Identification and structural characterization of anthrax toxin receptor 2 as the Clostridium perfringens NetF receptor

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: minor_revision

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.

Introduction
Clostridium perfringens is a versatile pathogen, causing wound infections, septicemia, enterotoxemia or enteritis in animals and humans1–3. Especially, intestinal diseases in animals such as pigs, cattle, sheep, goats, chickens, dogs, and horses result in significant losses of livestock and companion animals worldwide. C. perfringens induces tissue damage via highly potent exotoxins of which hemolysin β-pore-forming toxins (βPFTs) represent the largest group, with currently 11 identified members4,5. Two of these, C. perfringens beta-toxin (CPB) and necrotic enteritis toxin B (NetB), are essential virulence factors in necrotic enteritis in pigs and humans and poultry, respectively. Our knowledge about other C. perfringens hemolysin βPFTs, however, is limited. In 2015, a hemolysin βPFT called NetF was identified in C. perfringens isolates from dogs with hemorrhagic gastroenteritis and foals with necrotic enterocolitis6. Subsequent epidemiological evidence substantiated, that the toxin plays an important role in C. perfringens induced enteric disease in these animals7–11. NetF molecular weight is 34.4 kDa, it shares 30%, 33% and 48% sequence identity with S. aureus α-hemolysin (Hla), CPB and NetB, respectively. It was shown to bind to the membrane of susceptible cells through interaction with an undefined sialylated receptor and to form oligomers with varying stoichiometries on liposomes12.

Hemolysin βPFTs are mostly secreted by the bacteria as soluble monomers. They then bind to receptors on target cells, oligomerize into pre-pores and subsequently undergo conformational change to insert into the plasma membrane as ion permissive pores13. Thus, identifying membrane receptors and the structural basis of the interaction between the toxin, the receptor and the plasma membrane is key to understanding the function of these toxins. So far, only two receptors for clostridial hemolysin βPFTs have been identified: CD31 for C. perfringens beta-toxin (CPB) and ganglioside GM2 for delta-toxin14,15. In addition, oligomeric pore-structures have only been determined for CPB and NetB14,16,17, whereas precise structural information about clostridial toxin-receptor and toxin-membrane interaction is still lacking.

Here, we elucidate the structure of NetF in its oligomeric pre-pore and its membrane-inserted pore state. We identify anthrax toxin receptor 2 (ANTXR2), also known as capillary morphogenesis gene 2 (CMG2), as a receptor for NetF and show that the toxin can efficiently target human, bovine, equine, and canine ANTXR2. In addition, we determine the co-structure of NetF oligomer in complex with the extracellular part of ANTXR2, revealing insights into the interaction between a clostridial hemolysin βPFT and its membrane receptor.

Results
Structural characterization of NetF
Previous research demonstrated that NetF oligomerizes in different stoichiometries, forming complexes composed of six to nine protomers12. We recombinantly expressed the toxin in Escherichia coli and confirmed that NetF binds and oligomerizes on synthetic membranes. Cholesterol increased membrane binding of NetF (Supplementary Fig. 1A–C). To optimize the conditions for cryo-EM characterization of NetF oligomeric form, we generated pores following two distinct methods: (1) incubation of NetF monomers with liposomes, leading to the formation of oligomeric pores, which were subsequently solubilized (Supplementary Fig. 1D), and (2) incubation of NetF monomers with pre-formed lipid nanodiscs (Supplementary Fig. 1E). Surprisingly, the latter led predominantly to nonameric pre-pores (Fig. 1A, B), while detergent-solubilized oligomers mostly adopted an octameric pore conformation (Fig. 1C, D), as monitored by cryo-EM. The structures of NetF pre-pore and pore represent a hemolysin-like oligomer organized into three distinct regions: the cap, the rim, and the stem (Fig. 1C, E). The high resolution cryo-EM maps enable precise atomic modeling of the backbone and most sidechains, except for the first eleven N-terminal amino acids, which are partially unstructured (Supplementary Fig. 2).

