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

Journal name:
Nature Chemistry
Year published:
Published online


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.

At a glance


  1. Natural and recapitulated export of K30 CPS through the membrane protein Wza.
    Figure 1: Natural and recapitulated export of K30 CPS through the membrane protein Wza.

    a, Schematic of the structure of the cell surface in E. coli E69 and the associated machinery for K30 CPS export. The structure of a 16-repeating-unit fragment of K30 CPS was generated using SWEET219, 41, 42 and superimposed on the cross-sectional structure of wild-type Wza in PyMOL. This structure was used to generate the sphere ‘cartoon’ representation shown. The glycosyl units that make up K30 are represented by coloured spheres (green, α-D-mannosyl; magenta, β-D-galactosyl; blue, α-D-galactosyl; brown, β-D-glucuronyl). b, Chemical structure of K30 CPS. The two repeating oligosaccharidic units chosen as ‘monomer’ tetrasaccharides for polyglycosylation are shown highlighted by the grey boxes in the K30 polymer and are also drawn next to their corresponding structures (K30-1 and K30-2). The glycosyl units are represented by coloured spheres (green, α-D-mannosyl; magnenta, β-D-galactosyl; blue, α-D-galactosyl; brown, β-D-glucuronyl). c, A combined polyglycosylation and DIB-fusion strategy for the recapitulation of K30 sugar export through membrane protein Wza, allowing the detection of the Wza–sugar substrate interaction. Steps i to v represent a generalizable polyglycosylation synthetic strategy to create a defined polysaccharide substrate for analysis: (i) analysis of K30 CPS structures (see b); (ii) chemical synthesis of polymerizable partially protected repeating units of K30 CPS; (iii) polyglycosylation of polymerizable partially protected repeating units of K30 CPS; (iv) isolation and deprotection (represented by removal of sphere ‘shells’) of polysaccharides to afford representative K30 CPS fragments after separation; (v) formation of droplets containing fragments of K30 CPS suitable for analysis. Steps vi to viii represent the creation of a nanolitre single-molecule transport system: (vi) analysis of membrane protein Wza; (vii) protein engineering to afford constitutively open Wza pores; (viii) recapitulation of active Wza in a two-droplet bilayer (DIB) system. Step (ix) is the fusion of the K30 CPS fragment droplet (from step v) to either the cis or trans droplet of the DIB droplet pair. In the example shown, the cis droplet is fused with the substrate droplet, but the trans droplet could also be fused.

  2. Retrosynthetic analysis of K30 polysaccharide synthesis.
    Figure 2: Retrosynthetic analysis of K30 polysaccharide synthesis.

    a,b, Two repeating oligosaccharide units were chosen as ‘monomers’ for polyglycosylation. Tetrasaccharides 3 and 6 were chosen for the preparation of two K30 types: K30-1 from 3 (a) and K30-2 from 6 (b). These polymerizable repeating unit ‘monomers’ 3 and 6 were prepared using ‘2+2’ glycosylation strategies, 4+5 and 7+8, from disaccharide building blocks.

  3. Synthesis of disaccharide building blocks 4+5 and 7+8 and their assembly into tetrasaccharide ‘monomers’ by 2+2 glycosylation.
    Figure 3: Synthesis of disaccharide building blocks 4+5 and 7+8 and their assembly into tetrasaccharide ‘monomers’ by 2+2 glycosylation.

    Reagents and conditions. (a) PhCH(OMe)2, camphor sulfonic acid (CSA), dimethylformamide (DMF), 70%. (b) BnBr, dichloromethane (DCM), n-Bu4NHSO4, NaOH, aq. reflux, 23% 10, 35% 21 (Supplementary Fig. 2). (c) 11,TMSOTf, DCM, −20 °C. (d) 80% AcOH aq. 60 °C, 6 h, 75%. (e) Ac2O, pyr., 93%. (f) NIS, Tf2O, DCM/water (100:1, vol/vol), 62%. (g) CCl3CN, 1,8-diazabicycloundec-7-ene (DBU), DCM, 45% 4, 31% 7. (h) FmocCl, pyr., overnight. (i) Ac2O, pyr. (j) Et3N, DCM, 57% for three steps. (k) (step 1) 14, TMSOTf, DCM, −20 °C, 81%. (step 2) NaOMe, MeOH, 72%. (l) PhCH(OMe)2, CSA, DMF, 50%. (m) 80% AcOH aq. 70 °C, 3 h. (n) Ac2O, pyr., 69% over two steps. (o) (step 1) NIS, trifluoroacetic acid (TFA), DCM/water, 1 h; (step 2) CCl3CN, DCM, DBU, 71% for two steps. (p) NaH, BnBr, DMF, 64%. (q) (step 1) NIS, TFA, DCM/water, 1 h; (step 2) CCl3CN, DCM, DBU, 56% for two steps. (r) TMSOTf, HSCHMe2, DCM, 65%. (s) Pd/C, H2, 86%. (t) Butanedione, CSA, CH(OMe)3, DCM, 35%. (u) TBSOTf, 2,6-lutidine, DCM, 99%. (v) TBSOTf, 2,6-lutidine, DCM, 99%. (w) TMSOTf, DCM, −20 °C. (x) TFA/water (9:1, vol/vol), 29% for two steps. (y) TBSOTf, 2,6-lutidine, DCM, 100%. (z) TMSOTf, DCM, −20 °C. See Supplementary Fig. 2 for additional details and compounds. Although different trichloroacetimidate anomers were used here (indicated by the added α and β symbols for the cartoon representations of 4 and 7, respectively), there was no significant difference in the reactivity of anomers in these TMSOTf-activated ‘2+2’ glycosylations, although the variation of conditions with other activators allowed variation of stereoselectivity (see main text).

