Czeslaw Lugowski

Identification of a trisaccharide repeating-unit in the enterobacterial common-antigen

Abstract In addition to the previously reported 2-acetamido-2-deoxy- d -glucose and 2-acetamido-2-deoxy- d -mannuronic acid, 4-acetamido-4,6-dideoxy- d -galactose ( d -Fuc4NAc) is a major component of the enterobacterial common-antigen. The trisaccharide repeating-unit →4)-β- d -Man p NAcA-(1→4)-α- d -Glc p NAc-(1→3)- d -Fuc p 4NAc-(1→ constitutes 70% or more of this antigen.

The enterobacterial common-antigen, a cyclic polysaccharide.

Structural studies of the enterobacterial common-antigen, using chemical methods and fast-atom-bombardment mass spectrometry, indicate that it is a cyclic polysaccharide, composed of four, five, and, to a smaller extent, six trisaccharide repeating-units. In the structure of the antigen, given below, D-Fuc4NAc stands for 4-acetamido-4,6-dideoxy-D-galactose.

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]

A complex of lactoferrin with monophosphoryl lipid A is an efficient adjuvant of the humoral and cellular immune response in mice.

Our recent investigations demonstrated adjuvant properties of lactoferrin (LF). Other studies proved efficacy and safety of monophosphoryl lipid A (MPL) as an adjuvant in humans. In an attempt to construct more efficient and safer adjuvants, we evaluated the activity of LF-MPL complex, formed by incubation of LF and MPL from Hafnia alvei at 20:1xa0w/w ratio, and verified its characteristics by SDS-PAGE analysis. Binding kinetics was determined by surface plasmon resonance analysis using a BIAcoreTM 1000 biosensor system. The efficiency of the complex in enhancing the humoral and cellular immune responses was analyzed in BALB/c mice. The complex stimulated the humoral immune response to ovalbumin (OVA) and sheep red blood cells significantly stronger than both components separately, used at respective doses. In addition, the complex increased the serum levels of IgG, IgG2a and IgG1 OVA-specific antibodies as compared to the actions of LF or MPL alone. In the model of delayed type hypersensitivity (DTH) the strongest immune response was demonstrated with OVA administered subcutaneously, admixed with the complex. Administration of the complex in incomplete Freund’s adjuvant, together with a sensitizing dose of antigen, was similarly effective as immunization with complete Freund’s adjuvant. The complex also significantly enhanced the DTH response to orally administered Calmette-Guérin bacilli. In summary, the new type of adjuvant, the LF-MPL complex, was described. Its activity surpassed the adjuvant action of both constituents tested separately in the humoral and cellular immune responses in mice. The plausible mode of action of the new adjuvant is discussed.

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 studies of the o-specific side-chains of the Escherichia coli O 10 lipopolysaccharide

The structure of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide has been investigated, different 1H- and 13C-n.m.r. techniques being the main methods used. It is concluded that they are composed of pentasaccharide repeating-units having the following structure, in which D-Fuc3NAc is 3-acetamido-3,6-dideoxy-D-galactose. ----4)-beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----2)-alpha-L-Rh ap-(1----3)-beta-L-Rhap-(1----2 increases 1 alpha-D-Fucp3NAc.

Structural analysis of the O-specific polysaccharide isolated from Plesiomonas shigelloides O51 lipopolysaccharide

Plesiomonasshigelloides strain CNCTC 110/92 (O51) was identified as a new example of plesiomonads synthesising lipopolysaccharides (LPSs) that show preference for a non-aqueous surrounding during phenol/water extraction. Chemical analyses combined with (1)H and (13)C NMR spectroscopy, MALDI-TOF and ESI mass spectrometry showed that the repeating units of the O-specific polysaccharides isolated from phenol and water phase LPSs of P. shigelloides O51 have the same structure: -->4)-beta-D-GlcpNAc3NRA-(1-->4)-alpha-L-FucpAm3OAc-(1-->3)-alpha-D-QuipNAc-(1-->, containing the rare sugar constituent 2,3-diamino-2,3-dideoxyglucuronic acid (GlcpNAc3NRA), and substituents such as D-3-hydroxybutyric acid (R) and acetamidino group (Am). The HR-MAS NMR spectra obtained for the isolated LPSs and directly on bacteria indicated that the O-acetylation pattern was consistent throughout the entire preparation. The (1)H chemical shift values of the structure reporter groups identified in the isolated O-antigens matched those present in bacteria. We have found that the O-antigens recovered from the phenol phase showed a higher degree of polymerisation than those isolated from the water phase.

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.

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.

First Evidence for a Covalent Linkage between Enterobacterial Common Antigen and Lipopolysaccharide in Shigella sonnei Phase II ECALPS

Background: Enterobacterial common antigen (ECA) is a surface antigen of all enteric bacteria. Results: ECA polysaccharide substitutes the outer core region of Shigella sonnei lipopolysaccharide (LPS). Conclusion: First structural evidence for the existence of ECA covalently associated with LPS (ECALPS). Significance: ECALPS is the only immunogenic form of ECA and could be a target for therapeutic strategies against nosocomial infections. Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, occurs as a cyclic form (ECACYC), a phosphatidylglycerol (PG)-linked form (ECAPG), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECALPS). Since the discovery of ECA in 1962, the structures of ECAPG and ECACYC have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECALPS to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECALPS. ECALPS consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECALPS in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECALPS among Enterobacteriaceae species.