Fig. 2: NLRC4 is the primary inflammasome responsible for P. aeruginosa recognition and it is inhibited by ExoT.

a–j BMDMs (from C57BL/6) were infected with wild-type P. aeruginosa PA103 and the indicated T3SS isogenic mutants for 1-2 h (1 h for IF microscopy and 2 h for ELISA and Western blotting). a, b Culture supernatants were assessed for IL-1β or IL-18 by ELISA and the tabulated data are shown as the mean ± SEM. (N = 3; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). c, d BMDMs of caspase-1 or Caspase-11-knockout mice (Cas-1^−/− or Cas-11^−/−) were infected with PA103. Culture supernatants were assessed for IL-1β or IL-18 by ELISA and the tabulated data are shown as the mean ± SEM. (N = 4; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). e, f Cell lysates and supernatants were assessed for caspase-1 activation by Western blotting (e) and the corresponding densitometer data are shown as the mean ± SEM in (f) (N = 3, each experiment in e & f was repeated independently 2 times. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (g, h) BMDMs of indicated inflammasome knockout mice were infected with PA103. Culture supernatants were assessed for IL-1β or IL-18 by ELISA and the tabulated data are shown as the mean ± SEM (N = 4; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (i–j) BMDMs (from C57BL/6) were infected with PA103 and the indicated T3SS isogenic mutants. They were then fixed and stained for p-NLRC4 (green), ASC (red), and nucleus/DAPI (blue). Colocalized p-NLRC4/ASC foci were assessed by IF microscopy. Representative images are shown in (i), and the tabulated data are shown as the mean ± SEM in (j) (N = 3 replicates, ≥7 random fields per replicate. ns, not significant; p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Arrows point to representative p-NLRC4/ASC foci within BMDMs. k–n Wounds of C57BL/6, ASC, Caspase-1 or NLRC4 knockout mice were infected with 10³ of PA103 and the indicated T3SS isogenic mutants. Bacterial burden in wounds were determined by serial dilution and plating, 24 h after infection and the tabulated data are shown as the mean ± SEM. (N = 5 mice/group for C57BL/6, N = 4 mice/group for ASC and NLRC4 knockout mice and N = 8 mice/group for Caspase-1 knockout mice. Statistical analyses were determined by one-way ANOVA with post hoc testing; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Exact P values are presented in Supplementary Data 1. Source data are provided as a Source Data file.