Structural characterization reveals heterogeneity in the O-antigen polysaccharide of Escherichia coli O101 O-serogroup variants.

Isolates of the E. coli O101 serogroup collected from bacteremia patients were demonstrated to harbor different glycoforms of the O-antigen polysaccharide. Investigation by high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy of the O-antigen structure of the reference E. coli O101 strain H510a and of one naturally occurring variant (Onovel32) confirmed the previously reported simultaneous presence of O-polysaccharide repeat units made up of disaccharides PS1 and PS2. A newly discovered O101 glycoform was shown to be composed of the same backbones modified with a previously unrecognized structure having an additional terminal 4-O-methyl N-acetyl-galactosamine (4-O-Me-GalNAc) residue on the non-reducing end of the PS2 backbone. Another naturally occurring variant (Onovel32MT-) does not contain any PS2 repeat units, nor any 4-O-Me-GalNAc capping. Analyses indicated that the polysaccharide glycoforms from these three O101 strains varied in terms of their ratios of PS1 to PS2 repeat units and the amount of 4-O-Me-GalNAc terminal residue. From the Smith degradation data of the reference O101 strain and the Onovel32MT- variant, it is hypothesized that the PS1 and PS2 repeat units join into a PS1-PS2 block copolymer, which is formed by the addition of the PS2 backbone on the already existing PS1 core backbone. Finally, 4-O-Me-GalNAc acts as a terminal capping residue on the PS2 backbone. Such block copolymer structures have hitherto been reported in galactans of Klebsiella and in Brucella. To our knowledge, this is the first reported occurrence of such a block copolymer O-antigen in E. coli.