Figure 4.

P. aeruginosa ExoU phospholipase activity is required for inhibition of caspase-1 activation. (A and B) LPS-stimulated WT macrophages were either left untreated or pretreated with 100 μM AACOCF₃ for 30 min, and then infected with the P. aeruginosa strain PA103 or PA103ΔU at a MOI of 20 bacteria per macrophage. 1 h after infection, culture supernatants were collected. Parallel wells of untreated or AACOCF₃-pretreated LPS-stimulated WT macrophages were stimulated with 5 mM ATP for 30 min, and then culture supernatants were collected. Cytotoxicity was measured by LDH release into culture supernatants, and IL-1β release was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± the SD. *, P = 0.03; **, P = 0.0001. Results are representative of two separate experiments. (C) Nonstimulated WT macrophages were either left untreated or pretreated with 100 μM AACOCF₃ for 30 min, and then infected with the P. aeruginosa strain PA103 or PA103ΔU at a MOI of 20 bacteria per macrophage. 90 min after infection, the combined lysate of cells and supernatant were immunoblotted with antibodies against the p10 subunit of caspase-1. (D) Nonstimulated WT macrophages were infected with the P. aeruginosa strain PA103, PA103ΔUT, PA103ΔU, PA103ΔT, PA103ΔUT/LS608, or PA103ΔUT/S142A at a MOI of 20 bacteria per macrophage. 3 h after infection, culture supernatants were collected and IL-1β release was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± the SD. Results are representative of three separate experiments.