Adaptation of BioID for Toxoplasma bradyzoite GRAs

Our previous success with using BioID to identify secreted GRAs in tachyzoites suggested that this approach would also work for bradyzoites. To improve our changes of obtaining bradyzoite GRAs, we made several modifications to our approach. First, we used as our background the PrugniaudΔku80 strain of T. gondii that contains GFP driven by the bradyzoite specific promoter LDH2, which has previously been shown to be effective at monitoring switching to bradyzoites [37]. Second, we sought to improve the efficiency of in vitro switching to bradyzoites, thus we deleted AP2IV-4 (S1 Fig), which encodes one of the nuclear Apetala-2 (AP2) proteins which has previously been shown to be important for the maintenance of the tachyzoite phenotype in vitro [38]. Lastly, we used the bradyzoite-upregulated GRA protein MAG1 [39,40] as our bait which we fused to the biotin ligase BirA* (Fig 1A). Deletion of AP2IV-4 and obtaining the PruΔku80::Δap2iv-4 and MAG1-BirA*::Δap2iv-4 parasites enables increased bradyzoite switching efficiency without disrupting the lytic cycle of the tachyzoites as previously described [38].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. MAG1-BirA* localizes to the PV and biotinylates proteins in the cyst vacuole.

(A) Diagram of the construct encoding the promoter and full genomic sequence of MAG1 fused to BirA* along with a 3xHA C-terminal epitope tag. (B) IFA of MAG1-BirA* showing that the fusion protein appropriately traffics to the tachyzoite PV and colocalizes with the known protein GRA14. Scale bar: 5 μm (applicable to all panels). (C) IFA of MAG1-BirA*-expressing parasites, showing the bradyzoite PV is labeled in a biotin-dependent manner (yellow arrowheads, +Biotin row). Endogenously biotinylated apicoplasts are observed with and without biotin (white arrows, -Biotin row). Scale bar: 5 μm (applicable to all panels in C). (D) Western blot of whole-cell lysates of parental (PrugniaudΔku80Δhxgprt) and MAG1-BirA*-expressing parasites -/+ biotin. Lysates were probed with streptavidin-HRP, revealing an increase in biotinylated proteins in MAG1-BirA*-expressing parasites upon addition of biotin. The MAG1-BirA* fusion protein is predicted to be ~105 kDa (arrowhead). (E) Western blot showing that MAG1-BirA* fusion migrates to ~105 kDa as detected by antibody against HA epitope tag.

https://doi.org/10.1371/journal.pone.0232552.g001

When we expressed the MAG1-BirA* fusion driven from its endogenous promoter, the fusion was readily detectable in tachyzoites and trafficked appropriately to the PV when examined by immunofluorescence assay (IFA) (Fig 1B). While MAG1 is considered a bradyzoite GRA, this detection was expected as it is clearly expressed in tachyzoites and is one of the stronger hits in our tachyzoite GRA BioID experiments [36]. Upon in vitro switching to bradyzoites, the fusion expressed in bradyzoites and trafficked appropriately to cyst vacuole where it co-localized with the known GRA marker GRA14 (Fig 1B) [41]. The fusion protein migrated at a molecular weight of ~105 kDa as predicted [42]

In vivo biotinylation of the PV using MAG1-BirA* parasites

After producing switch-efficient MAG1-BirA* parasites, we assessed whether the fusion protein was active in the PV. To do this, we switched MAG1-BirA* parasites in vitro using high pH media for 5 days and supplemented biotin to the media on the final day [43]. Transition from the tachyzoite to bradyzoite stage was confirmed by the presence of GFP in the parasite cytosol (Fig 1C). As a control, MAG1-BirA* parasites were switched on a separate monolayer, without biotin supplementation. While the control parasites just showed the expected apicoplast background staining, we observed streptavidin staining overlapping with the fusion protein in the parasite vacuole when we supplemented biotin (Fig 1C). This demonstrated that the biotin was able to penetrate the PV compartment and the MAG1-BirA* was active during the induced bradyzoite stage. We additionally demonstrated by western blot that there was an increase in the number of biotinylated proteins in MAG1-BirA* lysates when excess biotin was supplemented to the media (Fig 1D). We then proceeded with a large-scale MAG1-BioID experiment by infecting human foreskin fibroblasts with a high multiplicity of infection, switching them in vitro to bradyzoites over the course of 5 days, supplementing biotin on day 4. We then harvested the cells, lysed the samples using stringent conditions, and purified the biotinylated protein fraction using streptavidin-affinity chromatography.

