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. 2022 Jun 29;10(3):e0070222.
doi: 10.1128/spectrum.00702-22. Epub 2022 Jun 2.

Restriction Checkpoint Controls Bradyzoite Development in Toxoplasma gondii

Affiliations

Restriction Checkpoint Controls Bradyzoite Development in Toxoplasma gondii

Anatoli V Naumov et al. Microbiol Spectr. .

Abstract

Human toxoplasmosis is a life-threatening disease caused by the apicomplexan parasite Toxoplasma gondii. Rapid replication of the tachyzoite is associated with symptomatic disease, while suppressed division of the bradyzoite is responsible for chronic disease. Here, we identified the T. gondii cell cycle mechanism, the G1 restriction checkpoint (R-point), that operates the switch between parasite growth and differentiation. Apicomplexans lack conventional R-point regulators, suggesting adaptation of alternative factors. We showed that Cdk-related G1 kinase TgCrk2 forms alternative complexes with atypical cyclins (TgCycP1, TgCycP2, and TgCyc5) in the rapidly dividing developmentally incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. Examination of cyclins verified the correlation of cyclin expression with growth dependence and development capacity of RH and ME49 strains. We demonstrated that rapidly dividing RH tachyzoites were dependent on TgCycP1 expression, which interfered with bradyzoite differentiation. Using the conditional knockdown model, we established that TgCycP2 regulated G1 duration in the developmentally competent ME49 tachyzoites but not in the developmentally incompetent RH tachyzoites. We tested the functions of TgCycP2 and TgCyc5 in alkaline induced and spontaneous bradyzoite differentiation (rat embryonic brain cells) models. Based on functional and global gene expression analyses, we determined that TgCycP2 also regulated bradyzoite replication, while signal-induced TgCyc5 was critical for efficient tissue cyst maturation. In conclusion, we identified the central machinery of the T. gondii restriction checkpoint comprised of TgCrk2 kinase and three atypical T. gondii cyclins and demonstrated the independent roles of TgCycP1, TgCycP2, and TgCyc5 in parasite growth and development. IMPORTANCE Toxoplasma gondii is a virulent and abundant human pathogen that puts millions of silently infected people at risk of reactivation of the chronic disease. Encysted bradyzoites formed during the chronic stage are resistant to current therapies. Therefore, insights into the mechanism of tissue cyst formation and reactivation are major areas of investigation. The fact that rapidly dividing parasites differentiate poorly strongly suggests that there is a threshold of replication rate that must be crossed to be considered for differentiation. We discovered a cell cycle mechanism that controls the T. gondii growth-rest switch involved in the conversion of dividing tachyzoites into largely quiescent bradyzoites. This switch operates the T. gondii restriction checkpoint using a set of atypical and parasite-specific regulators. Importantly, the novel T. gondii R-point network was not present in the parasite's human and animal hosts, offering a wealth of new and parasite-specific drug targets to explore in the future.

