Demonstration and Characterization of the Cell Wall Carbohydrate and Protein Antigens from Clostridium Botulinum Type E Saroma

Abstract

Two different cell wall antigens, carbohydrate (CHO) and protein (P), from Clostridium botulinum type E Saroma were extracted with sodium dodecyl sulfate (SDS) and purified by chromatography on DEAE-Sepharose CL-6B and Sephadex G-75 or G-100.The CHO antigen was composed of glucose, galactose, glucosamine, galactosamine, alanine and phosphorus with a molar ratio of 1.5: 1.5: 0.25: 0.25: 1: 1. The P antigen was an acidic protein with a molecular weight of 60 kDa, in which the major amino acids were aspartate, glutamate and serine, while the minor ones were cysteine and methionine.Thin sections of the intact or SDS-extracted cells of the organism demonstrated that the cell wall was composed of a two-layered structure, an inner layer about 20 nm thick and an outer layer about 10 nm, and by the extraction with SDS, the outer layer disappeared from the cell surface, leaving the inner layer.Immunogel diffusion tests demonstrated that either CHO antigen or P antigen was common among the nonproteolytic strains of C. botulinum.C. botulinum is classified into toxigenic types A through G depending on the antigenic specificity of the toxin produced.Biochemical activity of the organism, however, separates its strains into proteolytic and nonproteolytic groups regardless of the toxin type. On the other hand, it is well established that carbohydrate or protein confers antigenic specificity to many cell surface macromolecular components of bacteria.Solomon et al (13) showed that C. botulinum type E shared common somatic antigens among the nonproteolytic strains of the organism. While non-peptidoglycan carbohydrate components of the cell wall of these strains were hexoses (glucose and galactose (3)) and hexosamine (glucosamine and galactosamine (5)), none of the antigens of these strains have yet been isolated, nor have their composition and antigenic specificity been elucidated.Our previous data (14) demonstrated that the proteolytic strains of C. botulinum type A contained a protein antigen which was common among them.The present work was aimed at purification and characterization of the cell wall antigen of the nonproteolytic botulinal strains.