As with previous experiments, mass spectrometry analysis revealed a large number of tachyzoite and bradyzoite GRAs highly ranked by peptide spectrum matches when we examined the MAG1-BirA* lysate in comparison to the parental lysate (Table 1 and S1 Table) [36]. We found the bait protein (MAG1) as well as almost all numbered GRAs, MYR proteins, TgIST1, WNG1, WNG2, bradyzoite GRAs (such as CST proteins, MCP3, MCP4, and BPK1), cyclophilin, NTPaseI and NTPaseII. BPK1 did appear, although slightly lower ranking in abundance. There were several expected proteins that were missing from our dataset including the exported proteins GRA19, GRA20 and GRA21 [44]. In total, the MAG1-BioID experiment revealed 537 proteins that were not present in the control lysate, 675 proteins in total including some GRAs that were present in comparatively low levels in the control lysate (S1 Table). GRAs constituted many of the highest-ranking proteins in this experiment, however, as expected many other secreted proteins including rhoptry proteins (ROPs/RONs), and microneme proteins (MICs) were also found concentrated in the top hits of the dataset.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Summary of known and novel GRAs found by MAG1-BioID.

https://doi.org/10.1371/journal.pone.0232552.t001

Identification of novel bradyzoite dense granule proteins from BioID datasets

Our dataset also contained a large number of hypothetical proteins. We narrowed this list of proteins as likely GRAs by selecting those proteins that were highly ranked in the dataset and lacked a C-terminal endoplasmic reticulum retention signal (K/HDEL) [11,45]. We prioritized those proteins with predicted signal peptides, however as many secreted proteins do not contain these sequences, we considered GRA candidates from the hypothetical proteins that also lacked this feature. Because we wished to focus on those proteins that were upregulated in bradyzoites, we also narrowed our candidate pool by focusing on proteins that were shown to be highly transcribed in bradyzoites. However, we also chose some candidates whose expression was similar between tachyzoites and bradyzoites [46]. We chose 9 candidates and used endogenous gene tagging to recombine a C-terminal 3xHA epitope tag for each gene product and examined their localization by IFA. We found that 5 of these trafficked to the PV in both tachyzoites and bradyzoites (Fig 2). We named these GRA55 (TgME49_309760), GRA56 (TgME49_309930), GRA57 (TgME49_217680), and GRA58 (TgME49_268790). In addition, while reviewing earlier datasets from a prior GRA-BioID experiments [36], we found another GRA–GRA59 (TgME49_313440)–which was also upregulated in bradyzoites. Of these confirmed GRA proteins, only GRA56 had an identifiable domain which has homology to melibiase, an alpha-galactosidase that catabolizes the disaccharide melibiose.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Identification of novel dense granule proteins, GRA55-GRA59 by MAG1-BioID.

