EatA mediated degradation of intestinal mucus is species-specific and driven by MUC2 structural features

Domain: biology
Published: 2025-12-31
Editorial outcome: minor_revision

Abstract
Enterotoxigenic Escherichia coli (ETEC) infections are a leading cause of diarrheal illness, responsible for an estimated 100,000 deaths annually. ETEC pathogenesis is driven by various virulence factors, including toxins, adhesins, and noncanonical factors such as the protease EatA. The first line of host defense against intestinal pathogenic bacterial infections is the protective intestinal mucus layer. Here, we demonstrate the mechanism by which EatA degrades the core mucus component MUC2, thereby facilitating access to the epithelial cell surface and promoting infection. We identify the specific cleavage site region localized at the C-terminal of MUC2. EatA’s protease activity depends on the interaction between two distinct, uniquely spaced domains in human MUC2, which defines species specificity. We confirm this using a novel transgenic mouse model exclusively expressing human MUC2, which allows us to study the role of the mucus layer in the infection by human intestinal pathogens. These findings highlight how ETEC is adapted to specifically degrade the mucus layer of its human host.

Introduction
Enterotoxigenic Escherichia coli (ETEC) infections are one of the major causes of diarrheal illness in low- and middle-income countries1. While most of the population in endemic regions encounters ETEC, its effects are particularly severe for vulnerable groups. In particular, children under the age of five are especially vulnerable to acute symptomatic infections2. ETEC infections in young children are also significantly associated with long-term sequelae, including malnutrition and stunted growth3. ETEC infections are transmitted through contaminated food and water, and subsequently colonize the ileum by adhering to glycolipids and glycoproteins on the epithelial surface, enabling the efficient delivery of heat-labile and/or heat-stable enterotoxins4,5. Activation of cyclic nucleotide pathways by these enterotoxins leads to modulation of cellular ion channels and disruption of water and electrolyte balances, resulting in diarrhea6. Beyond the adhesins and toxins, additional colonization factors contribute to ETEC virulence7. These include secreted proteases belonging to the serine protease autotransporters of Enterobacteriaceae (SPATE) family8. Importantly, SPATE expression by diarrheagenic E. coli pathovars, including enteroaggregative E. coli (EAEC) and ETEC, has been associated with virulence in young children9–11.

The main physical barrier in the intestine preventing direct interaction between pathogenic microorganisms and the host epithelium is the mucus layer12. As proximity to the intestinal epithelium is important for adhesion and subsequent toxin delivery, bacteria equipped with a strategy to overcome the mucus layer may exhibit enhanced virulence. We have previously determined that ETEC secretes EatA, a member of the SPATE family that can degrade the MUC2 mucin, the main structural protein component of the intestinal mucus layer13,14. The MUC2 mucin is characterized by its central mucin domains, which are composed of multiple repeated sequences composed almost exclusively of proline, serine and threonine (PTS-tandem repeats) that serve as attachment sites for O-glycans essential for providing mucus its gel-like properties15. The central mucin domains are flanked by two terminal protein assemblies that are highly stabilized via disulfide bridges and form large oligomeric net-like structures through additional intermolecular disulfide bonds. These distinct features render the protein highly resistant to degradation by members of the commensal microbiota, and only pathogens, including bacteria and parasites, have been demonstrated to produce proteases that act on MUC2 and the oligomeric mucus gel16–18. This highlights the necessity of intestinal pathogens to adapt strategies to degrade the mucus to facilitate access to the underlying tissue to increase virulence.

In the present study, we provide a detailed mechanistic insight into how the ETEC protease EatA degrades the intestinal mucus layer. Identifying the proteolytic cleavage site and demonstrating the importance of the MUC2 protein structure for substrate recognition, a process dependent on the interaction between multiple domains. Using structural modeling and a novel transgenic human MUC2 expressing mouse model, we demonstrate how variations in the protein structure drive species specificity. Altogether, these data provide evidence that pathogenic E. coli have adapted to degrade the mucus specific to their host.

Results
The ETEC serine protease EatA acts on the C-terminal protein region of MUC2

Previous studies have demonstrated that EatA is proteolytically active on the MUC2 mucin13. To identify the region of MUC2 susceptible to cleavage we used recombinant proteins spanning different regions of the protein (Fig. 1A). When N- and C-terminal regions of MUC2 (hereafter referred to as MUC2N and MUC2C) were treated with EatA in an oxidizing environment and analyzed by SDS–PAGE under reducing conditions, activity was observed only on the C-terminal region (MUC2C). EatA reduced the apparent molecular weight from >250 to ~170 kDa, consistent with the generation of a large proteolytic fragment (Fig. 1B). The terminal regions of the MUC2 protein facilitate dimerization through intermolecular disulfide bonds. These dimers then assemble into large oligomeric structures, which are further stabilized internally by additional disulfide bonds19–21. To determine if EatA can dissociate dimerized MUC2N or MUC2C, the protease assay was performed under oxidizing conditions and analyzed by SDS–PAGE under non-reducing conditions. The results confirmed the resistance of the N-terminal, while in contrast, the C-terminal multimeric structure was degraded (Fig. 1C). To narrow down the region susceptible to proteolytic cleavage, an additional C-terminal protein (MUC2C-NC) was tested spanning the region between the von Willebrand CN domain (VWCN) and the cystine-knot (CK) at the C-terminus (Fig. 1A)20. Analysis by protein gel electrophoresis of the truncated C-terminus of MUC2 upon EatA treatment resulted in an unaltered band migration (Fig. 1D). This result indicates that the EatA cleavage site is before the VWCN domain. In support of this finding, an additional protein, MUC2C-N, was used. This protein was truncated at the C-terminus, containing only the N-terminus of the MUC2 C-terminal protein region of MUC2 (PTS3, VWCN, D4, and VWC1). Gel analysis revealed that EatA treatment resulted in a reduced band size, emphasizing that the approximate region of the cleavage site is in the PTS3-VWCN region (Fig. 1E). To confirm that the observed activity on MUC2C-N was specifically due to EatA and not a contaminating protease, a non-catalytic mutant of EatA was generated by substituting histidine 134 with an arginine. Additionally, the serine protease nature of EatA was validated by treatment with PMSF, a serine protease inhibitor. Neither the mutant EatA or PMSF-treated EatA were able to degrade MUC2C-N (Fig. 1E)13. The analyses were performed under non-reducing conditions, as the EatA and MUC2C-N protein are similar in size under reducing conditions.

The MUC2N and MUC2C-NC/MUC2C-N proteins lack the central mucin domain of the MUC2 mucin composed of multiple variable number tandem repeats (VNTR). These repeats are predominantly composed of proline, threonine, and serine (PTS) residues and are the main site of O-glycosylation. Protease activity was validated on two proteins composed of varying number of the PTS1 (nine repeats) or PTS2 (seven repeats) domains modified with either only GalNAc residues or extended to Core-1 or -2 structures with additional terminal sialylation (Fig. 1F)22. EatA treatment of the two PTS domains did not result in degradation (Fig. 1F). These results indicate that EatA has proteolytic activity specifically targeted towards the MUC2 C-terminal in the region between the PTS2 and VWCN domain. Cleavage in this region results in the degradation of the complex oligomeric mucin structures.

The EatA cleavage site is in the C-terminal PTS3 domain of MUC2

Proteases of the SPATE family have a broad substrate specificity, predominantly towards O-glycosylated secreted and cell surface proteins23. One of the first established substrates of SPATEs was IgA1, which can be cleaved in the hinge region between the Fab and Fc domains. The protease responsible for cleaving IgA1 was shown to be the immunoglobulin A protease (IgAP), which is secreted by various pathogens including Haemophilus influenza. When testing if IgA1 was an EatA substrate, we observed that EatA is not proteolytically active on IgA1 as demonstrated by gel electrophoresis (Supplementary Fig. S1A). The IgA1 hinge region is predominantly composed of serine and threonine residues and modified with three to six Core-1 O-glycans24. This region resembles the PTS repeats in MUC2, and a sequence similarity search against the human proteome with the IgA1 hinge region identified MUC2 as the protein with the highest local alignment, with 73% sequence similarity to the tandem repeat PTS3 region in the C-terminal region of MUC2 (amino acid 4329 to 4348) (Supplementary Fig. S1B). The high sequence similarity at the IgA1 cleavage site, coupled with the lack of EatA activity on IgA1, suggests that other regions of the molecule are important for substrate recognition.

To determine the cleavage site in the MUC2 C-terminus, we used mass spectrometry to detect the neo-N-terminus generated by EatA digestion of MUC2C. The method is based on α-amine specific labeling with the TMPP reagent, which retains the positive charge essential for peptide sequencing18,25. The EatA-treated MUC2C protein was labeled with TMPP prior to analysis by protein gel electrophoresis and staining with Coomassie. The 170 kDa MUC2C band was excised, and the protein was digested using trypsin. Resulting peptides were analyzed by mass spectrometry, and the TMMP-labeled dipeptide SK was identified as the neo-N-terminal formed after EatA cleavage (Fig. 2A). The SK motif appears three times within the PTS3 region, indicating that EatA acts on this region of MUC2. However, due to the repetitive nature of the sequence, the exact cleavage site could not be determined. (Fig. 2B). To refine mapping of the cleavage site, a recombinant non-O-glycosylated 34-mer peptide covering the cleavage site was synthesized corresponding to amino acid residues 4328 to 4362 (denoted as PTS3-pep). Incubation of the PTS3 peptide with EatA, and subsequent mass spectrometry analysis yielded only peaks representing the multiply charged intact peptide and no cleaved product (Fig. 2C). Together, these results localize the EatA activity to the PTS3 region but indicate that cleavage depends on determinants absent in the recombinant PTS3 peptide.

The PTS3 region of MUC2 is likely glycosylated; thus, the EatA activity may be dependent on the presence of O-glycans. To address this, we modified the PTS3 peptide using a combination of recombinant GalNAc-tranferases, introducing between 7 and 13 GalNAcs to the 34-mer peptide (Fig. 2E). Glycosylated residues were localized by mass spectrometry after digesting the modified peptide with the O-glycoprotease OgpA (Fig. 2D and Supplementary Data 1)26. Treatment of the GalNAc-transferase modified peptide with EatA for up to 24 h did not result in any cleavage product (Fig. 2E). Thus, EatA activity on the PTS3 region alone is not dependent on O-glycosylation.

We next evaluated the role of O-glycosylation for EatA activity on the MUC2C protein rather than the PTS3 peptide. To assess whether the O-glycosylation of the recombinantly GalNAc-transferase modified PTS3 peptide reflects the native cellular state, we characterized the modified sites and O-glycan composition on MUC2C. Glycopeptides were generated by desialylation and digestion with OgpA and analyzed by mass spectrometry. The results showed that MUC2C was O-glycosylated at 10 serines and threonines by either GalNAc or GalNAc-Gal, confirming a similar site occupancy as observed for the glycosylated PTS3 peptide (Fig. 2F and Supplementary Data 2). As expected for proteins produced by CHO-K1 cells, only Core-1 glycans were observed on MUC2C, and site occupancy varied, reflecting the CHO-K1 cells' limited capacity to generate extended O-glycan structures27. Stepwise O-glycan removal from MUC2C using sialidases, galactosidase and endo-N-acetylgalactosaminidase did not prevent cleavage by EatA (Fig. 2G). Both partial and complete removal of the glycan residues enhanced the protease kinetics, specifically following desialylation, which is in line with sialic acid's role in protecting glycoproteins from proteases, as demonstrated for other SPATEs28,29. These results confirm that EatA cleaves in a region of MUC2 that is highly O-glycosylated, but is not depending on O-glycans for activity or substrate recognition. Nevertheless, cleavage of MUC2C requires the intact protein, as the PTS3 peptide alone was not cleaved.

EatA activity is dependent on the structure of MUC2

The inability of EatA to degrade the recombinant peptides spanning the identified cleavage site indicates that the protease depends on the secondary protein structure and folding of MUC2 for protease activity. In addition to the protease domain, EatA contains additional smaller domains extending from the long beta-helix region of the passenger domain, which have been demonstrated by us and others to be essential for substrate recognition13,30. The frequency and size of these domains varies between SPATE classes, indicating a specific role31. The largest domain with unknown function (DUF) lies within the stalk region of EatA and is a feature common to other members of the SPATE family (Fig. 3A). To determine the function of the previously identified DUF domain for protease activity on MUC2, an EatA mutant lacking the DUF domain was tested13. MUC2C was incubated with decreasing concentrations of EatA or EatAΔDUF and analyzed by gel electrophoresis (Fig. 3B). EatA was able to degrade MUC2C even at a 1:10,000 protease to substrate ratio, while EatAΔDUF was unable to degrade MUC2C, not even when using a large excess of the protease. This highlights that the DUF domain is essential for the recognition of MUC2.

