Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling

Abstract

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

“…At reduced proteins concentrations, however, it showed reduced activity compared to the wild‐type protein (Fig 4E and F). Consistent with these data and previously published work (Kayagaki et al , 2015), we found that GSDMD I104N could also induce cell death of HEK293T cells when expressed by a doxycycline‐inducible promoter, although significantly less than the WT protein (Appendix Fig S4D). Similarly, expression of the I104N mutant of human GSDMD in immortalized Gsdmd ‐deficient mouse macrophages partially restored pyroptosis after Salmonella infection when compared to wild‐type human GSDMD (Appendix Fig S4C).…”

“…In a next experiment, we examined the functionality of the I105N mutant of GSDMD. This mutant had played a key role in the discovery of GSDMD, since it had previously been identified as a loss‐of‐function mutant in mouse models (Kayagaki et al , 2015). We generated the analogous mutation I104N in GSDMD and expressed and purified the mutant protein with the same biochemical protocols as the wild‐type protein.…”

“…Although the morphological features of pyroptotic cell death indicated the formation of a plasma membrane pore and subsequent lysis of the cell (Lamkanfi, 2011), the molecules involved in pyroptosis induction had remained elusive. The recent identification of GSDMD as an essential mediator of pyroptosis significantly expanded our understanding of pyroptotic cell death (He et al , 2015; Kayagaki et al , 2015; Shi et al , 2015), but whether after cleavage by caspases GSDMD executes pyroptosis directly, or by initiating other signaling events remained unclear. In this work, we have shown that the N‐terminal fragment of GSDMD, GSDMD Nterm , can form pores with average diameters of 21 nm in artificial liposomes, as well as large pores in the plasma membrane of cells (Figs 5 and 6).…”

“…Several landmark studies have recently identified GSDMD (gasdermin D), a member of the gasdermin protein family, as an essential mediator of pyroptosis in human and murine cells (He et al , 2015; Kayagaki et al , 2015; Shi et al , 2015). GSDMD is required for pyroptosis induction after canonical and non‐canonical inflammasome activation and is processed by caspase‐1, caspase‐11, caspase‐4, and caspase‐5, but not by apoptotic caspases.…”

“…GSDMD is required for pyroptosis induction after canonical and non‐canonical inflammasome activation and is processed by caspase‐1, caspase‐11, caspase‐4, and caspase‐5, but not by apoptotic caspases. The N‐terminal fragment of GSDMD (GSDMD Nterm ) was found sufficient to induce cell death with the morphological features of pyroptosis (Kayagaki et al , 2015; Shi et al , 2015), and overexpression of the C‐terminal domain GSDMD Cterm was found to block GSDMD Nterm ‐dependent cell death (Shi et al , 2015). These results gave rise to the hypothesis that caspase‐dependent cleavage releases the N‐terminal domain from an inhibitory interaction with the C‐terminus, allowing GSDMD Nterm to induce cell death by a yet undefined mechanism.…”