Keywords: Toxoplasma gondii; apicomplexan; bradyzoite; cyclin; cyclin-dependent kinase; development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
TgCrk2 interacts with three atypical cyclins. (A) Replication and differentiation rates of the parental RHΔKu80TIR1 and ME49ΔKu80TIR1 strains. Replication rates are shown on the left graph as the number of the parasites per vacuole after 1-day growth in pH 7.4 medium, 5%CO2. The percentage of the DBA-positive cysts after 3 days of growth in pH 8.2 medium, ambient CO2 conditions represents the rate of bradyzoite differentiation and is shown on the right graph. Mean values ± SD of three replicates are shown. Unpaired t test returned P values of 6.4 ×ばつ 10−4 and 8.8 ×ばつ 10−6. (B) Immunofluorescent images of TgCrk2myc expressed in ME49ΔKu80TIR1 tachyzoites (pH 7.4 medium, 2 days) or developing bradyzoites (pH 8.2 medium, 3 days). Parasites were labeled with α-myc/α-rabbit IgG Fluor 568, α-IMC1/α-mouse IgG Fluor 488. Developing bradyzoites were identified by costaining with DBA/7-amino-4-methylcoumarin-3-acetic acid methanethiosulfonate (AMCA) Streptavidin 350. Scale bar 5 μm. (C) Immunoblot image of TgCrk2 isolated from ME49ΔKu80TIR1 TgCrk2myc tachyzoites grown in pH 7.4 medium at 5% CO2. Equal fractions of the soluble proteins pre- (IN: input) and post pulldown (NB: not bound), and immunoprecipitated TgCrk2myc complexes (B: bound) were analyzed. Western blot was probed with α-myc (α-mouse IgG-HRP) to detect TgCrk2myc and confirm pulldown efficiency. (D) Summary of the mass-spectrometry analysis of TgCrk2 complexes purified from RH and ME49 parasites. RHΔKu80 TgCrk2HA and ME49ΔKu80TIR1 TgCrk2myc tachyzoites were grown in pH 7.4 medium at 5% CO2 for 2 days and ME49ΔKu80TIR1 TgCrk2myc bradyzoites were grown in pH 8.2 medium in ambient CO2 for 3 days. Table shows detected cyclin proteins. The intensity of the gray bars reflects the probability of cyclin interaction with TgCrk2 kinase. (E) Phylogenetic analysis of apicomplexan P/U type cyclins. Protein sequences of cyclins related to P/U or PHO80 type from representatives of the superphylum alveolate: Toxoplasma gondii (Tg), Hammondia hammondia (Hh), Plasmodium falciparum (PF3D7), Babesia bovis (BBOV), Cryptosporidium parvum (Cgd), Chromera velia (Cvel), Vitrella brassicaformis (Vbra); kinetoplastids: Trypanosome cruzi (Tc); fungi: Saccharomyces cerevisiae (Sc), Candida viswanathii (Cv), Aspergillus niger (An); and land plants: Arabidopsis thaliana (At) were analyzed in R package ‘phangorn’. Branch support was determined in 100 bootstraps and the nodes supported by a higher than 82% value are indicated with a filled circle. T. gondii P-cyclins are shown in red.
FIG 2
FIG 2
Expression of TgCycP1 negatively affects bradyzoite differentiation. (A) Immunofluorescent images of the RH Tet-OFF TgCycP1 mutant grown under tachyzoite (pH 7.4, 5% CO2, 2 days), bradyzoite differentiation (pH 8.2 ambient CO2, 3 days), and either in the presence or absence of 2 μM anhydrotetracycline (ATc). Individual parasites and tissue cysts were visualized with α-IMC1/α-rabbit IgG Alexa 488 and DBA/Texas Red Streptavidin 595, respectively. (B) Quantifications of cysts formed by RH Tet-OFF TgCycP1 mutant under indicated conditions. An unpaired t test returned a P value of 6.4 ×ばつ 10−8.
FIG 3
FIG 3
TgCycP2 regulates the G1 phase in type II ME49 parasites. (A) Immunofluorescent microscopy of TgCycP2AID-HA expression in RHΔKu80TIR1 and ME49ΔKu80TIR1 tachyzoites. TgCycP2AID-HA was visualized with α-HA antibody (α-rat IgG Alexa Fluor 568) and costained with α-IMC1 (α-rabbit IgG Alexa Fluor488) and DAPI (nucleus). Scale bar 5 μm. (B) Immunoblot of the total lysates of RHΔKu80TIR1 TgCycP2AID-HA and ME49ΔKu80TIR1 TgCycP2AID-HA parasites treated with vehicle (−) or 500 μM auxin (+) for 1 h. Western blots were probed with α-HA (α-rat IgG-HRP) to detect P-cyclins, and with α-Tubulin A (α-mouse IgG-HRP) to confirm equal sample loading. (C) Quantifications of plaques formed by RHΔKu80TIR1 TgCycP2AID-HA and ME49ΔKu80TIR1 TgCycP2AID-HA lines. Parasites were grown with vehicle or 500 μM auxin for 6 (RH strain) or 9 (ME49 strain) days. The percentage of plaques formed by parasites in the presence of auxin relative to plaques formed by parasites treated with the vehicle is plotted on the graph. Data were analyzed by unpaired t test on three independent experiments (P = 4.7 ×ばつ 10−4). (D) Immunofluorescence microscopy analysis of TgCycP2AID-HA expression in ME49ΔKu80TIR1 tachyzoites. To reveal cell cycle-dependent expression, TgCycP2AID-HA (α-HA/α-rat IgG Fluor 568) was costained