In the absence of X-ray data, the NetF soluble monomeric structure was predicted using AlphaFold318 (Fig. 1E, Supplementary Fig. 3A). A comparison between the AlphaFold3 monomer prediction and a protomer extracted from the NetF pre-pore indicates that AlphaFold3 prediction is extremely accurate with a root-mean-square deviation (RMSD) of 1.9 Å across all amino acids. Although the overall prediction confidence score is high (pTM of 0.91), the N-terminal region, the pre-stem loop, and one loop of the rim domain display lower confidence (Supplementary Fig. 3A). The differences between the predicted monomer and the protomer, as well as comparison with other hemolysin βPFTs, suggest that multiple conformational changes are necessary for oligomerization (Fig. 1E). The oligomerization of NetF into pre-pore conformation involves extensive interactions (Supplementary Table 1 and 2), engaging a large area on each side of the cap and rim domains, measuring approximately 1400 Å2 and encompassing more than 50 residues (Fig. 1B, Supplementary Fig. 2B). The N-terminus, however, remains flexible and unresolved (residues 1–20). In the pre-pore conformation, the pre-stem loop is partially visible except for five residues (Fig. 1E). Notably, in contrast to the AlphaFold3 prediction of NetF monomer and to the monomer structure of other known hemolysins, the pre-stem of the pre-pore is moved upwards, away from its position on the cap domain. This structural rearrangement may play a critical role in pore formation, potentially priming the protein for membrane insertion.

The NetF pore structure reveals additional conformational changes required for membrane insertion. In the pre-pore, the pre-stem loop is folded against the cap domain, and its extraction during pore formation triggers a second rearrangement of the N-terminus. Although unresolved in both structures, the N-terminus position can be inferred from its orientation and diffuse density of the backbone and N-terminal His-tag (Fig. 2A–C). In the pre-pore, it points outward, but upon pre-stem loop unfolding, it shifts toward the cavity formed by the cap domain, loosely occupying the site of the pre-stem (Fig. 2B). A focused 3D classification of the upper part of the pre-pore and pore confirmed this difference (Fig. 2D). As the β-barrel forms, inter-protomer interactions expand by 50% to ~2100 Å2 (Supplementary Table 1 and 2), involving 90 residues (Fig. 1B, Supplementary Fig. 2D). Notably, the cap and rim domains remain largely unchanged during this transition.

The most significant rearrangement during pore formation is the refolding of the pre-stem loop into a 50 Å-long transmembrane β-barrel, characterized by a hydrophilic lumen and a hydrophobic exterior. The barrel has a diameter of approximately 10 Å, with two constrictions that narrow the lumen to 6.7 Å on the cis (i.e., extracellular) side and 8.2 Å on the trans (i.e., cytoplasmic) side (Fig. 2E, F). The lumen of the transmembrane pore is polar but contains no charged residues, except for an arginine residue (Arg113) at the cis entrance and two lysine residues (Lys130 and 131) on the trans side, both of which contribute to the constriction sites. While the Lys130 points away from the lumen it bends the β-turn inwards creating a second constriction. An aromatic belt formed by residues Tyr120 and Phe141 is positioned on the cis side oriented towards the lipids, consistent with the typical features of transmembrane β-barrels (Fig. 2C, E).

NetF interacts with lipids through the rim and stem domains
The cryo-EM maps also reveal substantial extra densities surrounding the rim and stem domains of NetF oligomer, in particular in the pore conformation (Fig. 2A–C). Based on their shape and chemical environment, these densities correspond to bound lipid and detergent molecules. They are located around loops containing aromatic and charged residues: Tyr74, Tyr75, and Trp254 bind lipids on the outer rim domain; Tyr184, Trp259, His199 interact with lipids at the protomer-protomer interface. Furthermore, clear lipid/detergent densities are visible tightly interacting with the aromatic belt (Tyr120 and Phe141) as well as the middle and trans parts of the transmembrane β-barrel (Fig. 2B, C). It has been previously demonstrated for S. aureus α-hemolysin and related toxins that a loop in the rim domain interacts with lipid headgroups19,20. The equivalent loop is however shortened in NetF and lacks the tryptophan residue required for lipid binding (Trp179, Supplementary Fig. 3B), as also observed in C. perfringens β-toxin and NetB17,21. The presence of NetF-bound lipids and detergents suggest that the rim domain is partially embedded in the membrane tightly interacting with lipids and cholesterol.

