-100 in PBS and appropriate combinations of anti-rabbit Alexa Fluor 488, 555 or 594 (Invitrogen, A11070, A32794 or A11072) with anti-mouse Alexa Fluor 488, 594 or 647 (Invitrogen, A11017, A11005 or A21235) or anti-rat Alexa Fluor 488 (Invitrogen, A11006) with anti-rabbit Alexa Fluor 594 (Invitrogen, A11072) for 1 h at room temperature. After three washes, samples were mounted with Vectashield and imaged either with a Leica DMI 6000 B epi-fluorescence microscope or a Leica SP8 confocal microscope. Confocal images were deconvoluted using SVI Huygens Professional. Maximum-intensity projections were carried out using FIJI 2.9.1.
Transmission electron microscopy
For ultrastructural observations, Toxoplasma-infected HFFs grown as monolayers on a 6-well dish were exposed to 500 µM IAA or ethanol solvent as described above before fixation 24 h or 40 h post-infection in 2.5% glutaraldehyde in 0.1 mM sodium cacodylate (pH 7.4) and processed as described previously44. Ultrathin sections of infected cells were stained with osmium tetraoxide before examination with a Hitachi 7600 electron microscope under 80 kV equipped with a dual AMT CCD camera system.
Western blot
Immunoblot analysis of protein was carried out as described in ref. 45. Briefly, about 107 cells were lysed and sonicated in 50 μl lysis buffer (10 mM Tris-HCl, pH 6.8, 0.5% SDS (v/v), 10% glycerol (v/v), 1 mM EDTA and protease inhibitor cocktail). Proteins were separated using SDS–polyacrylamide gel electrophoresis, transferred by liquid transfer to a polyvinylidene fluoride membrane (Immobilon-P; EMD Millipore), and western blots were probed with the appropriate primary antibodies and alkaline phosphatase-conjugated or horseradish peroxidase-conjugated secondary goat antibodies. Signals were detected using NBT-BCIP (Amresco) or an enhanced chemiluminescence system (Thermo Scientific).
Plasmid construction
The plasmids and primers used in this work for the genes of interest (GOIs) are listed in Supplementary Table 1. To construct the vector pLIC-GOI-HA-Flag, pLIC-GOI-mAID-HA or pLIC-GOI-mAID-(MYC)2, the coding sequence of the GOI was amplified with the primers LIC-GOI-Fwd and LIC-GOI-Rev using genomic Toxoplasma DNA as a template. The resulting PCR product was cloned into the vector pLIC-HF-dhfr or pLIC-mCherry-dhfr using the ligation-independent cloning (LIC) method. Specific guide RNA for the GOI, based on the CRISPR–cas9 editing method, was cloned into the plasmid pTOXO_Cas9-CRISPR1. Twenty-base oligonucleotides corresponding to specific GOIs were cloned using the Golden Gate strategy. Briefly, the primers GOI-gRNA-Fwd and GOI-gRNA-Rev containing the single guide RNA targeting the genomic sequence of the GOI were phosphorylated, annealed and ligated into the pTOXO_Cas9-CRISPR plasmid linearized with BsaI, resulting in pTOXO_Cas9-CRISPR::sgGOI.
Toxoplasma transfection
Parasite strains were electroporated with vectors in Cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4 and KH2PO4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl2) using a BTX ECM 630 machine (Harvard Apparatus). Electroporation was carried out in a 2-mm cuvette at 1,100 V, 25 Ω and 25 µF. Antibiotics (concentration) used were chloramphenicol (20 µM), mycophenolic acid (25 µg ml−1) with xanthine (50 µg ml−1), pyrimethamine (3 µM) or 5-fluorodeoxyuracil (10 µM) as needed. Stable transgenic parasites were selected with the appropriate antibiotic, cloned in 96-well plates by limiting dilution, and verified by immunofluorescence assay or genomic analysis.
