The protein phosphatase 2A holoenzyme is a key regulator of starch metabolism and bradyzoite differentiation in Toxoplasma gondii

Abstract

Phenotypic switching between tachyzoite and bradyzoite is the fundamental mechanism underpinning the pathogenicity and adaptability of the protozoan parasite Toxoplasma gondii. Although accumulation of cytoplasmic starch granules is a hallmark of the quiescent bradyzoite stage, the regulatory factors and mechanisms contributing to amylopectin storage in bradyzoites are incompletely known. Here, we show that T. gondii protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit PP2A-C, a scaffold subunit PP2A-A and a regulatory subunit PP2A-B. Disruption of any of these subunits increased starch accumulation and blocked the tachyzoite-to-bradyzoite differentiation. PP2A contributes to the regulation of amylopectin metabolism via dephosphorylation of calcium-dependent protein kinase 2 at S679. Phosphoproteomics identified several putative PP2A holoenzyme substrates that are involved in bradyzoite differentiation. Our findings provide novel insight into the role of PP2A as a key regulator of starch metabolism and bradyzoite differentiation in T. gondii.

Fig. 1. Characterization of PP2A holoenzyme of Toxoplasma gondii.

a Indirect immunofluorescence of T. gondii RH tachyzoites expressing PP2A-C-6HA, PP2A-B-6HA and PP2A-A-6HA under the control of their endogenous promoters. Cells were fixed at 24 h post infection and stained with anti-IMC1 (green) antibody, anti-HA (red) antibody, and Hoechst DNA-specific dye (blue). Scale bar, 10 µm. b Indirect immunofluorescence of Pru bradyzoites expressing PP2A-C-6myc, PP2A-B-6myc and PP2A-A-6myc under the control of their endogenous promoters. Parasites were grown in HFF cells under bradyzoite-inducing alkaline conditions for 7 days and stained with FITC-Dolichos biflorus lectin (DBL) (green), anti-Myc (red) antibody and anti-IMC1 (magenta) antibody. Scale bar, 10 µm. c The overlap of Homo sapiens HsPP2A (PDB: 2iae, https://www.rcsb.org/structure/2IAE) structure and the predicted T. gondii TgPP2A holoenzyme. The protein structure homology-modeling program SWISS-MODEL was used to construct the structure of the T. gondii PP2A holoenzyme. Homo sapiens PP2A-A, PP2A-B, and PP2A-C were used as templates for TgPP2A-A, TgPP2A-B, and TgPP2A-C modeling, respectively. The three predicted PP2A subunits were constructed using ZDOCK module in the Discovery Studio.

Fig. 2. PP2A holoenzyme is essential for starch metabolism and lytic life cycle of Toxoplasma gondii.

a The wild-type Pru, PruΔcdpk2, Δpp2a-c and the complemented strains were inoculated into HFF cells under normal culture conditions and the amylopectin was stained with PAS (red), followed by immunofluorescence detection of tachyzoites with anti-IMC1 antibody (green). The third row showes the amylopectin accumulation in the extracellular or newly invaded PruΔpp2a-c tachyzoites. Scale bar, 10 µm. b Quantification of amylopectin levels in the indicated strains. Data represents the mean ± SD from five independent experiments. Statistical significance tested by two-tailed, unpaired t test with Welch’s correction. ***P value = 0.0001 (Pru vs PruΔpp2a-c), ***P value = 0.0001 (Pru vs PruΔpp2a-b), ***P value = 0.0001 (Pru vs PruΔpp2a-a), ***P value = 0.0003 (Pru vs PruΔcdpk2). c Representative images of the plaque assays of the indicated strains grown under normal culture conditions for 7 days. Images are representative of three independent experiments. d Relative size of the plaques detected in (c). Data represents the mean ± SD from three independent experiments. Statistical significance tested by two-tailed, unpaired t test with Welch’s correction and the indicated strains were compared with Pru ****P value < 0.0001 or PruΔcdpk2 strain ^####P value < 0.0001. e Western blotting analysis of amylose binding of PP2A-C-6HA, PP2A-B-6HA, PP2A-A-6HA, and CDPK2-3HA. Input, unbound, and amylose-bound fractions were stained with anti-HA antibody. CDPK2 was used as a positive control for amylose binding. Source data are provided as a Source data file.

