Analysis of the common polysaccharide antigens from the cell envelope of Clostridium perfringens type A

Abstract

The major, common antigen of Clostridium perfringens type A, isolated and purified independently from three selected strains (Hobbs 5, Hobbs 9, and Hobbs 10), is composed of equimolar amounts of 2-acetamido-2-deoxy- d-mannose (ManNAc) and 2-acetamido-2-deoxy- d-glucose (GlcNAc). The purified antigen gave a strong immunoprecipitin line by double immunodiffusion in gel. Smith degradation of the major, common antigen caused decomposition of all of the GlcNAc, without concomitant loss in ManNAc, or a perceptible change in serological activity. Therefore, the serological activity of the major, common antigen depended solely on the presence of ManNAc. Data obtained by the 13C-n.m.r.-spectral analysis of the Smith-degradation product revealed that it was a linear-backbone polysaccharide analogous to a Rhodotorula glutinis mannan, but composed of pairs of 2-acetamido-2-deoxymannopyranosyl residues alternately linked β-(1→3) and β-(1→4). The one-bond, carbon-hydrogen coupling-constant of 162 Hz for both anomeric centers was consistent with the proposed β-linkages. A similar, 13C-n.m.r.-spectral analysis of the native, common antigen indicated that the GlcNAc residues were randomly connected to three of the four hydroxyl groups not already involved in linking the ManNAc backbone, the 4-hydroxyl group being the exception. A second, serologically inactive, polysaccharide composed of rhamnose, GalNAc, and galactose was identified, but not obtained in homogeneous state. The rhamnosyl residues were probably situated as nonreducing antennae, as they were quantitatively removed by Smith degradation without concomitant decomposition of the polymeric structure of the remaining residues.