Fig. 2: Genetic ablation of ROCY1 T. gondii VEG and PRU strains disrupts stress-induced cyst formation. | Nature Communications

Fig. 2: Genetic ablation of ROCY1 T. gondii VEG and PRU strains disrupts stress-induced cyst formation.

From: A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease

Fig. 2

a Representative images of T. gondii VEG-containing vacuoles exposed to alkaline pH stress for 48 h. DNA was stained with DAPI (blue), the parasitophorous vacuole membrane (PVM) was stained with an anti-MAF1b mouse polyclonal antibody (green), and tissue cysts were stained with rhodamine-labeled Dohlichos biflorus Agglutinin (DBA) (red). Scale bar represents 10 μm. b Quantification of tissue cyst formation in TgVEGΔROCY1, TgVEGΔBFD1, and passaged matched TgVEG WT strains after growth in control conditions or alkaline pH. The mean and standard deviation (N = 3) of the percentage of DBA positive vacuoles observed in 15 parasite containing fields of view is plotted. Statistical significance was determined using ANOVA and Tukey’s multiple comparisons tests on arcsine transformed data. *p = 0.025, ****p < 0.0001 . c Quantification of fluorescence intensity in TgVEGΔROCY1 and TgVEGΔBFD1 from B. The mean +/- SD of the fluorescence intensity (background-corrected) is plotted. Each point represents a single vacuole. Black, gray, and white symbols indicate from which replicate. Statistical significance was determined using a Brown-Forsythe and Welch ANOVA and Dunn’s multiple comparisons (2 sided).***p = 0.0006 ****p < 0.0001. d Representative images of T. gondii PRU-containing vacuoles exposed to alkaline pH stress for 72 h. Staining as in A. Scale bar represents 10 µm. e Quantification of T. gondii infection described in D quantifying the percentage of DBA+ vacuoles in infected coverslips (N = 3 per clone and condition; mean and standard deviation shown). Statistical significance was determined as in panel B and each coverslip was considered a biological replicate. f Mean length (in arbitrary units) +/- standard deviation of individual plaques of each strain after 7 days of growth in vitro. N = 29 vacuoles per parasite strain. g Detection and quantification of DBA fluorescent staining within all parasites (regardless of DBA-positivity; mean intensity) using KNIME software from all vacuoles categorized in e. Each data point represents and individual vacuole (N = 78, 75, 63, 55, 62, and 51 vacuoles per strain/condition). Statistical significance was determined as in panel (****P < 0.0001, **P ≤ 0.01). Data are provided as a Source Data File.

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