MAG2, a Toxoplasma gondii Bradyzoite Stage-Specific Cyst Matrix Protein

Toxoplasma gondii cell culture and differentiation.Host cells consisting of human foreskin fibroblasts (HFF) were cultured at pH 7 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% EquaFETAL bovine serum (Atlas Biologicals) and 5% penicillin-streptomycin (Life Technologies) at 5% CO₂ and 37°C. Confluent HFF cells were inoculated with parasite strains for passaging using standard techniques (51). For differentiation into bradyzoites, parasites were inoculated into HFF cells and incubated for 2 h before replacement of the culture media with pH 8 DMEM containing 50 mM HEPES (Sigma-Aldrich), 1% penicillin-streptomycin, and 1% FBS and lacking sodium bicarbonate. Induced cultures were incubated at 37°C in the absence of CO₂ for 3 to 7 days.

Hybridoma generation and screening.The bradyzoite antigen hybridoma library was generated as previously described (7). Briefly, BALB/c^dm1 mice (52) were infected with ME49 parasites; after 4 weeks, brain cysts from infected mice were isolated by Percoll gradient ultracentrifugation (53). Isolated cysts were fractured by freeze-thaw cycles and emulsified with Freund’s complete adjuvant before subcutaneous injection into BALB/c mice. After 2 months, spleens from immunized mice were fused with myeloma cells to generate a hybridoma library (54). Using immunofluorescence assays (IFA), hybridoma supernatant was screened for reactivity against cysts from ΔCST1 parasites (7). Positive hybridoma clones were subcloned on agar plates and cultured in a CELLine Bioreactor (Wheaton) to increase antibody production.

Immunoprecipitation (IP) of the 20C3 MAb antigen.HFF cells were infected with Pru Δku80 Δhxgprt parasites at a multiplicity of infection (MOI) of 1. Parasites were differentiated to bradyzoites and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40). Bradyzoite lysates were precleared by incubation with protein G agarose beads for 30 min at 4°C to remove nonspecifically bound proteins. Then, precleared samples were incubated with protein G agarose beads covalently cross-linked with clone 20C3 monoclonal antibody (MAb) by the use of bis(sulfosuccinimidyl)suberate (BS³; Thermo Scientific) according to the manufacturer’s instructions. Precleared samples were incubated with 20C3 MAb-bound beads overnight at 4°C with rotation. Beads were washed twice with RIPA buffer before elution in Laemmli sample buffer. 20C3 MAb, lysate input, bead flowthrough, RIPA washes, and bead eluate were resolved on a 4% to 15% polyacrylamide gel (Bio-Rad) and stained with Coomassie blue. Bands in the eluate lanes were excised and analyzed by mass spectrometry.

Mass spectrometry (MS) analysis.Excised gel bands were reduced, alkylated, and digested with trypsin. LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analysis of the peptide digests was done by C₁₈ reversed-phase (RP) chromatography using an Ultimate 3000 RSLCnano system (Thermo Scientific, USA) equipped with an Acclaim PepMap rapid-separation liquid chromatography (RSLC) C₁₈ column (Thermo Scientific, USA) (2-μm pore size, 100 Å, 75 μm by 15 cm). The ultra-high-performance liquid chromatography (UHPLC) instrument was connected to a TriVersa NanoMate nano-electrospray source (Advion, USA) and a linear ion trap LTQ-XL (Thermo Scientific, USA) mass spectrometer with ESI source operated in positive-ionization mode. Automated protein identification was performed by the use of Mascot search engine v. 2.5.1 (Matrix Science) against the ToxoV12_uniprot_20150225 database (27,608 entries) with the following search parameters: trypsin; three missed cleavages; peptide charges of +2 and +3; peptide tolerance of 2.0 Da; MS/MS tolerance of 0.8 Da; carbamidomethylation (Cys) for fixed modifications; deamidation (Asn and Gln) and oxidation (Met) for variable modifications. A decoy database search was also performed to measure the false-discovery rate. Mascot protein identification results were further analyzed by the use of Scaffold software v. 4.4.5 based on 99% protein and 95% peptide probabilities.

