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dc.contributor.authorCzuchry, Dianaen
dc.description.abstractEscherichia coli serotype O104:H4 (ECO104) is an intestinal pathogen that causes severe bloody diarrhea and hemolytic-uremic syndrome, which can lead to kidney failure and death. Lipopolysaccharides (LPS) are important virulence factors in Gram-negative bacteria and our goal is to understand the enzymes and mechanisms involved in the assembly of the outer O antigenic polysaccharides of the ECO104 LPS. The O antigen repeating unit of ECO104 has the structure [4-Galα1-4Neu5,7,9Ac3α2-3Galβ1-3GalNAcβ1-]n, which contains a mimic of the human sialyl-T-antigen found on certain types of cancer cells. Each monosaccharide subunit of the repeating structure is added sequentially by glycosyltransferases. These enzymes catalyze the transfer of a donor sugar from a nucleotide sugar onto a nucleophilic acceptor. Our aim was to biochemically characterize the second, third and fourth glycosyltransferases that synthesize the repeating unit of ECO104 by expressing the enzymes transgenically in non-pathogenic E. coli BL21, by purifying the enzymes and performing enzyme assays to determine substrate specificity, co-factor requirements, and enzyme kinetics, as well as creating single point mutations in the enzymes to identify key residues. The second enzyme, WbwC, a β1,3-Gal-transferase, has an absolute requirement for a diphosphate in the acceptor. Remarkably, the third enzyme, α2,3-sialyltransferase WbwA, and the fourth enzyme, α1,4-Gal-transferase WbwB, also have this requirement. This finding is unique in that glycosyltransferases further down the O antigen synthesis pathway usually do not require a diphosphate in the acceptor substrate. WbwB does not require any divalent metal ions for activity and several synthetic bis-imidazolium salts could inhibit the enzymes. This work provides insight into the biosynthesis of bacterial polysaccharides and identifies potential anti-bacterial targets and a strategy for vaccine synthesis.en
dc.relation.ispartofseriesCanadian thesesen
dc.rightsAttribution-NonCommercial-NoDerivs 3.0 United Statesen
dc.rightsQueen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canadaen
dc.rightsProQuest PhD and Master's Theses International Dissemination Agreementen
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dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectO Antigenen
dc.subjectE. coli O104en
dc.titleAssembly of the O Antigen in the Highly Pathogenic Escherichia Coli Serotype O104:H4en
dc.contributor.supervisorBrockhausen, Inkaen
dc.contributor.supervisorAllingham, Johnen
dc.contributor.departmentBiochemistryen's University at Kingstonen

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