Structure of O-specific polysaccharide chains of Pseudomonas aeruginosa II (Sandwick) and V (Verder-Evans) lipopolysaccharides containing N-acetyl-D-galactosaminuronic acid

Acidic polysaccharides were isolated from the Pseudomonas aeruginosa II (Sandvik) and V (Verder-Evans) lipopolysaccharides on mild acid hydrolysis followed by gel filtration on Sephadex G-50. The Sandvik II polysaccharide consists of 2-acetamido-2-deoxy-D-galacturonic acid, 2-acetamido-2,6-dideoxy-D-glucose, and L-rhamnose in the ratio 1:1:2. The Verder-Evans V polysaccharide contained the same monosaccharides and, in addition, a D-glucose residue. On the basis of 13C NMR data, methylation analysis, Smith degradation and solvolysis with hydrogen fluoride, the following structures were determined for the repeating units of the polysaccharides: (Formula: see text).     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of P. aeruginosa O:3a, b and O:3a, d lipopolysaccharides

On mild acid degradation of Pseudomonas aeruginosa O:3a,b and O:3a,d lipopolysaccharides O-specific polysaccharides were isolated. Both polysaccharides were found to contain 2-acetamido-2,6-dideoxy-D-galactose, identified as fucosamine hydrochloride formed after hydrolysis with a very low yield. The other two components of the trisaccharide repeating unit, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid and 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, were identified without isolation in their free state directly in the course of structural investigation of the polysaccharides. Both these monosaccharides have never before been found in nature. Solvolysis of either O:3a,b or O:3a,d polysaccharides with liquid hydrogen fluoride resulted in the formation of the same trisaccharide, N-acetylfucosamine residue being the reducing end. The structure of this trisaccharide, which is the repeating unit of both polysaccharides, was deduced from the results of successive chemical modifications and 13C-nuclear magnetic resonance spectra recorded for every oligosaccharide formed. As a result, the acidic diaminosugars were converted into 2,3-diacetamido-2,3-dideoxy-D-mannose indistinguishable from authentic sample. The O-specific polysaccharides O:3a,b and O:3a,d differed in the configuration of the glycosidic bond of N-acetylfucosamine residue only and had the following structures: leads to 4)DManImU(beta 1 leads to 4)DMan(NAc)2U (beta 1 leads to 3)DFucNAc(beta 1- leads to 4)DManImU(beta 1 leads to 4)DMan(NAc)2U (beta 1 leads to 3)DFucNAc(alpha 1- where DManImU = 2.3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2, 3-dideoxy-D-mannuronic acid, DMan(NAc)2U = 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, DFucNAc = 2-acetamido-2,6-dideoxy-D-galactose. The structures established were in agreement with optical rotations and assignments of all the signals in the 13C-nuclear magnetic resonance spectra of the polysaccharides.     

Antigenic polysaccharides of bacteria. 21. The structure of O-specific polysaccharide chains and serological specificity of lipopolysaccharides from 7 Pseudomonas aeruginosa immunotypes

On mild acid degradation on lipopolysaccharides of seven Pseudomonas aeruginosa immunotypes, O-specific polysaccharides were obtained and their structures established. A peculiar feature of the polysaccharides is the presence of various, mostly acidic, mono- and diaminosugars, many of which have not previously been found in nature. The absence of serological cross-reactions (inhibition of passive haemagglutination) between lipopolysaccharides of seven immunotypes correlates with the absence of any common oligosaccharide fragments in their O-specific chains. The data obtained revealed structural and serological interrelations between O-antigens of seven immunotypes and P. aeruginosa O-serotypes, and showed that immunotypes 1 and 7 should be included into the serological classification scheme as individual O-serotypes.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the polysaccharide chain of Ps. aeruginosa O-serogroup 7 (Lanyi) lipopolysacharide

