Immunochemistry of K Antigens of <em>Escherichia coli</em>.

An acidic polysaccharide which consists of D­glucuronic acid. D­glucose, D­galactose and L­fucose in a molar ratio of 1:1:1:1 and which contains 5.2% of O-acetyl groups was isolated from Escherichia coli 08:K 27 (A):H- cells. About 80% of the total polysaccharide present on the bacterial cell could not be extracted with saline at 60°. This polysaccharide fraction was solubilized with 45% aqueous phenol at 65°. Purification of the acidic polysaccharide was achieved with cetavlon precipitation. It was shown to be homogeneous by ultracentrifugation analysis. The acidic polysaccharide has a high molecular weight (mean value 3 × 10⁶), gives an aqueous solution with high viscosity ([η] = 5.0 dl/g) and possesses a very large form factor (ƒ/ƒ_o = 6.5). Treatment of the acidic polysaccharide with dilute alkali produced smaller molecules of a molecular weight of about 100,000. Alkali treatment did not alter the sugar composition of the acidic polysaccharide nor did it impair its serological properties. The acidic polysaccharide was oxidized by sodium periodate with concomitant destruction of the galactose moiety. Glucuronic acid, glucose and fucose in the polysaccharide resisted periodate oxidation. When the periodate oxidized and sodium borohydride reduced polysaccharide was hydrolyzed, 0.8 moles of glycerol per mole of the oxidized galact, ose was detected in the hydrolyzate. The methylated acidic polysaccharide was subjected to methanolysis and the methyl glycosides of the methylated sugar constituents were investigated with the aid of gas liquid chromatography. Methyl-tetra-O-methyl-D-galactoside was found to be present in the methanolyzate. Acid hydrolysis of the acidic polysaccharide gave rise to the following charged oligosaccharides: (i) glucuronosyl-(l:3)-fucose, (ii) glucosyl-(l:3)-glucuronosyl-(l:3)-fucose and (iii) galactosyl-(1:3)-glucosyl-(1:3)-glucuronosyl-(l:3)-fucose. No neutral oligosaccharides were detected in hydrolyzates of the acidic polysaccharide. It is concluded that the acidic polysaccharide of E. coli 08:K27 (A):H- consists of subunits of a molecular weight of about 100,000. These subunits are probably linked through ester bonds between carboxyl groups of glucuronic acid and hydroxyl groups of sugar constituents. It is suggested that the polysaccharide chains of the subunits consist, of glucose, glucuronic acid and fucose in repeating order to which galactose is bound in 1:3-linkage, with glucose as the branch point. The acidic polysaccharide absorbes directly onto erythrocytes. In passive haemagglutination tests the polysaccharide-sensitized red cells reacted strongly with E. coli 08:K27 anti OK and anti K sera but very weakly with anti O serum. After absorption of anti OK serum with the purified acidic polysaccharide the antiserum no longer agglutinated the encapsulated strain. The acidic polysaccharide was precipitated by 08:K27 antiserum and by K27 antiserum. This precipitation was inhibited by the tetrasaccharide (iii) and to a lesser extent by the trisaccharide (ii), but not by disaccharide (i) and glucuronic acid. After periodate oxidation—which destroyed the galactose moiety—the acidic polysaccharide was no longer reactive in serological tests. From these results it is concluded that the acidic polysaccharide isolated from E. coli 08:K27 (A):H- is the K antigen of this strain. Galactose appears to be the immunodominant sugar constituent of the K27 antigen, but the whole determinant group consists (at least) of the galactose-1:3-glucose disaccharide. [ABSTRACT FROM AUTHOR]