Parasites and human cell culture
Human primary fibroblasts (HFFs, ATCC® CCL-171™) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 10 mM (4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid) (HEPES) buffer pH 7.2, 2 mM l-glutamine, and 50 μg/mL of penicillin and streptomycin (Invitrogen). Cells were incubated at 37 °C in 5% CO2. The Toxoplasma strains used in this study and listed in Additional file 2: Table S1 were maintained in vitro by serial passage on monolayers of HFFs. The 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 (RRID: AB_2713903), rabbit anti-H4K31ac (RRID: AB_2811024), rabbit anti-H4K31me1 (RRID: AB_2811025), rat anti-CC2 (gift from W. Bohne and U. Gross), mouse anti-TgBAG1, mouse anti-HA tag (Roche, RRID: AB_2314622), rabbit anti-HA Tag (Cell Signaling Technology, RRID: AB_1549585), rabbit anti-acetyl-Histone H4, and pan (Lys 5,8,12) (Millipore, RRID:AB_310270). Immunofluorescence secondary 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) or horseradish peroxidase. We also raised homemade BCLA-specific antibodies in rabbit against linear peptides of BCLA, i.e., peptide 1: C-EMERPAAGSMEK and peptide 2: C-VLPKHETKPALT. They were produced by Eurogentec and used for immunofluorescence and immunoblotting.
Mouse infection and experimental survey
Six-week-old BALB/c, CBA, NMRI, C57BL/6, or Swiss mice were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France). Mouse care and experimental procedures were performed 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 (agreement n°B3851610006). Female mice were used for all studies. For intraperitoneal (i.p.) infection; tachyzoites were grown in vitro and extracted from host cells by passage through a 27-gauge needle, washed three times in phosphate-buffered saline (PBS), and quantified with a hemocytometer. Parasites were diluted in Hank’s Balanced Salt Solution (Life), and mice were inoculated by the i.p. route with tachyzoites of each strain (in 200 μl volume) using a 28-gauge needle. For oral gavage of infectious cysts, the brains from chronically infected mice (76K-GFP-luc and 76K-GFP-luc-Δbcla) were crushed in PBS, the number of cysts was microscopically quantified, and the mice were forced fed with 100 μl of brain homogenate containing 20 to 40 cysts using ball-tipped feeding needle. Blood was collected by caudal puncture or by intracardiac puncture when the mice were euthanized. Animal euthanasia was completed in an approved CO2 chamber. For histological analysis of ileum and immunolabeling on histological sections of the brains, the ilea and brains were removed from mice, entirely embedded in a paraffin wax block and cut in 5-μm-thick layers using microtome. For statistical analysis of mouse survival data, the Mantel-Cox and Gehan-Breslow-Wilcoxon tests were used.
Auxin-induced degradation
Depletion of MORC-AID-HA was achieved with 3-indoleacetic acid (IAA, Sigma-Aldrich # 45533) used at 500 μM final concentration from a 500-mM stock solution prepared in EtOH. To monitor the degradation of AID-tagged proteins, parasites grown in HFF monolayers were exposed to auxin, or the ethanol vehicle alone, for different time intervals at 37 °C before parasites were harvested and analyzed by immunofluorescence or Western blotting.
HDAC3 inhibition using FR235222
FR235222 was provided by Astellas Pharma Inc. (Osaka, Japan) and dissolved into DMSO, and the final concentration in the culture medium was either 25 or 50 ng/mL. Sixteen hours after infection of HFF monolayers, FR235222 was added to the medium and cells cultivated for 24 h to 7 more days.
Immunofluorescence microscopy
T. gondii-infected HFF monolayers grown or cysts purified from mouse’s brains 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 PBS containing 3% (w/v) bovine serum albumin (BSA). For immunolabeling on histological sections of the brains, the brain layers spotted on glass slides were first solvent-dewaxed using toluene for 3 times 10 min and absolute alcohol for 3 times 10 min. The slides were then treated with citrate buffer pH 6, heated at 100 °C during 1 h, rinsed extensively with water and blocked in PBS containing 3% (v/v) BSA. The infected cells or brain layers were then incubated for 1 h with the primary antibodies indicated in the figures followed by the addition of secondary antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes) at a 1:1000 dilution for 1 h. The nuclei of both host cells and parasites were stained for 10 min at room temperature with Hoechst 33258 at 2 μg/mL in PBS. After four washes in PBS, coverslips were mounted on a glass slide with Mowiol mounting medium; images were acquired with a fluorescence ZEISS ApoTome.2 microscope and processed with the ZEN software (Zeiss).
