A high frequency chromosomal gene replacement method of general utility was developed for Salmonella enteritidis. This method uses a segregation-deficient temperature-sensitive replicon, pHSG415, as a carrier of the recombinant gene of interest. It allows for site-specific replacement of chromosomal genes without the need for antibiotic resistance markers in the recombinant genes or the use of specific bacterial strains. This gene replacement strategy was used to investigate the foreign antigen-carrying potential of SefA and AgfA, the major fimbrin subunit proteins of Salmonella SEF14 and SEF17 fimbriae. On the basis of epitope mapping and structural predictions, ten different sites within each fimbrin protein were selected for replacement with PT3, an immunoprotective T-cell epitope from the gp63 protein of Leishmania major. PCR-generated sefA and agfA fimbrin genes containing the 48 bp DNA fragment encoding PT3 were used to replace the native fimbrin genes in the chromosome. PCR and DNA sequence analysis confirmed that 10–20% of potential clones contained the corresponding chimeric fimbrin gene. Fimbrial expression and assembly in the chimeric S. enteritidis strains was analyzed by Congo red binding, Western blotting and immunoelectron microscopy using immune serum raised to whole SEF14, whole SEF17 or PT3 peptide. Remarkably, all ten AgfA chimeric fimbrin proteins were expressed under normal conditions and eight were effectively assembled into external SEF17 fimbrial fibers. In contrast, none of the chimeric SefA proteins were expressed and no assembled SEF14 fimbriae were detected. This represents the first fimbrial epitope replacement system in the Salmonellae and the first chimeric fimbrin genes to be reconstituted into a wild-type genetic background. Results are presented from a preliminary vaccine trial in which BALB/c mice were immunized with a PT3-expressing S. enteritidis strain and challenged with virulent L. major friedlin. This model represents a promising “organelle” expression system for epitope display with broad applications as subunit or attenuated vaccines. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9300 |
| Date | 30 April 2018 |
| Creators | White, Aaron Paul |
| Contributors | Kay, William Wayne |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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