Assembly of the O Antigen in the Highly Pathogenic Escherichia Coli Serotype O104:H4

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Czuchry, Diana
Glycobiology , O Antigen , E. coli O104 , Glycosyltransferase
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.
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