Article Figures & Data
Figures
- FIG 1
Ingestion of host cytosolic material by tachyzoites under bradyzoite-inducing conditions and doxycycline treatment. (A) Representative images of tachyzoites with ingested host cytosolic mCherry under bradyzoite-inducing conditions. Bar, 1 μm. (B) Ingestion of host cytosolic mCherry by T. gondii tachyzoites (Tz) under bradyzoite-inducing conditions. Three independent experiments were performed for each strain. The following parasites were enumerated for each experiment: ME49 for 4 h (280, 265, and 256 parasites), MΔcpl (304, 271, and 303), ME49 overnight (O/N) (199, 228, and 220), and MΔcpl overnight (236, 195, and 287). An unpaired t test was performed to compare genotypes within each time point. * denotes a P value of <0.05, and ** denotes a P value of <0.005. (C) Ingestion of host cytosolic mCherry by T. gondii tachyzoites under CHO cell culture conditions. Three or five independent experiments were performed. The following parasites were enumerated for each experiment: ME49 for 4 h (281, 227, 259, 244, and 262 parasites), MΔcpl (163, 228, 263, 283, and 272), ME49 overnight (194, 180, 220, 340, 293, and 529), and MΔcpl overnight (218, 295, 231, 328, 238, and 322). A Mann-Whitney U test was performed to compare genotypes within each time point. * denotes a P value of <0.05, and ** denotes a P value of <0.005. (D) Ingestion of host cytosolic mCherry by T. gondii tachyzoites after doxycycline treatment. Four independent experiments were performed. The following parasites were enumerated for each experiment: ME49 −DOX (318, 271, 258, and 210 parasites), MΔcpl −DOX (214, 277, 230, and 292), ME49 +DOX (226, 338, 248, and 206), and MΔcpl +DOX (324, 235, 222, and 266). An unpaired t test was performed to compare strains within each treatment. ** denotes a P value of <0.005, and *** denotes a P value of <0.0005.
- FIG 2
Bradyzoites can ingest host-derived mCherry. (A) Representative fields of view of bradyzoites with ingested host cytosolic mCherry. Bar, 10 μm. White arrowheads point to bradyzoites containing host-derived mCherry. (B) Bradyzoites (Bz) from mouse brain cysts can ingest host cytosolic mCherry. Three to six independent experiments were performed. The following parasites were enumerated for each experiment: ME49 with DMSO for 4 h (236, 210, and 277 parasites), ME49 with LHVS for 4 h (10 μM) (255, 299, and 245), ME49 with DMSO overnight (O/N) (282, 229, 217, 245, 228, and 245), and ME49 with LHVS (3 μM) overnight (266, 394, 234, 245, 202, and 217). An unpaired t test was performed to compare treatments within each time point. ** denotes a P value of <0.005, and **** denotes a P value of <0.0001. (C) Ingestion of host cytosolic mCherry by in vitro bradyzoites. Three to five independent experiments were performed. The following GFP+ parasites were enumerated for each experiment: Pru with DMSO for 4 h (365, 231, and 214 parasites), Pru with LHVS for 4 h (1 μM) (213, 238, and 175), Pru with DMSO overnight (231, 189, 154, 212, and 231), and Pru with LHVS (1 μM) overnight (318, 245, 213, 200, and 242). An unpaired t test was performed to compare treatments within each time point. ** denotes a P value of <0.005, and *** denotes a P value of <0.0005.
- FIG 3
In vitro bradyzoite cysts contain host-derived mCherry. (A) Ingestion of host cytosolic mCherry by T. gondii in vitro bradyzoites (Bz) converted in iCHO cells for 12 days and treated with LHVS for 2 days. BAG1+ stained parasites were enumerated. Four to five independent experiments were performed for each strain and treatment. The following parasites were enumerated for each experiment: ME49 with DMSO (262, 342, 272, 237, and 272 parasites), ME49 with LHVS (231, 195, 328, and 231), and MΔcpl (95, 254, 282, 135, and 251). A Mann-Whitney U test was used to compare groups. * denotes a P value of <0.05, and ** denotes a P value of <0.005. NS, not significant. (B) Ingestion of host cytosolic mCherry by T. gondii in vitro bradyzoites converted in iCHO cells for 12 days and treated with LHVS for 2 days. GFP+ parasites were enumerated. Four independent experiments were performed for each strain and treatment. The following parasites were enumerated for each experiment: Pru with DMSO (85, 277, 264, and 298 parasites), Pru with LHVS (209, 215, 223, and 317), and PΔcpl (270, 432, 232, and 276). An unpaired t test was used to compare all groups. * denotes a P value of <0.05, and ** denotes a P value of <0.005. (C) Ingestion of host cytosolic mCherry by T. gondii in vitro bradyzoites converted in iCHO cells for 8 days and treated with LHVS for 2 days. GFP+ parasites were enumerated. Three independent experiments were performed for each strain and treatment. The following parasites were enumerated for each experiment: Pru with DMSO (270, 210, and 209 parasites), Pru with LHVS (357, 227, and 209), and PΔcpl (464, 198, and 224). An unpaired t test was used to compare all groups. * denotes a P value of <0.05, and ** denotes a P value of <0.005. (D) Ingestion of host cytosolic mCherry by T. gondii in vitro bradyzoites converted in HFFs for 7 days, harvested, and then converted in iCHO cells for 3 days with 1 μM LHVS for 2 days. BAG1+ ME49 and GFP+ Pru bradyzoites were enumerated. Three independent experiments were performed for each strain and treatment. The following parasites were enumerated for each experiment: ME49 with DMSO (234, 97, and 205 parasites), ME49 with LHVS (281, 112, and 220), Pru with DMSO (230, 144, and 156), and Pru with LHVS (212, 186, and 209). (E) Schematic of the experimental design for panel D. (F) Representative LHVS-treated bradyzoites from panel D. The bradyzoite marker is BAG1 for ME49 and LDH2 promoter-driven expression of GFP for Pru. Bar, 1 μm. (G) Representative images of intracellular ME49 and Pru cysts at 24 h postinfection. Bars, 5 μm. (H) Percentage of dolichos-positive vacuoles at 24 and 48 h postinfection. The total numbers of GRA7 vacuoles counted are listed in parentheses. Data are from one biological replicate.