To determine how important the structure of MUC2 is for substrate recognition, we unfolded the MUC2 by reducing all disulfide bonds, followed by alkylation of the free sulfhydryl groups to prevent refolding. EatA was unable to degrade MUC2C after disrupting the disulfide bonds indicating that the protease is dependent on secondary protein structure in addition to the presence of a specific cleavage site (Fig. 3C). The interaction between the protein and the protease was demonstrated under native conditions by size-exclusion chromatography after incubation of MUC2C-N and the catalytically impaired H134R EatA mutant for 1 h prior to separation. The partial overlap between the elution of MUC2C-N and EatA indicates that the two proteins exist as a complex (Fig. 3D). In contrast, when the DUF domain was removed, the interaction was not observed (Fig. 3E). The elution profiles of the individually analyzed proteins show that the observed overlap between H134R EatA and MUC2C-N arises from their interaction. (Supplementary Fig. 2A, B). The size-exclusion chromatography results indicate an interaction between protease and substrate. To further demonstrate the direct interaction between MUC2 and EatA, the protein complex was chemically crosslinked using glutaraldehyde and analyzed by gel electrophoresis. The crosslinking formed protein complexes far exceeding the size of the MUC2C-N dimer observed around 250 kDa, indicating oligomerization between the two proteins (Fig. 3F). Mass spectrometry analysis of the protein composition of the four main bands observed on the gel confirmed the presence of both MUC2 and EatA in the three high molecular weight bands (Fig. 3G and Supplementary Data 3). To determine the interaction between EatA and MUC2C-terminal beyond the PTS3 domain, we crosslinked the mutant protease and the MUC2C-NC protein composed only of the C-terminal beyond the cleavage site (Fig. 1A). We observed that the combination still resulted in a reduction of the EatA band around 100 kDa and an increase in higher molecular weight bands. While no crosslinking was observed in the protease lacking the DUF domain (Fig. 3H). This suggests that, in addition to the PTS3, the DUF domain is required for protease interactions between the MUC2 C-terminal region after the cleavage site, which include the von Willebrand factor CN, D4, C1, and C3 domains and cystine-knot. These results demonstrate that coordinated interactions between distinct regions of EatA and MUC2 are essential for protease activity, involving the DUF domain on the stalk region of EatA and MUC2 domains C-terminal to the cleavage site.

EatA selectively acts on the human intestinal mucus layer

The MUC2 mucin is the major structural component of the mucus layer covering the epithelial surface in the small and large intestine. By creating large oligomeric mesh-like structures, it limits the penetration of the commensal microbiota residing in the intestinal lumen32. The pathogenesis of ETEC depends on adherence to intestinal epithelial cells, followed by the secretion of enterotoxins, a process that is normally limited by the protective mucus layer. To determine if EatA can degrade the mucus layer to facilitate access in vivo, we applied our ex vivo mucus measurement setup33. Colonic tissue was mounted horizontally in a perfusion chamber kept at 37 °C. The epithelium was visualized using a DNA stain, and fluorescent beads in the size range of bacteria were applied apically to visualize the top of the mucus layer. The protease was added to the apical tissue surface, and the thickness of the mucus layer was monitored over time by confocal microscopy.

Ex vivo mucus measurements of colonic tissue from wild-type C57BL/6N mice treated with EatA for up to 20 minutes had no effect on the thickness or penetrability of the mucus layer as indicated by the constant distance between the cell surface and the beads on top of the mucus (Fig. 4A, B and Supplementary Videos 1, 2). These results indicate that the mucus in mice is not degraded by EatA. Since mice are not susceptible to ETEC infection, we hypothesized that this is potentially due to an inability of ETEC to degrade the mouse Muc2 mucin. To address the role of species specificity for EatA substrate recognition, we generated a transgenic mouse model in which we introduced human MUC2. Human MUC2-expressing mice were crossbred with Muc2 knockout animals to generate the MUC2+/Muc2−/− mice, which have a humanized mucus layer. Treatment of mucus in the MUC2+/Muc2−/− mice with EatA resulted in a rapid degradation of the mucus layer, as shown by the decreased distance between the beads and the epithelial surface (Fig. 4C and Supplementary Fig. 3A). Beads were observed to settle in clusters and float away in the absence of the mucus layer (Supplementary Video 3). Mucus analysis without EatA addition demonstrates that the mucus of MUC2+/Muc2−/− mice maintained its barrier function over time (Fig. 4D, Supplementary Fig. 3B, and Supplementary Video 4). Repeated ex vivo analysis corroborated that only the MUC2+/Muc2−/− mucus is susceptible to degradation by EatA (Fig. 4E). To confirm that the transgenic mice generate a functional mucus layer that is maintained over time, we used an additional mucus measurement technique in which the thickness is measured using a penetrating needle while the mucus surface is visualized by beads34. Both initial mucus thickness and growth over time were similar in C57BL/6N and MUC2+/Muc2−/− mice; however, only mucus produced by MUC2+/Muc2−/− mice was degraded by EatA (Fig. 4F).

To further validate the EatA observations in the humanized mucus mouse model, we repeated the analysis using human colonic biopsies. Biopsies were obtained from control patients without a history of intestinal disease and analyzed in the same way as the mice. EatA treatment of the mucus resulted in degradation of the layer, while the mucus layer remained intact when treated with PMSF inhibited EatA (Fig. 4G–I and Supplementary Videos 5, 6). These results demonstrate that EatA selectively degrades the human MUC2 mucin, but not murine Muc2.

MUC2/Muc2 PTS3 sequence variation dictates species-selective mucus degradation

The cleavage site region comparison between human MUC2 and mouse Muc2 shows a clear variation in both length and amino acid composition (Fig. 5A). The identified cleavage site in MUC2 is present twice in Muc2 with the same residues in the P1-P1’ position (P/S) but without a lysine in the P2’ position. The lack of a positively charged residue in this position potentially impairs substrate recognition. We assessed EatA activity on the mouse C-terminal of Muc2 using a recombinant protein spanning the same region of the protein as the human MUC2C-N protein (Fig. 1A). Western blot analysis targeting the N-terminal FLAG-tag demonstrates that only human MUC2 is degraded while mouse Muc2 remains intact (Fig. 5B). To determine if the mouse Muc2C-N binds to EatA we crosslinked the two proteins with increasing concentrations of glutaraldehyde. This combination resulted in a decreased EatA band intensity, indicating complex formation, which was dependent on the presence of the DUF domain (Fig. 5C). In addition to humans, pigs, cattle, sheep, and dogs are susceptible to ETEC infection, especially during the neonatal period35. By contrast, mice and rats are not. When comparing the amino acid sequences of the PTS3 domain in the affected species to that of rodents, the major variation was observed in the length of the region (Fig. 5D). While the terminal regions are generally conserved, an ~18 amino acid insertion is present in both mouse and rat Muc2. These additional residues will affect the spatial arrangement of the cleavage site region and potential accessibility for the protease. These results indicate that EatA activity is tailored to degrade the mucus of its specific host species. Providing an insight into one of the determinants of why human colonizing ETEC strains are ineffective in mice.

Model of the EatA binding to MUC2

To gain insight into the molecular mechanism of EatA-MUC2 recognition and cleavage, we performed docking simulations using a model based on the MUC2 C-terminal homodimer (vWCN–D4–VWC1) cryo-EM structure (PDB: 7QCN) and an AlphaFold2 model of EatA (UniProt: Q84GK0)20. The AlphaFold2 EatA model showed very high confidence except for the DUF domain, the region suggested to drive the interaction between the two proteins. The DUF prediction presented low confidence areas in the regions connecting to the passenger domain. The predicted alignment error (PAE) between the DUF domain and the rest of the molecule was high, indicating low confidence in the relative position of the domain, typical for flexible regions. Consequently, the docking simulations were conducted using the isolated DUF domain first, then extended with the remaining part of EatA to generate a model of the full complex with the MUC2 C dimer. Models with major steric impediments were removed, whereas minor clashes were tolerated and subsequently relaxed by (5 ns) molecular dynamics simulation. The best-scoring complexes showed virtually the same interaction mechanism, with the DUF domain inserted into the cavity formed by the two MUC2 VWCN-D4 chains of the homodimer (Fig. 6A, B). This region of MUC2 is not protected by N- or O-glycans that could affect the dimer formation and present a hydrophobic region between the VWCN(B) and the VWD(B) domains, where the hydrophobic residues from the tip of the DUF domain are predicted to interact. Notably, the N-terminus of the VWCN from chain A of the MUC2 dimer points towards the catalytic domain at ~45 Å from the catalytic serine. Consistent with this arrangement, the EatA-MUC2 interface closely resembles the IgA1 interface described for IgA protease from Haemophilus influenzae (Fig. 6C)36.

The MUC2 PTS3 was modeled by extending it from the VWCN domain until it reached the EatA active site based on the observation that PTS domains extend linearly due to the extensive O-glycosylation (Fig. 6A)37. According to the model, the most C-terminal of the three putative cleavage sites (Pro4352-Ser4353) is too close to the VWCN domain to be cleaved. The first cleavage site (Pro4337-Ser4338) cannot be excluded, but it is the second site (Pro4344-Ser4345) that matches the distance to the active site. Therefore, the Pro4344 was manually placed in the S1 pocket and Ser4345 in S1’ (Fig. 6D, 0 ns). O-glycans were added to all serines and threonines in PTS3, including Ser4345 in P1 position, and the model was analyzed by molecular dynamics to evaluate the docking prediction, determine the stability of the complex, and to improve the model. Obvious interactions between glycans and the protease domain were not observed, indicating that glycans near the cleavage site are tolerated without compromising activity. The molecular dynamics simulation disclosed that the interaction between the catalytic domain and the PTS3 is mainly stabilized by Lys4346 in P2’, forming a salt bridge with Asp120 in S2’ (Fig. 6D, 50 ns). Homology modeling of the mouse PTS3 using the human structure as a template indicates that all lysines were positioned too far from the active site (Fig. 6D, 0 ns). Mouse Asp3794 occupied a position equivalent to human K4345 and was therefore modeled inside the S2’ pocket. However, the MD simulation showed that after 10 ns, that Asp3794 had moved out of the pocket and instead pointed toward the solvent (Fig. 6D, 10 ns).

The EatA-MUC2 model only suffered small rearrangements during the simulation; the interaction surfaces involving the DUF, passenger and protease domains were preserved (Supplementary Fig. 4A, B). The current model thus supports EatA recognition of a lysine in position P2’ and cleavage of the PTS3 not closer than ~15 amino acids N-terminally of the VWCN domain in an O-glycosylation-independent manner (Supplementary Video 7).

Methods
Mouse and human subject details
All experimental procedures involving mice were performed in accordance with protocols approved by the laboratory animal ethics committee guidelines at the University of Gothenburg (ethical permit: 2285/19). Mice were housed in a specific pathogen-free facility with access to standard chow and water ad libitum with a 12 h light/dark cycle, maintained at 22 °C ± 1 °C at 40–70% humidity. All mouse strains used were on a C57BL/6N background originating from Taconic. The Muc2 knockout strain has been previously described50. The MUC2 transgenic mice were generated via a pronuclear injection of a BAC clone (RP13-870H17) containing a segment of chromosome 11p15.5, including MUC2 and MUC651. The MUC6 gene was silenced by introducing a TetR cassette52. The mice expressing human MUC2 were crossbred with the Muc2 knockout to generate the MUC2+/Muc2−/− strain. All animals used for experiments were in the age range between 8 and 12 weeks, and both males and females were included. Approval for the study of patient intestinal biopsies was granted by the human research ethical committee at the Sahlgrenska University Hospital, Gothenburg and written informed consent was obtained from all study subjects. Biopsies were obtained from the distal colon of patients undergoing colonoscopy with no history of inflammatory bowel disease. Only macroscopically normal tissue was included from patients undergoing cancer screening due to occult blood in stool without signs of intestinal disease.

Expression vectors, constructs, and recombinant proteins
The MUC2 N- and C-terminal recombinant proteins were expressed in CHO-K1 cells (CCL-61, ATCC) and purified from spent media by size-exclusion chromatography on a Superose 6 HR 10/30 column53,54. Expression and design of the truncated MUC2C protein MUC2C-CN is fully described in detail20. The pcDNA3.1(+) plasmid vector for the MUC2C-N construct was composed of an N-terminal Igk signal sequence followed by a HIS-tag, a FLAG-tag site and the MUC2 sequence between amino acid 4256 and 4803 (GenBank: AZL49145.1) with the cysteine residue at position 4786 mutated to an alanine. The mouse Muc2C-N vector was composed similar to the MUC2C-N including the Muc2 sequence between amino acid 3691 and 4248 (UniProt: Q80Z19v2023_01) with the cysteine residue at position 4231 mutated to an alanine. CHO-S cells (R80007, Life Technologies) cultured in serum-free FreeStyleTM CHO growth medium (Gibco) were used for transient protein expression for 72 h. The spent media was concentrated, and buffer exchanged by tangential flow filtration and the protein was subsequently purified by immobilized metal affinity chromatography on a 1 mL HisTrap™ HP column (Cytiva) using an Ettan LC system (Amersham Bioscience). The eluted protein was concentrated and buffer exchanged to 20 mM HEPES, 150 mM NaCl, pH 7.4 using 50 kDa molecular weight cut-off filters (Vivaspin, Sartorius). The mucin tandem repeats VNTR-PTS1 (nine repeats, position in MUC2: 1406–1554) and VNTR-PTS2 (seven repeats, position in MUC2: 1916–2064) were expressed and purified from genetically-engineered HEK293 cells allowing the control of the addition of O-glycan modifications, limited to Tn (KOC1GALT1) or STn antigen (KOCOSMC/KI ST6GALNAC1) as described in detail22. EatA and the non-catalytic H134R mutant were cloned into a pBAD vector, expressed in E. coli and purified from concentrated spent LB media by ion exchange chromatography followed by size-exclusion chromatography as described in detail14. Design and purification of the EatA protein in which the DUF domain spanning amino acid residue 514 to 616 was replaced by eight HIS residues are described in detail previously13. The 34-mer peptide spanning the VNTR-PTS3 between the amino acid 4328 to 4362 of MUC2 was synthesized (JPT Peptide technologies) and N-acetylgalactosamine modification were introduced using recombinant GalNAc-Ts transferases 2, 3, and 755.

