Development of a recombinant pertussis toxin operon: expression and characterisation in Escherichia coli and Salmonella typhimurium aroA

Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated bouts of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A/B bacterial toxin consisting of five subunits S1-S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organised into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. In this research the five PT subunits were individually expressed in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli 8-lactamase leader sequence. In E. coli CAG629 all five PT subunits were at least partially processed, with S5 being completely processed. A stepwise cloning method has then been developed to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, SI was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble SI and S3; however the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side effects of some vaccine preparations presently in use. Current vaccines against whooping cough are administered parenterally, and generate a systemic immune response. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunisation with live vaccine strains of Salmonella spp. In this research, the five PT subunits have been individually expressed in Salmonella spp. and the synthetic PT operon (pDP16) has then been expressed in attenuated aroA mutants of S. typhimurium , SL3261 and SL7207, via the use of a temperature inducible expression system. In SL3261 containing individual subunit expression plasmids the S5 subunit was found to be completely processed, while all other subunits were partially processed. In SL3261 (pDP16) and SL7207 (pDP16) S5 was completely processed, SI was partially processed, whilst S2, S3 and S4 remained unprocessed. Induction of the heat inducible synthetic PT operon at 39.5°C decreased the growth and in vitro invasiveness of SL3261 and SL7207, compared to uninduced (30°C) and partially induced (37°C) bacterial cells. Antibody responses were monitered following oral immunisation of BALB/c mice with the live vaccine strain SL7207 (pDP16) induced at 39.5°C for 1 h. Mice orally immunised with SL7207 (pDP16) primarily produced a systemic antibody response which recognised the S1 subunit of PT. The protectiveness of this vaccine strain was assessed in a mouse intracerebral challenge assay. Mice immunised with one dose of S. typhimurium SL7207 (pDP16) were not protected following intracerebral challenge with live B. pertussis. Future work will be aimed at improving the processing of PT subunits and immunogenicity of this vaccine strain, using chaperone proteins and in vivo promoters.