FIG 4.

ExoU inhibits caspase-1 activation at the mitochondria. (A) WT BMDMs were treated with sterile saline (C) or inoculated with P. aeruginosa strains PA103 or PA103 ΔexoU exoT::Tc. At 60 min postinoculation, enriched mito-MAM fractions were isolated, and pro-caspase-1, active caspase-1, and prohibitin were measured by Western blotting. (B) Densitometry analysis of blots shown in panel A, where pro-caspase-1 and active caspase-1 levels were normalized to the loading control (prohibitin). (C) WT and Nlrc4^−/− BMDMs were treated with sterile saline or inoculated with P. aeruginosa strain PA103, PA103 exoT::Tc, PA103 ΔexoU exoT::Tc, or PA103 ΔpcrV. At 180 min postinoculation, enriched mito-MAM fractions were isolated for Western blot analysis. (D) Densitometry analysis of blots shown in panel C, where pro-caspase-1 and active caspase-1 levels were normalized to the loading control (prohibitin). *, P value = 0.03. (E) WT BMDMs were preincubated with DMSO (vehicle control) or the ExoU-specific inhibitor PsA (50 μM), where compounds were added 60 min prior to inoculation. Subsequently, cultures were treated with sterile saline or inoculated with P. aeruginosa strain PA103 for 180 min (DMSO or PsA was maintained throughout the time course). Enriched mito-MAM fractions were isolated, and pro-caspase-1, active caspase-1, active IL-1β, and prohibitin were measured by Western blotting. (F) Densitometry analysis of caspase-1 (top) and IL-1β (bottom) from the blots in panel E (normalized to prohibitin). **, P value = 0.0014. (G) NLRC4-FLAG BMDMs were treated as described for panel A. Enriched mito-MAM fractions were isolated and assayed for NLRC4-FLAG (112 kDa), pro-caspase-1, active caspase-1, IL-1β, and prohibitin, measured by Western blotting. (H) Densitometry analysis of NLRC4-FLAG (top), caspase-1 (middle), and IL-1β (bottom) from the blots in panel G (normalized to prohibitin). *, P value = 0.0126. All blots and densitometry analyses are representative of 3 independent experiments.