TgPP6 is expressed during the asexual development of T. gondii tachyzoites

Based on the ToxoDB database (https://toxodb.org), T. gondii PP6 has a potential catalytic subunit (TgPP6C, TGGT1_301010), a regulatory subunit (TgPP6R, TGGT1_319930) and a structural subunit (TgPP6S, TGGT1_216680) [16]. To determine the localization of the putative subunits of TgPP6, we generated strains (TgPP6C::6HA, TgPP6R::6HA and TgPP6S::6HA) with a C-terminal epitope tag in T. gondii type I RHΔku80 (WT) strain by using CRISPR-Cas9 (S1 Fig). We found that TgPP6C and TgPP6R were localized in the cytoplasm of tachyzoites, whereas TgPP6S was observed in the endoplasmic reticulum (ER) (Fig 1A). To further confirm the localization of this protein, we co-localized TgPP6S with the ER marker sarcoplasmic-endoplasmic reticulum calcium ATPase (TgSERCA) [25], and found that TgPP6S is indeed located in the ER of tachyzoites (Fig 1B).

To investigate potential interactions between the putative TgPP6 protein subunits in T. gondii, we immunoprecipitated lysates generated from the TgPP6C::6HA, TgPP6R::6HA and TgPP6S::6HA strains with anti-HA-conjugated magnetic beads, which were subjected to LC-MS analysis. The immunoprecipitation (IP) results were consistent with TgPP6C and TgPP6R interacting with each other, while TgPP6S did not appear to interact with TgPP6C or TgPP6R because no TgPP6C or TgPP6R peptides were detected in the TgPP6S::6HA group. This result suggests that TgPP6S may not be part of the TgPP6C and TgPP6R complex (Fig 1C and S1 Table). The low homology between TgPP6S and human PP6 structural subunits further implies that TgPP6S may function in a distinct way from the other TgPP6 subunits. Interestingly, both TgPP6C::6HA and TgPP6R::6HA groups identified the hypothetical protein TGGT1_203150 as a potential interaction partner, suggesting that TGGT1_203150 may form a heterotrimer with TgPP6C and TgPP6R (Fig 1C). Subsequently, a TGGT1_203150::6HA strain was generated, and immunofluorescence staining confirmed its subcellular localization in the parasite cytoplasm (Fig 1D).

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Fig 1. Characterization of the putative PP6 of Toxoplasma gondii.

(A) The subcellular localization of TgPP6C and TgPP6R by endogenous tagging shows two subunits localized in the cytoplasm, while TgPP6S was located in the endoplasmic reticulum. Green, TgIMC1 stain denotes the outline of the parasite. Red, mouse anti-HA antibody. Scale bars: 2 μm. (B) IFA shows TgPP6S::6HA (green) colocalized with the endoplasmic reticulum protein TgSERCA (red). Scale bars: 2 μm. (C) Immunoprecipitation (IP) assays were performed to identify TgPP6::6HA subunits (TgPP6C, TgPP6R and TgPP6S) interacting proteins, WT strain as a control. Selected hits identified by LC-MS analyses are shown and the numerical annotations of each gene show the count of unique peptides originating from the MS analysis that matched the corresponding hits. A full list of the hits is provided in S1 Table. (D) TGGT1_203150::6HA parasites were stained with antibodies against TgIMC1 (green) and HA (red). TGGT1_203150 was located in the cytoplasm of the parasite. Scale bars: 2 μm.

https://doi.org/10.1371/journal.ppat.1011831.g001

TgPP6C and TgPP6R are indispensable for T. gondii replication

To elucidate the biological significance of each putative subunit of TgPP6 to parasite fitness, CRISPR-Cas9-mediated gene homologous recombination was used to disrupt TgPP6C, TgPP6R, TgPP6S and TGGT1_203150, respectively (S2A Fig). Single knock-out clones were screened using diagnostic PCRs, with PCR3 and PCR5 confirming the replacement of homologous fragments. These PCRs amplified a ~1200–1600 bp fragment, which was absent in the WT strain (S2B Fig). In contrast, the expected coding sequence of PCR4 (~ 600–800 bp) was amplified in the WT strain but was absent in the RHΔpp6 strains (S2B Fig). To explore the roles of these four genes in the growth of parasites in vitro, plaque assays were performed using WT and knock-out strains. There was no significant difference in the size and number of plaques formed by the WT, RHΔpp6s and RHΔ203150, however RHΔpp6c and RHΔpp6r strains formed smaller plaques in HFFs compared to those in WT after 7 days of incubation (Fig 2A and 2B), suggesting that both TgPP6C and TgPP6R are important for the parasite growth. This result is consistent with the low phenotypic score (–4.05 for TgPP6C and –3.76 for TgPP6R) in T. gondii genome-wide CRISPR-Cas9 screens [24]. To further confirm that the growth defect of the parasite was caused by the deletion of TgPP6C and TgPP6R, RHΔpp6c or RHΔpp6r strains were complemented by inserting TgPP6C or TgPP6R fused with a C-terminal 3×HA epitope tag driven by β-tubulin promoter into the UPRT locus using CRISPR-Cas9 directed gene integration (S2C Fig). Complemention of RHΔpp6c strain with TgPP6C (RHΔpp6c-cp) or RHΔpp6r strain with TgPP6R (RHΔpp6r-cp) successfully restored the parasite growth.

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Fig 2. Phenotypic analyses of TgPP6 subunits.