  4. Polyglycosylation of tetrasaccharide ‘monomer’ 6 for K30.
    Figure 4: Polyglycosylation of tetrasaccharide ‘monomer’ 6 for K30.

    a, Polyglycosylation of tetrasaccharide monomer 6 yielded K30-2n=y where y = 1,2,3,4,5,6… Reagents and conditions: (i) NIS, TMSOTf, 3 h, −30 to −20 ° C, 55%; X = NHC(O)CH2CH2COOH, see Supplementary Section ‘Characterization’ for details. (ii) 0.5 N NaOH, MeOH-DCM, 1 day, 100%. b, MALDI-MS spectrum of ‘monomer’ 6. c, MALDI-MS spectrum of the product of polyglycosylation shows a distribution with iterative increases in mass consistent with the expected polyglycosylation.

  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 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.

    A three-droplet DIB-fusion system based on a bilayer interface between cis and trans droplets was constructed. An aperture (∼200 μm in diameter) in the Teflon film (red and red arrow) restricts the size of the bilayer and provides stability. Oil is shown in light blue. Steps (1) and (2) show the fusion of a third droplet, containing the oligosaccharide substrate, to the cis droplet. The distance between the cis (orange) and trans (dark blue) electrodes was kept constant. The substrate droplet was fused with the cis droplet using a translational manipulator (purple) in the substrate droplet to mechanically drive the fusion process. The mutant Wza pore ΔP106-A107 (dark blue) was inserted into the lipid bilayer from the cis droplet (buffer, 300 mM KCl with 5 mM HEPES, pH 7.5).

  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.
    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.

    ad, Scatter plots of mutant Wza pore ΔP106-A107 interaction with K30-1n=1 (10 mM), K30-2n=1 (100 mM), K30-2n=2 (5 mM) and K30-2n=3 (1 mM) in 300 mM KCl buffer at +25 mV, respectively. e, Lifetimes of K30-1n=1 (black, 10 mM), K30-2n=1 (red, 100 mM), K30-2n=2 (blue, 5 mM) and K30-2n=3 (purple, 1 mM) associated with the mutant Wza pore ΔP106-A107 versus the applied potential. Each point is derived from the mean of three experiments and the standard deviation (s.d.) is shown (mean ± s.d., n = 3) fi, Representative interaction events of K30-1n=1 (f), K30-2n=1 (g), K30-2n=2 (h) and K30-2n=3 (i) with mutant Wza pore ΔP106-A107 in 300 mM KCl buffer. The volume of each droplet was ∼200 nl. The applied electrical potential was +35, +50, +75 and +75 mV, respectively. Dashed line shows 0 pA. The sampling rate was 5 kHz (filtering 1 kHz). Note that events are less frequent for lower concentrations of sugar substrate but the lifetimes analysed here are independent of concentration. j, Radii of gyration of K30-2n=1 (red), K30-2n=2 (green) and K30-2n=3 (blue) predicted by MD simulation. k, Aligned simulated conformers of K30-2n=1, K30-2n=2 and K30-2n=3. Ten predicted conformers of each substrate were aligned. Each conformer was generated after every 1 µs in a 10 µs continuous molecular dynamics simulation. l, Pore radii of Wza pore ΔP106-A107 determined with HOLE (ref. 19).


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  1. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK

    • Lingbing Kong,
    • Hagan Bayley &
    • Benjamin G. Davis
  2. School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK

    • Andrew Almond


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.

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