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. 2020 Dec 28;16(12):e1008771. doi: 10.1371/journal.ppat.1008771

Fig 4. ΔTgPPM3C parasites exhibit partial defects in protein effector export.

Fig 4

(A) Violin plots of GRA16-3xHA, GRA24-3xHA, GRA28-3xHA, and TgIST-3xHA accumulation in host nuclei infected with either PruQ or ΔTgPPM3C parasites, transiently transfected with epitope tagged effector constructs and allowed to infect HFF monolayers for 24 hours. Effector protein fluorescence was quantified from fibroblast nuclei containing a single HA-positive parasite vacuole. Violin plots of uninfected host nuclei in the same monolayer are provided to demonstrate background. A significant decrease in GRA16 and GRA28, but not GRA24 and TgIST, host nuclear accumulation is observed during ΔTgPPM3C infection, likely indicating defects in effector export from the parasitophorous vacuole. Violin plots represent three independent experiments. Representative images from which effector intensity was quantified are shown on the right. Antibody to SAG1 was used as a parasite marker, DAPI as a host nucleus marker, and anti-HA antibody was used to detect epitope tagged effectors. The white asterisks indicate uninfected fibroblast nuclei. (B) Violin plots of c-Myc induction in host nuclei infected with either PruQ, ΔTgPPM3C, or TgPPM3C-COMP parasites for 24 hours. Host c-Myc fluorescence was quantified from fibroblast nuclei containing a single parasite vacuole. A significant decrease in host c-Myc induction is observed during ΔTgPPM3C infection compared to PruQ and TgPPM3C-COMP infections. Notably, significant induction of host c-Myc expression over uninfected host cells are observed during infection with all three strains (asterisks above each plot). Violin plots represent three independent experiments. Representative images from which c-Myc intensity were quantified are shown on the right. Antibody to SAG1 was used as a parasite marker and DAPI as a host nucleus marker. Violin plots of uninfected host nuclei in the same monolayer are provided to demonstrate background. The white asterisk indicates an uninfected fibroblast nucleus. Non-specific labeling of parasite vacuoles can be observed with the c-Myc antibody used to label host cells. (C) Violin plots of IRF1 induction in host nuclei infected with either PruQ, ΔTgPPM3C, or TgPPM3C-COMP parasites and stimulated with IFN-γ for six hours prior to fixation at 24 hours post-infection. Host IRF1 fluorescence was quantified from fibroblast nuclei containing a single parasite vacuole. Although each strain significantly attenuated IRF1 induction compared to uninfected cells (asterisks above each plot), no significant differences in the extent of IRF1 suppression are observed between any of the strains. Violin plots of uninfected host nuclei in the same monolayer are provided to demonstrate background. Violin plots represent three independent experiments. A representative image from a PruQ infected fibroblast and uninfected fibroblast (white asterisk) is shown beside the violin plots. Antibody to SAG1 was used as a parasite marker and DAPI as a host nucleus marker. (D) Violin plots of EZH2 induction in host nuclei infected with either TgPPM3C-HA, ΔTgPPM3C, or TgPPM3C-COMP parasites for 24 hours. Host EZH2 fluorescence was quantified from fibroblast nuclei containing a single parasite vacuole. Each strain significantly induces host EZH2 compared to uninfected cells (asterisks), although no significant differences in host EZH2 induction were observed between any of the strains. Violin plots of uninfected host nuclei in the same monolayer are provided to demonstrate background. Violin plots represent three independent experiments. A representative image from a TgPPM3C-HA infected fibroblast and uninfected fibroblast (white asterisk) is shown beside the violin plots. Antibody to IMC3 was used as a parasite marker and DAPI as a host nucleus marker. The white asterisk indicates an uninfected fibroblast nucleus. Non-specific parasite labeling is evident with the EZH2 antibody used to label host cells. For all violin plots in this figure, **** asterisks indicate p < 0.0001 and ns indicates no significant difference. Kruskal-Wallis and Dunn’s multiple comparisons tests were performed to calculate p-values. All scale bars in this figure indicate 10μm.