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Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

Haemophilus parasuis is a Gram-negative bacterium from the family Pasteurellaceae and a swine pathogen. H. parasuis is found in the upper respiratory tract of piglets and produces Glässer's disease, an invasive disease characterized by polyserositis. H. parasuis contains a short lipopolysaccharide (LPS) or lipooligosaccharide (LOS) reported to play a partial role in interaction with host cells. The presence of capsule has been phenotypically demonstrated in certain H. parasuis strains and its role in virulence has been suggested, but the chemical structure of the surface polysaccharides of this bacterium was unknown. The structure of capsular polysaccharide (CPS) and LOS from virulent strains ER-6P and Nagasaki was studied by NMR spectroscopy, mass spectrometry and chemical methods. CPS from both strains had the same main chain with disaccharide repeating unit, substituted with α-Neu5R-(2-3)-α-GalNAc-(1-P-(strain ER-6P) or α-Neu5R-(2-3)-α-Gal-(1-P-strain Nagasaki) side chains, where R is the N-acetyl or N-glycolyl group. Glycolyl-neuraminic acid is widely found in animal glycoproteins, but it apparently has not been found in bacteria before, and might be important for the biology of this microorganism. Ac and Gc were present in equal amounts in the strain ER-6P but Nagasaki contained only about 20% of Gc substituent. Both strains produced the same LPS of a rough type with a single phosphorylated Kdo linking core and lipid A parts. LOS structure was similar to some strains of H. influenzae and contained a globotetraose terminal sequence. Crown Copyright © 2013 Published by Elsevier Ltd.

Atypical Actinobacillus pleuropneumoniae serotype 13 strains present in North America are described here for the first time. Different from serotype 13 strains described in Europe, North America strains are biotype I and antigenically related to both, serotypes 13 and 10. Chemical and structural analysis of the capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of a representative strain revealed that the CPS is almost identical to that of the reference strain of serotype 13, having a slightly higher degree of glycose O-acetylation. However, it produces an O-PS within the LPS antigenically and structurally identical with that of the reference strain of A. pleuropneumoniae serotype 10. The O-PS was characterized as a homopolymer of 1,2 linked β-d-galactofuranosyl residues, a structure unrelated to that of the O-PS produced by the reference strain of serotype 13. Strains from Canada and United States are antigenically, phenotypically and genotypically similar. Animals infected by one of these strains induced antibodies that were detected by a LPS-based ELISA diagnostic test using either the homologous antigen or that of serotype 10. Based on the LPS and toxin profile, these strains might be misidentified as A. pleuropneumoniae serotype 10.

Cronobacter dublinensis (formerly Enterobacter sakazakii) HPB 3169 is a pathogenic Gram-negative bacterium that produces a smooth-type lipopolysaccharide in which the antigenic O-polysaccharide component was determined to be a repeating pentasaccharide unit composed of L-rhamnose; 2-acetamido-2-deoxy-D-glucose; 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-glucose; and 3-deoxy-manno-oct-2-ulosonic acid in the respective molar ratio 2:1:1:1. Chemical and 2D NMR analyses of the O-polysaccharide and a pentasaccharide derived by the mild acid hydrolysis of the ketosyl linkage of the Kdo (3-deoxy-D-manno-2-octulosonic acid) residue in the O-polysaccharide established that the O-antigen is a high molecular mass unbranched polymer of a repeating pentasaccharide unit and has the structure [see formula in text] where Bu is a (R)-3-hydroxybutanoyl substituent. The O-antigen is structurally similar to that of the recently reported Cronobacter sakazakii strain G706 (designated as serotype O5), except that in strain G706 the d-Qui3N is in its N-acetyl form, in contrast to its presence as a 3-deoxy-3-(R)-3-hydroxybutyramido derivative in the C. sakazakii HPB 3169 strain O-antigen.

Endotoxin (lipopolysaccharide, LPS) is, in general, composed of two moieties: a hydrophilic polysaccharide linked to a hydrophobic lipid A terminal unit and forms a major surface component of Gram-negative bacteria. The structural features of LPS moieties play a role in pathogenesis and also involve immunogenicity and diagnostic serology. The major toxic factor of LPS resides in the lipid A moiety, anchored in the outer layer of the bacterium, and its relative biological activity is critically related to fine structural features within the molecule. In establishing relationships between structural features and biological activities of LPS it is of the utmost importance to develop new analytical methods that can be applied to the complete unambiguous characterization of a specific LPS molecule. Herein is presented a practical rapid and sensitive analytical procedure for the mass spectral screening of LPS using triethylamine citrate as an agent for both disaggregation and mild hydrolysis of LPS. It provides improved matrix-assisted laser desorption/ionization (MALDI) mass spectra and, in particular, affords the identification of fragments retaining labile substituents present in the native macromolecular LPS structures. The methods were developed and applied using purified LPS of Escherichia coli and Salmonella enterica, as well as more complex LPS of Actnobacillus pleuropneumoniae. Copyright © 2011 John Wiley & Sons, Ltd.

