Abstract
Hemolysin β-pore-forming toxins (βPFTs) are key virulence factors of Clostridium perfringens, associated with severe diseases in humans and animals. Yet, the mechanisms by which Clostridium βPFTs recognize and engage specific target cells remain poorly understood. Here, we identify the cellular receptor for C. perfringens necrotizing enteritis toxin F (NetF), a recently discovered toxin implicated in severe enteritis in dogs and foals. We show that NetF binds to the same receptor as anthrax toxin, namely ANTXR2. Using cryo-electron microscopy, we determined the structure of the oligomeric NetF pre-pore as well as the transmembrane pore, both alone and in complex with the extracellular domain of ANTXR2. Unlike anthrax toxin, which binds to the apical MIDAS motif of ANTXR2 – as does the natural ANTXR2 ligand collagen type VI – NetF engages the receptor laterally, spanning both the von Willebrand A and the Ig-like domains. This interaction positions the toxin near the membrane, facilitating contact with membrane lipids and promoting transmembrane pore formation. Our findings uncover key principles of hemolysin βPFT-receptor recognition and advance our understanding of how pathogenic bacteria use these toxins to breach host defenses.
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Data availability
The RNA-seq data generated in this study have been deposited in the European Nucleotide Archive under accession PRJEB78819. The cryo-EM maps and their respective models generated in this study have been deposited are available in the worldwide Protein Data Bank and the EM Data Bank under accession 9RSM (NetF prepore model) EMD-54221 (NetF prepore map) 9RSU (NetF pore model) EMD-54226 (NetF pore map) 9RT2 (NetF-ANTXR2 C4 model) 9RT4 (NetF-ANTXR2 local refinement focused model) EMD-54238 (NetF-ANTXR2 C4 map) EMD-54245 (NetF-ANTXR2 local refinement focused map). The AlphaFold predictions used in this study are available as additional data in the supplemental material and supplemental source data file. Source Data are provided as a Source Data file. Source data are provided with this paper.
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Acknowledgements
We thank Prof. P. Plattet, University of Bern, for providing canine cell cultures. EM data were acquired on an instrument of the Dubochet Center for Imaging in Bern and supported by the Microscopy Imaging Center (MIC) of the University of Bern. We gratefully acknowledge Marek Kaminek and David Kalbermatter for their assistance with EM. We thank Sylvia Ho for providing the plasmids for protein expression in Expi293. This study was funded by a University of Bern ID Grant (H.P., B.Z.), SNSF grant 310030_212837 (H.P.), and SNSF sinergia grant 10000175 (B.Z., H.P.).
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H.P. and B.Z. conceptualized the study, supervised the research, and secured funding. C.W., F.C., I.I., and A.N. designed and performed experiments and analyzed the data. F.F. and J.F. performed experiments. L.A. performed experiments under the supervision of F.G.v.d.G. F.C., I.I., and H.P. wrote the initial draft with input from C.W. F.C., I.I., H.P., and B.Z. substantially revised and refined the manuscript. All authors reviewed and approved the final version.
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Wang, C., Cattalani, F., Iacovache, I. et al. Identification and structural characterization of anthrax toxin receptor 2 as the Clostridium perfringens NetF receptor. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69526-6
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DOI: https://doi.org/10.1038/s41467-026-69526-6