with centrosomes (α-Centrin1/α-mouse IgG Fluor 488), centrocone (α-MORN1/α-rabbit IgG Flour 488) or alveolar protein IMC1 (α-IMC1/α-rabbit IgG Fluor 488). Cell cycle phases were determined based on the number and morphology of the reference structures indicated with arrows. Percentage of parasites with single centrosome, expressing or not expressing TgCycP2AID-HA were calculated from 3 independent experiments. Mean value ± SD is shown. Scale bar 5 μm. (E) Degradation of TgCycP2 slows replication of ME49ΔKu80TIR1 tachyzoites. ME49ΔKu80TIR1 TgCycP2AID-HA parasites were grown with vehicle or 500 μM auxin for 24 h and visualized with α-IMC/α-rabbit IgG Fluor 488 antibodies. The number of parasites per vacuole was quantified in three independent experiments. Mean values ± SD are plotted on the graph. The unpaired t test showed nonsignificant changes between the two conditions. (F) Quantification of IFA images of ME49ΔKu80TIR1 TgCycP2AID-HA parasites treated with vehicle (−auxin) or with 500 μM auxin for 24 h and labeled with α-Centrin1 antibodies. The ratio of parasites with single (G1 phase, light green) or duplicated (S-phase/mitosis/budding, dark green) centrosomes is shown on the graph. The results of three independent experiments are shown. The unpaired t test showed nonsignificant changes at no auxin conditions and returned a P value of 4.2 ×ばつ 10−4 in the presence of auxin. (G) Quantification of IFA images of ME49ΔKu80TIR1 TgCycP2AID-HA parasites treated with vehicle (−auxin) or with 500 μM auxin for 24 h and labeled with α-IMC1 antibodies. The ratio of parasites with (light gray, see [E]) or without internal buds (dark gray) is plotted on the graph. An unpaired t test showed nonsignificant changes in the budding populations of parasites expressing or lacking TgCycP2. (H) Schematics of cell cycle-dependent TgCycP2 expression. Red cytoplasmic stain marks cell cycle phases of maximum TgCycP2 expression. Blue, nucleus; green dots, centrosomes. (I) Transcriptome analysis of ME49ΔKu80TIR1 TgCycP2AID-HA tachyzoites. Transcripts with >1.5 log2 change in differential expression caused by auxin-induced TgCycP2AID-HA degradation were sorted into G1, S/M/C, or noncyclical expression groups based on the predicted peak of the mRNA expression. A plot of the values at ± auxin conditions is shown. (J) The pie chart shows cellular pathways affected by TgCycP2 deficiency in ME49ΔKu80TIR1 tachyzoites. The table on the left lists the transcripts found in the gene expression category.
FIG 4
FIG 4
TgCycP2 and TgCyc5 play opposite roles in T. gondii differentiation. (A) IF analysis of ME49ΔKu80TIR1 TgCycP2AID-HA mutant differentiation. Images show cyst expansion from day 2 to day 5 in a pH 8.2 medium. TgCycP2AID-HA was detected with α-HA/α-rat IgG Fluor 568. Individual parasites were labeled with α-IMC1/α-mouse IgG Fluor 488 and the cyst wall was visualized using DBA/AMCA Streptavidin 350. Clusters of parasites undergoing the budding process are indicated with arrows on the IMC1 graph. (B) IFA images of day 2 and day 5 in vitro cysts formed by the ME49ΔKu80TIR1 TgCyc5AID-HA mutant in a pH 8.2 medium. TgCyc5AID-HA was labeled with α-HA/α-rat IgG Fluor 568, parasites with α-IMC1/α-mouse IgG Fluor 488, and cyst wall with DBA/AMCA Streptavidin 350. (C) IFA images of ME49ΔKu80TIR1 TgCycP2AID-HA and ME49ΔKu80TIR1 TgCyc5AID-HA mutants grown for 3 days in pH 8.2 medium with vehicle (−auxin) or with 500 μM auxin. The cyclins, parasites, and cysts were labeled with α-HA/α-rat IgG Fluor 568, α-IMC1/α-mouse IgG Fluor 488, and DBA/AMCA Streptavidin 350, respectively. The enlarged image on the right of each subfigure shows the expression or degradation of the corresponding AID-3xHA-fused cyclin. (D) Western blot of the total lysates of ME49ΔKu80TIR1 TgCycP2AID-HA and ME49ΔKu80TIR1 TgCyc5AID-HA tachyzoites (pH 7.4 medium for 2 days) and bradyzoites (at pH 8.2 medium for 3 days). Western blots were probed with α-HA (α-rat IgG-HRP) to detect cyclins, and with α-Tubulin A (α-mouse IgG-HRP) to confirm equal loading of lysates. (E) Quantification of cysts formed by the parental ME49ΔKu80TIR1 parasites and ME49ΔKu80TIR1 TgCycP2AID-HA and ME49ΔKu80TIR1 TgCyc5AID-HA mutants grown for 3 days in the pH 8.2 medium with vehicle (−auxin) or with 500 μM auxin. Cysts were identified by costaining of cyst wall (DBA/AMCA Streptavidin 350) and parasites (α-IMC1/α-mouse IgG Fluor 488). Bars show a mean value ± SD from three independent experiments. Unpaired t test returned P values of 3.6 ×ばつ 10−8 and 8.9 ×ばつ 10−10. (F) Quantification of cysts formed by the parent and ME49ΔKu80TIR1ΔTgCyc5AID-HA mutant grown in pH 8.2 medium for the indicated time. Cysts were identified by costaining of the cyst wall (DBA/AMCA Streptavidin 350) and parasites (α-IMC1/α-mouse IgG Fluor 488). Bars show a mean value ± SD from three independent experiments. Note a steady temporal increase of cyst number formed by TgCyc5-deficient parasites.
