title: Functional expression of adhesive peptides as fusions to Escherichia coli flagellin.
abstract: An expression system for studying epitopes of adhesion proteins based on fusion of gene fragments into fliC(H7) of Escherichia coli is described. We constructed the system by an in-frame insertion of DNA fragments encoding one, two or three of the fibronectin-binding D repeats present in the fibronectin-binding protein A (FnBPA) of Staphylococcus aureus, into the fliC(H7) gene region encoding the variable domain of the H7 flagellin. The constructs were expressed by in trans complementation in the E. coli strain JT1 which harbours knock-out mutations for the expression of FliC as well as of the mannoside-binding fimbrial adhesin. The resulting chimeric flagella, which contained 39, 77 or 115 heterologous amino acid residues, efficiently bound soluble and immobilized human plasma and cellular fibronectin, and the binding was most efficient with the flagella containing the three D repeats of FnBPA. The chimeric flagella bound to frozen sections of human kidney and to cultured human cells. Antibodies raised against the chimeric flagella bound to Protein A-deficient S. aureus cells and inhibited the binding of staphylococci to immobilized fibronectin. We also expressed peptides, ranging in size between 48 and 302 amino acids, of the collagen-binding YadA adhesin of Yersinia enterocolitica. A fragment of 302 amino acids representing the middle region of YadA was needed for collagen binding. Chimeric flagellar filaments expressing hundreds of intimately associated adhesive epitopes offer versatile tools to analyze adhesin-receptor interactions and functional epitopes of adhesion proteins.
authors.affiliation: Department of Biosciences, University of Helsinki, Finland.
authors.forename: B
authors.initials: B
authors.lastname: Westerlund-Wikström
authors.forename: J
authors.initials: J
authors.lastname: Tanskanen
authors.forename: R
authors.initials: R
authors.lastname: Virkola
authors.forename: J
authors.initials: J
authors.lastname: Hacker
authors.forename: M
authors.initials: M
authors.lastname: Lindberg
authors.forename: M
authors.initials: M
authors.lastname: Skurnik
authors.forename: T K
authors.initials: TK
authors.lastname: Korhonen
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004926:Escherichia coli / Q000737:chemistry*; D005353:Fibronectins / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D005408:Flagellin / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid; D013211:Staphylococcus aureus / Q000737:chemistry
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005353:Fibronectins; D011993:Recombinant Fusion Proteins; C064832:YadA protein, Yersinia; C078635:fibronectin-binding proteins, bacterial; D005408:Flagellin