Fig. 3. PP2A holoenzyme is essential for bradyzoite differentiation in cell culture.

a The indicated parasite strains were allowed to infect HFF cells for 4 h followed by incubation in an alkaline culture medium without CO₂ for 4 days for induction of bradyzoites. Parasites were stained with anti-IMC1 antibody (red) and bradyzoite cyst wall was detected by FITC-Dolichos biflorus lectin (DBL) (green). PruΔBfd1 was used as a negative control for stage conversion. Scale bar, 10 µm. b Quantification of differentiation in wild-type, knockout, and complemented strains following 4 days of exposure to alkaline stress. The mean ± SD was plotted for six biological replicates, with percentage of DBL positive vacuoles calculated based on at least 100 vacuoles per replicate. Statistical significance tested by Student’s one-tailed t test and the indicated strains were compared with Pru strain ****P value < 0.0001, n.s. = not significant. c Western blotting analysis of the tachyzoites lysates (incubated for 2–3 days under normal culture conditions) and bradyzoites (incubated for 4 days under high alkaline culture conditions) by anti-BAG1 antibody. d The indicated parasites were added to HFF cells for 4 h to allow the parasites to invade host cells, followed by incubation in an alkaline culture medium without CO₂ for 4 days for induction of bradyzoites. Parasites were detected by anti-GAP45 antibody (green) and bradyzoite cysts were stained with succinylated wheat germ agglutinin (s-WGA) (red) and the bradyzoites specific anti-BAG1 antibody (magenta). Scale bar, 10 µm. e Transmission electron micrograph of parasites incubated for 4 days under high alkaline culture conditions. Arrowheads point to cyst wall (CW) or parasitophorous vacuole membrane (PVM) in the insert. f Quantification of spontaneously differentiated bradyzoites of Pru and PruΔpp2a-c strains following 48 h growth in differentiated myotubes. The mean ± SD was plotted for three biological replicates, with the percentage of DBL positive vacuoles calculated based on examining at least 100 vacuoles per sample. (Student’s one-tailed t test, *P value = 0.0159). Source data are provided as a Source data file.

Fig. 4. PP2A holoenzyme is necessary for formation of brain cysts in mice.

a–c C57BL/6 female mice were infected intraperitoneally with the designated doses of the indicated strains (n = 8 mice / strain), and the survival of mice was monitored for 30 days. Statistical significance tested by log rank Mantel-Cox test. Compared with Pru strain, for (a) group, *P value =  0.024 (Pru vs PruΔcdpk2), **P value = 0.003 (Pru vs PruΔpp2a-c), **P value = 0.003 (Pru vs PruΔpp2a-b), **P value = 0.003 (Pru vs PruΔpp2a-a); For (b) group, n.s. = not significant, ****P value < 0.0001; for (c) group, ***P value = 0.0007 (Pru vs PruΔcdpk2), ***P value = 0.0002 (Pru vs PruΔpp2a-c), ***P value = 0.0002 (Pru vs PruΔpp2a-b), ***P value = 0.0002 (Pru vs PruΔpp2a-a). d Number of Pru cysts in the brain tissue of the survived mice. At 30 days’ post-infection, tissue cyst burden in the brain of the survived mice form (a–c) was determined by FITC-Dolichos biflorus lectin (DBL) staining. Cyst burden was estimated based on the number of cysts detected in one cerebral hemisphere per mouse brain. e Representative images of the plaque assays of the ME49 and ME49Δpp2a-c strains grown under normal culture conditions for 8 days. Images are representative of three independent experiments. f, g C57BL/6 female mice were infected intraperitoneally with the designated doses of the ME49 and ME49Δpp2a-c strains, and the survival of mice was monitored for 30 days. Statistical significance tested by log rank Mantel-Cox test. ****P value < 0.0001. (n = 20 mice / strain for the 10² dose; n = 15 mice / strain for the 10⁵ dose). h The number of ME49 cysts in the brain tissue of the survived mice. At 30 days’ post-infection, tissue cyst burden in the brain of the survived mice form (f, g) was determined by DBL staining. Cyst burden was estimated based on the number of cysts detected in one cerebral hemisphere per mouse brain. Source data are provided as a Source data file.

Fig. 5. Functional analysis of the consensus sequences of the core catalytic motif of PP2A-C.

a Schematic representation of the gene model for PP2A-C with the six consensus sequences of the core catalytic motif. b PAS staining of PruΔpp2a-c tachyzoites expressing the wild-type or mutant version of PP2A-C fused with 3Myc tags under control of endogenous promoters, under normal culture (pH 7.4) or alkaline culture conditions (pH 8.2). The amylopectin was stained with PAS and the tagged protein was detected using anti-Myc antibody (green or red), followed by immunofluorescence detection of the parasites using anti-IMC1 antibody (magenta). Scale bar, 10 µm.