Protein gel electrophoresis and Western blot analysis
MUC2C was deglycosylated with β1–3 Galactosidase (P0726, New England Biolabs), α2-3,6,8 Neuraminidase (P0720, New England Biolabs) and O-glycosidase (P0733, New England Biolabs) either as a single reaction or sequentially. All reactions were set up according to the manufacturer’s instructions without the addition of denaturing buffer and performed overnight at 37 °C. For the protease assays, recombinant MUC2 was combined with EatA and incubated at 37 °C for 1 h on a thermal shaker (300 rpm) if not otherwise stated. The protease activity was quenched by the addition of SDS–PAGE sample buffer (4% SDS, 125 mM Tris-HCl, pH 6.8, 30% (v/v) glycerol, 5% (v/v) bromophenol blue, supplemented with 200 mM DTT for reducing conditions) and heated for 5 min at 95 °C. Protein gel electrophoresis was performed on gradient NuPAGE Bis-Tris mini gels (Invitrogen) with a 4–12 or 4–15% polyacrylamide gradient and stained by Coomassie (Imperial, Thermo). The protein molecular weight marker used was either PageRuler pre-stained (26619, Thermo Fisher), Dual Color (1610374, Bio-Rad) or HiMark pre-stained (LC5699, Invitrogen). Western blot analysis was performed after transfer to PVDF by electroblotting for 10 min at 1.3 A/25 V (Trans-blot Turbo, Bio-Rad). Blots were blocked with 5% BSA in PBS-T for 1 h, probed with anti-FLAG antibody M2 (1:1000, F3165, Sigma, validated for western blot) for 2 h, and detected with anti-mouse IgG1-HRP (1:2000, 4030-05, Santa Cruz). Proteins on blots were visualized by chemiluminescence after the addition of ECL ultra western HRP substrate (Millipore) and the band intensities were analyzed using Image Lab (v6.1, Bio-Rad).

Protein interaction analysis
To determine complex formation, the non-catalytic EatA variant H134R or EatA lacking the DUF domain were incubated with MUC2C-N at an equal molar ratio for 1 h at 37 °C. The reaction mixture was applied to a Superdex 200 size-exclusion column (Cytiva) and eluted isocratically with 20 mM Tris, 150 mM NaCl, pH 7.4, monitoring UV at 215 and 280 nm using an Ettan LC system (Amersham Bioscience). Eluting fractions were analyzed by SDS–PAGE as described. For chemical protein crosslinking analysis, EatA H134R was incubated with MUC2C-N or Muc2C-N as above at a concentration of 0.5 µg/µL. Glutaraldehyde solution was added to a final concentration of 0.1, 0.2, or 0.4% and the reaction was terminated after 20 min by the addition of Tris buffer to a final concentration of 50 mM. Crosslinked protein samples were analyzed by SDS–PAGE and visualized by Coomassie (24615, Thermo).

Sample preparation and mass spectrometry analysis
Trimethoxyphenyl phosphonium (TMPP) protein labeling was performed for the identification of the newly generated MUC2C-N-terminus after EatA cleavage. The protease reaction was quenched by the addition of 1 mM phenylmethylsulfonyl fluoride, the protein solution was titrated to pH > 8 and < 8.5 with Na2HPO4 and TMPP was added to a final concentration of 100 µM. The reaction was incubated at room temperature for 1 h and subsequently quenched by the addition of 0.1 M hydroxylamine and analyzed by SDS–PAGE. To determine the protein composition and identify TMPP or O-glycan modified sites, bands of interest were excised from the gel and destained, proteins were reduced, alkylated, and digested using trypsin (V5111, Promega) overnight at 37 °C, as described in detail18. For glycopeptide analysis, the protein was first desialylated with a combination of two broad-specific neuramidases (SialEXO, Genovis) and digested into peptides with a Core-1 specific glycoprotease (OpeRATOR, Genovis) in 25 mM ammonium bicarbonate, incubated overnight followed by trypsin for 2 h, all at 37 °C. The extracted peptides were dried under vacuum and dissolved in 0.2% trifluoroacetic acid and analyzed by mass spectrometry. Mass spectrometric analysis was performed by LC/MS/MS using an Easy-nLC 1200 (Thermo) coupled to a HF-X Orbitrap mass spectrometer (Thermo). Peptides were separated using in-house packed columns (150 × 0.075 mm) packed with Reprosil-Pur C18-AQ 3-μm particles (Dr. Maisch). Peptides were separated using a 5 to 35% gradient (A 0.1% formic acid, B 0.1% formic acid, 80% acetonitrile) over 30 minutes. Full mass spectra were acquired over a mass range of minimum 350 m/z and maximum 1600 m/z, with a resolution of at least 60,000 at 200 m/z. The 12 most intense peaks with a charge state ≥ 2–5 were fragmented by higher-energy collision dissociation (HCD) with a normalized collision energy of 27%, and tandem MS was acquired at a resolution of 17,500 and subsequently excluded for selection for 20 s. Peak lists were generated from raw mass spectrometry data using MSConvert (v3.0) and searched using Mascot (v2.2.04, Matrix Science) for peptide identification and Byonic (v2.10.21, Protein Metrics) for glycopeptide identification. Mascot was used for standard protein identification with the following settings: fixed modification carbamidomethylation (C), variable modification oxidation (M), enzyme trypsin with a maximum number of two missed cleavages, a mass tolerance of 10 ppm for precursor mass and 20 ppm for fragment ions. For newly generated N-termini identification, the search settings were adapted to include TMPP (N-terminal, Y and K) and semi-trypsin for enzyme specificity. All peptide searches were performed against an in-house protein database (MucinDB v2), including the recombinant proteins and EatA, only peptides at 1% FDR were considered. Byonic parameters were set to semi-specific for C-terminal cleavage trypsin (KR) and N-terminal OpeRATOR (ST) with a maximum number of missed cleavages up to two, mass tolerance of 10 ppm for precursor mass and 10 ppm for fragment ions. These searches were performed against the mucinDB, and a list of 70 common human O-glycans was selected as the set of considered glycan modifications; only glycopeptides and peptides at 1% FDR were considered. The unmodified or GalNAc-modified 34-mer PTS3 peptide (JPT Peptide Technologies) was dissolved in PBS and further diluted with 0.2% trifluoracetic acid to 2 µM for analysis by LC-MS/MS using the same instrument settings as described. Protease cleavage was validated by combining 100 µM PTS3 with up to 1 µg EatA, incubated at 37 °C overnight and up to 24 h. The reaction was quenched by the addition of 0.2% trifluoracetic acid and analyzed by LC-MS/MS.

Ex vivo analysis of EatA mucus degradation
Ex vivo mucus analysis34,56 of EatA activity was performed as following after euthanasia, mouse distal colon was dissected and collected into ice-cold oxygenated (95% O2 and 5% CO2) Kreb’s buffer (116 mM NaCl, 1.3 mM CaCl2, 3.6 mM KCl, 1.4 mM KH2PO4, 23 mM HCO3−, and 1.2 mM MgSO4). The colon was opened along the mesenteric border, fecal material was removed, the longitudinal muscle tissue was stripped off, and the tissue was mounted in a horizontal perfusion chamber34. Human sigmoid colon biopsies were collected into Krebs’ buffer and mounted in a horizontal perfusion chamber as for mouse tissue. Tissue was maintained at 37 oC throughout the duration of each experiment and was perfused basolaterally with Kreb’s glucose buffer (Kreb’s buffer + 10 mM D-glucose, 5.1 mM sodium L glutamate, and 5.4 mM sodium pyruvate) with Kreb’s mannitol buffer (Kreb’s buffer + 10 mM D-mannitol, 5.1 mM sodium L glutamate, and 5.4 mM sodium pyruvate) added to the apical chamber. EatA activity on colonic mucus was evaluated by confocal microscopy to assess mucus penetrability, and by measurements of mucus thickness over time. For the penetrability assessment, human and mouse tissue were counterstained with Syto9 (10 µM; Thermo Fisher) for 5 min, and the mucus layer was visualized by the addition of 1 µm FluoSpheres™ carboxylate-modified microspheres (625/645; Thermo Fisher). EatA (10 ng/mL) was added to the apical Krebs' mannitol buffer, and the integrity of the mucus layer was monitored on an upright LSM900 confocal microscope (Carl Zeiss) using a Pan-Apochromat x20/1.0 DIC 75 mm lens (Carl Zeiss). Following the acquisition of an initial scan before the addition of the protease, z-stacks were acquired every 3–5 min for up to 45 min using Zen Blue software (version 3.1; Carl Zeiss). Control analyses were performed on paired mouse or human tissue treated with the non-catalytic EatA variant H134R.

To quantify mucus integrity, beads and tissue surfaces were mapped to isosurfaces using Imaris (version 9.5; Bitplane)52. Data regarding the bead position in relation to the tissue surface over time was then extracted and analyzed to generate mucus thickness and normalized bead positional data (Prism version 10.3.1; Graphpad). For the thickness measurements, the mucus surface was visualized by the addition of 10 µm black FluoSpheres™ carboxylate-modified microspheres (Thermo Fisher) to the apical chamber. The thickness of the mucus layer was measured every 15 min for 60 min using a micropipette mounted to a micromanipulator and viewed through a stereomicroscope. The thickness of the mucus layer was obtained by measuring the distance between the mucus surface and the epithelial surface. Following the 60 min measurement, 10 ng/mL EatA was added to the apical chamber, incubated for 15 min followed by one additional measurement of the mucus thickness.