The antigenic O-polysaccharide component of the lipopolysaccharide produced by Escherichia coli serotype O71:H12 was analyzed by chemical composition, nuclear magnetic spectroscopy, and Smith-type periodate oxidation methods. It was determined to be a partially O-acetylated unbranched polymer of a repeating tetrasaccharide unit composed of L-rhamnose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and 3-acetamido-3-deoxy-D-quinovose (1:1:1:1) residues having the following structure: →3)-α-L-Rhap-(1→4)-β-D-Galp-(1→3)-α-D-GalpNAc-(1→4)-α-D-Quip3NAc-(1→ 2,3 ↑ (Ac)0.3

The structure of the antigenic O-polysaccharide isolated from the lipopolysaccharide produced by enterohemorrhagic Escherichia coli O103:H2 was determined and shown to be composed of d-glucose (1 part), 2-acetamido-2-deoxy-d-glucose (2 parts), 2-acetamido-2-deoxy-d-galactose (1 part), and 3-deoxy-3-(R)-3-hydroxybutyramido-d-fucose (1 part). From the results of methylation analysis, Smith-type periodate oxidation degradation studies, and the use of one- and two-dimensional1H and13C NMR spectroscopy, the O-polysaccharide antigen was found to be an unbranched polymer of a repeating pentasaccharide unit having the following structure: →2)-β-d-Glcp-(1→2)-β-d-Fucp3NBu-(1→6)-α-d-GlcpNAc-(1→4)-α-d-GalpNAc-(1→3)-β-d-GlcpNAc-(1→, where Bu is (R)-3-hydroxybutyramido.

Cronobacter malonaticus, a Gram-negative bacterium previously known as Enterobacter sakazakii, is an opportunistic pathogen known to cause serious infection in infants and neonates. To provide aid for the serological and chemical identification of clinical, environmental, or food isolates of this emerging pathogen, the characterization of the lipopolysaccharide (LPS) O-polysaccharide (O-PS) antigens of Cronobacter spp. is being undertaken. The structural analysis of the O-PS, obtained by hydrazinolysis of the lipopolysaccharide produced by Cronobacter malonaticus HPB 3267, was investigated by composition, methylation, and two-dimensional high-resolution nuclear magnetic resonance methods, and was found to be a polymer of a branched pentasaccharide unit. This unit is composed of d-glucose (d-Glc), d-galactose (d-Gal), 2-amino-2-deoxy-d-glucose (d-GlcN), 2-amino-2-deoxy-d-galactose (d-GalN) and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues (1: 1: 1: 1: 1), forms the repeating oligosaccharide in the O-PS antigen, and has the structure: [Formula: see text]

Lipopolysaccharide (LPS) is an important virulence factor of Burkholderia cepacia, an opportunistic bacterial pathogen that causes life-threatening disease in cystic fibrosis patients and immunocompromised individuals. B. cepacia LPS comprises an O-specific polysaccharide covalently linked to a core oligosaccharide (OS) which in turn is attached to a lipid A moiety. The complete structure of the LPS core oligosaccharide from B. cepacia serotype O4 was investigated by detailed NMR and mass spectrometry (MS) methods. High- (HMW) and low-molecular-weight (LMW) OSs were obtained by deacylation, dephosphorylation, and reducing-end reduction of the LPS. Glycan and NMR analyses established that both OSs contain a common inner-core structure consisting of D-glucose, L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, 3-deoxy-D-manno-octulsonic acid, and D-glycero-D-talo-2-octulosonic acid. The structure of the LMW OS differed from that of the HMW OS in that it lacks a tetra-rhamnosyl GlcNAc OS extension. These structural conclusions were confirmed by tandem MS analyses of the two OS fractions as well as an OS fraction obtained by alkaline deacylation of the LPS. The location of a phosphoethanolamine substituent in the core region was determined by ESI-MS and methylation analysis of O-deacylated LPS and core OS samples. A polyclonal antibody to B. cepacia serotype O4 core OS was cross-reactive with several other serotypes indicating common structural features.

Taylorella equigenitalis is a Gram-negative bacterium that causes venereally transmitted contagious equine metritis (CEM), and its identification and differentiation from other bacteria and Taylorella species is an important requirement for the control of CEM infection. Based on the results of NMR and MS analysis, the antigenic O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) produced by the type strain T. equigenitalis (ATCC 35865) was found to be a linear polymer composed of a repeating disaccharide unit, containing partially amidated 2,3-diacetamido-2,3-dideoxy-α- l -guluronic and 2,3-diacetamido-2,3-dideoxy-β- d -mannuronic acids, terminated with a 4-O-methylated non-reducing Gulp-NAc3NAcA residue, and has the structure. Download : Download full-size image The O-PS of the type strain T. equigenitalis LPS provides a specific antigenic marker for the discrimination of the pathogen from the related type strain of T. asinigenitalis sp. nov, a phenotypically indistinguishable non-pathogenic bacterium having a serologically and structurally unrelated LPS O-antigen. The analysis of a structurally unusual core oligosaccharide of the LPS is also reported.

