Interaction of Mannose-Binding Lectin With Lipopolysaccharide Outer Core Region and Its Biological Consequences.

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.

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins.

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.

Fractionation and analysis of lipopolysaccharide-derived oligosaccharides by zwitterionic-type hydrophilic interaction liquid chromatography coupled with electrospray ionisation mass spectrometry.

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.

Ficolin-3 activity towards the opportunistic pathogen, Hafnia alvei.

Ficolin-3 (also called H-ficolin or Hakata antigen) is a complement-activating pattern recognition molecule, possessing a fibrinogen-like domain involved in carbohydrate binding. Amongst human ficolins, Ficolin-3 has the highest concentration in serum and is the most potent lectin pathway activator in vitro. Evidence for its physiological function is sparse, although its deficiency has been suggested to increase susceptibility to infections. The specificity of Ficolin-3 is poorly characterized and currently few ligands are known. Here we report agglutination of Hafnia alvei, a Gram-negative enteric commensal bacterium and opportunist pathogen, in the presence of recombinant Ficolin-3 and calcium. Ficolin-3 also augmented phagocytosis of H. alvei by macrophages and displayed bactericidal activity. Additionally, Ficolin-3 inhibited host cells' response to TLR4/MD-2/CD14-LPS dependent NF-κB activation. This is the first demonstration of protective activity of Ficolin-3 against a human bacterial pathogen. Although human Ficolin-3 does not recognise and bind to common pathogenic bacteria, it could be an important component of innate immunity providing protection, for example, from commensal flora that can cause extraintestinal, opportunistic infections.

Occurrence of glycine in the core oligosaccharides of Hafnia alvei lipopolysaccharides--identification of disubstituted glycoform.

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.

Selective detection of carbohydrates and their peptide conjugates by ESI-MS using synthetic quaternary ammonium salt derivatives of phenylboronic acids.

We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

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 (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.