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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
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
Similar content being viewed by others
References
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).
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).
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).
Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444, 226–229 (2006).
Whitfield, C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu. Rev. Biochem. 75, 39–68 (2006).
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).
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).
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).
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).
Adamo, R. et al. Synthetically defined glycoprotein vaccines: current status and future directions. Chem. Sci. 4, 2995–3008 (2013).
Boltje, T. J., Buskas, T. & Boons, G.-J. Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research. Nature Chem. 1, 611–622 (2009).
Wang, L.-X. & Davis, B. G. Realizing the promise of chemical glycobiology. Chem. Sci. 4, 3381–3394 (2013).
Hudak, J. E. & Bertozzi, C. R. Glycotherapy: new advances inspire a reemergence of glycans in medicine. Chem. Biol. 21, 16–37 (2014).
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).
Schuster, H. J., Vijayakrishnan, B. & Davis, B. G. Chain-growth polyglycosylation: synthesis of linker-equipped mannosyl oligomers. Carbohydr. Res. 403, 135–141 (2015).
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).
Kochetkov, N. K. Tetrahedron report number 218: synthesis of polysaccharides with a regular structure. Tetrahedron 43, 2389–2436 (1987).
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).
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).
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.
Holden, M. A., Needham, D. & Bayley, H. Functional bionetworks from nanoliter water droplets. J. Am. Chem. Soc. 129, 8650–8655 (2007).
Hwang, W. L., Chen, M., Cronin, B., Holden, M. A. & Bayley, H. Asymmetric droplet interface bilayers. J. Am. Chem. Soc. 130, 5878–5879 (2008).
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).
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).
Lein, M., Huang, J. & Holden, M. A. Robust reagent addition and perfusion strategy for droplet-interface bilayers. Lab Chip 13, 2749–2753 (2013).
Li, Z. & Gildersleeve, J. C. Mechanistic studies and methods to prevent aglycon transfer of thioglycosides. J. Am. Chem. Soc. 128, 11612–11619 (2006).
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).
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).
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).
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).
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).
Kullman, L., Winterhalter, M. & Bezrukov, S. M. Transport of maltodextrins through maltoporin: a single-channel study. Biophys. J. 82, 803–812 (2002).
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).
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).
Gu, L.-Q., Cheley, S. & Bayley, H. Capture of a single molecule in a nanocavity. Science 291, 636–640 (2001).
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).
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).
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).
Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).
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).
Bohne, A., Lang, E. & von der Lieth, C. W. W3-SWEET: carbohydrate modeling by internet. J. Mol. Model. 4, 33–43 (1998).
Bohne, A., Lang, E. & von der Lieth, C. W. SWEET—WWW-based rapid 3D construction of oligo- and polysaccharides. Bioinformatics 15, 767–768 (1999).
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
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
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary information
Supplementary information (PDF 9778 kb)
Rights and permissions
About this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchem.2487
This article is cited by
-
Studying glycobiology at the single-molecule level
Nature Reviews Chemistry (2018)