High efficiency gene replacement in Salmonella enteritidis: chimeric fimbrins containing a T-cell epitope from Leishmania major

A simple, high frequency chromosomal gene replacement method of general uti lity was developed for Salamonella enteritidis. This system uses an unstabl e, imperfectly segregating, temperature-sensitive replicon, pHSG415, as a c arrier of the recombinant gene of interest. It also allows for site-specifi c replacement of chromosomal genes without the need for antibiotic resistan ce markers in the recombinant genes or the use of specific bacterial strain s. This strategy was used to replace the chromosomal sefA and agfA fimbrin genes of S. enteritidis 3b with recombinant genes containing a 48 bp DNA fr agment encoding PT3, an immunoprotective T-cell epitope from GP63 of Leishm ania major. The fidelity of chimeric fimbrial replacements were confirmed b y DNA sequence analysis. Nearly 30% of the S. enteritidis clones selected i n the final stage of sefA mutagenesis contained the sefA::PT3 recombinant g ene, whereas for agfA the efficiency was as high as 10%. To our knowledge, this is the first report of fimbrial epitope replacement in the Salmonellae and the first chimeric fimbrin genes that have been reconstituted into a w ild-type genetic background for any organism. As such, this model represent s a promising 'organelle' expression system for epitope display in vaccinol ogy. (C) 1999 Elsevier Science Ltd. All rights reserved.