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Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin [1]

['Zachary T. Berndsen', 'Department Of Integrative Structural', 'Computational Biology', 'Scripps Research', 'La Jolla', 'California', 'United States Of America', 'Marjahan Akhtar', 'Department Of Medicine', 'Division Of Infectious Diseases']

Date: 2024-09

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as volunteers challenged with ETEC, diarrheal severity is significantly increased in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

Enterotoxigenic E. coli (ETEC), a leading cause of diarrhea disproportionately affecting young children in low-income regions, are a priority for vaccine development. Individuals possessing A blood-type are more susceptible to severe cholera-like disease. EtpA, a secreted, immunogenic, blood group A binding protein, is a current vaccine target antigen. Here, we determined the structure of EtpA in complex with protective as well as non-protective monoclonal antibodies targeting two different domains of the protein, pinpointing key regions involved in blood-group A antigen recognition and uncovering the mechanism of antibody-based protection. In addition, we show through mass-spectrometry that EtpA is extensively and heterogeneously glycosylated at surface-exposed asparagine residues by a promiscuous and low-fidelity glycosyltransferase, EtpC, and that this unique form of bacterial glycosylation is critical for to development of protective immune responses. Lastly, polyclonal antibodies from vaccinated mice as well as monoclonal antibodies obtained from ETEC-infected volunteers revealed that the highly antigenic surface of EtpA exhibits both protective and non-protective epitopes. These results greatly expand our understanding of ETEC pathogenesis, and the immune responses elicited by these common infections, providing valuable information to aid in the rational design and testing of subunit vaccines.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JMF is listed as the "Inventor" on U.S. patent 8323668 assigned to the University of Tennessee Research Foundation on December 4, 2012 that relates to use of EtpA related antigens in vaccine development. The other authors have declared that no competing interests exist.

Funding: This work was supported by National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) R01 AI089894, and R01 AI126887 to JMF and by funding from the Department of Veterans Affairs (5I01BX001469-05) to JMF. Research conducted by AS was also supported by National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number T32AI007172. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, or the Department of Veterans Affairs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Here we provide the complete structure of EtpA determined by cryo-EM, both alone and complexed to anti-EtpA monoclonal antibodies produced by vaccination, as well as high-resolution mass-spectrometry (MS) analysis of EtpA glycosylation. These data define regions of the molecule that are required for activity and are targets for antibody neutralization, provide a detailed profile of its extensive antigenic glycosylation, and show that alterations in glycosylation can impact antibody function. We identified a diverse set of epitopes from polyclonal sera of vaccinated mice, indicating that EtpA possesses a large and variable immunogenic surface with numerous potential neutralizing and decoy epitopes. We anticipate that the elucidation of the antigenic structure of this important virulence factor will afford insights into molecular correlates of protection and help guide further development of EtpA and similar proteins as vaccine immunogens.

Several structures of truncated N-terminal (TPS) domains required for secretion of TpsA molecules [ 41 – 45 ] as well as a single full-length TpsA exoprotein [ 46 ], have been solved by X-ray crystallography. The highly homologous TPS domain structures all adopt a similar fold, specifically, an extended 3-sided β-helix. These N-terminal TPS domains may be followed by a series of repeat modules, with some such as HxuA [ 46 ] containing short extra-helical loops or motifs thought to contribute to function [ 47 ]. The N-terminal secretion domain of EtpA was previously shown to be sufficient for export as well as binding to flagella [ 19 ], while the function of a series of four C-terminal repeats was unknown.

In exploring the utility of EtpA as a potential vaccine antigen, studies to date have demonstrated that the etpBAC locus is highly conserved across ETEC from geographically disparate origins [ 25 – 30 ], is immunogenic following natural [ 27 , 30 ] and human experimental challenge [ 31 , 32 ] infections, and that immunization with recombinant EtpA (rEtpA) affords considerable protection against intestinal colonization [ 19 , 21 , 27 , 29 , 33 – 37 ]. In addition, EtpA expression by ETEC strains is strongly associated with the development of diarrheal illness in young children, while antibodies against EtpA are associated with protection [ 30 ]. Despite enthusiasm for targeting EtpA in next-generation ETEC vaccines [ 38 – 40 ], relatively little is known about its structure, antigenicity, or the mechanisms by which antibodies targeting this molecule mediate protection.

