Utilization of anion-exchange chromatography and monoclonal antibodies to characterize multiple pilus types on a uropathogenic Escherichia coli O6 isolate.

摘要

Multiple pilus types from a uropathogenic strain of Escherichia coli O6, strain 6260, were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography, binding assays, and erythrocyte adsorption. In addition, monoclonal antibodies were raised against purified pili of E. coli 6260 and used for immunological characterization. SDS-PAGE analysis of the purified pili showed at least three different subunits with molecular weights of 15,700, 17,800, and 19,300. SDS-PAGE analysis of four protein peaks from anion-exchange chromatography of intact pili showed polypeptides with molecular weights of 19,300 (fraction 1), 15,700 (fraction 2), and 17,800 and 15,700 (both fractions 3 and 4). Erythrocyte adsorption of the whole-pilus preparation removed the 17,800-molecular-weight subunit (17.8K subunit) and reduced the 15.7K subunit. Pili from an isogenic hemagglutination-negative variant of E. coli 6260, showing only the 15.7K and 19.3K subunits by SDS-PAGE, lacked the 17.8K subunit of fractions 3 and 4 present in the parent high-pressure liquid chromatography profile. Our data suggest that two of the pilus subunits, the 15.7K and 17.8K subunits, mediate mannose-resistant agglutination of human erythrocytes. Pili in fractions 1 and 2 from the parent strain bound specifically to mannose residues, while pili in fraction 4 bound to P-coated horse erythrocytes; no receptor specificity was identified for pili in fraction 3. Immunological analysis by the immunoblot technique showed that monoclonal antibody 11-2 reacted with the 19.3K subunit, monoclonal antibodies 34-3 and 73-3 reacted with the 15.7K subunit, and monoclonal antibodies 81-1, 82-1, and 91-1 reacted with polymers of subunits retained in the stacking gel. Intact pili precipitated by any of the six monoclonal antibodies showed two polypeptides by SDS-PAGE: 15.7K and 19.3K polypeptides for monoclonal antibody 11-2, and 15.7K and 17.8K polypeptides for monoclonal antibodies 34-3, 73-3, 81-1, 82-1, and 91-1. The cross-reactivity of the monoclonal antibodies with purified pili from other E. coli strains was determined by enzyme-linked immunosorbent assay. Monoclonal antibody 11-2 showed no significant cross-reactivity with heterogeneous pili. In contrast, the other monoclonal antibodies showed equivalent or greater reactivity with P pili from heterologous strains as compared with reactivity with E. coli 6260 pili.(ABSTRACT TRUNCATED AT 400 WORDS)