title: Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background.
abstract: The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested with respect to host background in three different Escherichia coli strains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenous fim gene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized cholera toxin B chain, confirming the utility of the employed strategy.
authors.affiliation: Department of Microbiology, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.
authors.forename: Bodil
authors.initials: B
authors.lastname: Stentebjerg-Olesen
authors.affiliation: Department of Microbiology, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Pallesen
authors.affiliation: Department of Microbiology, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.
authors.forename: Lars Bogø
authors.initials: LB
authors.lastname: Jensen
authors.affiliation: Department of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Gunna
authors.initials: G
authors.lastname: Christiansen
authors.affiliation: Department of Microbiology, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.
authors.forename: Per
authors.initials: P
authors.lastname: Klemm
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000502:physiology; D018933:Pili, Sex; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D012107:Research Design
chemical_list: D001426:Bacterial Proteins; D000939:Epitopes; D011993:Recombinant Fusion Proteins; C063055:fimbrillin; D036781:Fimbriae Proteins; D002772:Cholera Toxin; D004262:DNA Restriction Enzymes