Once secreted, the high molecular weight (~170 kDa) EtpA glycoprotein serves as a unique molecular bridge between the bacteria and intestinal mucosal surfaces [ 19 ], essential to pathogen-host interactions required for delivery of both LT [ 20 , 21 ] and ST [ 22 ]. On host epithelia, EtpA binds to N-acetylgalactosamine (GalNAc) residues on enterocyte surfaces as well as secreted mucins including MUC2, interactions that are critical for efficient adhesion, toxin delivery, and intestinal colonization [ 23 ]. EtpA preferentially engages GalNAc as the terminal sugar of human A blood group presented on enterocytes. Importantly, volunteers challenged with the EtpA-producing H10407 strain of ETEC were significantly more likely to develop moderate-severe diarrhea if they were blood group A [ 24 ], recapitulating earlier observations that young bgA+ children in Bangladesh were more likely to develop diarrhea with ETEC infection [ 9 ].

Given the persistent and pervasive impacts of ETEC infections, these pathogens have remained a high priority for vaccine development [ 14 – 16 ]. Efforts to identify novel surface-expressed molecules that might be targeted in ETEC vaccine development led to the identification of the plasmid-borne etpBAC two-partner secretion (TPS) locus responsible for export of EtpA, an extracellular adhesin [ 17 ]. At a minimum, TPS loci are comprised of a transmembrane polypeptide-transport-associated (POTRA) [ 18 ] domain (TpsB) protein responsible for secretion of a cognate (TpsA) exoprotein. The corresponding components of the etpBAC locus include EtpB the transmembrane protein required for secretion of the extracellular EtpA adhesin, as well as EtpC, a glycosyltransferase responsible for glycosylation of EtpA [ 17 ]. All three genes are required for optimal secretion of EtpA. The EtpA molecule is typically heavily glycosylated and etpC mutants exhibit dramatically reduced production of EtpA, as well as altered tropism for target epithelial cells, suggesting that glycosylation of EtpA may be important for proper folding and function of the adhesin [ 17 ].

Enterotoxigenic Escherichia coli (ETEC) are diarrheal pathogens defined by their production of heat-labile (LT) and heat-stable (ST) enterotoxins [ 1 ]. ETEC, an exceedingly common cause of infectious diarrhea in areas where clean water and sanitation remain limited, accounts for hundreds of millions of cases of acute diarrheal illness each year [ 2 ]. In addition, these pathogens are a leading cause of more severe diarrhea and death [ 3 , 4 ] among young children of low-income regions and are associated with long-term sequelae including poor growth [ 5 – 9 ] and malnutrition [ 10 – 13 ].

Results

EtpA repeat regions direct blood group A binding on target host cells Like many bacterial adhesins, EtpA is a lectin, or a carbohydrate binding protein [23]. Similar to another TpsA protein, filamentous hemagglutinin of Bordetella pertussis [48,49], EtpA also possesses the ability to agglutinate erythrocytes. Hemagglutination activity of lectins typically arises when these molecules possess two or more carbohydrate binding sites permitting cross-linking of cells [50]. While individual interactions may be of low affinity, high avidity can be achieved through tandem repetition of lectin-binding regions [51–53]. To determine whether the C-terminal region of EtpA, comprised of 4 repeat modules (Fig 1A), was involved in blood group A glycan recognition, we first examined a truncated version of recombinant EtpA (rEtpA 1-1086 , Fig 1A), lacking the full complement of repeat modules. We found that while the truncated molecule was efficiently secreted, it was incapable of binding efficiently to blood group A glycans on the surface of intestinal epithelial cells (Fig 1B), on solid substrates (Fig 1C), or erythrocytes (Fig 1D) suggesting that the repeats act in concert to engage target carbohydrates. PPT PowerPoint slide