Chromatographic purification of Flag-tagged proteins
Toxoplasma extracts from RH∆ku80 or Pru∆ku80 cells stably expressing HA–Flag-tagged AP2XII-1 or AP2XI-2 proteins, respectively, were incubated with anti-Flag M2 affinity gel (Sigma-Aldrich) for 1 h at 4 °C. Beads were washed with 10 column volumes of BC500 buffer (20 mM Tris-HCl, pH 8.0, 500 mM KCl, 20% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.5% NP-40 and protease inhibitors). Bound polypeptides were eluted stepwise with 250 μg ml−1 Flag peptide (Sigma-Aldrich) diluted in BC100 buffer. For size-exclusion chromatography, protein eluates were loaded onto a Superose 6 HR 10/30 column equilibrated with BC500. The flow rate was set at 0.35 ml min−1, and 0.5-ml fractions were collected.
MS-based quantitative analyses of parasite proteomes and AP2 interactomes
Sample preparation
For proteome-wide analyses, HFF cells were grown to confluence, infected with the RH (AP2XII-1 and AP2XI-2 KD) strain and treated with IAA for 24 h, 32 h and 48 h or mock-treated. Proteins were extracted using cell lysis buffer (Invitrogen). Three biological replicates were prepared and analysed for each condition. For characterization of HA–Flag-tagged AP2XII-1 or AP2XI-2 interactomes, eluted proteins were solubilized in Laemmli buffer. Three biological replicates were prepared for each bait protein and for the negative control. Proteins were stacked in the top of a 4–12% NuPAGE gel (Invitrogen) and stained with Coomassie blue R-250 (Bio-Rad) before in-gel digestion using modified trypsin (Promega, sequencing grade) as previously described1.
Nanoliquid chromatography coupled to MS/MS analyses
The resulting peptides were analysed by online nanoliquid chromatography coupled to an MS/MS instrument (Ultimate 3000 RSLCnano and Q-Exactive HF, Thermo Fisher Scientific) using a 360-min gradient for proteome-wide analysis and a 200-min gradient for interactome characterization. For this, peptides were sampled on a 300 μm × 5 mm PepMap C18 precolumn and separated in a 200 cm µPAC column (PharmaFluidics) or a 75 μm × 250 mm C18 column (Aurora Generation 2, 1.7 µm, IonOpticks) for, respectively, proteome-wide and interactome analyses. MS and MS/MS data were acquired using Xcalibur software version 4.0 (Thermo Scientific).
Protein identification and quantification
Peptides and proteins were identified by Mascot (version 2.8.0, Matrix Science) through concomitant searches against the T. gondii database (ME49 taxonomy, version 58 downloaded from ToxoDB), the Uniprot database (Homo sapiens taxonomy) and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (human keratins, trypsin and so on). Trypsin was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set, respectively, at 10 and 20 ppm. Peptide modifications allowed during the search were: carbamidomethyl (C, fixed), acetyl (protein amino terminus, variable) and oxidation (M, variable). The Proline software (version 2.2.0) was used for the compilation, grouping and filtering of the results (conservation of rank 1 peptides, peptide length ≥ 6 amino acids, false discovery rate of peptide-spectrum-match identifications < 1% and minimum of one specific peptide per identified protein group). Proline was then used to carry out an MS1 label-free quantification of the identified protein groups based on razor and specific peptides.
Statistical analyses
Statistical analyses were carried out using ProStaR46. Proteins identified in the reverse and contaminant databases or matched to human sequences were discarded. For proteome-wide analyses, only proteins identified by MS/MS in a minimum of two replicates of one condition and quantified in the three replicates of one condition were conserved. After log2 transformation, abundance values were normalized using the variance-stabilizing normalization method, before missing-value imputation (structured least squares algorithm (SLSA) for partially observed values in the condition and DetQuantile algorithm for totally absent values in the condition). For comparison of each IAA-treated condition to the mock-treated condition, statistical testing was conducted with limma, whereby differentially expressed proteins were selected using a log2[FC] cutoff of 1 and a P-value cutoff of 0.01, allowing one to reach a false discovery rate inferior to 5% according to the Benjamini–Hochberg estimator. Proteins found differentially abundant but identified by MS/MS in fewer than two replicates, and detected in fewer than three replicates, in the condition in which they were found to be more abundant were invalidated (P value = 1). Protein abundances measured in the four different conditions were also compared globally by ANOVA using Perseus; q values were obtained by Benjamini–Hochberg correction.
