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. 2020 Aug 11;32(6):108008. doi: 10.1016/j.celrep.2020.108008

Figure 1.

Figure 1

GBP1 Disrupts Toxoplasma Vacuoles and Parasite Membrane

(A) AiryScan immunofluorescence images of type I or type II Toxoplasma gondii (Tg) decorated with mCH-GBP1 in IFNγ- and Doxycycline (Dox)-treated THP-1 ΔGBP1+Tet-mCH-GBP1 cells. Red: mCH-GBP1; white: Tg. White arrowhead indicates GBP1 on the parasite; yellow arrowhead indicates GBP1 on the vacuole membrane. Scale bar, 4 μm.

(B) Left: illustration of the high-throughput imaging assay to measure parasitophorous vacuole (PV) integrity by CellMask flooding. Right: representative immunofluorescence images from proof-of-principle experiment using THP-1 WT infected with type I Tg for 18 h and stained with CellMask but not permeabilized (no perm.; top) or fully permeabilized with saponin (full perm.; bottom) and corresponding rainbow intensity diagram to illustrate the CellMask signal from images used for quantification; AU: arbitrary units for fluorescence intensity values. Red: CellMask; gray: Tg; blue: nuclei. Scale bars, 10 μm.

(C) Representative quantification of CellMask fluorescence intensities within vacuoles (PV) of type I or type II Tg infected THP-1 ΔGBP1+Tet-mCH-GBP1 pre-treated with IFNγ and Dox to induce GBP1 expression. Plotted depending on whether PVs were decorated with GBP1 (+) or not (−). N = number of vacuoles.

(D) Illustration of the high-throughput imaging assays to measure Tg integrity with the split-GFP system.

(E and F) Example immunofluorescence image (E) and quantification (F) of disrupted and thus green-fluorescing type II Tg parasites expressing GFP1-10 fragment (Pru ΔHpt+GFP1-10) infecting IFNγ-primed THP-1+GFP11 or IFNγ and Dox-primed THP-1 ΔGBP1+Tet-GBP1+GFP11 cells stained for all Tg using anti-surface-antigen 1 (SAG1). Data plotted as proportion of all parasites (left), proportion of all PVs containing at least one green parasite (middle), or proportion of green parasites within the same PV (right). Red: SAG1; green: holo-GFP; blue: nuclei. Scale bar, 20 μm.

Graphs in (F) show mean ± SEM from n = 3 independent experiments. Graphs in (C) representative of n = 3 independent experiments. ∗∗∗p ≤ 0.001 or ∗∗∗∗p ≤ 0.0001 in (C) from nested Student’s t test comparing GBP1+ to GBP1− objects from the three experiments following adjustment for multiple comparisons and in (F) from two-way ANOVA comparing to untreated (UT) condition.

See also Figures S1 and S2.