A loosely bound lipopolysaccharide-protein complex was extracted from cells of Pseudomonas aeruginosa strain 170015 (O:7ab; Lanyi classification) by saline solution and purified from contaminant nucleic acid by Cetavlon treatment followed by precipitation in an ultracentrifuge. The saline-treated cells were re-extracted with hot aqueous phenol to give firmly bound lipopolysaccharide which was isolated from the phenol layer and purified by ultracentrifugaiton. The identity of both lipopolysaccharide preparations was proved by serological and chemical evidence. Mild acid degradation of the lipopolysaccharide resulted in the splitting off of a lipid component and led to polysaccharide which was purified by gel-filtration on a Sephadex G-50 column. The polysaccharide consisted of N-acetyl-D-fucosamine, N-acetyl-L-fucosamine and D-glucose in the ratio 1:1:1. On the basis of nuclear magnetic resonance spectra, results of methylation analysis and two sequential Smith degradations, the following structure can be assigned to the repeating unit of the polysaccharide: -3)LFucNAc(alpha 1-3)DFucNAc(beta 1-2)DGlc(beta 1-. The polysaccharide did not show serological activity whereas alkali-treated lipopolysaccharide readily sensitised sheep erythrocytes and inhibited the passive haemagglutination reaction with anti-(O:7a,b)serum. Evidence is presented that the oligosaccharide repeating units of the polysaccharide and alkali-treated lipopolysaccharide are indistinguishable. Ps. aeruginosa strain 170016 (O:7a,c) was shown to have the O-specific lipopolysaccharide identical with that from strain 170015. The presented data show that subfactors 7b and 7c in the Lanyi classification of Ps. aeruginosa O-antigens seem to relate to components of the bacterial surface other than lipopolysaccharides.     

The structure of O-specific polysaccharide chains of lipopolysaccharides from Citrobacter 032 and Salmonella arizonae 064 (Arizona 29)

On the basis of acid hydrolysis, methylation, Smith degradation, selective cleavage with anhydrous hydrogen fluoride, and 13C NMR analysis, the repeating unit of the O-specific polysaccharide of Citrobacter O32 was concluded to have the following structure: (Formula: see text). The repeating unit of the Salmonella arizonae O64 O-specific polysaccharide has the same structure lacking the O-acetyl group.     

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens IMV 2366 (biovar C)

The O-specific polysaccharide of P. fluorescens IMV 2366 was studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D gsCOSY, TOCSY, gsNOESY, H-detected 1H,(13)C gsHSQC, HMQC-TOCSY, and gsHMBC experiments. The polysaccharide contains L-rhamnose, 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) and 3-acylamido-3,6-dideoxy-D-glucose (D-Qui3NAcyl, where Acyl is 3-hydroxy-2,3-dimethyl-5-oxoprolyl). The structure 1 of the polysaccharide was found to be similar to the structure 2 of a 6-deoxy-L-talose (L-6dTal)-containing O-specific polysaccharide of a non-classified P. fluorescens strain, 361, studied earlier [Khomenko, V. A.; Naberezhnykh, G. A.; Isakov, V. V.; Solov'eva, T. F.; Ovodov, Y. S.; Knirel, Y. A.; Vinogradov, E. V. Bioorg. Khim. 1986, 12, 1641-1648; Naberezhnykh, G. A.; Khomenko, V. A.; Isakov, V. V., El'kin, Y. N.; Solov'eva, T. F.; Ovodov, Y. S. Bioorg. Khim. 1987, 13, 1428-1429]. --> 2)-beta-D-Quip3NAcyl-(1 --> 3)-alpha-L-Rhap-(1 --> 3)-alpha-D-FucpNAc-(1 --> 1. --> 4)-beta-D-Quip3NAcyl-(1 --> 3)-alpha-L-6dTalp4Ac-(1 --> 3)-alpha-D-FucpNAc-(1 -->2.     

The lipopolysaccharide from Pseudomonas aeruginosa NCTC 8505. Structure of the O-specific polysaccharide

Structural studies have been carried out on the O-specific fraction from the lipopolysaccharide of Pseudomonas aeruginosa NCTC 8505, Habs serotype 03. The O-specific polysaccharide has a tetrasaccharide repeating-unit containing residues of L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and 2,4-diacetamido-2,4,6-trideoxy-D-glucose (BacNAc2). The following structure has been assigned to the repeating-unit: leads to 3)Rhap(beta 1 leads to 6)GlcpNAc(alpha 1 leads to 4)GalpNAcA(alpha 1 leads to 3)BacpNAc2(alpha 1 leads to. The parent lipopolysaccharide is a mixture of S, R, and SR species, and its high phosphorus content is partly due to the presence of triphosphate residues, as found for other lipopolysaccharides from P. aeruginosa. In addition to phosphorus, heptose, a 3-deoxyoctulosonic acid, and amide-bound alanine, the core oligosaccharide contains glucose, rhamnose, and galactosamine (molar proportions 3:1:1). The rhamnose and part of the glucose are present as unsubstituted pyranoside residues: other glucose residues are 6-substituted.     

Antigenic polysaccharides of bacteria. 32. The structure of O-specific polysaccharide chains of Pseudomonas cepacia serotype B and E lipopolysaccharides containing D-fucose

On the basis of acid hydrolysis, methylation, 1H and 13C NMR analysis, and calculation of specific optical rotation, the following structures were established for O-specific polysaccharides of Pseudomonas cepacia serotypes B and E: ----3)-beta-D-Galf-(1----3)-alpha-D-Fucp-(1----serotype B ----3)-beta-D-GlcpNAc-(1----3)-alpha-D-Fucp-(1----serotype E A characteristic feature of the polysaccharides is the presence of D-fucose, rather rare for bacterial antigens.     