NetF resistance is associated with reduced ANTXR2 expression
We next set out to identify the cell surface receptor for NetF. To determine suitable cell lines, we performed viability assays on different human, murine, canine, equine, and bovine cells. NetF was toxic to human U937 and HFF-1 cells, canine MDCK and glial J3T cells, primary equine fibroblasts, and bovine endothelial BUcEC cells. In contrast, other cell lines from these species as well as two murine cell lines were resistant to NetF (Fig. 3A). We additionally determined that NetF rapidly leads to LDH release, shrinkage, and lysis in susceptible J3T cells (Supplementary Fig. 4A, B).

A small fraction of cells in several susceptible cell lines consistently survived NetF exposure, regardless of the toxin concentration applied. We chose the human U937 and the canine J3T cell lines, which showed less than 10% viability levels after 24 h of toxin incubation (Fig. 3A), to evaluate whether surviving cells were NetF-resistant subpopulations. Following six passages under constant toxin pressure, we isolated resistant subpopulations of both cell lines (U937-R and J3T-R) (Supplementary Fig. 4C). Compared with the parental cell populations mRNA expression profiles of these resistant subclones showed four significantly downregulated genes in both U937-R and J3T-R cells: ANTXR2, PRDM8, CREB5, and ZNF790 (Fig. 3B and Supplementary Fig. 4D). ANTXR2 was the only gene encoding a transmembrane protein. PRDM8, encoding an intracellular histone methyltransferase, is a neighboring gene to ANTXR2 and is co-regulated with it in the same transcriptional unit22. CREB5 encodes a transcription factor that binds to cis-responsive elements (CRE) in the promoter region of the ANTXR2 transcriptional unit22. No link has been identified between ANTXR2 expression and zinc finger protein 790 (ZNF790), a nuclear protein predicted to be involved in transcriptional regulation23,24. Consistent with cell viability data, qRT-PCR analyses in human, canine, and bovine cells showed that ANTXR2 mRNA levels were higher in susceptible than in non-susceptible cells (Supplementary Fig. 4E). Interestingly, despite showing resistance to NetF, ANTXR2 mRNA was detectable in murine endothelial cells.

ANTXR2 is one of two receptors for anthrax toxin protective antigen (PA). The other PA receptor is ANTXR1, also known as tumor endothelial marker 8 (TEM8)25. ANTXR1 and ANTXR2 are single pass transmembrane proteins with two extracellular domains: an N-terminal von Willebrand A (vWA) domain containing a metal ion-dependent adhesion site (MIDAS) motif and a membrane proximal immunoglobulin (Ig) domain26. Additionally, they contain a C-terminal cytoplasmic domain involved in ligand-mediated signaling. ANTXR2 binds collagen VI via the MIDAS motif and participates in extracellular matrix homeostasis. Our RNA-sequencing (RNA-seq) data indicated that ANTXR1 expression was unchanged in U937-R and J3T-R compared to their susceptible counterparts (Supplementary Data 1). These results led us to hypothesize that ANTXR2 serves as the membrane receptor for NetF.

