BALB/c mice were obtained from Japan SLC. Vero cells (RIKEN BioResource Research Center: RCB0001) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) containing 10% heat-inactivated fetal bovine serum (FBS; JRH Bioscience), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. HFFs (ATCC; SCRC-1041) were maintained in RPMI (Nacalai Tesque) supplemented with 2% heat-inactivated FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Nacalai Tesque).

T. gondii strain PruΔku80Δhxgprt or PruΔku80Δhxgprt LDH2-GFP, which expresses GFP under the control of a bradyzoite-specific gene LDH2 promoter (Fox et al., 2011) was maintained in Vero cells in DMEM containing 10% heat-inactivated FBS (JRH Bioscience), 100 U/mL penicillin, and 0.1 mg/mL streptomycin, as previously described (Bando et al., 2019) and was used as a parental strain for protein tagging and gene disruption.

An antibody against rabbit anti-MIC2-associated protein (M2AP) was kindly provided by Dr. Vern B. Carruthers (John Hopkins University, Baltimore, MD, USA). Mouse anti-CST1 (SalmonE) was kindly provided by Dr. Louis M. Weiss (Albert Einstein College of Medicine, Bronx, NY, USA). DBA-FITC was obtained from EY Laboratories. Succinylated wheat germ agglutinin (sWGA)-FITC was obtained from Vector Laboratories. Anti-SAG1 monoclonal antibody (mAb; TP3) was obtained from HyTest. Anti-GAP45 polyclonal antibody (pAb) was kindly provided by Dr. Dominique Soldati-Favre (University of Geneva, Geneva, Switzerland). Anti-GFP pAb was obtained from MBL.

In vitro bradyzoite differentiation was performed as previously described (Bando et al., 2021). Briefly, confluent HFFs were infected with tachyzoites and incubated in DMEM (pH 7.2) supplemented with 5% FBS, L-glutamine, penicillin, and streptomycin (normal media) with 5% CO₂. Three hours post-infection, wells were washed with PBS and cultured in DMEM adjusted to pH 8.3 with 25 mM HEPES, 1% FBS, and penicillin–streptomycin (induction media) for 3–7 days without CO₂ supplementation.

RNA was extracted using Trizol (Life Technologies) and isolated with the SV Total RNA Isolation System (Promega) according to the manufacturer’s protocol. Then, the cDNA library was created by using SuperScript III Reverse Transcriptase (Invitrogen). The C-terminal 900 bp of the TgCLP1 coding sequence were amplified by polymerase chain reaction (PCR) from cDNA from the PruΔku80Δhxgprt strain and inserted into the pGEX-6P-2 vector. BL21 competent Escherichia coli were transformed with the TgCLP1-fused vector and grown in LB medium supplemented with 50 μg/mL ampicillin for 3 h at 37°C, then IPTG (final concentration: 1 mM) was added and incubated for 16 h at 28°C. The cells were centrifuged at 1,100 ×g for 5 minutes, resuspended in sonication buffer (Triton X-100 diluted 1:100 in PBS), and sonicated on ice (30 s of sonication followed by 30 s on ice, repeated 8 times). GST tag-fused recombinant CLP1 was expressed in E. coli and purified by GST pull-down using Glutathione Sepharose 4B (GE Healthcare) according to the manufacturer’s protocol.

The recombinant protein (50 μg) was emulsified in an equal volume of TiterMax Gold (TiterMax) and subcutaneously injected into 6-week-old female BALB/c mice three times every week. Antisera were collected 4 weeks after the first immunization and used for western blotting.

Genomic DNA was isolated from the PruΔku80Δhxgprt or PruΔku80Δhxgprt LDH2-GFP strain with a QIAamp DNA Mini Kit (QIAGEN). To generate TgCLP1-HA or Myc-TgCLP1-HA parasites, 2 kb of the TgCLP1 sequence from the 3’ end were amplified by PCR and cloned into the pMini.ht. C-HA vector between the EcoRI and EcoRV sites (pTgCLP1-HA). Then, a Myc tag was added to the pMini.ht. C-HA vector (pMyc-TgCLP1-HA). pTgCLP1-HA or pMyc-TgCLP1-HA was linearized, and then transfected as previously described (Bando et al., 2018b). T. gondii tachyzoites were transfected with the linearized vector by electroporation. TgCLP1-HA or Myc-TgCLP1-HA parasites were selected based on the presence of a selectable marker, the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) cassette, in DMEM containing 20 µg/mL mycophenolic acid and 50 µg/mL xanthine. Selected parasites were cloned by limiting dilution, and integration of the vector at the TgCLP1 locus was verified by PCR.

