Carbohydrate analysis and serological classification of typical and atypical isolates of Aeromonas salmonicida: a rationale for the lipopolysaccharide-based classification of A. salmonicida

The cell envelope of Aeromonas salmonicida contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of bacterial cell membrane. Using a recently developed in-source fragmentation technique, we screened 39 typical and atypical isolates of A. salmonicida and established their O-chain polysaccharide structure by capillary electrophoresis-mass spectrometry (CE-MS), compositional and linkage analyses and comparison to the previously determined O-chain polysaccharide structure of A. salmonicida strain A449. These studies have demonstrated that A. salmonicida isolates fall into three distinct structural types, types A-C, based on chemical structures of their respective O-chain polysaccharide components. Subsequent immunoblotting and serological studies with salmon polyclonal antisera produced to formalin-fixed cells of A. salmonicida strains A449, N4705 and 33659 representing three structural types A-C revealed that variations in the O-chain polysaccharide structure have led to significant serological differences between strains belonging to type A and non-type A, where non-type A species include chemically separated structural types B and C. Due to the presence of common antigenic determinants shared by their respective O-chain polysaccharide components, serological cross-reactions were observed between A. salmonicida strains belonging to structural types B and C. These findings suggest the possibility of developing LPS-based classification system of A. salmonicida sub-species consisting of two serologically distinct types, type A and non-type A.     

Structural studies of the capsular polysaccharide and lipopolysaccharide O-antigen of Aeromonas salmonicida strain 80204-1 produced under in vitro and in vivo growth conditions.

Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3-deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated.     

Structural characterization of the O-chain polysaccharide isolated from Bordetella avium ATCC 5086: variation on a theme(1)

The O-chain polysaccharide (O-PS) of Bordetella avium was isolated from the lipopolysaccharide by mild acid hydrolysis to remove the lipid A, followed by hydrofluorolysis to remove the lipopolysaccharide core oligosaccharide leaving a residual O-PS for structural analysis. High resolution (1)H and (13)C NMR and MALDI studies showed the O-chain to be a polymer composed of 1,4-linked 2-acetamidino-3-[3-hydroxybutanamido]-2,3-dideoxy-beta-D-glucopyranosyluronic acid residues.     

Structural and serological characterization of the O-chain polysaccharide of Aeromonas salmonicida strains A449, 80204 and 80204-1

The O-chain polysaccharide (O-PS) of Aeromonas salmonicida was studied by a combination of compositional, methylation, CE-ESMS and one- and two-dimensional NMR analyses. It was found to be a branched polymer of trisaccharide-repeating units composed of L-rhamnose (Rha), D-glucose (Glc), 2-acetamido-2-deoxy-D-mannose (ManNAc) and O-acetyl group (OAc) and having the following structure: CE-ESMS analysis of A. salmonicida cells from strains A449, 80204 and 80204-1 grown under different conditions confirmed that the O-PS structure was conserved. ELISA-based serological study with native LPS-specific antisera performed on the native O-PS and its O-deacetylated and periodate-oxidized derivatives confirmed the importance of the O-PS backbone structure as an immunodominant determinant.     

Structure of the lipopolysaccharide O-chain of Yersinia enterocolitica serotype O:5,27

The cellular lipopolysaccharide produced by Yersinia enterocolitica serotype O:5,27 was of the S-type and composed of an antigenic O-chain polysaccharide linked through a core oligosaccharide region, which in turn was linked through 3-deoxy-D-manno-octulonosyl units to a lipid A moiety. The O-chain polysaccharide was composed of equal molar amounts of L-rhamnose and D-xylulose. By partial hydrolysis, periodate oxidation, methylation, specific optical rotation, and 13C and 1H nuclear magnetic resonance studies, the structure of the O-chain was established as being a linear backbone of alternating 1,3-linked alpha-L-rhamnopyranosyl and beta-L-rhamnopyranosyl units, to which 2,2-linked beta-D-threo-pent-2-ulofuranoside (D-xylulofuranoside) units were present on every L-rhamnopyranosyl residue, as shown below. (Formula: see text)     

The O-chain structure from the LPS of the endophytic bacterium Burkholderia cepacia strain ASP B 2D

The O-chain polysaccharide of the lipopolysaccharide from the endophytic bacterium Burkholderia cepacia strain was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be the following: -->2)-beta-D-Ribf-(1-->6)-alpha-D-Glcp-(1-->.     

Structural characterization of the O-chain polysaccharide of Aeromonas caviae ATCC 15468 lipopolysaccharide

The O-chain polysaccharide produced by a mild acid degradation of Aeromonas caviae ATCC 15468 lipopolysaccharide was found to be composed of L-rhamnose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose and phosphoglycerol. Subsequent methylation and CE-ESIMS analyses and 1D/2D NMR ((1)H, (13)C and (31)P) spectroscopy showed that the O-chain polysaccharide is a high-molecular-mass acidic branched polymer of tetrasaccharide repeating units with a phosphoglycerol substituent having the following structure: [structure: see text] where Gro represents glycerol and P represents a phosphate group.     

