Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules

Abstract

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-β-d-manno-oct-2-ulosonic acid (β-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5′-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal β-(2→4) Kdo transferase from KpsC reveals two α/β domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal β-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 β-Kdo transferases. This enzyme provides the prototype for the GT107 family.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Unrooted phylogenetic tree of KpsS and KpsC homologs from T. dismutans and pathogens of humans and livestock.
Fig. 2: Biochemical characterization of KpsC-NTd/KpsC-CTd and KpsCEc.
Fig. 3: Structural characterization of the in vitro KpsS product.
Fig. 4: KpsC structure analysis.
Fig. 5: E. coli KpsC-N active site organization.
Fig. 6: Model for assembly of CPS in a KpsS- and KpsC-dependent pathway.

Data availability

Crystallographic datasets have been deposited in the PDB repository under accession codes 6MGB (KpsC-NTd), 6MGC (KpsC-NEc) and 6MGD (KpsC-CTd). Datasets will become publicly available upon manuscript acceptance. Raw NMR and MS data are available from the corresponding authors upon request.

References

  1. 1.

    Willis, L. M. & Whitfield, C. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways. Carbohydr. Res. 378, 35–44 (2013).

    CAS  Article  Google Scholar 

  2. 2.

    Lambris, J. D., Ricklin, D. & Geisbrecht, B. V. Complement evasion by human pathogens. Nat. Rev. Microbiol. 6, 132–142 (2008).

    CAS  Article  Google Scholar 

  3. 3.

    Micoli, F., Costantino, P. & Adamo, R. Potential targets for next generation antimicrobial glycoconjugate vaccines. FEMS Microbiol. Rev. 42, 388–423 (2018).

    CAS  Article  Google Scholar 

  4. 4.

    Cress, B. F. et al. Masquerading microbial pathogens: capsular polysaccharides mimic host-tissue molecules. FEMS Microbiol. Rev. 38, 660–697 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Gao, Z. et al. High-throughput ‘FP-tag’ assay for the identification of glycosyltransferase inhibitors. J. Am. Chem. Soc. 141, 2201–2204 (2019).

    CAS  Article  Google Scholar 

  6. 6.

    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  Article  Google Scholar 

  7. 7.

    Toukach, P. V. Bacterial carbohydrate structure database 3: principles and realization. J. Chem. Inf. Model. 51, 159–170 (2011).

    CAS  Article  Google Scholar 

  8. 8.

    Holst, O. The structures of core regions from enterobacterial lipopolysaccharides—an update. FEMS Microbiol. Lett. 271, 3–11 (2007).

    CAS  Article  Google Scholar 

  9. 9.

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

    CAS  Article  Google Scholar 

  10. 10.

    Willis, L. M. et al. Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens. Proc. Natl Acad. Sci. USA 110, 7868–7873 (2013).

    CAS  Article  Google Scholar 

  11. 11.

    Willis, L. M. & Whitfield, C. KpsC and KpsS are retaining 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules. Proc. Natl Acad. Sci. USA 110, 20753–20758 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    Ovchinnikova, O. G. et al. Biochemical characterization of bifunctional 3-deoxy-β-d-manno-oct-2-ulosonic acid (β-Kdo) transferase KpsC from Escherichia coli involved in capsule biosynthesis. J. Biol. Chem. 291, 21519–21530 (2016).

    CAS  Article  Google Scholar 

  13. 13.

    Lin, C.-H., Murray, B. W., Ollmann, I. R. & Wong, C.-H. Why is CMP-ketodeoxyoctonate highly unstable? Biochemistry 36, 780–785 (1997).

    CAS  Article  Google Scholar 

  14. 14.

    Slobodkin, A. I. et al. Thermosulfurimonas dismutans gen. nov., sp. nov., an extremely thermophilic sulfur-disproportionating bacterium from a deep-sea hydrothermal vent. Int. J. Syst. Evol. Microbiol. 62, 2565–2571 (2012).

    CAS  Article  Google Scholar 

  15. 15.

    Itabashi, Y. & Kuksis, A. Reassessment of stereochemical configuration of natural phosphatidylglycerols by chiral-phase high-performance liquid chromatography and electrospray mass spectrometry. Anal. Biochem. 254, 49–56 (1997).

    CAS  Article  Google Scholar 

  16. 16.

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

    CAS  Article  Google Scholar 

  17. 17.

    Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44, W351–W355 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Zheng, L., Lin, Y., Lu, S., Zhang, J. & Bogdanov, M. Biogenesis, transport and remodeling of lysophospholipids in Gram-negative bacteria. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1862, 1404–1413 (2017).

    CAS  Article  Google Scholar 

  19. 19.

    Rigg, G. P., Barrett, B. & Roberts, I. S. The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex. Microbiology 144, 2905–2914 (1998).

    CAS  Article  Google Scholar 

  20. 20.

    McNulty, C. et al. The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell. Mol. Microbiol. 59, 907–922 (2006).

    CAS  Article  Google Scholar 

  21. 21.

