Fungal gasdermin-like proteins are controlled by proteolytic cleavage

TLDR

Gasdermins are pore‑forming proteins that trigger inflammatory cell death in mammals, activated by proteolytic removal of an inhibitory C‑terminal domain, and fungal gasdermin‑like proteins have been identified as cell‑death toxins in allorecognition, yet the regulatory pathways in fungi remain largely unknown. The study aims to characterize a gasdermin‑based cell death reaction in Podospora anserina regulated by het‑Q allorecognition genes and to demonstrate that HET‑Q1 cytotoxicity is controlled by proteolysis. The authors show that HET‑Q2, a subtilisin‑like serine protease, directly cleaves HET‑Q1, and this cleavage induces cell death, as confirmed by mutational analysis and heterologous reconstitution in yeast and human cells. The study demonstrates that HET‑Q1 loses a ~5‑kDa C‑terminal fragment during cell death mediated by HET‑Q2, that most fungal gasdermin genes cluster with protease genes, and that proteolytic regulation of gasdermins in fungi parallels mammalian pathways.

Abstract

Gasdermins are a family of pore-forming proteins controlling an inflammatory cell death reaction in the mammalian immune system. The pore-forming ability of the gasdermin proteins is released by proteolytic cleavage with the removal of their inhibitory C-terminal domain. Recently, gasdermin-like proteins have been discovered in fungi and characterized as cell death-inducing toxins in the context of conspecific non-self-discrimination (allorecognition). Although functional analogies have been established between mammalian and fungal gasdermins, the molecular pathways regulating gasdermin activity in fungi remain largely unknown. Here, we characterize a gasdermin-based cell death reaction controlled by the het-Q allorecognition genes in the filamentous fungus Podospora anserina We show that the cytotoxic activity of the HET-Q1 gasdermin is controlled by proteolysis. HET-Q1 loses a ∼5-kDa C-terminal fragment during the cell death reaction in the presence of a subtilisin-like serine protease termed HET-Q2. Mutational analyses and successful reconstitution of the cell death reaction in heterologous hosts (Saccharomyces cerevisiae and human 293T cells) suggest that HET-Q2 directly cleaves HET-Q1 to induce cell death. By analyzing the genomic landscape of het-Q1 homologs in fungi, we uncovered that the vast majority of the gasdermin genes are clustered with protease-encoding genes. These HET-Q2-like proteins carry either subtilisin-like or caspase-related proteases, which, in some cases, correspond to the N-terminal effector domain of nucleotide-binding and oligomerization-like receptor proteins. This study thus reveals the proteolytic regulation of gasdermins in fungi and establishes evolutionary parallels between fungal and mammalian gasdermin-dependent cell death pathways.