The O-chain structure from the LPS of marine halophilic bacterium Pseudoalteromonas carrageenovora-type strain IAM 12662T

The O-chain polysaccharide of the lipopolysaccharide from the halophilic marine bacterium Pseudoalteromonas carrageenovora IAM 12662T was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy of the de-O-acylated lipopolysaccharide and shown to be the following:Col is colitose, 3,6-di-deoxy-L-xylo-hexose.     

Rhizobium etli CE3 bacteroid lipopolysaccharides are structurally similar but not identical to those produced by cultured CE3 bacteria

Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin.     

O-antigen structure in a virulent strain of Aeromonas hydrophila

Abstract Lipopolysaccharide (LPS) was isolated from a strain of Aeromonas hydrophila which had displayed serological, bacteriophage attachment and virulence properties similar to those found in strains of Aeromonas salmonicida . The structure of the O-antigen was determined and had many points of similarity with that previously elucidated for the O-antigen of A. salmonicida . Methylation analysis, chromium trioxide oxidation and ¹H-n.m.r. were used to confirm that the repeating unit of the O-chain had the following structure:
     

Differential partition of virulent Aeromonas salmonicida and attenuated derivatives possessing specific cell surface alterations in polymer aqueous-phase systems.

Two-polymer aqueous-phase systems were used to compare via partitioning the surface properties of strains of the fish pathogen Aeromonas salmonicida which differed in their ability to produce the surface protein array known as the A layer and in their ability to produce smooth lipopolysaccharide. In these two-phase systems, biological particles are known to partition between the phases in a manner related to a variety of surface properties, including hydrophobicity, charge, and lipid composition. Both the presence of the superficial protein layer and the O polysaccharide chains of lipopolysaccharide were shown to play an important role in the partitioning behavior of A. salmonicida cells. The presence of the A layer, which is crucial to the virulence of A. salmonicida, appeared to decrease the surface hydrophilicity of this pathogen and to increase, in a somewhat specific manner, its surface affinity for fatty acid esters of polyethylene glycol. The ability of two-polymer aqueous-phase systems to differentially partition A. salmonicida cells on the basis of differences in surface architecture suggests their general usefulness for the analysis of surface properties important in bacterial virulence and should permit their use in the selection of strains and mutants exhibiting specific surface characteristics.     

Differential partition of virulent Aeromonas salmonicida and attenuated derivatives possessing specific cell surface alterations in polymer aqueous-phase systems

Two-polymer aqueous-phase systems were used to compare via partitioning the surface properties of strains of the fish pathogen Aeromonas salmonicida which differed in their ability to produce the surface protein array known as the A layer and in their ability to produce smooth lipopolysaccharide. In these two-phase systems, biological particles are known to partition between the phases in a manner related to a variety of surface properties, including hydrophobicity, charge, and lipid composition. Both the presence of the superficial protein layer and the O polysaccharide chains of lipopolysaccharide were shown to play an important role in the partitioning behavior of A. salmonicida cells. The presence of the A layer, which is crucial to the virulence of A. salmonicida, appeared to decrease the surface hydrophilicity of this pathogen and to increase, in a somewhat specific manner, its surface affinity for fatty acid esters of polyethylene glycol. The ability of two-polymer aqueous-phase systems to differentially partition A. salmonicida cells on the basis of differences in surface architecture suggests their general usefulness for the analysis of surface properties important in bacterial virulence and should permit their use in the selection of strains and mutants exhibiting specific surface characteristics.     

Structural determination of the O-specific polysaccharide from Aeromonas hydrophila strain A19 (serogroup O:14) with S-layer

Bacteria belonging to the genus Aeromonas are Gram-negative mesophilic and essentially ubiquitous in the microbial biosphere; moreover they are considered very important pathogens in fish and responsible for a great variety of human infections.The virulence of Gram-negative bacteria is often associated with the structure of lipopolysaccharides, which consist of three regions covalently linked: the glycolipid (lipid A), the oligosaccharide region (core region) and the O-specific polysaccharide (O-chain, O-antigen).The O-chain region seems to play an important role in host-pathogen interaction. In the case of Aeromonas hydrophila the majority of pathogenic strains belongs to serogroups O:11, O:16, O:18 and O:34. In this paper, we report the complete structure of the O-chain of A. hydrophila strain A19 (serogroup O:14), a pathogenic strain isolated from European eels, which showed high virulence when tested in trout or mice. Dried cells were extracted by the PCP (phenol/chloroform/petroleum ether) method obtaining the lipopolysaccharide. After mild acid hydrolysis the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by means of chemical analysis and one- and two-dimensional NMR spectroscopy. All the data collected are directed towards the following structure:     

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.     