(A) IFA of tachyzoites with rabbit anti-HA antibodies shows strong staining of the parasitophorous vacuole for each novel GRA that colocalizes with GRA14, demonstrating that these are novel dense granule proteins. Gene numbers and novel designations are as follows: GRA55 (TgME49_309760), GRA56 (TgME49_309930), GRA57 (TgME49_217680), GRA58 (TgME49_268790), and GRA59 (TgME49_313440). Scale bar: 5 μm. (B) IFA of bradyzoites with mouse anti-HA antibodies shows staining of each GRA at the cyst periphery and vacuolar space. Transgenic parasites expressed GFP, driven by LDH2 promoter, upon successful bradyzoite conversion. Images were taken three days after growth under alkaline-stressed conditions. Scale bar: 5 μm.

https://doi.org/10.1371/journal.pone.0232552.g002

Targeted deletion of novel GRA proteins and functional characterization

Once we identified these novel GRAs, we assessed their function by gene deletion using CRISPR/Cas9 and homologous recombination [47–49]. Gene knockouts were assessed by loss of the epitope tag from the parental line and replacement of the DHFR selectable marker used in tagging for HXGPRT used in the knockout (e.g. by growth in mycophenolic acid and xanthine but failure to grow in pyrimethamine) (Fig 3A). Each of the knockouts was then confirmed by PCR (Fig 3B). We examined the deletion mutants by plaque assay and discovered none of the lines had impaired in vitro growth (Fig 4, S2 Fig). We then proceeded with a pilot study of each of the knockouts to identify severe changes in cyst burden. This pilot study suggested that Δgra55 parasites produced substantially lower cyst numbers per mouse brain, thus we focused on this strain for complementation and more detailed in vivo analyses.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Deletion of GRAs 55–59 using CRISPR/Cas9.

(A) IFA shows the absence of HA signal in the PV. GRA14 is a marker of the PV and is shown as a control. Scale bar: 5 μm. (B) PCR verification of the deletion of GRAs 55–59. Attempted amplification of genes in knockout parasite strains show an absence of the coding sequence in the knockout strain but present in the wild-type control (gene check). The insertion of HXGPRT into the correct locus is verified by PCR from upstream of the gene of interest into the selectable marker (KO check) which is present only in the knockouts (PruΔku80 genomic DNA used as positive control).

https://doi.org/10.1371/journal.pone.0232552.g003

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. GRA55 is a novel GRA which is not important for in vitro growth or short-term virulence.

(A) Complementation strategy of GRA55 into the UPRT locus. The complemented gene contains a C-terminal 3xHA epitope tag and is driven by the native promoter. (B) IFA of GRA55-HA, Δgra55, and GRA55c parasites. Rabbit anti-HA shows strong staining in the vacuole of GRA55-HA and GRA55c parasites, colocalizing with GRA14. HA signal is absent in Δgra55 parasites. Scale bar: 5 μm. (C) Western blot demonstrating GRA55-HA migrating at the predicted size of 35.2 kDa (the size of GRA55-HA without the predicted signal peptide) and showing similar expression levels to the endogenously tagged strain. As expected, there is no HA signal in the Δgra55 strain. RON5C is used as a loading control [50]. (D) Quantitation of plaques formed by GRA55-HA, Δgra55, and GRA55c parasites. Results are shown as box-whisker plot with the middle line representing the median, the bottom and top of box representing the 25^th and 75^th percentile, and whiskers corresponding to smallest and largest plaques. (E, F, G) C57BL/6 female mice were infected with indicated doses of GRA55HA, Δgra55, and GRA55c parasites and Kaplan-Meier survival curves were generated. There was no statistical difference between the virulence of Δgra55 (F) vs. GRA55-HA (E) or GRA55c (G) parasites. (H) CBA/J mice were infected with 250 parasites each of the indicated strain. Mice were euthanized and mouse brains were examined by IFA after 30 days infection to enumerate T. gondii cyst burden. Δgra55 caused 10-fold lower burden of cysts when compared to GRA55-HA infection and GRA55c infection (p < 0.05 by two-tailed T-test). (I) Numerical counts of cysts per mouse brain (* = mouse dead prior to end of experiment).

https://doi.org/10.1371/journal.pone.0232552.g004

GRA55 is not important for in vitro growth or virulence but affects chronic infection of Type II T. gondii

