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.
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Brockhausen, I., Czuchry, D. (2019). Enzymatic Synthesis of Repeating Unit Oligosaccharides of Escherichia coli O104. In: Brockhausen, I. (eds) Bacterial Polysaccharides. Methods in Molecular Biology, vol 1954. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9154-9_15
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DOI: https://doi.org/10.1007/978-1-4939-9154-9_15
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