Characterization of Two UDP-Gal:GalNAc-Diphosphate-Lipid β1,3-Galactosyltransferases WbwC from Escherichia coli Serotypes O104 and O5

Abstract

c Escherichia coli displays O antigens on the outer membrane that play an important role in bacterial interactions with the environment. The O antigens of enterohemorrhagic E. coli O104 and O5 contain a Gal␤1-3GalNAc disaccharide at the reducing end of the repeating unit. Several other O antigens contain this disaccharide, which is identical to the mammalian O-glycan core 1 or the cancer-associated Thomsen-Friedenreich (TF) antigen. We identified the wbwC genes responsible for the synthesis of the disaccharide in E. coli serotypes O104 and O5. To functionally characterize WbwC, an acceptor substrate analog, GalNAc␣-diphosphate-phenylundecyl, was synthesized. WbwC reaction products were isolated by high-pressure liquid chromatography and analyzed by mass spectrometry, nuclear magnetic resonance, galactosidase and O-glycanase digestion, and anti-TF antibody. The results clearly showed that the Gal␤1-3GalNAc␣ linkage was synthesized, confirming WbwC ECO104 and WbwC ECO5 as UDP-Gal:GalNAc␣-diphosphate-lipid ␤1,3-Gal-transferases. Sequence analysis revealed a conserved DxDD motif, and mutagenesis showed the importance of these Asp residues in catalysis. The purified enzymes require divalent cations (Mn 2؉ ) for activity and are specific for UDP-Gal and GalNAc-diphosphate lipid substrates. WbwC was inhibited by bis-imidazolium salts having aliphatic chains of 18 to 22 carbons. This work will help to elucidate mechanisms of polysaccharide synthesis in pathogenic bacteria and provide technology for vaccine synthesis.

Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP- d -Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3- d -Rhamnosyltransferase WbpZ

“…a Neutral compounds were assayed by the AG1x8 method, and sugar-diphosphatelipids were assayed by the C 18 Sep-Pak method (23). Radioactive product was quantified by scintillation counting.…”

“…After culturing at 6 h with either 100 g/ml ampicillin (GMD) or 50 g/ml kanamycin (RMD), IPTG (isopropyl-␤-D-thiogalactopyranoside) was added to make a 1 mM solution and proteins expressed overnight at room temperature. SDS-PAGE and Western blots (14,23) showed that the 4,6-dehydratase GMD and reductase RMD were well expressed. Isolated bacteria were sonicated in 90% phosphate-buffered saline (PBS)-10% glycerol.…”

“…Site-directed mutagenesis (SDM) was performed following the QuikChange mutagenesis protocol provided by the manufacturer (Agilent) with the primers listed in Table S2 in the supplemental material and pET-wbpZ as the template. Purification of wild-type or mutant forms of the C-terminally His-tagged WbpZ was performed using Ni 2ϩ -nitrilotriacetate (Ni-NTA) affinity chromatography, and SDS-PAGE and Western blots were carried out as described previously (23). To test the in vivo activity of the site-directed mutants of wbpZ in P. aeruginosa, the broadhost-range vector pHERD20T (32) was used.…”

“…GlcNAc and GalNAc derivatives were synthesized as reported earlier (23)(24)(25). Anthracenyl derivatives (24) and potential inhibitors were synthesized as described pre-viously (25)(26)(27).…”

Identification and Biochemical Characterization of the Novel α2,3-Sialyltransferase WbwA from Pathogenic Escherichia coli Serotype O104

“…Radioactive nucleotide sugars were from American Radiolabeled Chemicals. GalNAc and GlcNAc derivatives and inhibitors were synthesized as described previously (17)(18)(19)(20)(21). Glycopeptides and Gal␤1-3GalNAc␣-Bn derivatives were synthesized and kindly donated by Hans Paulsen, University Hamburg, Germany.…”