We engineered GRA55-complemented parasites by re-introducing the wild-type gene driven from its endogenous promoter into the uracil phosphoribosyltransferase (UPRT) locus of the Δgra55 parasite genome [51] (Fig 4A). We verified that GRA55 targeted properly to the PV by IFA and showed similar expression levels to wild type parasites by western blot and thus we named this strain GRA55c (Fig 4B and 4C). We verified that that Δgra55 parasites did not have any decreased growth by plaque assays (Fig 4D) compared to wild-type and complemented strains, confirming that this protein does not meaningfully participate in parasite growth in vitro. Upon examination of the role of GRA55 in acute infection in mice, we found that Δgra55 parasites killed mice in a dose-dependent manner, identical to that of mice infected with wild-type and GRA55c (Fig 4E, 4F and 4G). We then conducted an investigation into whether GRA55 was important for chronic toxoplasmosis in mice. We injected 3 separate groups of mice intraperitoneally with 250 parasites per mouse, allowed the infection to proceed to 30 days, and then euthanized the mice and examined tissue cyst burden in the brains. We found that mice infected with Δgra55 parasites suffered a consistent 10-fold decrease in cyst burden compared to the mice infected with wild-type and GRA55c parasites (Fig 4H and 4I). In summary, this data demonstrated that although GRA55 is not important for the acute infection and mortality, it is an important secreted protein for the establishment or maintenance of the chronic infection in mice.

Host cell and parasite cultures

PruΔku80Δhxgprt (Type II, strain Prugniaud) and modified strains of T. gondii were used to infect human foreskin fibroblast (HFF) cells. HFFs were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 2 mM glutamine, and maintained as previously described [70].

Antibodies

Primary antibodies used in immunofluorescence assays (IFAs) and Western blot: mouse polyclonal anti-GRA14 (1:1000), rabbit anti-HA (Covance, 1:2000), rabbit polyclonal anti-ROP13 (1:500).

IFA and Western blot

For IFA, HFFs were grown to confluence on coverslips and infected with T. gondii parasites. After 18 to 36 h, the coverslips were fixed and processed for indirect immunofluorescence as previously described [71]. Primary antibodies were detected by species-specific secondary antibodies conjugated to Alexa 594/488. The coverslips were mounted in ProLong Gold antifade reagent (ThermoFisher) and viewed with an Axio Imager Z1 fluorescence microscope (Zeiss) as previously described [41]. For Western blotting, parasites were lysed in Laemmli sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.1% bromophenol blue), and lysates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were probed with the indicated primary antibodies, followed by secondary antibodies conjugated to horseradish peroxidase (HRP). Target proteins were visualized by chemiluminescence.

Generation of Δap2IV-4 parasites using CRISPR/Cas9 and homologous recombination

An sgRNA sequence in the coding sequence of AP2IV-4 was chosen using the EuPaGDT tool (http://grna.ctegd.uga.edu/) and the gRNA scaffold forward and reverse sequences were engineered with BsaI ends (primers p1-p2 –S2 Table). This sequence was cloned into the pu6-Universal [48,49] plasmid at the same BsaI site. Separately, an HXGPRT cassette (driven by a Neospora GRA7 promoter) was amplified from the pJET1.2:NcGRA7pro:HXGPRT. The primers contained 39 bp homology to the 5’ UTR (forward) and 3’ UTR (reverse) regions of AP2IV-4 (primers: p3-p4 –S2 Table). PrugniaudΔku80 parasites were transfected with 30 μg of closed-circular pU6-universal/sgRNA with 60 μg of the HXGPRT/AP2IV-4-flanks double-stranded oligonucleotide. The transfected parasites were grown in medium containing 50 μg /ml MPA (mycophenolic acid) and 50 μg /ml xanthine and cloned by limiting dilution. Knockout clones were verified by PCR (primers: gene check: p44-p45, KO check: p43, p51 –S2 Table).