ANTXR2 interacts with NetF and is essential for its cytotoxicity
To validate the role of ANTXR2 in NetF toxicity, we performed CRISPR-Cas9-mediated knockouts (KOs) of ANTXR2 in U937 cells using two single-guide RNAs (sgRNAs) which led to a high number of frameshift and/or in-frame mutations (Fig. 3C). Viability assays showed that sgRNA1- and sgRNA2-targeted cells became resistant to NetF treatment (Fig. 3D) even though NetF binding and oligomerization were still detectable by western blot analysis on U937-R (Supplementary Fig. 4F)

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Wang and colleagues have described the pore-forming toxin NetF, a member of the beta-PFT family and\nidentified its cellular receptor, ANTXR2. The structures of NetF in the pre-pore and pore conformation were solved to high\nresolution using Cryo-EM as well as the structure of the pore form in complex with the extracellular domain of ANTXR2. In\naddition, the authors have characterised in much detail, the binding properties if the receptor and identified the part of the\nreceptor responsible for cell susceptibility. The paper is very well written and contains numerous controls that validate the\nauthors conclusions. Furthermore, the structure of the complex is one of a few available describing the detailed molecular\nstructure of a toxin and its receptor and is the first structure of the soluble part of the ANTXR2 receptor in the literature.\nIn my opinion, this study justifies publication in Nature Communications. Besides being a elegant and very complete study of\nthe system, it provides important insights into the mode of action of these beta-PFTs and paves the way to design molecules\nto inhibit binding to susceptible cells. It would be good however, for the authors to clarify the points below for the readers:\n1. The authors describe seeing binding and oligomerisation on U937 wt and resistant cells lines as well as the KO cell lines\nto similar levels. This observation is very interesting and implies that oligomerisation it-self, is not a pre-requisite for pore\nformation. However, the percentage of oligomers compared to monomer is very little. It would be useful for the authors to\ncomment on this. Is the majority of monomer seen due to oversaturation of the cells ? For example, in Sup.Fig1B the majority\nof NetF has oligomerised on liposomes with cholesterol in the absence of a receptor and has presumable formed pores\nsince they go on to solve the pore structure from these particles.\n2. The extra densities seen for the N-terminus and suggested by the authors to be the His-tag is somewhat speculative and\nweakens an otherwise solid study. Of course, a conclusive experiment here would be to cleave the his-tag and see if this\ndensity disappears. In the absence of this experiment, I don’t think one cannot draw conclusions as to the location of the N-\nterminus and the text should be more indicative of the presumption taken here. In addition, in Figure 2A, a ring of flat density,\npresumably lipid, is shown below the pre-pore but nor mentioned in the text. Is this noise from the nanodisc?\n3. In figure 5B, the map is described as a local resolution map? Should that read local resolution filtered map as the\ncolouring is not indicative of local resolution?", "reviewer_2": "This study provides remarkable structural evidence on the pore formation and binding of NetF, belonging to the beta-\nhaemolysin family. There are few limitations regarding the cell specificity of NetF that require clarification which could be\nsubstantiated by necessary controls.\na) More information on the toxicity kinetic would complete the information provided in Figures 3a and 3b. 24h incubation with\nNetF is not informative enough to grasp the extent of NetF toxicity activity. A kinetic is required to provide more information\non the dynamic of NetF toxicity, and to better introduce the notion of specificity to ANTRX2\nb) Additional information should be provided to support the Figures 3a and 3b. Figure 3f shows that the authors have used\nan antibody targeted against endogenous ANTRX2.\nAlthough the authors have shown that ectopic human ANTRX2 expression in HAP1 cells (but not murine) render them\nsensitive to NetF toxicity, these data should be further completed with evidence showing the levels of ANTRX2 in the tested\ncell lines. The same antibody used in Figure 3f should be used against the cell lines tested in Figures 3a and 3b to\nsubstantiate and/or corroborate their insensitivity to NetF.