Western blot
Immunoblot analysis of protein was performed as described [32]. Briefly ~ 107 cells were lysed in 50 μl lysis buffer (10 mM Tris-HCl, pH 6.8, 0.5% SDS [v/v], 10% glycerol [v/v], and 1 mM EDTA and protease inhibitors cocktail) and sonicated. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (PVDF, Immobilon-P; EMD Millipore) by liquid transfer 1h30 at 100 V; blotted membranes were then probed using appropriate primary antibodies followed by alkaline phosphatase or horseradish peroxidase-conjugated goat secondary antibodies (Life technologies). Band revelation was detected using NBT-BCIP (Amresco) or enhanced chemiluminescence system (Thermo Scientific).
Cyst purification
Cysts were isolated from the brains of mice chronically infected with the 76k-GFP-luc or the 76k-GFP-luc-Δbcla strains for at least 6 weeks, either using the Percoll gradient method as described previously [33] either directly harvesting the cysts using a 10-μl pipet for the dyes experimentation in order not to deteriorate the cyst wall for permeability studies. Neither saponin nor trypsin was added at the end of the experiment.
Cyst quantification
Five to 12 weeks post-infection, the brain of each of the recipient mouse was homogenized in 2 mL of PBS. The numbers of cysts in three or ten aliquots (200 μl each) of the brain suspensions were counted microscopically. For statistical analysis of cyst quantification differences between mice infected with 76K-GFP-luc and 76K-GFP-luc-Δbcla, the non-parametric Wilcoxon-Mann-Whitney test was applied.
Cyst characterization
Images of semi-purified cysts were acquired between slide and slip cover with a fluorescence ZEISS ApoTome.2 microscope. Cysts areas and GFP signals were quantified using the ZEN software (Zeiss). For statistical analysis of cysts areas and GFP-fluorescence differences between 76K-GFP-luc and 76K-GFP-luc-Δbcla cysts, the non-parametric Wilcoxon-Mann-Whitney test was applied.
Histological analysis of ileum
Fragments of the small intestines of mice infected with WT or bcla-deficient cysts were harvested on day 8 post-infection, fixed in 10% buffered formalin, and paraffin processed. Tissue sections that were 5-μm-thick were mounted on slides and stained with hematoxylin and eosin. The histological score was analyzed with the following parameters: intensity of the lamina propria inflammatory infiltration, thickening of lamina propria, and destruction of the villi and necrosis, which were evaluated by intensity, represented as arbitrary units ranging from zero (less intense or absent) to six (highly intense) for each parameter.
Quantitative PCR
The parasite loads in the brain or ileum were quantified following DNA extraction (QiAmp DNA mini kit, Qiagen) using the quantitative PCR targeting of the Toxoplasma-specific 529-bp repeat element [34]. For statistical analysis of parasitic load differences between mice infected with 76K-GFP-luc and 76K-GFP-luc-Δbcla, the non-parametric Wilcoxon-Mann-Whitney test was applied.
Quantitative RT-PCR analysis of interleukins in the brain and ileum
Total RNA was isolated from brains or ilea using TRIzol (Thermo Fisher Scientific). cDNA was synthesized with random hexamers by using the high Capacity RNA-to-cDNA kit (Applied Biosystems). Samples were analyzed by real-time quantitative PCR for appropriate probes (brain: TNF-α, INF-γ, IL-6, and IL-22β; ileum: INF-γ, CCL2, IL-22β, IL-18, and IL-1β) using the TaqMan Gene Expression Master Mix (Applied Biosystems). RNA levels were normalized using TBP levels. Quantitative RT-PCR was repeated for three independent biological replicates of each sample. For statistical analysis of RNA levels between mice infected with 76K-GFP-luc and 76K-GFP-luc-Δbcla, the non-parametric Wilcoxon-Mann-Whitney test was applied.
DBA lectin labeling on in vitro FR235222 parasites and ex vivo cysts
T. gondii-infected HFF cells grown on coverslips or cysts purified from the brains of mice 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 PBS containing 3% (v/v) BSA. The infected cells or cysts were stained with 1:100-diluted Dolichos lectin for 30 min. The stained vacuoles or cysts were examined with a fluorescence ZEISS ApoTome.2 microscope, and images were processed by the ZEN software (Zeiss).