Generation of MAG1-BirA* fusion

To generate the MAG1-BirA* fusion, the MAG1 gene and promoter was PCR amplified from genomic DNA (primers: p5-p6 –S2 Table). This was cloned into the p.LIC.BirA*.3xHA.DHFR vector by ligation independent cloning as previously described [35,72]. 30 μg of the construct was linearized with HpaI and transfected into PruΔku80Δhxgprt::Δap2IV-4.hxgrprt parasites. The parasites were selected with medium containing 1 μM pyrimethamine, cloned by limiting dilution, and screened by IFA and Western blot against the HA tag. A clone expressing the fusion protein was selected and designated MAG1-BirA*:Δap2IV-4.hxgrprt.

Affinity capture of biotinylated proteins

HFF monolayers were infected with parasites expressing MAG1-BirA* fusions or the parental line (PruΔku80Δhxgprt:::Δap2IV-4.hxgrprt) for 3h. Media was changed to bradyzoite “switch” media (DMEM with 1%FBS, 50mM HEPES, 1%PSG, pH 8.1). [35,72]. Parasites were incubated at 37° C in ambient air (0.05% CO2) for 5 days to induce switch to tissue cysts, exchanging media every 2 days to maintain alkaline pH [33,43]. 150 μM biotin was supplemented in the media on the final 24 hrs. Cyst formation was verified by expression of LDH2-GFP. Intracellular parasites were collected, washed in PBS, and lysed in radioimmunoprecipitation assay (RIPA) buffer (50mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) supplemented with Complete Protease Inhibitor Cocktail (Roche) for 30 min on ice. Lysates were centrifuged for 15 min at 14,000 × g to pellet insoluble material and the supernatant was incubated with Streptavidin Plus UltraLink Resin (Pierce) at room temperature for 4 h under gentle agitation. Beads were collected and washed five times in RIPA buffer, followed by three washes in 8M urea buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl). Ten percent of each sample was boiled in Laemmli sample buffer, and eluted proteins were analyzed by Western blotting by streptavidin-HRP while the remainder was used for mass spectrometry.

Mass spectrometry

The proteins bound to streptavidin beads were reduced and alkylated via sequential 20 minute incubations of 5mM TCEP 10mM iodoacetamide at room temperature in the dark while being mixed at 1200 rpm an Eppendorf thermomixer. Proteins were then digested by the addition of 0.1μg Lys-C (FUJIFILM Wako Pure Chemical Corporation, 125–05061) and 0.8μg Trypsin (Thermo Scientific, 90057) while shaking 37°C overnight. The digestions were quenched via addition of formic acid to a final concentration of 5% by volume. Each sample was desalted via C18 tips (Thermo Scientific, 87784) and then resuspended in 15μL of 5% formic acid before analysis by LC-MS/MS. Peptide samples were then separated on a 75μM ID, 25cm C18 column packed with 1.9μM C18 particles (Dr. Maisch GmbH) using on a 140-minute gradient of increasing acetonitrile and eluted directly into a Thermo Orbitrap Fusion Lumos instrument where MS/MS spectra were acquired by Data Dependent Acquisition (DDA). Data analysis was performed using the ProLuCID and DTASelect2 algorithms as implemented in the Integrated Proteomics Pipeline—IP2 (Integrated Proteomics Applications, Inc., San Diego, CA). Protein and peptide identifications were filtered using DTASelect and required a minimum of two unique peptides per protein and a peptide-level false positive rate of less than 1% as estimated by a decoy database strategy.

Generation of GRA-HA parasites (epitope tagging of BioID hits)

For GRA55, GRA56 and 59, LIC methodology was used: To epitope tag the novel GRA candidates, ~1500 bp of the 3’ genomic region of each gene was amplified using primers p7-p8 (GRA55), p15-16 (GRA66), p37-p38 (GRA59) (S2 Table) from PruΔku80Δhxgprt genomic DNA. The products were cloned into the p.LIC.3xHA.DHFR vector to add a 3xHA tag on the target gene as previously described [73–75]. The constructs were linearized and 50 μg of DNA was transfected by electroporation into PruΔku80Δhxgprt strain T. gondii.

