Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Chemical polyglycosylation and nanolitre detection enables single-molecule recapitulation of bacterial sugar export

Abstract

The outermost protective layer of both Gram-positive and Gram-negative bacteria is composed of bacterial capsular polysaccharides. Insights into the interactions between the capsular polysaccharide and its transporter and the mechanism of sugar export would not only increase our understanding of this key process, but would also help in the design of novel therapeutics to block capsular polysaccharide export. Here, we report a nanolitre detection system that makes use of the bilayer interface between two droplets, and we use this system to study single-molecule recapitulation of sugar export. A synthetic strategy of polyglycosylation based on tetrasaccharide monomers enables ready synthetic access to extended fragments of K30 oligosaccharides and polysaccharides. Examination of the interactions between the Escherichia coli sugar transporter Wza and very small amounts of fragments of the K30 capsular polysaccharide substrate reveal the translocation of smaller but not larger fragments. We also observe capture events that occur only on the intracellular side of Wza, which would complement coordinated feeding by adjunct biosynthetic machinery.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Natural and recapitulated export of K30 CPS through the membrane protein Wza.
Figure 2: Retrosynthetic analysis of K30 polysaccharide synthesis.
Figure 3: Synthesis of disaccharide building blocks 4+5 and 7+8 and their assembly into tetrasaccharide ‘monomers’ by 2+2 glycosylation.
Figure 4: Polyglycosylation of tetrasaccharide ‘monomer’ 6 for K30.
Figure 5: Development of DIB-fusion system to allow analysis of the interaction between Wza pore ΔP106-A107 and K30 fragments K30-1n=1, K30-2n=1, K30-2n=2 and K30-2n=3.
Figure 6: Analysis of the interaction between Wza pore ΔP106-A107 and K30 fragments K30-1n=1, K30-2n=1, K30-2n=2 and K30-2n=3 using the DIB-fusion system.

Similar content being viewed by others

References

  1. Lebeer, S., Vanderleyden, J. & De Keersmaecker, S. C. Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nature Rev. Microbiol. 8, 171–184 (2010).

    CAS  Google Scholar 

  2. Bushell, S. R. et al. Crystallization and preliminary diffraction analysis of Wzi, a member of the capsule export and assembly pathway in Escherichia coli. Acta Crystallogr. F 66, 1621–1625 (2010).

    CAS  Google Scholar 

  3. Cuthbertson, L., Mainprize, I. L., Naismith, J. H. & Whitfield, C. Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in Gram-negative bacteria. Microbiol. Mol. Biol. Rev. 73, 155–177 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444, 226–229 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Whitfield, C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu. Rev. Biochem. 75, 39–68 (2006).

    CAS  PubMed  Google Scholar 

  6. Nickerson, N. N. et al. Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes. Proc. Natl Acad. Sci. USA 111, 8203–8208 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Bushell, S. R. et al. Wzi is an outer membrane lectin that underpins group 1 capsule assembly in Escherichia coli. Structure 21, 844–853 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 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  PubMed  Google Scholar 

  9. Micoli, F. et al. Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X. Proc. Natl Acad. Sci. USA 110, 19077–19082 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Adamo, R. et al. Synthetically defined glycoprotein vaccines: current status and future directions. Chem. Sci. 4, 2995–3008 (2013).

    CAS  PubMed  Google Scholar 

  11. Boltje, T. J., Buskas, T. & Boons, G.-J. Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research. Nature Chem. 1, 611–622 (2009).

    CAS  Google Scholar 

  12. Wang, L.-X. & Davis, B. G. Realizing the promise of chemical glycobiology. Chem. Sci. 4, 3381–3394 (2013).

    CAS  PubMed  Google Scholar 

  13. Hudak, J. E. & Bertozzi, C. R. Glycotherapy: new advances inspire a reemergence of glycans in medicine. Chem. Biol. 21, 16–37 (2014).