Tachyzoites were obtained from infected cells by syringe lysis and passage through a filter (5 µM). Bradyzoites were obtained by syringe lysis of parasite-infected cells that had been prepared as described above. The parasites were centrifuged at 2,000 ×g for 5 minutes, then the pellets were resuspended in sodium dodecyl sulfate (SDS) sample buffer after washing with PBS, and boiled for 5 minutes. The samples were separated by SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane for protein blotting (Bio-RAD). The membranes were blocked with Blocking One reagent (Nacalai Tesque), following the manufacturer’s instructions, and incubated with the primary antibodies anti-HA (3F10; Roche), anti-BAG1, anti-ALD1, or anti-TgCLP1 in 5% Blocking One reagent in PBS (BO-PBS) overnight at 4°C. After they were washed three times with 0.1% Tween in PBS, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rat, donkey anti-rabbit, or sheep anti-mouse antibodies (GE Healthcare) for 1 h at room temperature.

Infected cells were fixed on coverslips with 4% paraformaldehyde in phosphate buffer (PFA) for 30 minutes at 4°C and permeabilized with 0.2% Triton X-100, 0.1% glycine, and 0.2% bovine serum albumin in PBS for 5 minutes at room temperature and then blocked with 8% FBS in PBS for 1 h at room temperature. Next, the cells were incubated with the indicated primary antibodies for 1 h at 37°C, followed by incubation with secondary antibodies (Alexa 488- or Alexa 546-conjugated goat anti-rat, anti-mouse, or anti-rabbit IgG; Invitrogen) and DAPI. Coverslips were mounted with Fluorescence Mounting Medium (Dako). Photomicrographs were taken with a TCS SP5 confocal microscope (Leica) or BZ-X800 All-in-One Fluorescence Microscope (KEYENCE).

The plasmid pSAG1::Cas9-U6::sgUPRT, which encodes the Cas9 nuclease (GFP fusion) under the control of the T. gondii SAG1 promoter, was obtained from Addgene (plasmid 54467). The TgCLP1-targeting CRISPR/Cas9 plasmid (pSAG1::Cas9-U6::sgTgCLP-1) was constructed as previously described (Bando et al., 2018b). To obtain the targeting vector, we amplified 2 kb of the upstream and downstream sequences of the TgCLP1 gene from the genomic DNA of PruΔku80Δhxgprt LDH2-GFP strain and the drug-resistant marker HXGPRT cassette was amplified from the pMini.ht.C-HA vector. These fragments were fused into the pBluescript KS (-) vector with the In-Fusion HD Cloning Kit (Clontech). Before transfection, the vector was linearized with SpeI and KpnI. T. gondii tachyzoites were transfected with the linearized vector using Basic Parasite Nucleofector Kit 1 and protocol U-030 (Lonza). TgCLP1 knock-out (TgCLP1-KO) parasites were selected based on the presence of the HXGPRT cassette, as described above. Selected parasites were cloned by limiting dilution, and integration of the vector at the TgCLP1 locus was verified by PCR. Deletion of the TgCLP1 protein was confirmed by western blotting with anti-TgCLP1 mouse antisera.

Two plasmids were used to generate the TgCLP1 complement plasmid. Plasmid I was pSAG1::cas9-U6::UPRT. Plasmid II was the pBluescript-UPRT vector containing the whole TgCLP1 coding sequence amplified by PCR from the cDNA of the PruΔku80Δhxgprt LDH2-GFP strain. After PCR and sequence checking, these plasmids were transfected into the TgCLP1-KO parasite. To select TgCLP1 complemented parasites, the transfected parasites were cultured with medium containing 5-fluorouracil (final concentration: 5 µM). After three passages, parasites were cloned by limiting dilution. Integration of the vector at theTgCLP1 locus was verified by PCR. Expression of the TgCLP1 protein was confirmed by western blotting with anti-TgCLP1 mouse antisera.

The invasion assay was performed as previously described (Bando et al., 2018a). Briefly, tachyzoites purified from host cells were inoculated into HFFs in 12-well plates containing coverslips. The plates were incubated at 4°C for 15 minutes and then 37°C for 3 h to allow parasite invasion. After incubation, the cells were fixed with 4% PFA. A standard non-permeabilized immunofluorescence assay (IFA) was performed by incubating the extracellular parasites with an anti-SAG1 mAb, and then the cells were permeabilized as described above and incubated with the anti-GAP45 pAb. After being washed with 0.1% Tween in PBS, the cells were incubated with secondary antibodies: Alexa 546-conjugated goat anti-mouse IgG for extracellular parasites and Alexa 488-conjugated goat anti-mouse IgG for intracellular parasites. For each experiment, at least 100 parasites were counted and the numbers of extracellular parasites (red) and intracellular parasites (green) were calculated.

