The ability of Edwardsiella tarda to induce pyroptosis in human macrophages.

(A) The schematic of experimental design. (B-E) dTHP-1 cells were infected with E. tarda for the indicated hours and then subjected to microscopy (B), measurement of cell death (C), IL- β release(D), and Western blot (E) using antibodies against Casp1, GSDMD, and β-actin (loading control). In (B), red arrowheads indicate pyroptotic cells; scale bar, 10 μm. (F-I) dTHP-cells in the form of wild type (WT), knockout (KO) variants (Aim2-KO, NLRC4-KO, ASC-KO, Casp4-KO, and GSDMD-KO), and knockdown (KD) variants (NLRP3-KD and Casp1-KD) were infected with or without E. tarda for 2 or 4 h, and then assessed for cell death (F, H) and IL-1β release (G, I). For panels C, D, and F-I, data were the means of triplicate assays and shown as means ±SD. ns, not significant, ***p<0.001, ****p<0.0001, one-way ANOVA with Dunnett’s multiple-comparison test.

The importance of the translocon for Edwardsiella tarda-induced pyroptosis.

(A) A diagram showing the in-frame deletion (red curved line) of eseB-D, escA, eseB, eseC and eseD. (B, C) dTHP-1 cells were infected with wild type (WT) or mutant E. tarda for 1h. The intracellular bacteria (B) and the total bacteria associated with the cells (i.e., both the cell-attached and the intracellular bacteria) (C) were determined by plate count. (D-F) dTHP-1 cells were treated with or without E. tarda variants for 2 or 4 h, and then subjected to cell death analysis (D), IL-1β release measurement (E), and immunoblot (F) using antibodies against Casp1, GSDMD, and β-actin (loading control). For panels B-E, data are the means of triplicate assays and shown as means ± SD. ns, not significant, ****p<0.0001, one-way ANOVA with Dunnett’s multiple-comparison test.

The pyroptotic effect of the translocon proteins and its dependence on the inflammasomes.

(A-C) To determine the extracellular and intracellular effects of EscA, EseB, EseC, and EseD, each of the proteins was added into the culture medium of THP-1 cells (extracellular) or electroporated into THP-1 cells (intracellular). The control cells were mock treated with PBS. The cells were subjected to microscopy (A), cell death analysis (B), and immunoblot using antibodies against Casp1, GSDMD, and β-actin (loading control) (C). In (A), red arrowheads indicate pyroptotic cells; scale bar, 10 μm. (D) The wild type (WT) and knockout (KO) THP-1 cells were treated with or without extracellular and intracellular EseB as above and then examined for cell death. (E) The control THP-cells (Null) and the NLRP3/Casp1 knockdown (KD) THP-cells were treated with or without extracellular and intracellular EseB as above and then examined for cell death. (F) THP-1 cell treated with or without (Control, Ctrl) NAIP-targeting shRNA or scramble RNA (negative control RNA) were examined for NAIP expression by qRT-PCR. (G) THP-1 cells administered with or without NAIP-targeting or scramble RNA were treated or without extracellular and intracellular EseB as above and then examined for cell death. For panels B, and D-F, data are the means of triplicate assays and shown as means ± SD. ns, not significant, ***p<0.001, ****p<0.0001.

Identification of the functional important region in EseB.

(A) Sequence alignment of EseB and T3SS needle proteins with NLRC4/NAIP-stimulating activity. (B) A diagram showing EseB wild type (WT) and truncates. (C-E) THP-1 cells were electroporated with or without (Mock) EseB WT or truncate. The cells were subjected to microscopy (C), cell death analysis (D), and immunoblot with antibodies against Casp1, GSDMD, and β-actin (loading control) (E). In (C), red arrowheads indicate pyroptotic cells; scale bar, 10 μm. For panel D, data are the means of triplicate assays and shown as means ± SD. ns, not significant, ****p<0.0001. (F) A diagram showing detection of the activating effect of EseB on NAIP/NLRC4 in NLRC4 inflammasome-reconstituted HEK293T cells by determining proIL-1β cleavage. (G) HEK293T cells were transfected with or without the indicated combination of vectors expressing Flag-tagged NLRC4, HA-tagged NAIP, Myc-tagged EseB, proCasp1, and proIL-1β for 24 h. The cells were subjected to immunoblot using antibodies against the tags or the proteins with β-actin as a loading control. (H) HEK293T cells were transfected with the indicated combination of vectors expressing Flag-tagged NLRC4, HA-tagged NAIP, and Myc-tagged EseB. The cells were subjected to immunoprecipitation (IP) using antibodies against the tags with β-actin as a loading control. (I) HEK293T cells were transfected with the indicated combination of vectors expressing HA-tagged NAIP and Flag-tagged EseB variants. IP was performed as above.

The ability of the EseB homologs to activate the NLRC4/NAIP inflammasome.

(A) NLRC4 inflammasome-reconstituted HEK293T cells were transfected with or without the EseB homologs from the indicated bacteria (Table S2). The cells were immunoblotted with antibodies against IL-1β and β-actin (loading control). (B) Sequence alignment of the C-terminal regions of the EseB homologs from various bacteria (Table S2). Red stars indicate the EseB selected for mutation analysis. (C) NLRC4 inflammasome-reconstituted HEK293T cells were transfected with or without wild type (WT) or mutant (M) EseB homologs from the indicated bacteria. The cells were immunoblotted as above.