Structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: (struture: see text). The main D-mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.

Structure of an acidic O-specific polysaccharide of the bacterium Providencia alcalifaciens O7.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O7 and purified by gel chromatography followed by anion-exchange chromatography. On the basis of full acid hydrolysis, methylation, carboxyl reduction, selective cleavage with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional 1H,1H homonuclear and H-detected 1H,13C heteronuclear correlation spectroscopy and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: [figure], where Rhap2Ac is 2-O-acetylrhamnopyranose.

Structure of a neutral O-specific polysaccharide of the bacterium Providencia alcalifaciens O5.

A neutral polysaccharide containing D-galactose, 2-acetamido-2-deoxy-D-glucose, and 3-acetamido-3,6-dideoxy-D-glucose (Qui3NAc) in the ratios 2:1:1 was obtained by mild acid degradation of lipopolysaccharide of the bacterium Providencia alcalifaciens O5 followed by gel chromatography and ion-exchange chromatography or treatment with anhydrous hydrogen fluoride. On the basis of full acid hydrolysis, methylation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), H-detected heteronuclear 1H,13C single-quantum coherence (HSQC), and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established:

[An antigenic analysis and the protective properties of the R-form lipopolysaccharides of gram-negative bacteria]. / Antigennyi analiz i protektivnye svoistva lipopolisakharidov R-form nekotorykh gramotritsatel'nykh bakterii.

The antigenic and immunogenic properties of the R-form lipopolysaccharides (R-LPS) of Pseudomonas aeruginosa, Salmonella minnesota, Escherichia coli and Shigella were studied. The results of the study revealed the existence of antigenic relationship between P.aeruginosa R-LPS and R-LPS Escherichia and Shigella. In serological tests no antigenic relationship between P. aeruginosa R-LPS and Salmonella R-LPS was revealed, but as shown in earlier experiments of the protection of mice, Re-glycolipid stimulated protective immunity against Pseudomonas infection in the animals. On the basis of R-LPS obtained from selected P. aeruginosa and Salmonella strains a vaccine was prepared which proved to be effective against infection caused by P. aeruginosa S-strain in experiments on mice. The vaccine induced protection in 40-100% of immunized mice, depending on the scheme of immunization. The vaccine may probably be effective against infections caused by other gram-negative bacteria.

Structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c containing D-mannose and D-rhamnose was established using sugar analysis and NMR spectroscopy, including computer-assisted analysis of the 13C NMR spectrum, 2D COSY, H,H-relayed COSY, heteronuclear 13C, 1H correlation (HETCOR), and rotating-frame NOE spectroscopy (ROESY):-->4)-alpha-D-Manp-(1-->3)-beta-D-Rhap-(1-->4) -beta-D-Rhap-(1-->.

R-form lipopolysaccharides (LPS) of Gram-negative bacteria as possible vaccine antigens.

The antigenic and immunogenic properties of R-form lipopolysaccharides (LPS) of Pseudomonas aeruginosa, Salmonella spp., Escherichia coli and Shigella spp. were studied. The results showed the presence of antigenic relationships among P. aeruginosa R mutants with different structures of the LPS core lipid A region and also among E. coli, Shigella and P. aeruginosa R-LPS, but not with S. minnesota Re-LPS. Vaccines prepared with R-LPS proved to be effective preparations for the active immunization of mice against P. aeruginosa infection. The vaccine stimulated 40-100% protection in mice depending upon the scheme of immunization.

Structure of the O-specific polysaccharide of the bacterium Providencia alcalifaciens O23 containing a novel component: an amide of D-glucuronic acid with N(epsilon)-(1-carboxyethyl)lysine.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O23 and found to contain D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and N epsilon-(1-carboxyethyl)-N alpha-(D-glucuronoyl)lysine. On the basis of full and partial acid hydrolyses, selective solvolysis with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H, 13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), the following structure of the liner tetrasaccharide repeating unit of the polysaccharide was established [structure: see text]

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella.

Structural analysis using 13C NMR spectroscopy and methylation showed that lipopolysaccharides (LPSs) of Citrobacter freundii O35 and Salmonella arizonae O59 have structurally identical O-specific polysaccharide chains, and those of C. freundii O38 and Salmonella kentucky differ only in the presence of O-acetyl groups in the former. Serological relationships between the structurally similar LPSs were demonstrated using inhibition of ELISA, rocket immunoelectrophoresis, double gel diffusion, and immunoblotting. The O-acetyl groups present in C. freundii O38 LPS are of little importance for its serological specificity. A cross-reaction was observed in immunoblotting between O-antisera to C. freundii O35 and S. arizonae O59 and a structurally related LPS of Pseudomonas aeruginosa O11a, 11b (Lányi-Bergan classification).

