Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi‐protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill‐characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N‐terminal fragment of caspase‐1‐cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N‐terminal fragment of caspase‐1‐cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome‐inserted GSDMD at nanometer resolution by cryo‐electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.

Pyroptosis is a lytic form of cell death that is induced by inflammatory caspases upon activation of the canonical or noncanonical inflammasome pathways. These caspases cleave gasdermin D (GSDMD) to generate an N-terminal GSDMD fragment, which executes pyroptosis by forming membrane pores. We found that calcium influx through GSDMD pores serves as a signal for cells to initiate membrane repair by recruiting the endosomal sorting complexes required for transport (ESCRT) machinery to damaged membrane areas, such as the plasma membrane. Inhibition of the ESCRT-III machinery strongly enhances pyroptosis and interleukin-1β release in both human and murine cells after canonical or noncanonical inflammasome activation. These results not only attribute an anti-inflammatory role to membrane repair by the ESCRT-III system but also provide insight into general cellular survival mechanisms during pyroptosis.

The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines IL-1β and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella novicida. AIM2 activation by F. novicida requires bacteriolysis, yet whether this process is accidental or a host-driven immune mechanism remained unclear. Using siRNA screening for nearly 500 interferon-stimulated genes, we identified guanylate-binding proteins GBP2 and GBP5 as key AIM2 activators during F. novicida infection. Their prominent role was validated in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs target cytosolic F. novicida and promote bacteriolysis. Thus, besides their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.

A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASCPYD into filaments and cross-linking of these filaments by ASCCARD. ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASCPYD is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production.