K88 fimbriae as carriers of heterologous antigenic determinants.
The K88 fimbriae of enterotoxigenic Escherichia coli are strongly immunogenic antigens that can be used to evoke protective immunity. To find out whether these fimbriae can be used as carriers for foreign epitopes, a highly variable region present in the primary structure of the different K88 variants was replaced with five different heterologous epitopes to investigate to what extent these insertions affected the expression, assembly (biogenesis), stability and immunogenic properties of the resulting hybrid fimbriae. Amino acid residues 163-173, were replaced using site-directed in vitro mutagenesis and the hybrid fimbriae were tested for these aspects using ELISA, immunoelectronmicroscopy and immunoblotting. Replacement of this highly variable region did not affect the biosynthesis of fimbriae, although all mutations tested resulted in a reduced expression depending on the epitope inserted. Testing of the different hybrid fimbriae with a panel of monoclonal antibodies raised against the various K88 serotypes K88ab, K88ac and K88ad indicated that replacement of amino acid sequence 163-173 did not affect conserved or K88ab specific epitopes but the K88ac and K88ad specific conformation was lost. Immunization with hybrid fimbriae raises antibodies specific for the inserted heterologous epitopes.
Department of Molecular Microbiology, Faculty of Biology, Vrije Universiteit, Amsterdam, The Netherlands.
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Bakker
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van Zijderveld
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van der Veen
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Oudega
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de Graaf
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004274:DNA, Recombinant; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D015870:Gene Expression; D015151:Immunoblotting; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D004274:DNA, Recombinant; D000939:Epitopes; D029968:Escherichia coli Proteins; C026888:K88 antigen, E coli; D036781:Fimbriae Proteins