Toxoplasma gondii transfection
T. gondii strains were electroporated with vectors in cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/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 performed in a 2 mm cuvette at 1.100 V, 25 Ω and 25 μF. When needed, the antibiotic (concentration) used for drug selection was chloramphenicol (20 μM), mycophenolic acid (25 μg/mL) with xanthine (50 μg/mL), pyrimethamine (3 μM), or 5-fluorodeoxyuracil (10 μM). Single-clone of stable transgenic tachyzoites were obtained by limiting dilution in 96-well plates and verified by immunofluorescence assay or genomic analysis.
Plasmid construction
The plasmids and primers for the genes of interest (GOI) used in this work are listed in Additional file 2: Table S1. To construct the vector pLIC-GOI-HAFlag, the coding sequence of GOI was amplified using primers LIC-GOI-Fwd and LIC-GOI-Rev using T. gondii genomic DNA as template. The resulting PCR product was cloned into the pLIC-HF-dhfr or pLIC-mCherry-dhfr vectors using the ligation-independent cloning (LIC) cloning method [35]. The plasmid pTOXO_Cas9-CRISPR was described previously [36]. Twenty mer-oligonucleotides corresponding to specific GOI were cloned using the Golden Gate strategy. Briefly, primers GOI-gRNA-Fwd and GOI-gRNA-Rev containing the sgRNA targeting GOI genomic sequence were phosphorylated, annealed, and ligated into the pTOXO_Cas9-CRISPR plasmid linearized with BsaI, leading to pTOXO_Cas9-CRISPR::sgGOI.
Recombinant expression of the C-terminal domain of BCLA (rBCLA)
Design and cloning
To recombinantly express the domain, the N-terminal boundary was chosen at the methionine 1089 and the original C-terminal end was conserved. DNA synthesis was performed by Genscript to generate a fusion construct composed of rBCLA (1089-1275) with a TEV cleavable N-terminal His-tag (Fig. 1d and Additional file 2: Table S1). Codon optimization for E. coli was performed, and the gene was cloned by Genscript within a pet30-(a) vector (Addgene) using NdeI and XhoI sites.
Recombinant expression
Transformation was performed using BL21(DE3)-CodonPlus—RIL chemically competent E. coli (Stratagene) which were incubated on ice with 1 μg of the pET30-(a) rBCLA plasmid for 10 min heat shocked at 42 °C for 45 s, pre-incubated 45 min in LB at 37 °C then spread on a LB agar plate containing kanamycin (Kan) and chloramphenicol (Chlo) and incubated for 12 h. A single colony was then picked to inoculate a LB/Kan/Chlo 50 mL pre-culture grown for 16 h; 5 mL of grown pre-culture was then used to inoculate 1-L flasks of Terrific Broth medium (Formedium) containing Chlo/Kan. Cultures were grown at 37 °C until reaching an OD600 of 0.5–0.8 then induced by adding 0.7 mM IPTG (VWR) and further incubated at 18 °C ON. After incubation, cells were centrifuged 25 min at 3000g, the supernatant was discarded, and the pellet flash-frozen in liquid nitrogen and kept at − 80 °C.
Bacteria lysis
Purification was performed on 3 pellets of 1 L culture, each resuspended in 50 mL of lysis buffer containing (600 mM NaCl, 50 mM Tris pH 8, 5 mM beta-mercaptoethanol (BME), 0.2% w/v N-Lauryl Sarkozine and 1 complete anti-protease cocktail (Roche) tab per 50 mL) and subjected to a 10-min pulsed sonication (15 s ON, 30 s OFF) at 50° amplitude over ice to remain below 13 °C. After sonication, the lysate was centrifuged at 4 °C for 1 h at 15,000g, and the pellet was discarded. All the following steps were subsequent at 4 °C. Prior to incubation with 5 mL of pre-equilibrated Ni-NTA resin, the clarified lysate was supplemented with 30 mM imidazol. Batch incubation was performed for 30 min at 4 °C with a gentle stirring. After incubation, the resin was retained on a vertical column then washed with 4 column volumes (CV) of wash buffer (600 mM NaCl, 50 mM Tris pH 8, 5 mM BME, 0.2% w/v N-Lauryl Sarkosine and 30 mM Imidazole). Elution was then performed stepwise by collecting fraction of 1.5 mL using the elution buffer (300 mM NaCl, 50 mM Tris pH 8, 5 mM BME, and 300 mM imidazole). Fractions of interest were then pooled and dialyzed in 50 mM NaCl, 50 mM Tris pH 8.5 mM BME using a 10-kDa cutoff dialysis cassette (Thermo Scientific).
