FIG 7.

T. gondii ASP1 has a limited role in protein turnover of autophagosomes. (A) The rate of parasite differentiation from the tachyzoite stage to the bradyzoite stage was determined in vitro by assessment of the tachyzoite-specific antigen SAG1 and GFP under the control of a bradyzoite LDH2 promoter. Infected monolayers were cultured for the indicated days, fixed, probed for SAG1, and quantified. Bars indicate means ± standard deviations from 3 biological replicates. Experiments analyzed 105, 106, 153, and 151 cysts for Pru, 75, 108, 155, and 130 cysts for PΔcpl, 119, 124, 189, and 146 cysts for PΔasp1 and 78, 110, 112, and 161 cysts for PΔasp1Δcpl on days 1, 2, 3, and 4, respectively. All strains were compared, and only statistical significance is shown. Unpaired two-tailed t test with Welch’s corrections was performed on the means across 3 biological replicates. (B) Phase-contrast microscopy was used to image bradyzoites in vitro following 7- and 14-day culture under differentiation conditions. Enlarged dark puncta indicative of defective protein degradation in VACs are visible in PΔcpl bradyzoites and PΔasp1Δcpl cysts (arrows). Scale bars, 5 μm. (C) The size of the punctae in bradyzoites was measured. The following numbers of cysts across 3 biological replicates were used to analyze each strain: Pru (day 7, 109 cysts; day 14, 98 cysts), PΔcpl (day 7, 105 cysts; day 14, 97 cysts), PΔasp1 (day 7, 85 cysts; day 14, 97 cysts), PΔasp1Δcpl (day 7, 90 cysts; day 14, 97 cysts). Lines represent medians with 95% confidence intervals. For this data set, ROUT with a Q value of 0.1% was used to identify and remove 1 outlier from PΔasp1 for 7 days postdifferentiation and 1 outlier each for Pru and PΔcpl for 14 days postdifferentiation. Since the data did not fit a normal distribution, we used a nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons to compare the medians of data combined from 3 biological replicates. All strains were compared, and only significant differences are shown.