In silico docking and molecular dynamics
The EatA-DUF domain structure was predicted using AlphaFold257. The cryo-EM structure of MUC2 VWCN-D4-VWC1 (PDB: 7QCN) was modified with Modloop58 in order to complete the missing loops. The glycosylation sites were modified, ensuring that all of them were occupied with two sugar molecules, two N-acetylglucosamines (GlcNAcβ1-4GlcNAc) on N-glycosylation sites (Asn4389, Asn4453, Asn4567, Asn4578, Asn4703, and Asn4738) and galactose and N-acetylgalactosamine (Core-1: Galβ1-3GalNAc) in O-glycosylation sites (Thr4632 and Thr4633). The simplified glycans circumvented the risk of masking interaction surfaces due to their high flexibility, not being considered by the docking programs, while blocking the regions where they are directly attached. The in-silico docking was performed based on shape complementarity using PatchDock59 and further refined and ranked with FireDock60. The EatA structure without signal peptide and β-barrel predicted by AlphaFold2 and a model of MUC2 from the VWCN domain to CK were aligned to the docking models to check their feasibility20. The DUF domain was replaced by the full-length EatA in the best-ranked model. The small clashes were solved by a short molecular dynamics simulation (MD). The complex was prepared for MD using the Glycan Reader and Modeler tool from CHARMM-GUI61. It was solvated with the TIP3P water model in a cubic simulation box under periodic boundary conditions, neutralized and adjusted to 150 mM NaCl. The generated CHARMM36m force field parameters were used in GROMACS (v2022.4)62 to perform the energy minimization, equilibration, and production simulations at 303.15 K. The production was simulated for 5 ns. The PTS3 region of MUC2 chain B was modeled in Coot63, extending it from the VWCN N-terminal residue towards the EatA active site. A mouse PTS3 model was constructed by replacing the human PTS3 sequence in the modeled structure with the corresponding mouse sequence (UniProt: Q80Z19, residue 3760–3812) and subsequently expanded to include all lysine residues. The PTS was glycosylated using GLYCAM, and the resulting model was manually curated in order to remove ring-piercing artifacts. The lateral chains of affected residues were rebuilt, and glycans involved in ring penetrations were removed. A 50 ns MD simulation using CHARMM-GUI and GROMACS. The simulation analysis and figure generation were performed using PyMOL (v2.5) and UCSF Chimera (v1.19). The PTS3 of MUC2 chain A was modeled by applying symmetry operations to the chain B PTS3 only for illustrative purposes.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes the study of the target proteolytic site for the EatA protease from enterotoxigenic Escherichia coli on the MUC2 mucin. Relevant questions are where in MUC2 the protease cuts, whether glycosylation is required for cleavage, and how the protease recognizes MUC2. Overall, the information presented appears reliable and interesting. However, some improvements to the experiments and presentation are necessary. For example, the proper controls (individual proteins alone) are not presented for the gel filtration. Moreover, the story seem relatively straightforward, but the text is overly wordy and poorly organized at points. As noted below, the part describing the modelling of the interaction should be drastically shortened.\nAdditional comments:\nLine 66: “porcess” should be “process”\nLine 259: “EatA was only active on the protein C-terminal, resulting in protein fragment of approximately 170kDa (Fig. 1B).”\nFirst, no band of size 170 kDa is labeled in Fig. 1B. Second, the significance of this size is not evident from the text. What was the starting size of the protein fragment? What does it mean about the possible cleavage site if a fragment of 170 kDa is generated? What about the complementary fragment resulting from the cleavage?\nLine 262: The issue of the reducing vs. non-reducing conditions is not clear. There is the redox status of the reaction during which the proteolysis is taking place, and then there is the redox status of the gel used to assess cleavage. These are two separate things. Whether each of them was oxidizing or reducing should be specified.\nLine 267: Here the span of the C-terminal fragment chosen is specified and named (MUC2C-NC), but the first fragment used (line 259) was not specified or named (was it MUC2C?).\nLine 273: “only the active form of EatA” This statement is confusing, since no reference to any other form of EatA was presented in the text or used in the previous experiments. The mutant needs to be properly introduced.\nFigure 1: The legend says that the lines to the sides of the gel are “color coded,” but what is the code? The legend says the lines represent the position of the indicated protein, but the protein is not in fact indicated.\nLine 292: “intramolecular” disulfide bonds required for dimerization? Do the authors mean “intermolecular”?\nLine 322: “indicating the potential for multiple cleavages” How can the authors distinguish between multiple cleavage sites and ambiguity in site identification? Just because the SK sequence appears multiple times does not mean that all such sites are cleaved. As the authors point out later in the text, other factors in addition to the local sequence determine the actual site of cleavage.\nLine 323: “To confirm the cleavage site” The experiment with the peptide does not actually end up confirming the cleavage site, so best not to start the sentence this way. Also, it should be specified that the synthetic peptide is not glycosylated.\nLine 327: The topic is the peptide (which is not glycosylated; see previous note), so when the text says, “As this region of the protein is likely to be glycosylated...” the reader is confused for a second until he/she realizes that the authors have jumped to the full protein. In fact, the whole logical progression of this section could be improved. The glycan analysis of MUC2 produced in CHO cells is stuck in the middle. In this reviewer’s opinion, it would be more logical to say, “... no cleaved product. We hypothesized that EatA may required O-glycans for activity. We therefore used recombinant GalNAc transferases to add GalNAc to the PTS3 peptide... [describe analysis of the resulting degree and variability of glycosylation]. However, GalNAc addition did not render the peptide cleavable by EatA. To confirm that the degree of glycan modification resembles that of MUC2 produced in CHO cells... Moreover, deglycosylation of MUC2 enhanced proteolysis, indicating that differences in glycosylation between the peptide and native MUC2 are unlikely to account for the differences in cleavage susceptibility. Therefore, another determinant, present in MUC2 but absent from the PTS3 peptide, appears to facilitate cleavage. [start a new paragraph here] EatA has additional domains, outside the catalytic core, which may contribute to substrate recognition. A requirement for binding via these domains to segments of MUC2 that are absent from the PTS3 peptide may explain the inability of EatA to cleave the peptide. To test this hypothesis...”\nLine 385: reducing disulfides will likely destroy both secondary and tertiary structure, so best to say, “To determine whether structure is important for substrate recognition, we unfolded...”\nLine 389: No need to invoke the PTS3 peptide here. The authors already determined that the peptide lacks important substrate recognition sites.\nLine 392: To establish interaction by gel filtration, it is absolutely essential to show the elution profile of each protein alone as well as the mixture of both.\nLine 395: The authors cannot conclude anything about whether the interaction is transient or long-lived. It could be that only a fraction of the recombinant EatA is capable of binding and the rest is defective but binds stably (if indeed there is even any binding at all; see previous note).\nLine 416: The title of the figure is a bit odd. Yes, EatA and MUC2 domains are involved in substrate recognition, and unfolding of the domains destroys recognition, but domains and folding in general don’t “drive” recognition. Also, again the little lines on the sides of the gels are unlabeled and unexplained.\nLine 432: “limits” should be “limit”\nRegarding the supplementary videos, this reviewer had a difficult time extracting any information from them. Also, at least one of them has repetitive patterns of red blobs appearing and disappearing that look highly artifactual. What is the origin of the uniform spacing seen in these rows of blobs?\nLines 429-486: This paragraph is two pages long. Can it be broken into bite-sized pieces, for example at the beginning of the colon biopsy section?\nLine 471: again, “validated” is not the correct word in, “We validated EatA activity on the mouse C-terminal of Muc2...” since there was no activity to validate.\nLine 476: “reduction of the EatA band” is not clear. Decrease in intensity of the band?\nLine 512: In the opinion of this reviewer, the whole section on the modelling should be drastically shortened and focused. The spatial relationship between putative DUF binding sites on MUC2 and the cleavage site is an interesting question, and the docking is useful for hypothesis generation. However, the docking and related analyses do not “elucidate the molecular mechanism” or justify the mass of detail presented in Fig. 6. One short paragraph and a simple figure discussing/illustrating the spatial arrangements of the relevant parts of the proteins and their proposed interaction modes would be much more effective at this point. (It should also be stated or shown what the differences were between the ranked models.)", "reviewer_2": "General comments:\nThe experimental sections of the manuscript are well presented in an easy-to-follow manner. However, the English could use improvement.\n1. There are several fragmented sentences in the publication, as such it could use revision.\n2. Accordingly, terminus = noun, terminal = adjective; the 2 are not interchangeable.\nIntroduction:\n35-36: The sentence would read better as “In particular, children under the age of five are especially vulnerable to acute symptomatic infections”.\n38: Spacing surrounding the punctuation following “..stunted growth..”\n40: change “and promote” to “..the epithelial surface, enabling the efficient delivery…”\n43: Should use “Beyond the adhesins..” rather than “Besides the adhesins”\nComment:\nThe first paragraph of the introduction talks of the vulnerability and risks to children. As a general question here, what is known about the mucus layer in children? There appears to be a jump in reasoning, without addressing the possible gaps in knowledge.\n66: typo: “process”\n70: might be better as “..the mucus specific to their host”, rather than “its host”.\n76: ad libitum should be in italics.\n77: double space following “cycle”\n77: “All mouse strains..” rather than “mice strains”\n77: “were derived from a C57BL/6N”\n78: via in italics.\n80: General grammar – when giving further examples, a comma should be used before the examples provided. I.e. “..segment of chromosome 11p15.5, including MUC2 and MUC6.”\n98: “serum-free”\n104: “cut-off”\n105: Are there expected sizes / molecular weights of the constructs?\nThis will tie into a question later in the manuscript regarding the decoration with more elaborate sugars, but demonstration of a lower molecular weight.\n105: “genetically-engineered”\n107: “non-catalytic”\n113: Should use the long form for JPT, as it only seems to be used twice in the manuscript, and lacks the need to be abbreviated.\n121: “For the protease assays, recombinant..” a comma is needed here.\n122: rpm is generally not in all caps (RPM)\n127: abbreviation MW not introduced.\n129-130: Please include the transfer conditions, as mucins are not the easiest to transfer quantitatively.\n130: Trans-blot Turbo?\n131: This section would benefit from antibody concentrations.\n133: “..the band intensities were analyzed” not “was”.\n138: Sentence fragmented or information lacking. The “reaction mixture” was applied to the superdex column?\n150: N-terminus\n161: the word “resolved” is often used in place of “dissolved”.\n162: Mass spectrometric analysis\n165: (Dr. Maisch), this either needs more information or to be removed.\n166: “Peptides.. over 30 minutes” rather than “in 30 minutes”\n169: Define HCD.\n170: Clarification regarding “subsequent excluded for selection for 20 seconds”\n173-176: This could use some organization / clarification.\n177: semi trypsin? Please clarify.\n178: “..performed using”, rather than “performed towards”\n181: missed cleavages\n183: The searches ”were” performed.\n183-184: Needs work/clarification.\n184: >300 were “retained / analyzed”?\n185: “resolved” versus “dissolved”\n195-197: The sentence needs clarification / better punctuation.\nGeneral comment for the Ex vivo analysis of EatA mucus degradation: This section could be simplified with subheadings for “Mucus penetrability” and “Mucus integrity quantification”\n228: Could you specify the number of sites occupied / involved?\n229: N-acetylglucosamine, capital A not needed.\n232: “while blocking”\n237: “full-length”\nResults:\nMain concerns regarding the results section:\n1) Line 259 says digestion of C-terminus results in a fragment of 170kDa. The problem here is that the 170 kDa fragment is present in MUC2C without EatA (image to the left). As such, the statement doesn’t make sense, as this does not appear to represent a digestion fragment. The manuscript should make note of the size shift in the MUC2C, rather than the presence of a digestion product.\n2) Figure 1C. The red bar indicates the presence of the EatA protein. However, in the 3rd lane (MUC2N + EatA), when EatA is added, there is no band detected by Coomassie blue. This is an issue with the manuscript that needs to be resolved.\n3) In Figure 1A, the limits of the MUC2C-N construct need to be corrected, as I don’t believe they span the correct domains (VWC1 should be included in the shaded rectangle under the image).\n4) Is there an explanation for the differences in the size of the differentially glycosylated PTS domains? Specifically, why does the addition of a larger glycan result in a reduced size / increased electrophoretic migration? Do you hypothesize an effect on molecular structure or blocking of neighboring glycosylation sites?\n5) Regarding the modelling, we’re assuming a 1-to-1 interaction in the in vitro studies with expressed mucins, as the cysteines are removed, and thus no dimerization. Is there an explanation why in the modelling the models demonstrate the interaction of EatA with 2 MUC2 chains?\n6) This is related to a statement on line 483, indicating that the additional 18 amino acid residues in the rodent Muc2 “will affect the spatial arrangement of the cleavage site region and potential accessibility for the protease.” Without direct evidence of this, it should be modified to “will potentially affect…”.\n7) Otherwise, it would be beneficial to see a modelling/docking interaction of the EatA protein with the rodent Muc2, to demonstrate that the catalytic domain would no longer have access to the cleavage site.\n8) Supplemental data Figure 1: Resolution of the image/text is poor, renders the figure unreadable.\n259: C-terminus, not C-terminal\n261: intermolecular, rather than intramolecular (for dimerization)\n263: “resolve” should be switched to “dissociate / digest”\n263: N-terminus and C-terminus, not N-/C-terminal\n268: C-terminus\n271: C-terminus (last time pointing it out, but there are other occurrences in the manuscript)\n285-286: This sentence requires clarification in the context of the study. This implies that the enzyme will not be able to cleave native MUC2, while EatA digestion of native MUC2 is demonstrated later in the paper.\n288: Terminus\n306: Pathogen name incorrect.\n303-316: This section introduces a characterized target of SPATEs (IgA), shows similarity of IgA hinge region to MUC2, and goes on to show that EatA does not degrade IgA. What is needed in this section is an explanation of why EatA is active on MUC2 PTS which resembles the hinge region. This needs to be tied back in, as otherwise is leaves the reader confused.\nIf the IgA hinge region resembles TR-PTS3, and EatA does not digest IgA, then why did you continue to investigate the activity of EatA on the PTS region? This section needs to be re-worked to facilitate easier reader comprehension.