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Expression of a synthetic pertussis toxin operon in Escherichia coli
1Department of Biological Sciences, University of Wollongong, New South Wales, Australia.
Insights
Researchers engineered Escherichia coli to produce pertussis toxin (PT) subunits for whooping cough vaccines. This synthetic PT operon allows for improved vaccine development by expressing PT components in a purified, E. coli-based system.
Area of Science:
- Microbiology
- Bacterial Toxin Research
- Vaccine Development
Background:
- Bordetella pertussis causes whooping cough, a severe infant illness.
- Pertussis toxin (PT) is a key virulence factor, but its genes are not expressed in E. coli, hindering vaccine production.
- Current vaccines may have side effects linked to other pertussis virulence factors.
Purpose of the Study:
- To develop a method for expressing all five PT subunits in E. coli.
- To construct a synthetic PT operon for simultaneous expression of PT subunits.
- To investigate the potential of E. coli for producing detoxified PT for vaccine use.
Main Methods:
- Individual expression of PT subunits in E. coli, modifying leader sequences for S4.
- Stepwise cloning to create a synthetic PT operon enabling simultaneous expression.
- Western blot analysis to assess subunit processing and periplasmic extraction to determine solubility.
Main Results:
- The synthetic PT operon successfully expressed PT subunits in E. coli KS476.
- Subunits S4 and S5 were fully processed, S1 partially processed, while S2 and S3 were largely unprocessed.
- Soluble S1 and S3 were found in periplasmic extracts, but processed S2, S4, and S5 were not detected, suggesting membrane association or insolubility.
Conclusions:
- A synthetic PT operon allows for the expression of PT subunits in E. coli.
- This system facilitates the study of E. coli as a platform for producing detoxified PT.
- This approach could lead to safer pertussis vaccines free from other bacterial virulence factors.
Abstract:
Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated 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 organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually 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 beta-lactamase leader sequence. We have developed a stepwise cloning method 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, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 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 currently in use.