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Glycoproteins are processed endosomally prior to presentation to T cells and subsequent induction of specific antibodies. The sugar part of glycoconjugate may be degraded while the type of the process depends on the features of the particular structure. The generated carbohydrate epitopes may differ from native structures and influence immunogenicity of the antigens. We have devised a model of endosomal-like pre-processing of Bordetella pertussis 186 oligosaccharides (OSs) to verify how it affects the immunogenicity of their conjugates. The glycoconjugates of structurally defined forms of the dodecasaccharide OS were synthesized and their immunogenicity was assessed using immunochemical methods. The structural features of the oligosaccharides and their sensitivity to deamination were analyzed by NMR spectroscopy. The distal trisaccharide-comprising pentasaccharide conjugated to a protein was the most effective in inducing immune response against the B. pertussis 186 LOS and the immune response to the complete OS conjugates was significantly lower. This could be explained by the loss of the distal trisaccharide during the in-cell deamination process suggesting that the native structure is not optimal for a vaccine antigen. Consequently, our research has shown that designing of new glycoconjugate vaccines requires the antigen structures to be verified in context of possible endosomal reactions beforehand.

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: &rarr, 3)-&alpha, D-Fucp4NAc-(1&rarr, 4)-&beta, D-ManpNAcA-(1&rarr, 4)-&alpha, D-GlcpNAc-(1&rarr, and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide &minus, endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC&reg, HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

Plesiomonas shigelloides is a Gram-negative, rod-shaped bacterium which causes foodborne intestinal infections, including gastroenteritis. It is one of the most frequent causes of travellers&rsquo, diarrhoea. Lipopolysaccharide (LPS, endotoxin), an important virulence factor of the species, is in most cases characterised by a smooth character, demonstrated by the presence of all regions, such as lipid A, core oligosaccharide, and O-specific polysaccharide, where the latter part determines O-serotype. P. shigelloides LPS is still a poorly characterised virulence factor considering a &ldquo, translation&rdquo, of the particular O-serotype into chemical structure. To date, LPS structure has only been elucidated for 15 strains out of 102 O-serotypes. Structures of the new O-specific polysaccharide and core oligosaccharide of P. shigelloides from the Czechoslovak National Collection of Type Cultures CNCTC 90/89 LPS (O22), investigated by chemical analysis, mass spectrometry, and 1H,13C nuclear magnetic resonance (NMR) spectroscopy, have now been reported. The pentasaccharide repeating unit of the O-specific polysaccharide is built of one d-QuipNAc and is rich in four d-GalpNAcAN residues. Moreover, the new core oligosaccharide shares common features of other P. shigelloides endotoxins, i.e., the lack of phosphate groups and the presence of uronic acids.

Lipopolysaccharides are the main surface antigens and virulence factors of gram‑negative bacteria. Removal of four ester‑bound fatty acid residues from hexaacyl lipid A of Escherichia coli lipooligosaccharide (LOS) resulted in the de‑O‑acylated derivative E. coli LOS‑OH (LOS‑OH). This procedure caused a significant reduction in the toxicity of this compound compared to the native molecule. We investigated the effect of such a structural LOS modification on its biological activity using in vitro assays with monocytic cells of the RAW264.7 line, dendritic cells of the JAWS II line, bone marrow‑derived dendritic cells (BM‑DCs), and spleen cells. Furthermore, in in vivo experiments with a melanoma B16 metastasis model, the anti‑metastatic activity of the compounds and spleen cell reactivity mediated by them representing a systemic response were analyzed. The results revealed that LOS‑OH demonstrated weaker ability than LOS to stimulate and polarize an immune response both in vitro and in vivo. It induced lower cytokine production by cells of myeloid lines. Multiple applications of LOS‑OH into mice injected intravenously with B16 cells significantly (P