Somatic antigens of the Brucella genus. The structure of the O-specific polysaccharide chain of Brucella melitensis lipopolysaccharide

The phenol-phase soluble antigenic lipopolysaccharide was isolated from Brucella melitensis, strain 565, by the routine phenol/water procedure followed by chromatography on Sepharose 4B. After mild acid hydrolysis and chromatography on Sephadex G-50, the lipopolysaccharide yielded a linear O-specific polysaccharide built up from 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The structure of the polysaccharide was deduced mainly from the nuclear magnetic resonance and methylation analyses. The phenol-soluble lipopolysaccharide, isolated from commercial vaccine strain B. abortus 19-BA, on mild hydrolysis afforded material, 13C and 1H-NMR spectra of which were identical to those of the O-specific polysaccharide from B. melitensis 565.     

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----.     

Antigenic polysaccharides of bacteria. 16. The structure of O-specific polysaccharide chain of Pseudomonas aeruginosa 025 (Wokatsch) lipopolysaccharide

O-Specific polysaccharide built up of trisaccharide repeating units containing 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid (ManNAcAmA), 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (Man(NAc)2A), N-acetyl-D-fucosamine (FucNAc), and O-acetyl group was obtained on mild acid hydrolysis of P. aeruginosa O25 (Wokatsch classification) lipopolysaccharide. Basing on de-O-acetylation of polysaccharide with aqueous triethylamine accompanied by hydrolysis of acetamidino group to acetamido group, as well as on the 1H and 13C NMR data, the following structure of the repeating unit of the polysaccharide was established: (Formula: see text) P. aeruginosa O25 polysaccharide has the same carbohydrate skeleton as that of P. aeruginosa O3a,b (Lányi classification) and differs from the latter only by the presence of the O-acetyl group at position 4 of N-acetylfucosamine.     

Somatic antigens of Shigella and Escherichia coli. Determination of the structure of O-specific polysaccharide from Shigella boydii type 12 and its serological properties compared with the O-specific polysaccharide from Escherichia coli

The structure of the O-specific polysaccharide of the somatic antigen (lipopolysaccharide) of Shigella boydii, type 12, was established by 1H- and 13C-NMR, methylation analysis and partial acid hydrolysis methods. The polysaccharide consists of pentasaccharide repeating units of the following structure: (formula; see text) The amount of O-acetyl groups was far less than stoichiometric, only about 2 for 3-4 repeating units. Nevertheless, the results of serological studies revealed 3-O-acetyl-alpha-L-rhamnose residue to be the major immunodominant group. In spite of the presence of similar trisaccharide fragments, the lipopolysaccharide and polysaccharide from Shigella boydii type 12 gave no crossreaction with lipopolysaccharide and polysaccharide from Escherichia coli 07. The possible reasons of the absence of serological relatedness between the Sh. boydii, type 12, and E. coli 07 cells were discussed.     

Antigenic polysaccharides of bacteria. 34. Structure of O-specific polysaccharide chains of lipopolysaccharides from Pseudomonas cepacia strains IMV 4207 (Serotype A) and IMV 598/2

On the basis of non-destructive analysis by means of 1H and 13C NMR spectroscopy and calculation of specific optical rotation, it was concluded that O-specific polysaccharide of Pseudomonas cepacia strain IMV 4207 (serotype A) has the structure (I): (formula; see text) Two structurally different polysaccharides were found in the ratio of approximately 2.5:1 in P. cepacia strain IMV 598/2 which is serologically related to serotype A in Nakamura classification and serotype 2 in Heidt classification. The minor polysaccharide has the structure (I) whereas the major one possesses the structure (II) which is characteristic of the formerly studied O-specific polysaccharide of P. cepacia strain IMV 4137 belonging to serotype 2: ----4)-beta-D-Galp-(1----2)-alpha-L-Rhap-(1----.     

The structure of the O-specific chain of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584)

Structural studies were carried out on the O-polysaccharide fraction obtained from the lipopolysaccharide of Pseudomonas aeruginosa IID 1008 (ATCC 27584). The O-polysaccharide comprises L-rhamnose, N-acetyl-D-quinovosamine, N-acetyl-D-galactosaminuronic acid, and N-formyl-D-galactosaminuronic acid. The characterization of oligosaccharide fragments resulting from acid hydrolysis, Smith degradation and alkaline degradation of the O-polysaccharide, together with 1H-NMR and 13C-NMR spectroscopic data of the polysaccharide, led to the following structure for the repeating units: ----3)Rha(alpha 1----4)GalNAcA(alpha 1----4 GalNFoA(alpha 1----3)QuiNAc(alpha 1----. Almost all of the carboxyl groups of the N-acetylgalactosaminuronic acid residues and about half of the same groups of the N-formylgalactosaminuronic acid residues were in an amide form.     