The intracellular replication assay was performed as described previously (Bando et al., 2019). Briefly, freshly egressed parasites were allowed to invade HFFs grown in 12-well plates containing coverslips for 1 h. The wells were then washed with normal medium to remove extracellular parasites. The parasites were allowed to grow for 24 h at 37°C and then the cells were fixed with 4% PFA. An IFA using anti-GAP45 pAb was performed to stain the intracellular parasites, and the number of parasites per vacuole was scored. For each condition, 100 vacuoles were counted in three independent replicates.

The plaque assay was performed as described previously (Bando et al., 2021). Briefly, confluent monolayers of HFFs grown in 6-well plates were infected with 100 parasites/well and incubated for 8 days at 37°C in 5% CO₂. Then, the cells were washed twice with PBS, fixed with 2.5% glutaraldehyde–2% paraformaldehyde in phosphate buffer, fixed with 8% PFA, and stained as described above. After they were washed three times, the cells were stained with 0.1% crystal violet at room temperature for 10 minutes. Plaque size was analyzed using Image J. PV size was measured using a BZ-X800 All-in-One Fluorescence Microscope (KEYENCE).

To induce in vitro bradyzoite differentiation, parasites were grown in induction medium as described above. After 4 days, the incubation medium was changed and the T. gondii was incubated for 24 h in normal medium to induce bradyzoite reactivation. Then, the cells were fixed with 4% PFA and permeabilized as described above for IFA. Tachyzoites were stained with anti-SAG1 mAb (red). Bradyzoites were stained with α-GFP mAb (green). RFP-positive parasites are defined as reactivated parasites. GFP-positive parasites are defined as non-reactivated parasites. The cyst wall was stained with α-CST mAb. To measure the rate of non-reactivated parasites, at least 100 vacuoles were counted, and the numbers of GFP-positive parasites per CST1-positive vacuoles were calculated.

Mice were intraperitoneally infected with 2.0 × 10³ T. gondii tachyzoites in 200 μL of PBS per mouse. Survival rates (25 days) and weight (10 days) were measured daily.

All statistical analyses were performed using Prism 7 (GraphPad) or Excel (Microsoft). All experimental points and n values represent the average of three biological replicates (three independent experiments). The statistical significance of differences in mean values was analyzed by using an unpaired two-tailed Student’s t-test. P values less than 0.05 were considered statistically significant. The statistical significance of differences in survival times of mice between two groups was analyzed by using the Kaplan-Meier survival analysis log-rank test.

A previous study predicted the existence of hundreds of bradyzoite-secreted proteins based on a transcriptomic analysis of in vivo and in vitro T. gondii bradyzoites (Buchholz et al., 2011). However, analyses of the protein expression patterns, localization, and functions of these proteins have not been conducted. Then, we performed localization analyses by selecting 14 genes with high expression levels in bradyzoite. As a result, we successfully generated four kinds of genetically modified parasites. Thus, we focused on four uncharacterized bradyzoite-secreted proteins: gene accession numbers TGME49_093770, TGME49_022330, TGME49_062930, and TGME49_112950. To determine the expression patterns of these proteins, we inserted an HA-epitope tag endogenously into the C-terminus of each target gene using T. gondii type II strain PruΔku80Δhxgprt LDH2-GFP ( Figure 1A ). Then, the expression of these C-terminal HA-tagged proteins in tachyzoites and bradyzoites was analyzed ( Figure 1B ). We first confirmed that the proteins encoded by TGME49_022330, TGME49_062930, and TGME49_112950 were expressed in both the tachyzoite and bradyzoite stages ( Figure 1B ). TGME49_093770, also called T. gondii chitinase-like protein 1 (TgCLP1), has previously been detected in only tachyzoites (Huynh et al., 2009); however, we confirmed that it is also expressed in bradyzoites ( Figure 1B ). The properties of TGME49_022330, TGME49_062930, and TGME49_112950 are largely unknown, whereas TgCLP1 has a chitinase-like domain. Previous studies suggest that an interaction between the T. gondii cyst wall, which contains chitin, and host chitinase is important to control chronic infection (Nance et al., 2012). Therefore, we hypothesized that TgCLP1 has an important role in the parasite life cycle, especially in chronic infection and focused it in this study.

