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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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.
Custom Python scripts used in this study are available at https://github.com/bclarke2/wbbM_glf_scripts.
Whitfield, C. & Trent, M. S. Biosynthesis and export of bacterial lipopolysaccharides. Annu. Rev. Biochem. 83, 99–128 (2014).
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).
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).
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).
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).
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).
Rollenske, T. et al. Cross-specificity of protective human antibodies against Klebsiella pneumoniae LPS O-antigen. Nat. Immunol. 19, 617–624 (2018).
Follador, R. et al. The diversity of Klebsiella pneumoniae surface polysaccharides. Microbial Genomics 2, e000073 (2016).
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).
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).
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).
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).
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).
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).
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).
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).
Lukose, V., Walvoort, M. T. & Imperiali, B. Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface. Glycobiol. 27, 820–833 (2017).
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).
Liu, D. & Reeves, P. R. Escherichia coli K12 regains its O antigen. Microbiol. 140, 49–57 (1994).
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).
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).
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).
Lairson, L. L., Henrissat, B., Davies, G. J. & Withers, S. G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 77, 521–555 (2008).
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).
Chaikuad, A. et al. Conformational plasticity of glycogenin and its maltosaccharide substrate during glycogen biogenesis. Proc. Natl Acad. Sci. USA 108, 21028–21033 (2011).
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).
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).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
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).
Ishida, T. & Kinoshita, K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res. 35, W460–W464 (2007).
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).
Toukach, P. V. & Egorova, K. S. Carbohydrate structure database merged from bacterial, archaeal, plant and fungal parts. Nucleic Acids Res. 44, D1229–D1236 (2016).
Zhang, H. et al. The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold. Nat. Commun. 5, 4339 (2014).
Geno, K. A. et al. Pneumococcal capsules and their types: past, present, and future. Clin. Microbiol. Rev. 28, 871–899 (2015).
Zeidan, A. A. et al. Polysaccharide production by lactic acid bacteria: from genes to industrial applications. FEMS Microbiol. Rev. 41, S168–S200 (2017).
Zhou, Y., Cui, Y. & Qu, X. Exopolysaccharides of lactic acid bacteria: structure, bioactivity and associations: A review. Carbohydr. Polym. 207, 317–332 (2019).
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).
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).
Guachalla, L. M. et al. Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3. Sci. Rep. 7, 6635 (2017).
Ovchinnikova, O. G. et al. Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99). Proc. Natl Acad. Sci. USA 113, E3120–E3129 (2016).
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).
Ninomiya, T. et al. Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4. J. Biol. Chem. 277, 21567–21575 (2002).
Litschko, C. et al. A new family of capsule polymerases generates teichoic acid-like capsule polymers in Gram-negative pathogens. mBio. 9, 35 (2018).
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).
Osawa, T. et al. Crystal structure of chondroitin polymerase from Escherichia coli K4. Biochem. Biophys. Res. Commun. 378, 10–14 (2009).
Doyle, L. et al. Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules. Nat. Chem. Biol. 15, 632–640 (2019).
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).
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).
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Meth. 9, 671–675 (2012).
Tsai, G. M. & Frasch, C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115–119 (1982).
Galanos, C., Lüderitz, O. & Westphal, O. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9, 245–249 (1969).
Kabsch, W. XDS. Acta Crystallogr. D. 66, 125–132 (2010).
Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D. 58, 1948–1954 (2002).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. 60, 2126–2132 (2004).
Marchler-Bauer, A. et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 45, D200–D203 (2017).
Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 30, 3059 (2017).
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).
Gouet, P. & Robert, X. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
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.
The authors declare no competing interests.
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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
Annual Review of Biochemistry (2020)