Structure–Activity Relationship of Plesiomonas shigelloides Lipid A to the Production of TNF-α, IL-1β, and IL-6 by Human and Murine Macrophages

Plesiomonas shigelloides is a Gram-negative bacterium that is associated with diarrheal disease in humans. Lipopolysaccharide (LPS) is the main surface antigen and virulence factor of this bacterium. The lipid A (LA) moiety of LPS is the main region recognized by target cells of immune system. Here, we evaluated the biological activities of P. shigelloides LA for their abilities to induce the productions of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) by human and murine macrophages [THP-1 macrophages and immortalized murine bone marrow-derived macrophages (iBMDM)]. Four native P. shigelloides LA preparations differing in their phosphoethanolamine (PEtn) substitution, length, number, and saturation of fatty acids were compared with Escherichia coli O55 LA. The bisphosphorylated, hexaacylated, and asymmetric forms of the P. shigelloides and E. coli LA molecules had similar activities in human and murine macrophages, indicating that shortening of the acyl chains in P. shigelloides LA had no effect on its in vitro activities. The PEtn decoration also had no impact on the interaction with the toll-like receptor 4/MD-2 receptor complex. The heptaacylated form of P. shigelloides LA decorated with 16:0 exhibited strong effect on proinflammatory activity, significantly decreasing the levels of all tested cytokines in both murine and human macrophages. Our results revealed that despite the presence of shorter acyl chains and an unsaturated acyl residue (16:1), the bisphosphorylated, hexaacylated, and asymmetric forms of P. shigelloides LA represent highly immunostimulatory structures

First Report on the Streptococcus gallolyticus (S. bovis Biotype I) DSM 13808 Exopolysaccharide Structure

Streptococcus gallolyticus subspecies gallolyticus, known as Streptococcus bovis biotype I, is a facultative pathogen causing bacteraemia, infective endocarditis and sepsis that has been linked with colorectal cancer (CRC), but this correlation is still unclear. Bacterial surface structures, such as the major sugar antigens exposed to the outside of the microorganism, are potential virulence factors. One of the primary sugar antigens loosely attached to the cell surface is the biofilm component, exopolysaccharide (EPS). EPSs of S. bovis are poorly characterized molecules. Until now, only one S. macedonicus Sc136 EPS structure was known to the entire S. bovis group. The S. gallolyticus DSM 13808 EPS was investigated by chemical analysis, mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hexasaccharide repeating unit of the EPS, containing four Glc, two Rha residues and one phosphate group, has been described “ →6)-α-d-Glcp-(1→3)-β-l-Rhap-(1→4)-β-d-Glcp-(1→3)-[β-d-Glcp-(1→2)]-α-l-Rhap-(1→2)-α-d-Glcp-(1→P→”

Structure and Immunogenicity of the Bordetella pertussis LOS-Derived Oligosaccharides in the Endosomal-Like Pre-Processing Mice Model

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

Conjugation of the meningococcal lipopolysaccharide R-type oligosaccharides to tetanus toxoid as a route to a potential vaccine against Group B Neisseria meningitidis

NRC publication: Ye

The New Structure of Core Oligosaccharide Presented by Proteus penneri 40A and 41 Lipopolysaccharides

The new type of core oligosaccharide in Proteus penneri 40A and 41 lipopolysaccharides has been investigated by 1H and 13C NMR spectroscopy, electrospray ionization mass spectrometry and chemical methods. Core oligosaccharides of both strains were chosen for structural analysis based on the reactivity of LPSs with serum against P. penneri 40A core oligosaccharide–diphtheria toxoid conjugate. Structural analyses revealed that P. penneri 40A and 41 LPSs possess an identical core oligosaccharide

A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − 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®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

A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − 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®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

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-α-d-Hepp-(1→4)-[α-d-Galp6OAc-(1→7)]-α-Kdop-(2→} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between →4,7)-α-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