\n321: Punctuation would help: “TMMP-labelled peptide, SK, was identified”, or even to say dipeptide, which would better explain the “SK”.\nThe supplementary tables could be better organized / titled to help the reader.\n349: “Generally” not “general”.\n358: Mass spectrum (as it is singular)\n364: spectrum\n377: Why are there parentheses around DUF?\n388: dependent, not depended\n391: “..of and MUC2C-N”, remove the “and”\n394: Punctuation will help to separate ideas sentences. “…DUF domain was lacking, the interaction…”\n396: sensitive “to” dilution.\n408: This suggest(s) that..\n409: “between the region C-terminal to the cleavage site..” would give better context, rather than “beyond”.\n412: “..mucin and EatA is essential” not “are essential”\n413: For clarity, the DUF interaction domain is C-terminal to the cleavage site, it is not correct to say that it is “beyond”, as this lacks context.\nFigure 3C: While there is no decrease in size of the principal MUC2 band, there is a reduction in intensity (it seems) and there is an increase in the intensity bands between 150-250kDa.\n419: This would represent “decreasing” concentrations, as the amount of enzyme to MUC2 is decreasing from left to right in the image. Or its better just to say “treated at varying protease to substrate ratios”.\n431: Punctuation: it would read better as “..mesh-like structures, it limits the penetration of the…”\nLack of continuity in the use of “mouse Muc2” and “murine Muc2”.\n480: “affected species”, not “effected species”\n485: determinants\n501: unpaired Student’s t-test?\n505: determines species specificity\n510: Error in description of the figure. Grey = unaffected (mouse / rat), Red = affected.\n522: It should likely be “clashes”, rather than “crashes”.\n556: Please clarify, central region between (not “among”) the DUF domain and the C-terminus?\n558: suggests\n565: got worse?, decreased?\n566: remove “so”, use “as a result”.\n570: over time\n588 + 590: “shown”\n593: “..in size with respect to the central figure”\n594: Interacting residues?\nDiscussion:\nConcerns:\n1) Lack of discussion regarding interaction with either monomers or dimers / oligomers of MUC2.\n613: Remove “resolve”, use “degrade” or “dissolve”\n618: “localized in a mucin domain”\n621: Punctuation for readability. A common would help this sentence.\n628: grammar – glycan structures\n631: “alone, suggesting”\n632: “Previously, a putative binding domain”\n634-635: discussion of binding in the cleft of homodimers (modelling data), but these were not present in the digestion studies. This needs to be addressed here in the discussion.\n635-638: Punctuation for readability. Need commas to separate ideas or sections.\nData availability: The identifier given does not work. PXDXXXX. Need to replace the place-holder in the text.\nSupplementary tables.\nWhen possible, headers in the tables should give more information, such as Band 1, rather than Exp1.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the Editor and Reviewers for their positive review of our manuscript and constructive feedback. Please find below our point-by-point responses to each of the Reviewers’ comments.\nRevisions made to the manuscript and supplemental data are indicated in the respective documents in red.\nReviewer #1:\nThis manuscript describes the study of the target proteolytic site for the EatA protease from enterotoxigenic Escherichia coli on the MUC2 mucin. Relevant questions are where in MUC2 the protease cuts, whether glycosylation is required for cleavage, and how the protease recognizes MUC2. Overall, the information presented appears reliable and interesting. However, some improvements to the experiments and presentation are necessary. For example, the proper controls (individual proteins alone) are not presented for the gel filtration. Moreover, the story seem relatively straightforward, but the text is overly wordy and poorly organized at points. As noted below, the part describing the modelling of the interaction should be drastically shortened.\nWe thank Reviewer #1 for their thoughtful comments and efforts. In the revised manuscript, we have addressed the concerns raised by improving the clarity and conciseness of the text, adding appropriate controls for the gel filtration analysis and shortening the interaction modeling results section. Below, we provide a point-by-point response to each comment.\nLine 66: “porcess” should be “process”\nCorrected in the revised manuscript\nLine 259: “EatA was only active on the protein C-terminal, resulting in protein fragment of approximately 170kDa (Fig. 1B).” First, no band of size 170 kDa is labeled in Fig. 1B. Second, the significance of this size is not evident from the text. What was the starting size of the protein fragment? What does it mean about the possible cleavage site if a fragment of 170 kDa is generated? What about the complementary fragment resulting from the cleavage?\nWe agree with the reviewer that the results description lack sufficient detail required for correct data interpretation. In the revised manuscript for clarity, we have replaced the gel electrophoresis image by a repeating the analysis on a gradient gel with a higher resolving power in region of interest, as used for all other analysis throughout the paper. The text has been altered to include the mW of the initial noncleaved molecule and describes the significance of the cleavage product upon EatA treatment, and now reads as following:\nLine 262- 266\n“When N- and C-terminal regions of MUC2 (hereafter referred to as MUC2N and MUC2C) were treated with EatA in an oxidizing environment and analyzed by SDS–PAGE under reducing conditions, activity was observed only on the C-terminal region (MUC2C). EatA reduced the apparent molecular weight from >250 kDa to ~170 kDa, consistent with the generation of a large proteolytic fragment (Fig. 1B).”\nThe fragment complementary to the 170 kDa band is predominantly composed of the PTS domains, which are heavily O-glycosylated and therefore not readily visualized by Coomassie staining. We have made this observation in previous work using the MUC2 protein construct when studying protease activity, see (PMID:1482604 (Fig. 1D) and PMID:23546879 (Fig. 7B).\nUpdated figure 1B and 1C:\nLine 262: The issue of the reducing vs. non-reducing conditions is not clear. There is the redox status of the reaction during which the proteolysis is taking place, and then there is the redox status of the gel used to assess cleavage. These are two separate things. Whether each of them was oxidizing or reducing should be specified.\nWe clarified the redox status of the proteolysis assay and the gel electrophoresis in the revised manuscript, indicating that only protein separation was performed under either reducing or none-reducing conditions. In addition, we now highlight the oligomeric state in figure 1C and 1D.\nLine 267: Here the span of the C-terminal fragment chosen is specified and named (MUC2C-NC), but the first fragment used (line 259) was not specified or named (was it MUC2C?).\nWe reviewed the paragraph and now included the specific recombinant protein used for the initial experiment (MUC2N and MUC2C). To match the naming scheme in figure 1A.\nLine 273: “only the active form of EatA” This statement is confusing, since no reference to any other form of EatA was presented in the text or used in the previous experiments. The mutant needs to be properly introduced.\nIn the revised manuscript we justify the reason for using the non-catalytic and inhibitor treated protease by including the following sentence.\nLine 282 - 286\n“To confirm that the observed activity on MUC2C-N was specifically due to EatA and not a contaminating protease, a non-catalytic mutant of EatA was generated by substituting histidine 134 with an arginine. Additionally, the serine protease nature of EatA was validated by treatment with PMSF, a serine protease inhibitor. Neither the mutant EatA nor PMSF-treated EatA was able to degrade MUC2C-N (Fig. 1E)¹³.”\nFigure 1: The legend says that the lines to the sides of the gel are “color coded,” but what is the code? The legend says the lines represent the position of the indicated protein, but the protein is not in fact indicated.\nWe agree with the Reviewer that the description in legend was unclear on how the lines were matched to the proteins used for analysis. Figure legend 1 is now updated with the following appended sentence.\nFigure legend 1\n“Colored lines underneath the protein names color-match with the lines on the gel image representing the position of the specific protein.”\nLine 292: “intramolecular” disulfide bonds required for dimerization? Do the authors mean “intermolecular”?\nCorrected in the revised manuscript.\nLine 322: “indicating the potential for multiple cleavages” How can the authors distinguish between multiple cleavage sites and ambiguity in site identification? Just because the SK sequence appears multiple times does not mean that all such sites are cleaved. As the authors point out later in the text, other factors in addition to the local sequence determine the actual site of cleavage.\nIn the revised manuscript we emphasize that the results of the assay demonstrate that EatA acts on this specific region of MUC2, but that based on results we can’t determine which of the sites in PTS3 is the actual cleavage site.\nLine 335 - 338\n“This motif appears three times within the TR-PTS3 region, indicating that EatA acts on this region of MUC2. However, due to the repetitive nature of the sequence, the exact cleavage site could not be determined.”\nLine 323: “To confirm the cleavage site” The experiment with the peptide does not actually end up confirming the cleavage site, so best not to start the sentence this way. Also, it should be specified that the synthetic peptide is not glycosylated.\nIn the revised manuscript we use the term “assess” instead of “confirm” and indicate that the synthetic peptide was non-O-glycosylated.\nLine 327: The topic is the peptide (which is not glycosylated; see previous note), so when the text says, “As this region of the protein is likely to be glycosylated...” the reader is confused for a second until he/she realizes that the authors have jumped to the full protein. In fact, the whole logical progression of this section could be improved. The glycan analysis of MUC2 produced in CHO cells is stuck in the middle. In this reviewer’s opinion, it would be more logical to say, “... no cleaved product. We hypothesized that EatA may required O-glycans for activity. We therefore used recombinant GalNAc transferases to add GalNAc to the PTS3 peptide... [describe analysis of the resulting degree and variability of glycosylation]. However, GalNAc addition did not render the peptide cleavable by EatA. To confirm that the degree of glycan modification resembles that of MUC2 produced in CHO cells... Moreover, deglycosylation of MUC2 enhanced proteolysis, indicating that differences in glycosylation between the peptide and native MUC2 are unlikely to account for the differences in cleavage susceptibility. Therefore, another determinant, present in MUC2 but absent from the PTS3 peptide, appears to facilitate cleavage. [start a new paragraph here] EatA has additional domains, outside the catalytic core, which may contribute to substrate recognition. A requirement for binding via these domains to segments of MUC2 that are absent from the PTS3 peptide may explain the inability of EatA to cleave the peptide. To test this hypothesis...”\nWe thank the reviewer for their insightful feedback on how to improve the readability of this section and the order of data representation. The results section has been updated and now starts with describing the analysis of the glycosylated synthetic peptide followed by the analysis of MUC2 protein.\nLine 385: reducing disulfides will likely destroy both secondary and tertiary structure, so best to say, “To determine whether structure is important for substrate recognition, we unfolded...”\nWe agree and adapted the sentence stating “structure” instead of “secondary structure”.\nLine 389: No need to invoke the PTS3 peptide here. The authors already determined that the peptide lacks important substrate recognition sites.\nThe sentence was removed from revised manuscript.\nLine 392: To establish interaction by gel filtration, it is absolutely essential to show the elution profile of each protein alone as well as the mixture of both.\nWe included the size exclusion elution profile of the individual proteins to the revised manuscript. The data demonstrates that EatA when combined with MUC2 will elute earlier than when analyzed alone which suggest partial complex formation. The results are included as supplementary figure 2A and B and the following sentence has been added to the main manuscript:\nLine 408 - 410\n“The elution profiles of the individually analyzed proteins show that the observed overlap between H134R EatA and MUC2C-N arises from their interaction. (Extended figure 2A and B).”\nFigure S2A and B:\nLine 395: The authors cannot conclude anything about whether the interaction is transient or long-lived. It could be that only a fraction of the recombinant EatA is capable of binding and the rest is defective but binds stably (if indeed there is even any binding at all; see previous note).\nBased on the gel filtration results we can indeed not conclude the nature of the EatA:MUC2 interaction, as emphasized by the reviewer. In the revised manuscript we have removed the claims about the nature of the interaction and the potential reason for why we did not observed a higher proportion of complex formation. The updated sentence reads as follows:\nLine 410 - 411\n“The size exclusion chromatography results indicate interaction between protease and substate.”\nLine 416: The title of the figure is a bit odd. Yes, EatA and MUC2 domains are involved in substrate recognition, and unfolding of the domains destroys recognition, but domains and folding in general don’t “drive” recognition. Also, again the little lines on the sides of the gels are unlabeled and unexplained.\nWe agree with the reviewer that the title was too specific on aspects not directly responsible for MUC2 recognition and changed the title of the figure legend to:\n“MUC2 structural features are critical for recognition by EatA”\nTo clarify the color coding of each protein on the gel the figure legend was appended with the following sentence.\n“Colored lines underneath the protein names color-match with the lines on the gel image representing the position of the specific protein.”\nLine 432: “limits” should be “limit”\nWe corrected the error in the revised manuscript.\nRegarding the supplementary videos, this reviewer had a difficult time extracting any information from them. Also, at least one of them has repetitive patterns of red blobs appearing and disappearing that look highly artifactual. What is the origin of the uniform spacing seen in these rows of blobs?\nStreaks of beads were observed upon EatA treatment in video 3 and are a technical artifact of the confocal microscopy analysis. Dislodged beads are no longer retained by the mucus layer, and will float away, and can therefore be observed in multiple z-stack during acquisition (see example still below). We agree that the video provided to little experimental details and have now included explanatory text in each video describing the genotype, treatment, and a description of what is visualized in each fluorescent channel.\nLines 429-486: This paragraph is two pages long. Can it be broken into bite-sized pieces, for example at the beginning of the colon biopsy section?\nWe agree with the reviewer that this section is unnecessary long. We therefor introduced a second result paragraph with the results describing the differences in the cleavage site region with the following title:\nLine 485\n“Species-Specific variation in EatA cleavage of MUC2/Muc2”\nLine 471: again, “validated” is not the correct word in, “We validated EatA activity on the mouse C-terminal of Muc2...” since there was no activity to validate.\nIn the revised manuscript we use the term “assessed” instead “validated”.\nLine 476: “reduction of the EatA band” is not clear. Decrease in intensity of the band?\nWe agree with the reviewer and changed reduction to “decreased EatA band intensity”.