Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: Calcium binding property of TgpCaBP. ( A ) Western blot analysis of native, HIS- and GST-tagged TgpCaBP using a rat anti-TgpCaBP antibody. ( B ) Calcium-dependent solubility was detected using western blotting. Parasites were lysed in 1% Triton X-100 in 5 mM EGTA or 5 mM CaCl 2 and fractionated by centrifugation. TgpCaBP was detected with a rat anti-TgpCaBP antibody, a mouse anti-IMC1 antibody was used as a control for the protein in the pellet (P) and a rabbit β-Tubulin monoclonal antibody was used as a control for the protein in the supernatant (S). ( C ) TgpCaBP migrated faster in the presence of Ca 2+ . Ca 2+ was replaced by Mg 2+ and K + in the control group, and EGTA was used to chelate-free calcium in the extracellular environment (Fig. S3A and B). Parasites were lysed in 1% Triton X-100 and incubated with EGTA, a combination of EGTA and CaCl 2 , or CaCl 2 . The native protein was extracted from T. gondii RH tachyzoites. ( D ) The expression of TgpCaBP in the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites (with two batches of proteins from each parasite line) was analyzed using western blotting with a rat anti-TgpCaBP antibody to verify the presence and absence of TgpCaBP protein in different parasite lines.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Binding Assay, Western Blot, Solubility, Centrifugation, Control, Incubation, Expressing

Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: TgpCaBP was associated with intracellular growth and gliding motility of T. gondii . ( A ) Plaque assays were conducted for parental (P), ΔTgpCaBP ( Δ ), and ΔTgpCaBP-C (C) parasites. Quantification of plaque sizes from three independent biological experiments using one-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). P > 0.05: not significant. Scale bar, 2 mm. ( B ) Red/green assays of parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites calculating the invasion efficiency. The invasion capacity of the ΔTgpCaBP parasites was significantly lower than that of the parental and the gene-complemented parasites. Quantification of invasion from three independent biological experiments using one-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). P > 0.05: not significant. ( C ) Egress assays of the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites stimulated by 3 µM A23187. The egress of the ΔTgpCaBP parasites was significantly lower than the other two parasite lines. Quantification of egress from three independent biological experiments using two-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). P > 0.05: not significant. ( D ) Effect of A23187 on gliding of T. gondii . Indirect immunofluorescence microscopy demonstrated that the length of trails deposited during gliding with A23187 treatment in the parental and ΔTgpCaBP-C parasite was longer and more complete than the ΔTgpCaBP parasite. Trails were visualized with a mouse anti-SAG1 antibody and conjugated to Alexa 488. Scale bar, 5 µm. ( E ) Quantification and statistical analysis of the trail length of the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites treated with A23187 and dimethyl sulfoxide control. The motility was impaired in the ΔTgpCaBP parasites and rescued in the ΔTgpCaBP-C parasites. Values are means ± SEM; ( n = 3). P > 0.05: not significant. ( F ) Replication assays of the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites. There was no significant difference between the replication of the ΔTgpCaBP parasites and the other two parasite lines. Quantification of replication from three independent biological experiments using two-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). Ns: not significant. ( G ) Survival of mice challenged with 50, 500, 5 × 10 3 , or 5 × 10 5 parental and ΔTgpCaBP parasites. ( H ) Survival of mice challenged with 50 parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites. The virulence of the ΔTgpCaBP parasites decreased significantly compared to the parental parasites. The virulence was rescued in the ΔTgpCaBP-C parasites. All mice were monitored for 28 days. All data were analyzed using simple survival analysis (Kaplan-Meier), P < 0.0001.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Immunofluorescence, Microscopy, Control

Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: Effect of the Ca 2+ ionophore A23187 on microneme secretion of T. gondii . ( A ) Western blot of supernatants secreted by parasites after stimulation with A23187 at 37°C for 5 min, showing a significant difference in MIC2 secreted by the parental and the ΔTgpCaBP parasites. The pellets were monitored by a mouse anti-IMC-1 monoclonal antibody. Stimulation with 2% ethanol was used as a positive control, and DMSO was used as a solvent control. ( B ) Quantification and statistical analysis of MIC2 secreted by the parental and the ΔTgpCaBP parasites after treatment with 2% ethanol, DMSO, and different concentrations of A23187. Secretion of MIC2 was inhibited in the ΔTgpCaBP parasites. Values are means ± SEM; ( n = 3). P > 0.05: not significant.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Western Blot, Positive Control, Solvent, Control

Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: Role of TgpCaBP in the efflux of cytosolic Ca 2+ of T. gondii . ( A ) Intracellular calcium concentration ([Ca 2+ ] i ) (nM) of the parental parasites, the ΔTgpCaBP and the ΔTgpCaBP-C parasites in the presence of 100 µM EGTA or 2 mM CaCl 2 . Values are means ± SEM; ( n = 6–10). P > 0.05: not significant. ( B ) Measurements of dynamic cytosolic Ca 2+ in the presence of Fura-2AM in tachyzoites of the parental (RH), the ΔTgpCaBP and the ΔTgpCaBP-C parasites. The buffer contains 100 µM EGTA to chelate contaminating Ca 2+ , and 2 mM Ca 2+ was added to the suspension at 120 s. The pink box indicates the area used for the quantification presented in ( C ). ( C ) Quantification and statistical analysis of the change in cytosolic Ca 2+ during the first 20 s after the addition of extracellular Ca 2+ . Values are means ± SEM; ( n = 7–12). P > 0.05: not significant. ( D ) Cytosolic Ca 2+ increases after adding Thap (1 µM), following Ca 2+ influx resulting from adding 2 mM extracellular Ca 2+ at 260 s. Thap: thapsigargin. The pink boxes indicate the area used for the quantification presented in ( E ) and ( F ). ( E ) Quantification and statistical analysis of the change in cytosolic Ca 2+ (Δ[Ca 2+ ] cyt ) at 50 s after adding Thap. Values are means ± SEM; ( n = 9–12). P > 0.05: not significant. ( F ) Quantification and statistical analysis of the Δ[Ca 2+ ] cyt at 20 s after adding 2 mM of Ca 2+ . Values are mean ± SEM; ( n = 9–16). P > 0.05: not significant. ( G ) Quantification and statistical analysis of the Δ[Ca 2+ ] cyt at 20 s after adding Ca 2+ in the presence or absence of Thap in the parental (P) and the ΔTgpCaBP parasites. Values are means ± SEM; ( n = 7–12). P > 0.05: not significant. ( H ) Quantification and statistical analysis of cytosolic Ca 2+ increase stimulated by Zap (100 µM) in the presence of 2 mM extracellular Ca 2+ . Values are means ± SEM; ( n = 5–6). P > 0.05: not significant. Zap: zaprinast. ( I ) Quantification and statistical analysis of the Δ[Ca 2+ ] cyt during the 20 s after adding Ca 2+ without additions (ND) or after adding Thap or Zap. Values are means ± SEM; ( n = 5–15). P > 0.05: not significant. Two-way ANOVA with Tukey’s multiple comparison test for A and I and one-way ANOVA with Tukey’s multiple comparison tests for C, E, F, G, and H.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Concentration Assay, Suspension, Comparison