Caulobacter crescentus CB15 is a dimorphic bacterium that is best known as a prokaryotic model for cell development. However, it is also being exploited in biotechnology, where the crystalline surface (S-layer) protein secretion system has been adapted for heterologous protein display or secretion. Because the S-layer attaches to the cell surface via lipopolysaccharide (LPS) and since the LPS represents a potential endotoxin contaminant of recombinant proteins, the lipid A component was examined in detail. LPS was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. The structure of the major lipid A of C. crescentus comprised the tetrasaccharide backbone α-D-GalpA-(1→4)-β-D-DAG-(1→6)-α-D-DAG-(1→1)-α-D-GalpA substituted with six fatty acids, and a molecular mass of 1875 (GalpA, galactopyranuronic acid; DAG, 2,3-diamino-2,3-dideoxyglucopyranose). No phosphate residues were detected. The major lipid A component had 12:0[3-O[Δ5-12:1(3-OH)]] and 12:0[3-O(Δ5-12:1)] fatty acyl chains at either the 3′- or the 2′ positions of the distal subunit DAG B, and 12:0(3OH) and 12:0[3,6-(OH) 2] fatty acyl chains at 3- and 2- positions of the reducing end subunit DAG A, respectively. In addition, several other variations in the structure were observed. The LPS was evaluated for TNF-α inducing activity and consistent with its unusual lipid A structure (relative to that of enteric bacteria), the activity was reduced by greater than 100-fold as compared to Escherichia coli ReLPS. This and other evidence suggests the potential application of this lipid A as a vaccine adjuvant or the suitability of Caulobacter displaying antigens for formulation of whole cell vaccines.

The structure and serological specificities of the lipopolysaccharides (LPSs) from Salmonella enterica serovar Gallinarum biovar Pullorum were studied to provide an improved basis for the distinction between antigenic types and the development of improved diagnostic tests. The structure of the LPS O-polysaccharide (O-PS) from S. Pullorum standard, intermediate and variant antigenic type strains was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and chemical analysis. The LPS of the three strains shared a common structural repeating oligosaccharide unit containing d-mannose, l-rhamnose, d-galactose and d-tyvelose (1:1:1:1). The O-PS of the variant type LPS contained an additional d-glucose residue linked to the O-4 position of the d-galactose residue. The O-PS of the intermediate type LPS was partially the same as that of the variant LPS, however, the molar ratio of the d-glucose component was lower with respect to the other glycose components. Serological specificities of the three antigenic type LPSs were examined with anti-S. Pullorum LPS monoclonal antibodies (Mabs). On immunoblots, Mabs to the standard type O-PS reacted with high molecular mass (HMM) and low molecular mass (LMM) LPS from the standard strain, and with LMM but not HMM LPS from the variant strain. Monoclonal antibodies to the variant type O-PS reacted with HMM but not LMM LPS from the variant strain, and did not react with HMM or LMM LPS from the standard strain. On ELISA, the standard, intermediate and variant antigenic type strains were differentiated by the relative reactivity with the anti-LPS O-PS Mabs. Several of the anti-LPS O-PS Mabs were specific for S. Pullorum and other serogroup D1 Salmonella, and are potentially useful for the development of improved diagnostic tests for these organisms.

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.

Most field isolates of the swine pathogen Actinobacillus pleuropneumoniae form tenacious biofilms on abiotic surfaces in vitro. We purified matrix polysaccharides from biofilms produced by A. pleuropneumoniae field isolates IA1 and IA5 (serotypes 1 and 5, respectively), and determined their chemical structures by using NMR spectroscopy. Both strains produced matrix polysaccharides consisting of linear chains of N-acetyl- d -glucosamine (GlcNAc) residues in β(1,6) linkage (poly-β-1,6-GlcNAc or PGA). A small percentage of the GlcNAc residues in each polysaccharide were N-deacetylated. These structures were nearly identical to those of biofilm matrix polysaccharides produced by Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. PCR analyses indicated that a gene encoding the PGA-specific glycoside transferase enzyme PgaC was present on the chromosome of 15 out of 15 A. pleuropneumoniae reference strains (serotypes 1–12) and 76 out of 77 A. pleuropneumoniae field isolates (serotypes 1, 5 and 7). A pgaC mutant of strain IA5 failed to form biofilms in vitro, as did wild-type strains IA1 and IA5 when grown in broth supplemented with the PGA-hydrolyzing enzyme dispersin B. Treatment of IA5 biofilms with dispersin B rendered them more sensitive to killing by ampicillin. Our findings suggest that PGA functions as a major biofilm adhesin in A. pleuropneumoniae. Biofilm formation may have relevance to the colonization and pathogenesis of A. pleuropneumoniae in pigs.

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 was obtained by phenol-water extraction of bacterial cells. Structural investigations were made on the lipid A free saccharide moiety released from the lipopolysaccharide by mild acetic acid hydrolysis. Nuclear magnetic resonance, gas liquid chromatography-mass spectrometry and fast atom bombardment-mass spectrometry were employed in the characterization of products from chemical manipulations. A monoclonal antibody specific for blood group A reacted strongly with lipopolysaccharide of H. mustelae . Chemical and serological data showed that the outer core region of the lipopolysaccharide from H. mustelae ATCC 43772 expresses the monofucosyl A type 1, α- d -GalNAc-(1→3)-[α- l -Fuc-(1→2]-β- d -Gal-(1→3)-β- d -GlcNAc, blood group determinant, a mimic of animal cell surface glycolipids and glycoproteins.