Lipopolysaccharide (LPS, endotoxin) is a main surface antigen and virulence factor of Gram-negative bacteria. Regardless of the source of LPS, this molecule, isolated from the smooth forms of bacteria, is characterised by a general structural layout encompassing three regions: (i) an O-specific polysaccharide (O-PS) - a polymer of repeating oligosaccharide units, (ii) core oligosaccharide (OS), and (iii) the lipid A anchoring LPS in the outer membrane of the cell envelope of Gram-negative bacteria. Structural analysis usually requires degradation of LPS and further efficient separation of various poly- and oligosaccharide glycoforms. The hydrophilic interaction liquid chromatography (HILIC) was shown as an efficient technique for separation of labelled or native neutral and acidic glycans, glycopeptides, sialylated glycans, glycosylated and nonglycosylated peptides. Herein we adopted ZIC(®) (zwitterionic stationary phase covalently attached to porous silica)-HILIC technology in combination with electrospray ionisation mass spectrometry to separate different LPS-derived oligosaccharides. As a result three effective procedures have been developed: (i) to separate different core oligosaccharides of Escherichia coli R1 LOS, (ii) to separate RU-[Hep]-Kdo oligosaccharides from core OS glycoforms of Hafnia alvei PCM 1200 LPS, and (iii) to separate Hep and Kdo-containing mono, di-, tri- and tetrasaccharides of H. alvei PCM 1200 LPS. Moreover, some of developed analytical procedures were scaled to semi-preparative protocols and used to obtain highly-purified fractions of the interest in larger quantities required for future evaluation, analysis, and biological applications.

The structure of the repeating unit of O-antigen of Plesiomonas shigelloides serotype O36 has been investigated by 1H and 13C NMR spectroscopy, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and chemical methods. The new structure of trisaccharide has been established: →4)-β-Pse5Ac7(R3Hb)-(2 → 4)-β-D-Galp-(1 → 3)-β-D-GlcpNAc-(1→ These trisaccharide O-antigen units substitute the core undecasaccharide at C-4 of the β-D-GlcpNAc residue. The core oligosaccharide and lipid A are identical with these of the serotype O17 (PCM 2231) (Maciejewska, A., Lukasiewicz, J., Kaszowska, M., Jachymek, W., Man-Kupisinska, A.; Lugowski, C. Mar. Drugs.2013, 11 (2), 440–454; Lukasiewicz, J., Dzieciatkowska, M., Niedziela, T., Jachymek, W., Augustyniuk, A., Kenne, L., Lugowski, C. Biochemistry, 2006, 45, 10434–10447).

Lipopolysaccharide (LPS, endotoxin), the main surface antigen and virulence factor of Gram-negative bacteria, is composed of lipid A, core oligosaccharide, and O-specific polysaccharide (O-PS) regions. Each LPS region is capable of complement activation. We have demonstrated that LPS of Hafnia alvei, an opportunistic human pathogen, reacts strongly with human and murine mannose-binding lectins (MBLs). Moreover, MBL–LPS interactions were detected for the majority of other Gram-negative species investigated. H. alvei was used as a model pathogen to investigate the biological consequences of these interactions. The core oligosaccharide region of H. alvei LPS was identified as the main target for human and murine MBL, especially l-glycero-d-manno-heptose (Hep) and N-acetyl-d-glucosamine (GlcNAc) residues within the outer core region. MBL-binding motifs of LPS are accessible to MBL on the surface of bacterial cells and LPS aggregates. Generally, the accessibility of outer core structures for interaction with MBL is highest during the lag phase of bacterial growth. The LPS core oligosaccharide–MBL interactions led to complement activation and also induced an anaphylactoid shock in mice. Unlike Klebsiella pneumoniae O3 LPS, robust lectin pathway activation of H. alvei LPS in vivo was mainly the result of outer core recognition by MBL; involvement of the O-PS is not necessary for anaphylactoid shock induction. Our results contribute to a better understanding of MBL–LPS interaction and may support development of therapeutic strategies against sepsis based on complement inhibition.

