Lipolysaccharides were isolated from dry bacterial cells of Pseudomonas aeruginosa OSa,b,c, OSa,b,d, 05a,d (Lanyi classification) and immunotype 6 (Fisher classification) by the Westphal procedure. Their polysaccharide chains were built up of trisaccharide repeating units containing ~-xylose, 2-acetamido-2,6-dideoxy-~-galactose and a new sialic acid-like sugar, the di-N-acyl derivative of 5,7-diarnino-3,5,7,9-tetradeoxy-~-glycero-~-mannononulosonic (pseudaminic) acid. Formyl, acetyl and (R)-3-hydroxybutyryl groups were identified as the N-acyl substituents of the last monosaccharide; O5a,b,c and O5a,b,d lipopolysaccharides also contained 0-acetyl groups.The glycosidic linkage of pseudaminic acid was extremely labile towards acids, and mild acid degradation of the lipopolysaccharides produced, instead of the 0-specific polysaccharides, their trisaccharide fragments with pseudaminic acid at the reducing terminus. Similar degradation of immunotype 6 lipopolysaccharides, followed by oxidation with sodium metaperiodate, resulted in a disaccharide fragment due to destruction of xylose. In contrast the glycosidic linkage of pseudaminic acid proved to be more stable towards treatment with hydrogen fluoride than those of xylose and N-acetylfucosamine. As a result, solvolysis of immunotype 6 lipopolysaccharide with hydrogen fluoride in methanol gave methyl glycosides of a disaccharide and a trisaccharide with pseudaminic acid at the non-reducing terminus. Mild acid hydrolysis of these oligosides afforded free 5-N-acetyl-7-Nformylpseudaminic acid, which was identified by the 'H and 13C nuclear magnetic resonance data, as well as by the mass spectrum of the corresponding fully methylated aldonic acid.As a result of the identification of all oligosaccharides obtained and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and lipopolysaccharides the following structures were estab- +~) -D -X~~~-(~~+~) -D -F~C N A C -(~~+~) -P S~~N A C~N F~-(~where D-xyl = ~-xylose, D-FUCNAC = 2-acetamido-2,6-dideoxy-~-galactose, Pse5N7NFm = 5-amino-3,5,7,9-tetradeoxy-7-formamido-~-glycero-~-manno-nonulosonic acid (7-N-formylpseudaminic acid). All the polysaccharides have an identical carbohydrate skeleton and differ from each other by the acyl substituent at N-5 of pseudaminic acid [acetyl or (R)-3-hydroxybutyryl group] or by the presence or absence of the 0-acetyl group at position 4 of N-acetylfucosamine. The data obtained account properly for the 0 specificity of the studied P. aeruginosa strains.The data on composition and structure of lipopolysaccharides, determining the 0 specificity of gram-negative bacteria, are employed to work out intraspecies chemotype schemes. Investigation of the lipopolysaccharides of an opportunistic bacterial pathogen P. aeruginosa showed many [2, 81, acetamidino [5, 6, 91, (R)- [7] and (S)-3-hydroxybutyryl [3] groups.In the present communication we report the establishing of the structures of the 0-specific polysaccharide chains of P. aeruginosa 0 5 (Lanyi classifi...

Pseudomonas aeruginosa is one of the most important opportunistic bacterial pathogens in humans and animals. This organism is ubiquitous and has high intrinsic resistance to antibiotics due to the low permeability of the outer membrane and the presence of numerous multiple drug efflux pumps. Various cell-associated and secreted antigens of P. aeruginosa have been the subject of vaccine development. Among pseudomonas antigens, the mucoid substance, which is an extracellular slime consisting predominantly of alginate, was found to be heterogeneous in terms of size and immunogenicity. High molecular mass alginate components (30^300 kDa) appear to contain conserved epitopes while lower molecular mass alginate components (10^30 kDa) possess conserved epitopes in addition to unique epitopes. Surface-exposed antigens including Oantigens (O-specific polysaccharide of LPS) or H-antigens (flagellar antigens) have been used for serotyping due to their highly immunogenic nature. Chemical structures of repeating units of O-specific polysaccharides have been elucidated and these data allowed the identification of 31 chemotypes of P. aeruginosa. Conserved epitopes among all serotypes of P. aeruginosa are located in the core oligosaccharide and the lipid A region of LPS and immunogens containing these epitopes induce crossprotective immunity in mice against different P. aeruginosa immunotypes. To examine the protective properties of OM proteins, a vaccine containing P. aeruginosa OM proteins of molecular masses ranging from 20 to 100 kDa has been used in pre-clinical and clinical trials. This vaccine was efficacious in animal models against P. aeruginosa challenge and induced high levels of specific antibodies in human volunteers. Plasma from human volunteers containing anti-P. aeruginosa antibodies provided passive protection and helped the recovery of 87% of patients with severe forms of P. aeruginosa infection. Vaccines prepared from P. aeruginosa ribosomes induced protective immunity in mice, but the efficacy of ribosomal vaccines in humans is not yet known. A number of recent studies indicated the potential of some P. aeruginosa antigens that deserve attention as new vaccine candidates. The outer core of LPS was implicated to be a ligand for binding of P. aeruginosa to airway and ocular epithelial cells of animals. However, heterogeneity exists in this outer core region among different serotypes. Epitopes in the inner core are highly conserved and it has been demonstrated to be surface-accessible, and not masked by O-specific polysaccharide. The use of an in vivo selection/expression technology (IVET) by a group of researchers identified a number of P. aeruginosa proteins that are expressed in vivo and essential for virulence. Two of these in vivo-expressed proteins are FptA (ferripyochelin receptor protein) and a homologue of an LPS biosynthetic enzyme. Our laboratory has identified a highly conserved protein, WbpM, and P. aeruginosa with a deficiency in this protein produces only rough LPS and became serum sensit...