For interactome analysis, only proteins identified by MS/MS in the three replicates of one condition and proteins quantified with a minimum of five peptides were conserved. After log2 transformation, abundance values were normalized by condition-wise median centring, before missing-value imputation (SLSA algorithm for partially observed values in the condition and DetQuantile algorithm for totally absent values in the condition). Statistical testing was conducted with limma, whereby differentially expressed proteins were selected using a P-value cutoff of 0.01 and FC cutoffs of 5 and 3, respectively, for comparison of each AP2 interactome with negative control and AP2 interactomes together, allowing one to reach a false discovery rate inferior to 1% according to the Benjamini–Hochberg estimator. The relative abundances of AP2-associated proteins were determined using the iBAQ metrics; only proteins with an iBAQ ratio of at least 0.1 in relation to the bait protein were considered.
MS-based proteomic analyses of SEC fractions
Protein bands were excised from colloidal blue-stained gels (Thermo Fisher Scientific) before in-gel digestion using modified trypsin (Promega, sequencing grade) as previously described1. Resulting peptides were analysed by online nanoliquid chromatography coupled to MS/MS (UltiMate 3000 RSLCnano and Orbitrap Exploris 480, Thermo Scientific). Peptides were sampled on a 300 µm × 5 mm PepMap C18 precolumn and separated on a 75 µm × 250 mm C18 column (Aurora Generation 2, 1.6 µm, IonOpticks) using a 25-min gradient. MS and MS/MS data were acquired using Xcalibur version 4.0 (Thermo Scientific). Peptides and proteins were identified using Mascot (version 2.8.0) through concomitant searches against the T. gondii database (ME49 taxonomy, version 58 downloaded from ToxoDB), the Uniprot database (T. ni taxonomy) and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (human keratins, trypsin and so on). Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set, respectively, at 10 and 20 ppm. Peptide modifications allowed during the search were: carbamidomethyl (C, fixed), acetyl (protein N terminus, variable) and oxidation (M, variable). The Proline software (version 2.2.0) was used for the compilation, grouping and filtering of the results (conservation of rank 1 peptides, peptide length ≥ 6 amino acids, false discovery rate of peptide-spectrum-match identifications < 1% and minimum of one specific peptide per identified protein group). iBAQ values were calculated for each protein group in Proline using MS1 intensities of specific and razor peptides.
ChIP coupled with Illumina sequencing
ChIP
HFF cells were grown to confluence and infected with KD strains as indicated in the figure legends. Collected intracellular parasites were crosslinked with formaldehyde (final concentration 1%) for 8 min at room temperature, and crosslinking was stopped by addition of glycine (final concentration 0.125 M) for 5 min at room temperature. The parasites were lysed in ice-cold lysis buffer A (50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.125% Triton X-100 and protease inhibitor cocktail) and after centrifugation, crosslinked chromatin was sheared in buffer B (1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 10 mM Tris pH 8.0 and protease inhibitor cocktail) by sonication with a Diagenode Biorupter. Samples were sonicated for 16 cycles (30 s on and 30 s off) to achieve an average size of 200–500 base pairs. Sheared chromatin, 5% BSA, a protease inhibitor cocktail, 10% Triton X-100, 10% deoxycholate, magnetic beads coated with DiaMag protein A (Diagenode) and antibodies to epitope tags (HA or MYC) or the protein of interest (MORC or HDAC3) were used for immunoprecipitation. A rabbit IgG antiserum served as a control mock. After overnight incubation at 4 °C on a rotating wheel, chromatin–antibody complexes were washed and eluted from the beads using the iDeal ChIP–seq kit for transcription factors (Diagenode) according to the manufacturer’s protocol. Samples were de-crosslinked by heating for 4 h at 65 °C. DNA was purified using the IPure kit (Diagenode) and quantified using Qubit Assays (Thermo Fisher Scientific) according to the manufacturer’s protocol. For ChIP–seq, the purified DNA was used for library preparation and subsequently sequenced by Arraystar (USA).
