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Characterization of GSDME in Amphioxus Provides Insights Into the Functional Evolution of GSDM-mediated Pyroptosis

Xinli Wang, Xuxia Wei, Yan Lu, Qinghuan Wang, Rong Fu, Yin Wang, Qin Wang, Xiangyan Wang, Shangwu Chen, Anlong Xu, Shaochun Yuan


Members of the gasdermin (GSDM) family are pore-forming effectors that cause membrane permeabilization and pyroptosis, a lytic proinflammatory type of cell death. To reveal the functional evolution of GSDM-mediated pyroptosis at the transition from invertebrates to vertebrates, we conducted functional characterization of amphioxus GSDME (BbGSDME) and found that it can be cleaved by distinct caspase homologs, yielding the N253 and N304 termini with distinct functions. The N253 fragment binds to cell membrane, triggers pyroptosis, and inhibits bacterial growth, while the N304 performs negative regulation of N253-mediated cell death. Moreover, BbGSDME is associated with bacteria-induced tissue necrosis and transcriptionally regulated by BbIRF1/8 in amphioxus. Interestingly, several amino acids that are evolutionarily conserved were found to be important for the function of both BbGSDME and HsGSDME, shedding new lights on the functional regulation of GSDM-mediated inflammation.


Cytokines of the interleukin-1 (IL-1) family are essential determinants of inflammation. Due to the absence of an amino-terminal secretion signal, the mechanisms underlying their protein secretion from phagocytes have attracted intense attention for more than 30 years. In 2015, 3 independent research groups using genetic screening or quantitative mass spectrometry-based analysis identified the pore-forming protein gasdermin D (GSDMD) as the conduit for IL-1 secretion. GSDMD is a substrate of activated caspase-1 (CASP1) or CASP4/5/11, allowing it to release the N-terminus and to form inner diameters of 10 to 14 nm oligomeric pores in the cell membranes. In addition to inflammasome-mediated pore formation in macrophages, GSDMD can mediate neutrophil pyroptosis, which is essential in NETosis and antimicrobial NET extrusion, revealing fundamental differences in GSDMD trafficking between neutrophils and macrophages.

Materials and method


HEK 293T and HeLa cells were purchased from ATCC and preserved in our lab. HeLagsdmd/e DKO cells were kindly presented by Prof.Feng Shao (National Institute of Biological Sciences, Beijing, China). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Life Technologies) and antibiotics (streptomycin and penicillin; Life Technologies).


Evolution of the GSDM family in Metazoans

To shed light on the origin and evolution of GSDM-mediated pyroptosis, we used BLAST programs to search the public genome, expressed sequence tag (EST), and protein databases of the available species. We found that GSDME homologs are widely distributed in multicellular organisms except jellyfish, nematodes, insects, and ascidians (S1 Table). Based on the protein alignment of the GSDM full-length (FL) sequences, we then constructed a maximum-likelihood (ML) tree by taking the GSDM-like molecules from bacteria and fungi as the outgroup (S1 Fig). To better understand the evolution of GSDM family, we also completed the intron phase and gene collinearity analyses of some GSDM representatives among species. In agreement with the previous studies [27,28], the derived tree suggested that GSDMs can be divided into 2 major clades, the GSDMA/B/C/D and GSDME/PJVK clades (S1 Fig). The former is specific to jawed vertebrates, while the latter is distributed widely from cnidaria to mammals (S1 Fig)


Targeting GSDM execute pyroptosis should be an important means in controlling microbial infection during evolution

Pyroptosis, a new type of programmed cell death mediated by GSDM, is characterized by the swelling and rupture of cells, release of cellular contents, and a strong inflammatory response [12]. GSDM-mediated pyroptosis has been shown to be critical for controlling microbial infection and tumor growth in mammals [12]. Recently, a coral GSDME homolog was reported to be cleaved by coral CASP3 to induce pyroptosis [24]. Coral can be protected from bacterial infection when coral GSDME cleavage was inhibited by a CASP3 inhibitor [24]. By establishing a zebrafish crispant in vivo analysis model, Yang’s group revealed that the zebrafish caspy2-GSDMEb axis contributes to lethal LPS-induced septic shock and NETosis for bacterial clearance in vivo. Similar to its counterparts, amphioxus BbGSDME is cleaved by CASPs to release its BbGSDME-N253, which can form oligomers and bind to membrane lipids in a similar fashion as mammalian GSDMD-NT. Moreover, cleavage of BbGSDME and tissue damage induced by bacterial infection can be alleviated by the pan CASP inhibitor Z-VAD-FMK and the specific peptide Ac-VHTD-CHO in amphioxus. Since GSDMD emerged for the first time in primitive mammals, studies from coral, amphioxus, and zebrafish GSDME indicated that GSDME may concurrently function as mammalian GSDMD in bony fish and other aquatic invertebrates .

Citation: Wang X, Wei X, Lu Y, Wang Q, Fu R, Wang Y, et al. (2023) Characterization of GSDME in amphioxus provides insights into the functional evolution of GSDM-mediated pyroptosis. PLoS Biol 21(5): e3002062.

Academic Editor: Feng Shao, National Institute of Biological Sciences, CHINA

Received: July 30, 2022; Accepted: March 6, 2023; Published: May 3, 2023

Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files. All FCS files are available from the FlowRepository database (FR-FCM-Z642).

Funding: This work was supported by Ministry of Science and Technology of the People's Republic of China (2018YFD0900502 to SC.Y), National Natural Science Foundation of China (31770943 and 31970852 to SC.Y), the Guangdong Science and Technology Department (2020B1212060031 to SC.Y) and Innovation Group Project of Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) (311021006 to SC.Y). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: Bb, Branchiostoma belcheri; CASP1, caspase-1; CASP3, caspase 3; CHX, cycloheximide; DED, death effector domain; Dr, Danio rerio; EST, expressed sequence tag; FL, full-length; GAS, group A Streptococcus; GSDM, gasdermin; HE, hematoxylin–eosin; IL-1, interleukin-1; IL-6, interlukin 6; IPTG, isopropyl β-D-thiogalactoside; IRF1, interferon regulatory factor 1; ISRE, IFN-stimulated response element; κB, kappa B; LB, Luria-Bertani; LDH, lactate dehydrogenase; LOF, loss-of-function; LPS, lipopolysaccharide; ML, maximum-likelihood; PI, propidium iodide; PJVK, Pejvakin; RT, room temperature; Rcd-1, regulator cell death; SNP, single nucleotide polymorphism; SpeB, streptococcal pyrogenic exotoxin B; TNFα, tumor necrosis factor alpha; WB, western blot; WCL, whole cell lysate

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