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Uncovering bacterial pseudaminylation with pan-specific antibody tools

Nature Chemical Biology (2026)Cite this article

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Abstract

Pseudaminic acids (Pse) are a family of carbohydrates found within bacterial lipopolysaccharides, capsular polysaccharides and glycoproteins that are critical for the virulence of human pathogens. However, a dearth of effective tools for detecting and enriching Pse has restricted study to only the most abundant Pse-containing glycoconjugates. Here we devise a synthesis of α- and β-O-pseudaminylated glycopeptides to generate ‘pan-specific’ monoclonal antibodies (mAbs) that recognize α- and β-configured Pse with diverse N7 acyl groups, as well as its C8 epimer (8ePse), presented within glycans or directly linked to polypeptide backbones. Structural characterization reveals the molecular basis of Pse recognition across a range of diverse chemical contexts. Using these mAbs, we establish a glycoproteomic workflow to map the Pse glycome of Helicobacter pylori, Campylobacter jejuni and Acinetobacter baumannii strains. Finally, we demonstrate that the mAbs recognize diverse capsule types in multidrug-resistant Acinetobacter baumannii and enhance phagocytosis to eliminate infections in mice.

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Fig. 1: Synthesis of Pse-containing glycopeptides.
Fig. 2: Generation of Pse-specific mAbs.
Fig. 3: Structural insights into the recognition of Pse5Ac7Ac by mAb 1E8.
Fig. 4: Enrichment of Pse-modified proteins in H. pylori and C. jejuni.
Fig. 5: Detection and enrichment of Pse-containing glycopeptides from A. baumannii strains.
Fig. 6: Pse antibodies as tools for detecting and treating A. baumannii infections.

Data availability

Structure coordinates have been deposited in the Protein Data Bank (https://www.rcsb.org/) under accession code 9PQS. All mass spectrometry data (RAW files, FragPipe outputs, Rmarkdown scripts and input tables) have been deposited into the PRIDE ProteomeXchange repository with the following data identifiers: PXD056875 for proteomic studies of H. pylori flagellin; PXD046836 for H. pylori P12 enrichments; PXD048493 for H. pylori 26695 enrichments; PXD046858 for C. jejuni NCTC 11168 enrichments; PXD046859 for A. baumannii BAL062 enrichments; and PXD053904 for A. baumannii A74 enrichments. Source data are provided with this paper.

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Acknowledgements

E.D.G.-B. acknowledges support from The Walter and Eliza Hall Institute of Medical Research; a Victorian State Government Operational Infrastructure support grant; Rebecca Cooper Fellowship; and National Health and Medical Research Council (NHMRC) Ideas Grants (GNT2027601 and GNT2000517) and Investigator Grant (GNT 2033340). N.E.S. was supported by an Australian Research Council (ARC) Future Fellowship (FT200100270), an ARC Discovery Project Grant (DP210100362) and NHMRC Ideas Grant (GNT2018980). R.J.P. acknowledges funding from an NHMRC Investigator Grant (GNT1174941) and the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012). D.H.D. acknowledges funding from the National Institutes of Health (NIH) Award R15 (GM109397). We thank the Melbourne Mass Spectrometry and Proteomics Facility of The Bio21 Molecular Science and Biotechnology Institute for access to mass spectrometry instrumentation. We also thank R. Haas for the generous gift of the H. pylori P12 strain.

Author information

Author notes

  1. These authors contributed equally: Arthur H. Tang, Niccolay Madiedo Soler, Kristian I. Karlic.

Authors and Affiliations

  1. School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia

    Arthur H. Tang, Leo Corcilius, Caitlin E. Clarke-Shepperson, Christopher Lehmann & Richard J. Payne

  2. Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, Sydney, New South Wales, Australia

    Arthur H. Tang, Leo Corcilius, Caitlin E. Clarke-Shepperson, Christopher Lehmann & Richard J. Payne

  3. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Niccolay Madiedo Soler, Chenzheng Guan & Ethan D. Goddard-Borger