For GRA57 and GRA58, CRISPR/Cas9 approach was used: An sgRNA sequence in the coding sequence of the intended GRA protein was chosen using the EuPaGDT tool (http://grna.ctegd.uga.edu/) and the gRNA scaffold forward and reverse sequences were engineered with BsaI ends. This sequence was cloned into the pU6-universal plasmid at the same BsaI site: p21-22 (GRA57), p29-p30 (GRA58) (S2 Table). Separately, a 3xHA:DHFR region was amplified from the p.LIC.3xHA.DHFR plasmid. The primers contained a 40 bp homology to a region just upstream of the gene-of-interest STOP codon (forward) and a 38 bp homology to a region in the gene-of-interest 3’ UTR (reverse) regions of the GRA gene: p23-24 (GRA57), p31-p32 (GRA58) (S2 Table). PrugniaudΔku80Δhxgprt parasites were transfected with 30 μg of closed-circular pU6-universal/sgRNA with 30 μg of the 3xHA-DHFR/GOI-C-term-flanks double-stranded oligonucleotide [49,72].

The transfected parasites were grown in media containing 1 μM pyrimethamine and selected parasites were cloned by limiting dilution. Clones were designated as GRA(X)-HA (e.g. GRA55-HA). All confirmed GRAs were examined in silico by BLAST to search for other T. gondii proteins with sequence similarity.

Deletion of GRAs 55–59 using CRISPR/Cas9

An sgRNA sequence in the coding sequence of the intended GRA protein was chosen using the EuPaGDT tool (http://grna.ctegd.uga.edu/) and the gRNA scaffold forward and reverse sequences were engineered with BsaI ends. This sequence was cloned into the pU6-universal plasmid at the same BsaI site [48,49]. Oligos: p9-p10 (GRA55), p17-18 (GRA46), p25-26 (GRA57), p33-p34 (GRA58), p39-p40 (GRA59) (S2 Table). Separately, an HXGPRT cassette (using the Neospora GRA7 promoter) was amplified from the pJET1.2:NcGRA7pro:HXGPRT. The primers contained 39 bp homology to the 5’ UTR (forward) and 3’ UTR (reverse) regions of the GRA gene. Primers: p11-p12 (GRA55), p19-20 (GRA56), p27-28 (GRA57), p35-p36 (GRA58), p41-p42 (GRA59) (S2 Table) PrugniaudΔku80Δhxgprt GRA-HA-tagged parasites were transfected with 30 μg of closed-circular pU6-universal/sgRNA with 30 μg of the HXGPRT/GRA-flanks double-stranded oligonucleotide. The transfected parasites were grown in medium containing 50 μg /ml MPA (mycophenolic acid) and 50 μg /ml xanthine and cloned by limiting dilution. Knockout clones were selected by loss of HA tag (IFA) and verified by PCR to examine for the absence of the gene of interest and presence of the HXGPRT cassette at the predicted recombination site (KO check primers: p46-p50, each paired with p51), (gene check: p7-p8 [GRA55], p15-16 [GRA56], p52-p53 [GRA57], p54-55 [GRA58] p37-p38 [GRA59]) (S2 Table).

Complementation of GRA55

GRA55-HA was re-introduced into the genome of the Δgra55 line in the uracil phosphoribosyltransferase (UPRT) locus [51]. This was done by amplifying the GRA55-3xHA gene as well as its native promoter from genomic DNA of the GRA55-HA parasite line (p13-p14) (S2 Table). The PCR product was cloned into the UPRT knockout vector with a tubulin promoter added using PacI and SpeI, forming a construct with the GRA55-3xHA cassette inserted between the UPRT 5’ and 3’ flanking regions. This plasmid was linearized using SacI-HF and transfected into Δgra55 parasites and selected with 5-Fluoro-5’-deoxyuridine (FUDR) to isolate parasites with the construct integrated at the UPRT locus [76]. Clones were selected by limiting dilution, examined by IFA (anti-HA antibody), and a positive clone was designated GRA55c.