The structure of Escherichia coli B strain PCM 1935 core oligosaccharide has been investigated by 1H and 13C NMR spectroscopy, MALDI-TOF MS and ESI MSn. It was concluded that the core oligosaccharide is a pentasaccharide with the following structure: ESI MS/MS analysis revealed that the glycine (a minor component) is linked to the →3,7)- l -α- d -Hepp-(1→ residue.

The chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharide of pathogenic Edwardsiella tarda strain EIB 202 were studied for the first time. The complete gene assignment for all LPS core biosynthesis gene functions was acquired. The complete structure of core oligosaccharide was investigated by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry MSn, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. The following structure of the undecasaccharide was established: The heterogeneous appearance of the core oligosaccharide structure was due to the partial lack of β-d-Galp and the replacement of α-d-GlcpNAcGly by α-d-GlcpNGly. The glycine location was identified by mass spectrometry.

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 (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) 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 ECA(LPS) 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 ECA(LPS). ECA(LPS) 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 ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.

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.)

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 (1)H, (13)C 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.

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.

Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3.

Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.

Serum H-ficolin (ficolin-3) concentrations (n=613) and FCN3 genotypes (n=529) from a large group of neonates are presented. Both pre-term deliveries and low birthweight (independently of gestational age) were significantly associated with low H-ficolin concentrations but not with heterozygosity for the FCN3 1637delC frameshift mutation. The presence of the variant allele, however, apparently influenced the protein level. No association of FCN3 gene heterozygosity or relative functional H-ficolin insufficiency (determined as serum level ≤8.6 μg/ml) with perinatal infections was found. One premature newborn, with confirmed infection caused by Streptococcus agalactiae, was H-ficolin-deficient (FCN3 variant homozygote, no detectable protein). We present what is only the fourth case report of total H-ficolin deficiency in the world literature. This neonate was however previously found to be mannan-binding lectin (MBL) as well as MBL-associated serine protease-2 (MASP-2) deficient and also had low serum L-ficolin.

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 beta-D-GlcpN4P-(1-->6)-alpha-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-MS(n) 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 alpha-GlcpN residue. All the studied strains of H. alvei have an ability to modify their lipid A structure by palmitoylation.

The lipopolysaccharide (LPS) of Hafnia alvei strain PCM 1195 was obtained by the hot phenol/water method. The O-specific polysaccharide was released by mild acidic hydrolysis and isolated by gel filtration. The structure of the O-specific polysaccharide was investigated by 1 H, 13 C, and 31 P NMR spectroscopy, MALDI-TOF MS, and GC–MS, accompanied by monosaccharide and methylation analysis. It was concluded that the O-specific polysaccharide is composed of a hexasaccharide repeating units interlinked with a phosphate group: {→4-α- d -Glc p -(1→3)-α- l - Fuc p NAc-(1→3)-[α- d -Glc p -(1→4)]-α- d -Glc p NAc-(1→3)-α- l - Fuc p NAc-(1→4)-α- d -Glc p -(1→P} n .

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 someH. alveilipopolysaccharides (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 ofH. alveiPCM 32 LPS. It was established that the trisaccharide {l-α-d-Hepp-(1→4)-[α-d-Galp6OAc-(1→7)]-α-Kdop-(2→} is an integral part of the outer-core oligosaccharide ofH. alvei32 LPS. The very labile ketosidic linkage between →4,7)-α-Kdopand →2)-Glcpin the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes ofH. alveiLPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides ofH. alvei2, 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.

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [--2)-beta-d-Quip3NAcyl-(1--3)-alpha-l-Rhap2OAc-(1--3)-alpha-d-FucpNAc-(1--] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--residue and the --3)-beta-d-FucpNAc-(1--4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The --3)-beta-d-FucpNAc-(1--residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.