Ion exchange and size exclusion chromatography
The entire sample was then directly pumped through the chromatography system (Akta Pure, GE healthcare) onto a HL-Mono-Q (GE healthcare) 5 mL column pre-equilibrated with the same buffer as for dialysis. The column was washed with 2 CV then eluted by a salt gradient (50 mM to 2 M NaCl) over 40 mL, 1.5 mL fractions and 280 nm absorbance monitoring was performed over the whole elution period. SDS PAGE analysis revealed that the sample was purified in the later stages of the gradient elution and that the early elution fractions presented most of the bacterial contaminants visible at higher molecular weight. Desired fractions were collected, pooled, and concentrated to 600 μl using a 10-kDa cutoff concentrator (Amicon-Ultra, Millipore). After concentration, the sample was injected on a S75 (GE healthcare) with a running buffer containing 150 mM NaCl, 50 mM Tris pH 8, and 5 mM BME and eluted in a heterogeneous peak consistent with a multimeric state, starting close to the void volume and eluting over 3 mL. All elution fractions were pooled to generate the final sample.
Ammonium sulfate precipitation
To avoid nucleic acid contamination, an ammonium precipitation was performed by adding 15% w/v of ammonium sulfate (Sigma), gentle rolling at 4 °C for 1 h then 30 min of centrifugation at 10,000g. The supernatant was discarded, and the pellet resuspended in the same initial volume of buffer. To clear all ammonium sulfate, the sample was dialyzed in the same buffer as for the size exclusion.
Identification of anti-BCLA circulating antibodies by Western blot
Single Western blot strips were prepared using 15-well 4–12% NuPage gels (Life technologies) loaded with 5 μl of sample at 0.1 mg/mL. The gels were run at 185 V for 40 min in MES buffer then electro-transferred at 105 V for 1.5 h on PVDF membranes. The transferred lanes were then cut out into individualized strips. The strips were then blocked in Tris buffer saline with Tween (TTBS) with 5% powdered milk (w/v) for 1 h. Serum testing was then performed in TTBS with a dilution of 1:400 of the serum for 1 h at 4 °C. The strips were then extended washed in TTBS and further incubated 1 h with a 1:7500 dilution of secondary antibody targeting either mouse IgGs or human IgGs and coupled with a phosphatase alkaline enzyme (Promega). Following extended TTBS wash steps, the blots were revealed by the addition of the chromogenic substrate at room temperature (RT) (Invitrogen). Bands in the positive sera appear within 1 to 5 min. In addition to the serum testing, a single strip was always used as an internal antigen control for each blot set. After blocking, this strip was incubated for 1 h with a peroxidase coupled anti poly-histidine monoclonal antibody (Sigma) diluted 1:2000 in TTBS. After extended wash steps in TTBS, the blot was revealed using the SigmaFast DAB with metal enhancers (Sigma). For each series of i.p. or orally infected mice, a serum of at least one mouse was checked for Toxoplasma antibodies using Western blot analysis of the IgG immune response using the commercial kit LD bio Toxoplasma mouse IgG (LD bio), with the same anti-mouse IgG-alkaline phosphatase conjugate and chromogenic substrate previously described as for BCLA.
BCLA ELISA
Peptide synthesis
The following BCLA peptides were synthetized by Genscript with N-terminal Acetyl groups:
AB_F: Nter-MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP-Cter.
A3_B: Nter-AAGSMEKDKLVLPGE-Cter.
Plate preparation
Midisorp plates (Nunc) were coated overnight (O.N) at 4 °C with rBCLA, peptides AB_F and A3_B all at 2 μg/mL in 100 mM calcium carbonate buffer pH 9.6 with 100 μl per well. After coating, plates were washed twice with 350 μl of DPBS 0.05% Tween 20 (DPBS/Tween) then blocked for at least 2 h with 300 μl Superblock blocking buffer (Thermo Fisher) after which the buffer was removed and the plates dried upside down. Once dried, the plates could be stored for extended periods of time at 4 °C with no loss in serological reactivity.
Sample preparation
All serum dilutions were prepared in DPBS 0.05% Tween 20, 0.1% BSA no more than 2 h prior to the assay. For both mouse and human tested sera, 1:400 dilutions were prepared. Eleven standards were also freshly prepared in both tests, consisting of 10 serial dilutions of a positive frozen stock serum set at 100 UI. Starting at a dilution 1:200 and following a ¾ dilution increment, the following titration points were prepared: 200 UI (1:200), 150 UI (1:266), 112.5 UI (1:356), 84.4 UI (1:474), 63.3 UI (1:632), 47.5 UI, (1:843) 35.6 UI (1:1124), 26.7 UI (1:1498), 20 UI (1:1998), and 15 UI (1:2663). A 0 UI standard was prepared with a seronegative serum diluted at 1:400.