Mild acid degradation of lipopolysaccharides from Pseudomonas aeruginosa 0 1 Oa and 010a,b (Lanyi classification) resulted in 0-specific polysaccharides built up of trisaccharide repeating units containing 2-acetamido-2,6-dideoxy-o-glucose (N-acetylquinovosamine, DQuiNAc), 2-acetamido-2,6-dideoxy-~-galactose (N-acetylfucosamine, DFucNAc), and 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-3-hydroxybutyramido]-~-glycero-~-munnononulosonic acid. The latter is a di-N-acyl derivative of a new sialic-acid-like sugar which was called by US pseudaminic acid (PseN2). A 3-hydroxybutyric acid residue was also found in natural carbohydrates for the first time. In the 010a,b polysaccharide pseudaminic acid carried an 0-acetyl group at position 4.For selective cleavage of the 0 1 Oa polysaccharide, solvolysis with hydrogen fluoride was employed which, owing to the relatively high stability of the glycosidic linkage of pseudaminic acid, led to the disaccharide with this sugar on the non-reducing terminus. Performing the solvolysis in methanol afforded the methyl glycoside of this disaccharide which proved to be more advantageous for further analysis. Carboxyl-reduction made the glycosidic linkage of pseudaminic acid extremely labile, and mild acid hydrolysis of the carboxyl-reduced 01 Oa polysaccharide afforded the trisaccharide with a ketose derivative on the reducing terminus. Establishing the structure of the oligosaccharide fragments obtained and interpreting the 13C nuclear resonance spectra of the polysaccharides allowed to determine the following structure for their repeating units:In the polysaccharides the N-acetylquinovosamine residue is attached not to pseudaminic acid itself, but to its N-acyl substituent, 3-hydroxybutyryl group, and thus the monomers are linked via both glycosidic and amidic linkages.Pseudomonas aeruginosa is an opportunistic pathogen causing heavy surgical infections. In the serological sense. it is a complex species, counting by different data from 8 to 32 0-serotypes [I, 21; a unified serological classification for P. aeruginosu is still lacking. Attempts to create a chemical foundation for such a classification on the basis of the composition and structure of 0-antigens [3-51 ran into difficulties connected with the occurrence in them of highly unusual constituents. Thus, P. aeruginosa 0-specific polysaccharides involved derivatives of 2,4-diamino-2,4,6-trideoxy-D-glUCOSe 161, 2-amino-2-deoxy-~-and -L-galacturonic acid [6 -81, and 2,3-diamino-2,3-dideoxyuronic acids [9 -1 I], the latter sugars not being found in other sources. Only quite recently, has the data on the structure of the polysaccharides containing these rarely occurring aminosugars become available [6 -1 I].Not long ago we found that some 0-specific polysaccharides from P. aeruginosa involve monosaccharides of another hitherto unkown type. These are sialic-acid-like MATERIALS AND METHODS Miscellaneous methods'H and I3C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in D20 at 60°C for polysaccharides ...