The rough mutants of Gram-negative bacteria are widely used to induce protective antisera but the nature of the target epitope for such antibodies is not precisely defined. Endotoxin is one of several antigens present on the surface of bacterial cells, which are able to elicit specific antibodies. We studied the specificity of antibodies produced against a conjugate of E. coli J5 endotoxin core oligosaccharide with tetanus toxoid. The use of chemically defined antigen for immunisation excludes the possibility of production of antibodies against other cell surface antigens. A comparison of this monospecific anti-endotoxin serum with antiserum against E. coli J5 whole cells was performed in order to distinguish the role that endotoxin core oligosaccharide plays in the interaction with humoral host defences from that of other potentially important Gram-negative bacterial surface antigens. The reactivity of both sera with smooth and rough lipopolysaccharides was determined in ELISA, immunoblotting and by flow cytometry. Both antisera reacted with similar specificity with most lipopolysaccharides of identical or related core type. Less distinct reactions with endotoxins of the antibacterial serum in comparison with the anti-conjugate serum were found in all serological tests. LPS of E. coli O100 that showed the strongest reactions with both sera was used to stimulate IL-6, TNFalpha and nitric oxide production by the J-774A.1 cell line. Both sera were used to inhibit that stimulation and no inhibitory effects of the examined sera in comparison with non-immune serum were observed.

The structure of the core oligosaccharide moiety of the lipopolysaccharide (LPS) ofPlesiomonas shigelloides O54 (strain CNCTC 113/92) has been investigated by 1H and 13C NMR, fast atom bombardment mass spectrometry (MS)/MS, matrix-assisted laser-desorption/ionization time-of-flight MS, monosaccharide and methylation analysis, and immunological methods. It was concluded that the main core oligosaccharide of this strain is composed of a decasaccharide with the following structure: in which l-α-d-Hepp isl-glycero-α-d-manno-heptopyranose. The nonasaccharide variant of the core oligosaccharide (∼10%), devoid of β-d-Glcp substituting the α-d-GlcpN at C-6, was also identified. The core oligosaccharide substituted at C-4 of the outer core β-d-Glcp residue with the singleO-polysaccharide repeating unit was also isolated yielding a hexadecasaccharide structure. The determination of the monosaccharides involved in the linkage between theO-specific polysaccharide part and the core, as well as the presence of →3)-d-β-d-Hepp-(1→ instead of →3,4)-d-β-d-Hepp-(1→ in the repeating unit, revealed the structure of the biological repeating unit of the O-antigen. The core oligosaccharides are not substituted by phosphate residues and represent novel core type of bacterial LPS that is characteristic for the Plesiomonas shigelloides serotype O54. Serological screening of 69 differentO-serotypes of P. shigelloides suggests that epitopes similar to the core oligosaccharide of serotype O54 (strain CNCTC 113/92) might also be present in the core region of the serotypes O24 (strain CNCTC 92/89), O37 (strain CNCTC 39/89) and O96 (strain CNCTC 5133) LPS.

Endotoxins (lipopolysaccharides, LPS) are the main surface antigens and virulence factors of Gram-negative bacteria involved for example in the development of nosocomial infections and sepsis. They consist of three main regions: O-specific polysaccharide, core oligosaccharide, and lipid A. Bacteria modify LPS structure to escape the immune defence, but also to adapt to environmental conditions. LPS's structures are highly diversified in the O-specific polysaccharide region to evade bactericidal factors of immune system, but retain some common epitopes that are potential candidates for therapeutic strategies against bacterial infections. Common occurrence of glycine within the structure of LPS is a known phenomenon and was previously reported for variety of species. Since glycine residue substitutes mainly core oligosaccharide of LPS, especially inner core region, it was also considered as a part of common epitope for broad-reactive antimicrobial antibodies. Herein, we used multiple-stage electrospray ionisation mass spectrometry to identify glycine substitution in core oligosaccharide type characteristic for Hafnia alvei LPS, and isolated from five strains of different O-serotypes: 32, PCM 1190, PCM 1192, PCM 1200, and PCM 1209. The location of glycine in core oligosaccharide was determined in detail for LPS 1190 using ESI-MS(n). Three glycoforms were identified, including two mono-glycinylated and one diglycinylated core oligosaccharides.