Plaque assay

Serial dilutions of parasites were infected into separate wells of a 6-well plate with an HFF monolayer and allowed to form plaques. The dilutions were calibrated to infect 100 parasites/well, 200 parasites/well and 300 parasites/well. 6 days after infection, HFF monolayers were fixed in 3.7% formaldehyde for 15 minutes, washed with PBS, dried and stained with safranin. Plaques were then visualized with a Zeiss upright light microscope (Zeiss Axio Imager Z1). Plaque areas were measured using ZEN software (Zeiss) and plaque sizes were compared. 2 separate experiments of 30 plaques for each parasite line were measured to generate a mean plaque size along with interquartile ranges. Statistical significance comparing lines was calculated using two-sample two-tailed t-test.

Quantitation of in vitro bradyzoite differentiation efficiency in GRA55-HA, Δgra55, and GRA55c parasites

HFFs were grown on coverslips for seven days and infected with GRA55-HA, Δgra55 or GRA55c parasites for 3h. Media was changed to bradyzoite “switch” media (DMEM with 1%FBS, 50mM HEPES, 1%PSG, pH 8.1). [35,72] and parasites were incubated at 37°C in ambient air (0.05% CO₂) for 3 days to induce switch to tissue cysts [33,43]. The Coverslips were then fixed for IFA analysis. To quantify bradyzoite switch efficiency, GFP positive and negative vacuoles were counted and percentages of GFP positive vacuoles were calculated. Two biological replicates were reported for each strain. Statistical significance was calculated comparing the means of GRA55-HA with Δgra55 or GRA55c with Δgra55 using a one-way ANOVA test (using p < 0.05 for significance).

Mouse virulence assays

Intracellular GRA55-HA, Δgra55 and GRA55c parasites mechanically liberated from infected HFF monolayers and resuspended in Opti-MEM Reduced Serum Medium (ThermoFisher) prior to intraperitoneal injection into groups of 4 female C57BL/6 mice each. Injected parasites were confirmed to be live and viable parasites by plaque assays with HFF monolayers. Mice were monitored for symptoms of infection, weight loss, and mortality for 21 days. Survival was plotted on a Kaplan-Meier curve. To confirm infection, surviving mice were sacrificed at 30 days after infection, and mouse brains were collected in aseptic fashion, homogenized, and examined by phase microscopy and fluorescence microscopy (for detection of GFP under LDH2 promoter for parasite encystation). In parallel, 1/3 of each brain was homogenized and incubated with HFF monolayers to qualitatively assess viability of parasites.

Mouse brain cyst quantitation

Intracellular GRA55-HA, Δgra55, and GRA55c parasites were mechanically liberated from infected HFF monolayers and resuspended in Opti-MEM prior to intraperitoneal injection into groups of 4 female CBA/J mice each. 250 parasites/mouse of GRA55-HA, Δgra55 and GRA55c were injected. The mice were monitored for 30 days after infection, and then sacrificed. Mouse brains were collected, homogenized and examined for T. gondii cysts (as above). Quantitation of cysts was performed by examining 25 μL aliquots of homogenate by fluorescence microscopy until approximately 25% (by volume) of each brain was examined. Total cyst burden was then extrapolated. Statistical significance of cyst burden between GRA55-HA, Δgra55, and GRA55c experiments was calculated by two-sample 2-tailed t-test [77].

Animal experimentation ethics statement

Specific details of our protocol were approved by the UCLA Institutional Animal Care and Use Committee, known as the Chancellor’s Animal Research Committee (protocol: 2004–005). Mice were euthanized for two purposes. In the acute infection model, they were euthanized when the animals reached a moribund state. For the chronic infection model, these mice were sacrificed at the conclusion of the experiment at 30 days postinfection. Euthanasia per AVMA guidelines—slow (20–30% per minute) displacement of chamber air with compressed CO2. This is followed by confirmatory method of cervical dislocation’.