Antigenic polysaccharides of bacteria. 31. The structure of the O-specific polysaccharide chain of Pseudomonas aurantiaca IMB 31 lipopolysaccharide

The O-specific polysaccharide chain of the Pseudomonas aurantiaca IMV 31 lipopolysaccharide contains N-acetyl-L-fucosamine (FucNAc) and di-N-acetyl-D-bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose, Bac(NAc)2) in the ratio 2:1. On the basis of methylation, solvolysis with anhydrous hydrogen fluoride, and computer-assisted analysis of 13C-NMR spectrum, it was concluded that the trisaccharide repeating unit of the polysaccharide possesses the following structure: structure: ----3)-beta-D-Bac(NAc)2-(1----3)-alpha-L-FucNAc-(1----3)-alpha-L- FucNAc-(1----.     

Structure of the O-antigen of Francisella tularensis strain 15.

The O-specific polysaccharide, obtained by mild acid degradation of the lipopolysaccharide of Francisella tularensis strain 15, contained 2-acetamido-2,6-dideoxy-D-glucose (D-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (D-Qui4NFm), and 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) in the ratios 1:1:2. Tri- and tetra-saccharide fragments were obtained on treatment of the polysaccharide with anhydrous hydrogen fluoride and partial hydrolysis with 0.1 M hydrochloric acid, respectively. On the basis of 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and the saccharides, it was concluded that the O-antigen had the structure: ----4)-alpha-D-GalpNAcAN-(1----4)-alpha-D-GalpNAcAN-(1----3) -beta-D-QuipNAc-(1----2)-beta-D-Quip4NFm-(1----. This O-antigen is related in structure to those of Pseudomonas aeruginosa O6, immunotype 1, and IID 1008, and Shigella dysenteriae type 7.     

The structure of O-specific polysaccharide of Pseudomonas aeruginosa immunotype 3; revision of the structure of acetoamidine derivative of 2,3-diamino-2,3-dideoxy-D-mannuronic acid

O-Specific side chain of P. aeruginosa immunotype 3 lipopolysaccharide is composed of N-acetyl-D-fucosamine (FucNAc), 2,3-diacetamido-2,3-dideoxy-L-guluronic acid (GulN2Ac2A) and 3-acetamidino = 2-acetamido = 2,3 = dideoxy = D-mannuronic acid (ManNAcAmA). The latter sugar is identified on the basis of solvolysis with anhydrous hydrogen fluoride, 13C NMR spectroscopy and fast-atom bombardment mass spectrometry analysis, as well as of reactions of acetamidino function (alkaline hydrolysis to acetamido group and reductive deamination to ethylamino group). Earlier, in the course of investigation of P. aeruginosa O3 lipopolysaccharides, the structure of 1-methyl-2-imidazoline was erroneously ascribed to the acetamidino group. The following structure was established for the repeating unit of immunotype 3 polysaccharide which is identical to P. aeruginosa O3(a),3c polysaccharide: ----4)-beta-D-ManNAcAmA-(1----4)-alpha-L-GulN2Ac2A-(1----3)- beta-D-FucNac-(1----.     

Antigenic bacterial polysaccharides. 23. The structure of the O-specific polysaccharide chain of Salmonella arizonae 059 lipopolysaccharide

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.     

Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).

The lipopolysaccharide (LPS) of Klebsiella serotype O2 is antigenically heterogeneous; some strains express multiple antigenic factors. To study this heterogeneity, we determined the structure of the O-antigen polysaccharides in isolates belonging to serotypes O2(2a), O2(2a,2b), and O2(2a,2c), by using composition analysis, methylation analysis, and both 1H and 13C nuclear magnetic resonance spectroscopy. The repeating unit structure of the 2a polysaccharide was identified as the disaccharide [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] and was identical to D-galactan I, one of two O polysaccharides present in the LPS of Klebsiella pneumoniae serotype O1 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). LPS from serotype O2(2a,2b) also contained D-galactan I as the only O polysaccharide, suggesting that the 2b antigen is not an O antigen. The LPS of serotype O2(2a,2c) contained a mixture of two structurally distinct O polysaccharides and provides a second example of this phenomenon in Klebsiella spp. One polymer was identical to D-galactan I, and the other polysaccharide, the 2c antigen, was a polymer with a disaccharide repeating unit structure, [----3)-beta-D-GlcpNAc-(1----5)-beta-D-Galf-(1----]. The 2c structure does not resemble previously reported O polysaccharides from Klebsiella spp. Periodate oxidation confirmed that D-galactan I and the 2c polysaccharide are distinct glycans, rather than representing domains within a single polysaccharide chain. Monoclonal antibodies against the 2c antigen indicated that only LPS molecules with the longest O-polysaccharide chains contained the 2c epitope.     

