Ancient pyroptotic machinery via GSDMA/B cleavage by LPS-activated caspase-1 in cartilaginous fish

Technical Summary: Ancient Pyroptotic Machinery via GSDMA/B Cleavage by LPS-Activated Caspase-1 in Cartilaginous Fish

Problem and Context
Pyroptosis, a lytic and proinflammatory form of programmed cell death, is executed by gasdermin (GSDM) family proteins. While the evolutionary history of the GSDME/PJVK branch is well-traced back to metazoans, the functional origins of the GSDMA/B/C/D branch remain poorly understood. In mammals, this branch has diversified significantly, with GSDMD serving as the primary executor of caspase-1-mediated pyroptosis, while GSDMA-C exhibit tissue-restricted roles often activated by non-canonical mechanisms. The evolutionary transition from an ancestral pyroptosis executioner to these multifunctional regulators is unclear. Specifically, the mechanism by which the ancestral GSDMA/B lineage interacts with inflammatory caspases and responds to pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS) in basal jawed vertebrates has not been characterized.

Methodology
The study utilized the elephant shark (Callorhinchus milii), a basal representative of cartilaginous fish and the GSDMA-D lineage, to investigate the ancestral state of this pathway.

• Bioinformatics and Structural Analysis: The authors reconstructed maximum likelihood phylogenetic trees of GSDM and caspase families using bacterial and fungal homologs as outgroups. They performed structural modeling (AlphaFold3, PyMOL) to compare C. milii GSDMA/B (CmiGSDMA/B) and Caspase-1 (CmiCASP1) with mammalian counterparts, analyzing substrate-binding pockets (S1-S4) and hydrophobicity.
• Cellular Assays: Using GSDMD/GSDME double-knockout HeLa cells (HeLagsdmd/e DKO) and HEK293T cells, the authors co-expressed CmiCASP1 and CmiGSDMA/B (and various mutants) to assess pyroptotic morphology, membrane permeability (Annexin V/PI staining), LDH release, and ATP depletion.
• Biochemical Characterization: Western blotting was used to identify cleavage sites and products. Site-directed mutagenesis (D241A, D288A, C279A, CARD deletion, and charge-reversal mutants) was employed to map cleavage sites and functional domains.
• LPS Sensing Mechanisms: To determine if CmiCASP1 is activated by LPS, the authors performed biotinylated LPS pull-down assays using catalytically inactive CmiCASP1 mutants (C279A) and CARD domain deletions. Caspase activity was measured using the fluorogenic substrate Z-VAD-AMC.
• Antibacterial Activity: Cell lysates containing full-length CmiGSDMA/B or its N-terminal fragments (N241, N288) were incubated with Gram-negative (E. coli, E. tarda, V. parahaemolyticus) and Gram-positive (S. aureus, L. monocytogenes, B. megaterium) bacteria. Viability was assessed via colony-forming unit (CFU) assays and live/dead staining (DMAO/PI).
• Lipid Binding: Lipid strip assays were conducted to determine the binding specificity of the N241 fragment to various phospholipids.

Key Results

1. CmiCASP1 Activates CmiGSDMA/B: Co-expression of CmiCASP1 and CmiGSDMA/B induced robust pyroptosis, characterized by cell swelling, membrane blebbing, and LDH release. This process was dependent on caspase activity, as it was abolished by the pan-caspase inhibitor Z-VAD-FMK.
2. Dual Cleavage Sites: CmiCASP1 cleaves CmiGSDMA/B at two distinct aspartate residues: D241 and D288. This generates two N-terminal fragments: N241 (residues 1–241) and N288 (residues 1–288).
3. Antagonistic Regulation:
   • N241: This fragment is the active pyroptosis executor. It localizes to the plasma membrane and induces cell death.
   • N288: This fragment is intrinsically inactive. Structural modeling suggests the C-terminal extension (242–288) folds back to mask the membrane-insertion motif (β1-β2 loop) of N241. N288 physically interacts with N241, sequestering it and preventing membrane association, thereby suppressing N241-induced pyroptosis.
4. Non-Canonical LPS Sensing: Unlike mammals where LPS sensing typically requires an inflammasome complex (NLRs, ASC), C. milii lacks canonical inflammasome components (NLRP3, ASC). Instead, CmiCASP1 directly binds LPS via its CARD domain. This binding, dependent on conserved positively charged residues, activates CmiCASP1 protease activity, leading to CmiGSDMA/B cleavage.
5. Bactericidal Activity: The N241 fragment exhibits direct bactericidal activity against Gram-negative bacteria, disrupting their membrane integrity. It shows no activity against Gram-positive bacteria. This activity is dependent on basic residues within N241 that facilitate binding to acidic phospholipids (e.g., cardiolipin, PtdIns(4,5)P2).

Significance and Claims
The paper claims to uncover an ancestral, non-canonical pyroptosis pathway in cartilaginous fish that predates the specialization seen in mammals.

• Evolutionary Insight: The findings suggest that the coupling of inflammatory caspases (CASP1) to GSDMA/B effectors and direct LPS sensing via CARD domains existed in early jawed vertebrates. This represents a pre-tetrapod state where a single inflammatory caspase could drive both host cell death and direct bacterial killing.
• Regulatory Complexity: The study reveals an ancient regulatory mechanism where a single proteolytic event generates both an active effector (N241) and a regulatory inhibitor (N288), fine-tuning the pyroptotic response before the lineage-specific expansion of GSDM paralogs in mammals.
• Dual Functionality: The research supports the hypothesis that antimicrobial activity was an early feature of the GSDMA-D lineage, with the N-terminal domain retaining the capacity to directly kill Gram-negative bacteria alongside its role in host cell lysis.

In summary, the study defines a functional LPS-responsive CASP1-GSDMA/B axis in Callorhinchus milii, providing a model for the evolutionary transition from primitive cytolysis to the sophisticated mucosal immunity observed in higher vertebrates.