{"id":{"repo_id":"queens","oai_identifier":"oai:queensu.scholaris.ca:1974/24274"},"canonical_url":"https://search.dev.ndltd.org/etd/queens/oai:queensu.scholaris.ca:1974/24274","repository":{"repo_id":"queens","name":"Queens University","base_url":"https://qspace.library.queensu.ca/server/oai/request"},"display":{"title":"Assembly of the O Antigen in the Highly Pathogenic Escherichia Coli Serotype O104:H4","abstract":"Escherichia 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.","abstract_html":"Escherichia 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.","abstract_has_math":false,"creators":["Czuchry, Diana"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Biochemistry","school":null,"contributors":[],"advisors":["Brockhausen, Inka","Allingham, John"],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-06-06","date_published":"2018-06-06","updated_at":"2026-07-27T20:35:21Z","subjects":["Glycobiology","O Antigen","E. coli O104","Glycosyltransferase"],"languages":["eng"],"rights":["Attribution-NonCommercial-NoDerivs 3.0 United States"],"rights_urls":["http://creativecommons.org/licenses/by-nc-nd/3.0/us/"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1974/24274","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.department","label":"Department","values":["Biochemistry"]},{"key":"dc:contributor.supervisor","label":"Supervisor","values":["Brockhausen, Inka","Allingham, John"]},{"key":"dc:creator","label":"Author","values":["Czuchry, Diana"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2018-06-06T15:54:10Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2018-06-06T15:54:10Z"]},{"key":"dc:date.issued","label":"Date","values":["2018-06-06"]},{"key":"dc:type","label":"Dc Type","values":["thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Glycobiology","O Antigen","E. coli O104","Glycosyltransferase"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Attribution-NonCommercial-NoDerivs 3.0 United States"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://creativecommons.org/licenses/by-nc-nd/3.0/us/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1974/24274"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Escherichia 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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["PhD"]},{"key":"dc:title","label":"Title","values":["Assembly of the O Antigen in the Highly Pathogenic Escherichia Coli Serotype O104:H4"]}]}],"canonical_facts":{"dc:contributor.department":["Biochemistry"],"dc:contributor.supervisor":["Brockhausen, Inka","Allingham, John"],"dc:creator":["Czuchry, Diana"],"dc:date.accessioned":["2018-06-06T15:54:10Z"],"dc:date.available":["2018-06-06T15:54:10Z"],"dc:date.issued":["2018-06-06"],"dc:description.abstract":["Escherichia 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."],"dc:description.degree":["PhD"],"dc:identifier.uri":["http://hdl.handle.net/1974/24274"],"dc:language.iso":["eng"],"dc:rights":["Attribution-NonCommercial-NoDerivs 3.0 United States"],"dc:rights.uri":["http://creativecommons.org/licenses/by-nc-nd/3.0/us/"],"dc:subject":["Glycobiology","O Antigen","E. coli O104","Glycosyltransferase"],"dc:title":["Assembly of the O Antigen in the Highly Pathogenic Escherichia Coli Serotype O104:H4"],"dc:type":["thesis"]},"updated_at":"2026-07-27T20:35:21Z"}