The structure of the capsular polysaccharide of Escherichia coli O8:K43:H11.

The primary structure of the acidic capsular polysaccharide of Escherichia coli 08:K43:H11 was shown by monosaccharide analysis, methylation analysis, beta-elimination, and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of branched pentasaccharide repeating units with the structure.

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques.

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and 1H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage phi SK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the parent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide. [Formula: see text] The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.

Structural determination of the capsular polysaccharide produced by Klebsiella pneumoniae serotype K40. NMR studies of the oligosaccharide obtained upon depolymerisation of the polysaccharide with a bacteriophage-associated endoglycanase.

The Klebsiella pneumoniae K40 capsular polysaccharide has been isolated and investigated by use of methylation analysis, specific degradations and NMR spectroscopy. The polysaccharide was depolymerised by a bacteriophage-associated endogalactosidase, and the resulting oligosaccharide was characterised by one-dimensional and two-dimensional NMR spectroscopy and direct chemical ionisation MS. The repeating unit of the K40 capsular polysaccharide was shown to be a linear hexasaccharide with the composition-->3)- alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GlcpA++ +-(1-->2-)- alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Galp-(1--> (Rha, rhamnose).

Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.

Determination of the structure of the capsular antigen of Escherichia coli O8:K46:H30, using FABMS and 2D-NMR spectroscopy.

The structure of the capsular antigen from Escherichia coli O8:K46:H30 was elucidated by methylation analysis and 1D and 2D 1H- and 13C-NMR spectroscopy, and by methylation analysis, 1D- and 2D-NMR spectroscopy, and FABMS of the oligosaccharide-alditol obtained after dephosphorylation of the polymer with aqueous hydrofluoric acid. The capsular polymer is of the teichoic acid type and has the following repeating unit. [Formula: see text]

Analysis of pyruvic acid acetal containing polysaccharides by methanolysis and reductive cleavage methods.

The mass spectra of permethylated methyl 4,6-O-(1-carbomethoxyethylidene)-D-hexopyranoside and 1,5-anhydro-D-hexitol of glucose, galactose, and mannose and permethylated methyl 5,6-O-(1-carbomethoxyethylidene)-D-galactofuranoside and 1,4-anhydro-D-galactitol have been determined. The stability of each compound toward methanolysis and reductive cleavage is discussed. These techniques permit the identification of the acetalic linkages of pyruvic acid present in polysaccharides.

Identification, isolation, and structural studies of extracellular polysaccharides produced by Caulobacter crescentus.

Caulobacters are adherent prosthecate bacteria that are members of bacterial biofouling communities in many environments. Investigation of the cell surface carbohydrates produced by two strains of the freshwater Caulobacter crescentus, CB2A and CB15A, revealed a hitherto undetected extracellular polysaccharide (EPS) or capsule. Isolation and characterization of the EPS fractions showed that each strain produced a unique neutral EPS which could not be readily removed from the cell surface by washing. Monosaccharide analysis showed that the main CB2A EPS contained D-glucose, D-gulose, and D-fucose in a ratio of 3:1:1, whereas the CB15A EPS fraction contained D-galactose, D-glucose, D-mannose, and D-fucose in approximately equal amounts. Methylation analysis of the main CB2A EPS showed the presence of terminal glucose and gulose groups, 3-linked fucosyl, and two 3,4-linked glucosyl units, thus confirming the pentasaccharide repeating unit indicated by 1H nuclear magnetic resonance analysis. Similar studies of the CB15A EPS revealed a tetrasaccharide repeating unit consisting of terminal galactose, 4-linked fucosyl, 3-linked glucosyl, and 3,4-linked mannosyl residues. EPS was not detectable by thin-section electron microscopy techniques, including some methods designed to preserve or enhance capsules, nor was the EPS readily detected on the cell surface by scanning electron microscopy when conventional fixation techniques were used; however, a structure consistent with EPS was revealed when samples were prepared by cryofixation and freeze-substitution methods.

Structure of the glycocalyx polysaccharide of Pseudomonas fragi ATCC 4973.

The structure of the exocellular glycocalyx polysaccharide of Pseudomonas fragi ATCC 4973, a bacterium implicated in the spoilage of meat, has been determined using hydrolysis, methylation analysis and 1D- and 2D-n.m.r. spectroscopy. The polysaccharide, which aids in the adhesion of the cells to each other and to the meat tissue, has the regular repeating unit ----4)-3-O-[(R)-1-carboxyethyl]-alpha-D-Glcp-(1----3)-beta-D-ManpNAc+ ++- (1----4)-beta-D-Glcp-(1----. Random partial O-acetylation occurred in some preparations of the polysaccharide.

Lectinophagocytosis of encapsulated Klebsiella pneumoniae mediated by surface lectins of guinea pig alveolar macrophages and human monocyte-derived macrophages.

Macrophages express a mannose/N-acetylglucosamine-specific lectin which serves as a receptor for nonopsonic phagocytosis of mannose-coated particles. We have examined the binding to guinea pig alveolar macrophages in a serum-free medium of 16 Klebsiella pneumoniae serotypes and of the capsular polysaccharides isolated from 7 of these serotypes. Only five polysaccharides containing the repeating sequence Man alpha 2/3Man or L-Rha alpha 2/3-L-Rha bound to the macrophages. Of the 11 bacterial serotypes expressing such disaccharides in their capsular polysaccharides, 7 bound efficiently, 2 bound poorly, and 2 did not bind at all. No binding occurred with five serotypes lacking these disaccharides. Binding of the bacteria was inhibited by homologous and heterologous capsular polysaccharides that contain the disaccharide sequences, by mannan, and by (Man)25BSA (where BSA is bovine serum albumin). Man alpha 2/3Man-containing oligosaccharides were potent inhibitors compared with monosaccharides. Binding was dependent on Ca2+, modulated by cultivating the macrophages on mannan-coated surfaces, and increased in human monocyte-derived macrophages compared with monocytes. The bulk of the bacteria bound to the macrophages was internalized and killed. The data taken together suggest that Klebsiella pneumoniae cells undergo lectinophagocytosis mediated by capsular disaccharides recognized by the mannose/N-acetylglucosamine-specific lectin of macrophages. This may enhance clearance of the organisms from the serum-poor environment of the lung.