\nLine 512: In the opinion of this reviewer, the whole section on the modelling should be drastically shortened and focused. The spatial relationship between putative DUF binding sites on MUC2 and the cleavage site is an interesting question, and the docking is useful for hypothesis generation. However, the docking and related analyses do not “elucidate the molecular mechanism” or justify the mass of detail presented in Fig. 6. One short paragraph and a simple figure discussing/illustrating the spatial arrangements of the relevant parts of the proteins and their proposed interaction modes would be much more effective at this point. (It should also be stated or shown what the differences were between the ranked models.)\nBelow a point-by-point reply to the comments concerning the results section and figure describing the EatA:MUC2 interaction model.\n We agree with the reviewer and have reduced the level of detail considerably and thereby shorten the length of the paragraph substantially.\n The term “elucidate” was too strong for a hypothesis generating model and replaced with “to gain insight”.\n We decided to move supportive results to the supplement limiting the panels in figure 6 only showing the key aspects of the model (see below). Upon request of reviewer 2 the panel now includes the results of the MD analysis of the mouse Muc2 cleavage site region in the active site of EatA (Fig 6C).\n The presented model was the only model without major clashes between the two proteins, due to these unrealistic close contacts no other models were considered.\nReviewer #2:\nGeneral comments:\nThe experimental sections of the manuscript are well presented in an easy-to-follow manner.\nHowever, the English could use improvement.\n1. There are several fragmented sentences in the publication, as such it could use revision.\n2. Accordingly, terminus = noun, terminal = adjective; the 2 are not interchangeable.\nWe appreciate Reviewer #2’s constructive feedback and the time they dedicated to our manuscript. In the revised manuscript, we have addressed the concerns raised by adding additional gel analysis of MUC2 treated with the EatA proteases to clarify the gel electrophoresis results in figure 1B and 1C. The section describing the interaction model between MUC2 and EatA now includes the results of the analysis of the human MUC2 with the mouse PTS3 regions covering the cleavage site. Demonstrating that the difference in spacing of the lysine residues is likely responsible for the lack of EatA activity on mouse Muc2. In addition, we have implemented the various suggestion made to improve the flow and grammar.\nIntroduction:\n35-36: The sentence would read better as “In particular, children under the age of five are especially vulnerable to acute symptomatic infections”.\nAdapted\n38: Spacing surrounding the punctuation following “..stunted growth..”\ncorrected\n40: change “and promote” to “..the epithelial surface, enabling the efficient delivery…”\nAdapted\n43: Should use “Beyond the adhesins..” rather than “Besides the adhesins”\ncorrected\nComment:\nThe first paragraph of the introduction talks of the vulnerability and risks to children. As a general question here, what is known about the mucus layer in children? There appears to be a jump in reasoning, without addressing the possible gaps in knowledge.\nWe technically only have this information from rodents where the mucus layer is developing in the first few week postnatal (PMID:40323318).\n66: typo: “process”\nCorrected\n70: might be better as “..the mucus specific to their host”, rather than “its host”.\nAdapted\n76: ad libitum should be in italics.\nCorrected\n77: double space following “cycle”\nCorrected\n77: “All mouse strains..” rather than “mice strains”\nCorrected\n77: “were derived from a C57BL/6N”\nWe appreciate the reviewer’s suggestion, as the mice genetic background of all mice in the study was C57BL/6N we prefer to use the term “on” over “from”.\n78: via in italics.\nVia is in generally not italicize in scientific writing. Nature publishing considers it a normal word during typesetting.\n80: General grammar – when giving further examples, a comma should be used before the examples provided. I.e. “..segment of chromosome 11p15.5, including MUC2 and MUC6.”\nCorrected\n98: “serum-free”\nCorrected\n104: “cut-off”\nCorrected\n105: Are there expected sizes / molecular weights of the constructs?\nIn the methods section of the revised manuscript we have now included the region of MUC2 covered by the two PTS tandem repeats.\nThis will tie into a question later in the manuscript regarding the decoration with more elaborate sugars, but demonstration of a lower molecular weight.\nThe addition of multiple negatively charged sialic acids can alter protein migration, resulting in a substantially lower apparent molecular weight compared to the protein modified with only a T- or Tn antigen.\n105: “genetically-engineered”\nCorrected\n107: “non-catalytic”\nCorrected\n113: Should use the long form for JPT, as it only seems to be used twice in the manuscript, and lacks the need to be abbreviated.\nJPT refers to the company that performed the custom peptide synthesis, in the revised manuscript we written out the full name of the company.\n121: “For the protease assays, recombinant..” a comma is needed here.\nCorrected\n122: rpm is generally not in all caps (RPM)\nCorrected to lower case\n127: abbreviation MW not introduced.\nIn the revised manuscript, ‘MW’ has been spelled out as ‘molecular weight’.\n129-130: Please include the transfer conditions, as mucins are not the easiest to transfer quantitatively.\nIn the revised manuscript the details of the blotting conditions are included.\n130: Trans-blot Turbo?\nCorrected\n131: This section would benefit from antibody concentrations.\nThe antibody concentrations are included in the revised manuscript\n133: “..the band intensities were analyzed” not “was”.\nCorrected\n138: Sentence fragmented or information lacking. The “reaction mixture” was applied to the superdex column?\nClarified\n150: N-terminus\nCorrected\n161: the word “resolved” is often used in place of “dissolved”.\nCorrected\n162: Mass spectrometric analysis\nAdapted\n165: (Dr. Maisch), this either needs more information or to be removed.\nDr. Maisch revers to the manufacturer of the chromatography material\n166: “Peptides.. over 30 minutes” rather than “in 30 minutes”\nCorrected\n169: Define HCD.\nDefined\n170: Clarification regarding “subsequent excluded for selection for 20 seconds”\nThis refers to the .\n173-176: This could use some organization / clarification.\nRephrased as follows: “Mascot was used for standard protein identification with the following settings: fixed modification carbamidomethylation (C), variable modification oxidation (M), enzyme trypsin with a maximum number of two missed cleavages, a mass tolerance of 10 ppm for precursor mass and 20 ppm for fragment ions.”\n177: semi trypsin? Please clarify.\nThis is a general enzyme cleavage rule frequently used in proteomics when identifying peptides for which only one end must follow the trypsin K/R cleavage rule.\n178: “..performed using”, rather than “performed towards”\nChanged to “performed against”\n181: missed cleavages\nCorrected\n183: The searches ”were” performed.\nCorrected\n183-184: Needs work/clarification.\nRephrased as follows: “The searches were performed against the mucinDB, and a list of 70 common human O-glycans was selected as the set of considered glycan modifications, only glycopeptides with a score. >300 were included.” Line 184 - 186\n184: >300 were “retained / analyzed”?\nThis is a probability score above which glycopeptides were considered an accurate match.\n185: “resolved” versus “dissolved”\nCorrected\n195-197: The sentence needs clarification / better punctuation.\nRephrased as follows: “The colon was opened along the mesenteric border, fecal material was removed, the longitudinal muscle tissue was stripped off and the tissue was mounted in a horizontal perfusion chamber.” Line 197 - 199\nGeneral comment for the Ex vivo analysis of EatA mucus degradation: This section could be simplified with subheadings for “Mucus penetrability” and “Mucus integrity quantification”\nIn the revised manuscript we introduced an indentation before the data analysis section and introduced a heading to break up the section. Line 232\n228: Could you specify the number of sites occupied / involved?\nThe exact modified sites are described in the revised manuscript.\n229: N-acetylglucosamine, capital A not needed.\nCorrected\n232: “while blocking”\nCorrected\n237: “full-length”\nCorrected\nResults:\nMain concerns regarding the results section:\n1) Line 259 says digestion of C-terminus results in a fragment of 170kDa. The problem here is that the 170 kDa fragment is present in MUC2C without EatA (image to the left). As such, the statement doesn’t make sense, as this does not appear to represent a digestion fragment. The manuscript should make note of the size shift in the MUC2C, rather than the presence of a digestion product.\nWe agree with the reviewer that the results presentation and description lack sufficient detail required for correct data interpretation. In the revised manuscript for clarity, we have replaced the gel electrophoresis image by a repeating the analysis on a gradient gel with a higher resolving power, as used for all other analysis throughout the paper. The text has been altered to include the mW of the initial noncleaved molecule and describes the significance of the cleavage product upon EatA treatment. See updated figure 1B and C below:\n2) Figure 1C. The red bar indicates the presence of the EatA protein. However, in the 3rd lane (MUC2N + EatA), when EatA is added, there is no band detected by Coomassie blue. This is an issue with the manuscript that needs to be resolved.\nIn the new figure 1B and C the EatA is clearly visible in the protease treated conditions.\n3) In Figure 1A, the limits of the MUC2C-N construct need to be corrected, as I don’t believe they span the correct domains (VWC1 should be included in the shaded rectangle under the image).\nWe are grateful to the reviewer for pointing out our mistake in the annotation of Figure 1A. The MUC2C-N construct indeed includes the complete VWD domain and almost the complete VWC domain, which is corrected in the revised version of the figure.\n4) Is there an explanation for the differences in the size of the differentially glycosylated PTS domains? Specifically, why does the addition of a larger glycan result in a reduced size / increased electrophoretic migration? Do you hypothesize an effect on molecular structure or blocking of neighboring glycosylation sites?\nThe more extensively O-glycosylated PTS domains are sialylated. The addition of multiple negatively charged sialic acids can alter protein migration, resulting in a substantially lower apparent molecular weight compared to the protein modified with only a T- or Tn antigen.\nConcerning the blocking of neighboring sites by the proximity of extensive glycosylation, this is unlikely as the synthesis occurs sequentially and the glycosyltransferases responsible for extension are distributed further distally in the Golgi.\n5) Regarding the modelling, we’re assuming a 1-to-1 interaction in the in vitro studies with expressed mucins, as the cysteines are removed, and thus no dimerization. Is there an explanation why in the modelling the models demonstrate the interaction of EatA with 2 MUC2 chains?\nTo clarify we didn’t mutate or remove any cysteines in the recombinant proteins included in the study. The C-terminus of MUC2 is always occurs as a dimer stabilized by four intermolecular disulfide bonds, three between the CK domains and one in the VWCN domain (Fig. 1A and PMID:37031240), as demonstrated in figure 1C and 3D. For that reason, all modelling was performed on the dimer. EatA interacts through its MUC2-binding domains with both chains of the MUC2C dimer indicating a 1-to-2 interaction.\n6) This is related to a statement on line 483, indicating that the additional 18 amino acid residues in the rodent Muc2 “will affect the spatial arrangement of the cleavage site region and potential accessibility for the protease.” Without direct evidence of this, it should be modified to “will potentially affect…”.\nIn support of our claim, we have now preformed the modelling and molecular dynamics of the mouse PTS3 region within the active site of EatA. As the structure of the mouse MUC2 has not been resolved we replaced the PTS3 domain in the human structure with the mouse sequence. The results of these analysis indicate that three potential cleavage sites are out of range of the active site (Fig. 6D). The site at the correct distance was used for modelling, but loss of interfacial contacts was observed already after ~2 ns, indicating an unstable interaction (Fig. 6D). The simulation indicates that the lysine in the P1’ position which is replaced with a glutamic acid in mice dictates the stability of the interaction.\n7) Otherwise, it would be beneficial to see a modelling/docking interaction of the EatA protein with the rodent Muc2, to demonstrate that the catalytic domain would no longer have access to the cleavage site.\nFor detailed answer see point 6\n8) Supplemental data Figure 1: Resolution of the image/text is poor, renders the figure unreadable.\nIn the revised manuscript we have increased the resolution of the supplementary figures.\n259: C-terminus, not C-terminal\nCorrected\n261: intermolecular, rather than intramolecular (for dimerization)\nCorrected\n263: “resolve” should be switched to “dissociate / digest”\nChanged to dissociated\n263: N-terminus and C-terminus, not N-/C-terminal\nThe sentence was adapted on request of reviewer #1 and now reads as follows: “To determine if EatA can dissociate dimerized MUC2N or MUC2C the protease assay was performed under oxidizing conditions and analyzed by SDS-PAGE under non-reducing conditions.” Line 282 - 284\n268: C-terminus\nCorrected\n271: C-terminus (last time pointing it out, but there are other occurrences in the manuscript)\nCorrected\n285-286: This sentence requires clarification in the context of the study. This implies that the enzyme will not be able to cleave native MUC2, while EatA digestion of native MUC2 is demonstrated later in the paper.\nWe agree with the reviewers and removed the statement concerning the disulfide bonds.\n“Cleavage in this region results in the degradation of the complex oligomeric mucin structures”\n288: Terminus\nWe adapted the figure panel title to indicate that EatA acts on the C-terminal protein region of MUC2\n306: Pathogen name incorrect.\nCorrected Haemophilus\n303-316: This section introduces a characterized target of SPATEs (IgA), shows similarity of IgA hinge region to MUC2, and goes on to show that EatA does not degrade IgA. What is needed in this section is an explanation of why EatA is active on MUC2 PTS which resembles the hinge region. This needs to be tied back in, as otherwise is leaves the reader confused.\nIf the IgA hinge region resembles TR-PTS3, and EatA does not digest IgA, then why did you continue to investigate the activity of EatA on the PTS region? This section needs to be re-worked to facilitate easier reader comprehension.\nWe agree with the reviewer and now included a statement at the end of the paragraph to indicate that likelihood that even though the cleavage site region is similar other regions of EatA are specifically tailored for the degradation of MUC2. This will be later in the results section discussed.\nLine 324 - 326\n“The similarity at the cleavage site, coupled with the lack of EatA activity on IgA1 suggests that other regions of the molecule are important for substrate recognition.”\n321: Punctuation would help: “TMMP-labelled peptide, SK, was identified”, or even to say dipeptide, which would better explain the “SK”.\nCorrected to “TMMP-labelled dipeptide”\nThe supplementary tables could be better organized / titled to help the reader.\nThe tables have been revised and now include a separate tab in the xlxs file with a description of the results presented in each table and a reference to the figure in the main text. In addition, we adapted the same naming scheme for the column as in the manuscript for figure 3F-G.\n349: “Generally” not “general”.