Fig. 3: EseJ inhibits pyroptosis by deactivating caspase-8 and NLRP3 inflammasome.

a Immunoblotting of cleaved caspase-1 (p20), cleaved GSDMD and cleaved caspase-8 from J774A.1 cells infected with E. piscicida strains at 3 hpi. J774A.1 cells were pre-treated with the caspase-8 inhibitor Z-IETD-fmk for 1 h prior to infection with the ΔeseJ strain. J774A.1 cells infected with WT strain and ΔeseJ strain were used as negative and positive controls, respectively. Actin was used to confirm that similar amounts of protein were loaded per lane. b Confocal images of J774A.1 cells infected with E. piscicida strains. J774A.1 cells were pretreated with 50 μM caspase-8 inhibitor Z-IETD-fmk for 1 h prior to infection with the ΔeseJ strain. The morphology of cell rounding in J774A.1 cells infected with ΔeseJ and WT strains was observed at 1 hpi. Infected J774A.1 cells were live stained with PI at 3 hpi. Black arrows and white arrows indicate cell rounding and cell rupture, respectively. c Percentage of PI-positive cells at 3 h post infection. Twelve images per infection were quantified. Means ± SD for a representative experiment are shown. ***P < 0.001. d Immunoblotting of cleaved caspase-1 (p20), cleaved GSDMD, cleaved caspase-3 and cleaved caspase-8 from J774A.1 cells infected with E. piscicida ΔfimH, ΔeseJΔfimH, and ΔeseJΔfimH + Z-IETD-fmk. J774A.1 cells were pretreated with the caspase-8 inhibitor Z-IETD-fmk for 1 h prior to infection with the ΔeseJΔfimH strain. Actin was used to confirm that similar amounts of protein were loaded per lane. e Immunoblotting of cleaved caspase-1 (p20), cleaved GSDMD and cleaved caspase-8 from J774A.1 cells infected with E. piscicida strains. J774A.1 cells were pretreated with 650 μM NLRP3 inhibitor MCC950 for 1 h prior to infection with the ΔeseJΔfimH strain. J774A.1 cells infected with ΔfimH and ΔeseJΔfimH strains were used as negative and positive controls, respectively. Actin was used to confirm that similar amounts of protein were loaded per lane. f Confocal images of PI-positive J774A.1 cells infected with E. piscicida strains. J774A.1 cells were pretreated with 650 μM NLRP3 inhibitor MCC950 for 1 h prior to infection with the ΔeseJΔfimH strain. J774A.1 cells infected with ΔfimH and ΔeseJΔfimH strains were used as negative and positive controls, respectively. J774A.1 cells infected with the different E. piscicida strains were stained with PI at 3 hpi. g Quantification of cells that were PI-positive at 3 hpi. Twelve views per infection were quantified from (f). Means ± SD for one representative experiment are shown. ***p < 0.001. Quantification of infected cells that were PI positive at 3 hpi. HEK293T cells were co-transfected with pLVX-caspase-8 siRNA (h), pLVX-NLRP3 siRNA (i) or pLVX-control siRNA, as well as pMD2.g (envelope plasmid) and pSPAX2 (packing plasmid). 48 h later, the cell culture supernatant was collected to harvest the lentiviral particles. J774A.1 cells were infected with the concentrated lentivirus for 48 h before being infected with ΔfimH and ΔeseJΔfimH strains. Three hours post-infection, both cells and cell culture media were harvested and precipitated, followed by immunoblotting for caspase-8 (h), NLRP3 (i), and cleaved caspase-1 (p20). Actin was used as a loading control. j Leukocytes were isolated from the head kidney of mandarin fish, treated with either the caspase-8 inhibitor z-IETD-fmk or the NLRP3 inhibitor MCC950 for one hour, and then infected with the ΔeseJΔfimH strain at an MOI of 2.5, 5 or 10. Three hours post-infection, the culture supernatant was collected and the amount of LDH released was measured. Means ± SD from four samples for a representative experiment are shown. *P < 0.05; **P < 0.01; ***P < 0.001.