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The capsular polysaccharide was isolated from Escherichia coli 010:K5:H4; it could not be obtained from a uncapsulated (K5-) mutant. It contains N-acetylglucosamine and glucuronic acid in a molar ratio of 1:1. Acid hydrolysis of the acidic polysaccharide as well as Smith degradation and degradation by deamination of the carboxyl-reduced polysaccharide suggested that the polysaccharide is composed of a disaccharide repeating unit. The data obtained by methylation analysis and nuclear magnetic resonance spectroscopy indicated that the repeating sequence of the capsular polysaccharide is the 4-beta-glucuronyl-1,4-alpha-N-acetylglucosaminyl unit. This structure is similar to that of desulfo-heparin.

The Escherichia coli K2 capsular antigen is known to be composed of alpha-D-galactopyranosyl(1--2)glycerophosphate and alpha-D-galactofuranosyl(1--2)glycerophosphate units which are connected by phosphodiester bonds to C-4 of the galactopyranosyl and C-5 or C-6 of the galactofuranosyl moieties. In the present study the glycerophosphates were released by two different procedures and shown to have the sn-glycero-3-phosphate stereochemical configuration. In the first, the chain was fragmented by Smith degradation to glycerophosphothreitol from which the glycerophosphate was released by alkali hydrolysis. The structure-dependent low recovery of alpha-glycerophosphate (less than 10%) initiated the development of another degradative sequence which consisted of periodate oxidation, beta elimination, hydrazinolysis, and alkaline treatment. This way, approximately 90% of the glycerophosphate was released as sn-glycero-3-phosphate. beta elimination revealed in addition that most of the galactofuranosyl residues carry the phosphodiester bond at position 5. Separation by gel permeation chromatography and analysis of the fragments obtained by beta elimination showed that pyranosidic and furanosidic galactosyl residues alternate in the same chain and suggested the sequences Galf(p)GroP-(GalpGroP)n-Galf- and -GalfGroP-(GalpGroP)n-Galf-, where n is 6, 4, and 3, respectively.

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/ƒo = 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]

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 front 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, serotogical specificity, pK value, neutral equivalent and intrinsic viscosity. In the ultracentrifuge, the non-extractable polysaccharide showed one peak, whereas 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 lite 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 test 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. [ABSTRACT FROM AUTHOR]