Mouse protection induced by Pseudomonas aeruginosa PAC1R and its defective mutants, Salmonella minnesota Re-mutant and Escherichia coli O14.

Pseudomonas aeruginosa PAC1R and its defective mutants (acetone-killed bacteria), Salmonella minnesota Re mutant (acetone-killed bacteria and Re-LPS) and Escherichia coli O14 (acetone-killed bacteria and enterobacterial common antigen, ECA) were studied in a mouse active protection test. Immunized mice were challenged with wild-type P. aeruginosa strains. It was established that P. aeruginosa LPS-defective mutants induced cross-immunity against different Fisher immunotypes of P. aeruginosa. S. minnesota Re-LPS and ECA gave mice protection against P. aeruginosa.

The structure of the O-specific polysaccharide of Salmonella arizonae O21 (Arizona 22) containing N-acetylneuraminic acid.

The O-specific polysaccharide of S. arizonae O21 was found to contain 2-acetamido-2-deoxy-D-glucose, 2-acetamidino-2,6-dideoxy-L-galactose, N-acetylneuraminic acid, and O-acetyl groups. On the basis of 1H and 13C NMR studies of the intact and O-deacetylated polysaccharide and oligosaccharide fragments obtained by solvolysis with anhydrous hydrogen fluoride, partial methanolysis and partial hydrolysis, it was concluded that the O-specific polysaccharide has the following structure: [formula: see text]

The structure of the O-specific polysaccharide of Citrobacter O16 containing glycerol phosphate.

The O-specific polysaccharide, obtained by mild acid degradation of Citrobacter O16 lipopolysaccharide, consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, glycerol and phosphate in the ratios 2:2:2:1:1. Selective cleavage of the polysaccharide was carried out by Smith degradation, N-deacetylation-deamination and dephosphorylation with 48% hydrofluoric acid, which was accompanied by unexpected splitting of one of the glycosidic linkages. The structures of the oligosaccharides thus obtained were established using 1H- and 13C-NMR spectroscopy, including one-dimensional NOE, two-dimensional rotating-frame NOE, homonuclear and heteronuclear 13C, 1H correlation spectroscopy, and, for the Smith degradation product, positive- and negative-ion-mode fast-atom-bombardment MS and MS/MS with collision-induced dissociation. On the basis of these data and the results of methylation analysis, it was concluded that the O-specific polysaccharide has the following repeating unit structure: [formula: see text]

Immunological studies of an artificial antigen with specificity of a common polysaccharide antigen of Pseudomonas aeruginosa.

Synthetic D-rhamnan, with the structure of Pseudomonas aeruginosa common polysaccharide antigen (CPA), was conjugated with BSA. The artificial antigen obtained, and the natural antigens, lipopolysaccharides (LPS) of P. aeruginosa and Pseudomonas cerasi with rhamnan chains of the same structure, were studied by ELISA with rabbit antibodies to the D-rhamnan-BSA conjugate and to the P. cerasi O-antigen. Immunological relations between the LPS of P. aeruginosa and P. cerasi determined by CPA as well as between these LPS and D-rhamnan-BSA were revealed by ELISA. O-antiserum to P. cerasi possesses protective activity in the mouse passive protection test when mice are challenged with some P. aeruginosa strains; the antiserum to the D-rhamnan-BSA does not possess protective activity in mice.

Structure of the O-specific polysaccharide of Salmonella arizonae O45.

The O-specific polysaccharide of Salmonella arizonae O45 (Arizona 11) is acidic and has a branched hexasaccharide repeating unit containing two residues of L-fucose, one residue each of D-galactose, D-ribose, D-glucuronic acid, and 2-acetamido-2-deoxy-D-glucose, and an O-acetyl group. It was studied with the help of 1H and 13C NMR spectroscopy, including 1D selective spin-decoupling and homonuclear Hartmann-Hahn spectroscopy, 2D homonuclear and 13C-1H heteronuclear shift-correlated (COSY) and NOE (ROESY) spectroscopy, as well as by methylation analysis, and selective cleavages with anhydrous HF (or dilute HCl) and lithium in ethylenediamine to yield two different tetrasaccharide fragments. As a result, the following structure of the polysaccharide was established: [formula: see text] Anomalous 13C chemical shifts were observed in the spectrum of the trisaccharide fragment alpha-L-Fucp-(1-->2)-beta-D-Gal p-(1-->3)-beta-D-Glc pNAc, structurally related to the Le(d) blood-group determinant, and rationalised by inter-residue proton-proton interactions.