Library preparation, sequencing and data analysis (Arraystar)
ChIP–seq libraries were prepared according to the Illumina protocol “Preparing Samples for ChIP Sequencing of DNA”. For library preparation, 10 ng of DNA from each sample was converted to blunt-end phosphorylated DNA fragments using T4 DNA polymerase, Klenow polymerase and T4 polymerase (NEB); an ‘A’ base was added to the 3′ end of the blunt-end phosphorylated DNA fragments using the polymerase activity of Klenow (Exo-Minus) polymerase (NEB); Illumina genomic adapters were ligated to the A-tailed DNA fragments; PCR amplification to enrich the ligated fragments was carried out using Phusion High Fidelity PCR Master Mix with HF Buffer (Finnzymes Oy). The enriched product of about 200–700 bp was excised from the gel and purified. For sequencing, the library was denatured with 0.1 M NaOH to generate single-stranded DNA molecules and loaded into flow cell channels at a concentration of 8 pM and amplified in situ using TruSeq Rapid SR cluster kit (number GD-402-4001, Illumina). Sequencing was carried out for 100 cycles on the Illumina HiSeq 4000 according to the manufacturer’s instructions. For data analysis, after the sequencing platform generated the sequencing images, the stages of image analysis and base calling were carried out using Off-Line Basecaller software (OLB V1.8). After passing the Solexa CHASTITY quality filter, the clean reads were aligned to the T. gondii reference genome (TGME49) using BOWTIE V2 and then converted and sorted using Bamtools. Aligned reads were used for peak calling of the ChIP-enriched peaks using MACS v2.2 with a cutoff P value of 10−4. For Integrated Genome Browser visualization and gene-centred analysis using Deeptools, MACS2-generated bedgraph files were processed with the command ‘sort -k1,1 -k2,2n 5_treat_pileup.bdg > 5_treat_pileup-sorted.bdg’, and then converted using the BedGraphToBigWig program (ENCODE project). The Deeptools analysis was generated using the command computeMatrix reference point, with the following parameters: –minThreshold 2, –binSize 10 and –averageTypeBins sum. Plotprofile or heat map was then used with k-means clustering when applicable. Inter-sample comparisons were obtained using the nf-core ChIP–seq workflow with standard parameters47. From this pipeline, HOMER (annotatePeaks) was used to analyse peak distribution relative to gene features. All of these raw and processed files can be found at GSE222819.
RNA-seq and sequence alignment
Total RNAs were extracted and purified using TRIzol (Invitrogen) and RNeasy Plus Mini Kit (Qiagen). RNA quantity and quality were measured using a NanoDrop 2000 (Thermo Scientific). For each condition, RNAs were prepared from three biological replicates. RNA integrity was assessed by standard non-denaturing 1.2% TBE agarose gel electrophoresis. RNA-seq was carried out following standard Illumina protocols, by Novogene (Cambridge, UK). Briefly, RNA quantity, integrity and purity were determined using the Agilent 5400 Fragment Analyzer System (Agilent Technologies). The RNA quality number ranged from 7.8 to 10 for all samples, which was considered sufficient. mRNAs were purified from total RNA using poly-T oligonucleotide-attached magnetic beads. After fragmentation, the first-strand cDNA was synthesized using random hexamer primers. Then the second-strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification and purification. The library was checked with Qubit and real-time PCR for quantification and a bioanalyser for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. The samples were sequenced on the Illumina NovaSeq platform (2 × 150 bp, strand-specific sequencing) and generated about 40 million paired-end reads for each sample. The quality of the raw sequencing reads was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC. For quantification and normalization of the expression data, the FASTQ reads were aligned to the ToxoDB-49 build of the T. gondii ME49 genome using Subread version 2.0.1 with the following options: subread-align -d 50 -D 600–sortReadsByCoordinates. Read counts for each gene were calculated using featureCounts from the Subread package. Differential expression analysis was conducted using DESeq2 and default settings within the iDEP.96 web interface48. Transcripts were quantified and normalized using TPMCalculator. The Illumina RNA-seq dataset generated during this study is available at the National Center for Biotechnology Information: BioProject number PRJNA921935.
