Structural elucidation of the O-antigen polysaccharide from shigatoxin-producing E. coli O179 using genetic...

The serological properties of the O-antigen polysaccharide region of the lipopolysaccharides are used to differentiate E. coli strains into serogroups. In this study, we report the structure elucidation of the O-specific chain of E. coli O179 using NMR data, the program CASPER and analysis of biosynthetic information available in the E. coli O-antigen Database (ECODAB). The presence of genes that encode enzymes involved in the biosynthesis of the GDP-Man and UDP-GlcA within the O-antigen gene cluster of the bacteria indicates that the corresponding residues could be present in the polysaccharide. Furthermore, the occurrence of four genes that encode for glycosyltransferases indicates that the polysaccharide is composed of pentasaccharide repeating units; a bioinformatics approach based on predictive glycosyltransferase functions present in ECODAB revealed that the β-d-Manp-(1→4)-β-d-Manp-(1→3)-d-GlcpNAc structural element could be present in the O-specific chain. NMR spectroscopy data obtained from homonuclear and heteronuclear 2D NMR spectra (¹H,¹H-TOCSY, ¹H,¹³C-HSQC, ¹H,¹³C-H2BC and ¹H,¹³C-HMBC) were analyzed using the CASPER program, revealing the following arrangement of monosaccharide residues as the most probable structure: →4)-α-d-GlcpA-(1→3)-[β-d-Glcp-(1→2)]β-d-Manp-(1→4)-β-d-Manp-(1→3)-β-d-GlcpNAc-(1→, which was further confirmed using 2D homonuclear ¹H,¹H-COSY and ¹H,¹H-NOESY spectra. The functions of the α-gluconosyltransferase and the β-glucosyltransferase were predicted using structural alignment of AlphaFold-predicted 3D structures. This O-antigen polysaccharide shares structural similarities with those of E. coli O6 and O188, S. boydii type 16, and the capsular polysaccharide of E. coli K43, explaining the serological cross-reactivities observed with strains belonging these O- and K-antigen groups.