\nCorrected\n358: Mass spectrum (as it is singular)\nCorrected\n364: spectrum\nCorrected\n377: Why are there parentheses around DUF?\nTo indicate the abbreviation of domain with unknown function (DUF)\n388: dependent, not depended\nCorrected\n391: “..of and MUC2C-N”, remove the “and”\nCorrected\n394: Punctuation will help to separate ideas sentences. “…DUF domain was lacking, the interaction…”\nIntroduced a comma after “lacking” as suggested by the reviewer\n396: sensitive “to” dilution.\nThis sentence was revised following the suggestion of reviewer #1, as no evidence was provided to support the claim in the original submission.\n408: This suggest(s) that..\nCorrected\n409: “between the region C-terminal to the cleavage site..” would give better context, rather than “beyond”.\nChanged to “C-terminal to the cleavage site”\n412: “..mucin and EatA is essential” not “are essential”\nCorrected\n413: For clarity, the DUF interaction domain is C-terminal to the cleavage site, it is not correct to say that it is “beyond”, as this lacks context.\nCorrected “beyond” to “C-terminal of”\nFigure 3C: While there is no decrease in size of the principal MUC2 band, there is a reduction in intensity (it seems) and there is an increase in the intensity bands between 150-250kDa.\nWe interpret these bands as minor impurities that remain present after protein purification by ion exchange chromatography and subsequent size exclusion chromatography. In all other assays EatA activity resulted in a complete absence of the high molecular weight band (~250kDa), and repeated analysis yielded the same results for the unfolded molecule.\n419: This would represent “decreasing” concentrations, as the amount of enzyme to MUC2 is decreasing from left to right in the image. Or its better just to say “treated at varying protease to substrate ratios”.\nIn the revised manuscript the figure legend is adapted according to the reviewers suggestion.\nLine 434\n“Protein gel electrophoresis of MUC2C treated at varying protease to substrate ratios of EatA or EatA-DUF lacking a domain extending from the helical stalk region.”\n431: Punctuation: it would read better as “..mesh-like structures, it limits the penetration of the…”\nAdapted\nLack of continuity in the use of “mouse Muc2” and “murine Muc2”.\nIn the remaining paragraph the distinction between mouse Muc2 and human MUC2 is indicated by the use of lowercase and uppercase.\n480: “affected species”, not “effected species”\nCorrected\n485: determinants\nCorrected\n501: unpaired Student’s t-test?\nCorrected\n505: determines species specificity\nCorrected\n510: Error in description of the figure. Grey = unaffected (mouse / rat), Red = affected.\nCorrected\n522: It should likely be “clashes”, rather than “crashes”.\nCorrected\n556: Please clarify, central region between (not “among”) the DUF domain and the C-terminus?\nWe altered the statement to be more precise about the region of the EatA protein involved in the interaction: “The model reveals a secondary interaction surface that spans around 600 Å2 between MUC2 C8 (A) and the EatA passenger domain central region C-terminally of the DUF domain (Extended figure 4C).”\n558: suggests\nCorrected\n565: got worse?, decreased?\nCorrected to “decreased”\n566: remove “so”, use “as a result”.\nCorrected to “as a result”\n570: over time\nWe corrected the statement: “The root mean square deviation (RMSD) stabilized between 15 and 20 ns, indicating that the system reached equilibrium (Extended figure 4D).”\n588 + 590: “shown”\nCorrected\n593: “..in size with respect to the central figure”\nAdapted\n594: Interacting residues?\nTo clarify the terminology used. The residues highlighted in the figure panel are the once on the interface between the surface of the two proteins, therefor we use the word “interfacing residues”.\nDiscussion:\nConcerns:\n1) Lack of discussion regarding interaction with either monomers or dimers / oligomers of MUC2.\nThe C-terminus of MUC2 always occurs as a dimer supported by four intermolecular disulfide bonds, so we have not considered interaction or degradation with monomers. Our interaction model indicates that the MUC2-binding domain (DUF) interacts with both MUC2 chains of the dimer. Suggesting that it depends on oligomerization for substrate recognition. In the revised manuscript we have added the following sentence to the discussion to clarify this:\nLine 651 - 652\n“Importantly, proteolysis depends on dimerization of the MUC2C-terminus, as the DUF engages the interface formed by both chains.”\n613: Remove “resolve”, use “degrade” or “dissolve”\nCorrected to “dissolve”\n618: “localized in a mucin domain”\nCorrected to “localized in a mucin domain”\n621: Punctuation for readability. A common would help this sentence.\nThe sentence has been modified to improve readability: “In contrast to other recently identified O-glycoproteases acting on mucin domains, EatA is not dependent on glycosylation but tolerates glycans in proximity, as supported by molecular dynamics analysis”\n628: grammar – glycan structures\nCorrected to “glycan”\n631: “alone, suggesting”\nCorrected\n632: “Previously, a putative binding domain”\nCorrected\n634-635: discussion of binding in the cleft of homodimers (modelling data), but these were not present in the digestion studies. This needs to be addressed here in the discussion.\nThe C-terminus of MUC2 always occurs as a dimer. The location of the intermolecular disulfide bonds has been confirmed by Cryo-EM (PMID:37031240), which was used for modeling and data interpretation. This point is hopefully sufficiently clarified in response to reviewer 2 at point 5.\n635-638: Punctuation for readability. Need commas to separate ideas or sections.\nThe punctation of the sentence was corrected as follow: “In the absence of the DUF, no cleavage activity was observed, and binding to MUC2 was reduced, highlighting the importance of the DUF domain for initiating interactions with its substrate.”\nData availability: The identifier given does not work. PXDXXXX. Need to replace the place-holder in the text.\nThe mass spectrometry data and search results are deposited to PRIDE and can be accessed using the login details below:\nRepository: https://www.ebi.ac.uk/pride/login\nProject accession: PXD068558\nToken: crG1mfKvn9zz\nAlternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details:\nUsername: reviewer_pxd068558@ebi.ac.uk\nPassword: fzrG4HRdQCnj\nSupplementary tables.\nWhen possible, headers in the tables should give more information, such as Band 1, rather than Exp1.\nWe have updated the headers in the supplementary table to match the titles used in the manuscript.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe are grateful for your time and effort."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made some improvements to the manuscript, but they seem to have ignored many of the reviewers’ requests. Though both reviewers pointed out the general sloppiness of the original version, the revised version has both many of the old typos as well as new typos throughout the added sections. It is frustrating having to go through all the mess again. The lack of care by the authors is a shame, because the results are valuable. I am re-stating my point that the description of the research is still not logical and effective. For example, right after the authors note that they cannot precisely identify the cleavage site because the SK sequence appears multiple times, they write, “To assess the cleavage site…” But then the story goes in the direction of the glycans and never returns to which SK site (if there is actually any selectivity) gets cleaved. It would be better to start a new paragraph when there is a new question, state the question clearly, then describe the experiment done to address it, then report the results, then state the conclusion from those results before starting a new paragraph with a new question. It is not clear why the authors are having a hard time with this.\nThis reviewer did not think that the description of the modeling was reader-friendly the first time, and, despite claims to the contrary, the authors have not improved the presentation. Yes, some panels were removed from Fig. 6, but other, equally unclear, panels were added. What is the reader supposed to see in panels C and D? For example, this reviewer zoomed in as much as possible and still cannot see the lysine that is referred to in the main text and supposedly labeled in the figure.\nLines 312-367: Again, despite both reviewers pointing out the poor editing of the text in the first round, the authors do not seem to have done much to improve readability. This whole section is one long, unbroken paragraph that is unpleasant to wade through. The scientific questions are straightforward:\n1) Where is the cleavage site?\n2) Is glycosylation required?\n3) Are regions outside the cleavage region itself required for recognition?\nThese questions were actually addressed in a relatively logical manner experimentally. The text should be re-written so that the logic is clearer to the reader.\nBelow is a very partial list of remaining or new typos, and additional potential mistakes:\nLine 315: should be “between the Fab and Fc domains”\nLine 344: should be “GalNAc-transferases”\nLine 344: Can the authors please clarify the lengths of the peptides? The text says that a 34-mer was made, but the residue numbers 4328 to 4362 correspond to 35 amino acids and do not match the termini shown in Fig. 2B. Then, what is the “38-mer peptide”?\nLine 347: should be “did not result in any”\nLine 352: should be “The results highlight the extent of”\nLine 366: should be “highly O-glycosylated but is not dependent on glycosylation”\nLine 403: “secondary protein structure”?\nLine 424: “between the region to the cleavage site”?\nLine 425: The CTCK domain is a “cystine” knot, not a “cysteine” knot.\nLine 454: A general comment on punctuation: Compound sentences (i.e., two full sentences fused into one with “and” or “but”) should have a comma between the two halves. For example, a comma should be added to yield: “The protease was added to the apical tissue surface, and the thickness of the mucus layer was monitored…” (“The protease” is the subject of the first part of the compound sentence, and “the thickness” is the subject of the second part.) The text should be corrected throughout to apply this rule.\nLine 497: should be “addition to humans, pigs, cows, sheep, and dogs are susceptible to ETEC infections, especially”\nLine 498: “but not mouse and rat” is awkward tacked onto the end of the sentence. It could be replaced by a new, short sentence saying, “Mouse and rat are not susceptible.”\nLine 499: No reason to remind the reader that the rodents are resistant, since this will be stated clearly in the preceding sentence.\nLine 532: should be “To gain insight into”\nLine 545: should be “the DUF domain inserted into the cavity”\nLine 546: should be “the N-terminus of VWCN from chain A” Despite the explicit request by the other reviewer to check “terminus” vs. “terminal” for proper usage throughout the text, the authors did not make this correction.\nLine 561: should be “all lysines”\nLine 613 what is “(50-10 ns)”? Do the authors mean the panels that are labeled either 10 ns or 50 ns?\nLine 629: do the authors mean “degrade” rather than “dissolve”?\nLines 633-637: The first sentence says that mucin domains were considered to be resistant to proteases due to glycosylation, while the second sentence immediately refers to enzymes that cleave mucin domains. Contradiction.\nLines 676-678: How the two parts of this sentence are connected (both logically and grammatically) is unclear.\nLines 678-681: Not a well-formed sentence.\nLine 687: There seems to be a period in the middle of the (long, run-on) sentence.\nLines 694-696: This is not a complete sentence.\nLines 698-700: This is a poorly constructed sentence. “limited to” follows “mucus” but actually refers to “proteases”\nIt is unclear why identification of the cleavage site is necessary for vaccine strategies.\nThe supplementary figures showing the gel filtration of the two proteins separately are labeled “None reduced” instead of “non-reduced.” Also, the red mark in Figure S2B is above the 100 kDa marker while the protein seems to run below 100 kDa.\nThe new supplementary figures showing the gel filtration of the two proteins separately (which were added at this reviewer's request-- thank you) are labeled “None reduced” instead of “non-reduced.” Also, the red mark in Figure S2B is above the 100 kDa marker while the protein seems to run below 100 kDa.", "reviewer_2": "(please see the attached document regarding formatting colors of the comments)\nWe greatly appreciate the effort of the authors to update the manuscript addressing our questions and concerns. Nearly all points have been addressed to our satisfaction.\nWe would like to come back to a response of the authors to look for further clarification.\nResponse (including reviewer’s question):\n“5) Regarding the modelling, we’re assuming a 1-to-1 interaction in the in vitro studies with expressed mucins, as the cysteines are removed, and thus no dimerization. Is there an explanation why in the modelling the models demonstrate the interaction of EatA with 2 MUC2 chains?\nTo clarify we didn’t mutate or remove any cysteines in the recombinant proteins included in the study. The C-terminus of MUC2 is always occurs as a dimer stabilized by four intermolecular disulfide bonds, three between the CK domains and one in the VWCN domain (Fig. 1A and PMID:37031240), as demonstrated in figure 1C and 3D. For that reason, all modelling was performed on the dimer. EatA interacts through its MUC2-binding domains with both chains of the MUC2C dimer indicating a 1-to-2 interaction.”\nIt was stated that the cysteines were not mutated or removed, however, in the materials and methods ‘lines 95-98 of the updated manuscript”:\n“with the cysteine residue at position 4786 mutated to an alanine. The mouse Muc2C-N vector was composed similar to the MUC2C-N including the Muc2 sequence between amino acid 3691 and 4248 (UniProt: Q80Z19 v2023_01) with the cysteine residue at position 4231 mutated to an alanine.”\nIt was due to this information in the materials and methods that the reviewers assumed the experimental conditions were assessing a monomeric MUC2 structure, as removing the cysteine would likely impair dimer formation. Could you please clarify the confusion surrounding this? Specifically addressing the purpose of the removal of the cysteine.\n  Would it be possible to clarify in the manuscript that the EatA or DUF domain interacts with MUC2 dimers. This would remove the confusion encountered by the reviewers in the context of the interaction conditions, i.e. protein:monomer or protein:dimer\nMinor comments.\nThe response to the following comment appears to be unclear or imcomplete.\n170: Clarification regarding “subsequent excluded for selection for 20 seconds”", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Degradation of the intestinal mucus layer by the ETEC protease EatA is species-specific\nand determined by the structure of the MUC2 mucin\nTrillo-Muyo, S., et al.\nCorresponding Author: Sjoerd van der Post\nWe thank the Editor and Reviewers for their positive review of our manuscript and constructive\nfeedback. Please find below our point-by-point responses to each of the Reviewers’ comments.\nRevisions made to the manuscript and supplemental data are indicated in the respective\ndocuments in blue.\nReviewer #1:\nThe authors have made some improvements to the manuscript, but they seem to have ignored\nmany of the reviewers’ requests. Though both reviewers pointed out the general sloppiness of\nthe original version, the revised version has both many of the old typos as well as new typos\nthroughout the added sections. It is frustrating having to go through all the mess again. The lack\nof care by the authors is a shame, because the results are valuable. I am re-stating my point\nthat the description of the research is still not logical and effective. For example, right after the\nauthors note that they cannot precisely identify the cleavage site because the SK sequence\nappears multiple times, they write, “To assess the cleavage site…” But then the story goes in\nthe direction of the glycans and never returns to which SK site (if there is actually any\nselectivity) gets cleaved. It would be better to start a new paragraph when there is a new\nquestion, state the question clearly, then describe the experiment done to address it, then report\nthe results, then state the conclusion from those results before starting a new paragraph with a\nnew question. It is not clear why the authors are having a hard time with this.