Structure of the O-polysaccharide leads to classification of Proteus penneri 31 in Proteus serogroup O19

O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C and 1H,31P HMQC experiments, demonstrated the following structure of the polysaccharide: [carbohydrate structure: see text] where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and alpha-L-FucNAc-(1-->3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31.     

Antigenic polysaccharides of bacteria. 35. Establishment of the structure of polysaccharide chains of lipopolysaccharides of Pseudomonas cepacia IMV 4176 and IMV 4202 (Serotype 3)

On mild acid degradation of the Pseudomonas cepacia strain IMV 4176 lipopolysaccharide, two polysaccharides were obtained, one of which is a homopolymer of N-acetyl-D-galactosamine and the other is composed of equal amounts of N-acetyl-D-galactosamine and D-ribose. Partial hydrolysis with aqueous oxalic acid caused depolymerization of the heteropolysaccharide, and the homopolysaccharide was isolated in the individual state. On the basis of methylation and 13C NMR analysis, it was concluded that both polysaccharides are built up of disaccharide repeating units having the following structures: ----4)-alpha-D-GalpNAc-(1----4)-beta-D-GalpNAc-(1---- and ----4)-alpha-D-GalpNAc-(1----2)-beta-D-Ribf-(1----. The heteropolysaccharide from P. cepacia strain 4176 is identical by the structure of the repeating unit to the O-specific polysaccharide of P. cepacia strain IMV 4202 (serotype 3), Pseudomonas aeruginosa O12 and Serratia marcescens O14.     

Human low-molecular-mass kininogen. Amino-acid sequence of the light chain; homology with other protein sequences

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.     

Antigenic polysaccharides of bacteria. 25. Structure of the O-specific polysaccharide chain of Pseudomonas cepacia 673/2 lipopolysaccharide containing L-glycero-D-manno-heptose

O-Specific polysaccharide, consisting of D-rhamnose and L-glycero-D-manno-heptose (LD-Hep) in a 2 : 1 ratio, was obtained on the mild acid degradation of the Pseudomonas cepacia IMV 673/2 lipopolysaccharide; monosaccharide LD-Hep has not previously been found in O-specific chains of lipopolysaccharides. On the basis of methylation and 13C-NMR data, it was concluded that the polysaccharide is composed of trisaccharide repeating units having the following structure: ----3)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1----2)-alpha-LD-Hep-(1----     

Structure of the O-specific polysaccharide of Vibrio fluvialis serovar 3]

O-Specific polysaccharide composed of L-rhamnose and 2-acetamido-2-deoxy-D-mannose was obtained on mild acid degradation of the V. fluvialis lipopolysaccharide. On the basis of the 13C-NMR data and methylation studies, the following structure was suggested for the polysaccharide repeating unit: ----4)-alpha-L-Rhap-(1----3)-beta-D-ManpNAc-(1---- This structure was confirmed by calculations using known glycosidation effects on 13C chemical shifts.     

Antigenic polysaccharides of bacteria of the genus Shigella. Determination of the structure of polysaccharide chains of Shigella boydii type 9 lipopolysaccharide and detection of unusually high molecular weight glycolipid

Specific acidic polysaccharide has been isolated from the Shigella boydii type 9 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and L-rhamnose. From the results of methylation analysis, partial acid hydrolysis and 13C NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [----4)DGlcp(alpha 1----4)DGlcAp(beta 1----3)DGlcNAcp(alpha 1----3)LRhap(alpha 1----]n. The lipopolysaccharide from Sh. boydii 9 was fractionated by gel chromatography on the Sephadex G-200 column in a buffer containing sodium deoxycholate into three fractions. PAGE-SDS of the fractions obtained, 13C NMR- and chromato-mass-spectrometry data indicated that the three fractions contained the O-specific polysaccharide as the only carbohydrate component. The substance from the most high-molecular weight fraction contained unusually long O-specific chains (60,000 dalton). In the fat acid composition this fraction differed from other lipopolysaccharides by absence of beta-hydroxymyristic acid.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from P. aeruginosa O13 (Lányi)

