Receptor-specific binding of purified F4 to isolated villi.
Different procedures for preparing and purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26 (O149:K91:F4ac LT+Sta+STb+) were performed and the purity and yield of F4ac were compared. Fimbriae were prepared by either mechanical shearing or heatshock treatment of concentrated bacterial suspensions (10(11) bacteria/ml). The mechanical shearing procedure resulted in approximately 1.7 mg fimbriae (i.e. 74.4% of the isolated protein) and 0.6 mg (25.6%) contaminating proteins per 10(12) bacteria, whereas the yield of fimbriae following heatshock treatment was lower (0.3 mg per 10(12) bacteria, i.e. 26.2%) and the relative contamination higher (1.0 mg per 10(12) bacteria, i.e. 73.8%). A further purification consisted of either anion exchange chromatography (AEC) or electro-elution from SDS-polyacrylamide gels. The electroelution procedure was performed under reducing and denaturing conditions, so that purified FaeG subunits, the major subunit of F4, were finally obtained. The binding activity of fimbriae, nonpurified as well as purified, and FaeG to F4-specific receptors on isolated intestinal villi was assessed in an inhibition adhesion assay. Native fimbriae as well as major subunits were able to bind to the receptors, and the specificity of the binding was demonstrated by blockage with F4ac-specific MAb.
Van den Broeck
W
W
Laboratory of Veterinary Immunology, Faculty of Veterinary Medicine, Universiteit Gent, Merelbeke, Belgium. wim.vandenbroeck@rug.ac.be
Cox
E
E
Goddeeris
B M
BM
D000818:Animals; D000911:Antibodies, Monoclonal; D001422:Bacterial Adhesion / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification*; D001428:Bacterial Vaccines / Q000276:immunology; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000276:immunology* / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000276:immunology / Q000662:veterinary*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000276:immunology*; D007114:Immunization / Q000662:veterinary; D007422:Intestines / Q000276:immunology / Q000382:microbiology; D008871:Microvilli / Q000276:immunology / Q000382:microbiology; D013552:Swine; D013553:Swine Diseases / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; C022924:colonization factor antigens; D036781:Fimbriae Proteins