\nc) In Figure 3c and 3d, the authors have used a CRISPR-Cas9 strategy to ablate ANTRX2 in U937 cells. In addition to\nsequencing results shown in Figure 3c, a western-blot using the ANTRX2 antibody should be shown to support the further\nsupport the Figure 3c and 3d data.", "reviewer_3": "This manuscript presents a comprehensive study combining structural biology and cellular approaches to identify and\ncharacterize ANTXR2 (also known as CMG2) as the receptor for NetF, a β-pore-forming toxin (βPFT) from Clostridium\nperfringens. The authors provide high-resolution cryo-EM structures of NetF in both pre-pore and pore conformations, as\nwell as in complex with the PMT-modified extracellular domain of ANTXR2. They demonstrate that NetF engages ANTXR2\nthrough a unique binding mode involving primarily the Ig-like domain but also the vWA domain, distinct from the interaction\nof anthrax protective antigen (PA) with the same receptor. Functional assays, including CRISPR-Cas9 mutagenesis and\nreceptor ortholog screening, support the conclusion that ANTXR2 is essential for NetF-mediated cytotoxicity.\nMajor comment:\nThe authors convincingly demonstrate the importance of the Ig-like domain of ANTXR2 in NetF binding and toxicity.\nHowever, the mutagenesis data (i.e., in-frame mutations from the CRISPR-Cas9 experiments) should be summarized in a\ndedicated table, along with interaction data between ANTXR2 and NetF. This would clarify the correlation between mutation\ntypes and resistance phenotypes, especially in light of the structural findings that highlight the Ig domain as a key interaction\nsite. A table listing key amino acid residues involved in the ANTXR2–NetF interaction should also be provided. Furthermore, the conservation of critical residues in the vWA domain across species should be discussed in relation to the observed\nresistance of murine ANTXR2-expressing cells. It is important to discuss how ANTXR2 and NetF interaction data correlate\nbetween mutagenesis, structure, and species-specific variants of ANTXR2.\nOther comments:\nPoint 1 : The intracellular retention of canine ANTXR2 in HAP1 cells is shown, but its membrane localization in HEK293\ncells is less evident. The authors should perform plasma membrane fractionation or surface biotinylation assays to confirm\nsurface expression levels. Additionally, a non-transfected HAP1 control is missing in the relevant figure.\nPoint 2 (Figure 4A and 4C) : There are noticeable differences in ANTXR2 expression levels across species. The authors\nshould discuss whether these differences could influence the observed susceptibility to NetF, particularly for murine\nANTXR2, which is expressed at lower levels. Moreover, given the low membrane expression of cANTXR2 and its inverse\ncorrelation with high cell susceptibility to NetF, the lack of correlation between expression levels and susceptibility should\nbe addressed, as this is unexpected for a toxin receptor.\nPoint 3 (Figure 4D–4E): The chimeric receptor experiments highlight the importance of the Ig-like domain. However, the\ncryo-EM structure also implicates specific residues in the vWA domain. The authors should discuss whether these residues\nare conserved between human and murine ANTXR2.\nPoint 4 : The comparison between NetF and anthrax PA is somewhat speculative. While both toxins use ANTXR2, their\nmechanisms of action and physiological effects differ significantly. This section should be shortened and more cautiously\nframed.\nPoint 5 (Sup. Fig. 1B) : provide quantifications showing that cholesterol increases membrane binding of NetF.\nOverall, this is a well-executed and thorough study that significantly advances our understanding of clostridial βPFTs and\ntheir receptor interactions. The structural and functional data are robust and well-integrated. Addressing the major comment\nand clarifying the minor points will further strengthen the manuscript."}, "author_response": "Point-by-point answer to reviewers comments\nReviewer #1 (Remarks to the Author):\nIn this study, Wang and colleagues have described the pore-forming toxin NetF, a\nmember of the beta-PFT family and identified its cellular receptor, ANTXR2. The\nstructures of NetF in the pre-pore and pore conformation were solved to high\nresolution using Cryo-EM as well as the structure of the pore form in complex with\nthe extracellular domain of ANTXR2. In addition, the authors have characterised in\nmuch detail, the binding properties if the receptor and identified the part of the\nreceptor responsible for cell susceptibility. The paper is very well written and contains\nnumerous controls that validate the authors conclusions. Furthermore, the structure\nof the complex is one of a few available describing the detailed molecular structure of\na toxin and its receptor and is the first structure of the soluble part of the ANTXR2\nreceptor in the literature.