Brockhausen Inka, Czuchry Diana
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada.
Methods Mol Biol. 2019;1954:187-202. doi: 10.1007/978-1-4939-9154-9_15.
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-]. 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.
大肠杆菌血清型O104:H4（ECO104）是一种强效肠道病原体，可导致严重的血性腹泻和溶血尿毒综合征。ECO104的O抗原多糖由结构为[4Galα1-4Neu5,7,9Ac3α2-3Galβ1-3GalNAcβ1-]的重复单元组成。这些重复单元通过特定的糖基转移酶在二磷酸十一碳烯醇中间体上依次组装。内部结构包括人类T抗原和唾液酸-T抗原的模拟物。本方案描述了由β1,3-半乳糖转移酶WbwC、α2,3-唾液酸转移酶WbwA和α1,4-半乳糖转移酶WbwB体外合成重复单元的过程。所有这些酶都需要基于GalNAc-二磷酸-脂质的受体底物。这些方法适用于需要糖二磷酸中间体的革兰氏阴性菌细菌多糖的组装，是疫苗合成的基础。
