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Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor

Abstract

Capsular polysaccharides form the outermost protective layer around many Gram-negative bacteria. Antibiotics aimed directly at weakening this layer are not yet available. In pathogenic Escherichia coli E69, a protein, Wza, forms a pore in the outer membrane that transports K30 capsular polysaccharide from its site of synthesis to the outside of the cell. This therefore represents a prospective antibiotic target. Here we test a variety of grommet-like mimics of K30 capsular polysaccharide on wild-type Wza and on mutant open forms of the pore by electrical recording in planar lipid bilayers. The most effective glycomimetic was the unnatural cyclic octasaccharide octakis(6-deoxy-6-amino)cyclomaltooctaose (am8γCD), which blocks the α-helix barrel of Wza, a site that is directly accessible from the external medium. This glycomimetic inhibited K30 polysaccharide transport in live E. coli E69. With the protective outer membrane disrupted, the bacteria can be recognized and killed by the human immune system.

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Figure 1: Models of sections of WT Wza, Wza Y110G/K375C and Wza ΔP106-A107 and their pore radii.
Figure 2: Preparation and electrical properties of WT Wza and Wza mutants.
Figure 3: Reaction of the Wza mutant Y110G/K375C with MTSES.
Figure 4: Screening of blockers against WT Wza and Wza mutants.
Figure 5: Interaction of compounds 10 and 13 with Wza mutants.
Figure 6: Interaction of am8γCD 13 with the α-helix barrel of Wza mutants and proteolysed WT Wza.
Figure 7: Effects of am8γCD 13 on K30 CPS and O9a LPS expression in E. coli E69, O9a LPS exposure to O9a-specific LPS antibodies and complement-mediated killing of E69.

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Acknowledgements

The authors thank J. Naismith and G. Hageluken for the pWQ126 (pBAD-Wza) plasmid, WT Wza protein and the E69 strain, C. Whitfield for the CWG281 strain and O9a antiserum, and N. Rust, C.M. Tang and Min Chen for discussions and advice. The authors thank the Medical Research Council and the Engineering and Physical Sciences Research Council for financial support. L.K. received a Wellcome Trust Value in People (VIP) award and a UK/China Postgraduate Research Scholarship. L.H. was supported by a Biotechnology and Biological Sciences Research Council studentship. B.G.D. is a Royal Society Wolfson Research Merit Award recipient.

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L.K., S.C., B.G.D. and H.B. designed the experiments. L.K. performed protein engineering, single-channel recording, molecular modelling, microbiology, cell wall extraction, flow cytometry, complement-mediated killing and cytotoxicity experiments. Q.L. performed protein engineering. L.H. performed molecular modelling. L.K., B.G.D. and H.B. analysed results. L.K., B.G.D. and H.B. wrote the paper.

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Correspondence to Benjamin G. Davis or Hagan Bayley.

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Kong, L., Harrington, L., Li, Q. et al. Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor. Nature Chem 5, 651–659 (2013). https://doi.org/10.1038/nchem.1695

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