A previous study revealed that C-terminal-tagged TgCLP1 localizes to the microneme in tachyzoites (Huynh and Carruthers, 2009); however, the localization of TgCLP1 in bradyzoites is unknown. To determine the localization of TgCLP1 in bradyzoites, we generated C-terminal HA-tagged TgCLP1 (TgCLP1-HA) parasites ( Figure 2A ). We confirmed that TgCLP1-HA, albeit partially, colocalized with microneme marker protein M2AP in tachyzoites ( Figure 2B and Supplementary Figure S1A ), as previously reported (Huynh and Carruthers, 2009). We also found that TgCLP1-HA localized to the microneme in bradyzoites ( Figure 2B and Supplementary Figure S1A ). We performed western blot analyses with an anti-HA antibody to confirm the protein expression and molecular weight of HA-tagged TgCLP1 ( Figure 2C ). Interestingly, two band sizes were detected despite the predicted mass of TgCLP1 being approximately 75 kDa (ToxoDB). One was a thick band with a molecular mass of approximately 30 kDa, whereas the other was a thin band with a molecular mass of approximately 75 kDa ( Figure 2C ). To exclude the possibility that the HA tag was expressed with an extra protein, we performed western blotting using anti-TgCLP1 antisera, which identified bands of three sizes: 30 kDa, 45 kDa, and 75 kDa ( Supplementary Figure 3C ), suggesting that although multiple introns exist in TgCLP1, the TgClp1 protein is cleaved after translation rather than being alternative splicing. We then performed a CLP1 domain analysis using ToxoDB, CDD/SPARCLE: the conserved domain database in 2020, and SignalP 5.0. We found the predicted signal peptide (residues 1–26) at the N-terminus, and two additional domains: a chitinase domain-like domain (cd00325; residues 130–322: e-value = 4e-19 by the CDD) and a large tegument protein UL36-like domain (PHA03247; residues 321–542: e-value = 4e-04 by the CDD) ( Supplementary Figure S2A ). The observed molecular weight of each fragment obtained by western blotting and the predicted motifs indicated that the cleavage site lies within the C-terminal UL36-like domain, but no definite cleavage site on the domain was detected. The previous study (Huynh and Carruthers, 2009) and our TgCLP1-HA localization findings ( Figure 2B ), may have shown only the processed C-terminal-domain protein.

Since our western blotting suggested that TgCLP1 is cleaved into two fragments of 45 kDa and 30 kDa, we investigate whether each fragment shows different subcellular localization. To determine the localization of the N- or C-terminal-domain-containing TgCLP1 proteins, we generated N-terminal Myc-tagged and C-terminal HA-tagged TgCLP1 (Myc-TgCLP1-HA) parasites ( Figures 3A , B ). Then, we performed an IFA to detect the localization of each protein ( Figure 3C ). We found that the N-terminal-domain-containing TgCLP1 protein (Myc-TgCLP1) did not localize with the C-terminal-domain-containing TgCLP1 protein (TgCLP1-HA). Myc-TgCLP1 was found in the cytosol in both tachyzoites and bradyzoites ( Figure 3C ), whereas TgCLP1-HA colocalized with M2AP ( Figure 2B ). These results suggest that TgCLP1 does not exist exclusively within micronemes, in addition, TgCLP1 undergoes cleaving after which the 30-kDa C-terminal fragment containing the large tegument protein UL36-like domain remains in the microneme and the 45-kDa fragment containing the chitinase-like protein is secreted into the cytosol.

The function of TgCLP1 is unclear in both the tachyzoite and bradyzoite stages. To clarify the role of TgCLP1 during the tachyzoite stage, we generated TgCLP1 knock-out (TgCLP1-KO) parasites ( Figure 4A ) and TgCLP1-KO parasites complemented with wild-type TgCLP1 (TgCLP1-KO+TgCLP1) ( Supplementary Figures S3A, B ). The amplification of the coding region of TgCLP1 was not observed in the TgCLP1-KO parasite, and the inserted allele containing HXGPRT was only amplified from the TgCLP1-KO parasite ( Figure 4A ). The deletion of TgCLP1 protein was confirmed by western blotting with anti-TgCLP1 antisera ( Supplementary Figure 3C ). The tachyzoite stage can be divided into three main steps: invasion of the host cell, intracellular replication, and egress from the host cell (Dubey, 2009). First, we tested whether TgCLP1 affected the invasion of T. gondii into the host cell. We observed no difference in the numbers of wild-type, TgCLP1-KO, and TgCLP1-KO+TgCLP1 T. gondii inside host cells 3 h post-infection ( Figure 4B ), suggesting that TgCLP1 is not involved in parasite invasion. Next, we tested whether TgCLP1 affects the intracellular replication of T. gondii. The number of T. gondii per vacuoles at 24 h post-infection did not differ among the wild-type, TgCLP1-KO, and TgCLP1-KO+TgCLP1 parasites ( Figure 4C ), which suggests that TgCLP1 is also not involved in intracellular replication.