Immunofluorescence assay (IFA).Pru Δku80 Δhxgprt or transgenic parasite strains were inoculated into HFF cells on coverslips and incubated in pH 7 media for 1 day for tachyzoite staining or were differentiated to bradyzoites. Infected coverslips were rinsed twice with phosphate-buffered saline (PBS) before fixation was performed with 4% paraformaldehyde for 20 min followed by permeabilization in 0.2% Tween containing 0.1% glycine for 20 min to quench the fixative. Fixed and permeabilized cells were washed three times with PBS before blocking was performed with 1% bovine serum albumin (BSA)–PBS at room temperature for 1 h. 20C3 MAb, TgME49_209755 PAb, fluorescein isothiocyanate (FITC)-conjugated Dolichos biflorus agglutinin (DBA) (Vector Lab), ALD1 (Kentaro Kato), and SalmonE (7) were used as primary antibodies. Anti-mouse or anti-rabbit goat antibodies conjugated with Alexa Fluor 488 or 594 were used as secondary antibodies. All antibodies were diluted 1:500 in 1% BSA and incubated for 60 min at 37°C followed by three washes with PBS. Coverslips were mounted with ProLong Gold with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies), and images were acquired using a Leica TCS SP5 confocal microscope.

Polyclonal antibody production.The N-terminal coding region of MAG2 (aa 284 to 407) was PCR amplified using Q5 High-Fidelity DNA polymerase (New England Biolabs) and MAG2 N-terminal specific primers and ligated into pET32 vector. The resulting vector was transformed into BL21(DE3) competent Escherichia coli (NEB). Transformed bacteria were grown in Luria broth (LB; Sigma-Aldrich) containing ampicillin (100 μg/μl) for 5 to 6 h at 37°C and were induced with isopropyl β-d-1-thiogalactopyranoside (0.1 mM) for protein expression. The bacterial cells were pelleted, resuspended, and sonicated in lysis buffer (1% Triton X-100–10 mM imidazole–7 mM beta-mercaptoethanol–PBS), and the resulting supernatant was purified with nickel beads. Purified peptides were emulsified with Titer Max Gold and injected intraperitoneally into BALB/c^Δdm1 mice. Immunized mice were boosted every 14 days before sera were collected 1 month after initial immunization and checked for reactivity against in vitro T. gondii cysts by IFA. All primers used are listed in Table 2.

Endogenous knockout, complementation, and tagging strategy using CRISPR-Cas9.To knock out MAG2, p-HXGPRT-Cas9-GFP containing a single guide RNA (sgRNA) targeting the N terminus of MAG2 was prepared as previously described (55). Pru Δku80 Δhxgprt or ME49 Δhxgprt Δku80 parasites were cotransfected with 20 μg of uncut circular p-HXGPRT-Cas9-GFP and 280 pmol of each unannealed donor oligonucleotides in incomplete cytomix (56). Donor DNA consisted of 100 bp of oligonucleotides (Thermo Fisher) of the forward and reverse sequences of the N terminus of MAG2 containing four in frame stop codons. After transfection, transgenic parasites were selected with 25 μg/ml mycophenolic acid and 50 μg/ml xanthine (MPA-XA) for 10 days followed by immediate subcloning. Clones of MAG2-negative (ΔMAG2) parasites were screened by IFA for loss of 20C3 MAb signal. Because circular p-HXGPRT-Cas9-GFP was transfected into the parasites, sensitivity to MPA-XA was restored after loss of the HXGPRT resistance cassette after multiple rounds of parasite replication. In addition, loss of pHXGPRT-Cas9-GFP prevents constitutively expressed Cas9-mediated toxicity (57, 58).

To generate the MAG2 complement (MAG2-COMP) parasite line, a different sgRNA targeting the N terminus of MAG2 was ligated into p-HXGPRT-Cas9-GFP. The resulting plasmid was cotransfected with donor oligonucleotides, reversing the stop codons to synonymous mutations into ΔMAG2 parasites by the use of the same transfection and selection method as that described above. Loss of the HXGPRT resistance cassette was confirmed when the negative-control parasites (ΔMAG2 parasites transfected without p-HXGPRT-Cas9-GFP) did not grow under conditions of selection. Sanger sequencing of the N terminus of MAG2 and IFA were used to confirm successful knocking out and complementation of the MAG2 endogenous locus.