Structure of the amino acid-containing capsular polysaccharide from Escherichia coli O8:K49:H21.

The structure of the capsular antigen of E. coli K49 and the oligosaccharides derived from it by partial acid hydrolysis were studied by 1D- and 2D-n.m.r. spectroscopy, g.l.c.-c.i.-mass spectrometry, and methylation analysis. The K49 polysaccharide consists of the repeating unit----4)-beta-D-GlcpA-(1----6)-beta-D-Galp-(1----6)-beta-D-Glcp- (1----3)-beta - D-GalpNAc-(1----. The glucuronic acid residues are substituted, in the apparent molar ratio of 4:1, with L-threonine and L-serine linked amidically to the carboxyl group.

The structure of the capsular antigen from Escherichia coli O8:K87:H19.

The structure of the capsular polysaccharide from Escherichia coli O8:K87:H19 was investigated by methylation analysis and by one- and two-dimensional 1H- and 13C-n.m.r. spectroscopy. The repeating unit was shown to be a branched pentasaccharide with the structure (formula; see text)

A structural investigation of the capsular polysaccharide of Escherichia coli O9:K57:H32.

The primary structure of the acidic capsular polysaccharide of Escherichia coli K57 was elucidated by methylation analysis and 1D- and 2D-n.m.r. spectroscopy. The repeating unit was identified as a linear tetrasaccharide having the structure shown. ----2)-beta-D-Ribf-(1----4)-beta-D-Galp-(1----3)-alpha-D-GlcpNAc-( 1----4)-alpha - D-GalpA-(1----.

The structure of Escherichia coli K26 antigen.

The structure of the capsular antigen of E. coli K26 has been found by a combination of chemical and spectroscopic techniques to be of the "5 + 1" type shown. An important step was the simultaneous separation and identification of a mixture of neutral and acidic oligosaccharides by g.l.c.-c.i.-m.s. [formula: see text]

The structure of Escherichia coli K31 antigen.

The capsular polysaccharide of Escherichia coli K31 has been found by methylation analysis and n.m.r. spectroscopy to be based on the hexasaccharide shown. The sequence of the repeating unit was deduced from the combined results of beta-elimination, lithium-ethylenediamine degradation, and hydrogen-fluoride and selective hydrolyses. The nature of the anomeric linkages, established by chromic acid oxidation, was confirmed by 1H-coupled 13C-n.m.r. spectroscopy. Two dimensional n.m.r. studies on a low molecular weight polymer obtained by bacteriophage depolymerization are also reported. (formula; see text)

A re-investigation of the structure of the capsular polysaccharide of Klebsiella K10.

The capsular polysaccharide of Klebsiella K10 was investigated by methylation analysis and 1H-n.m.r. spectroscopy, and the deacetylated bacteriophage-degraded polysaccharide by 1D- and 2D-n.m.r. spectroscopy. The repeating unit was shown to be a branched hexasaccharide (see text). OAc substituents were located on the terminal and 2-linked galactopyranosyl residues by Prehm methylation of a low-molecular-weight fraction obtained by bacteriophage degradation.

Structure of the capsular antigen of Escherichia coli O8:K8:H4.

The structure of the capsular polysaccharide from Escherichia coli O8:K8:H4 has been elucidated, using mainly methylation analysis, Smith degradation, and 1D- and 2D-n.m.r. spectroscopy. The polysaccharide, after removal of bound lipid, was found to be composed of repeating units of the linear tetrasaccharide. (sequence; see text)

Proof of the occurrence of 5,6-O-(1-carboxyethylidene)-D-galactofuranose units in the capsular polysaccharide of Klebsiella K12.

The 13C-n.m.r. spectra of the capsular polysaccharide of Klebsiella K41 and phage-derived oligosaccharides K41-P1 and K41-P2 were compared with spectra from the structurally similar polysaccharide of Klebsiella K12 and oligosaccharides K12-P1 and K12-P2. This led to the conclusion that K41 and K12 contain one and two galactofuranose residues per repeating unit, respectively, and that the terminal, lateral residue in K12 has the 5,6-O-(1-carboxyethylidene)-D-galactofuranose structure rather than that of a 4,6-acetal of D-galactopyranose as originally stated. This is the first reported occurrence in Nature of such a structural unit.

Homologous and heterologous reactions of bacteriophages phi 41 and phi 12 on the capsular polysaccharides from Klebsiella K41 and K12.

The structures of the capsular polysaccharides from Klebsiella K41 and K12 are very similar and differ only in the lateral, terminal group of their respective repeating units. The bacteriophages phi 41 and phi 12 are shown to hydrolyze the same alpha-galactopyranosyl bond in each of the polysaccharides, giving rise to an oligosaccharide characteristic of the starting polysaccharide, irrespective of the phage employed. The presence of the uronic acid function is essential for the phages to be active, but the carboxyl group of the pyruvic acetal in K12 does not appear to play a role in the recognition process.