A bacterial toxin co-opts caspase-3 to disable active gasdermin D and limit macrophage pyroptosis

Abstract

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1β maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

Keywords: CP: Immunology; CP: Microbiology; EHEC; Shiga toxin; caspase-11; caspase-3; enterohemorrhagic E. coli; gasdermin D; noncanonical inflammasome; pyroptosis.

Figure 1.. Stx induces further processing of caspase-11-generated NT-GSDMD

(A–F) GSDMD cleavage in the lysates of Pam3CSK4-primed C57BL/6 or Casp11^−/− BMDMs treated with 4 μg/mL purified Stx2 or enzymatically inactive Stx2 (toxoid) at the time of transfection (Tra) with LPS (1 μg/10⁶ cells) complexed with lipofectamine 2000 for 16 h (A and D) or infection with EHEC (B and E) or H₂O- or IPTG-treated BL21/pEmpty or BL21/pStx2 (C and F) for 16 h. Note the appearance of a p23 fragment of GSDMD in indicated conditions. (G) Coomassie-blue-stained SDS-PAGE gel of recombinant GSDMD subjected to cleavage by recombinant active caspase-11 (p20/p10) in the presence or absence of Stx2. Data are representative of 3 experiments.

Figure 2.. Caspase-3 activation corresponds to the appearance of p23 GSDMD

(A–C) Caspase-3 (A–C) and GSDMD (C) cleavage in the lysates of BMDMs treated with Stx2 (4 μg/mL) or infected with H₂O- or IPTG-treated BL21/pEmpty or BL21/pStx2 for 6 h or infected with EHEC for the indicated time. (D and E) Pyroptosis and IL-1β secretion at the indicated times post-infection with EHEC as assessed by lactate dehydrogenase (LDH) assay and ELISA, respectively, in the supernatants of BMDMs. Mean ± SEM of combined data from 3 biological replicates, p < 0.05 is considered significant; two-way ANOVA with Tukey’s post-test. (A–C) Data are representative of 3 experiments. See also Figure S1 and Figure S2.

Figure 3.. Caspase-3 is essential for Stx-mediated inflammasome suppression

(A–C, E, and F) GSDMD and caspase-3 cleavage in the lysates of Pam3CSK4-primed C57BL/6 or Casp3^−/− BMDMs treated with Stx2 (4 μg/mL) at the time of EHEC infection (A) or infected with H₂O- or IPTG-treated BL21/pEmpty or BL21/pStx2 (B) or treated with 4 μg/mL of Stx2, Stx2d, or Stx1 at the time of LPS Tra (C, E, and F) for 16 h. (D) Pyroptosis and IL-1β secretion at 16 h as assessed by LDH assay and ELISA, respectively, in the supernatants of BMDMs treated as in (C). Mean ± SEM of combined data from 3 biological replicates, p < 0.05 is considered significant; two-way ANOVA with Tukey’s post-test. (A–C, E, and F) Data are representative of 3 experiments.

Figure 4.. Caspase-3 cleaves NT-GSDMD at D88

(A and B) Caspase-3 cleavage site (D88) and predicted cleaved fragments for FL-GSDMD or NT-GSDMD (A) and dox-inducible BFP-tagged FL-GSDMD or BFP-tagged NT-GSDMD (B) (created with BioRender.com). (C and D) Lysates of Dox-treated Gsdmd^−/− iBMDMs expressing FL-GSDMD-BFP or NT-GSDMD-BFP were incubated with 3 units of recombinant active or inactive (C163A) caspase-3 at 37C for 3 h and the cleavage of BFP-tagged FL-GSDMD (C) or NT-GSDMD (D) was assessed. Note in (D) the appearance of a p47 band corresponding to caspase-3-cleaved NT-GSDMD-BFP. (E) Cell death in Gsdmd^−/− iBMDMs expressing NT-GSDMD-BFP or FL-GSDMD-BFP treated with 0.5 μg/mL Dox for 4 h. Mean ± SEM of combined data from 3 biological replicates, p < 0.05 is considered significant; two-way ANOVA with Tukey’s post-test. (F) Coomassie-blue-stained SDS-PAGE gel of recombinant human GSDMD subjected to cleavage by recombinant active caspase-3 and/or active caspase-11 as indicated by “+.” (C, D, and F) Data are representative of 3 experiments.