(A) Representative images of the plaque assays of the indicated strains grown for 7 days, showing that the ability of RHΔpp6c and RHΔpp6r to form plaques is almost completely inhibited. The arrows indicate the plaques formed by RHΔpp6c and RHΔpp6r. (B) The relative size of the plaques produced by each strain. Data represents mean ± SD of three independent experiments and statistical differences were analyzed by the unpaired t-test. ****P < 0.0001; n.s., not significant. (C) Deletion of TgPP6C and TgPP6R resulted in tachyzoites with dysregulated endodyogeny and morphological defects. Green, TgIMC1. Scale bar: 2 μm. (D) The validation of the complemented strain RHΔpp6c-cp and RHΔpp6r-cp shows that both strains regained their normal endodyogeny. Green, TgIMC1. Red, HA. Scale bar: 2 μm. (E) Intracellular replication of the WT, RHΔpp6c, RHΔpp6c-cp, RHΔpp6r and RHΔpp6r-cp strains. The intracellular replication rate and the percentage of asynchronous division of RHΔpp6c and RHΔpp6r were significantly different from that of the WT strain. Data are mean ± SD from three independent experiments, > 200 vacuoles were counted per replicate and statistical differences were analyzed by a two-way ANOVA. **P = 0.0012; ****P < 0.0001; n.s., not significant.

https://doi.org/10.1371/journal.ppat.1011831.g002

The significant reduction in plaque formation suggests some defects in one or multiple stages in the parasite lytic cycle. Therefore, we examined the invasion, intracellular replication and egress properties of RHΔpp6c and RHΔpp6r. The deletion of TgPP6C and TgPP6R did not significantly affect the egress of tachyzoites, but slightly affected their ability to invade host cells (S2D and S2E Fig). Interestingly, both RHΔpp6c and RHΔpp6r exhibited a significant reduction in replication efficiency compared with WT strain. In addition, an abnormal phenotype with asynchronous division of the intracellular tachyzoites was observed in the RHΔpp6c strain (Fig 2C). While a small number of the RHΔpp6r strain also exhibited this asynchronous division, it was less pronounced than that seen in the RHΔpp6c strain (Fig 2C). Based on this results, the study was we focused on TgPP6C. When the number of tachyzoites in the parasitophorous vacuoles (PVs) reached “2”, the division of certain RHΔpp6c tachyzoites became asynchronous and when it divided further to “4”, or more (i.e., 4–8, 8–16, 16–32 and > 32 divisions) the tachyzoites were unable to form normal "rosettes". This abnormal phenotype was restored to the wild-type level in the complemented strain (Fig 2D).

Therefore, we infer that RHΔpp6c and RHΔpp6r mainly affects the tachyzoite replication. To verify our hypothesis, intracellular replication assays were performed. We cultured the parasites for a longer period of time compared to normal replication assays because most of the PVs of RHΔpp6c and RHΔpp6r strains contained only two or four tachyzoites 24 h post infection. We quantified the replication rate of RHΔpp6c and RHΔpp6r, and quantified the asynchronous division events which appeared in PVs containing > 2 tachyzoites. Thus, all the strains were cultured for 32 h, the proportion of PVs with 1–3 (13.67 ± 2.52%), 4–7 (22.33 ± 1.61%) and 8–15 (37.5 ± 1%) tachyzoites was significantly higher in the RHΔpp6c strain compared to the WT strain and RHΔpp6c-cp strain, while the proportion of PVs containing 16–32 (24.5 ± 2.29%) and > 32 (2 ± 0.87%) tachyzoites were significantly lower in RHΔpp6c strain compared to the WT and RHΔpp6c-cp strains (Fig 2E). Additionally, the proportion of irregular PVs (i.e., containing asynchronously dividing parasites) of RHΔpp6c (64.17 ± 1.76%) was significantly higher compared to the other strains (Fig 2E). As for the RHΔpp6r strain, the replication ability was also significantly reduced, and the anomalous division (10.67 ± 1.61%) occurred on a notably smaller scale compared to that of the RHΔpp6c strain (Fig 2E).

Given the crucial role of TgPP6C and TgPP6R in parasite repliaction, their functions were also examined using a conditional protein depletion approach. To achieve this, the endogenous TgPP6C and TgPP6R genes were tagged with an indole-3-acetic acid (IAA)-inducible degradation domain (mAID-6HA) at the C-terminus in the RHΔku80::TIR1 (RH::TIR1) strain. As expected, after treatment with IAA, PP6C-mAID and PP6R-mAID parasites exhibited growth defects, asynchronous division and reduced replication rate, similar to that observed in RHΔpp6c and RHΔpp6r (S3 Fig). These results show the significant involvement of TgPP6C and TgPP6R in parasite replication.

TgPP6C is required for synchronous daughter budding and mitosis

As explained earlier, TgPP6C plays an indispensable role in promoting the growth of type I RH parasites, particularly during their replication stage, while concurrently affecting endodyogeny. To assess endodyogeny changes in TgPP6C knock-out parasites, we stained intracellular parasites separately with IMC1/ISP1 (IMC sub-compartment protein 1) and IMC1/GAP45 combinations. TgISP1, as an early progenitor IMC protein, is present at the apical cap of both parental and progeny parasites, whereas the expression of TgGAP45 is confined solely to the parental parasite. We noted an increase in the proportion (18.67 ± 2.57%) of RHΔpp6c parasites exhibiting endopolygeny, together with impairment in the divisional capacity of some parental parasites (Fig 3A to 3C). However, absence of PP6C did not affect the integrity of the IMCs and pellicles. Additionally, in up to 10.67 ± 2.47% of PVs of RHΔpp6c strain (Fig 3B, 3D to 3F), nuclear segregation defects (e.g. abnormal segregation or inability to segregate) were observed. These nuclear phenotypic chnages were also observed in RHΔpp6c by electron microscopy (Fig 3E). The detection of multinucleated parasites confirmed that some RHΔpp6c undergo endopolygeny replication.

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Fig 3. TgPP6C plays a critical role in daughter budding and nuclear segregation.