  4. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

    Niccolay Madiedo Soler, Chenzheng Guan & Ethan D. Goddard-Borger

  5. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

    Kristian I. Karlic, Lauren Zavan, Lucy Li, Christopher McCrory, Maria Kaparakis-Liaskos, Benjamin P. Howden, Norelle L. Sherry, Linda M. Wakim, Glen P. Carter & Nichollas E. Scott

  6. Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, The University of Western Australia, Nedlands, Western Australia, Australia

    Aleksandra W. Debowski

  7. School of Molecular Sciences, The University of Western Australia, Crawley, Western Australia, Australia

    Aleksandra W. Debowski & Keith A. Stubbs

  8. School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia

    Ashleigh L. Dale & Stuart J. Cordwell

  9. Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia

    Ashleigh L. Dale, Michelle Cielesh, Stuart J. Cordwell & Mark Larance

  10. School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia

    Michelle Cielesh & Mark Larance

  11. Department of Chemistry and Biochemistry, Bowdoin College, Brunswick, ME, USA

    Adedunmola P. Adewale, Karen D. Moulton & Danielle H. Dube

  12. Centre for Pathogen Genomics, The University of Melbourne, Melbourne, Victoria, Australia

    Benjamin P. Howden, Norelle L. Sherry & Glen P. Carter

  13. Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, People’s Republic of China

    Ruohan Wei & Xuechen Li

  14. School of Life and Environmental Science, The University of Sydney, Sydney, New South Wales, Australia

    Ruth M. Hall

  15. Institute for Biomedicine and Glycomics, Griffith University, Southport, Queensland, Australia

    Johanna J. Kenyon

  16. Infection Program and Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    Francesca L. Short

  17. Sydney Mass Spectrometry, The University of Sydney, Sydney, New South Wales, Australia

    Stuart J. Cordwell

Authors
  1. Arthur H. Tang
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  2. Niccolay Madiedo Soler
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  3. Kristian I. Karlic
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  4. Leo Corcilius
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  5. Caitlin E. Clarke-Shepperson
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  6. Christopher Lehmann
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  7. Aleksandra W. Debowski
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  8. Ashleigh L. Dale
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  9. Lauren Zavan
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  10. Michelle Cielesh
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  11. Adedunmola P. Adewale
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  12. Karen D. Moulton
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  13. Lucy Li
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  14. Chenzheng Guan
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  15. Christopher McCrory
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  16. Maria Kaparakis-Liaskos
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  17. Benjamin P. Howden
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  18. Norelle L. Sherry
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  19. Ruohan Wei
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  20. Xuechen Li
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  21. Ruth M. Hall
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  22. Johanna J. Kenyon
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  23. Linda M. Wakim
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  24. Francesca L. Short
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  25. Danielle H. Dube
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  29. Glen P. Carter
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  30. Nichollas E. Scott
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  31. Ethan D. Goddard-Borger
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  32. Richard J. Payne
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Contributions

A.H.T., L.C., C.E.C.-S., C.L., R.W. and X.L. planned and performed all chemical syntheses; A.W.D. and K.A.S. cultured H. pylori P12 samples; M.K.-L. and L.Z. cultured H. pylori 26695 samples; M.C. and M.L. performed proteomic analyses of H. pylori P12 flagellin; N.M.S. and C.G. raised/characterized mAbs, collected and analyzed all SPR/BLI data, and performed all protein expression and structural biology; A.P.A., K.D.M. and D.H.D. made H. pylori Pse-knockout strains; N.L.S., R.M.H. and J.J.K. sourced A. baumannii strains with diverse K types; L.M.W., C.M. and F.L.S. performed all fluorescence-activated cell sorting experiments; A.L.D. and S.J.C. cultured C. jejuni strains and purified flagellin from these samples; K.I.K. and N.E.S. cultured A. baumannii strains and performed in vitro infection assays; K.I.K., N.M.S. and N.E.S. performed glycopeptide enrichments and glycoproteomic analyses; L.L., G.P.C. and B.P.H. performed in vivo infection assays; N.E.S., E.D.G.-B. and R.J.P. conceived and coordinated the project, and cowrote the paper.

Corresponding authors

Correspondence to Nichollas E. Scott, Ethan D. Goddard-Borger or Richard J. Payne.

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Competing interests

The authors declare no competing interests.