The structural characterization of the antigenic O-polysaccharide component of the lipopolysaccharide produced by the fish pathogenic bacterium Edwardsiella ictaluri MT104 was undertaken by the application of NMR spectroscopy and chemical analysis. The O-chain was found to be a linear polymer of a repeating tetrasaccharide unit composed of d -glucose, 2-acetamido-2-deoxy- d -galactose, and d -galactose in a 1:2:1 ratio having the structure: [→4)-α- d -Glc p -(1→4)-α- d -Gal p NAc-(1→3)-β- d -Gal p NAc-(1→4)-β- d -Gal p -(1→] n

Edwardsiella tarda , a Gram-negative bacterium, is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. The lipopolysaccharide produced by the fish pathogenic strain E. tarda MT 108 was isolated and the structure of its antigenic O-polysaccharide component determined by the application of chemical analyses, high-resolution 1D and 2D nuclear magnetic resonance spectroscopy, and mass spectrometry. The polysaccharide was found to be a polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy- d -glucose ( d -GlcNAc), 2-acetamido-2-deoxy- d -galactose ( d -GalNAc), d -galactose ( d -Gal), l -rhamnose ( l -Rha), d -galacturonic acid ( d -GalA) and (2 S ,3 R )-threonine (1:1:1:1:1:1) having the structure

The antigenic O-polysaccharide of the lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 14 was shown by chemical analysis and 1D and 2D nuclear magnetic resonance methods to be a high-molecular-mass polymer of a repeating disaccharide unit composed of a chain of (1--5)-linked beta-D-galactofuranose (beta-D-Galf) residues substituted at their O-2 positions by alpha-D-galactopyranose residues (D-Galp) (1:1): [Formula: see text].

Francisella novicida (U112), a close relative of the highly virulent bacterium F. tularensis, was shown to produce a lipopolysaccharide in which the antigenic O-polysaccharide component was found by chemical, 1H and 13C NMR and MS analyses to be an unbranched neutral linear polymer of a repeating tetrasaccharide unit composed of 2-acetamido-2-deoxy- d -galacturonamide ( d -GalNAcAN) and 2,4-diacetamido-2,4,6-trideoxy- d -glucose ( d -Qui2NAc4NAc, di-N-acetylbacillosamine) residues (3:1) and had the structure: →4) -α- d -GalNAcAN- (1→4) -α- d -GalNAcAN- (1→4) -α- d -GalNAcAN- (1→3) -α- d -QuiNAc4NAc- (1→ With polyclonal murine antibody, the F. novicida O-antigen did not show serological cross-reactivity with the O-antigen of F. tularensis despite the occurrence of a common → 4)- d -GalpNAcAN-(1 → 4)-α- d -GalpNAcAN-(1 → disaccharide unit in their respective O-antigens. Thus, O-PS serology offers a practical way to distinguish between the two Francisella species.

Proteolytic digestion of the phenol–water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4- O -methyl-2-acetamido-2-deoxy- d -glucuronic acid (GlcNAcA), d -glucuronic acid ( d -GlcA), 2,3-di- O -acetyl- d -xylose ( d -Xyl), 2- O -methyl- d -glucuronic acid ( d -GlcA), d -mannose ( d -Man), and 2- O -methyl- l -rhamnose ( l -Rha). By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: Download full-size image where all monosaccharides have the d -configuration except for 2- O -methyl- l -rhamnose; and were in the pyranose form. Only one carbohydrate structure was found. The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.

Flexibacter maritimus, a Gram-negative bacterium, is a fish pathogen responsible for disease in finfish species and a cause of cutaneous erosion disease in sea-caged salmonids. For the development of serology based diagnostics, protective vaccines, and a study of pathogenesis, the structural analysis of the lipopolysaccharide (LPS) produced by the bacterium has been undertaken. We now report that an acidic O-specific polysaccharide, obtained by mild acid degradation of the F. maritimus LPS was found to be composed of a disaccharide repeating unit built of 2-acetamido-3-O-acetyl-4-[(S)-2-hydroxyglutar-5-ylamido]-2,4,6-trideoxy-beta-glucose and 5-acetamido-7-[(S)-3-hydroxybutyramido]-8-amino-3,5,7,8,9-pentadeoxynonulopyranosonic acid (Sug) having the structure: The configuration of the C-2-C-7 fragment of the latter monosaccharide (B) was assigned beta-manno; however, the configuration at C-8 could not be established. NMR data indicate that the two monosaccharides have opposite absolute configurations. The repeating unit includes a linkage via a (S)-2-hydroxyglutaric acid residue, reported here for the first time as a component of a bacterial polysaccharide. The LPS was also found to contain a minor amount of a disaccharide beta-Sug-(2-3)-l-Rha, isolated from the products of the acidic methanolysis of the LPS.

Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens. Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. A unique antigen from F. psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test. LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests. Rabbit polyclonal anti-O-PS serum was highly specific for F. psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test. Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F. psychrophilum.

