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-1β 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, one-way ANOVA with Dunnett’s multiple-comparison test.

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, one-way ANOVA with Dunnett’s multiple-comparison test. (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 EseB homologs (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. Red stars indicate the EseB selected for mutation analysis. (C) NLRC4 inflammasome-reconstituted HEK293T cells expressing or not expressing the indicated EseB homologs or their mutants were immunoblotted as panel A. (D) THP-1 cells were electroporated with the indicated EseB homologs or their mutants or PBS (mock) and then determined for cell death. Data are the means of triplicate assays and shown as means ± SD. ns, not significant, ***p<0.001, ****p<0.0001, Student’s t-test.