\nThis reviewer did not think that the description of the modeling was reader-friendly the first time,\nand, despite claims to the contrary, the authors have not improved the presentation. Yes, some\npanels were removed from Fig. 6, but other, equally unclear, panels were added. What is the\nreader supposed to see in panels C and D? For example, this reviewer zoomed in as much as\npossible and still cannot see the lysine that is referred to in the main text and supposedly\nlabeled in the figure.\nWe appreciate the reviewer’s careful reading. We acknowledge and regret the errors in the\nprevious revision and have amended the text accordingly. The results section describing the\ncleavage site region identification and the role of O-glycosylation on protease activity is\nrestructured and clarified. In addition, the result section describing the EatA-MUC2 interaction is\nshortened by excluding multiple figures and restructured to only focus on our key findings.\nLines 312-367: Again, despite both reviewers pointing out the poor editing of the text in the first\nround, the authors do not seem to have done much to improve readability. This whole section is\none long, unbroken paragraph that is unpleasant to wade through. The scientific questions are\nstraightforward:\n1) Where is the cleavage site?\n2) Is glycosylation required?\n3) Are regions outside the cleavage region itself required for recognition?\nWe apologize that our previous revision did not sufficiently improve this section. We have now\nsubstantially edited it to enhance clarity:\n Split the former single paragraph into subsections, each addressing one question and\nincluded transition sentences.\n Removed redundancies, shortened sentences, and moved technical detail to keep the\nnarrative concise.\nThese questions were actually addressed in a relatively logical manner experimentally. The text\nshould be re-written so that the logic is clearer to the reader.\nBelow is a very partial list of remaining or new typos, and additional potential mistakes:\nLine 315: should be “between the Fab and Fc domains”\nWe changed domain to plural “domains”.\nLine 344: should be “GalNAc-transferases”\nWe have added “transferase” after GalNAc.\nLine 344: Can the authors please clarify the lengths of the peptides? The text says that a 34-mer was made, but the residue numbers 4328 to 4362 correspond to 35 amino acids and do not match the termini shown in Fig. 2B. Then, what is the “38-mer peptide”?\nThe mention of a 38-mer is an error and is corrected to 34-mer in the revised manuscript.\nTo clarify the length and region of MUC2 that was covered by the recombinant peptide in the\nvarious figure panels:\n- Figure 2B: Depicting the region of MUC2 that we defined as PTS3 comprising residue\n4328 to 4362 is 34 residues in length.\n- Figure 2D: The section of the PTS3 region on full length MUC2 for which we identify\nglycopeptides by mass spectrometry analysis\n- Figure 2E: Glycopeptide coverage of the recombinant 34-mer peptide spanning the\nsame region as depicted in 2B (4328 – 4362).\n- Figure 5A and D: The PTS3 domain is indicated to be 35 residues long as it includes\nresidue 4363. For sequence comparison between species the PTS3 borders were\ndefined as the region between the last cysteine in the domain before and the first\ncysteine in the domain after the PTS3 region, justifying the slight ambiguity in length.\nLine 347: should be “did not result in any”\nWe changed results to singular “result\".\nLine 352: should be “The results highlight the extent of”\nThe spelling of extent was corrected.\nLine 366: should be “highly O-glycosylated but is not dependent on glycosylation”\nThe comma after but was removed.\nLine 403: “secondary protein structure”?\nIncluded “secondary” to protein structure.\nLine 424: “between the region to the cleavage site”?\nWe corrected the sentence as following:\nThis suggests that in addition to the PTS3, the DUF domain is required for protease interactions\nbetween the MUC2 C-terminal region after the cleavage site, which includes the von Willebrand\nfactor CN, D4, C1, C3 domains and cysteine knot.\nLine 425: The CTCK domain is a “cystine” knot, not a “cysteine” knot.\nCorrected to “cystine” throughout the revised manuscript\nLine 454: A general comment on punctuation: Compound sentences (i.e., two full sentences\nfused into one with “and” or “but”) should have a comma between the two halves. For example,\na comma should be added to yield: “The protease was added to the apical tissue surface, and\nthe thickness of the mucus layer was monitored…” (“The protease” is the subject of the first part\nof the compound sentence, and “the thickness” is the subject of the second part.) The text\nshould be corrected throughout to apply this rule.\nWe have systematically reviewed the manuscript and corrected punctuation in compound\nsentences throughout, inserting commas before coordinating conjunctions when they join two\nindependent clauses.\nLine 497: should be “addition to humans, pigs, cows, sheep, and dogs are susceptible to ETEC\ninfections, especially”\nA comma was introduced after infections.\nLine 498: “but not mouse and rat” is awkward tacked onto the end of the sentence. It could be\nreplaced by a new, short sentence saying, “Mouse and rat are not susceptible.”\nWe moved the statement to the next sentence as suggested.\nIn addition to humans, pigs, cattle, sheep, and dogs are susceptible to ETEC infection,\nespecially during the neonatal period. By contrast, mice and rats are not.\nLine 499: No reason to remind the reader that the rodents are resistant, since this will be stated\nclearly in the preceding sentence.\nWe removed the repeated statement in the revised manuscript.\nLine 532: should be “To gain insight into”\nCorrected to “To gain insight into”\nLine 545: should be “the DUF domain inserted into the cavity”\nRemoved “was”\nLine 546: should be “the N-terminus of VWCN from chain A” Despite the explicit request by the\nother reviewer to check “terminus” vs. “terminal” for proper usage throughout the text, the\nauthors did not make this correction.\nCorrected to “terminus”\nLine 561: should be “all lysines”\nCorrected to “lysines”\nLine 613 what is “(50-10 ns)”? Do the authors mean the panels that are labeled either 10 ns or\n50 ns?\nThis indicates the length of the molecular dynamics simulation variation between the human and\nmouse PTS3 with the active site of EatA. The human PTS3 domain remained in the pocket and\nthe simulation was performed for up to 50ns while for the mouse PTS3 the side chain already\nleft the pocket after less than 10 ns so the simulation time was shortened to 10 ns. We have\nnow clarified this in the figure legend as following:\n“EatA active site with human or (E) mouse MUC2 PTS3 detailed interaction before (0 ns) and\nafter (50 ns human PTS3 and 10 ns for mouse PTS3) MD simulation.”\nLine 629: do the authors mean “degrade” rather than “dissolve”?\nCorrected to “degrade”\nLines 633-637: The first sentence says that mucin domains were considered to be resistant to\nproteases due to glycosylation, while the second sentence immediately refers to enzymes that\ncleave mucin domains. Contradiction.\nWe agree with the reviewer that the section was unclear and the second sentence contradicting.\nWe aimed to acknowledge the existence of O-glycoprotease acting on mucin domains, but lack\nof evidence that these act on MUC2 and/or can degrade the mucus layer. This is likely limited\nby the complex and dense O-glycosylation observed in vivo. We updated the section to clarify\nthis as following:\nThe EatA cleavage site is localized in a mucin domain in the C-terminal part of the protein\nclosely resembling the central mucin domain, which has been considered to be proteolytically\nresistant due to its extensive O-glycosylation. Various O-glycoproteases acting on mucin\ndomains in other proteins have been identified but none has been demonstrated to actively\ndegrade MUC2, in contrast EatA is not dependent on glycosylation but tolerates glycans in\nproximity, as supported by molecular dynamics analysis.\nLines 676-678: How the two parts of this sentence are connected (both logically and\ngrammatically) is unclear.\nThe second sentence has been rephrased in the revised manuscripts as following:\n“The colonic mucus layer shares protein composition and O-glycosylation with the small\nintestine, but is thicker, providing superior visualization of mucus degradation.”\nLines 678-681: Not a well-formed sentence.\nSee above comment.\nLine 687: There seems to be a period in the middle of the (long, run-on) sentence.\nWe corrected the sentence to improve readability:\n“This shared virulence axis among related E. coli pathotypes offers a potential avenue for the\nrational development of broadly protective vaccine strategies designed to block the initial\nbinding to MUC2 thereby preventing mucus degradation and colonization 14. Progress toward\nsuch vaccines requires mapping the regions that mediate substrate recognition, so that\ninhibitory epitopes can be prioritized for immunogen design.”\nLines 694-696: This is not a complete sentence.\nThe sentence was rephrased for clarity to:\n“We localized the cleavage site to a susceptible region of MUC2 that mediates mucus-gel\ndegradation, and show that interspecies variation in this region underlies host selectivity.”\nLines 698-700: This is a poorly constructed sentence. “limited to” follows “mucus” but actually\nrefers to “proteases”\nWe reconstructed the sentence according to the reviewers suggestion to clarify our statement.\n“The combined results underscore that mucus degradation requires highly specialized\nproteases, whose expression appears restricted to intestinal pathogens, highlighting a potential\nstrategy to prevent ETEC infection.”\nIt is unclear why identification of the cleavage site is necessary for vaccine strategies.\nWe agree with the reviewer that identification of the cleavage site is not essential knowledge\nrequired for the development of a vaccine. Its more important to identify the domains essential\nfor interaction. In the revised manuscript the part about the cleavage site is removed and now\nreads as follows:\n“Progress toward such vaccines requires mapping the regions that mediate substrate\nrecognition, so that inhibitory epitopes can be prioritized for immunogen design.”\nThe supplementary figures showing the gel filtration of the two proteins separately are labeled\n“None reduced” instead of “non-reduced.” Also, the red mark in Figure S2B is above the 100\nkDa marker while the protein seems to run below 100 kDa.\nWe agree with the reviewer and corrected the figure annotation to “nonreduced” in the revised\nmanuscript. In our experience EatA runs around 100 kDa when analysed by protein gel\nelectrophoresis, but agree that the position of the marker was off and corrected this in the\nrevised manuscript.\nReviewer #2 (Remarks to the Author):\n(please see the attached document regarding formatting colors of the comments)\nWe greatly appreciate the effort of the authors to update the manuscript addressing our\nquestions and concerns. Nearly all points have been addressed to our satisfaction.\nWe would like to come back to a response of the authors to look for further clarification.\nResponse (including reviewer’s question):\nWe are grateful for your thoughtful review and are glad that nearly all points have been\naddressed to your satisfaction. Below we restate the remaining question and provide\nclarification.\n“5) Regarding the modelling, we’re assuming a 1-to-1 interaction in the in vitro studies with\nexpressed mucins, as the cysteines are removed, and thus no dimerization. Is there an\nexplanation why in the modelling the models demonstrate the interaction of EatA with 2 MUC2\nchains?\nTo clarify we didn’t mutate or remove any cysteines in the recombinant proteins included in the\nstudy. The C-terminus of MUC2 is always occurs as a dimer stabilized by four intermolecular\ndisulfide bonds, three between the CK domains and one in the VWCN domain (Fig. 1A and\nPMID:37031240), as demonstrated in figure 1C and 3D. For that reason, all modelling was\nperformed on the dimer. EatA interacts through its MUC2-binding domains with both chains of\nthe MUC2C dimer indicating a 1-to-2 interaction.”\nIt was stated that the cysteines were not mutated or removed, however, in the materials and\nmethods ‘lines 95-98 of the updated manuscript”:\n“with the cysteine residue at position 4786 mutated to an alanine. The mouse Muc2C-N vector\nwas composed similar to the MUC2C-N including the Muc2 sequence between amino acid 3691\nand 4248 (UniProt: Q80Z19 v2023_01) with the cysteine residue at position 4231 mutated to an\nalanine.”\nIt was due to this information in the materials and methods that the reviewers assumed the\nexperimental conditions were assessing a monomeric MUC2 structure, as removing the\ncysteine would likely impair dimer formation. Could you please clarify the confusion surrounding\nthis? Specifically addressing the purpose of the removal of the cysteine.\nWould it be possible to clarify in the manuscript that the EatA or DUF domain interacts with\nMUC2 dimers. This would remove the confusion encountered by the reviewers in the context of\nthe interaction conditions, i.e. protein:monomer or protein:dimer\nOur excuses for the confusion. We indeed mutated a cysteine to alanine in two of the\nrecombinant proteins used in the study, but this did not affect the dimer formation. Cysteine\n4786 was mutated in the recombinant MUC2C-N and Muc2-N proteins which forms an\nintramolecular disulfide bond with 4830. The mutation allowed us to generate a truncated\nversion of the protein lacking a large part of the c-terminus with good expression. In figure 1E its\nshown that the MUC2C-N with the mutated cysteine still forms dimers.\nIn the revised results section describing the EatA–MUC2 model, we aim to clarify that MUC2 is\nhomodimeric in the proposed complex. We have revised the following sentences accordingly:\nMain text:\n“To gain insight into the molecular mechanism of EatA-MUC2 recognition and cleavage, we\nperformed docking simulations using a model based on the MUC2 C-terminal homodimer\n(vWCN–D4–VWC1) cryo-EM structure (PDB: 7QCN) and an AlphaFold2 model of EatA\n(UniProt:Q84GK0) 24.”\n“Consequently, the docking simulations were conducted using the isolated DUF domain first,\nthen extended with the remaining part of EatA to generate a model of the full complex with the\nMUC2C dimer.”\nFigure legend:\n“Chains A and B of the MUC2 homodimer are labeled A and B, glycans on chain A are shown in\nmagenta and those on chain B in orange.”\nMinor comments.\nThe response to the following comment appears to be unclear or incomplete.\n170: Clarification regarding “subsequent excluded for selection for 20 seconds”\nAuthors’ response: “This refers to the .”\nOur excuses for the incomplete statement in the previous response. The 20 seconds dynamic\nexclusion means that once a precursor ion is selected for MS/MS, the instrument places that\nm/z (within a set tolerance) on an exclusion list for 20 seconds, preventing it from being\nreselected for further fragmentation so other precursors can be sampled.\nThe corrected sentence below needs a slight adjustment.\n“domain central region C-terminal to the DUF domain”, rather than “C-terminally”.\n556: Please clarify, central region between (not “among”) the DUF domain and the C-terminus?\nWe altered the statement to be more precise about the region of the EatA protein involved in the\ninteraction: “The model reveals a secondary interaction surface that spans around 600 Å2\nbetween MUC2 C8 (A) and the EatA passenger domain central region C-terminally of the DUF\ndomain (Extended figure 4C).”\nIn the revised manuscript, we removed the figure at the request of Reviewer 1 to clarify the\nmodeling section and maintain focus on the key interaction site.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate your time and commitment to this initiative."}]

Decision letter