\nIn my opinion, this study justifies publication in Nature Communications. Besides\nbeing a elegant and very complete study of the system, it provides important insights\ninto the mode of action of these beta-PFTs and paves the way to design molecules\nto inhibit binding to susceptible cells. It would be good however, for the authors to clarify\nthe points below for the readers:\nWe are grateful for the reviewer positive feedback and constructive comments.\n1. The authors describe seeing binding and oligomerisation on U937 wt and resistant\ncells lines as well as the KO cell lines to similar levels. This observation is very\ninteresting and implies that oligomerisation it-self, is not a pre-requisite for pore\nformation. However, the percentage of oligomers compared to monomer is very little.\nIt would be useful for the authors to comment on this. Is the majority of monomer\nseen due to oversaturation of the cells ? For example, in Sup.Fig1B the majority\nof NetF has oligomerised on liposomes with cholesterol in the absence of a receptor\nand has presumable formed pores since they go on to solve the pore structure from\nthese particles.\nThis is an interesting observation. NetF can efficiently oligomerize on liposomes,\nparticularly when they contain a high fraction of cholesterol (see revised Sup. Fig. 1).\nIn contrast to liposomes, approximately 50% of the mass of plasma membranes\nconsist of proteins (Alberts et al., 2022, Molecular biology of the cell, 7th ed.). We\nspeculate that this high protein content may hinder NetF access to, and insertion into\nthe membrane, and that a receptor could serve to dock the forming oligomer. In the\nabsence of a receptor it is possible that some NetF molecules form oligomers at the\ncell surface, but these may detach before their beta-barrel can insert into the\nmembrane. An experimental validation of this hypothesis is beyond the scope of the\npresent study but we mention it in the Discussion.\n2. The extra densities seen for the N-terminus and suggested by the authors to be\nthe His-tag is somewhat speculative and weakens an otherwise solid study. Of\ncourse, a conclusive experiment here would be to cleave the his-tag and see if this\ndensity disappears. In the absence of this experiment, I don’t think one cannot draw\nconclusions as to the location of the N-terminus and the text should be more\nindicative of the presumption taken here. In addition, in Figure 2A, a ring of flat\ndensity, presumably lipid, is shown below the pre-pore but nor mentioned in the text.\nIs this noise from the nanodisc?\nWe performed focused classification on the membrane-distal region of the pre-pore\nand pore datasets. The backbone of some additional N-terminal residues is now\nresolved (Figure 2D, red density). A clear conformational change can be observed: in\nthe pre-pore, the residues protrude from the vestibule, whereas in the pore they form\npart of its inner surface. The most N-terminal residues remain unresolved, suggesting\nthat this region is highly flexible. The ring of flat density below the pre-pore indeed\noriginates from the nanodisc, as confirmed by the image. We now mention it in the\nfigure legend.\n3. In figure 5B, the map is described as a local resolution map? Should that read\nlocal resolution filtered map as the colouring is not indicative of local resolution?\nWe thank the reviewer for noticing this. This was a mistake and it has been\ncorrected. The coloring indicates the different domains rather than the local\nresolution.\nReviewer #2 (Remarks to the Author):\nThis study provides remarkable structural evidence on the pore formation and\nbinding of NetF, belonging to the beta-haemolysin family. There are few limitations\nregarding the cell specificity of NetF that require clarification which could be\nsubstantiated by necessary controls.\nWe thank the reviewer for the encouraging feedback and helpful remarks.