Stability of short and long O-chain lipopolysaccharide types in Rhizobium galegae and their correlation with symbiotic properties and growth conditions, tolerance of low pH, aluminum and salt in the growth medium

We recently showed that Rhizobium galegae strains had two different lipopolysaccharide types, short and long O-chain. In the present study we observed that the lipopolysaccharide type was a stable feature of the strain. Both types persisted in cells at all phases of the growth cycle, during differentiation of bacteria into bacteroids and when the cells were grown under environmental stress. When R. galegae strains were grown at low pH or in medium containing aluminum or salt, simulating conditions in acid soils of temperate regions and osmotic stress, respectively, the tolerance of low pH was associated with the long O-chain lipopolysaccharide and abundant acidic polysaccharide production.     

Occurrence of a nontypable Helicobacter pylori strain lacking Lewis blood group O antigens and DD-heptoglycan: evidence for the role of the core alpha1,6-glucan chain in colonization

The cell envelope of Helicobacter pylori contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of the bacterial cell membrane. The O-chain of this LPS frequently expresses type 2 Lewis x (Lex) and Lewis y (Ley) blood group antigens that mimic human gastric mucosal cell-surface glycoconjugates. This article describes the isolation and structural analysis of the LPS from a clinical isolate of H. pylori strain PJ2 that lacks Le antigens but is still capable of colonization. Subsequent composition, methylation, and CE-ESMS analyses of LPS revealed its core oligosaccharide structure to be consistent with the previously proposed structural model for H. pylori LPS. In addition, it carries an unusually long side branch alpha1,6-glucan and was devoid of Le O-chain polysaccharide. Its ability to colonize the mouse stomach was essentially identical to that of DD-heptoglycan- and Le antigen- producing H. pylori strains.     

Structure of the O-chain of the lipopolysaccharide of Haemophilus pleuropneumoniae serotype 1

By phenol-water extraction an aqueous-phase soluble cellular lipopolysaccharide was isolated from Haemophilus pleuro-pneumoniae serotype 1. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hydrolysis, methylation, and both one- and two-dimensional 1H and 13C nuclear magnetic resonance studies to be an S-type lipopolysaccharide, which could be cleaved to yield a lipid A and an O-chain polysaccharide identified as a high molecular weight branched polymer of a tetrasaccharide repeating unit having the structure: (Formula: see text).     

The O-chain structure from the LPS of the bacterium Naxibacter alkalitolerans YIM 31775T

The O-chain polysaccharide of the lipopolysaccharide from the bacterium Naxibacter alkalitolerans strain YIM 31775(T) was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be built up by the following tetrasaccharide repeating unit: -->3)-alpha-D-FucpNAc-(1-->2)-beta-D-Quip3NHBu-(1-->2)-alpha-D-Rhap-(1-->)-beta-D-Galp-(1--> where HBu is hydroxy-butanoyl.     

Capsule structure of Proteus mirabilis (ATCC 49565).

Proteus mirabilis 2573 (ATCC 49565) produces an acidic capsular polysaccharide which was shown from glycose analysis, carboxyl reduction, methylation, periodate oxidation, and the application of one dimensional and two-dimensional high-resolution nuclear magnetic resonance techniques to be a high-molecular-weight polymer of branched trisaccharide units composed of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine), 2-acetamido-2,6-dideoxy-L-galactose (N-acetyl-L-fucosamine), and D-glucuronic acid, having the structure: [formula: see text] P. mirabilis 2573 also produces an O:6 serotype lipopolysaccharide in which the O-chain component has the same structure as the homologous capsular polysaccharide. This is the first report of a defined capsular polysaccharide in this bacterial genus.     

O-Specific chain structure from the lipopolysaccharide fraction of Pseudomonas reactans: a pathogen of the cultivated mushrooms

An O-specific polysaccharide containing 2-acetamidino-2-deoxy-beta-D-glucopyranose (Glcp2Am), 2,4-diacetamido-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAc4NAc, bacillosamine) and 2,4-di-(N-acetyl-L-alanylamino)-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAlaAc4NAlaAc) was isolated from the phenol-soluble lipopolysaccharide fraction of the mushroom-associated bacterium Pseudomonas reactans. The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a linear trisaccharide-repeating unit, as shown below:-->3)-beta-D-QuipNAlaAc4NAlaAc-(1-->3)-alpha-D-Glcp2Am-(1-->3)-alpha-D-QuipNAc4NAc(1-->To our knowledge, this is the first complete O-chain structure reported for the lipopolysaccharide of a mushroom-associated bacterium.