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a beta-GlcN-(1-6)-GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O-1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O-3 and O-4 of beta-GlcN, and no substituent at O-4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure: where Delta-GalNA-(1-3)-beta-QuiNAc represents a modified fragment of the O-chain repeating unit. Two shorter oligosaccharides lacking the O-chain fragment were also identified. A minor amount of the disaccharide beta-GlcN-(1-6)-alpha-GlcN-1-P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end. The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2-N-(3-O-acyl4-acyl2)-beta-GlcN-(1-6)-2-N-acyl1-3-O-acyl3-GlcN where acyl1, acyl2 and acyl3 are 3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH-1 of the reducing end glucosamine, and OH-3 and OH-4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.

Previous studies from our laboratory have indicated that the lipopolysaccharide (LPS) from Mannheimia haemolytica serotype A1 contains both L-glycero-D-manno-heptose and D-glycero-D-manno-heptose residues. NMR methods making use of 1D 1H selective excitation and 2D (1H, 13C) and (1H, 31P) heteronuclear experiments were used for the structural determination of the major core oligosaccharide components of the deacylated low-molecular-mass LPS obtained following sequential treatment with anhydrous hydrazine and aq KOH. The core oligosaccharide region was found to be composed of a branched octasaccharide linked to the deacylated lipid A moiety via a 3-deoxy-4-phospho-D-manno-oct-2-ulosonate residue having the structure,[Formula: see text]Heterogeneity was found to be present at several linkages. NMR methods were devised to distinguish between the diastereomeric forms of the heptose residues. Synthesized monosaccharides of L-D- and D-D-heptose were used as model compounds for analysis of the 1H and 13C NMR chemical shifts and proton coupling constants. Molecular modeling using a Monte Carlo method for conformational analysis of saccharides was used to determine the conformation of the inner core of the oligosaccharide and to establish the stereochemical relationships between the heptoses.Key words: LPS, NMR, conformation, oligosaccharide, heptose.

Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.

Citrobacter rodentium is the etiologic agent of transmissible murine colonic hyperplasia (TMCH) and is the only Citrobacter species known to possess virulence factors homologous to human enteropathogenic and enterohemorrhagic Escherichia coli. Members of this species are considered clonal and represent the only known attaching and effacing bacterial pathogen of mice and thus provides a useful animal model for studying the molecular basis of attaching and effacing pathology. The lipopolysaccharide (LPS) produced by C. rodentium has not been previously studied or its possible role as a virulence factor determined. The structure of the LPS has been undertaken as a first step in an investigation of its possible role in pathogenesis. The structure of C. rodentium (ATCC 51459, prototype TMCH isolate, original biotype 4280, previously designated DBS 100) LPS was determined from composition and methylation analyses, mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The antigenic O-polysaccharide was found to be a high molecular mass branched polymer of repeating pentasaccharide units composed of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), d-glucose (D-Glc), and L-rhamnose (L-Rha) in the molar ratio 2 : 2 : 1 linked through phosphate, and has the structure: [structure: see text]

The structure of the core region of the lipopolysaccharide (LPS) from the nontypable Haemophilus influenzae strain SB 33 was elucidated. The LPS was subjected to a variety of degradative procedures. The structures of the derived oligosaccharide products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. These analyses revealed a series of related phosphocholine (PCho) containing structures differing in the number of hexose residues. The results pointed to each species containing a conserved phosphoethanolamine (PEtn) substituted heptose-containing trisaccharide inner-core moiety. The major LPS glycoforms were identified as 2-Hex, 3-Hex and 4-Hex species according to the number of hexose residues present.

The core regions of the lipopolysaccharides (LPS) from Klebsiella pneumoniae serotypes O1, O2a, O2a,c, O3, O4, O5, O8, and O12 were analysed using NMR spectroscopy, ESI-MS spectroscopy, and chemical methods. All the LPSs had similar core structures, as shown below, differing only in the number and position of beta-D-galacturonic acid substituents: [carbohydrate structure: see text] where P is H or alpha-Hep, J, K is H or beta-GalA. LPS from all serotypes contained varying proportions of structures having additional or missing phosphate substituents. The core from serotype O1 contained a minor amount of a previously described variant with alpha-DD-Hep-(1--2)-alpha-DD-Hep-(1--6)-alpha-GlcN-(1--replacing the alpha-Hep-(1--4)-alpha-Kdo-(2--6)-alpha-GlcN-(1--component.

