Journal: Frontiers in Cellular and Infection Microbiology
Article Title: An Extracellular Redox Signal Triggers Calcium Release and Impacts the Asexual Development of Toxoplasma gondii
doi: 10.3389/fcimb.2021.728425
Figure Lengend Snippet: H 2 O 2 induces Ca 2+ release in intracellular parasites and triggers CDPK3-dependent egress. (A) Representative trace of 100 μM H 2 O 2 induction of Ca 2+ flux in intracellular RH-GFP-T2A-jRCaMP1b parasites. a – c: widefield microscopy images depicting changes in parasite fluorescence signal intensity for both GFP and the Ca 2+ sensor at baseline (a at 30s), the peak of Ca 2+ after H 2 O 2 addition (b at 270s), and peak of Ca 2+ after 1 μM A23187 addition (c at 460s). Data are representative of 27 infected vacuoles from four independent experiments. (B) Representative trace of 100 μM H 2 O 2 induction of Ca 2+ flux following pre-treatment of infected host cells with 50 μM α-tocopherol. a: baseline at 30s, b: trace after H 2 O 2 addition at 270s and c : peak of Ca 2+ triggered by A23187 at 520s. Data are representative of 14 infected vacuoles from three independent experiments. For (A, B) black arrows indicate the time of compound addition. Scale bar: 5 μM. (C) Intensity of parasite Ca 2+ signal increase in RH-GFP-T2A-jRCaMP1b parasites (expressed as a percentage over baseline) following addition of: 1 μM A23187 alone, 1 μM of A23187 following 100 μM H 2 O 2 pre-treatment, and 100 μM H 2 O 2 alone. Gray dots indicate vacuole data points where parasite egress was observed during the measurement period, blue dots represent vacuoles where egress was not observed. (D) Intensity of parasite Ca 2+ signal increase (%) over the baseline upon addition of 100 μM H 2 O 2 in RH-GFP-T2A-jRCaMP1b versus RH-GFP-T2A-jRCaMP1bΔ CDPK3 parasites. For (C, D) histograms present data mean ± SEM of three independent experiments (five vacuoles measured in each experiment), with individual vacuole data points also shown. (E, F) Egress assay measuring tachyzoite release after compound treatment in RH-GFP-T2A-jRCaMP1b and RH-GFP-T2A-jRCaMP1bΔ CDPK3 , respectively. Data represent the mean ± SEM of three independent experiments (except for DMSO that has two independent experiments), with six technical replicates for each. All data were normalised to the water control. Significance was calculated using one-way Anova, Bonferroni’s multiple comparisons test. P values: *< 0.05; **< 0.01, ***< 0.001 and ****< 0.0001.
Article Snippet: Parasites per vacuole were counted by excitation with 470nm laser (4% intensity) widefield Nikon Eclipse Ti-E inverted microscope equipped with an ORCA- Flash4.0 camera (Hamamatsu, Japan) and NIS-Elements Viewer software (Nikon), 60x-oil objective.
Techniques: Microscopy, Fluorescence, Infection, Control
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