Isolation and characterization of the a-sialyl-p-2,3-galactosyl- specific adhesin from fimbriated Escherichia coli

The a-sialyl-1J-2,3-gaIactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-flmbrial adhesin complex. A crude cell extract was partiaUy dissociated into fimbriae and an adhesin-enriched fraction by heating to 7O"C. From the latter, adhesin was purified to apparent homogeneity (by fast protein liquid chromatography, immunoblot, and NaDodSO .. /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 -10' that was made up of one type of 12-kDa polypeptide (flmbrillin is 16.5 kDa). It had pI value of 4.7 (fimbriae has a pI value of 6). Adhesin and fimbrillin had different amino add compositions. The purified adhesins agglutinated human and bovine erythrocytes with the same speclfkity as the whole bacteria; purified fimbriae were not adhesive. Monoclonal anti-adhesin and anti-fimbriae antibodies were obtained. Monoclonal antiadhesin, 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. Infections caused by pathogenic Escherichia coli are often initiated by their adherence to host cell surfaces. This cell interaction is mediated by lectin-like adhesive proteins associated with the bacterial surface (1). In many cases the adhesiveness of E. coli is correlated with the expression of supermolecular, extracellular structures, the fimbriae or pili. Since isolated fimbriae often cause the agglutination of erythrocytes (RBC), which is used as an in vitro assay for the adhesiveness of bacteria, they were recognized as adhesive bacterial appendages (2-8). Bacterial adhesins specifically recognize certain carbohydrate moieties of glycolipids or glycoproteins such as a-mannose (common type 1 fimbriae), a-galactosyl-l,4-!:J-galactose (p fimbriae), a-sialyl-2,3-!:J-galactose (S fimbriae), or galactose/N-acetylgalactosamine/ serin (M fimbriae) (2, 5, 9-12). Molecular analysis of the cloned determinants coding for common type 1 (13, 14), P (15-17), and S (18) fimbriae revealed that distinct regions of these direct fimbriation and adhesiveness. Transposon-insertional mutants and subclones were found that still expressed fimbriae but were no longer adhesive or that lacked fimbriae but were adhesive. These re suits indicated that adhesin and fimbriae subunits may be different molecules. In spite of the wealth of information on fimbriae, to our knowledge the nature of the adhesins and their correlation to fimbriae had not yet been defined. During our studies on fimbriae and adhesins of E. coli, we were able'to physically separate adhesin from the fimbriae of a pyelonephritogenic E. coli 06 strain. The adhesin was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be bereby marked "advertisement" in accordance with 18 U.S.C. U734 solely to indicate this fact. 3462 characterized inter alia with the help of monoclonal antiadhesin antibodies that do not cross-react with the fimbrial subunit. MATERIALS AND METHODS Bacteria. The strains used are listed in Table 1. E. coli wild-type strain 536 (serotype 06:KI5:H31) was isolated from a patient with a urinary tract infection (18). It expresses common type 1 fimbriae and the S-fimbrial adhesin complex (Sfa, previously termed X; see refs. 18 and 19) consisting of S fimbriae and S adhesin. The phenotype for S fimbriae is termed Fim + and that for S adhesive capacity (indicated by mannose-resistant hemagglutination) Adh +. The bacteria were grown at 37°C for at least 18 hr on Loeb agar containing 0.1% glucose and appropriate antibiotics (100 JLg/ml; see Table 1). Before use the bacteria were monitored for hemagglutination. With the exception of strain HBI0l (pANN801-13/Tn 5-38) the strains agglutinated human and bovine RBC; agglutination was more pronounced at 4°C. Isolation of Fimbriae and Adhesin from the Recombinant E. coli S~ HBIOI (pANNSOI-13). Agar-grown bacteria were collected and suspended in 50 mM Tris·HCI (PH 7.8) at a cell density of 1010 cells per ml and agitated twice for 5 min with an Omnimixer (setting 3) while cooling in an ice bath. The bacteria and debris, were removed by centrifugation (27,000 x g, 40 min), and the fimbriae were pelleted from the supematant by ultracentrifugation (140,000 x g, 120 min). The crude fimbriae were suspended to 1 mg of protein per ml in PBS/5 mM EDTA and heated to 700C for 1 hr. (pBS = 10 mM phosphate buffer containing 0.2 g of KCI and 8 g ofNaCI per liter, pH 7.4.) After a subsequent centrifugation (140,000 x g, 120 min), the pellet contained predominantly fimbriae, and the supematant was enriched in adhesin. The adhesincontaining supematant was dialyzed against 50 mM NH..HC03, lyophilized, suspended to 500 p.g of protein per ml in PBS, and precipitated with ammonium sulfate (10-40%