The O-specific polysaccharide, obtained on mild acid degradation of lipopolysaccharide of Pseudomonas aeruginosa O13 (Lányi classification), is built up of trisaccharide repeating units involving 2-acetamidino-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), 2-acetamidino-2,6-dideoxy-L-galactose (L-fucosacetamidine, L-FucAm), and a new sialic-acid-like sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso n ic acid (Sug), and thus contains simultaneously both acidic and basic functions. Cleavage of the polysaccharide with hydrogen fluoride in methanol revealed the high stability of the glycosidic linkage of the ulosonic acid and afforded methyl glycosides of a disaccharide and a trisaccharide. The structures of the new ulosonic acid and acetamidino group were established by analysing the oligosaccharide fragments by 1H, 13C nuclear magnetic resonance spectrometry, as well as on the basis of their chemical conversions: alkaline hydrolysis of the acetamidino group into acetamido group, reductive deamination with lithium borohydride into the ethylamino group and acetylation with acetic anhydride in pyridine accompanied by intramolecular acylation of the acetamidino function by the ulosonic acid to form a six-membered lactam ring. Identification of the oligosaccharide fragments and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and polysaccharide revealed the following structure of the repeating unit: ----3)D-QuiNAcp(alpha 1----3)Sugp(alpha 2----3)L-FucAmp(alpha 1----.     

Structure and cross-reactivity of the O-antigen of Proteus vulgaris O8.

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O8 lipopolysaccharide followed by gel permeation chromatography. Studies of the polysaccharide by sugar and methylation analyses and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, demonstrated the presence of a tetrasaccharide repeating unit having the following structure: [sequence: see text] The role of an epitope associated with the alpha-L-FucpNAc-(1-->3)-D-GlcpNAc disaccharide in serological cross-reactivity of P. vulgaris O8 is discussed.     

Antigenic polysaccharides of bacteria. 17. The structure of O-specific polysaccharide chain of Pseudomonas aeruginosa X (Meitert) lipopolysaccharide

O-Specific polysaccharide composed of L-rhamnose and 2-acetamido-2-deoxy-D-mannose was obtained on mild acid degradation of P. aeruginosa X (Meitert classification) lipopolysaccharide. On the basis of non-destructive analis using 1H, 13C NMR spectroscopy and Klyne's rule calculation, as well as chemical methods (acid hydrolysis, methylation, Smith degradation), it was established that the polysaccharide is built up of disaccharide repeating units of the following structure: ----4)-alpha-L-Rha-(1----3)-beta-D-ManNAc-(1----.     

Partial deletion of the cloned rfb gene of Escherichia coli O9 results in synthesis of a new O-antigenic lipopolysaccharide.

The rfb gene, involved in the synthesis of the O-specific polysaccharide (a mannose homopolymer) of Escherichia coli O9 lipopolysaccharide (LPS), was cloned in E. coli K-12 strains. The O9-specific polysaccharide covalently linked to the R core of K-12 was extracted from the K-12 strains harboring the O9 rfb gene. All the other genes required for the synthesis of rfe-dependent LPS are therefore considered to be present in the K-12 strains. It was found that bacteria harboring some clones with deletions of the ca. 20-kilobase-pair (kbp) BglII-StuI fragment no longer synthesized the O9-specific polysaccharide. However, bacteria harboring clones del 21, del 22, and del 25, which carry deletions of the 10-kbp PstI-StuI fragment, synthesized an O-specific polysaccharide antigenically distinct from E. coli O9 LPS. Although this new O-specific polysaccharide consisted solely of mannose and the mannose residues were combined only through alpha-1,2 linkage, it was still composed of a repeating oligosaccharide unit, possibly a trisaccharide unit,----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----. It is therefore likely that this new O-specific polysaccharide was derived from a part of the O9-specific polysaccharide----3)alpha Man-(1----3)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----and that the deleted part of the clones was responsible for the synthesis of alpha-1,3 linkages of the O9-specific polysaccharide.     