\na) More information on the toxicity kinetic would complete the information provided in\nFigures 3a and 3b. 24h incubation with NetF is not informative enough to grasp the\nextent of NetF toxicity activity. A kinetic is required to provide more information on the\ndynamic of NetF toxicity, and to better introduce the notion of specificity to ANTRX2\nWe performed the requested experiment and it is now included in Sup. Fig. 4 A and\nB.\nb) Additional information should be provided to support the Figures 3a and 3b. Figure\n3f shows that the authors have used an antibody targeted against endogenous\nANTRX2.\nAlthough the authors have shown that ectopic human ANTRX2 expression in HAP1\ncells (but not murine) render them sensitive to NetF toxicity, these data should be\nfurther completed with evidence showing the levels of ANTRX2 in the tested cell\nlines. The same antibody used in Figure 3f should be used against the cell lines\ntested in Figures 3a and 3b to substantiate and/or corroborate their insensitivity to\nNetF.\nThis is a valid point, and we have worked to address it, testing many primary\nantibodies. Unfortunately, antibody-based detection of endogenous ANTXR2 protein\nis extremely challenging, as confirmed by the long-standing experience of coauthors\nwho have worked on this protein for two decades (LA and FGvdG). To complement\nthese efforts, we quantified ANTXR2 mRNA levels by qRT-PCR (Sup. Fig. 4E).\nOverall, transcript abundance correlated with the sensitivity of cell lines to NetF, with\nthe exception of murine lines. This is consistent with our observation that ectopic\nexpression of murine ANTXR2 does not render cells susceptible to NetF.\nThe canine fibroblast line that we had originally established was unfortunately no\nlonger available at the time of revision. Because we were therefore unable to perform\na reliable quantification of ANTXR2 mRNA levels in this line, we chose to remove the\ncorresponding data from the manuscript to avoid presenting incomplete or non-\nverifiable results.\nc) In Figure 3c and 3d, the authors have used a CRISPR-Cas9 strategy to ablate\nANTRX2 in U937 cells. In addition to sequencing results shown in Figure 3c, a\nwestern blot using the ANTRX2 antibody should be shown to support the further\nsupport the Figure 3c and 3d data.\nAs noted above, endogenous ANTXR2 cannot be detected by Western blot.\nTherefore, we could not perform this additional control.\nReviewer #3 (Remarks to the Author):\nReview Report\nThis manuscript presents a comprehensive study combining structural biology and\ncellular approaches to identify and characterize ANTXR2 (also known as CMG2) as\nthe receptor for NetF, a β-pore-forming toxin (βPFT) from Clostridium perfringens.\nThe authors provide high-resolution cryo-EM structures of NetF in both pre-pore and\npore conformations, as well as in complex with the PMT-modified extracellular\ndomain of ANTXR2. They demonstrate that NetF engages ANTXR2 through a unique\nbinding mode involving primarily the Ig-like domain but also the vWA domain, distinct\nfrom the interaction of anthrax protective antigen (PA) with the same receptor.\nFunctional assays, including CRISPR-Cas9 mutagenesis and receptor ortholog\nscreening, support the conclusion that ANTXR2 is essential for NetF-mediated\ncytotoxicity.\nWe thank the reviewer for their positive assessment.\nMajor comment:\nThe authors convincingly demonstrate the importance of the Ig-like domain of\nANTXR2 in NetF binding and toxicity. However, the mutagenesis data (i.e., in-frame\nmutations from the CRISPR-Cas9 experiments) should be summarized in a\ndedicated table, along with interaction data between ANTXR2 and NetF. This would\nclarify the correlation between mutation types and resistance phenotypes, especially\nin light of the structural findings that highlight the Ig domain as a key interaction site.\nA table listing key amino acid residues involved in the ANTXR2–NetF interaction\nshould also be provided. Furthermore, the conservation of critical residues in the\nvWA domain across species should be discussed in relation to the observed\nresistance of murine ANTXR2-expressing cells. It is important to discuss how\nANTXR2 and NetF interaction data correlate between mutagenesis, structure, and\nspecies-specific variants of ANTXR2.\nWe thank the reviewer for helpful suggestions. We have expanded Table S3, which\noriginally showed residue pairs forming h-bonds and salt bridges. It now includes\nhydrophobic interactions as well. We have marked with a box all ANTXR2 residues\ndirectly interacting with NetF in ANTXR2 sequence in Sup. Fig. 7 A. Additionally this\nfigure shows all the interaction sites that comprise a mutation in the murine sequence\nand their location with respect to vWA and Ig-like domains. We have added a new\npanel to Sup. Fig. 7 showing murine amino acids that are predicted by Alphafold3 to\nprevent complex formation with human ANTXR2. And we have added a discussion\nabout this topic.