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TIFF original image Download: Fig 1. The repeat region of EtpA directs critical interactions with A blood group glycans. A. Schematic depicts molecular organization of the EtpA molecule from ETEC H10407 (top), recombinant EtpA encoded on pJY017, and the truncated recombinant antigens EtpA (1–1086) and the NTS domain (1–607) encoded on pMH004, and pQL211, respectively. Inset: anti-EtpA immunoblot of TCA-precipitated culture supernatants from H10407 wild type strain, and recombinant Top10 strains jf1696 and jf5090 carrying plasmids pJL017 and pMH004, respectively. B. Binding of full-length and truncated, mutant EtpA molecules to blood group A-expressing HT-29 cells. Nuclei, (white) are shown at left; center column: protein binding (yellow); right: cell membranes (CellMask Plasma Membrane Stain ThermoFisher). Shown at left are representative fields quantified in graph at right from n-20 replicate fields from two independent experiments. Bars indicate geometric mean fluorescence intensity per cell (****≤0.0001, ** = 0.0015 by Kruskal-Wallis nonparametric testing). C. Kinetic ELISA data reflect binding of full-length and truncated EtpA to blood group A ** = 0.0043 (Mann-Whitney, two-tailed). D. Blood group A1 erythrocyte (A1-RBC) pull-down assay with full-length and truncated EtpA. * = 0.0147, **0.0073 by Kruskal-Wallis. Results in C, D represent combination of technical duplicates from three independent experiments. E. Inhibition of EtpA-A1-RBC interactions with anti-EtpA monoclonal antibodies recognizing the repeat (mAb 1G05) and secretion (mAb 1G08) domains compared to anti-EtpA mouse polyclonal IgG, and negative IgG isotype control (-IgG) * = 0.0103, *** = 0.0002. F. mAb inhibition of blood group A-EtpA interaction ** = 0.0084 (Kruskal-Wallis). G. Anti-EtpA mAbs inhibit ETEC bacterial adhesion. Data reflect replicate experiments (closed and open symbols represent respective experiments) (n = 45 fields total) and the impact of anti-EtpA mAbs on ETEC adhesion to target blood group A expressing HT-29 cells ****<0.0001, **<0.001(Kruskal-Wallis). Grey bars throughout represent geometric mean values. H. mAb recognition of full-length rEtpA (blue bars) and NTS of EtpA (grey bars) in end-point ELISA. Data represent geometric mean of ≥ 6 technical replicates combined from 2 experimental replicates. ****<0.0001 by ANOVA. I. mAb inhibition of EtpA binding to human A blood group in kinetic ELISA assay. N = 8 technical replicates from 2 independent experiments (Ø = no antibody control; *<0.05, **<0.005, by Kruskal-Wallis). https://doi.org/10.1371/journal.ppat.1012241.g001

Antibodies targeting EtpA repeats interrupt bgA binding and ETEC adhesion To identify potential protective epitopes on EtpA, we examined the capacity of anti-EtpA monoclonal antibodies (mAb) to impair EtpA binding to A blood group glycans, and interrupt pathogen-host interactions. Two mAbs isolated from rEtpA-vaccinated mice, 1G05 and 1C08, both bound to EtpA with high affinity, (S1A Fig), but recognized distinct epitopes on EtpA (S1B–S1C Fig). The 1G05 mAb, which recognized the CTR domain (S1D Fig) significantly inhibited interactions with blood group A (Fig 1E–1F) and impaired bacterial adhesion (Fig 1G). Conversely, 1C08, which recognized the NTS domain (S1D Fig), exhibited no demonstrable impact on EtpA binding to target blood group A molecules or ETEC pathogen-host interactions. This pattern was also observed in monoclonals isolated from volunteers challenged with ETEC H10407, with the three monoclonals that recognized the NTS domain (Fig 1H) failing to inhibit EtpA-bgA interactions (Fig 1I). Conversely the single monoclonal (1F09) that bound the CTR significantly inhibited EtpA interactions with BgA. Collectively, these data indicate that the C-terminal repeat region of EtpA is essential to ETEC virulence, and that antibodies targeting this region can effectively inhibit interactions with the host.

