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A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family

Abstract

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-β-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.

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Fig. 1: Structures and biosynthesis of OPSs based on the O2a antigen.
Fig. 2: Identification of two GT modules in WbbM.
Fig. 3: Overall structure of WbbM.
Fig. 4: Details of the WbbM catalytic sites.
Fig. 5: Model for the membrane-tethered WbbM trimer.

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Data availability

The crystallographic dataset for native WbbM has been deposited in the PDB repository under accession code 6U4B. Raw NMR and mass spectrometry data are available from the corresponding authors upon request.

Code availability

Custom Python scripts used in this study are available at https://github.com/bclarke2/wbbM_glf_scripts.

References

  1. Whitfield, C. & Trent, M. S. Biosynthesis and export of bacterial lipopolysaccharides. Annu. Rev. Biochem. 83, 99–128 (2014).

    CAS  PubMed  Google Scholar 

  2. Szijártó, V. et al. Bactericidal monoclonal antibodies specific to the lipopolysaccharide O antigen from multidrug-resistant Escherichia coli clone ST131-O25b:H4 elicit protection in mice. Antimicrobial Agents Chemother. 59, 3109–3116 (2015).

    Google Scholar 

  3. Pennini, M. E. et al. Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae. Nat. Commun. 8, 1991 (2017).

    PubMed  PubMed Central  Google Scholar 

  4. Cohen, T. S. et al. Anti-LPS antibodies protect against Klebsiella pneumoniae by empowering neutrophil-mediated clearance without neutralizing TLR4. JCI Insight 2, e92774 (2017).

    PubMed Central  Google Scholar 

  5. Szijártó, V. et al. Endotoxin neutralization by an O-antigen specific monoclonal antibody: A potential novel therapeutic approach against Klebsiella pneumoniae ST258. Virulence 17, 1–13 (2017).

    Google Scholar 

  6. Hegerle, N. et al. Development of a broad spectrum glycoconjugate vaccine to prevent wound and disseminated infections with Klebsiella pneumoniae and Pseudomonas aeruginosa. PLoS ONE 13, e0203143 (2018).

    PubMed  PubMed Central  Google Scholar 

  7. Rollenske, T. et al. Cross-specificity of protective human antibodies against Klebsiella pneumoniae LPS O-antigen. Nat. Immunol. 19, 617–624 (2018).

    CAS  PubMed  Google Scholar 

  8. Follador, R. et al. The diversity of Klebsiella pneumoniae surface polysaccharides. Microbial Genomics 2, e000073 (2016).

    PubMed  PubMed Central  Google Scholar 

  9. Bronner, D., Clarke, B. R. & Whitfield, C. Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1. Mol. Microbiol 14, 505–519 (1994).

    CAS  PubMed  Google Scholar 

  10. Okuda, S., Sherman, D. J., Silhavy, T. J., Ruiz, N. & Kahne, D. Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model. Nat. Rev. Microbiol 14, 337–345 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Clarke, B. R. et al. Role of Rfe and RfbF in the initiation of biosynthesis of D-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1. J. Bacteriol. 177, 5411–5418 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Guan, S., Clarke, A. J. & Whitfield, C. Functional assignment of galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen in Klebsiella pneumoniae. J. Bacteriol. 183, 3318–3327 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kos, V. & Whitfield, C. A membrane-located glycosyltransferase complex required for biosynthesis of the d-galactan I lipopolysaccharide O antigen in Klebsiella pneumoniae. J. Biol. Chem. 285, 19668–19687 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kelly, S. D. et al. Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification. J. Biol. Chem. 294, 10863–10876 (2019).

    CAS  PubMed  Google Scholar 

  15. Clarke, B. R. et al. Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae. J. Biol. Chem. 293, 4666–4679 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kelly, R. F., MacLean, L. L., Perry, M. B. & Whitfield, C. Structures of the O-antigens of Klebsiella serotypes O2(2a,2e), O2(2a,2e,2h), and O2(2a,2f,2g), members of a family of related d-galactan O-antigens in Klebsiella spp. J. Endotox. Res. 2, 131–140 (1995).

    CAS  Google Scholar 

  17. Lukose, V., Walvoort, M. T. & Imperiali, B. Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface. Glycobiol. 27, 820–833 (2017).