(A) to (C) Immunofluorescence staining shows that RHΔpp6c exhibits significant increase in endopolygeny. Green, TgIMC1. Red, TgISP1/GAP45. White asterisks denote daughter buds. Red asterisk indicates the parasite nucleus. Arrows indicate the deformed parasite. Scale bar: 2 μm. Data are mean ± SD from three independent experiments (> 200 vacuoles were counted per replicate), and statistical differences were analyzed by a two-way ANOVA; ****P < 0.0001. (D) to (F) Immunofluorescence staining and electron micrographs show that lack of TgPP6C resulted in marked defects in the segregation of the parasite nucleus. White asterisks, red asterisks and white numbers denote daughter buds, nuclei and number of nuclei, respectively. TgIMC1 and DAPI stains were used to stain the outline and nuclei (DNA) of the parasites, respectively. Scale bar: IFA, 2 μm; Electron microscopy, 1 μm. Data shown in the graphs represent the mean ± SD of three independent experiments (> 200 vacuoles were counted per replicate) and statistical differences were analyzed by a two-way ANOVA; *P = 0.0204.

https://doi.org/10.1371/journal.ppat.1011831.g003

T. gondii’s cell cycle has a precisely timed and strictly regulated sequence of subcellular divisions [10]. The initial organelle to undergo duplication is the centrosome, which plays a key role in the division of T. gondii [4]. We used TgCentrin1 as a centrosome marker protein to examine its potential alterations in the absence of TgPP6C [4]. By staining TgCentrin1, we discovered that most TgCentrin1 exhibited a persistent inclination towards division, harboring the duplicated, awaiting to be duplicated or over-duplicated entities within abnormal PVs (Fig 4A). Further examination of tachyzoites revealed discrete, brightly marked centrosomes in close proximity to the nucleus in the WT strain, whereas the centrosomes in RHΔpp6c tachyzoites (comprising 15.17 ± 3.22%) exhibited anomalies (Fig 4A and 4B). These irregularities include impaired duplication within the parental parasites or instances where the daughter parasite nuclei were associated with duplicated centrosomes (Fig 4A and 4B). Considering the role of TgChromo1, which remains near the centrosome and centrocone throughout the cell cycle, it functions as a centromere marker [26]. In agreement with our reasoning, staining of TgChromo1 closely mirrored earlier findings from TgCentrin1 staining, however, the stability of centromere was not significantly altered in RHΔpp6c (Fig 4A). These results indicate that TgPP6C may contribute to the stability of the centrosome.

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Fig 4. TgPP6C is important for the stability of centrosome.

(A) Immunofluorescence staining shows that RHΔpp6c harboring the duplicated, awaiting to be duplicated or over-duplicated centrosome and centromere within an abnormal PV as revealed by Centrin1 and Chromo1 markers, respectively. White and yellow asterisks indicate a centrosome and a missing centrosome, respectively. Red, yellow and white arrows denote the duplicated, awaiting to be duplicated and over-duplicated centrosome and centromere, respectively. Red, TgIMC1/TgCentrin1. Green, TgIMC1/TgChromo1. Scale bar: 2 μm. (B) Data are mean ± SD from three independent experiments (> 200 vacuoles were counted per replicate), and statistical differences were analyzed by a two-way ANOVA; ***P = 0.0007.

https://doi.org/10.1371/journal.ppat.1011831.g004

Next, we examined the effect of TgPP6C deletion on other organelles, including rhoptries, dense granules, apicoplast, mitochondria and micronemes. Interestingly, tachyzoites continued to replicate despite considerable morphological defects (e.g., enlarged paternal parasites or abnormal endodyogeny) caused by TgPP6C deletion (S4 Fig). However there were no obvious effects on the other T. gondii organelles. These results suggest that TgPP6C plays a role in the modulation of T. gondii’s cell cycle-specific expression pattern, and its deletion affects parasite replication, from the inception of the S phase onwards.

TgPP6C core motifs are required for endodyogeny and function

TgPP6C possesses core motifs GDIHG, GDYVDRG, GNHE, HGG, RG and H, which are conserved in eukaryotes [15,16]. To assess the role of the predicted TgPP6C core motifs, we individually introduced mutations at every amino acid position within the six core motifs, by substituting them with alanine (MU-GDIHG/AAAAA, MU-GDYVDRG/AAAAAAA, MU-GNHE/AAAA, MU-HGG/AAA, MU-RG/AA and MU-H/A) in the vector of the complemented TgPP6C, which includes a C-terminus 3×HA epitope tag and is driven by β-tubulin promoter (Figs 5A and S2B). Each target fragment was inserted into the UPRT locus of RHΔpp6c by CRISPR-Cas9-mediated gene integration, and the clonal strains were successfully obtained by chloramphenicol selection and limited dilution. The mutation of each of the six motifs failed to complement the parasite’s phenotype, and the division remained asynchronous, indicating that all the TgPP6C motifs are indispensable for the intracellular division of T. gondii, but they appear to play no or minor role in TgPP6C localization (Fig 5B). Interestingly, RHΔpp6c and each mutant produced hardly visible plaques, which were significantly smaller than those of the WT and RHΔpp6c-cp (Fig 5C and 5D). These results show that mutation in any of the six motifs of TgPP6C could not rescue the TgPP6C knock-out, suggesting critical role of these motifs in the parasite’s endodyogeny.

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Fig 5. The TgPP6C core motif is essential for its function.

