The enterohaemorrhagic O157 strain of Escherichia coli displays its O-antigen on the extracellular face of the outer membrane. The O157 antigen is a virulence factor and consists of repeating units with the sequence [-2-D-Rha4NAca1-3-L-Fuca1-4-D-Glcb1-3-D-GalNAc1-]. In this work, we showed that wbdN found in the O-antigen synthesis gene cluster encodes a Glc-transferase that adds Glc to GalNAc as the second step in O-antigen repeat unit synthesis. The wbdN gene was expressed in E. coli BL21 bacteria; WbdN was purified and characterized using the donor substrate UDP-[14C]Glc and the synthetic acceptor substrate GalNAca-PO3-PO3-(CH2)11-O-Phenyl. WbdN has a DxD motif and requires Mn2+ ions for full activity. Mg2+ and Co2+ ions also activate the enzyme. WbdN activity has a broad pH optimum; the enzyme is stable at both 4 oC and -20 �C, and is highly specific for UDP-Glc as the donor substrate. GalNAca derivatives lacking the pyrophosphate group were inactive as substrates. Two imidazolium-lipid compounds strongly inhibited the activity. These inhibitors could be applied to block O-antigen synthesis in specific bacteria. This work was supported by an NSERC Discovery Grant (to I.B.).

Characterization and inhibition of a novel glucosyltransferase involved in O-antigen synthesis in enterohaemorrhagic <i>Escherichia coli </i>serotype O157

243rd National Meeting (2012)

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Characterization and inhibition of a novel glucosyltransferase involved in O-antigen synthesis in enterohaemorrhagic Escherichia coli serotype O157