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Enzymatic Synthesis of Repeating Unit Oligosaccharides of Escherichia coli O104

  • Inka BrockhausenEmail author
  • Diana Czuchry
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)

Abstract

Escherichia coli serotype O104:H4 (ECO104) is a potent intestinal pathogen that causes severe bloody diarrhea and hemolytic-uremic syndrome. The O antigenic polysaccharides of ECO104 consist of repeating units with the structure [4Galα1-4Neu5,7,9Ac3α2-3Galβ1-3GalNAcβ1-]n. These repeating units are assembled sequentially by specific glycosyltransferases on a diphosphate-undecaprenol intermediate. Internal structures include mimics of the human T and sialyl-T antigen. This protocol describes the in vitro synthesis of the repeating unit by β1,3-Gal-transferase WbwC, α2,3-sialyltransferase WbwA, and α1,4-Gal-transferase WbwB. All of these enzymes require acceptor substrates based on GalNAc-diphosphate-lipid. These methods are applicable for the assembly of bacterial polysaccharides of gram-negative bacteria that require sugar-diphosphate intermediates and are a basis for vaccine synthesis.

Key words

O antigen Escherichia coli O104 Repeating unit Glycosyltransferases Assays In vitro synthesis 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biomedical and Molecular SciencesQueen’s UniversityKingstonCanada

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