To tag MAG1 and MAG2 endogenously with mScarlet, the mScarlet coding sequence was amplified and ligated between bp 500 and 900 of the C terminus and 3’ untranslated region (3’UTR) of the respective genes with pUC19 as the backbone. Amplified donor plasmids were cut with SwaI and cotransfected with sgRNA targeting the C terminus of MAG1 or MAG2 on p-HXGPRT-Cas9-GFP into Pru Δku80 Δhxgprt parasites. Transfected parasites were selected with MPA-XA for 10 days and subcloned by screening for mScarlet fluorescent parasites. All primers and oligonucleotides used are listed in Table 2 and Table 3, respectively.

Expression of transgenes.The coding sequence of MAG2 and ∼2,400 bp upstream of its start codon were PCR amplified using Q5 High-Fidelity DNA polymerase (New England Biolabs) and placed into the pLIC-DHFR-BirA*-3×HA plasmid (19) with the BirA* tag attached to the C terminus of MAG2. Sanger sequencing of this plasmid revealed a truncated MAG2 sequence attached to BirA*-3×HA. The resulting plasmid was cut with PsiI and transfected into the Pru Δku80 Δhxgprt strain for random integration into the genome. Transfected parasites were selected with 1 μM pyrimethamine.

The CST1 promoter-driven mScarlet plasmid was constructed by placing the 5′UTR and signal peptide of CST1 before the N-terminal coding sequence of mScarlet followed by the 3′UTR of MAG1. This transgene was then ligated between (approximately) bp 500 and 750 of the 5′UTR and 3′UTR of the Ku80 gene (17) for direct integration of the mScarlet construct into the Ku80 locus. The CST1 promoter-driven mScarlet plasmid was cut with SwaI and cotransfected with p-HXGPRT-Cas9-GFP containing a sgRNA targeting the Ku80 locus into the Pru Δku80 Δhxgprt strain, and the transfected parasites were selected with MPA-XA. All primers used are listed in Table 2.

Immunoblotting.HFF cells that had been infected with T. gondii bradyzoites induced by 3 days of differentiation were harvested, centrifuged at 10,000 × g, and lysed in Laemmli buffer. The samples were resolved on a 4% to 15% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% BSA–Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature before being probed with 20C3 MAb or anti-HA rat monoclonal antibody conjugated with horseradish peroxidase (HRP; Roche) at 1:500 for 1 h at room temperature. Following 20C3 MAb primary antibody incubation, the membrane was washed three times with TBST and then incubated with sheep anti-mouse antibody conjugated with HRP (GE Healthcare) for 1 h at room temperature. The membrane was then washed and scanned using an Odyssey imaging system (Li-COR).

Fluorescence recovery after photobleaching.Parasites expressing mScarlet were inoculated into HFFs grown on a glass-bottom dish and either analyzed the next day for tachyzoite experiments or differentiated to bradyzoites for 4 days prior to analysis. Infected cells were viewed under a Zeiss LSM 5 Live duo-scan laser scanning microscope at a controlled temperature of 37°C. Images were acquired and analyzed using ZEN 2009 software. The sequence of the experiments consisted of an acquisition step of three images of unbleached vacuoles followed by 10 bleaching steps performed with a 100% laser setting at wavelengths 488 nm and 561 nm; fluorescence recovery images were taken every 2 s for 1 min. A total of 21 cysts/vacuoles were analyzed for each experimental group.

Cell fractionation.HFFs were infected and incubated with tachyzoites for 2 days or induced under bradyzoite induction conditions for 3 days. The infected cells were washed and scraped with cold PBS (containing 1× Roche cOmplete protease inhibitor cocktail [with EDTA], 5 mM NaF, and 2 mM activated Na₃VO₄). Cells were lysed by passage through 27.5-gauge needles, and intact parasites were separated by low-speed centrifugation at 2,500 × g for 10 min at 4°C. The resulting low-speed pellet (LSP) containing intact parasites was saved for further analysis, while the low-speed supernatant (LSS), which contained cyst or parasitophorous vacuole components, was separated into soluble and membrane-associated fractions by high-speed centrifugation at 100,000 × g for 1.5 h. The resulting high-speed supernatant (HSS) containing soluble cyst or parasitophorous vacuole components was saved, while the pellet was treated by resuspension in either PBS or 1 M NaCl to free peripheral membrane-associated proteins. The resuspended fractions were centrifuged at 100,000 × g to separate salt-released peripheral membrane proteins in the high-speed supernatant (HSS) from the membrane-bound proteins in the high-speed pellet (HSP). Before analysis by immunoblotting was performed, all soluble fractions were concentrated by acetone precipitation.

