Tomasz Niedziela

Structural studies of the O-specific chain and a core hexasaccharide of Hafnia alvei strain 1192 lipopolysaccharide

The structure of the O-specific side-chain and a core hexasaccharide of the Hafnia alvei strain 1192 lipopolysaccharide has been investigated. Methylation analysis, NMR spectroscopy, MALDI-TOF spectrometry, and various specific chemical degradations were the principal methods used. It is concluded that the polysaccharide is composed of hexasaccharide repeating-units having the following structure which is partially O-acetylated in the 2-position of the --> 4)-alpha-D-Glc pA-(1-->(70%) and on different positions of the L-Rha residues (50%). [Formula: see text] The core hexasaccharide was found to have the following structure: [Formula: see text]

New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei.

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.

Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides

Hafnia alvei, a Gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. The majority of clinical symptoms of diseases caused by this bacterium have a lipopolysaccharide (LPS, endotoxin)-related origin. The lipid A structure affects the biological activity of endotoxins predominantly. Thus, the structure of H. alvei lipid A was analyzed for the first time. The major form, asymmetrically hexa-acylated lipid A built of β-d-GlcpN4P-(1→6)-α-d-GlcpN1P substituted with (R)-14:0(3-OH) at N-2 and O-3, 14:0(3-(R)-O-12:0) at N-2′, and 14:0(3-(R)-O-14:0) at O-3′, was identified by ESI-MSn and MALDI-time-of-flight (TOF) MS. Comparative analysis performed by MS suggested that LPSs of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 share the identified structure of lipid A. LPSs of H. alvei are yet another example of enterobacterial endotoxins having the Escherichia coli-type structure of lipid A. The presence of hepta-acylated forms of H. alvei lipid A resulted from the addition of palmitate (16:0) substituting 14:0(3-OH) at N-2 of the α-GlcpN residue. All the studied strains of H. alvei have an ability to modify their lipid A structure by palmitoylation.

Structural studies of the O-specific chain of Hafnia alvei strain 32 lipopolysaccharide.

The structure of the O-specific side chain of the Hafnia alvei strain 32 lipopolysaccharide has been investigated. Methylation analysis, partial acid hydrolysis, Smith degradations, NMR spectroscopy, MALDI-TOF and FAB mass spectrometry in combination with collision-induced decomposition MS/MS were the principal methods used. It is concluded that the polysaccharide is composed of pentasaccharide repeating units having the following structure which is partially O-acetylated in the 2- (20%) and 3- (50%) position of the-->4)-alpha-D-GalpA-(1-->residue. [sequence :see text] A MALDI-TOF mass spectrum of the O-specific chains indicated that they consisted of up to 16 repeating units.

Two Kdo-Heptose Regions Identified in Hafnia alvei 32 Lipopolysaccharide: the Complete Core Structure and Serological Screening of Different Hafnia O Serotypes

Hafnia alvei, a gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. Various 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-containing fragments different from known structures of core oligosaccharides were previously found among fractions obtained by mild acid hydrolysis of some H. alvei lipopolysaccharides (LPSs). However, the positions of these segments in the LPS structure were not known. Analysis of de-N,O-acylated LPS by nuclear magnetic resonance spectroscopy and mass spectrometry allowed the determination of the location of a Kdo-containing trisaccharide in the structure of H. alvei PCM 32 LPS. It was established that the trisaccharide {L-alpha-D-Hepp-(1-->4)-[alpha-D-Galp6OAc-(1-->7)]-alpha-Kdop-(2-->} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between -->4,7)-alpha-Kdop and -->2)-Glcp in the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes of H. alvei LPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides of H. alvei 2, 32, 600, 1192, 1206, and 1211 LPSs. The weaker cross-reactions with LPSs of strains 974, 1188, 1198, 1204, and 1214 suggest the presence of similar structures in these LPSs, as well. Thus, we have identified new examples of endotoxins among those elucidated so far. This type of core oligosaccharide deviates from the classical scheme by the presence of the structural Kdo-containing motif in the outer-core region.

Epitope of the vaccine-type Bordetella pertussis strain 186 lipooligosaccharide and antiendotoxin activity of antibodies directed against the terminal pentasaccharide-tetanus toxoid conjugate

ABSTRACT Lipooligosaccharides (LOS) isolated from Bordetella pertussis strains 186 and 606 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution magic angle spinning nuclear magnetic resonsnace (NMR). These analyses distinguished between the LOS of strains 186 and 606, suggesting that the structure of LOS in B. pertussis is heterogeneous. The pentasaccharide was selectively cleaved from LOS of B. pertussis strain 186, purified, and covalently linked to a monomer fraction of tetanus toxoid. Injection of rabbits with the neoglycoconjugate emulsified in complete Freunds adjuvant yielded immunoglobulin G antibodies that were reactive with the LOS. These antibodies reacted strongly with B. pertussis LOS possessing the complete dodecasaccharide, as determined by an enzyme-linked immunosorbent assay, immunoblotting, and flow cytometry with intact, live bacterial cells. The binding epitope within the pentasaccharide was investigated by saturation transfer difference (STD) NMR spectroscopy. Protons H-1 and H-4 of the terminal α-d-GlcpNAc and proton H-6 and protons of an N-methyl group at H-4 of 3-substituted β-l-FucpNAc4NMe exhibited the largest saturation transfers. STD NMR experiments confirmed that the immunodominant epitope recognized by the antineoglycoconjugate antibodies is located predominantly in the distal trisaccharide of B. pertussis 186 LOS. The antipentasaccharide antibodies induced by the conjugate inhibited the secretion of tumor necrosis factor alpha, interleukin-6, and NO by LOS-stimulated J774A.1 cells.