    CAS  Google Scholar 

  18. Vinogradov, E. et al. Structures of lipopolysaccharides from Klebsiella pneumoniae. Elucidation of the structure of the linkage region between core and polysaccharide O chain and identification of residues at the non-reducing termini of the O chains. J. Biol. Chem. 277, 25070–25081 (2002).

    CAS  PubMed  Google Scholar 

  19. Liu, D. & Reeves, P. R. Escherichia coli K12 regains its O antigen. Microbiol. 140, 49–57 (1994).

    CAS  Google Scholar 

  20. Köplin, R., Brisson, J. R. & Whitfield, C. UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO1 gene. J. Biol. Chem. 272, 4121–4128 (1997).

    PubMed  Google Scholar 

  21. Persson, K. et al. Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nat. Struct. Biol. 8, 166–175 (2001).

    CAS  PubMed  Google Scholar 

  22. Jiang, Y.-L. et al. Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP. J. Biol. Chem. 292, 6213–6224 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lairson, L. L., Henrissat, B., Davies, G. J. & Withers, S. G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 77, 521–555 (2008).

    CAS  PubMed  Google Scholar 

  24. Hurley, T. D., Stout, S., Miner, E., Zhou, J. & Roach, P. J. Requirements for catalysis in mammalian glycogenin. J. Biol. Chem. 280, 23892–23899 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Chaikuad, A. et al. Conformational plasticity of glycogenin and its maltosaccharide substrate during glycogen biogenesis. Proc. Natl Acad. Sci. USA 108, 21028–21033 (2011).

    CAS  PubMed  Google Scholar 

  26. Wheatley, R. W., Zheng, R. B., Richards, M. R., Lowary, T. L. & Ng, K. K. S. Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial arabinogalactan. J. Biol. Chem. 287, 28132–28143 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Blackler, R. J. et al. Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site. Glycobiol. 276, 48608 (2016).

    Google Scholar 

  28. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    CAS  PubMed  Google Scholar 

  29. Gautier, R., Douguet, D., Antonny, B. & Drin, G. HELIQUEST: a web server to screen sequences with specific α-helical properties. Bioinf. 24, 2101–2102 (2008).

    CAS  Google Scholar 

  30. Ishida, T. & Kinoshita, K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res. 35, W460–W464 (2007).

    PubMed  PubMed Central  Google Scholar 

  31. Kos, V., Cuthbertson, L. & Whitfield, C. The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters. J. Biol. Chem. 284, 2947–2956 (2009).

    CAS  PubMed  Google Scholar 

  32. Toukach, P. V. & Egorova, K. S. Carbohydrate structure database merged from bacterial, archaeal, plant and fungal parts. Nucleic Acids Res. 44, D1229–D1236 (2016).

    CAS  PubMed  Google Scholar 

  33. Zhang, H. et al. The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold. Nat. Commun. 5, 4339 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Geno, K. A. et al. Pneumococcal capsules and their types: past, present, and future. Clin. Microbiol. Rev. 28, 871–899 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Zeidan, A. A. et al. Polysaccharide production by lactic acid bacteria: from genes to industrial applications. FEMS Microbiol. Rev. 41, S168–S200 (2017).

    PubMed  Google Scholar 

  36. Zhou, Y., Cui, Y. & Qu, X. Exopolysaccharides of lactic acid bacteria: structure, bioactivity and associations: A review. Carbohydr. Polym. 207, 317–332 (2019).

    CAS  PubMed  Google Scholar 

  37. Ati, J. et al. The LPG1x family from Leishmania major is constituted of rare eukaryotic galactofuranosyltransferases with unprecedented catalytic properties. Sci. Rep. 8, 1–8 (2018).

    CAS  Google Scholar 

  38. Greenfield, L. K. et al. Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens. J. Biol. Chem. 287, 38135–38149 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Guachalla, L. M. et al. Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3. Sci. Rep. 7, 6635 (2017).

    PubMed  PubMed Central  Google Scholar 

  40. Ovchinnikova, O. G. et al. Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99). Proc. Natl Acad. Sci. USA 113, E3120–E3129 (2016).

    CAS  PubMed  Google Scholar 

  41. Williams, D. M. et al. Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control. Proc. Natl Acad. Sci. USA 114, E1215–E1223 (2017).

