Lipooligosaccharides (LOS) isolated from Bordetella pertussis strains 186 and 606 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution magic angle spinning nuclear magnetic resonance (NMR). These analyses distinguished between the LOS of strains 186 and 606, suggesting that the structure of LOS in B. pertussis is heterogeneous. The pentasaccharide was selectively cleaved from LOS of B. pertussis strain 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 with B. pertussis LOS 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 an N-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 of B. pertussis 186 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
lipopolysaccharides, Bacterial, antibodies, Bordetella pertussis, tetanus toxoid, immunodominant epitopes, pertussis vaccine
NCBI PubMed ID: 16239537Publication DOI: 10.1128/IAI.73.11.7381-7389.2005Journal NLM ID: 0246127Publisher: American Society for Microbiology
Correspondence: tomasz.niedziela@iitd.pan.wroc.pl
Institutions: Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Wroclaw, Poland
Methods: NMR, MALDI-TOF MS, deamination, STD NMR, HR-MAS NMR
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