(A) Gene model for TgPP6C with the six predicted core catalytic motifs mutated to alanine. (B) Immunofluorescence staining revealed that six core motifs of TgPP6C are essential for T. gondii endodyogeny. Green, TgIMC1. Red, HA. Scale bar: 2 μm. (C) to (D) The size and number of plaques formed by RHΔpp6c and mutant strains with each core motif mutated to alanine were significantly reduced compared with the WT and RHΔpp6c-cp strains. Data represents mean ± SD of three independent experiments and statistical differences were analyzed by the unpaired t-test. The indicated strains were compared with WT. ****P < 0.0001; n.s., not significant. Scale bar: 0.5 mm.

https://doi.org/10.1371/journal.ppat.1011831.g005

Disruption of TgPP6C alters phosphorylation status of many proteins related to parasite cell cycle and division

To identify phosphorylation events mediated by TgPP6C, quantitative phosphoproteomic analysis was performed on TgPP6C knock-out parasites compared with the WT strain using label-free phosphoproteomics. This analysis identified 434 differentially regulated proteins between RHΔpp6c and WT, of those 226 proteins were upregulated in RHΔpp6c and 208 were upregulated in WT, as defined by a P < 0.05 and fold-change of < –1.5 or > 1.5 (S2 Table). Additionally, 8,748 phosphosites were identified in 2,390 proteins (S3 Table). To determine the changes in the phosphorylated proteins between RHΔpp6c and WT, we normalized the level of phosphorylated proteins relative to the level of all the identified phosphorylated peptides (2581 phosphorylation sites and 706 proteins) (S3 Table). After normalization, 777 unique phosphorylation sites on 375 T. gondii proteins exhibited significantly altered abundance in RHΔpp6c. Of these, 350 phosphopeptides on 162 phosphoproteins were upregulated, and 427 phosphopeptides on 213 phosphoproteins were downregulated in RHΔpp6c (Fig 6A and S3 Table). The down-regulated phosphopeptides may be a secondary effect to the loss of TgPP6C, which may have led to activation of other phosphatases or suppressing kinases.

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Fig 6. Functional annotation of the significantly regulated phosphoproteins.

(A) to (C) Volcano plots showing the significant fold changes of the phosphopeptides from RHΔpp6c strain compared with WT strain. Red and blue dots represent up-regulated (fold changes > 1.5 with P < 0.05) or down-regulated (fold changes < –1.5 with P < 0.05), and gray dots represent non-differentially regulated proteins. Proteins with predicted or known roles in chromosome organization, cell cycle progression and cell division are shown as red dots. Those proteins are plotted in (B), and some of them have more than one differentially phosphorylated site. Eleven phosphoproteins that have been previously shown to be associated with cell division during the tachyzoite lytic cycle were plotted in (C) and each phosphoprotein was shown in a different color. (D) The percentage of differential phosphoproteins relative to the total number of differential phosphoproteins, according to their predicted localization. (E) The top 10 significantly enriched GO terms of the differentially regulated phosphoproteins in the cellular component, biological process, and molecular function categories of RHΔpp6c vs WT.

https://doi.org/10.1371/journal.ppat.1011831.g006

A large number of proteins involved in the regulation of the cell cycle and most of the IMC proteins were differentially regulated in RHΔpp6c strain compared with WT strain (Fig 6A to 6C). In addition, deletion of TgPP6C resulted in both upregulated and downregulated phosphorylation sites in four proteins (TGGT1_265250, TGGT1_258870A, TGGT1_269180 and TGGT1_220240). Both TGGT1_265250 and TGGT1_269180 have predicted RNA binding activities, while TGGT1_258870A and TGGT1_220240 (GRA31) are integral components of the membrane.

An in-depth look at differentially phosphorylated proteins between RHΔpp6c strain and WT strain revealed that many of them are predicted or known to play roles in chromosome organization, cell cycle progression and cell division (Fig 6B). Specifically, IMC-related proteins accounted for the majority of the differentially phosphorylated proteins, including (TGGT1_235340, ISC1), (TGGT1_237820, ISP2), (TGGT1_220900, AC13), (TGGT1_231070, BCC2), (TGGT1_311230, BCC7), (TGGT1_230850, TSC3), (TGGT1_249850, GAP40), (TGGT1_206690, GAPM2B), (TGGT1_271970, GAPM3), (TGGT1_235470, Myosin A), (TGGT1_257680, MLC1), (TGGT1_231640, IMC1), (TGGT1_222220, IMC7), (TGGT1_226220, IMC9), (TGGT1_230210, IMC10), (TGGT1_260540, IMC14), (TGGT1_286580, IMC17), (TGGT1_295360, IMC18), (TGGT1_316340, IMC22) (TGGT1_258470, IMC24), (TGGT1_243200, IMC29), (TGGT1_293360, IMC38), and (TGGT1_255420, IMC39) (Fig 6B).

Focusing on the function of selected differential phosphoproteins, Cdk-related kinase 2 (TGGT1_218220, Crk2) plays a role in the regulation of the tachyzoite G1 phase [27]. Its absence induces the arrest of the G1 phase. Moreover, upon deletion of TgPP6C, two proteins were differentially phosphorylated (TGGT1_289100, HOOK and TGGT1_290160, SORTLR), which are involved in the parasite invasion, egress and virulence [28,29]. Some differentially phosphorylated proteins contribute to the division of T. gondii, including IMC proteins (IMC10, IMC14 and IMC29), IMC related proteins (ISP2, GAP40, GAPM2B and GAPM3), phosphatidic acid phosphatase (TGGT1_230690, LIPIN), myosin-specific co-chaperone of the UCS family (TGGT1_249480, UNC), cyclase associated protein (TGGT1_310030, CAP) and calmodulin (TGGT1_249240, CaM) [30–42] (Fig 6C and Table 1). The nucleus, cytosol and IMC were the main predicted locations for most of the differentially phosphorylated proteins based on the spatial data obtained from hyper LOPIT [43] (Fig 6D and S4 Table).

