Fig. 3: Locally acting epithelium-autonomous NAIP/NLRC4 drives caspase-dependent expulsion of enterocytes with an initially Draq7-impermeable membrane.

a, b Pan-caspase inhibition phenocopies NAIP1–6 deficiency with respect to epithelial S.Tm loads in mixed enteroid cultures. a Representative micrographs of S.Tm/pssaG-GFP-infected (MOI 100) WT and RFP-expressing NAIP1–6-deficient enteroids in the absence or presence of 50 µM pan-caspase inhibitor Z-VAD-FMK at 4 h p.i. Arrowheads indicate intracellular S.Tm-G⁺ infection foci. Scale bar: 50 µm. b Microscopy-based quantification of intracellular S.Tm-G⁺ infection foci per enteroid. In violin plot, line represents median and dashed line quartiles. c, d Cell lysis during early enterocyte expulsion. c Representative time-lapse microscopy image series of S.Tm-infected enteroid-derived 2D monolayers (MOI 2). Cell lysis was determined using the membrane impermeable nuclear dye Draq7. Example of an expelling enterocyte indicated by arrowhead - time point 0 represents the first sign of expulsion, as judged from differential interference contrast (DIC) microscopy. Star represents first sign of expulsion. Scale bar: 20 µm. d Quantification of Draq7 mean intensity of individual enterocytes during expulsion. Time point 0 represents the first signs of expulsion, as judged from DIC microscopy. e, f Chimeric 3D enteroids show higher S.Tm loads in NAIP1–6-deficient epithelial regions. e Representative micrographs of an S.Tm/pssaG-GFP-infected WTxNaip1–6^−/− (red; RFP^IECNaip1–6^Δ/ΔIEC) chimeric 3D enteroid (MOI 100) at 4 h p.i. Arrowheads indicate intracellular S.Tm-G⁺ infection foci. Scale bar: 50 µm. f Microscopy-based quantification of intracellular S.Tm-G⁺ infection foci density. Line at median. Each data point represents one enteroid. g, h Enterocyte death in WTxNaip1–6^−/− (red; RFP^IECNaip1–6^Δ/ΔIEC) chimeric enteroid-derived 2D monolayers. g Representative micrographs of S.Tm-infected WTxNaip1–6^−/− chimeric monolayers at MOI 2 over time in the absence or presence of 50 µM Z-VAD-FMK. Draq7 was added to the medium to visualize enterocyte cell death (end stage, see c above). Scale bar: 50 µm. h Quantification of Draq7⁺ pixel numbers relative to the start of the imaging series. Results in a–d and g, h representative for three independent infections. e, f Present data from one infection experiment (see also Fig S6B for an additional supporting experiment). b Two-way ANOVA with Tukey HSD and f Mann–Whitney U test (ns—not significant, *p < 0.05, ***p < 0.001).