The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of l -rhamnose and 2-acetamido-2-deoxy- d -galactose (molar ratio, 2:1) with the structure →2)-α- l -Rha p -(1–3)-2- O -(β- d -Gal p NAc)-α- l -Rha p -(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS 903 φ kan , and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

Helicobacter pylori is a widespread Gram-negative bacterium responsible for the onset of various gastric pathologies and cancers in humans. A familiar trait of H. pylori is the production of cell-surface lipopolysaccharides (LPSs; O-chain [Formula: see text] core [Formula: see text] lipid A) with O-chain structures analogous to some mammalian histo-blood-group antigens, those being the Lewis determinants (Lea, Leb, Lex, sialyl Lex, Ley) and blood groups A and linear B. Some of these LPS antigens have been implicated as autoimmune, adhesion, and colonization components of H. pylori pathogenic mechanisms. This article describes the chemical structures of LPSs from H. pylori isolated from subjects with no overt signs of disease. Experimental data from chemical- and spectroscopic-based studies unanimously showed that these H. pylori manufactured extended heptoglycans composed of 2- and 3-linked D-glycero-α-D-manno-heptopyranose units and did not express any blood-group O-antigen chains. The fact that another H. pylori isolate with a similar LPS structure was shown to be capable of colonizing mice indicates that H. pylori histo-blood-group structures are not an absolute prerequisite for colonization in the murine model also. The absence of O-chains with histo-blood groups may cause H. pylori to become inept in exciting an immune response. Additionally, the presence of elongated heptoglycans may impede exposure of disease-causing outer-membrane antigens. These factors may render such H. pylori incapable of creating exogenous contacts essential for pathogenesis of severe gastroduodenal diseases and suggest that histo-blood groups in the LPS may indeed play a role in inducing a more severe H. pylori pathology.Key words: lipopolysaccharide, carbohydrates, glycobiology, Helicobacter pylori, histo-blood groups.

The polysaccharides from the outer membrane of the Gram-negative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by size-exclusion chromatography in the presence of deoxycholate detergent into a lower-molecular-mass fraction designated 'glycolipid' and a high-molecular-mass 'capsular polysaccharide' fraction. Both fractions lacked typical lipopolysaccharide components including 2-keto-3-deoxyoctulosonic acid and 3-hydroxy fatty acids. Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures: : : where HEAEP is N-(2-hydroxyethyl)-2-aminoethylphosphonic acid, found for the first time in natural compounds. The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components. All constituent monosaccharides except L-rhamnose had a D-configuration. In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide-less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.

Flavobacterium psychrophilum, a Gram-negative bacterium, is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic infections in reared salmonids. In humans Flavobacterium spp. have been associated with neonatal meningitis and septicemia, catheter-associated bacteremia, and pneumonia. Recently, several F. psychrophilum surface molecules, including lipopolysaccharide (LPS), have been implicated in its pathogenesis and identified as potential vaccine and diagnostic candidate macromolecules. Studies on the LPS produced by the bacterium are reported herein. The structure of the antigenic O-polysaccharide contained in the LPS of F. psychrophilum was deduced by the application of analytical NMR spectroscopy, mass spectrometry, glycose and methylation analysis, and partial hydrolysis degradations, and was found to be an unbranched polymer of trisaccharide repeating units composed of L-rhamnose (L-Rhap), 2-acetamido-2-deoxy-L-fucose (L-FucpNAc) and 2-acetamido-4-((3S,5S)-3,5-dihydroxyhexanamido)-2,4-dideoxy-D-quinovose (D-Quip2NAc4NR, 2-N-acetyl-4-N-((3S,5S)-3,5-dihydroxyhexanoyl)-D-bacillosamine) (1 : 1 : 1) and having the structure: -->4)-alpha-L-FucpNAc-(1-->3)-alpha-D-Quip2NAc4NR-(1-->2)- alpha-L-Rhap-(1--> where R is (3S,5S)-CH3CH(OH)CH2CH(OH)CH2CO-.

The structures of the polysaccharide chains of the LPS from Bordetella bronchiseptica strains 110H and Bp512 were analysed by NMR spectroscopy and mass spectrometry. The polysaccharides consist of alpha-(1-4)-linked 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid repeating units. Polysaccharides from both strains have 2,3, 4-triamino-2,3,4-trideoxy-alpha-galacturonamide derivatives at their nonreducing ends, a monosaccharide identified for the first time in nature. The polymers from the two strains differ in the nature of the acylation of the amino groups of this monosaccharide. In the strain 110H, the residue is formylated at positions 3 and 4, and has N-formyl-L-alanyl or L-alanyl substituents at N-2. In the strain Bp512, the amino group at position 2 is acetylated, at position 3 it is formylated, and the amino group at position 4 bears a 2-methoxypropionyl substituent. The distribution of the acyl groups was determined from long range 1H-13C correlation (HMBC) NMR spectra. Measurement of the spectra under different pH conditions showed that carboxyl groups of the inner uronic acid residues of the polymeric chain are free, and that carboxyl groups of the terminal residues are amidated. These conclusions were confirmed by the results of mass spectrometric analysis.