    CAS  PubMed  Google Scholar 

  42. Ninomiya, T. et al. Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4. J. Biol. Chem. 277, 21567–21575 (2002).

    CAS  PubMed  Google Scholar 

  43. Litschko, C. et al. A new family of capsule polymerases generates teichoic acid-like capsule polymers in Gram-negative pathogens. mBio. 9, 35 (2018).

    Google Scholar 

  44. Mann, E., Kimber, M. S. & Whitfield, C. Bioinformatics analysis of diversity in bacterial glycan chain-termination chemistry and organization of carbohydrate binding modules linked to ABC transporters. Glycobiol. 29, 822–838 (2019).

    Google Scholar 

  45. Osawa, T. et al. Crystal structure of chondroitin polymerase from Escherichia coli K4. Biochem. Biophys. Res. Commun. 378, 10–14 (2009).

    CAS  PubMed  Google Scholar 

  46. Doyle, L. et al. Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules. Nat. Chem. Biol. 15, 632–640 (2019).

    CAS  PubMed  Google Scholar 

  47. Liston, S. D. et al. Domain interactions control complex formation and polymerase specificity in the biosynthesis of the Escherichia coli O9a antigen. J. Biol. Chem. 290, 1075–1085 (2015).

    CAS  PubMed  Google Scholar 

  48. Clarke, B. R., Greenfield, L. K., Bouwman, C. & Whitfield, C. Coordination of polymerization, chain termination, and export in assembly of the Escherichia coli O9a antigen in an ABC transporter-dependent pathway. J. Biol. Chem. 284, 30662–30672 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Meth. 9, 671–675 (2012).

    CAS  Google Scholar 

  50. Tsai, G. M. & Frasch, C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115–119 (1982).

    CAS  PubMed  Google Scholar 

  51. Galanos, C., Lüderitz, O. & Westphal, O. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9, 245–249 (1969).

    CAS  PubMed  Google Scholar 

  52. Kabsch, W. XDS. Acta Crystallogr. D. 66, 125–132 (2010).

    CAS  PubMed  Google Scholar 

  53. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D. 58, 1948–1954 (2002).

    PubMed  Google Scholar 

  54. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  55. Marchler-Bauer, A. et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 45, D200–D203 (2017).

    CAS  PubMed  Google Scholar 

  56. Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 30, 3059 (2017).

    Google Scholar 

  57. Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0NCBI. Syst. Biol. 59, 307–321 (2010).

    CAS  PubMed  Google Scholar 

  58. Gouet, P. & Robert, X. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors wish to thank S. Labiuk at the Canadian Light Source for help with data collection and S. Al-Abdul-Wahid and A. Lo for technical support with NMR spectroscopy. These studies were supported by individual Natural Sciences and Engineering Research Council Discovery Grants awarded to M.S.K., T.L.L., and C.W. T.L.L. and C.W. hold Canada Research Chairs and S.D.K. is a recipient of a Natural Sciences and Engineering Research Council Postgraduate Scholarship.

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B.R.C. performed all of the biochemical experiments and phylogenetics analysis shown. O.G.O. performed NMR experiments and participated in interpretation of mass spectrometry data. R.P.S. synthesized the synthetic oligosaccharide acceptors. E.R.K.-H. generated site-directed mutants and performed initial activity assessments. R.G., E.R.K.-H. and E.M. crystallized WbbM and collected X-ray data. S.D.K. purified LPS samples, performed mass spectrometry analysis, together with O.G.O. T.L.L. oversaw the work of R.P.S. and contributed to data analysis. M.S.K. oversaw the work of E.R.K.-H., R.G. and E.M. assisted with crystallographic refinements, identified targets for mutagenesis and performed bioinformatic analysis of the membrane-associated C terminus. C.W. conceived the project and oversaw the biochemical studies and data analysis performed by B.R.C., O.G.O. and S.D.K. The initial draft of the paper was prepared by B.R.C., O.G.O., M.S.K. and C.W., and all authors made contributions to the final version.

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Correspondence to Matthew S. Kimber or Chris Whitfield.

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Clarke, B.R., Ovchinnikova, O.G., Sweeney, R.P. et al. A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family. Nat Chem Biol 16, 450–457 (2020). https://doi.org/10.1038/s41589-020-0494-0

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