The complex cell-wall polysaccharide, C-substance, was isolated from Streptococcus pneumoniae type 1 and purified by DEAE-cellulose (HCO3(-) form) and Sephadex column chromatography. The complete structure of this antigen was obtained by the application of methylation and 1H NMR and 13C NMR spectroscopic techniques to a series of oligosaccharide fragments obtained by the selective degradation of the N-acetylated antigen. Native C-substance is composed of the following repeating unit: beta-D-Glup-1 leads to 3-alpha-AAT-Galp-1 leads to 4-alpha-D-GalNAcp-1 leads to 3-beta-D-GalNH2p-1 leads to 1'-ribitol-5-phosphate where AATGal is 2-acetamido-4-amino-2,4,6-trideoxygalactose. Phosphocholine substituents are situated at O(6) of the unacetylated galactosamine residues, and the repeating units are linked through a diphosphate ester from ribitol to O(6) of the beta-D-glucopyranose residue. This structure has also been shown to be common to C-substances prepared from a number of other pneumococcal types based on the criterion of their identical 13C NMR spectra.

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Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960–80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.