Molecular characterization of a fungal gasdermin-like protein

Abstract

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulatorof cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa. rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1–1 and rcd-1–2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1–1/rcd-1–2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1. This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

Fungal gasdermin-like proteins are controlled by proteolytic cleavage

“…The het-Q1 gene encodes a 278 amino acid long protein, HET-Q1, which belongs to the same protein family as RCD-1 from N. crassa (6) and thus is a fungal gasdermin-like protein ( SI Appendix , Fig. S2).…”

“…The cell death reaction limits the transmission of mycoviruses and other deleterious replicons, thus representing a defence reaction akin to an immune response (3, 4). The molecular characterization of cell death-pathways, regulating conspecific non-self discrimination (allorecognition) in fungi, has revealed evolutionary relations to cell death pathways operating in mammalian innate immunity, notably necroptosis and pyroptosis (5, 6). The extent of conservation of these pathways is gradually revealed as the characterization of fungal RCD pathways progresses.…”

“…The co-expression of antagonistic alleles of the rcd-1 gene (regulator of cell death) triggers rapid, lytic cell death in asexual spores and hyphae of the model ascomycete Neurospora crassa (22). RCD-1 is a distant homolog of the cytotoxic pore-forming GSDMD-NT domain and, like gasdermin, induces cell death by targeting the plasma membrane, while forming oligomers (6). However, in spite of the uncovered functional similarities between the mammalian gasdermins and fungal RCD-gasdermin-like protein, in the latter case, cytotoxic activity is not dependent on proteolytic activation but results instead from the interaction of antagonistic RCD-variants.…”

To Kill But Not Be Killed: Controlling the Activity of Mammalian Pore-Forming Proteins

“…In total there are six gasdermin paralogues in humans (GSDMA, GSDMB, GSDMC, GSDMD, GSDME/DFNA5, and PJVK/DFNB59) and 10 in mice (Gsdma1-3, Gsdmc1-4, Gsdmd, Gsdme, and Pjvk). Notably, gasdermin orthologues are also found in lower vertebrates, such as zebrafish (43), and more distant homologues are present in fungi (44). Mutations in gasdermin genes have been associated with a variety of disease phenotypes including skin and developmental defects (GSDMA3), susceptibility to asthma (GSDMA3 and GSDMB) (45)(46)(47), and autosomal dominant and recessive hearing loss (GSDME and DFNB59) (48,49).…”

The Predicted Mannosyltransferase GT69-2 Antagonizes RFW-1 To Regulate Cell Fusion in Neurospora crassa

“…The second checkpoint blocks the transition from cell adhesion to cell wall dissolution when adhered cells have nonidentity of cwr-1 and cwr-2 (cell wall remodeling) loci ( 29 ). The third checkpoint occurs postfusion and triggers a rapid cell death response in the fusion cells, which is determined by allelic differences at plp-1/sec-9 ( 30 ) or rcd-1 ( 27 – 30 ).…”