Glycosylation of EtpA by EtpC, a promiscuous low-fidelity N-linked glycosyltransferase Perhaps the most striking feature of the EtpA structure is its unique and extensive surface glycosylation. In addition to the etpBAC operon, other TPS loci from Yersinia, and Burkholderia spp. appear to encode glycosyltransferases related to HMW1C of H. influenzae [57–59]. The structure of the EtpC glycosyltransferase predicted by AlphaFold2 [60] shows high structural similarity (pruned Ca-RMSD = 1.1Å; all residue Ca-RMSD = 3.7Å) to the crystal structure of the closely related HMW1C glycosyltransferase from Actinobacillus pleuropneumoniae [61] (S4 Fig), a functional homolog to the HMW1C glycosyltransferase of H. Influenzae which shares ~40% sequence identity, and 56% similarity to EtpC. The HMW1 adhesin glycosylated by the HMW1C enzyme in H. influenzae exhibits a unique glycosylation profile consisting of asparagine-linked (N-linked) mono- and di-hexose glycans appearing predominately at canonical N-X-S/T sequences (where X is any amino acid expect proline), with a single modification of a non-canonical asparagine (Asn) residue [62]. Additional analysis identified the hexose residues as primarily glucose and sometimes galactose [58], with the HMW1 glycosyltransferase catalyzing the formation of both the Asn-hexose and hexose-hexose linkages. HMW1C exhibited no apparent selection for modification of distinct sequons with either mono or dihexose sugars. Glycosylation by HMWC1 likely stabilizes the HMW1A adhesin and is required to tether this molecule to the surface of H. influenzae. Similarly, EtpC is required efficient secretion and function of the EtpA exoprotein adhesin [17]. Presently however, neither the glycosylation profile conferred by EtpC or its precise impact on pathogen-host interactions are understood. To identify the location and type of glycan modifications on rEtpA we employed high- resolution site-specific mass spectrometry (MS) [63]. We analyzed potential glycosylation at 166 out of the 196 non-tandem Asn residues (we did not detect peptides associated with 30 Asn residues), of which 96 adhere to the canonical (N-X-S/T) N-linked glycosylation sequon, and we found evidence for hexose modification at 133 sites, 94 of which meet the stricter criteria of > = 25% occupancy (Fig 3A). Based on the occupancy across all potential N-glycosylation sites (PNGS), mature EtpA would have on average 61 glycans per molecule, meaning that ~1 in every 24 residues (~4%) of the EtpA exoprotein harbors an N-glycan modification. Comparatively, ≤ 2% of HMW1A [64] and 1.7% of the SARS-CoV2 spike protein [65] are glycosylated. Among sites with the highest occupancy (≥ 75%) only 4 out of the 29 are non-canonical Asn residues, (S5–S7 Figs), suggesting that like HMW1A, Asn residues within canonical sequons are glycosylated with higher fidelity. All 4 of these non-canonical glycosylation sites fall within the same repeating sequence/structural motif located on the last β-strand of each CTR in PB1. Though the majority of PNGS were found to be occupied with monohexose, dihexose was observed at 83 sites, but only 4 of those sites, N744, N972, N1200 and N1428, were found to have ≥ 50% dihexose, again all belonging to a common repeating structural motif located on the second short β-strand of each CTR in PB1 (Fig 3A and 3B). Altogether these data suggest that the surface glycan coat of EtpA is both dense and variable. PPT PowerPoint slide