    CAS  PubMed  Google Scholar 

  14. Hungerer, D., Jann, K., Jann, B., Orskov, F. & Orskov, I. Immunochemistry of K antigens of Escherichia coli. 4. The K antigen of E. coli O9:K30:H12. Eur. J. Biochem. 2, 115–126 (1967).

    CAS  PubMed  Google Scholar 

  15. Schuster, H. J., Vijayakrishnan, B. & Davis, B. G. Chain-growth polyglycosylation: synthesis of linker-equipped mannosyl oligomers. Carbohydr. Res. 403, 135–141 (2015).

    CAS  PubMed  Google Scholar 

  16. Kochetkov, N. K., Betaneh, V. I., Ovchmmkov, M. V. & Backmowsky, L. V. Synthesis of the O-antigenic polysaccharide of Salmonella newington and of its analogue differing in configuration at the only glycosidic centre. Tetrahedron 37, 149 (1981).

    Google Scholar 

  17. Kochetkov, N. K. Tetrahedron report number 218: synthesis of polysaccharides with a regular structure. Tetrahedron 43, 2389–2436 (1987).

    CAS  Google Scholar 

  18. Nifantiev, N. E., Backmowsky, L. V. & Kochetkov, N. K. Synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 14. Bioorg Khzm (USSR) 13, 273 (1987).

    Google Scholar 

  19. Kong, L. et al. Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor. Nature Chem. 5, 651–659 (2013).

    CAS  Google Scholar 

  20. Fischer, A., Holden, M. A., Pentelute, B. L. & Collier, R. J. Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers. Proc. Natl Acad. Sci. USA 108, 16577–16581.

  21. Holden, M. A., Needham, D. & Bayley, H. Functional bionetworks from nanoliter water droplets. J. Am. Chem. Soc. 129, 8650–8655 (2007).

    CAS  PubMed  Google Scholar 

  22. Hwang, W. L., Chen, M., Cronin, B., Holden, M. A. & Bayley, H. Asymmetric droplet interface bilayers. J. Am. Chem. Soc. 130, 5878–5879 (2008).

    CAS  PubMed  Google Scholar 

  23. Hwang, W. L., Holden, M. A., White, S. & Bayley, H. Electrical behavior of droplet interface bilayer networks: experimental analysis and modeling. J. Am. Chem. Soc. 129, 11854–11864 (2007).

    CAS  PubMed  Google Scholar 

  24. Syeda, R., Holden, M. A., Hwang, W. L. & Bayley, H. Screening blockers against a potassium channel with a droplet interface bilayer array. J. Am. Chem. Soc. 130, 15543–15548 (2008).

    CAS  PubMed  Google Scholar 

  25. Lein, M., Huang, J. & Holden, M. A. Robust reagent addition and perfusion strategy for droplet-interface bilayers. Lab Chip 13, 2749–2753 (2013).

    CAS  PubMed  Google Scholar 

  26. Li, Z. & Gildersleeve, J. C. Mechanistic studies and methods to prevent aglycon transfer of thioglycosides. J. Am. Chem. Soc. 128, 11612–11619 (2006).

    CAS  PubMed  Google Scholar 

  27. Kato, M., Hirai, G. & Sodeoka, M. Studies on the selectivity between glycosylation and intermolecular aglycone transfer of thioglucoside in synthesis of lactose derivatives. Chem. Lett. 40, 877–879 (2011).

    CAS  Google Scholar 

  28. Cheng, L., Chen, Q., Liu, J. & Du, Y. Synthesis of a fluorescence-labeled K30 antigen repeating unit using click chemistry. Carbohydr. Res. 342, 975–981 (2007).

    CAS  PubMed  Google Scholar 

  29. Bazin, H. G., Wolff, M. W. & Linhardt, R. J. Regio- and stereoselective synthesis of β-D-gluco-, α-L-ido-, and α-L-altropyranosiduronic acids from Δ(4)-uronates. J. Org. Chem. 64, 144–152 (1999).

