The Apoptotic Role of Metacaspase in Toxoplasma gondii

Abstract

Toxoplasma gondii is a major opportunistic pathogen that spreads in a range of animal species and human beings. Quite a few characterizations of apoptosis have been identified in T. gondii treated with apoptosis inducers, but the molecular mechanisms of the pathway are not clearly understood. Metacaspases are caspase-like cysteine proteases that can be found in plants, fungi, and protozoa in which caspases are absent. Metacaspases are multifunctional proteases involved in apoptosis-like cell death, insoluble protein aggregate clearance, and cell proliferation. To investigate whether T. gondii metacaspase (TgMCA) is involved in the apoptosis of the parasites, we generated TgMCA mutant strains. Western blot analysis indicated that the autoproteolytic processing of TgMCA was the same as that for metacaspases of some other species. Indirect immunofluorescence assay (IFA) showed that TgMCA was dispersed throughout the cytoplasm and relocated to the nucleus when the parasites were exposed to the extracellular environment, which indicated the execution of its function in the nucleus. The number of apoptosis parasites was significantly diminished in the TgMCA knockout strain and increased in the TgMCA overexpression strain after treatment with extracellular buffer, as determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The lack of TgMCA did not affect the parasite propagation in vitro and virulence in vivo, suggesting that it is probably redundant in parasite propagation. But overexpression of TgMCA reduced the intracellular parasites growth in vitro. The TgMCA knockout strain showed more viability in extracellular buffer compared to the parental and overexpression lines. In this study, we demonstrated that TgMCA contributes to the apoptosis of T. gondii.

Keywords: T. gondii; apoptosis; knockout; metacaspase; programmed cell death.

Figure 1

Sequences-structure alignment of metacaspases. (A) TGGT1_206490, TGGT1_278975, and TGGT1_243298 protein sequence were aligned with sequences of human Caspase3, Caspase9, and yeast Yca1. The positions of identical and conserved residues in p20 domain are indicated by red-filled and non-filled rectangular frames, respectively. The conserved histidine and cysteine are indicated with red asterisk. Dots indicate gaps or missing residues. (B) The ternary structure of TgMCA was predicted by a model based on Yca1 (C) according to SWISS-Model and exhibited by Pymol. (D) Model pattern of the three ICE family proteins containing p20 domains in T. gondii, from top to bottom of the pattern, in order, TGGT1_206490, TGGT1_278975, and TGGT1_243298.

Figure 2

Identification of TgMCA and cellular localization. (A) Western blot analysis of native TgMCA. Total antigens from cell cultured RHΔku80 tachyzoites. An expected band and two other bands were elicited by anti-rTgMCA polyclonal antibody. Pre-immune serum was used as control. (B) Total antigens from transgenic tachyzoites with TgMCA-HA and RHΔku80, two expected bands were revealed using anti-HA monoclonal antibody as primary antibody. (C) TgActin used as control for (B). (D) Schematic of experimental design of TgMCA endogenously marked HA. A knock-in vector was constructed to target TgMCA endogenously marked HA at its C-terminal. (E) IFA analysis of TgMCA localization. TgMCA was distributed in the cytoplasm of the intracellular parasites and relocated to the nucleus of tachyzoites in the extracellular medium. The tachyzoites were stained with mouse anti-rTgMCA serum (green) or rabbit anti-T. gondii serum (red, stain the shape of the parasites), and the nucleus DNA was stained with Hoechst (blue). Scale bar, 5 μm.

Figure 3

Generation of a TgMCA mutant strain. (A,B) Schematic of the experimental design of the TgMCA knockout strain. A knockout vector (PTCR-CD TgMCA KO) was constructed to target the TgMCA complete gene. (C) Genomic PCR analysis of ΔTgMCA strain. The position of the primers are shown in (B). P1, P2 and P3, P4 were used to amplify exon2 to exon3 and exon10 to exon11 of TgMCA, respectively. (D) Western blot performed with anti-rTgMCA antibody on total extracts from Δku80 and ΔTgMCA, with TgActin used as control. (E) Schematic of the complementary strain construction. Crispr/Cas9-UPRT was used to target the UPRT locus of ΔTgMCA, and TgMCA coding sequence fused with the HA-tag at the C-terminal under the GRA1 promoter was inserted into the gap of the UPRT locus. (F) Western blot analysis of the restoration of TgMCA with anti-rTgMCA serum and anti-HA antibody on total lysate of Δku80 and ΔTgMCA-cm, with TgActin used as control.

Figure 4

Extracellular ΔTgMCA is less sensitive to environmental stress. (A) Ratio of apoptotic parasites by TUNEL assay. Apoptotic ΔTgMCA was significantly decreased when treated with BAG buffer for 4 h (p ≤ 0.05). TgMCA OE ΔTgMCA-cm1 and ΔTgMCA-cm2 produced far more TUNEL-positive parasites than Δku80 (p ≤ 0.05, p ≤ 0.01). Asterisks indicate statistically significant results as determined by One-way ANOVA with Tukey's post-hoc comparison. (B) Ratio of apoptotic parasites pre-incubated in Ringer buffer for 1–6 h by TUNEL assay. Both strains showed increased apoptotic ratio. Apoptotic Δku80 was higher than ΔTgMCA after 2 h in extracellular buffer and significantly increased at 4 h (p ≤ 0.05). Asterisks indicate statistically significant results (p ≤ 0.05) as determined by Two-way ANOVA with Tukey's post-hoc comparison. Data are mean ± SD (error bars) of three independent experiment.

Figure 5

Lack ofTgMCA did not affect intracellular parasite replication but did affect parasite viability in the extracellular environment. (A) Plaque assay comparing growth of ΔTgMCA, Δku80, and TgMCA OE. Each well (HFF cell) was infected with 500 parasites, and plaques were stained 7 days later. Data were compiled from three independent assay. (B) The plaque areas were counting by randomly chosen at least 50 plaques and using the Pixel point in Photoshop C6S software (Adobe, USA), and the data were compiled from three independent experiment. Analysis of plaque area was performed using One-way ANOVA with Tukey's post-hoc comparison. Asterisks indicated significant results (p ≤ 0.05). (C) Intracellular parasite replication of ΔTgMCA, Δku80, and TgMCA OE. Data were compiled from three independent assay, and in each assay 100 total PVs of each strain were counted. Data were determined by Chi-square analysis. (D) Invasion ratio of ΔTgMCA, Δku80, and TgMCA OE, freshly isolated and pre-incubated in extracellular buffer for 1 or 2 h. The ratio was based on the number of cells infected with parasites divided by the number of total cells in one field of view. Asterisks indicated statistically significant results (p ≤ 0.05 as determined by Two-way ANOVA with Tukey's post-hoc comparison). Data are mean ± SD (error bars) of three independent experiment. (E,F) Mouse survival after infection with different doses of ΔTgMCA, Δku80, and TgMCA OE. Balb/c mouse were injected intraperitoneally (i.p.) with 100 or 10 indicated parasites. There were 5 female mice in each group, statistical analysis was performed using life test (life test data = surv) in statistical analysis system (SAS institute Inc., USA). The figures are representative of three experiments (a dose of 100 tachyzoites) and two experiments (a dose of 10 tachyzoites) with similar outcomes.