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides (LPSs) of Proteus mirabilis O48 and Proteus vulgaris O21 were found to have tetrasaccharide and pentasaccharide repeating units, respectively, interlinked by a glycosidic phosphate. Polysaccharides and an oligosaccharide were derived from the LPSs by various degradation procedures and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,13C and 1H,31P HMQC experiments. The following related structures of the repeating units of the O-antigens were established (top: Proteus mirabilis O48; bottom: Proteus vulgaris O21) The O-specific polysaccharide of P. vulgaris O21 has the same structure as that of Hafnia allvei 744 and PCM 1194 [Petersson C., Jachymek, W., Klonowska, A., Lugowski, C., Niedziela, T. & Kenne, L. (1997) Eur. J. Biochem., 245, 668–675], except that the GlcN residue carries the N-acetyl rather than the N-[(R)-3-hydroxybutyryl] group. Serological investigations confirmed the close relatedness of the Proteus and Hafnia O-antigens studied.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Le x ), Le y , and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Le x and Le y and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a serotype similar to the phase variant. Structural analysis of the NCTC 11637 LPS confirmed the loss of the H type I epitope. Sequencing of JHP563 in strains NCTC 11637, an H type I-negative variant, and an H type I-positive switchback variant showed a C14 (gene on), C13 (gene off), and C14 tract, respectively. Inactivation of strain G27, which expresses Le x , Le y , H type I, and Le a , yielded a transformant that expressed Le x and Le y . We conclude that JHP563 encodes a β3-galactosyltransferase involved in the biosynthesis of H type I and Le a and that phase variation in H type I is due to C-tract changes in this gene. A second H type I-negative variant (variant 3a) expressed Le x and Le a and had lost both H type I and Le y expression. Inactivation of HP093-HP094 resulted in a transformant expressing Le x and lacking Le y and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that the HP093-HP094 α2-fucosyltransferase (α2-FucT) gene product is involved in the biosynthesis of both Le y and Le x . Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Le x and Le a expression, which demonstrates that the HP0379 gene product is both an α3- and an α4-FucT. Our data provide understanding at the molecular level of how H. pylori is able to diversify in the host, a requirement likely essential for successful colonization and transmission.

A cell surface antigen-typing system was devised for the swine pathogen Actinobacillus suis and used to examine the prevalence of different lipopolysaccharide (O) types in healthy and diseased pigs. The strains examined in this study were isolated from a variety of locations in Canada and from Kansas. Lipopolysaccharide preparations of 151 isolates of A. suis were characterized by immunoblotting using polyclonal antisera generated to strains SO4 (O1/K1), H89-1173 (O2/K3), and VSB 3714, a rough strain. Approximately 54% (62 of 114) of A. suis isolates from diseased pigs, all (11 of 11) isolates from healthy pigs, and all (4 of 4) reference strains reacted with O1/K1 antiserum. More than 80% (18 of 22) of A. suis strains used for bacterin production and approximately 41% (47 of 114) of isolates from diseased pigs bound O2/K3 antiserum. One isolate appeared to be rough, and five were untypeable. O1/K1- and O2/K3-reactive strains were equally prevalent in Kansas, whereas O2/K3-reactive strains were more common in Québec and western Canada and O1/K1 strains were most common in Ontario. The fact that virtually all of the strains submitted for bacterin production were O2/K3-reactive strains is consistent with the notion that these strains may be more virulent than O1/K1 strains; alternatively, this may reflect geographic or other biases. In addition, we observed cross-reactivity between A. suis cell surface antigens and swine antisera to several other important pathogens. This finding may explain why previous attempts to develop a simple serodiagnostic test for A. suis have been unsuccessful.

A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freunndii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [ 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.

Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes and, infrequently, type 1 Le(a), Le(b), and Le(d) antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Le(a) without the presence of type 2 Le(x), strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Le(b) and type 2 Le(y). The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.

The structure of the O-antigenic polysaccharide (O-PS) component of the lipopolysaccharide produced by Yersinia enterocolitica serotype O:28 has been elucidated. From chemical methods involving glycose analysis, periodate oxidation, methylation and the use of one- and two-dimensional NMR spectroscopy, the O-PS was found to be a polymer of repeating branched hexasaccharide units composed of L-rhamnose (four parts), 2-acetamido-2-deoxy-D-glucose (one part), and 2-acetamido-2-deoxy-D-galacturonic acid (one part) having the following structure

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The structure of the lipid A of its endotoxin was investigated using chemical analyses, nuclear magnetic resonnance (NMR), gas liquid chromatography/mass spectrometry (GC/MS), plasma desorption mass spectrometry (PDMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The lipid A contains the classical bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid (C14OH) in amide linkages. The lipid A components of B. pertussis, B. bronchiseptica, and B. parapertussis all differ in their acylation pattern but share a residue of tetradecanoyl-3-hydroxytetradecanoic acid in amide linkage at the C-2' position. However, in the B. hinzii species, the tetradecanoic acid (C14) is stoichiometrically replaced by a 2-hydroxytetradecanoic acid (2-C14OH). In the few reported examples of a hydroxylated fatty acid in this position, the substitutions were only partial. The B. hinzii lipid A differs from that of B. pertussis also by replacement of the hydroxydecanoic acid (C10OH) by hydroxydodecanoic acid (C12OH) and by the presence of a hexadecanoic acid (C16) to give a sixth fatty acid. The lipid A was heterogeneous, being composed of three major molecular species: tetra-, penta- and hexaacylated. The fatty acids in ester linkage were localized by PDMS of the native and alkali-treated lipid A. The lipid A components isolated from the O-chain-linked lipopolysaccharides (LPSs) were shown to be more acylated than those from the O-chain-free LPSs.