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Table 1. List of the differentially phosphorylated proteins involved in tachyzoite division as revealed by phosphoproteome analysis.

https://doi.org/10.1371/journal.ppat.1011831.t001

We performed GO enrichment analysis to identify the function of the 375 differentially abundant phosphoproteins (Fig 6E). Prominent among the enriched GO terms, within the cellular components, are those associated with proteins residing in membrane-bound organelles, cytoplasm, nucleus and diverse protein-containing complexes (Fig 6E). In the biological process category, translation, protein phosphorylation and metabolic processes, were enriched (Fig 6E). In the molecular function category, the GO terms binding, catalytic activity and the structural constituents of the ribosome were enriched in the significantly up- or down-regulated proteins (Fig 6E). These results show that multiple cell cycle-associated phosphoproteins are differentially regulated in RHΔpp6c, and likely contribute to abnormal endodyogeny in RHΔpp6c mutant tachyzoites.

RHΔpp6c elicits protective immunity against acute and chronic toxoplasmosis

To determine the impact of TgPP6 deficiency on type I RH virulence, groups (n = 6/group) of Kunming mice were intraperitoneally (i.p.) injected with 100 tachyzoites in 200 μL phosphate-buffered saline (PBS) of the indicated strains (Fig 7A). Mice in the control (naïve) group were only injected with 200 μL PBS. In congruence with the in vitro parasite proliferation results, all mice infected by WT, RHΔpp6c-cp and RHΔpp6r-cp parasites reached their humane end-point within 8–10 days (Fig 7A). In contrast, a substantial extension in the survival was observed in mice infected by RHΔpp6r (Fig 7A). Notably, mice infected by RHΔpp6c remained healthy and did not exhibit any clinical signs (Fig 7A). Anti-T. gondii IgG and IgG subclasses (IgG1 and IgG2a) were analyzed at day 30 post-immunization with 200 RHΔpp6c tachyzoites to evaluate the Th1/Th2 polarization. The levels of anti-T. gondii IgG, IgG1 and IgG2a increased significantly in the immunized mice compared with mice in the naïve group (Fig 7B). These results suggest that RHΔpp6c induced a mixed anti-T. gondii Th1/Th2 response. In addition, IgG2a level was higher relative to IgG1, suggesting that RHΔpp6c induced a Th1-biased antibody response.

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Fig 7. Immunization of RHΔpp6c protects mice against acute and chronic infection by T. gondii.

(A) Mice in each group (6 mice/strain) were intraperitoneally (i.p.) injected with 100 tachyzoites of the indicated strains and monitored for 30 days. All RHΔpp6c-infected and control (naïve) mice remained alive without any signs of illness and RHΔpp6r-infected mice had significantly prolonged survival. However, all mice infected with WT, RHΔpp6c-cp and RHΔpp6r-cp reached their humane endpoint within 8–10 days. Statistical significance were tested by log rank Mantel-Cox test. ***P = 0.0005, n.s., not significant. (B) The levels of total IgG and IgG isotypes (IgG1 and IgG2a) were determined in the serum of mice immunized with 200 RHΔpp6c tachyzoites (6 mice/strain). Analysis was performed using ELISA at 30 days post-immunization, and the results show that RHΔpp6c-immunized mice developed a high level of IgG and IgG isotypes compared with the naïve mice. Data represnet mean ± SD and statistical differences were analyzed by a two-way ANOVA. ****P < 0.0001. (C) 30 days after immunization with RHΔpp6c, mice were injected i.p. with 1000 tachyzoites of RH or PYS (6 mice/strain) and monitored for 30 days. Survival curves indicated that RHΔpp6c-immunized mice were protected against challenge with RH or PYS compared with the naïve mice. Mice in the naïve group reached their humane endpoint within 8–10 days. Statistical significance was examined by log rank Mantel-Cox test. ***P = 0.0007 (Naïve+RH vs. RHΔpp6c+RH), ***P = 0.0006 (Naïve+PYS vs. RHΔpp6c+PYS). (D) 30 days after immunization with RHΔpp6c, mice (6 mice/strain) were orally gavaged with 20 Pru cysts and monitored for 30 days. No morbidity and death were detected in RHΔpp6c-immunized mice. However, half of the naïve mice have succumbed to illness and reached their humane endpoint between 10–13 days post challenge. The level of significance was analyzed by log rank Mantel-Cox test. n.s., not significant. (E) Parasite cyst burden was quantified in the brain of mice that remained alive at 30 days post infection (n = 3 for naïve and n = 6 for RHΔpp6c). The cysts in the brain of the RHΔpp6c-immunized mice were significantly lower than that in the naïve mice. Data represnet mean ± SD and Statistical differences were analyzed by the unpaired t-test. ***P = 0.0003.

https://doi.org/10.1371/journal.ppat.1011831.g007

To explore whether RHΔpp6c confers protection against acute and chronic infection by T. gondii, mice were i.p. injected with a single dose of 200 RHΔpp6c tachyzoites in 200 μL PBS or mock-immunized with 200 μL of PBS as the naïve group. Thirty days after immunization, six immunized mice per group were challenged with 10³ tachyzoites of type I strain RH or ToxoDB#9 strain PYS, and mouse survival was recorded for 30 days. The results showed that mice in the naïve group reached their humane endpoint within 8–10 days, whereas mice immunized with RHΔpp6c did not exhibit signs of illness for 30 days after challenge by the WT strains (Fig 7C). We also tested the protective efficacy of immunization with RHΔpp6c against chronic infection by orally challenging immunized mice with cysts (20 cysts/mice) of type II Pru strain 30 days post-immunization. As anticipated, 50% of mice in the naïve group succumbed to illness and reached their humane endpoint between 10–13 days post infection, however mice immunized with 200 RHΔpp6c tachyzoites remained alive and did not develop illness (Fig 7D). Parasite cyst burden in the survied mice was counted after 30 days of infection. The number of cysts in the naïve+challenged mice (3,290 ± 105) was significantly higher than that in the RHΔpp6c-immunized+challenged mice (35 ± 8) (Fig 7E). Taken together, these results showed that immunization with low doses of RHΔpp6c conferred significant protection against type I (RH) and ToxoDB#9 (PYS) strains and type II Pru cysts challenge and elicited a mixed anti-T. gondii Th1/Th2 responses, with a shift toward Th1-mediated cellular immunity axis.