FIG 5
FIG 5
TgCyc5 is required for activation of the bradyzoite differentiation program. (A) The pie chart shows classes of transcripts differentially expressed upon conditional degradation of TgCycP2AID-HA or TgCyc5AID-HA cyclins during 3-day in vitro bradyzoite differentiation. (B) Transcripts with >1.5 log2 change expression in 3-day ME49ΔKu80TIR1 bradyzoites expressing (−auxin) or deficient (+auxin) in TgCycP2 or TgCyc5 are plotted on the graphs. Transcripts were sorted based on peak mRNA expression at different life stages (ToxoDB). The comparative analysis showed the opposite gene expression response to the loss of TgCycP2 and TgCyc5 cyclins.
FIG 6
FIG 6
Function of the TgCycP2 and TgCyc5 in rat brain cells. (A) Costaining of the primary brain cells isolated from 18-day embryonic rats (E18). Neurons were detected using an antibody against microtubule-associated protein 2 (α-MAP2/α-chicken IgG Fluor 488). Copurified astrocytes were detected using an antibody targeting glial fibrillary acidic protein (α-GFAP/α-mouse IgG Fluor 568). Spontaneous cysts (DBA/AMCA Streptavidin 350) formed by ME49ΔKu80TIR1 parasites (α-IMC1/α-mouse IgG Fluor 568) in the isolated primary neurons (α-MAP2/α-chicken IgG Fluor 488) or astrocytes (α-GFAP/α-mouse IgG Fluor 488) are shown on the right. (B) Immunofluorescent images of the ME49ΔKu80TIR1 TgCycP2AID-HA and ME49ΔKu80TIR1 TgCyc5AID-HA cysts in embryonic rat neurons. The 3-day cysts/vacuoles formed in the presence or absence of 500 μM auxin are shown. Toxoplasma cyclins, individual parasites, and cysts were labeled with α-HA/α-rat IgG Fluor 568, α-IMC1/α-mouse IgG Fluor 488, and DBA/AMCA Streptavidin 350, respectively. (C) Quantification of the cyst sizes formed by ME49ΔKu80TIR1 TgCycP2AID-HA mutant in rat neurons after 3 days of growth with vehicle or 500 μM auxin. Cyst diameter is plotted. Black lines indicate a mean value ± SD. The unpaired t test returned a P value of 3.6 ×ばつ 10−9. (D) Quantification of the cysts formed by the ME49ΔKu80TIR1 TgCycP2AID-HA and ME49ΔKu80TIR1 TgCyc5AID-HA mutants after a 3-day infection of the rat brain cells in the presence of vehicle (−auxin) or 500 μM auxin. Cysts were identified by lectin binding with DBA/AMCA Streptavidin 350 and individual parasites with α-IMC1/α-mouse IgG Fluor 488. Cysts were quantified in 10 random microscopic fields. Bars show a mean value ± SD of the three independent experiments. An unpaired t test returned a P value of 8.3 ×ばつ 10−7.
FIG 7
FIG 7
Model of restriction checkpoint regulation in T. gondii. (A) Schematics show the central cell cycle mechanisms that control T. gondii progression through intermediate life cycle stages. Cell cycles are drawn as color arrow circles: growth phase, G1a/G1b (red); DNA replication phase, S (dark green); mitosis coupled to cytokinesis (budding), M/C (light green). Note the relative extension of the G1 phase during bradyzoite development. Tachyzoite to bradyzoite conversion is regulated by alternative TgCrk2/Cyclin complexes operating at the restriction checkpoint (stop sign) in the G1 phase. The complex of the G1 kinase TgCrk2 with TgCycP1 promotes rapid cell division of early tachyzoites by quickly passing parasites through the R-point. In preparation for development, tachyzoites’ slow division was caused by the replacement of the highly active TgCrk2/TgCycP1 with moderately active TgCrk2/TgCycP2 complexes. Developmental (external) signals activate the expression of bradyzoite-specific TgCyc5, which in complex with TgCrk2, acts as a repressor of the R-point progression. T. gondii strains and in vitro conditions representing specific life cycle stages are indicated in the corresponding cell cycle diagrams. (B) Integration of the restriction checkpoint into the current model of the T. gondii development. Key factors that induce (signals), transduce (transducer), and activate (regulatory hub) tachyzoite-to-bradyzoite conversion are shown. Various signaling kinases and the integrated stress response (ISR) directly or indirectly activate/inhibit the restriction checkpoint function, which culminates in the execution of alternative differentiation programs. Dashed arrows mark putative connections.

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