Presentation of foreign antigenic determinants at the cell surface of enteric bacteria using the trat protein

Harrison, Jacqueline Laura (1992) Presentation of foreign antigenic determinants at the cell surface of enteric bacteria using the trat protein. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The TraT protein is an oligomeric, highly cell-surface-exposedlipoprotein, specified by F-like plasmids. It confers resistance to the bactericidal effects of serum and blocks the conjugal transfer of plasmids to cells bearing identical or closely-related plasmids, a process known as surface exclusion. This thesis describes studies on the structure and function of TraT and on its use for presentation of foreign antigenic determinants at the bacterial cell surface. The protein specified by the antibiotic resistance plasmid R6-5 was purified to homogeneity by a new procedure. Circular dichroism spectroscopy indicated that the protein contained significant α-helical structure (22%), in contrast to other previously-studied outer membrane proteins. The purified protein had a protective effect on bacterial cells incubated in serum, suggesting that it does not have to be located on the cell surface to mediate serum resistance. Additionally, when added to mating mixtures, the purified TraT inhibited the conjugal transfer of plasmids belonging to surface exclusion group IV, but had no significant effect on the transfer of plasmids belonging to other groups, confirming that TraT is specific in its surface exclusion effect. Since the mature form of the R6-5 protein differs from that of F (which belongs to surface exclusion group I) by a single amino acid (Ala_120 rather than Gly_120), the result indicates that this residue is a critical determinant of surface exclusion specificity. A model for surface exclusion specificity is presented. The TraT protein structure and function was further probed, around this specificity region, by genetic insertion of a foreign antigenic determinant (the C3 epitope of poliovirus) at residue 125 of the protein. The hybrid protein was transported to the outer membrane and assembled into trypsin-resistant oligomers, characteristic of the wild-type protein. However, the C3 epitope inserted in this region was not recognised by monoclonal antibodies added to whole cells and disrupted the permeability barrier effect of the outer membrane. In parallel with these studies, plasmids specifying hybrid TraT proteins with the C3 epitope of poliovirus inserted at positions 61 or 180 were introduced into an aroA^- attenuated strain of Salmonella typhimurium. The plasmid-bearing derivatives which efficiently expressed the C3 epitope without detectably altering any of the important characteristics of the vaccine strain, were used for oral immunisation of BALB/c mice. The bacteria invaded the livers of the mice and were detectable until day 14 after inoculation. However, no immune response to C3 was elicited, probably due to the instability of the plasmids following inoculation. To circumvent these problems and to improve expression of the hybrid TraT proteins in vivo, a modified inducible expression system with improved stability was developed. The hybrid traT/C3 genes were placed under the control of the groEL promoter (which is strongly induced when S. typhimurium enters macrophages) and cloned into a vector that allows homologous recombination of the promoter-gene cassette into the aroC chromosomal locus. The TraT/C3 hybrid proteins accounted for more than 4% of the total cellular protein in the Salmonella vaccine strain.