Skip to main content
. Author manuscript; available in PMC: 2017 Dec 19.
Published in final edited form as: Nat Microbiol. 2017 Jun 19;2:17096. doi: 10.1038/nmicrobiol.2017.96

Figure 1. VAC proteolytic activity is required for Toxoplasma viability and persistence in vitro.

Figure 1

a, Extracellular tachyzoites were stained with α-CPB (red) to mark the VAC and DAPI (blue) to identify the nucleus. An arrow indicates the localization of the VAC. Scale bar, 2 μm. b, Violin plots of VAC size. Data are mean ± SEM of 3 biological replicates, with the following number of parasites evaluated: Pru, 317, 153, 154; PΔcpl, 271, 177, 176; PΔcpl:c/CPL, 293, 153, 153. Shape indicates distribution of the pooled data. ****, p<0.0001 Mann Whitney test. c, In vitro differentiation based on expression of the bradyzoite specific antigen BAG1. Cultures were differentiated for the indicated time, fixed, stained with α-BAG1 and enumerated by fluorescence microscopy. Bars are mean ± SEM of 2 biological replicates. Experiment 1 assessed for Pru 205, 214, 204, and 208 parasitophorous vacuoles (PV) on days 1, 2, 3, and 4, respectively; and for PΔcpl, 209, 211, 213, and 202 PV, respectively. Experiment 2 assessed for Pru 212, 202, 208, and 202 PV on days 1, 2,3, and 4, respectively; and for PΔcpl 205, 207, 214, and 208 PV, respectively. Viability of Pru (d) or PΔcpl (e) bradyzoites based on stage specific expression of cytosolic GFP under the LHD2 promoter8. Cultures were differentiated, and enumerated by fluorescence microscopy. Green bars indicate cysts that are uniformly GFP positive. Checkerboard bars specify cysts that contain GFP positive and GFP negative bradyzoites. Grey bars indicate cysts that are uniformly GFP negative. Data are mean ± SD of 3 biological replicates each with 100 cysts evaluated. **, p<0.001, two-tailed paired student’s t-test. f, Viability of CPL proficient and deficient strains based on plaquing efficiency. Strains were differentiated for 4 weeks, mechanically liberated from cysts, quantified by qPCR and inoculated onto fresh cell monolayers for plaque formation. PΔcpl:c/CPL* is complemented with catalytically inactive CPL. Bars are mean ± SEM of 3 (Pru, PΔcpl) or 4 (PΔcpl:c/CPL, PΔcpl:c/CPL*) biological replicates each with two technical replicates. **, p<0.01, Mann Whitney test. g, Viability of Pru bradyzoites differentiated for 1 week before treatment with solvent (DMSO) or 1 μM LHVS for 4 weeks. Viability was measure as in (f). n=2 biological replicates each with 3 technical replicates.