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Microbiology 143 (1997), 2027-2038; DOI  10.1099/00221287-143-6-2027
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Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

Bodil Stentebjerg-Olesen1, Lars Pallesen1,{dagger}, Lars Bogø Jensen1,{ddagger}, Gunna Christiansen2 and Per Klemm1

1 Department of Microbiology, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark
2 Department of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C, Denmark

Author for correspondence: Per Klemm. Tel: +45 45 25 25 06. Fax: +45 45 93 28 09. e-mail: pk@im.dtu.dk

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.

Keywords: surface display of foreign epitopes, chimeric proteins, effect of host background, type 1 fimbriae

{ddagger} Present address: Danish Veterinary Laboratory, DK-1790, Copenhagen, Denmark.

{dagger} Present address: Department of Research and Development, Division of Microbiology, State Serum Institute, DK-2300 Copenhagen S, Denmark.




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