Assay
All the subsequent steps were implemented on the Gemini ELISA automation platform (Stratec) but could also be performed manually at RT. Dried plates were first washed twice with 350 μl of DPBS/Tween. Dilutions of the tested sera and standards were then distributed in the plates as row duplicates with 100 μl per well. Plates were then incubated 1 h at RT. After the incubation period, plates were washed 4 times with 350 μl of DPBS/Tween, 100 μl of peroxidase coupled secondary antibody dilution (1:50,000 anti-mouse IgG or 1:60,000 anti-human IgG, Sigma Aldrich ref A0168 and A0170 respectively) in DPBS 0.05% Tween 20, 0.1% BSA were then rapidly distributed in all wells. After 1 h at RT, plates were washed 4 times in DPBS-Tween. Revelation reaction were performed by adding 100 μl of TMB Substrate (Thermo Fisher ref 34029) for 20 min precisely at RT then stopping the reaction with 50 μl of H2SO4 0.2 M followed by 30 s of mixing. Well absorbance measurement was then performed using the Gemini integrated spectrophotometer at 450 nm.
Data treatment
Blank subtractions were performed on duplicate blank wells for which where primary antibody/sera were omitted but treated similarly as the others for all subsequent steps (washes, secondary Ab, substrate). Standard serum dilutions were averaged and fitted with a 4-parameter logistic regression with the upper asymptote value (Di) fixed at 2.5 AU and all other variables (Ai, Bi, Ci) allowed to fit. From this regression, tested dilution duplicates could have their apparent UI calculated and averaged; if in a duplicate measurement the coefficient of variation was observed above 10%, then the sample would be re-tested. All the ELISA data presented in this work was obtained several times in independent titrations.
Peptide dot blot screening
Dot blot peptide assays were custom synthetized by JPT Peptide technologies on cellulose membranes with N-acetyl moieties on the N-terminus. Two sets of membranes were screened: (1) covering the rBCLA region (res 1089-1275) with a total of 59 peptides, each 15 aa long with an overlap of 12 and an offset of 3; (2) covering repeat 4 (res 446-493) with a total of 18 peptides, each 15 aa long with an overlap of 12 and an offset of 3. Dot blot assays were performed as described by the manufacturer. Briefly, the membranes were first activated 5 min in 100% ethanol then washed 3 times 3 min in DPBS-Tween. Blocked O. N at 4 °C in DPBS-Tween 0.5% powdered milk then washed again 3 times 3 min in DPBS-Tween. Tested sera were diluted to 1/400 in DPBS-Tween 0.1% BSA and incubated for 3 h at RT with the membrane. Following a 3 times 3 min DPBS-Tween wash, the membranes were incubated with anti-IgG peroxidase coupled Ab (Sigma A0170) diluted to 1/100,000 for 2 h at RT. Following a 3 times 3 min wash in DPBS-Tween, the membrane was briefly immerged in the freshly prepared SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) and revealed using the C-Digit (Licor) scanner. Dot intensity was integrated using ImageJ. For data analysis of independent dot blots, integrated intensities from every peptide dot [I(p)] were normalized into enrichment factors Fe(p) using the baseline integrated intensity of peptide 59 [I(p = 59)] which never reacts with any sera. The following can be expressed with the following equation:
$$ {\mathrm{Fe}}_{(p)}=\raisebox{1ex}{${I}_{(p)}$}\!\left/ \!\raisebox{-1ex}{${I}_{\left(p=59\right)}$}\right. $$
where p represents the peptide number.
In order to increase the reactivity score over 5 independent serum blots (from patients positive to rBCLA by Western blot) symbolized as (+), Fe(p) enrichment scores were summed with each other, and to subtract the non-specific reactivity, the same sum was performed on 5 negative serum (all from Sag1-negative patients) peptides, symbolized as (−) and subtracted. Peptide reactivity scores can be expressed though the following equation:
$$ {\mathrm{Rs}}_{(p)}=\left[\sum {\left({\mathrm{Fe}}_{(p)}\right)}^{\left(+\right)}-\sum {\left({\mathrm{Fe}}_{(p)}\right)}^{\left(-\right)}\right] $$
where Rs(p) is the total reactivity score at a specific peptide position.