Serological characterization of anti-endotoxin serum directed against the conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid

The covalent conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid was prepared using reaction of reductive amination. The neoglycoconjugate was a good immunogen in rabbits yielding a high level of anti-lipopolysaccharide (LPS) antibodies of the IgG class. It was found that antiserum was able to react with the smooth LPS molecules of identical (R4) or related (R1) core type. The reactions were shown in the enzyme-linked immunosorbent assay and the immunoblotting test. Flow cytometry showed that anti-core antibodies reacted with LPS present on intact, live, smooth bacteria labelling more than 90% of cells. The anti-OS R4-TT serum used for in vitro studies showed high endotoxin neutralization activity. The serum inhibited endotoxin-induced tumor necrosis factor alpha and nitric oxide synthesis by the J-774A.1 cell line and attenuated pulmonary retention of YAC-1 cells.

The novel structure of the core oligosaccharide backbone of the lipopolysaccharide from the Plesiomonas shigelloides strain CNCTC 80/89 (serotype O13)

The new structure of the core oligosaccharide of Plesiomonas shigelloides CNCTC 80/89 (serotype O13) lipopolysaccharide has been investigated by chemical methods, (1)H and (13)C NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight (MALDI-TOF). It was concluded that the core oligosaccharide of P. shigelloides CNCTC 80/89 is a nonasaccharide with the following structure: The position of glycine was determined by MALDI-TOF MS/MS analyses.

The unique structure of complete lipopolysaccharide isolated from semi-rough Plesiomonas shigelloides O37 (strain CNCTC 39/89) containing (2S)-O-(4-oxopentanoic acid)-α-D-Glcp (α-D-Lenose).

The complete structure of semi-rough lipopolysaccharide (SR-LPS) of Plesiomonas shigelloides CNCTC 39/89 (serotype O37) has been investigated by (1)H and (13)C NMR spectroscopy, matrix-assisted laser-desorption/ionization time-of-flight MS, and chemical methods. The following structure of the single unit of the O-antigen has been established: [formula see text] in which α-D-Lenp is (2S)-O-(4-oxopentanoic acid)-α-D-Glcp residue which has not been found in nature. The absolute configuration of oxopentanoic acid moiety in α-d-Lenose residue was determined by NOESY experiment combined with molecular modeling (MM2 force field). The decasaccharide core is substituted at C-4 of the β-D-Glcp residue with a single pentasaccharide unit. Lipid A is built of a β-D-GlcpN4P-(1→6)-α-D-GlcpN1P disaccharide asymmetrically substituted with fatty acids. It was concluded that the core oligosaccharide and the lipid A are identical with those in P. shigelloides CNCTC 113/92 Niedziela et al. (2002)(9) and Lukasiewicz et al. (2006).(10.)

Identification of d-Galactan-III As Part of the Lipopolysaccharide of Klebsiella pneumoniae Serotype O1

Klebsiella pneumoniae is a Gram-negative, ubiquitous bacterium capable of causing severe nosocomial infections in individuals with impaired immune system. Emerging multi-drug resistant strains of this species and particularly carbapenem-resistant strains pose an urgent threat to public health. The lipopolysaccharide (LPS) O-antigen is the main surface antigen. It contributes to the virulence of this species and determines the O-serotype of K. pneumoniae isolates. Among the nine main O-serotypes of K. pneumoniae, O1-and O2-type pathogens are causative agents of over 50% of all infections. Serotype O1, the most common O-serotype, expresses complex LPS consisting of d-galactan-I (a polymer built of → 3)-β-d-Galf-(1 → 3)-α-d-Galp-(1 → repeating units) capped by d-galactan-II (built of [ → 3)-α-d-Galp-(1 → 3)-β-d-Galp-(1 →] repeating units). Galactan-I is present as the sole polymer in O2 serotype. Recently, in case of serotype O2, conversion of galactan-I to galactan-III (→ 3)-β-d-Galf-(1 → 3)-[α-d-Galp-(1 → 4)]-α-d-Galp-(1 →) was reported. Substitution of → 3)-α-d-Galp by a branching terminal α-d-Galp was dependent on the presence of the gmlABC operon and had a major impact on the antigenicity of the galactan polymer. Genetic analysis indicated that 40% of the O1 clinical isolates also carry the gmlABC locus; therefore we aimed to characterize the corresponding phenotype of LPS O-antigens. The presence of galactan-III among O1 strains was proven using galactan-III-specific monoclonal antibodies and confirmed by structural analyses performed using sugar and methylation analysis as well as classical and high-resolution magic angle spinning NMR spectroscopy. By using an isogenic mutant pair, we demonstrated that galactan-III expression was dependent on the presence of glycosyltransferases encoded by gmlABC, as was shown previously for the O2 serotype. Furthermore, the galactan-II structures in O1gml+ strains remained unaffected corroborating no functional interactions between the biosynthesis of galactan-III and galactan-II polymers.