Peer review

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Nature Chemical Biology thanks Micha Fridman, Martin Pabst and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ExD enabled localisation of native H. pylori glycopeptides FlaA (206-211), FlaB (242-260) and FlgE(281-291).

Localised pseudaminylated events within tryptic digests of flagellin preparation of H. pylori P12 for (A) FlaA 206VSSSAGTGIGVLAEVINK211, (B) FlaB 242ASYNVMATGGTPVQSGTVR260 and (C) FlgE 281ISFTNDSAVSR291. For the FlgE glycopeptide 281ISFTNDSAVSR291 PRM EAD analysis was undertaken to allow localisation of glycosylation events while EThcD was undertaken on 206VSSSAGTGIGVLAEVINK211 and 242ASYNVMATGGTPVQSGTVR260.

Extended Data Fig. 2 Butterfly plots of synthetic verses native glycopeptide 281ISFTNDSAVSR291 of FlgE.

Butterfly plots comparing HCD fragmentation of synthetic glycopeptides bearing an (A) α- or (B) β-configured Pse residue with the identical authentic glycopeptides derived from tryptic digests of flagellin preparation of H. pylori P12. Spectral angle (SA) analysis comparing matched b- and y-ion intensities to native 281ISFTNDSAVSR291 of FlgE supports highest similarity to α-pseudaminylation compared to β-pseudaminylation.

Extended Data Fig. 3 ELISA screening of potential Pse-specific hybridoma supernatant.

(A) ELISA screen of hybridoma supernatant (1:1000) against: (i) control peptides 8, 9 and 10 conjugated to BSA (green), (ii) Pse peptides α-8, α-9 and α-10 conjugated to BSA (orange), and flagellin purified from C. jejuni 81-176, which is also pseudaminylated with Pse5Ac7Ac (blue). Data are presented as mean values ± SD for n = 2. (B) ELISA screen of hybridoma supernatants (1:2000) that are Pse-specific against: (i) α-configured Pse peptides α-8, α-9 and α-10 conjugated to BSA (orange) and (ii) β-configured Pse peptides β-8, β-9 and β-10 conjugated to BSA (blue). Data are presented as mean values ± SD for n = 2.

Extended Data Fig. 4 Binding affinity of Pse analogues to the 1E8 antibody.

SPR sensorgrams and fitted steady-state response data for Pse analogues binding to biotinylated 1E8 Fab’ immobilised on a streptavidin-coated chip.

Extended Data Fig. 5 Pse glycopeptide enrichment from H. pylori 26695.

(A) Volcano plot illustrating the enrichment of Pse5Ac7Ac-modified glycopeptides from H. pylori 26695. (B) EThcD / HCD spectra of the novel glycopeptides derived from Pgi, FlgE and HP_0564.

Source data

Extended Data Fig. 6 Pseudaminylation of C. jejuni glycoproteins.

MS/MS EThcD and HCD spectra of (A-B) 447KPSVFSNIWHK457 from FtsA (Q0PAI4_CAMJE) with pseudaminylation localised to Ser452; (C-D) 19KDATTATDAVISTITDVLAK38 from Hup (Q46121_CAMJE) with pseudaminylation localised to Ser30 and (E-F) 191GNDPHDSLVGIK202 from Maf3 (Q0P8S4_CAMJE) with pseudaminylation inferred to Ser197 by the absence of other hydroxyl containing amino acids.

Extended Data Fig. 7 Novel pseudaminic acid-containing glycopeptides identified in A. baumannii A74.

MS/MS EThcD and HCD spectra of unique the glycopeptides identified within A. baumannii A74 corresponding to (A-B) 374APAVANHASSVETK387 of the multidrug efflux RND transporter periplasmic adaptor subunit AdeA (MDW5585830.1); (C-D) 153DAASEALHQITSGGGVTR170 of the CvpA family protein (MDW5584485.1) and (E-F) 55VQQLMDEANATADNPNALASITASADK81 of the tetratricopeptide repeat protein (MDW5583480.1).

Extended Data Fig. 8 Pse-specific surface binding of mAb 1E8 to A. baumannii.

