Construction and characterization in vivo of Bordetella pertussis aroA mutants

M. Roberts, D. Maskell, P. Novotny, G. Dougan

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Abstract

A DNA fragment encoding a kanamycin resistance determinant was used to insertionally inactivate the cloned aroA gene of Bordetella pertussis in Escherichia coli K-12, and a conjugative shuttle vector system based on the suicide vector pRTP1 was used to deliver the mutations from E. coli into B. pertussis CN2992FS and BP1. The aroA mutation was introduced by allelic exchange into the chromosome of B. pertussis, resulting in otherwise isogenic parental and aorA mutant pairs. The B. pertussis aorA mutants grew well on laboratory medium supplemented with aromatic compounds but failed to grow on unsupplemented medium. The B. pertussis aorA mutants expressed the normal B. pertussis extracellular, virulence-associated proteins; inactivated, whole-cell vaccines prepared from the mutants protected mice as efficiently as vaccines made from the parent strains against intracerebral challenge with the virulent B. pertussis 18323. Live B. pertussis aroA bacteria inefficiently colonized the lungs of NIH/S mice after they were challenged with aerosol, unlike the wild-type B. pertussis organism. Mice exposed to three separate aerosols of live B. pertussis aroA bacteria were protected against lung colonization after being exposed to an aerosol containing the virulent parental B. pertussis strain. High-level antibodies against B. pertussis rapidly appeared in the sera of mice immunized by aerosol with the B. pertussis aroA strains and challenged with the virulent parent.

Original languageEnglish
Pages (from-to)732-739
Number of pages8
JournalInfection and Immunity
Volume58
Issue number3
Publication statusPublished - 1 Jan 1990
Externally publishedYes

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