    CAS  PubMed  Google Scholar 

  30. Matsuo, I., Isomura, M., Miyazaki, T., Sakakibara, T. & Ajisaka, K. Chemoenzymatic synthesis of the branched oligosaccharides which correspond to the core structures of N-linked sugar chains. Carbohydr. Res. 305, 401–413 (1997).

    CAS  PubMed  Google Scholar 

  31. de Kort, M. et al. Synthesis of potent agonists of the D-myo-inositol 1,4,5-trisphosphate receptor based on clustered disaccharide polyphosphate analogues of adenophostin A. J. Med. Chem. 43, 3295–3303 (2000).

    CAS  PubMed  Google Scholar 

  32. Kullman, L., Winterhalter, M. & Bezrukov, S. M. Transport of maltodextrins through maltoporin: a single-channel study. Biophys. J. 82, 803–812 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Dumas, F., Koebnik, R., Winterhalter, M. & Van Gelder, P. Sugar transport through maltoporin of Escherichia coli: role of polar tracks. J. Biol. Chem. 275, 19747–19751 (2000).

    CAS  PubMed  Google Scholar 

  34. Sanchez-Quesada, J., Ghadiri, M. R., Bayley, H. & Braha, O. Cyclic peptides as molecular adapters for a pore-forming protein. J. Am. Chem. Soc. 122, 11758–11766 (2000).

    Google Scholar 

  35. Gu, L.-Q., Cheley, S. & Bayley, H. Capture of a single molecule in a nanocavity. Science 291, 636–640 (2001).

    CAS  PubMed  Google Scholar 

  36. Sattelle, B. M. & Almond, A. Shaping up for structural glycomics: a predictive protocol for oligosaccharide conformational analysis applied to N-linked glycans. Carbohydr. Res. 383, 34–42 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Harvey, M. J., Giupponi, G. & Fabritiis, G. D. ACEMD: accelerating bio-molecular dynamics in the microsecond time-scale. J. Chem. Theory Comput. 5, 1632–1639 (2009).

    CAS  PubMed  Google Scholar 

  38. Schirmer, T., Keller, T. A., Wang, Y. F. & Rosenbusch, J. P. Structural basis for sugar translocation through maltoporin channels at 3.1 Å resolution. Science 267, 512–514 (1995).

    CAS  PubMed  Google Scholar 

  39. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Collins, R. F. et al. The 3D structure of a periplasm-spanning platform required for assembly of group 1 capsular polysaccharides in Escherichia coli. Proc. Natl Acad. Sci. USA 104, 2390–2395 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Bohne, A., Lang, E. & von der Lieth, C. W. W3-SWEET: carbohydrate modeling by internet. J. Mol. Model. 4, 33–43 (1998).

    CAS  Google Scholar 

  42. Bohne, A., Lang, E. & von der Lieth, C. W. SWEET—WWW-based rapid 3D construction of oligo- and polysaccharides. Bioinformatics 15, 767–768 (1999).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the Medical Research Council and the Engineering and Physical Sciences Research Council for financial support. L.K. has received a Wellcome Trust VIP award and a UK/China Postgraduate Research Scholarship. B.G.D. was a Royal Society Wolfson Research Merit Award recipient during this work.

Author information

Authors and Affiliations

Authors

Contributions

L.K., H.B. and B.G.D. designed the experiments. L.K. performed the experiments. A.A. designed, performed and analysed the MD simulations. L.K., H.B. and B.G.D. analysed the results. L.K., H.B. and B.G.D. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Hagan Bayley or Benjamin G. Davis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 9778 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kong, L., Almond, A., Bayley, H. et al. Chemical polyglycosylation and nanolitre detection enables single-molecule recapitulation of bacterial sugar export. Nature Chem 8, 461–469 (2016). https://doi.org/10.1038/nchem.2487

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2487

This article is cited by

Search

Quick links

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