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The structure of the O-specific side-chain of the Hafnia alvei strain PCM1207 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: -3)[aDGlcp(1-6),Ac(1-2)]bDGalpN(1-4)[xDGro(1-P-3),Ac(1-2)]bDGalpN(1-3)aDGalp(1-4)bDGalp(1-. The polysaccharide is partially (< 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.

Lipopolysaccharide, O-antigen, Hafnia alvei, MALDI-TOF, MALDI-TOF MS, HR-MAS NMR

NCBI PubMed ID: 10542050
Journal NLM ID: 0107600
Publisher: Oxford, UK: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
Correspondence: Lennart.Kenne@kemi.slu.se
Institutions: Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden, Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland
Methods: methylation, NMR-2D, partial acid hydrolysis, ELISA, MALDI-TOF MS, de-O-acetylation, FAB-MS/MS, TEMPO oxidation

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to contain D-glucose, D-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a beta configuration, except for the side-chain Glc, which is α. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti-C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue.

Lipopolysaccharide, O-antigen, polysaccharide structure, serological specificity, glycerol phosphate, Citrobacter werkmanii

NCBI PubMed ID: 18811720
Journal NLM ID: 9315554
Publisher: Elsevier
Correspondence: katzenel@iitd.pan.wroc.pl
Institutions: L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
Methods: 13C NMR, 1H NMR, methylation, GLC-MS, NMR-2D, SDS-PAGE, sugar analysis, mild acid hydrolysis, Smith degradation, serological methods

The lipopolysaccharide (LPS) is the major constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. Its lipid A moiety is embedded in the membrane and serves as an anchor for the rest of the LPS molecule. The outermost repetitive glycan region of the LPS is linked to the lipid A through a core oligosaccharide (OS), and is designated as the O-specific polysaccharide (O-polysaccharide, OPS) or O-antigen. The O-antigen is the most variable portion of the LPS and provides serological specificity, which is used for bacterial serotyping. The OPS also provides protection to the microorganisms from host defenses such as complement mediated killing and phagocytosis, and is involved in interactions of bacteria with plants and bacteriophages. Studies of the OPSs ranging from the elucidation of their chemical structures and conformations to their biological and physico-chemical properties help improving classification schemes of Gram-negative bacteria. Furthermore, these studies contributed to a better understanding of the mechanisms of pathogenesis of infectious diseases, as well as provided information to develop novel vaccines and diagnostic reagents.

Lipopolysaccharide, synthesis, lipopolysaccharides, structure, Bacterial, host, O-antigen, O antigen, cell, O antigens, O-antigens, chemical, interaction, cells, PDF, chemical synthesis, biogenesis

Publication DOI: 10.1007/978-3-7091-0733-1_3
Publisher: Springer
Correspondence: knirel@ioc.ac.ru
Editors: Knirel YA, Valvano MA
Institutions: Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia