Isolation and Characterization of the α -sialyl-β -2,3-galactosyl-Specific Adhesin from Fimbriated Escherichia coli

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 10; pp. 3462 - 3466
• Main Authors: Moch, Thomas, Hoschutzky, Heinz, Hacker, Jorg, Klaus-D. Kroncke, Jann, Klaus
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.05.1987; National Acad Sciences
• Subjects: adhesin; Adhesins, Escherichia coli; Adhesives; Agglutination; Amino acids; Animals; Antibodies; Antibodies, Monoclonal; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacteriology; Biological and medical sciences; Carbohydrate Conformation; Carbohydrate Sequence; Cattle; Chromatography; Escherichia coli; Escherichia coli - genetics; Escherichia coli - physiology; Fimbriae; Fimbriae, Bacterial - physiology; Fundamental and applied biological sciences. Psychology; Galactose; Hemagglutination Inhibition Tests; Hemagglutination Tests; Humans; Microbiology; Monoclonal antibodies; Morphology, structure, chemical composition; Sialic Acids; Spleen cells; Ungulates; Characterization; Cell-cell interaction; Escherichia coli; Bacteria; Isolation; Pilus; Glycoproteins; Escherichieae; Enterobacteriaceae
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.10.3462

The α -sialyl-β -2,3-galactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-fimbrial adhesin complex. A crude cell extract was partially dissociated into fimbriae and an adhesin-enriched fraction by heating to 70 degrees C. From the latter, adhesin was purified to apparent homogeneity (by fast protein liquid chromatography, immunoblot, and NaDodSO4/PAGE) by differential ammonium sulfate precipitation, dissociation in 8 M guanidine hydrochloride, and high-resolution anion-exchange chromatography in 8 M urea. The purified adhesin formed an aggregate of Mr≈ 106that was made up of one type of 12-kDa polypeptide (fimbrillin is 16.5 kDa). It had pI value of 4.7 (fimbriae has a pI value of 6). Adhesin and fimbrillin had different amino acid compositions. The purified adhesins agglutinated human and bovine erythrocytes with the same specificity as the whole bacteria; purified fimbriae were not adhesive. Monoclonal anti-adhesin and anti-fimbriae antibodies were obtained. Monoclonal anti-adhesin, but none of the anti-fimbriae, antibodies inhibited the agglutination of erythrocytes. The anti-adhesive antibodies were used in immuno-gold electron microscopy to localize adhesin exclusively on the fimbriae, with a possible preference to their tips.