The structure of the O-specific side chain of the lipopolysaccharide (LPS) of Plesiomonas shigelloides, strain CNCTC 113/92 has been investigated by NMR spectroscopy, matrix-assisted laser desorption/ionization time of flight mass spectrometry and sugar and methylation analysis. It was concluded that the polysaccharide is composed of a hexasaccharide repeating unit with the following structure: in which D-beta-D-Hepp is Dglycero-beta-Dmanno-heptopyranose and 6d-beta-D-Hep is 6-deoxy-beta-Dmanno-heptopyranose. This structure represents a novel hexasaccharide repeating unit of bacterial O-antigen that is characteristic and unique to the Plesiomonas shigelloides strain. Using the high-resolution magic angle spinning technique, 1H-NMR spectra were also obtained for the O-polysaccharide components of isolated LPS and in their original form directly on the surface of bacterial cells.

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS, fast atom bombardment (FAB)-MS/MS and 1H- and 13C-NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: The polysaccharide is partially (approximately 10%) substituted with O-acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR-MAS) NMR analysis, which showed both the signals of the O-specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.

The structures of the carbohydrate O-specific side-chain moiety of the lipopolysaccharides (LPS) of Yokenella regensburgei, strains PCM 2476, 2477, 2478, and 2494, have been investigated by (1)H and (13)C NMR, fast atom bombardment tandem mass spectrometry (FAB-MSMS), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, methylation analysis, partial acid hydrolysis, and immunological methods. It was concluded that the O-specific polysaccharides of strains 2476, 2477, 2478, and 2494 are composed of the same basic trisaccharide repeating unit having the structure -->3)-alpha-D-FucpNAc-(1-->2)-L-alpha-D-Hepp-(1-->3)-6-deoxy -alpha-L- Talp-(1-->, in which L-alpha-D-Hepp is L-glycero-alpha-D-manno-heptopyranose. The detailed analysis revealed, however, differences in O-acetylation patterns of the 6-deoxy-L-talose residue, with 2- and 4-O-acetyl disubstituted -->3)-6-deoxy-alpha-L-Talp-(1--> in strain PCM 2476 and a 2-O-acetylated residue in strains 2477, 2478, and 2494. These structures represent novel, trisaccharide repeating units of bacterial O-antigens that are characteristic and unique to the Y. regensburgeispecies. By use of the high-resolution magic-angle spinning (HR-MAS) technique, (1)H NMR spectra of the O-polysaccharides directly in isolated LPS were obtained. This allowed for almost full assignment and structural determination of the polysaccharide. By this technique the O-polysaccharide components were also observed in their original form directly on the surface of living bacterial cells.

The structures of the O-specific chains of the Hafnia alvei strain 744 and PCM strains 1194 and 1210 lipopolysaccharides have been investigated. Methylation analysis, dephosphorylation, NMR spectroscopy, matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and fast-atom-bombardment mass spectrometry were the principal methods used. It was concluded that the polysaccharides of strains 744 and PCM 1194 are composed of the same pentasaccharide repeating unit having the following structure: [structure in text] and that the polysaccharide of strain PCM 1210 is composed of a pentasaccharide repeating unit having the following structure: [structure in text].

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.

Immunochemical analysis of the Yokenella regensburgei lipopolysaccharides (LPS) indicated the presence of the core oligosaccharide-related immunotypes among the investigated strains. The structure of the core oligosaccharide segment of the Y. regensburgei LPS has been investigated using chemical methods, mass spectrometry, and (1)H, (13)C NMR spectroscopy. It was concluded that the core oligosaccharides of the strains PCM 2476 and PCM 2477 are composed of an undecasaccharide. The combined data revealed two immunotypes of the core oligosaccharide recognized by antibodies against the whole bacterial cells. The structural differences between the core oligosaccharides are limited to the outermost terminal hexopyranose residue. In the core oligosaccharide of the strain PCM 2476, it was identified as alpha-d-Glcp and in that of the strain PCM 2477 as alpha-d-Galp. This subtle difference between the glycoforms of the LPS core appeared to be essential for formation of the epitopes recognized by the specific antibodies directed against the Y. regensburgei whole bacterial cells. The oligosaccharides are not substituted by phosphate groups. Instead, the carboxyl groups of Kdo and galacturonic acid residues present in the core provide the negative charges. The undecasaccharides represent a novel core type of bacterial LPS, which is characteristic for Y. regensburgei.