The structure of lipid A core region of the lipopolysaccharides (LPS) from Proteus mirabilis serotypes O6, O57 and O48 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of LPS: Incomplete substitutions are indicated by bold italic type. All sugars are present in pyranose form, α-Hep is the residue of lglycero-α-dmanno-Hep, α-dd-Hep is the residue of dglycero-α-dmanno-Hep, l-Ara4N is 4-amino-4-deoxy-l-arabinose, Qui4NAlaAla is the residue of 4-N-(l-alanyl-l-alanyl)-4-amino-4,6-dideoxyglucose. All sugars except l-Ara4N have d-configuration. β-GalA* is partially present in the form of amide with 1,4-diaminobutane (putrescine)-HN(CH2)4NH2 or spermidine-HN(CH2)3NH(CH2)4NH2.

It has been postulated that a humoral immune response directed against the O157 antigen of Escherichia coli O157:H7, and expressed in the intestine, might afford protection from colonization and consequent infection by this enteric pathogen. The present study was conducted to determine whether such an immune response can be experimentally generated in mice. To this end, mice were orally immunized with a glycoconjugate vaccine consisting of horse serum albumin and the O157 polysaccharide admixed with the mucosal adjuvant, cholera toxin. Mice consistently developed robust local and systemic immune responses to the cholera toxin adjuvant, but were far from uniformly reactive to the test vaccine. Moreover, vaccinated mice were as susceptible to transient intestinal colonization following challenge with an isolate of E. coli O157:H7 as unvaccinated control mice. These results indicate that this vaccination approach is unlikely to be straightforward in target bovine or human hosts.Key words: Escherichia coli O157:H7, glycoconjugate vaccine, mucosal immunity, mice.

This study describes the molecular makeup of the cell-wall lipopolysaccharides (LPSs) (O-chain polysaccharide-->core oligosaccharide-->lipid A) from five Helicobacter pylori strains: H. pylori 26695 and J99, the complete genome sequences of which have been published, the established mouse model Sydney strain (SS1), and the symptomatic strains P466 and UA915. All chemical and serological experiments were performed on the intact LPSs. H. pylori 26695 and SS1 possessed either a low-Mr semi-rough-form LPS carrying mostly a single Ley type-2 blood-group determinant in the O-chain region covalently attached to the core oligosaccharide or a high-Mr smooth-form LPS, as did strain J99, with an elongated partially fucosylated type-2 N-acetyllactosamine (polyLacNAc) O-chain polymer, terminated mainly by a Lex blood-group determinant, connected to the core oligosaccharide. In the midst of semi-rough-form LPS glycoforms, H. pylori 26695 and SS1 also expressed in the O-chain region a difucosylated antigen, alpha-L-Fucp(1-3)-alpha-L-Fucp(1-4)-beta-D-GlcpNAc, and the cancer-cell-related type-1 or type-2 linear B-blood-group antigen, alpha-D-Galp(1-3)-beta-D-Galp(1-3 or 4)-beta-D-GlcpNAc. The LPS of H. pylori strain P466 carried the cancer-associated type-2 sialyl Lex blood-group antigen, and the LPS from strain UA915 expressed a type-1 Leb blood-group unit. These findings should aid investigations that focus on identifying and characterizing genes responsible for LPS biosynthesis in genomic strains 26695 and J99, and in understanding the role of H. pylori LPS in animal model studies. The LPSs from the H. pylori strains studied to date were grouped into specific glycotype families.

The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C-2′: this was dodecanoic acid (C12) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C14) in two other Y. enterocolitica and hexadecenoic acid (C16:1) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.

Helicobacter pylori can colonize the human stomach for prolonged periods of time, and this colonization uniformly leads to the development of chronic active gastritis. In a small percentage of individuals, gastric pathology progresses to peptic ulceration or more rarely certain gastric cancers. In addition to non-specific inflammation, specific systemic and local immunity develops in response to gastric colonization by this pathogen. However, these responses combined appear inadequate for eliminating H. pylori from the gastric mucosa. This is also the case in a mouse model of gastric colonization by H. pylori. In the present study, we attempted to determine whether the mammalian host response to infection with H. pylori exerts any overt antibacterial effects. To this end we examined H. pylori colonization in normal mice, and mice immunosuppressed by treatment with a corticosteroid. Despite obvious suppression of the immune response in the latter mice, H. pylori burdens remained similar in both groups after three months of colonization. This suggests that the murine host response, at least, exerts little obvious protection against H. pylori colonization.Key words: Helicobacter pylori, immunosuppression, mice.

The present study was performed to assess the potential of a humoral mucosal immune response directed against the O157 antigen of Escherichia coli O157:H7 to prevent intestinal colonization by the pathogen. To this end, mice were gavaged with inocula of Salmonella landau, a Salmonella strain that naturally expresses the O157 antigen. Salmonella landau was avirulent for mice. Despite this, mice exposed to S. landau developed high titres of serum and coproantibodies against the O157 antigen. These mice, compared with controls, demonstrated some ability to resist transient intestinal colonization by an oral inoculum of an isolate of E. coli O157:H7. These findings suggest that a local immune response directed against the O157 antigen might increase host resistance to this pathogen.Key words: Salmonella landau, Escherichia coli O157:H7, mucosal immunity, mice.