Human sera
Human sera were retrospectively selected from the biobank collection of the Parasitology-Mycology Clinical Laboratory in Grenoble Alpes University Hospital in France. This biobank is registered with the French Ministry of Health under the number DC-2008-582. The Quality Management System is applied in the Parasitology-Mycology Clinical Laboratory in the University Grenoble Alpes Hospital and our laboratory is accredited according to ISO 15189 standard. All the sera were collected in agreement with the institution and after no patient’s opposition. The selected sera were stored for toxoplasmosis serological routine analysis between January 1, 2014, and May 1, 2018. The analyses with Vidas® Toxo IgM and IgG (bioMérieux, France) and Architect Toxo IgG and IgM (Abbott, Germany) were performed in the Parasitology-Mycology Clinical Laboratory of Grenoble Alpes University Hospital. Briefly, the antibody titers for IgG were quantitatively expressed in IU/mL whereas IgM were expressed as an index. The cutoffs defined by Vidas®, bioMérieux manufacturer were as follows: (i) IgG (IU/mL): negative < 4.0; 4.0 ≤ equivocal (gray zone) < 8; ≥ 8 positive; (ii) IgM (index): negative < 0.55; 0.55 ≤ equivocal (gray zone) < 0.65; ≥ 0.65 positive. The cutoffs defined by Architect®, Abbott manufacturer were as follows: (i) IgG (IU/mL): negative < 1.6; 1.6 ≤ equivocal (gray zone) < 3.0; ≥ 3.0 positive; (ii) IgM (index): negative < 0.50; 0.50 ≤ equivocal (gray zone) < 0.60; ≥ 0.60 positive [29]. BCLA titers were measured on 156 selected patients’ sera corresponding to five different biological and clinical forms of toxoplasmosis (Additional file 7: Table S2 and Table 1): non-immunized patients against toxoplasmosis (seronegative, n = 43), patients with past immunity (chronic toxoplasmosis, n = 79), patients with proved ocular toxoplasmosis (ocular toxoplasmosis, n = 18), and immunocompromised patients with asymptomatic serological reactivation (serological reactivation, n = 5) or with toxoplasmosis disease (disseminated toxoplasmosis, n = 7). The absence of T. gondii immunity was concluded when the IgM- and IgG-specific antibody levels measured using Architect Toxo® IgG and Toxo® IgM assays were negative. Past immunity was considered when IgG were above the threshold of positivity with at least one method, and the IgM were negative or weakly positive (Architect® and Vidas®). Proved ocular toxoplasmosis (OT) was confirmed by detection of either Toxoplasma DNA using PCR or a local production of IgG and/or IgA antibodies by Western blot (LDBio®) [37]. Cerebral (n = 5), pulmonary (n = 2), disseminated (n = 1), and acute toxoplasmosis (n = 1) were diagnosed by PCR in cerebrospinal fluid, brain biopsy, bronchoalveolar fluid, and/or buffy coat in nine immunocompromised patients (acquired immunodeficiency syndrome, allogeneic hematopoietic stem cell transplant and kidney transplant recipients). These patients had clinical symptoms and radiological evidence of active disease [7, 38]. Asymptomatic serological reactivations were observed in immunocompromised patients during the serological follow-up. In these five patients, an increase of IgG levels compared to previous serological results were observed; furthermore, the Toxoplasma-PCR performed were negative, and these patients did not develop any clinical signs of toxoplasmosis [39].
Protein purification-, immunoblotting-, and mass spectrometry-based proteomic analysis
PruΔku80-BCLA-HAFlag-infected host HFF cell extracts containing Flag-tagged protein were incubated with anti-FLAG M2 affinity gel (Sigma-Aldrich) for 1 h at 4 °C. Beads were washed with a 10 CV of BC500 buffer (20% glycerol, 20 mM Tris-HCl pH 8.0, 500 mM KCl, 0.05% NP-40, 100 mM PMSF (phenylmethyl sulfonyl fluoride), 0.5 mM DTT, and 1× protease inhibitors). Bound peptides were eluted stepwise with 250 g/mL FLAG peptide (Sigma-Aldrich) diluted in BC100 buffer. Protein bands were excised from colloidal blue-stained gels (Thermo Fisher Scientific), treated with DTT and iodoacetamide to alkylate the cysteines before in-gel digestion using modified trypsin (Sequencing grade; Promega). The resulting peptides from individual bands were analyzed by online nanoLC-MS/MS (UltiMate 3000 coupled to LTQ-Orbitrap Velos Pro; Thermo Fisher Scientific) using a 25-min gradient. Peptides and proteins were identified and quantified using MaxQuant (version 1.5.3.17) through concomitant searches against ToxoDB (20151112 version), and the frequently observed contaminant database embedded in MaxQuant. The minimum peptide length was set to 7 amino acids. The minimum number of peptides, razor + unique peptides, and unique peptides was all set to 1. Maximum false discovery rates were set to 0.01 at peptide and protein levels.