    Steenbergen, S. M. & Vimr, E. R. Biosynthesis of the Escherichia coli K1 group 2 polysialic acid capsule occurs within a protected cytoplasmic compartment. Mol. Microbiol. 68, 1252–1267 (2008).

    CAS  Article  Google Scholar 

  22. 22.

    Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014).

    CAS  Article  Google Scholar 

  23. 23.

    Albesa-jové, D., Giganti, D. & Jackson, M. Structure–function relationships of membrane-associated GT-B glycosyltransferases. Glycobiology 24, 108–124 (2014).

    Article  Google Scholar 

  24. 24.

    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  Article  Google Scholar 

  25. 25.

    Zhang, G. et al. Cell-based screen for discovering lipopolysaccharide biogenesis inhibitors. Proc. Natl Acad. Sci. USA 115, 6834–6839 (2018).

    CAS  Article  Google Scholar 

  26. 26.

    Arshad, M., Goller, C. C., Pilla, D., Schoenen, F. J. & Seed, P. C. Threading the needle: small-molecule targeting of a xenobiotic receptor to ablate Escherichia coli polysaccharide capsule expression without altering antibiotic resistance. J. Infect. Dis. 213, 1330–1339 (2016).

    CAS  Article  Google Scholar 

  27. 27.

    Allen, R. C., Popat, R., Diggle, S. P. & Brown, S. P. Targeting virulence: can we make evolution-proof drugs? Nat. Rev. Microbiol. 12, 300–308 (2014).

    CAS  Article  Google Scholar 

  28. 28.

    Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. https://doi.org/10.1093/bib/bbx108 (2017).

  29. 29.

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

    CAS  Article  Google Scholar 

  30. 30.

    Lefort, V., Longueville, J.-E. & Gascuel, O. SMS: smart model selection in PhyML. Mol. Biol. Evol. 34, 2422–2424 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    Vimr, E. R. & Troy, F. A. Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli. J. Bacteriol. 164, 845–853 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Gronow, S., Brabetz, W. & Brade, H. Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from Escherichia coli. Eur. J. Biochem. 267, 6602–6611 (2000).

    CAS  Article  Google Scholar 

  33. 33.

    Makarova, O., Kamberov, E. & Margolis, B. Generation of deletion and point mutations with one primer in a single cloning step. Biotechniques 29, 970–972 (2000).

    CAS  Article  Google Scholar 

  34. 34.

    García-Nafría, J., Watson, J. F. & Greger, I. H. IVA cloning: a single-tube universal cloning system exploiting bacterial in vivo assembly. Sci. Rep. 6, 27459 (2016).

    Article  Google Scholar 

  35. 35.

    Guerrero, S. A., Hecht, H.-J., Hofmann, B., Biebl, H. & Singh, M. Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems. Appl. Microbiol. Biotechnol. 56, 718–723 (2001).

    CAS  Article  Google Scholar 

  36. 36.

    Klonis, N. & Sawyer, W. H. Spectral properties of the prototropic forms of fluorescein in aqueous solution. J. Fluorescence 6, 147–157 (1996).

    CAS  Article  Google Scholar 

  37. 37.

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

    CAS  Article  Google Scholar 

  38. 38.

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

    Article  Google Scholar 

  39. 39.

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

    Article  Google Scholar 

  40. 40.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

These studies were supported by funding from the Canadian Institutes of Health Research (C.W.), the Natural Sciences and Engineering Research Council (T.L.L., M.S.K. and C.W.) and the Canadian Glycomics Network (C.W.). T.L.L. and C.W. hold Canada Research Chairs and L.D. is a recipient of a Natural Sciences and Engineering Research Council Postgraduate Scholarship. The authors thank W. Wakarchuk for help and advice concerning the HPLC strategy to resolve product chain lengths.

Author information

Affiliations

Authors

Contributions

L.D. performed the cloning of constructs expressing KpsC, biochemically characterized and crystallized the enzymes and participated in determining and interpreting the structures. O.G.O. performed HPLC experiments and the structural analysis of the KpsS product. K.M. performed in vitro experiments with the NBD-PG lipid acceptor. E.M. collected X-ray data for KpsC and assisted L.D. with crystallographic refinements. B.-S.H. synthesized the synthetic oligosaccharide acceptors for KpsC. T.L.L. oversaw the work of B.-S.H. and contributed to data analysis; M.S.K. assisted with crystallographic refinements, oversaw the work of E.M. and the collection and interpretation of the crystallographic data. C.W. conceived the project and oversaw the biochemical studies and data analysis performed by L.D. and O.G.O. L.D., O.G.O., M.S.K. and C.W. prepared the initial draft of the paper and all authors made contributions to the final version.

Corresponding authors

Correspondence to Matthew S. Kimber or Chris Whitfield.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary

Supplementary Tables 1–6 and Supplementary Figures 1–10

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Doyle, L., Ovchinnikova, O.G., Myler, K. et al. Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules. Nat Chem Biol 15, 632–640 (2019). https://doi.org/10.1038/s41589-019-0276-8

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing