Fig. 5.

GBP1-dependent pyroptosis and restriction of S. flexneri growth is independent of GBP1 binding to bacteria. (A–D) Wild-type A549 or GBP1^KO cells expressing mCherry or mCherry-GBP1 mutants were unprimed or primed with 100 U/mL IFNγ overnight, where indicated. Cells were infected with S. flexneri expressing a bioluminescent reporter and cell death was measured over time using sytox green fluorescence (A). The sytox green signal at 4 h was used to determine statistical significance (B). Bacterial luminescence was measured over time (C). Luminescence measurements from the 6 h timepoint were used to calculate the growth of each strain in primed cells relative to unprimed cells (D). (E and F) Wild-type A549 or GBP1^KO cells expressing mCherry or mCherry-GBP1 mutants were primed overnight with IFNγ, then infected with GFP expressing S. flexneri ΔipaH9.8, and fixed at 2 h post infection. (E) Coverslips were imaged at 100× magnification using widefield microscopy. Images were deconvolved and z-projections are shown, scale bar are 5 μm. (F) Coverslips were imaged at 63× magnification, with images taken from five independent fields. Targeting of S. flexneri by each overexpressed protein was quantified using ImageJ. S. flexneri with indicated protein around at least 50% of the bacterial membrane were counted as targeted. (G) Expression levels of indicated overexpressed proteins determined by western blot. (H) Frames from timelapse microscopy at indicated time points for GBP1^KO cells expressing mCherry, mCherry-GBP1, or mCherry-GBP1^3R infected with GFP expressing S. flexneri ΔospC3ΔipaH9.8. Dying cells are shown in blue (sytox blue). All graphs show averages from three independent experiments and are represented by mean ± SD. (B and D) Significance was determined using two-way ANOVA with Tukey’s multiple comparisons test. Statistical comparisons are shown by letters, with bars sharing no matching letters being significantly different. Purple letters correspond to statistical comparisons for wild-type S. flexneri, and orange letters correspond to S. flexneri ΔipaH9.8ΔospC3. (F) One-way ANOVA with Tukey’s multiple comparisons test was used. All significant comparisons are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.