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. 2019 Jul 2;10(4):e00589-19. doi: 10.1128/mBio.00589-19

FIG 5.

FIG 5

Type II PruΔgra12 parasites exhibit a loss of PV viability in IFN-γ-activated host cells. (A) Survival of C57BL/6 IFN-γ−/− knockout mice infected intraperitoneally with 2 × 102 tachyzoites of PruΔgra12 or PruΔku80. ns, not significant. (B) Mouse embryonic fibroblasts (MEFs) were stimulated with IFN-γ, and PV viability (measured as PFU) was determined in comparison to the results for nonstimulated (no IFN-γ) MEFs. The results from at least 3 independent experiments are shown as mean values plus or minus standard errors of the means (± SEM). Significant P values were calculated with Student’s t test (****, P < 0.0001). (C) Quantification of Irgb6 coating of PVs 45 min after infection of IFN-γ-stimulated bone marrow-derived macrophages (BMDM) by PruΔgra12 or PruΔku80 parasites. Representative images of PVs stained with anti-Irgb6 antibody (green) and anti-GRA5 antibody (red) are shown above the graph. At least 500 PVs were scored to determine the significance. Significant P values were calculated with Student’s t test (ns, not significant). (D) MEFs were stimulated in vitro with IFN-γ and infected with PruΔgra12 or PruΔku80 mutant parasites or complemented PruΔgra12/GRA12I-HA or PruΔgra12/GRA12II-HA parasites. PV viability (measured as PFU) was determined in comparison to the results for nonstimulated (no IFN-γ) MEFs. Results from at least 4 independent experiments are shown as mean values ± SEM. Significant P values were calculated with Student’s t test (****, P < 0.0001). (E) Survival of C57BL/6 mice infected i.p. with 2 × 106 tachyzoites of PruΔgra12, parental PruΔku80, or complemented PruΔgra12/GRA12I-HA or PruΔgra12/GRA12II-HA strains. The data presented are the combined results of 2 independent experiments, each with 4 mice per group. The P value was calculated by log rank Mantel-Cox test, and a P value of <0.05 was considered significant (****, P < 0.0001; ns, not significant). (F) CD1 mice were infected i.p. with 2 × 102 tachyzoites of each designated strain, and chronic-stage cyst burdens were counted 21 days postinfection. The data are cumulative results from 2 to 3 independent experiments for each strain tested, as follows: PruΔku80 (3 experiments; n = 12 mice), PruΔgra12 (2 experiments; n = 8 mice), PruΔgra12/GRA12I-HA (2 experiments; n = 8 mice), and PruΔgra12/GRA12II-HA (2 experiments; n = 8 mice). ns, not significant.