Mass spectrometry-based proteome-wide analyses
HFF cells were grown to confluence and infected with type I (RHΔku80 or RHΔku80 MORC KD) or type II (PruΔku80 or PruΔku80 MORC KD) strains before lysis in 8 M urea and 50 mM HEPES. Extracted proteins were reduced using 20 mM of dithiothreitol for 1 h at 37 °C before alkylation with 55 mM of iodoacetamide for 45 min at room temperature in the dark. The samples were then diluted using ammonium bicarbonate to obtain a urea concentration of 4 M. Proteins were digested with LysC (Promega) at a ratio of 1:200 during 4 h at 37 °C. The samples were diluted again using ammonium bicarbonate to obtain a urea concentration of 1 M. Proteins were then digested with Trypsin (Promega) at a ratio of 1:200 overnight at 37 °C. The resulting peptides were purified by C18 reverse phase chromatography (Sep-Pak C18, Waters) before drying down before fractionation by tip-based strong cation exchange (3 M Empore). For this, peptides were dissolved in 5% acetonitrile, 1% TFA and eluted in 4 fractions (F1: 100 mM ammonium acetate, 20% ACN, 0.5% formic acid; F2: 175 mM ammonium acetate, 20% ACN, 0.5% formic acid; F3: 375 mM ammonium acetate, 20% ACN, 0.5% formic acid; F4: 80% acetonitrile, 5% ammonium hydroxide) before desalting using C18 reverse phase chromatography (Ultra-Micro SpinColumns, Harvard Apparatus). Technical triplicates were performed. NanoLC-MS/MS analyses were performed using an Ultimate 3000 RSLCnano coupled to a Q-Exactive Plus (Thermo Scientific). Peptides were sampled on a 300 μm × 5 mm PepMap C18 precolumn and separated on a 75 μm × 250 mm PepMap C18 column (Thermo Scientific) using a 120-min gradient. MS and MS/MS data were acquired using the Xcalibur software (Thermo Scientific). RAW files were processed using MaxQuant48 version 1.6.2.10. Spectra were searched against the Toxoplasma gondii database (ME49 taxonomy, version 30 downloaded from ToxoDB, the Uniprot database (Homo sapiens taxonomy, April 2019 version), the frequently observed contaminants database embedded in MaxQuant, and the corresponding reverse databases. Trypsin was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set at their default values. Peptide modifications allowed during the search were carbamidomethyl (C, fixed), acetyl (protein N-term, variable), and oxidation (M, variable). The minimum number of peptides and razor + unique peptides was set to 1. Maximum false discovery rates were set to 0.01 at PSM and protein levels. The match between runs option was activated. Statistical analyses were performed using ProStaR. Peptides and proteins identified in the reverse, and contaminant databases or matching to human sequences were discarded. Only proteins quantified in at least 3 replicates of one condition were conserved. After log2 transformation, protein intensities were normalized using the summed intensities of a pool of proteins for which transcripts were found invariable in RNA-seq dataset (0.91 < fold change < 1.1). Missing values were then imputed (slsa method for Partially Observed Values and DetQuantile with quantile set to 2.5 and factor set to 1 for missing entirely in the condition). Statistical testing was conducted using limma. Differentially abundant proteins were sorted out using the following cutoffs: log2(fold change) ≥ 0.8 or ≤ − 0.8 and P values allowing to reach an FDR ~ 1% according to the Benjamini-Hochberg estimator. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD016845.
Chromatin Immunoprecipitation and next-generation sequencing in T. gondii
HFF cells were grown to confluence and infected with type II (Pru∆ku80 or Pru∆ku80 MORC KD) strains. Harvested intracellular parasites were crosslinked with formaldehyde (final concentration 1%) for 8 min at room temperature, and the crosslinking was stopped by addition of glycine (final concentration 0.125 M) for 5 min at room temperature. Crosslinked chromatin was lysed in ice-cold lysis buffer (50 mM HEPES KOH pH 7.5, 14 0 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.125% Triton X-100, protease inhibitor cocktail) and sheared in shearing buffer (1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 10 mM Tris pH 8.0, protease inhibitor cocktail) by sonication using a Diagenode Biorupter. Samples were sonicated by 16 cycles of 30 s ON and 30 s OFF, to 200–500 base-pair average size. Immunoprecipitation was carried out using sheared chromatin, 5% BSA, protease inhibitor cocktail, 10% Triton X-100, 10% deoxycholate, DiaMag Protein A-coated magnetic beads (Diagenode), and antibodies targeting histone PTM or protein of interest. A rabbit IgG antiserum was used as a control mock. After overnight incubation at 4 °C on a rotating wheel, chromatin-antibody complexes were washed and eluted from beads by using iDeal ChIP-seq kit for Histones (Diagenode) according to the manufacturer’s protocol. Samples were de-crosslinked by heating for 4 h at 65 °C. DNA was purified by using the IPure kit (Diagenode) and quantified by using Qubit Assays (Thermo Fisher Scientific) according to the manufacturer’s protocol. For ChIP-seq, purified DNA was used to prepare libraries and then sequenced by Arraystar (USA).