Ethics statement

The study protocol was reviewed and approved by the Research Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Acdemy of Agricultural Sciences (Permit No. 2021–006). All experiments were carried out in accordance with the national guidelines.

Animals

Seven-to-eight week old Kunming mice were housed in environmentally enriched conditions with 12-h light/dark cycle. Mice were acclimatized to the local environment for at least one week prior to the experiment [17, 59]. Unless otherwise stated, six mice were used per group.

Cell line and parasite growth conditions

Tachyzoites of T. gondii type I RHΔku80 (referred to as WT), RHΔku80::TIR1 (RH::TIR1) and ToxoDB#9 (PYS) strains were maintained in monolayers of human foreskin fibroblasts (HFFs, ATCC, Manassas, VA, USA), as previously described [17,59]. HFFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C with 5% CO₂. Cysts of T. gondii type II Pru strain were obtained from the brain homogenates of Kunming mice as previously described [56,59].

Construction of the deletion strains and epitope tagging

Knock-out and epitope tagged strains were constructed using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system as previously described [17,59]. All the primers used are listed in S5 Table. For the CRISPR plasmid of each knock-out strain, pSAG1:CAS9-U6:sgUPRT was used as a template, and the corresponding sgRNA of each gene was replaced with sgUPRT, as previously described [59]. For the construction of 5HR-DHFR-3HR, according to the Gibson assembly kit protocol (New England Biolabs, USA), the 5’, 3’ homologous fragments and DHFR of the gene of interest (GOI) were amplified using T. gondii genomic DNA and pUPRT-DHFR-D plasmid, respectively. The three fragments were ligated to replace the coding region of GOI, as previously described [17,59]. For the construction of GOI epitope-tagged strain, using pLIC-6×HA-DHFR as the template, a fragment of about 42 bp containing the GOI gene and a tagged product containing 6×HA were amplified, as previously described [59]. Finally, the specific CRISPR plasmids (~40 μg) and the corresponding fragments (~15 μg) were co-transfected into freshly egressed WT tachyzoites. The transfectants were selected by 3 μM pyrimethamine and isolated via limiting dilution.

Construction of conditional knock-down strain

To construct conditional knock-down strains, mini-auxin-induced degradation system combined with CRISPR-Cas9 system were used as previously described [60]. Briefly, freshly egressed RH::TIR1 tachyzoite were co-transfected with ~15 μg amplicons flanked with short homology regions containing mAID-6HA and a DHFR maker, and ~40 μg CRISPR plasmids to tag the endogenous genomic locus of GOI at the C-terminus, and the transfectants were selected by limited dilution and grown in medium containing 3 μM pyrimethamine. All the primers used are listed in S5 Table. For knock-down, a final concentration of 500 μM indole-3-acetic acid (IAA) was added to the culture, while 0.1% ethanol was added to the culture for mock treatment.

Construction of the complemented and mutant strains

For the complete fragment complemented with TgPP6C and the six core motifs specific to the mutated TgPP6C, the complete TgPP6C coding region and the TgPP6C coding region of each core motif (GDIHG, GDYVDRG, GNHE, HGG, RG and H) mutated to alanine were amplified respectively for the construction of pTUB::PP6C::CAT and pTUB::MuPP6C::CAT plasmids, as previously described [17]. All the primers used are listed in S5 Table. About 40 μg pSAG1::Cas9-U6::sgUPRT plasmid and ~15 μg positive complementary or mutant fragment were co-transfected into fresh egressed RHΔpp6c tachyzoites. The transfectants were isolated by limited dilution and grown in medium containing 20 μM chloramphenicol and verified by immunofluorescence staining.

Immunofluorescence staining and Western blotting

For immunofluorescence, confluent HFF monolayers grown on coverslips were infected by tachyzoites of WT, TgPP6C::6HA, TgPP6S::6HA, TgPP6R::6HA, TGGT1_203150::6HA, RHΔpp6c, RHΔpp6c-cp, RHΔpp6r, RHΔpp6r-cp and PP6C mutants (MU-GDIHG, MU-GDYVDRG, MU-GNHE, MU-HGG, MU-RG and MU-H), and knock-down strains PP6C-mAID-HA and PP6R-mAID-HA with or without IAA, then incubated for 24–36 h. The infected HFFs were washed 3 times with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. Cells were permeabilized using 0.1% Triton X-100, blocked with 3% bovine serum albumin at room temperature for 30 min, and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study included mouse anti-SAG1 (1:1000), rabbit anti-IMC1 (1:500), mouse anti-IMC1 (1:500), rabbit anti-SERCA (1:500), rabbit anti-HA (1:1000), rabbit anti-Chromo1 (1:500), rabbit anti-Centrin1 (1:500), rabbit anti-GRA32 (1:500), rabbit anti-ISP1 (1:500), rabbit anti-ARO (1:500), rabbit anti-CPN60 (1:500), rabbit anti-HSP60 (1:500), rabbit anti-MIC2 (1:500) and rabbit anti-GAP45 (1:500) (prepared in our laboratory). The secondary antibodies conjugated with Alexa Fluor 488/594 (Invitrogen, USA) were added at appropriate dilutions in PBS for 1 h at 37 °C in the dark. The cell nuclei were counter-stained with DAPI (4’,6-diamidino-2-phenylindole), and the cells were photographed under a Leica confocal microscope (TCS SP8, Leica, Germany).

