O antigen Biosynthesis in Gram-Negative Bacteria: Characterization of Glycosyltransferases WclX and WclW from Escherichia coli O117 and WfbG from Salmonella enterica O43

Abstract

The outer membrane of Gram-negative bacteria is coated with complex polysaccharides, constituting primarily of lipopolysaccharide (LPS). LPS is an important virulence factor of Gram-negative bacteria essential to their survival and pathogenicity. The O antigen being the outermost component of LPS, plays crucial roles in immune evasion, adhesion, colonization, and biofilm formation. Understanding the enzymes and mechanisms involved in O antigen biosynthesis can lead to new antibacterial strategies to combat bacterial infections. Escherichia coli serotype O117 (ECO117) are pathogenic bacteria that produce Shiga toxin. The O antigen repeating unit of ECO117 has the structure [4-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-], similar to the ECO107 repeating unit that contains N-acetylglucosamine instead of glucose. Salmonella enterica serotype O43 (SO43) has the O antigen repeating unit structure [4-L-Fucα1-2-(D-Galα1-3)-D-Galβ1-3-D-GalNAcα1-3-D-GlcNAcβ1-], which contains a mimic of the human blood group B and E. coli O86 (ECO86) antigens. Compounds Galβ1-3-GalNAc-PO3-PO3-(CH2)11-O-phenyl, GlcNAc-PO3-PO3-(CH2)11-O-phenyl, and GalNAc-PO3-PO3-(CH2)11-O-phenyl were chemically synthesized as acceptor substrate analogs of the natural undecaprenyl phosphate intermediate in bacteria. Previously, WclY from ECO117 was shown to transfer glucose to Galβ1-3-GalNAc-PO3-PO3-(CH2)11-O-phenyl and that Arg194Cys mutants converted WclY to a highly active GlcNAc-transferase. We expressed the glycosyltransferases WclX and WclW in the pathway, demonstrating they complete the synthesis of the ECO117 and O107 repeating units. WclX strictly transfers L-rhamnose (L-Rha) from TDP-L-Rha with high specificity for the WclY enzyme product as the acceptor. Mutational analysis revealed several conserved Asp residues within WclX are essential for the catalytic of transfer of L-Rha. WclW strictly transfers N-acetylgalactosamine from UDP-GalNAc to a broad range of acceptors that contain L-Rha at the non-reducing end. Using L-Rhaα-p-nitrophenyl as an acceptor for WclW, nuclear magnetic resonance of the product revealed WclW is a β1-3-GalNAc-transferase. Additionally, we recombinantly expressed WfbG from SO43 and WbnH from ECO86 demonstrating both enzymes are specific for UDP-GalNAc as a donor. However, WfbG could use either GlcNAc-PO3-PO3-(CH2)11-O-phenyl or GalNAc-PO3-PO3-(CH2)11-O-phenyl as an acceptor, whereas WbnH was only active with GalNAc-PO3-PO3-(CH2)11-O-phenyl. Bioinformatics indicated the presence of a GlcNAc-diphosphate-undecaprenyl 4-epimerase gene that may explain the structural difference between the two repeating units. These studies set the foundation for discovery of antibacterial agents and preparation of O-antigenic oligosaccharides for glycoconjugate vaccines.