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

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. All six monoclonal antibodies inhibited agglutination of human erythrocytes by E. coli 6260; only monoclonal antibody 11-2 showed notable differences in reciprocal titers of hemagglutination inhibition (10 in the presence of mannose versus 1,000 in the absence of mannose). Our results suggest that E. coli 6260 pili are composed of at least four subunit types: 15.7 and 19.3K subunits involved in mannose-sensitive agglutination, and a second 15.7K subunit, in addition to a 17.8K subunit, involved in mannose-resistant agglutination.