(A) Flow cytometry analysis of A. baumannii strains ATCC19606 (K3 CPS lacking Pse) and A74 (K2 CPS with Pse) either unstained or stained with α-Pse mAb 1E8 alone, α-mouse-AlexaFluor647 alone, or both α-Pse mAb 1E8 and α-mouse-AlexaFluor647. Staining of A. baumannii with α-Pse mAb followed by α-mouse-AlexaFluor647 results in a shift in A647 intensity for A. baumannii A74, while no shift is observed for ATCC 19606. (B) Western blot analysis of lysates from A. baumannii strains to assess: (i) legionaminic acid reactivity using LUH5533 (K7), SDF (K5), LUH5551 (K63), NIPH-329 (K46), A74 (K2), and ATCC 19606 (K3); (ii) acinetaminic acid reactivity using D36 (K12), UMB001 (K13), SGH0703 (K73), A74 (K2), and ATCC 19606 (K3) and (iii) additional pseudaminic acid reactivity using AUSMDU00058681 (K90) and MRSN31468 (K58). The A74 (K2 CPS with Pse) and ATCC 19606 (K3 CPS lacking Pse) serve as positive and negative controls, respectively. (C) SNFG diagrams of the various A. baumannii CPS K-type monomers used to generate this figure.

Source data

Extended Data Fig. 9 Immunofluorescence imaging-based opsonization assays of A. baumannii A74.

(A) Density plots of biological replicates assessing the intracellular number of A. baumannii A74 in THP-1 macrophages treated in the absence of added antibody, or in the presence of Pse 1E8 or a mouse IgG1 isotype control with >200 cell assessed for each biological replicate. (B) Violin plots depicting the abundance of intracellular bacteria in THP-1 macrophages treated with A. baumannii A74 in the absence of added antibody, or in the presence of Pse 1E8 or a mouse IgG1 isotype control. Statistical analysis corresponding to pairwise two-sided student t-tests with p-values less then 0.001 denoted by *** and corresponding to p-values = 2.88×10−36 and 9.32×10−29. Statistical analysis was undertaken on the combined data from four biological replicates corresponding to n > 900 cells per group. The boxplot denotes the upper (Q3) and lower (Q1) quartiles of the observed data ranges, with the median denoted between these ranges. The whiskers denote 1.5 times the interquartile Range of Q1 and Q3, excluding outliers. (C) A representative immunofluorescence microscopy micrograph of THP-1 cells one hour after infection with A. baumannii A74. DNA has been stained with Hoechst 33342 (blue), actin with SiR-Actin (red), and Pse mAb 1E8 (green). Scale bar is 5 μm.

Source data

Extended Data Fig. 10 Surface-specific binding and opsonophagocytosis of H. pylori.

(A) In vitro gentamicin protection assays of H. pylori strains in response to pretreatment with 1E8 Pse-specific antibody, isotype control, or PBS (n = 3 independent experiments for each condition). Box and whisker plots indicate minimum, first quartile, median, third quartile, and maximum values. Kruskal–Wallis tests gave p-values < 0.05 (*) and <0.01 (**). (B) Flow cytometry analysis of H. pylori strains either unstained or stained with α-Pse mAb alone, α-mouse-AlexaFluor647 alone, or both α-Pse mAb 1E8 and α-mouse-AlexaFluor647.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–86, Tables 1–3, Data 1–6 captions and Note.

Reporting Summary

Supplementary Data 1

Glycoproteomic data for H. pylori P12 flagellin preparation.

Supplementary Data 2

Enriched H. pylori P12 glycopeptides.

Supplementary Data 3

Glycoproteomic data for H. pylori 26695.

Supplementary Data 4

Enriched C. jejuni glycopeptides.

Supplementary Data 5

Glycoproteomic data for A. baumannii A74.

Supplementary Data 6

Glycoproteomic data for A. baumannii BAL062.

Source data

Source Data Figs. 2 and 4–6 and Extended Data Figs. 5 and 8–10

Unprocessed western blots and gels, volcano plot source data, open search plot source data, SPR source data, BLI source data, ELISA plot source data, box-and-whisker plot source data, Kaplan–Meier curve and CFU source data.

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Tang, A.H., Soler, N.M., Karlic, K.I. et al. Uncovering bacterial pseudaminylation with pan-specific antibody tools. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-025-02114-9

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