The structure of the O-specific polysaccharide chain of the lipopolysaccharide of Salmonella arizonae O61.

The O-specific polysaccharide was obtained by mild degradation of the Salmonella arizonae O61 lipopolysaccharide with acid. It contained 2-acetamido-2-deoxy-D-glucose, 2-acetamidino-2,6-dideoxy-L-galactose (FucAm), and 7-acetamido-3,5,7,9-tetradeoxy-5-[(R)-3-hydroxybutyramido]-D- glycero-L-galacto-nonulosonic acid (Sug). On the basis of partial acid hydrolysis with 0.1 M HCl, solvolysis with anhydrous HF in methanol, and 1H- and 13C-NMR analysis (including 1H/13C inversely correlated spectroscopy for localisation of N-acyl substituents), it was concluded that the O-specific polysaccharide had the following structure. ----3)-alpha-L-FucAm-(1----3)-alpha-D-GlcNAc-(1----8)-beta-Sug+ ++-(2---- The O-antigen of S. arizonae O61 is structurally related to that of Pseudomonas aeruginosa O12, thus explaining the known serological cross-reactivity between these micro-organisms.

[The serological characteristics of the O antigens of Pseudomonas aeruginosa isolated from patients with a Pseudomonas infection during immunotherapy]. / Serologicheskaia kharakteristika O-antigenov Pseudomonas aeruginosa, vydelennykh ot bol'nykh sinegnoinoi infektsiei v protsesse immunoterapii.

Serologic characteristics of P. aeruginosa O-antigens isolated from patients with P. aeruginosa infection were studied over the course of treatment with anti-P. aeruginosa sheep immunoglobulin. The preparation was used in 54 patients with nongeneralized forms of P. aeruginosa infection (infected wounds, pleural empyema) externally or intraperitoneally. From the clinical material collected from the patients a total of 54 P. aeruginosa strains were isolated. Serologic typing of the isolated strains with factor or group diagnostic agglutinating sera has revealed the O-group composition of the isolated strains; 66% of them were classified with O-groups 2,3, and 6. Serologic variants of the strains isolated from patients proved to be stable over the course of the disease immunotherapy. Analysis of the results of bacteriologic control of immunotherapy. efficacy and the clinical data has demonstrated the efficacy of immunotherapy in 61.1% of cases and its partial effect in 20.4% of cases of P. aeruginosa infection.

[Immune response to a synthetic polysaccharide and protein conjugate]. / Immunnyi otvet k kon"iugatu sinteticheskogo polisakharida s belkom.

Synthetic polysaccharide (S-PS) containing aglycone-spacer with a free amino group was really alpha 1,6-mannan with Cn approximately 10. S-PS was transformed into isothiocyanate derivative by treating it with thiophosgene and engaged into reaction with amino group of bovine serum albumin (BSA) lysine residues. Rabbits were immunized with S-PS-BSA conjugate and antibodies to S-PS titres were estimated by means of ELISA. S-PS-BSA conjugate was proved to provoke specific anti-polysaccharide antibodies formation in rabbits.

[Experimental vaccinal prophylaxis of Pseudomonas aeruginosa burn sepsis]. / Vaktsinoprofilaktika sinegnoinogo ozhogovogo sepsisa v éksperimente.

After the injection of P. aeruginosa live culture under the burned skin of mice sepsis develops within the first 24 hours, finally leading to the death of the animals. The microorganisms can be isolated from the blood, liver, kidneys and mesenterial lymph nodes till day 3 and from the spleen till day 5. After the intraperitoneal injection of P. aeruginosa live culture into mice, sepsis also develops within 24 hours, and the culture can be isolated from the blood and parenchymatous organs till day 3. The LD50 of the culture is equal to 5.1 X 10(6) microbial cells when introduced intraperitoneally and to 30 microbial cells in experimental burn sepsis. Experimental burn sepsis clearly demonstrates the effectiveness of Pseudomonas acellular protein vaccine: its index of effectiveness exceeds 3,000.