Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

Microbiology. 1997 Jun;143 ( Pt 6):2027-38. doi: 10.1099/00221287-143-6-2027.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / genetics
  • Base Sequence
  • Cholera Toxin / genetics*
  • DNA Restriction Enzymes / genetics
  • Epitopes / genetics*
  • Epitopes / immunology
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Fimbriae Proteins*
  • Fimbriae, Bacterial / chemistry
  • Fimbriae, Bacterial / genetics*
  • Fimbriae, Bacterial / metabolism
  • Gene Expression / genetics*
  • Genes, Bacterial / genetics
  • Genetic Engineering / methods
  • Immunoblotting
  • Molecular Sequence Data
  • Mutagenesis, Insertional / genetics
  • Mutagenesis, Insertional / physiology
  • Pili, Sex
  • Plasmids / genetics
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / immunology
  • Research Design

Substances

  • Bacterial Proteins
  • Epitopes
  • Recombinant Fusion Proteins
  • fimbrillin
  • Fimbriae Proteins
  • Cholera Toxin
  • DNA Restriction Enzymes