Plaque assay.Fifty purified tachyzoites of each transgenic parasite were inoculated into triplicate wells in 6-well culture plates containing confluent host HFF cells. After 2 weeks of culture without disturbance, cells were fixed with a 20% methanol and 0.5% crystal violet solution for 30 min at room temperature. After fixation, cells were washed once with distilled water before being imaged (Alpha Innotech) and analyzed with ImageJ to measure the size of plaques.

In vivo murine infection for survival curve, cyst number, and cyst size quantification.C57BL/6 mice (Jackson) were injected intraperitoneally with 1,000 parasites of the Pru Δku80 Δhxgprt, ΔMAG2, or MAG2-COMP strains. Infected mice were monitored for 30 days and noted for death. After 30 days, brains were retrieved from mice that survived and were homogenized in PBS using a Wheaton Potter-Elvehjem tissue grinder (Thermo Fisher) (100-μm to 150-μm clearance). An aliquot of the brain homogenate was screened for GFP fluorescent cysts using a Microphoto-FXA epifluorescence microscope (Nikon). Images of these cysts were analyzed with ImageJ to determine their sizes.

Transmission electron microscopy (TEM) and immunoelectron microscopy (IEM).For ultrastructural analyses using a TEM, ME49 Δhxgprt Δku80 and ME49 Δhxgprt Δku80 Δmag2 parasites were injected into BALB/c^Δdm1 mice at 1,000 parasites per mouse, followed by cyst harvesting and purification 3 weeks postinfection. For the Pru Δku80 Δhxgprt, ΔMAG2, and MAG2-COMP strains, cysts were prepared in vitro by incubation under bradyzoite induction conditions for 7 days. Cysts were fixed with 2.5% glutaraldehyde–2% paraformaldehyde–0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide followed by 2% uranyl acetate, dehydrated through a graded ethanol series, and embedded in LX112 resin (Ladd Research Industries, Burlington, VT). Ultrathin sections were cut on a Leica Ultracut UC7 ultramicrotome, stained with uranyl acetate followed by lead citrate, and viewed on a JEOL 1400EX transmission electron microscope at 80 kV.

For IEM, ME49 parasites were injected into BALB/c^Δdm1 mice at 1,000 parasites per mouse, followed by cyst harvesting and purification 3 weeks postinfection. Purified cysts were fixed with 4% paraformaldehyde–0.05% glutaraldehyde–0.1 M sodium cacodylate buffer, dehydrated through a graded ethanol series, with a progressive lowering of the temperature to −50°C in a Leica EMAFS (electron microscopy automatic free substitution) system, embedded in Lowicryl HM-20 monostep resin (Electron Microscopy Sciences), and then polymerized using UV light. Thin sections were immunolabeled with or without 20C3 MAb and were then stained with uranyl acetate (Electron Microscopy Sciences). Stained sections were viewed on a JEOL 1400EX transmission electron microscope at 80 kV.

Statistics.Analysis of the mScarlet and GFP recovery curves was performed by fitting the mScarlet and GFP recovery values at each time point over all samples with a linear mixed-effects model using the R package lme4. Post hoc comparisons were performed to determine the time points that represented significant differences between mScarlet and GFP recovery percentages by the use of the R package emmeans. For analysis of the FRAP parameters obtained from the ZEN 2009 program, the Welch two-sample t test was used to determine whether there were significant differences in the immobile fractions or in the time of equilibrium between mScarlet and GFP.

Data obtained from counting cysts and determining cyst sizes were graphed in R as violin plots. To compare multiple groups, one-way analysis of variance (ANOVA) followed by a post hoc Tukey honestly significant difference (HSD) test was performed. Comparisons of survival curves for mice infected with different parasite strains were performed using the R packages survminer and survival. The statistical significance of the survival curves was determined by the Gehan-Wilcox test. Pairwise comparisons of mice infected by each strain were performed using the Peto & Peto test.