Nanopore direct RNA-seq
The mRNA library preparation followed the SQK-RNA002 kit (Oxford Nanopore)–recommended protocol; the only modification was the input mRNA quantity increased from 500 to 1,000 ng, and all other consumables and parameters were standard. Final yields were evaluated using the Qubit HS dsDNA kit (Thermo Fisher Scientific, Q32851) with minimum RNA preparations reaching at least 200 ng. For all conditions, sequencing was carried out on FLO-MIN106 flow cells using either a MinION MK1C or MinION sequencer. All datasets were subsequently base called (high-accuracy base calling) with a Guppy version higher than 5.0.1 with a Q score cutoff of >7. Long-read alignment was carried out by Minimap2 as previously described49. Sam files were converted to bam and sorted using Samtools 1.4. Alignments were converted and sorted using Samtools 1.4.1. For the three described samples, Toxoplasma aligned reads range between 600,000 and 800,000. The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935.
ATAC–seq
Intracellular tachyzoites (non-treated or IAA treated for 24 h) were prepared using HFF cell monolayers in a T175 format, which was freshly scraped, gently homogenized by pipetting and centrifuged at 500g. Before initiating the transposition protocol, the pellet was gently washed with warm Dulbecco’s PBS (Life technologies) and resuspended in 500 μl of cold PBS + protease inhibitor (Diagenode kit). Nuclei preparation, permeabilization, Tn5 transposition and library preparation was carried out following precisely the Diagenode ATAC–seq kit protocol (C01080002). Nucleus permeabilization was carried out on an estimated 100,000 tachyzoites by diluting 10 μl of Dulbecco’s cell suspension (from one T175 resuspended in 500 μl) in 240 μl of Dulbecco’s PBS + protease inhibitor (1/25 dilution). From this dilution, 50 μl was then taken to carry out the transposition reaction. Of note, the permeabilization protocol used a 3-min 0.02% digitonin (Promega) exposure. Following the Tn5 reaction, libraries were amplified using the Diagenode 24 UDI kit 1 (ref 01011034) following standard protocol procedures. Libraries were multiplexed and sequenced on a single Novaseq6000 lane by Fasteris (Genesupport SA) using 2 × 50 cycles, generating on average 27 million reads. Demultiplexing of raw reads was performed by bcl2fastq V3, and trimming, quality control, alignment to the ME49 reference genome (using bwa2) and duplicate read merging (using Picard) were carried out by the nf-core ATAQ-SEQ pipeline47. For Integrated Genome Browser visualization and gene-centred analysis using Deeptools, Picard merged bam files were converted to bigWig file format using a bin size of 5 by bamCoverage (Deeptools). The Deeptools analysis was then generated using ‘computeMatrix reference point’, with the following parameters: –minThreshold 2,–binSize 10 and –averageTypeBins sum. Quantitative analysis of untreated versus 24-h IAA conditions was carried out by nf-core through a broad peak calling and annotation (MACS2) followed by HOMER (annotatePeaks) to analyse peak distribution relative to gene features. Reads were counted on annotated peaks by featureCounts and counts were processed by DeSeq2 to generate global statistical analysis of peak intensities between conditions using biological duplicates. All of these raw and processed files can be found at GSE222832.
Gene synthesis for recombinant coexpression of T. gondii AP2XI-2 and AP2XII-I
Gene synthesis for all insect cell codon-optimized constructs was provided by GenScript. (Strep)2–APXI-2 and AP2XII-1–Flag or AP2IX-6–Flag genes were cloned within the coexpression donor vector pFastBac dual, which accepts two constructs. The (Strep)2–AP2XI-2 expression cassette was derived from the TGME49_310900 gene with a fused dual Strep tag and tobacco etch virus (TEV) site in the N terminus. AP2XII-1–Flag or AP2IX-6–Flag was derived from the full-length TGME49_218960 and TGME49_290180 genes, respectively, with an additional non-cleavable Flag tag on the carboxy terminus. The (Strep)2–AP2XI-2 expression cassette was under the control of the polyhedrin promoter; the AP2XII-1–Flag or AP2IX-6–Flag was under the control of the P10 promoter.
Generation of baculovirus
Bacmid cloning steps and baculovirus generation were carried out using EMBacY baculovirus (gift from I. Berger), which contains a yellow fluorescent protein reporter gene in the virus backbone. The established standard cloning and transfection protocols set up within the EMBL Grenoble eukaryotic expression facility were used. Although baculovirus synthesis (V0) and amplification (to V1) were carried out with SF21 cells cultured in SF900 III medium (Life Technologies), large-scale expression cultures were carried out with Hi-5 cells cultured in the protein-free ESF 921 insect cell culture medium (Expression System) and infected with 0.8–1.0% (v/v) of generation 2 (V1) baculovirus suspensions and collected 72 h after infection.
