Mapping the binding domain of the F18 fimbrial adhesin.
F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF.
Smeds
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A
Faculty of Veterinary Medicine, Department of Basic Veterinary Sciences, Section of Microbiology, 00014 University of Helsinki, Finland.
Pertovaara
M
M
Timonen
T
T
Pohjanvirta
T
T
Pelkonen
S
S
Palva
A
A
D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D001665:Binding Sites; D002478:Cells, Cultured; D004847:Epithelial Cells; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007421:Intestine, Small / Q000166:cytology; D008969:Molecular Sequence Data; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D013552:Swine
D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; C113053:FedA protein, E coli; C440653:FedF protein, E coli; D036781:Fimbriae Proteins