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TIFF original image Download: Fig 3. N-linked glycosylation of rEtpA. A. Site-specific mass-spectrometry data for rEtpA showing the % occupancy by monohexose, or dihexose, or no glycan (unoccupied) at all 196 asparagine residues analyzed along with the peptide count (right axis and blue line—number of peptides detected in the experiment) and corresponding domain diagram. The * under certain residues indicates a canonical PNGS sequon site. B. Structure of rEtpA with potential N glycosylation sites (PNGS) colored by % occupancy. C. Surface representation of rEtpA structure colored by local glycan density (20Å radius). D. Refined model of rEtpA colored by domain showing all modeled N-linked glucose residues (through CTR2) viewed from the side and looking down the core of the β-helix. E. Analysis of glycan-glycan interactions and (F) glycan-protein interactions from the refined model of rEtpA showing hydrogen bonds (blue) and contacts (yellow) along with summary tables and a histogram of all glycan-protein residue hydrogen bonding partners. BSA = Buried Surface Area. https://doi.org/10.1371/journal.ppat.1012241.g003 When viewed in a structural context, we see that the confirmed N-linked glycosylation sites (NGS) on EtpA are asymmetrically distributed across the protein (Fig 3B–3D). PNGS within the NTS domain are glycosylated with significantly higher fidelity and specificity (for the canonical N-linked glycosylation sequon) than Asn residues within the CTR domain. For example, the NTS accounts for ~39% of the protein (and ~39% of Asn residues), however, 22 out of the 33 PNGS (~67%) without any detected glycan modifications were within the NTS, and 19 of those were within the first ~400 residues. Further, the NTS domain contains the two NGS (N290 and N349) with the highest occupancies (≥ 95%). These data suggest that the EtpA sequence as well as structural determinants may dictate glycosylation by EtpC

N-linked glycan clustering and intramolecular interactions on rEtpA As illustrated by mapping the occupancy-weighted local glycan density onto the protein surface (Fig 3C), the NGS are more evenly dispersed across the CTRs, but significantly more abundant on the PB1 face of the β-helix (Fig 3C and 3D). Our cryo-EM maps confirm the location of many of these hexose modifications, as shown in the refined model (Fig 3D). Of the glycosylated residues up through CTR2 where the cryo-EM map permitted identification (84 >0%, 60 ≥25%, 43 ≥50%, 19 ≥75% occupancy), we were able to model hexose residues at 39. Although previous analytical studies of HMW1A reveal a mixture of glucose and galactose residues, we are unable to differentiate between glucose and galactose with MS alone, so all hexose residues were modeled as glucose for consistency (Fig 3D). We did not observe clear map density for dihexose modifications at any NGS. Given the extensive glycosylation of EtpA and the poor yields obtained in prior attempts to express the exoprotein without EtpC, we questioned whether the glycans might contribute to stabilization, folding and secretion of the protein. The stabilizing effect of N-linked glycans is at least partially mediated through favorable interactions with neighboring amino acid side chains, often via stacking with aromatic residues or hydrogen bonding with polar residues. This stabilizing interaction almost always involves the core N-acetylglucosamine, which in the case of EtpA would be equivalent to the N-linked hexose residue [66,67]. Although we did not find statistical enrichment of any aromatic residues around the NGS (S8A–S8C Fig), our structure did reveal numerous glycan-glycan, as well as glycan-amino acid interactions with other residue types (Fig 3E and 3F). Further, the NGS on EtpA have a tendency group into local clusters. For example, of the 11 glycans modeled on PB1, 5 of them, including 1 more from the final strand of the NTS domain, are located immediately adjacent to each other on the first residue of each β-strand and can be seen to form a chain of inter-glycan interactions (Fig 3E, top left). Other clusters of 2 or more glycans are observed throughout the structure on both the NTS and CTR domains (Fig 3E; bottom left, right). In total, there are 13 potential glycan-glycan hydrogen bonds and 47 contacts captured in our structure, resulting in 556Å of buried glycan surface area. In addition, we identified 63 potential glycan-amino acid hydrogen bonds, with the majority involving N residues as well as T and S (Fig 3F). Although individually weak, these numerous small interactions likely contribute collectively to overall EtpA stability. Finally, we sought to determine the glycosylation profile of native full-length EtpA from ETEC strain H10407. Despite inherent difficulties in protein purification and low peptide detection in MS, we were able to map the glycosylation state at 48 PNGS of the native protein, with variable occupancy similar to that exhibited by rEtpA. (S9 Fig).