Structure of a glycerol teichoic acid-like O-specific polysaccharide of Proteus vulgaris O12.

A phosphorylated O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of Proteus vulgaris O12 lipopolysaccharide and studied by sugar and methylation analyses, 1H-, 13C- and 31P-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H, 13C and 1H, 31P heteronuclear multiple-quantum coherence experiments. It was found that the polysaccharide consists of pentasaccharide repeating units connected via a glycerol phosphate group, and has the following structure: where FucNAc is 2-acetamido-2,6-dideoxygalactose and the degree of O-acetylation at position 4 of GalNAc is approximately 25%. Immunochemical studies with P. vulgaris O12 O-antiserum suggested that the lipopolysaccharide studied shares common epitopes with the lipopolysaccharide core of P. vulgaris O8 and with the O-antigens of P. penneri strains 8 and 63.     

Structure of a glucosyl phosphate-containing O-polysaccharide of Proteus vulgaris O42

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O42 and studied by sugar and methylation analyses along with 1H, 13C and 31P NMR spectroscopy. The following structure of the polysaccharide having a linear pentasaccharide phosphate repeating unit was established: -->3)-alpha-L-FucpNAc4Ac-(1-->4)-alpha-D-Glcp-1-P-(O-->4)-alpha-D-GlcpNAc-(1-->3)-alpha-L-FucpNAc4Ac-(1-->3))-alpha-D-GlcpNAc6Ac-(1--> where the degree of O-acetylation is approximately 80% on GlcNAc and approximately 40% on each of the FucNAc residues. A weak serological cross-reaction of anti-P. vulgaris O42 serum with the lipopolysaccharide of P. vulgaris O39 was observed and accounted for by the sharing of a disaccharide fragment of the O-polysaccharides.     

Antigenic bacterial polysaccharides. 13. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from Pseudomonas cepacia strain IMV 4137

On mild acid degradation of a lipopolysaccharide from Pseudomonas cepacia strain IMV 4137, a serologically active O-specific polysaccharide was obtained and shown to contain L-rhamnose and D-galactose. According to 1H- and 13C-NMR data as well as methylation analysis, the polysaccharide is made up of disaccharide repeating units of the following structure:----2)-alpha-L-Rhap-(1----4)-alpha-D-Galp-(1----.     

Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.

The lipopolysaccharide (LPS) molecule is an important virulence determinant in Klebsiella pneumoniae. Studies on the serotype O1 LPS were initiated to determine the basis for antigenic heterogeneity previously observed in the O1 side chain polysaccharides and to resolve apparent ambiguities in the reported polysaccharide structure. Detailed chemical analysis, involving methylation and 1H- and 13C-nuclear magnetic resonance studies, demonstrated that the O-side chain polysaccharides of serotype O1 LPS contained a mixture of two structurally distinct D-galactan polymers. The repeating unit structures of these two polymers were identified as [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] (D-galactan I) and [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] (D-Galactan II). D-Galactan I polysaccharides were heterogeneous in size and were detected throughout the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) profile of O1 LPS. In contrast, D-galactan II was confined to the higher-molecular-weight region. The structures of the two D-galactans were not influenced by simultaneous synthesis of a capsular K antigen. Apparently, neither of the D-galactans constitutes a common antigen widespread in Klebsiella spp. as determined by immunochemical analysis. Examination of the LPSs in mutants indicated that expression of D-galactan I can occur independently of D-galactan II. Transconjugants of Escherichia coli K-12 strains carrying the his region of K. pneumoniae were constructed by chromosome mobilization with RP4::mini-Mu. In these transconjugants, the O antigen encoded by the his-linked rfb locus was determined to be D-galactan I, suggesting that genes involved in the expression of D-galactan II are not closely linked to the rfb cluster.     

Structure of the O-specific polysaccharide of Proteus vulgaris O45 containing 3-acetamido-3,6-dideoxy-D-galactose

An O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O45 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established:-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Fucp3NAc4Ac-(1-->where Fuc3NAc4Ac is 3-acetamido-4-O-acetyl-3,6-dideoxygalactose. A cross-reactivity of anti-P. vulgaris O45 serum was observed with several other Proteus lipopolysaccharides, which contains Fuc3N derivatives.