Wojciech Jachymek

Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142.

This paper describes the structure of neutral exopolysaccharide (EPS) produced by Lactobacillus johnsonii 142, strain of the lactic acid bacteria isolated from the intestine of mice with experimentally induced inflammatory bowel disease (IBD). Sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY, and (1)H,(13)C HSQC experiments revealed that the repeating unit of the EPS is a pentasaccharide: -->3)-alpha-D-Galp-(1-->3)-beta-d-Glcp-(1-->5)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1--> The rabbit antiserum raised against whole cells of L. johnsonii 142 reacted with homologous EPS, and cross-reacted with exopolysaccharide from Lactobacillus animalis/murinus 148 isolated also from mice with IBD, but not reacted with EPS of L. johnsonii 151 from healthy mice.

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.

Distinct Immunomodulation of Bone Marrow-Derived Dendritic Cell Responses to Lactobacillus plantarum WCFS1 by Two Different Polysaccharides Isolated from Lactobacillus rhamnosus LOCK 0900

ABSTRACT The structures of polysaccharides (PS) isolated from Lactobacillus rhamnosus LOCK 0900 and results from stimulation of mouse bone marrow-derived dendritic cells (BM-DC) and human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR) upon exposure to these antigens were studied. L. rhamnosus LOCK 0900 produces PS that differ greatly in their structure. The polymer L900/2, with a high average molecular mass of 830 kDa, is a branched heteropolysaccharide with a unique repeating unit consisting of seven sugar residues and pyruvic acid, whereas L900/3 has a low average molecular mass of 18 kDa and contains a pentasaccharide repeating unit and phosphorus. Furthermore, we found that both described PS neither induce cytokine production and maturation of mouse BM-DC nor induce signaling through TLR2/TLR4 receptors. However, they differ profoundly in their abilities to modulate the BM-DC immune response to the well-characterized human isolate Lactobacillus plantarum WCFS1. Exposure to L900/2 enhanced interleukin-10 (IL-10) production induced by L. plantarum WCFS1, while in contrast, L900/3 enhanced the production of IL-12p70. We conclude that PS, probably due to their chemical features, are able to modulate the immune responses to third-party antigens. The ability to induce regulatory IL-10 by L900/2 opens up the possibility to use this PS in therapy of inflammatory conditions, such as inflammatory bowel disease, whereas L900/3 might be useful in reverting the antigen-dependent Th2-skewed immune responses in allergies.

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.

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.

Core Oligosaccharide of Plesiomonas shigelloides PCM 2231 (Serotype O17) Lipopolysaccharide — Structural and Serological Analysis

The herein presented complete structure of the core oligosaccharide of lipopolysaccharide (LPS) P. shigelloides Polish Collection of Microorganisms (PCM) 2231 (serotype O17) was investigated by 1H, 13C NMR spectroscopy, mass spectrometry, chemical analyses and serological methods. The core oligosaccharide is composed of an undecasaccharide, which represents the second core type identified for P. shigelloides serotype O17 LPS. This structure is similar to that of the core oligosaccharide of P. shigelloides strains 302-73 (serotype O1) and 7-63 (serotype O17) and differs from these only by one sugar residue. Serological screening of 55 strains of P. shigelloides with the use of serum against identified core oligosaccharide conjugated with bovine serum albumin (BSA) indicated the presence of similar structures in the LPS core region of 28 O-serotypes. This observation suggests that the core oligosaccharide structure present in strain PCM 2231 could be the most common type among P. shigelloides lipopolysaccharides.