Fig. 6. Comparative phosphoproteomic analysis of PruΔpp2a-c, PruΔcdpk2 strains, and their parental Pru strain under alkaline culture conditions.

a Schematic representation of the workflow of comparative proteomic and phosphoproteomic analyses. Parental Pru, PruΔcdpk2, and PruΔpp2a-c strains were grown in heavy, medium, or light SILAC labeling medium, respectively. After labeling, tachyzoites were allowed to differentiate to bradyzoites for 4 days under SILAC labeling medium, then the samples were harvested by needle passage, and proteins were mixed 1:1:1 before tryptic digestion. Phosphopeptides were enriched by I-MAC, and finally the samples were analyzed by LC/MS. b, c Volcano plots show significant fold changes in proteins in PruΔpp2a-c vs Pru and PruΔpp2a-c vs PruΔcdpk2 under alkaline culture conditions. Proteins with fold changes ≥2.0 or ≤−2.0 with P  < 0.05 are highlighted in orange and laurel-green color, respectively. d Venn diagram of the differential phosphopeptides of PruΔpp2a-c vs Pru and PruΔpp2a-c vs PruΔcdpk2. e The percentage of phosphoproteins from Supplementary Data 5 relative to the total number of phosphoproteins, according to their predicted localization.

Fig. 7. Dephosphorylation of CDPK2 is essential for the regulation of starch metabolism.

a PAS staining of PruΔcdpk2 tachyzoites expressing the wild-type or phosphomimetic mutant version of CDPK2 fused with 3HA tags under control of β-tubulin promoters and normal culture conditions. The amylopectin was stained with PAS (red), the tagged protein was detected using α-HA antibody (green) and parasites were detected with anti-IMC1 antibody (magenta). Scale bar, 10 µm. b PAS staining of the double knockouts of PruΔpp2a-cΔcdpk2 and PruΔpp2a-cΔgp strains. The amylopectin was stained with PAS (red), followed by immunofluorescence detection of parasites with anti-IMC1 antibody (green). Scale bar, 10 µm.

Fig. 8. Four hyperphosphorylated proteins in PruΔpp2a-c strain are important for parasite differentiation.

a Schematic representation of the domain architecture of four hyperphosphorylated proteins in PruΔpp2a-c strain. b Representative images showing the results of the plaque assay of the indicated strains grown under normal culture conditions for 7 days. Images are representative of three independent experiments. c Representative vacuoles of differentiation after 72 h exposure to alkaline medium. FITC-labeled Dolichos biflorus lectin (DBL) specifically stains the differentiated vacuoles and the parasites were stained with IMC1 and mAID-HA tagged proteins were detected with anti-HA antibody. Scale bar, 7.5 µm. d The percentages of DBL-positive cysts were calculated based on counting at least 100 vacuoles per sample treated as described in (c). Data represent the means ± SD from three independent experiments. The percentage of vacuoles scoring “DBL-high” was used for comparison. Statistical significance tested by Student’s one-tailed t test. ***P value = 0.0002 (Pru vs Δ311100), ***P value =  0.0003 (Pru vs Δ298610), ^###P value = 0.0004 (Pru::TIR Ctrl vs 298610-mAID IAA), ^###P value = 0.0002 (Pru::TIR Ctrl vs 224260-mAID IAA), ^#P value = 0.036 (Pru::TIR Ctrl vs 237520-mAID Ctrl), ^###P value = 0.0002 (Pru::TIR Ctrl vs 237520-mAID IAA). Source data are provided as a Source data file.

Fig. 9. Schematic representation of the PP2A holoenzyme as an integral component of the regulatory network mediating amylopectin metabolism and tachyzoite-to-bradyzoite transformation in Toxoplasma gondii.

T. gondii PP2A holoenzyme consists of a scaffolding subunit PP2A-A, a catalytic subunit PP2A-C and a regulatory subunit PP2A-B. The disruption of any of the PP2A subunits causes significant starch accumulation and blocks tachyzoite-bradyzoite differentiation via dephosphorylation of calcium-dependent protein kinase 2 and some bradyzoites regulators, respectively.