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.

Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1--6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS.

Plesiomonas shigelloides is a Gram-negative rod associated with episodes of intestinal infections and outbreaks of diarrhea in humans. The extraintestinal infections caused by this bacterium, for example, endopthalmitis, meningitidis, bacteremia, and septicemia, usually have gastrointestinal origin and serious course. The lipopolysaccharide (LPS, endotoxin) as virulence factor is important in enteropathogenicity of this bacterium. LPSs of P. shigelloides and especially their lipid A part, that is, the immunomodulatory center of LPS, have not been extensively investigated. The structure of P. shigelloides O54 lipid A was determined by chemical analysis combined with MALDI-TOF mass spectrometry, and the intact Kdo-containing core region was investigated by NMR spectroscopy on deacylated LPS. Products from alkaline deacylation of LPS, containing 4-substituted uronic acids, are usually very complex and difficult to separate. Since Kdo residues, like sialic acids, form complexes with serotonin, we used immobilized serotonin for one-step isolation of oligosaccharide containing the intact Kdo region from the reaction mixture by affinity chromatography. The major form of lipid A was built of β-D-GlcpN4PPEtn-(1[rightward arrow]6)-α-D-GlcpN1P disaccharide substituted with 14:0(3-OH), 12:0(3-OH), 14:0(3-O-14:0), and 12:0(3-O-12:0) acyl groups at N-2, O-3, N-2', and O-3', respectively. This is a novel structure among known lipid A molecules. Analysis of intact Kdo-lipid A region, lipid A and its linkage with the core oligosaccharide completes the structural investigation of P. shigelloides O54 LPS, resolving the entire molecule. Biological activities and observed discrepancy between in vitro and in vivo activity of P. shigelloides and Escherichia coli LPS are discussed.

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 20:1 w/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-Guerin 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.

Lipooligosaccharides (LOS) isolated fromBordetella pertussisstrains 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 inB. pertussisis heterogeneous. The pentasaccharide was selectively cleaved from LOS ofB. pertussisstrain 186, purified, and covalently linked to a monomer fraction of tetanus toxoid. Injection of rabbits with the neoglycoconjugate emulsified in complete Freund's adjuvant yielded immunoglobulin G antibodies that were reactive with the LOS. These antibodies reacted strongly withB. pertussisLOS 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 anN-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 ofB. pertussis186 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.

It has been known that administration of antibiotics may lead to excessive release of bacterial endotoxins and complicate clinical course of patients with Gram-negative infections. This concern may also apply to phages. Endotoxin may in turn activate neutrophils to produce reactive oxygen species (ROS) that are believed to play an important role in the pathogenesis of multiple organ dysfunction in the course of sepsis. We showed that a purified T4 phage preparation with low-endotoxin content could significantly diminish the luminol-dependent chemiluminescence (CL) of peripheral blood polymorphonuclear leukocytes (PMNs) both stimulated by lipopolysaccharides (LPSs) isolated from different Escherichia coli strains. This effect was also observed for live bacteria used for PMNs stimulation and was independent of bacterial susceptibility for T4-mediated lysis. Our data suggest, that phage-mediated inhibition of LPS- or bacteria-stimulated ROS production by PMNs may be attributed not only to phage-PMNs interactions, but also to phage-LPS interactions and bacterial lysis (in case of homologous phage). Interestingly, the T4 preparation did not influence ROS formation by PMNs stimulated with PMA. This suggests that the observed phenomena are also dependent upon the nature of activator. Bacteriophage-mediated inhibition of ROS formation by cells exposed to endotoxin provides new evidence for possible interactions between phages and mammalian cells. It helps in understanding the role of phages in our environment and may also be of important clinical significance.