For Western blot analysis, freshly egressed tachyzoites of the WT, TgPP6C::6HA, TgPP6S::6HA and TgPP6R::6HA strains were collected and lysed on ice using RIPA lysis buffer (Thermo Fisher Scientific, USA). The target protein was analyzed by SDS-PAGE and transferred to PVDF membrane (Immobilon, Millipore, USA). The Western blotting antibodies used in this study were rabbit anti-aldolase (1:500) and rabbit anti-HA (1:1000). Images were acquired on a Minichemi 610 chemiluminescence imager (Sagecreation, Beijing, China), as previously described [61].

Phenotypic characterization of the mutant strains

The standard plaque formation assay was performed as previously described [59]. Freshly egressed tachyzoites of the WT and mutant strains were collected, and diluted to 10³/mL or 2.5 × 10³/mL. According to the experimental requirements, and selecting the appropriate concentration, 200 μL of tachyzoites of the WT and mutant strains were added to monolayers of HFFs grown on 12-well tissue culture plastic plates. For conditional knock-down, 500 μM IAA or 0.1% ethanol was added to the medium before the addition of RH::TIR1 or mutant tachyzoites. The plates were incubated at 37 °C with 5% CO₂ for 7 days before fixation and staining with 0.5% crystal violet. The size and number of plaques were determined using a scanner as previously described [59]. All plaque assays were performed in triplicate.

The ability of tachyzoites of WT, RHΔpp6c, RHΔpp6c-cp, RHΔpp6r and RHΔpp6r-cp to invade host cells was determined by the invasion assay, as previously described [59]. Briefly, tachyzoites of WT, RHΔpp6c and RHΔpp6c-cp were harvested, counted, and diluted to a final concentration of 2 × 10⁷/mL. Then, 100 μL was added to coverslips positioned in the bottom of 12-well culture plates with confluent HFF monolayers and incubated for 40 min, followed by washing to remove the unattached tachyzoites. As for the knock-down and RH::TIR1 strains, tachyzoites were pretreated with 500 μM IAA or 0.1% ethanol for 16 h before being added to the well. The cells were fixed with 4% PFA. The mouse anti-SAG1 and Alexa Fluor 488 goat anti-mouse antibodies were added, followed by washing with PBS to remove unbound antibodies. Before the second round of immunolabeling, cells were permeabilized with 0.1% Triton X-100 in PBS. Then secondary antibodies mouse anti-SAG1 and Alexa Fluor 594 goat anti-mouse were added. The ratio of intracellular/total tachyzoites was used to determine parasite invasion efficiency, as previously described [59]. At least 20 random microscopic fields from three separate experiments per sample were examined.

For the intracellular replication assay, tachyzoites of WT, RHΔpp6c, RHΔpp6c-cp, RHΔpp6r and RHΔpp6r-cp were adjusted to a final concentration of 1 × 10⁶/mL. Then, 100 μL of tachyzoite suspension of each strain was inoculated into confluent HFFs grown in 6-well culture plates for 1 h. The tachyzoites remained attached to the cell surface were washed with PBS and the plates were further incubated at 37 °C with 5% CO₂ for 31 h. For knock-down and RH::TIR1 strains, parasites were pretreated with 500 μM IAA or 0.1% ethanol for 16 h before being used in the replication assay. The cells were fixed with 4% PFA, followed by addition of mouse anti-SAG1 and Alexa Fluor 488 goat-anti mouse as primary and secondary antibodies, respectively. For each strain, at least 200 PVs per well were counted to determine the number of PVs enclosing 1–3, 4–7, 8–15, 16–32 and >32 tachyzoites, and the number of abnormal PVs with asynchronous division was recorded. Replication assay data were obtained from three independent experiments.

For the egress assay, tachyzoites of WT, RHΔpp6c, RHΔpp6c-cp, RHΔpp6r and RHΔpp6r-c were adjusted to a final concentration of 1 × 10⁶/mL. Then, 100 μL of tachyzoite suspension of each strain was inoculated into 12 well culture plates containing monolayers of HFFs for 1 h. After washing the unbound tachyzoites and continued incubation for 36–48 h, tachyzoites were treated with DMEM (pre-warmed at 37 °C) containing 3 μM calcium ionophore A23187. For the knock-down and RH::TIR1, tachyzoites were grown in culture media containing 500 μM IAA or 0.1% ethanol for 36–48 h before the induction of egress. After treatment with calcium ionophore for 2 min, the cells were immediately fixed. For each strain, at least 200 PVs per well were counted to determine the number of PVs with egressed tachyzoites as a percentage of the total number of PVs [59]. Egress assays were performed three separate times.

For transmission electron microscopy, HFF monolayers were infected with WT and RHΔpp6c strains for 20 and 30 h, respectively, and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature. The samples were washed in cacodylate buffer, post-fixed in 1% osmium tetroxide, and processed as previously described [61]. Images were captured using a HITACHI HT7700 electron microscope under 80 kV. The data are obtained from three independent experiments.