\nOther comments:\nPoint 1 : The intracellular retention of canine ANTXR2 in HAP1 cells is shown, but its\nmembrane localization in HEK293 cells is less evident. The authors should perform\nplasma membrane fractionation or surface biotinylation assays to confirm surface\nexpression levels. Additionally, a non-transfected HAP1 control is missing in the\nrelevant figure.\nA surface biotinylation assay is now included in Sup. Fig. 6C. A non-transfected\nHAP1 control has been added.\nPoint 2 (Figure 4A and 4C) : There are noticeable differences in ANTXR2 expression\nlevels across species. The authors should discuss whether these differences could\ninfluence the observed susceptibility to NetF, particularly for murine ANTXR2, which\nis expressed at lower levels. Moreover, given the low membrane expression of\ncANTXR2 and its inverse correlation with high cell susceptibility to NetF, the lack of\ncorrelation between expression levels and susceptibility should be addressed, as this\nis unexpected for a toxin receptor.\nFigure 4A shows expression level of overexpressed ANTXR2 variants in HAP1 cells\nand the associated viability is shown in Figure 4B. Note that Figure 4C shows cells\nviability in another cell line. In HAP1 cells, cANTXR2 is expressed to low levels and is\nnot properly located at the plasma membrane (Sup. Fig. 6A). Consistently, cANTXR2\ndoes not make HAP1 cells susceptible to NetF (Figure 4B). Since cells endogenously\nexpressing cANTXR2 are highly susceptible to NetF (Figure 3A, Sup. Fig. 4E), we\nassessed expression of ectopic cANTXR2 in HEK293 cells. Surface expression was\nimproved (Sup. Fig. 6BC) and consistently led to NetF toxicity (Figure 4C).\nThe low susceptibility to NetF given by mANTXR2 may be explained by a lower\naffinity for NetF due to mutations in two binding sites located in the Ig-like domain\n(Sup. Fig. 7A). Corresponding point mutations significantly reduce binding affinity\naccording to AlphaFold predictions (Sup. Fig. 7B).\nPoint 3 (Figure 4D–4E): The chimeric receptor experiments highlight the importance\nof the Ig-like domain. However, the cryo-EM structure also implicates specific\nresidues in the vWA domain. The authors should discuss whether these residues\nare conserved between human and murine ANTXR2.\nWe have now indicated and discussed the conservation of residues between human\nand murine ANTXR2. We have also predicted the impact of point mutations on the\nbinding affinity (see our answer to previous reviewer)\nPoint 4 : The comparison between NetF and anthrax PA is somewhat speculative.\nWhile both toxins use ANTXR2, their mechanisms of action and physiological effects\ndiffer significantly. This section should be shortened and more cautiously framed.\nWe have carefully reviewed our discussion and found little to no speculation in the\ncomparison between NetF and PA. We highlight the differences in binding mode. PA-\nANTXR2 structure has been published. There is clearly only interaction with the most\nmembrane-distal part of ANTXR2 VWA domain and PA prepore (Lacy et al 2004).\nThe length difference of the respective beta-barrels is also well established based on\nour study and Jiang et al 2015 study. It is therefore clear that PA prepore has no\ninteraction with either membrane lipids or ANTXR2 Ig-like domain.\nPoint 5 (Sup. Fig. 1B) : provide quantifications showing that cholesterol increases\nmembrane binding of NetF.\nWe have now performed a quantification which is included in Sup. Fig 1.\nOverall, this is a well-executed and thorough study that significantly advances our\nunderstanding of clostridial βPFTs and their receptor interactions. The structural and\nfunctional data are robust and well-integrated. Addressing the major comment and\nclarifying the minor points will further strengthen the manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised version of the manuscript has dealt with my comments sufficiently and performed additional experiments to\nsupport the hypothesis. I have no further comments and support the publication in Nature Communications.", "reviewer_2": "The authors have provided a commendable revision. Well done all.", "reviewer_3": "The authors responded positively to all of my comments"}, "author_response": ""}]

Decision letter