The ability to egress from host cells can be assessed by examining the lytic capacity of host cells ( Figure 5A ). Accordingly, we performed a plaque assay and found that the TgCLP1-KO T. gondii produced smaller plaques than the wild-type or TgCLP1-KO+TgCLP1 parasites at 6 days post-infection ( Figure 5A ). This result suggests that TgCLP1 may be linked to egress during the tachyzoite stage. We showed that intracellular replication was not affected by the presence or absence of TgCLP1 ( Figure 4C ). Therefore, we hypothesized that parasitophorous vacuole (PV) size was different between Wild-type and TgCLP1-KO T. gondii. To test this hypothesis, we compared the PV size of T. gondii with or without TgCLP1 ( Figure 5B ). We found that the PV size of TgCLP1-KO T. gondii was larger than that of wild-type or TgCLP1-KO+TgCLP1 parasites ( Figure 5B ). In addition, TgCLP1-KO T. gondii spread slowly to host cells ( Supplementary Figure S4A ), supporting our finding on the effect of TgCLP1 deficiency on egress and PV size ( Figures 5A , B ). Finally, we examined the influence of TgCLP1 on T. gondii infection in mice using an in vivo acute infection model ( Figures 5C, D ). We found no difference in the survival ( Figure 5C ) or weight loss ( Figure 5D ) of the mice infected with T. gondii regardless of TgCLP1 expression. These results indicate that, although TgCLP1 is involved in egress during the tachyzoite stage, its deficiency results in a moderate phenotype and does not affect acute parasite infection in vivo.

To clarify the contribution of TgCLP1 during the bradyzoite stage, we assessed whether TgCLP1 expression is important for tachyzoite to bradyzoite conversion. T. gondii tachyzoites were cultured under alkaline stress conditions, and stage conversion was confirmed by staining of the cyst wall glycoprotein CST1, a bradyzoite-specific marker (Zhang et al., 2001). First, we compared the stage conversion rates of wild-type and TgCLP1-KO T. gondii by counting CST1-positive vacuoles ( Figures 6A, B ). We found that the ratio of CST1-positive vacuoles was comparable between wild-type and TgCLP1-KO parasites ( Figure 6B ). Furthermore, to confirm whether cyst wall formation was normal in the absence of TgCLP1, we investigated other cyst wall components by using sWGA and DBA, which bind to cyst wall lectins ( Figure 6C ). Both sWGA and DBA showed normal staining in wild-type and TgCLP1-KO parasites ( Figure 6C ). These results suggest that TgCLP1 is not involved in cyst formation in the bradyzoite stage.

In vitro bradyzoite reactivation for T. gondii has rarely been studied; therefore, in the present study, we established an assay to assess it ( Figure 7A ). Using our method, we confirmed that more than 90% of wild-type T. gondii differentiated into bradyzoites when cultured under induction conditions, and more than 60% of bradyzoites reactivated after reactivation stimulation ( Figure 7B ). Then, we first tested localization of TgCLP1 in each stage by using Myc-CLP1-HA T. gondii using this method ( Supplementary Figure S5 ). During the early to mid-tachyzoite stages, Myc-tagged CLP1 is present around the apical complex and on the surface of the parasite, while towards the end of egress from host cells, it is also located around the host cell membrane ( Supplementary Figure S5A ). On the other hand, during the bradyzoite stage, Myc-tagged CLP1 was only found around the apical complex or surface of parasite, but after reactivation stimulation, it also relocated to reside near the cyst wall ( Supplementary Figure S5B ). Additionally, HA-tagged CLP1 was found to be present at the apical complex throughout all stages ( Supplementary Figures S5A, B ). These results supported our finding about C-terminal CLP1 cleavage, and suggested the present of N-terminal CLP1 contains a signal peptide ( Figure 3B and Supplementary Figure S2A ). And also, these results suggested that CLP1 has some interaction with the cyst wall during reactivation. Then, we next tested whether TgCLP1 has an important role in reactivation from bradyzoite to tachyzoite ( Figure 7C ). After reactivation stimulation, approximately 50% of wild-type T. gondii and 80% of TgCLP1-KO+TgCLP1 T. gondii were reactivated ( Figure 7C ). In contrast, less than 10% of TgCLP1-KO T. gondii were reactivated ( Figure 7C ). Taken together, these results indicate that TgCLP1 has an important role in bradyzoite to tachyzoite conversion.