Virulence assessment in mice

To assess the role of TgPP6 in the virulence of T. gondii, groups (n = 6) of Kunming mice were injected with WT strain or mutant strains (RHΔpp6c, RHΔpp6c-cp, RHΔpp6r and RHΔpp6r-cp) intraperitoneally (i.p.) at an inoculum of 100 tachyzoites in a volume of 200 μL sterile PBS. Another group of mice was inoculated i.p. with 200 μL PBS only and served as a control group (naïve). The infected mice were monitored twice daily for signs suggestive of T. gondii infection for 30 days. Mice were euthanized once they reach their humane endpoint (e.g., body weight loss of ≥20%). The virulence of the different strains was compared using the survival curve of the mice. For immunization of mice using RHΔpp6c, the mice were inoculated i.p. with 200 μL PBS containing 200 RHΔpp6c tachyzoites or mock-immunized (naïve) with 200 μL PBS. To examine the immune responses induced by immunization using RHΔpp6c, enzyme-linked immunosorbent assay (ELISA) was used for quantification of anti-T. gondii total IgG and subclasses of IgG (IgG1, and IgG2a) antibodies 30 days after immunization, as previously described [56]. To assess the protective efficacy of immunization against acute infection, RHΔpp6c immunized and naïve mice (6 mice/group) were injected i.p. with a lethal challenge of type I strain RH or ToxoDB#9 strain PYS (10³ tachyzoites /mouse) in a volume of 200 μL PBS, 30 days after immunization. We also evaluated the efficacy of protection against chronic T. gondii, where immunized mice and naïve mice were gavaged orally with 20 cysts of type II Pru strain. All challenged mice were observed twice daily for morbidity (hunched posture, ruffled fur inactivity, body weight loss) to 30 d after re-challenge. The survived mice were euthanized to determine their brain cyst burden by homogenizing their brains, 1 brain in 1 mL of PBS, as previously described [56,59].

Immunoprecipitation assay

Immunoprecipitation assay was performed using the Pierce Magnetic HA-Tag IP/Co-IP kit (ThermoFisher Scientific, USA), as previously described [17]. In brief, the mixture of 50 μL of anti-HA beads and the supernatant of parasite lysates was incubated for 3 h at 4 °C under end-to-end rotation. Protein was obtained by adding 100 μL of elution buffer. The eluted proteins were separated by SDS-PAGE. After colloidal Coomassie staining, eight horizontal gel slices were excised in strip lanes and the proteins were processed with trypsin (Promega/Pierce) using a Janus liquid handling system (PerkinElmer) in-gel digestion. Subsequently, peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS), the RAW data were searched against the T. gondii databases in ToxoDB (https://toxodb.org). Peptides and proteins were identified with a 1% false discovery rate (FDR), and proteins with single modification sites, contaminants, and reversed proteins were all removed.

Phosphoproteomic analysis

Tachyzoites of the WT and RHΔpp6c were used to infect T-175 cell culture plastic flasks containing monolayers of HFFs using a multiplicity of infection (MOI) of 5. To compensate for the low rate of cell division in mutant strain, RHΔpp6c was cultured for a longer duration compared to wild type. Therefore, cells infected by WT or RHΔpp6c tachyzoites were incubated for 36 h or 48 h, respectively, to maximize the number of tachyzoites inside the PVs, followed by washing with PBS and storage at –80 °C. For protein extraction, lysis buffer (1% SDS, 1% protease inhibitor cocktail, 1% phosphatase inhibitor for phosphorylation) was added to each sample and vortexed for 5 min. The mixture was sonicated in an ice bath (sonication for 3 s, stop for 5 s, 25% power, power 220 W) and lysed for 3 min, then centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was collected and protein concentration of each sample was determined by BCA method.

For trypsin digestion and LC-MS/MS analysis, 30 μg of the protein solution was used for electrophoresis, followed by enzymatic digestion of the same amount of protein, and the volume was adjusted to the same volume with lysis buffer. Then, 1 volume of pre-cooled acetone was added, followed by vortexing and addition of 4 times of acetone at –20 °C/2 h. After centrifugation at 4500 g for 5 min, acetone was added for washing. After the precipitate was air-dried, triethylammonium bicarbonate was added, and the ultrasound machine was used to break up the precipitate. Trypsin was added at 1:50 protease-to-protein mass ratio and the enzyme was hydrolyzed overnight. Dithiothreitol was added to a final concentration of 5 mM at 56 °C for 30 min. Then, iodoacetamide was added to make a final concentration of 11 mM, followed by incubation at room temperature for 15 min in the dark. Finally, the peptides were desalted by Strata X SPE column. The peptides were dissolved in loading buffer solution (50% acetonitrile/0.5% acetic acid), the supernatant was transferred to IMAC microspheres suspension and incubated on a rotary shaker with gentle shaking. To remove non-specifically adsorbed peptides and elute the enriched phosphopeptides, IMAC microspheres were washed sequentially with 50% acetonitrile/0.5% acetic acid and 30% acetonitrile/0.1% trifluoroacetic acid, and then an elution buffer containing 10% NH₄OH was added.

The tryptic peptides were dissolved in solvent A, loaded onto a home-made reversed-phase analytical column (25-cm length, 75 μm i.d.). Solvent A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile, and solvent B was a solution containing 0.1% formic acid and 100% acetonitrile. The gradient settings were 0–70 min 6%~24%B, 70–84 min 24%~35%B, 84–87 min 35%~80%B and 87–90 min 80%B, and the flow rate was maintained at 500nL/min on an EASY-nLC 1200 system. The peptides were subjected to capillary ion source for analsyis using TimsTOF Pro mass spectrometer (Bruker Daltonics). The electrospray voltage and scan range were 1.60 kV and 100–1700 m/z, respectively.

For bioinformatics analysis, the resulting MS/MS spectra were searched against T. gondii GT1 database ToxoDB (https://toxodb.org) using MaxQuant search engine (v.1.6.15.0). Peptide and protein identifications were filtered to a 1% false discovery rate (FDR). Fold-Change (FC) > 1.5 or < –1.5 and P < 0.05 were set as the threshold for differentially phosphorylated proteins. The significantly enriched gene ontology (GO) terms of the differentially phosphorylated proteins were identified by comparing to the parasite genome in ToxoDB database (https://toxodb.org).

Statistical analysis

All statistical analyses were performed using GraphPad Prism software version 8.4.0 for MacOS (GraphPad, CA, USA) using two tailed unpaired Student’s t-test and two-way analysis of variance (ANOVA). Survival curves were compared using the log-rank (Mantel-Cox) test. Data are based on three independent biological replicates unless otherwise specified. P values < 0.05 were considered significant.