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Construction and characterization of Bordetella pertussis mutants lacking the vir-regulated P.69 outer membrane
M Roberts1, N F Fairweather, E Leininger
1Department of Molecular Biology, Wellcome Biotech, Beckenham, Kent, UK.
Abstract:
The Bordetella pertussis P.69 protein is an immunogen with vaccine potential. The role of this protein in pathogenesis is unclear; it has been associated with the toxic adenylate cyclase and adhesion to eukaryotic cells. For further analysis of the role of P.69 in the biology of B. pertussis, we have constructed strains which specifically lack P.69. The cloned P.69 (prn) gene of B. pertussis was insertionally inactivated with a kanamycin-resistance cassette. This inactivated gene was used to construct P.69- mutants of B. pertussis by allelic exchange using plasmid pRTP1. B. pertussis P.69- strains produced normal levels of other vir-regulated factors, including adenylate cyclase. The serotype of B. pertussis, determined by Eldering and Preston typing sera and monoclonal antibodies, was also unaffected by the presence or absence of P.69. The ability of a prn mutant to adhere to and invade HEp2 cells was not significantly different from that of its parent strain. A strain containing a mutation in fhaB was significantly less adhesive and invasive than its parent, and a prn fhaB double mutant exhibited an even greater reduction in adhesiveness and invasiveness down to levels comparable with a Vir- strain. However, strains harbouring mutations in FHA and/or P.69 were able to colonize or multiply in the murine respiratory tract, although a Vir- strain was unable to survive and proliferate in the same infection model.
Insights
Bordetella pertussis P.69 protein is not essential for virulence. Mutants lacking P.69 (prn) showed no defect in adenylate cyclase production, serotype, or initial adhesion and invasion, but reduced colonization in mice.
Area of Science:
- Microbiology
- Immunology
- Bacterial Pathogenesis
Background:
- The Bordetella pertussis P.69 protein is an immunogen with vaccine potential.
- Its precise role in pathogenesis, including contributions to toxicity and adhesion, remains unclear.
- Understanding P.69's function is crucial for developing effective pertussis vaccines and treatments.
Purpose of the Study:
- To elucidate the role of the P.69 protein in Bordetella pertussis pathogenesis.
- To construct and characterize B. pertussis strains lacking the P.69 protein.
- To assess the impact of P.69 deficiency on virulence factors, host cell interaction, and murine respiratory tract colonization.
Main Methods:
- Insertionally inactivated the cloned P.69 (prn) gene using a kanamycin-resistance cassette.
- Constructed P.69-deficient B. pertussis mutants via allelic exchange using plasmid pRTP1.
- Assessed adenylate cyclase levels, serotype, HEp-2 cell adhesion/invasion, and murine respiratory tract colonization in wild-type, mutant, and double-mutant strains.
Main Results:
- P.69-deficient B. pertussis strains produced normal levels of adenylate cyclase and maintained their serotype.
- P.69 mutants showed no significant difference in HEp-2 cell adhesion or invasion compared to the parent strain.
- A prn fhaB double mutant exhibited significantly reduced adhesion and invasion, while P.69 mutants could still colonize the murine respiratory tract.
Conclusions:
- The P.69 protein is not essential for Bordetella pertussis virulence-associated factors like adenylate cyclase or serotype.
- While P.69 contributes to adhesion and invasion, its absence is compensated by other factors like FHA.
- P.69 is not strictly required for B. pertussis colonization and multiplication in the murine respiratory tract, suggesting other virulence mechanisms are dominant.