Molecular interactions with the mAbs1C08 and 1G05 Our cryo-EM maps confirm that 1C08 monoclonal binds the NTS domain (Figs 4A and S3) while the neutralizing monoclonal 1G05 binds the CTR region (Figs 4A and S2). The 1G05 epitope encompasses PB1 and the inter-strand loops between PB1 and PB2 and is located at the interface of the repeat domains, allowing for the binding of up to 3 Fabs per molecule (Figs 4A and S2). The 1C08 epitope encompasses PB3 and the long inter-strand loops between PB3 and PB1, just before the H2 helix (Fig 4A). 1G05 and 1C08 share ~88% sequence identity and have similar structures (Cα-RMSD = 0.77Å, Fig 4B and 4C), despite being derived from different germline genes (S10 Fig). Both Fabs utilize their heavy chain (HC) complementarity determining region (CDR) loops extensively, while 1C08 also makes numerous contacts with its light chain (LC) CDR loops, and their binding interfaces bury 864Å2 and 1275Å2 of surface area, respectively (Fig 4D). The larger buried surface areas and more extensive interactions of 1C08 are consistent with the kinetics data showing tighter binding to rEtpA. The weaker binding of 1G05 is compensated for by avidity affects arising from the possible binding of both Fab arms of a single IgG to each EtpA molecule. Consistent with the different binding modes, analysis of somatic hypermutation from the predicted unmutated common ancestor (UCA) germline germline sequence using ARMADiLLO [68] shows that 1G05 harbors 11 mutations in its HC as opposed to 7 for 1C08, while 1C08 has a more mutated LC with 6 mutations compared to 1G05 with only 3 (S10 Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Molecular interactions between rEtpA and mAbs 1C08 and 1G05. A. Fabs Cryo-EM density maps of rEtpA in complex with 1C08 and 1G05 Fabs colored by domain. B. Structrual alignment of mAb Fc domains colored by heavy chain (HC) and light chain (LC) complementary-determining-region loops (CDRL) along with a Ca-RMSD. C. Pairwise sequence alignment of both Fv domains with CDRLs color coded. D. Table summarizing intermolecular interactions between rEtpA and mAbs calculated from PDBePISA. E. Intermolecular interactions between rEtpA and mAbs. F. Fabs 1 C08 and 1G05 projected onto glycan density maps of EtpA. https://doi.org/10.1371/journal.ppat.1012241.g004 A defining feature of both epitopes is the presence of a single centrally located N-linked hexose residue, N349 and N849 for the 1C08 and 1G05 epitopes, respectively (Fig 4E). Intriguingly, N349 has the second highest occupancy of any site as determined by MS (~96%) and N849 is in the top 17% of sites by occupancy. This could be taken as evidence that antibodies targeting PNGS with higher occupancy are enriched during affinity maturation, or conversely, that heterogeneity in glycosylation is being exploited as a mechanism of immune evasion. 1C08 targets an epitope with relatively low local glycan density in the NTS, while 1G05 targets an epitope with high local glycan density, however, 1G05 is oriented perpendicular to the long axis of the β-helix and utilizes a smaller HC dominant binding interface such that it positions its LC away from the heavily glycosylated surface of PB1, thus avoiding all but a single glycan residue. The local density analysis also reveals that it would be difficult for antibodies to target an entirely glycan-free epitope on the CTR domain, while there is ample glycan-free surface area for potential antibody binding on the NTD. Lastly, to determine the extent to which these glycans contribute to the affinity of mAb 1G05 for epitopes within the CTR, we mutated EtpA asparagine residues at N849, N1077, and N1305 to alanine. Affinity of the 1G05 mAb for the mutant protein was significantly diminished while binding of 1C08 was unimpaired (S11A and S11B Fig). Importantly however, these mutations within the CTR did not impact interaction with target A blood group glycans (S11C Fig).

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