Immunochemistry of K antigens of Escherichia coli. 4. The K antigen of E. coli O 9:K30:H12.

Abstract

An acidic polysaccharide which consists of glucuronic acid, galactose and mannose in a molar ratio of 1:1:1, and which contains 2.5%O‐acetyl groups, was isolated from Escherichia coli O 9:K 30:H 12 cells.Half the amount of the acidic polysaccharide present on the cell surface was extractable with saline at 60°. The residual polysaccharide was solubilized with 45% phenol at 65°. Purification of the two fractions was obtained by cetavlon precipitation.Saline‐extractable and non‐extractable polysaccharide fractions were identical with respect to chemical composition, serological specificity, pK value, neutral equivalent and intrinsic viscosity.In the ultracentrifuge, the non‐extractable polysaccharide showed one peak, wheareas the saline‐extractable fraction was inhomogenous. After treatment with diluted alkali a homogenous form of smaller particle size was obtained from both polysaccharide fractions. Alkali treatment did not alter the sugar composition of either fraction. The molecular weight of the alkali‐treated polysaccharide is about 150,000.The acidic polysaccharide was oxidized by sodium periodate with concomitant destruction of the glucuronic acid. Galactose and mannose in the polysaccharide chain were resistant to periodate oxidation.Acid hydrolysis gave rise to 3‐O‐β‐d‐glucuronosyl‐d‐galactose, which could be isolated in about 25% yield. No further oligosaccharides were detected in hydrolysates of the acidic polysaccharide.Acid hydrolysis of the polysaccharide in which the carboxyl groups had been reduced gave rise to glucosyl‐galactose and a second disaccharide which consisted of mannose and glucose, the latter being obtained at very low yield.It is concluded that the acidic polysaccharide of E. coli O 9:K 30: H 12 consists of linear subunits of a molecular weight of about 150,000. These subunits are joined through ester bonds between carboxyl groups of glucuronic acid residues and hydroxyl groups of sugar moieties. It is suggested that the trisaccharide mannosyl‐1,2‐glucuronosyl(β)1,3‐galactose is the repeating unit of the polysaccharide chain.The acidic polysaccharide has a high affinity for the surface of red blood cells. In passive haemagglutination tests the polysaccharide‐sensitized red cells react strongly with E. coli O 9: K 30: H 12 anti OK and anti K serum but not with anti O serum.After absorption of anti OK serum with the purified acidic polysaccharide the antiserum no longer agglutinates the encapsulated strain. The acidic polysaccharide is precipitated by K 30 antiserum. This precipitation is inhibited by glucuronic acid, β‐phenyl glucuronide and 3‐O‐β‐d‐glucuronosyl‐d‐galactose. After removal of the O‐acetyl groups the acidic polysaccharide shows reduced reactivity with K 30 antiserum. From these results we conclude that the acidic polysaccharide isolated from E. coli O 9: K 30:H 12 is the K antigen of this strain. The serologically determinant region of the K 30 antigen includes glucuronic acid and O‐acetyl groups.