The structure of the O-specific chain of lipopolysaccharide from Pseudomonas aeruginosa IID 1012 (ATCC 27588)

Structural studies were carried out on the O-polysaccharide fraction obtained from the lipopolysaccharide of Pseudomonas aeruginosa IID 1012, the standard strain of Homma serogroup K, by mild acid treatment. The O-polysaccharide was composed of L-rhamnose, N-acetyl-D-quinovosamine, and N-acetyl-D-galactosaminuronic acid. The results from analysis of fragments obtained by acid hydrolysis and Smith degradation of the O-polysaccharide, together with data on methylation analysis and nuclear magnetic resonance spectroscopic measurement of the polysaccharide, led to the most likely structure of the repeating units of the polymer chain, ----4)D-GalNAcA(alpha 1----3)D-QuiNAc(beta 1----2)L-Rha(alpha 1----3)L-Rha(alpha 1----, in which about 20% of the N-acetylgalactosaminuronic acid residues were in an amide form and about 75% of the same residues were O-acetylated at C-3.     

The structure of the O-specific polysaccharide from the lipopolysaccharide of Pseudomonas sp. OX1 cultivated in the presence of the azo dye Orange II

The Gram-negative bacterium Pseudomonas sp. OX1, previously known as Pseudomonas stutzeri OX1, is endowed with a high metabolic versatility. In fact, it is able to utilize a wide range of toxic organic compounds as the only source of carbon and energy for growth. It has been recently observed that, while growing on a glucose-containing liquid medium, Pseudomonas sp. OX1 can reduce azo dyes, ubiquitous pollutants particularly resistant to chemical and physical degradation, with this azoreduction being a process able to generate enough energy to sustain bacterial survival. We have found that, under these conditions, modifications in the primary structure of the O-specific polysaccharide (OPS) within the lipopolysaccharides occur, leading to remarkable changes both in the monosaccharide composition and in the architecture of the repeating unit, with respect to the polysaccharide produced in the absence of azo dyes. In the present paper, we present the complete structure of this O-specific polysaccharide, whose repeating unit is the following: [Formula: see text] This structure is totally different from the one determined from Pseudomonas sp. OX1 grown on rich medium.     

The structure of O-specific polysaccharide from Pseudomonas aeruginosa 013 containing 5,7-diacetoamido-3,5,7,9-tetradeoxy-D- glycero-L-galacto-nonulosonic acid and 2-acetamidino-2,6-dideoxy-L-galactose

O-Specific polysaccharide chain of P. aeruginosa 013 (Lányi) lipopolysaccharide is composed of N-acetyl-D-quinovosamine (QuiNAc), acetamidino derivative of L-fucosamine (FucNAm), and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso nic acid (Sug). On solvolysis with HF in methanol, the polysaccharide afforded methylglycosides of a disaccharide and a trisaccharide both containing fucosamine and ulosonic acid derivatives. Chemical transformations (alkaline hydrolysis, reductive deamination, acetylation accompanied by intramolecular acylation of acetamidino group by ulosonic acid), 1H and 13C NMR analysis and mass spectral data proved the following structure of the trisaccharide unit of the polysaccharide: -8)-beta-Sug-(1-3)-alpha-L-FucNAm-(1-3)-alpha-D-QuiNAc -(1-     

Immunologic study of artificial complex antigens obtained from P. aeruginosa lipopolysaccharides]

Artificial antigens were obtained on the basis of the polysaccharide component of P. aeruginosa complexed with an indifferent protein. Immunological study indicated that the specific polysaccharide of P. aeruginosa lipopolysaccharide contained two structures, high molecular and low molecular, having qualitative and quantitative differences in their hydrocarbon composition. Artificial complex antigens possessed serological and immunogenic properties, the low molecular polysaccharide fraction complexed with protein having less pronounced serological and immunogenic activity than polysaccharide and the high molecular fraction complexed with protein. Antificial complex antigens exerted no protective effect in generalized P. aeruginosa infection in rats.     

The structure of the O-specific polysaccharide from Ralstonia pickettii

The following structure of the Ralstonia pickettii have been determined using NMR and chemical methods: -->4)-alpha-D-Rha-(1-->4)-alpha-L-GalNAcA-(1-->3)-beta-D-BacNAc-(1-->.