Library preparation, sequencing, and data analysis (Arraystar)
ChIP-Sequencing library preparation was performed according to Illumina’s protocol Preparing samples for ChIP sequencing of DNA. Library preparation: 10 ng DNA of each sample was converted to phosphorylated blunt-ended with T4 DNA polymerase, Klenow polymerase, and T4 polymerase (NEB); An ‘A’ base was added to the 3′ end of the blunt phosphorylated DNA fragments using the polymerase activity of Klenow (exo minus) polymerase (NEB); Illumina’s genomic adapters were ligated to the A tailed DNA fragments; PCR amplification was performed to enrich ligated fragments using Phusion High Fidelity PCR Master Mix with HF Buffer (Finnzymes Oy). The enriched product of ~ 200–700 bp was cut out from the gel and purified. Sequencing: The library was denatured with 0.1 M NaOH to generate single-stranded DNA molecules and loaded onto channels of the flow cell at 8 pM concentration, amplified in situ using TruSeq Rapid SR cluster kit (#GD-402-4001, Illumina). Sequencing was carried out by running 100 cycles on Illumina HiSeq 4000 according to the manufacturer’s instructions. Data analysis: after the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using the Off-Line Basecaller software (OLB V1.8). After passing the Solexa CHASTITY quality filter, the clean reads were aligned to T. gondii reference genome (Tgo) using BOWTIE (V2.1.0). Aligned reads were used for peak calling of the ChIP regions using MACS V1.4.0. Statistically significant ChIP-enriched regions (peaks) were identified by comparison of two samples, using a P value threshold of 10−5. Then, the peaks in each sample were annotated by the overlapped gene using the newest T. gondii database. The EXCEL/BED format file containing the ChIP-enriched regions was generated for each sample. Data visualization: the mapped 100 bp reads represent enriched DNA fragments by the ChIP experiment. Any region of interest in the raw ChIP-seq signal profile can be directly visualized in a genome browser. We use 10-bp resolution intervals (10-bp bins) to partition the stacked reads region and count the number of reads in each bin. All the 10-bp resolution ChIP-seq profiles of each sample are saved as UCSC wig format files, which can be visualized in T. gondii Genome Browser (http://protists.ensembl.org/Toxoplasma_gondii/Info/Index). All these raw and processed files can be found at Series GSE136060.
RNA-seq and sequence alignment
Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA quantity and quality were measured by NanoDrop 2000 (Thermo Scientific). RNA integrity was assessed by standard non-denaturing 1.2% TBE agarose gel electrophoresis. The ribosomal large subunits from Toxoplasma and the host cells were used to verify that the ratio between Toxoplasma RNA versus host RNA was equivalent between the different biological samples, thus indicating that the samples had equivalent infection rates. For each condition, total RNAs from two independent biological replicates were pooled to prepare cDNA libraries which were then sequenced using Illumina technology in a single replicate dataset. RNA sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). Briefly, the RNA quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and Illumina TruSEQ RNA library prep and sequencing reagents were used following the manufacturer’s recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2 × 125 bp) on the Illumina Hiseq 2500 platform and generated at least 70 million reads for each sample. The RNA-seq reads (FASTQ) were processed and analyzed using the Lasergene Genomics Suite version 14 (DNASTAR, Madison, WI, USA) using default parameters. The paired-end reads were uploaded onto the SeqMan NGen (version 14, DNASTAR. Madison, WI, USA) platform for reference-based assembly using either the Mus musculus genome package (GRCm38.p3) or the Toxoplasma type II ME49 strain (ToxoDB-24, ME49 genome) as reference template. The ArrayStar module (version 14, DNASTAR. Madison, WI, USA) was used for normalization, differential gene expression and statistical analysis of uniquely mapped paired-end reads using the default parameters. The expression data quantification and normalization were calculated using the RPKM (reads per kilobase of transcript per million mapped reads) normalization method. All these raw and processed files can be found at Series GSE136123.