(Strep)2–AP2XI-2 and AP2XII-1–Flag or (Strep)2–AP2XI-2 and AP2XIX-6–Flag expression and purification
For purification, three cell pellets of bout 500 ml of Hi-5 culture were each resuspended in 50 ml of lysis buffer (50 mM Tris (pH 8.0), 400 mM NaCl and 2 mM β-mercaptoethanol (BME)) in the presence of an anti-protease cocktail (Complete EDTA-free, Roche) and 1 μl of Benzonase (Merck Millipore, 70746). Lysis was carried out on ice by sonication for 3 min (30-s on/ 30-s off, 45° amplitude). After the lysis step, 10% of glycerol was added. Clarification was then carried out by centrifugation for 1 h at 16,000g and 4 °C and vacuum filtration using 45-μm nylon filter systems (SteriFlip, Merck Millipore). Before purification, tetrameric avidin (Biolock, IBA Lifescience) was added to the clarified lysate (1/1,000 v/v), which was then batch incubated for 20 min with 3 ml of Strep-Tactin XT (IBA Lifescience). Following the incubation, the resin was retained on a glass column and washed three times using 6 ml of lysis buffer (W1), 6 ml of lysis buffer with NaCl content at 1 M (W2) and 6 ml of lysis buffer (W3). The elution was then carried out using 1× BXT buffer (IBA Lifescience) containing 50 mM biotin, 100 mM Tris pH 8 and 150 mM NaCl. This initial 1× solution was further supplemented with 300 mM NaCl, 2 mM BME and 10% glycerol. Following Strep-Tactin XT elution, the sample was concentrated to 500 μl using a 100-kDa concentrator (Amicon Ultra 4, Merck Millipore) injected on an ÄKTA pure FPLC using a Superose 6 Increase column 10/300 GL (Cytiva) running in 50 mM Tris pH 8, 400 mM NaCl and 1 mM BME.
Mouse infection and experimental survey
Six-week-old NMRI, CD1 or BALB/c mice were obtained from Janvier Laboratories. Female mice were used for all studies. For intraperitoneal infection, tachyzoites were grown in vitro and extracted from host cells by passage through a 27-gauge needle, washed three times in PBS, and quantified with a haemocytometer. Parasites were diluted in Hank’s balanced salt solution (Life), and mice were inoculated by the intraperitoneal route with tachyzoites of each strain (in 200 μl volume) using a 28-gauge needle. Animal euthanasia was completed in an approved CO2 chamber. For immunolabelling on histological sections of the brains, the brains were removed from mice, entirely embedded in a paraffin wax block and cut into 5-μm-thick layers using a microtome.
Statistics and reproducibility
Sample sizes were not predetermined and were chosen according to previous literature. Experiments were carried out in biological replicates and provided consistent statistically relevant results. No method of randomization was used. All experiments were carried out in independent biological replicates as stated for each experiment in the manuscript. All corresponding treatment and control samples from ChIP–seq and RNA-seq were processed at the same time to minimize technical variation. Investigators were not blinded during the experiments. Statistical significance was evaluated using P values from unpaired two-tailed Student t-tests. Data are presented as the mean ± s.d. Significance was set to a P value of <0.05. All of the micrographs shown are representatives from three independently conducted experiments, with similar results obtained.
Ethics statement
Mouse care and experimental procedures were carried out under pathogen-free conditions in accordance with established institutional guidance and approved protocols from the Institutional Animal Care and Use Committee of the University Grenoble Alpes (APAFIS number 4536-2016031 017075121 v5). Animal experiments were carried out under the direct supervision of a veterinary specialist, and according to Swiss law and guidelines on Animal Welfare and the specific regulations of the Canton of Zurich under permit numbers 130/2012 and 019/2016, as approved by the Veterinary Office and the Ethics Committee of the Canton of Zurich (Kantonales Veterinäramt Zürich).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.