An 0-antigenic lipopolysaccharide, manifesting a high serological activity in the reaction of passive haemagglutination and its inhibition, was isolated from Pseudomonas aeruginosa 0: 6 cells (Lanyi). Splitting-off of the lipid component by mild acid hydrolysis resulted in the polysaccharide deprived of 0-specific activity.The polysaccharide chain of the lipopolysaccharide was made up exclusively of the residues of amino sugars, among which have been identified: 2-acetamido-2-deoxy-~-galactose (GalNAc), 2-acetamido-2,6-dideoxy-~-glucose (QuiNAc), 2-acetamido-2,6-dideoxy-~-galactose (FucNAc) as well as 2,3-diacetamido-2,3-dideoxy-D-g1UCUrOniC acid [Glc(NAc)zA] found naturally for the first time. The principal methods of establishing the polysaccharide structure were I3C nuclear magnetic resonance, methylation analysis and solvolysis with hydrogen fluoride. Depending on solvolysis conditions, a disaccharide or a trisaccharide containing the diacetamidouronic acid residue was formed.From the results obtained it followed that the polysaccharide chain of the 0-antigenic Ps. aeruginosa 0 : 6 lipopolysaccharide represents an acidic hexosaminoglycan constructed of the repeating tetrasaccharide units of the following structure: +4)~GalNAc(al~4)~Glc(NAc)2A(~I -+ 3)~FucNAc(al+ 3)~QuiNAc(aI +.Chemical and immunochemical studies on the antigenic polysaccharides and lipopolysaccharides of serologically wellcharacterized bacteria, such as Salmonella, Escherichia coli, Klehsiella and Streptococcus pneumonia, are aimed, as a rule, at ascertaining the relationship between the structure of K and 0-antigens and the immunological properties of the microbial cell. In the case of species which are difficult to type, the information on the chemical structure of individual antigens can also serve as an important classificational criterion.Our attention has been centred on Pseudomonas aeruginosa bacteria for which, despite numerous studies, there is up to now no generally accepted serological classification [I], nor information on the structure of the 0-specific polysaccharides. In previous communications we presented the structural data for the polysaccharides of Ps. aeruginosa, serogroups 0 : 2 and 0 : 7 (Lanyi), and showed that the 0-specific polysaccharides of the 0 : 2a,b and 0 : 2a,c serotypes have a different, although similar, structure of the repeating units (preceding paper [2]), whereas the division of 0 : 7 group into two types, 0 : 7a,b and 0 : 7a,c, was not confirmed chemically as both these serotypes produce lipopolysaccharides with identical polysaccharide chains [3].In the present communication we report on the chemical structure of the polysaccharide chain of the 0-antigenic Ps. aeruginosa 0 : 6 lipopolysaccharide. The polysaccharide Abbreviations. GalNAc, 2-acetamido-2-deoxygdlactose; GI~(NA~)Z.A, 2,3-diacetamido-2,3-dideoxyglucuronic acid; FucNAc, N-acetylfucosamine, i.e. 2-acetamido-2,6-dideoxygalactose; QuiNAc, N-acetylquinovosamine, i.e. 2-acetamido-2,6-dideoxyglucose; Glc(NAc)z, 2,3-diacetamido-2,3-dideoxyglucose....

Structural and immunochemical studies have been performed on the 0-specific antigens of two serologically related types of Pseudomonas aeruginosa 0 : 2a,b and 0 : 2a,c (Lanyi's classification). The 0-specific polysaccharide chains of the two serotypes were shown to be acidic polysaccharides composed of repeating trisaccharide units consisting of L-rhamnose, N-acetyl-D-quinovosamine and N-acetyl-L-galactosaminuronic acid residues. Based on ' H and 13C nuclear magnetic resonance spectroscopy, data of methylation analysis and selective solvolysis with hydrogen fluoride, the repeating unit of the 0 : 2a,c 0-specific polysaccharide was assigned the following structure : -+ 4)~GalNAcA(stl+ 3)~QuiNAc(xl+ 3 )~R h a ( a l , where GalNAcA = N-acetylgalactosaminuronic acid, QuiNAc = N-acetylquinovosamine and Rha = rhamnose.The 0 : 2 a , b 0-specific polysaccharide had the same structure of the trisaccharide repeating unit but was distinguished by the presence of 0-acetyl groups on 70-80% of the rhamnose residues in position 2. The 0-acetyl groups were located both by methylation with methyltrifluoromethane sulphonate and by comparison of the 13C nuclear magnetic resonance spectra of the native and 0-deacetylated polysaccharides.Serological studies revealed the determinant role of the 0-acetyl groups in the 0-antigen 0 : 2a,b and suggested the 0-factor 2b to be related to the 2-0-acetyl-~-rhamnose residue, whereas the 0-factor 2c was probably determined by the nonacetylated rhamnose residue. The dominant moiety of the determinant 2a common to the two antigens was obviously presented by the N-acetyl-L-galactosaminuronic acid residue.Up to now, despite considerable efforts [l], no commonly adopted serological classification of Pseudomonas aeruginosa has been developed. In a previous communication [2] we proposed that the chemical structural data on the 0-specific chains of the corresponding somatic antigens (lipopolysaccharides) should be considered as a useful criterion for such a kind of classification. The absence of a common group-0 factor is typical for the Ps. aeruginosa 0-antigens but a serological relationship is known to exist for certain 0-serotypes. Thus, Lanyi [3] distinguished, along with insulated O-serotypes, several 0-serogroups involving from two to five crossreacting 0-serotypes. Chemical and immunochemical studies of these relating 0-serotypes seem to represent a primary interest for the rationalization of the serological properties of Ps. aeruginosa in terms of chemical structures of the corresponding 0-specific polysaccharides.Earlier, we established the structures of the 0-specific polysaccharide chains of the Ps. aeruginosa 0: MATERIALS A N D METHODS Miscellaneous MethodsDescending paper chromatography was performed on Filtrak FN-I1 paper in the solvent system ethyl acetate/ water/acetic acid/pyridine (5/3/1/5, v/v). Paper electrophoresis was carried out in a pyridine/acetate buffer, pH 5.5 at 28 V/cm. Ion-exchange chromatography of hexosamines and neutral sugars, as well as gas-liquid chromatography, ...