Construction and characterization in vivo of Bordetella pertussis aroA mutants

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 back 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 aroA mutant pairs. The B. pertussis aroA mutants grew well on laboratory medium supplemented with aromatic compounds but failed to grow on unsupplemented medium. The B. pertussis aroA 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.

Assessment of immunity against avian colibacillosis induced by an aroA mutant containing increased serum survival gene in broilers

“…Mutation of certain auxotrophic genes such as aroA has been employed to construct attenuated strains of various bacteria including APEC, Salmonella typhimurium, Salmonella choleraesuis, Bordetella pertussis (11,15,21,28). In this study, we evaluated the attenuation, immunogenecity and protective ability of the aroA mutant from a native E. coli O78:…”

AnaroAMutant ofEdwardsiella ictaluriIs Safe and Efficacious as a Live, Attenuated Vaccine

“…Exposure to bacterial auxotrophs, particularly those with defects in the aro genes that make up the common pathway of aromatic amino acid biosynthesis (the shikimate pathway), has conferred varying degrees of protection against subsequent challenge by virulent pathogens (Hoiseth and Stocker 1981; Smith et al 1984; ; Lindberg et al 1990; Roberts et al 1990; ; Vaughan et al 1993). Bacterial pathogens with stable defects in the aro genes are avirulent because chorismate, the end product of the shikimate pathway, is not produced.…”

Use ofBordetella bronchisepticaandBordetella pertussisas live vaccines and vectors for heterologous antigens

“…Signi¢cant anti-B. bronchiseptica serum antibody responses were detected before boosting even in the mice that received a single dose of vaccine, contrasting sharply with what was observed in B. pertussis [34,36]. This antibody response was directed against a variety of polypeptides ( Fig.…”

“…Despite B. bronchiseptica being able to colonise the respiratory tract more e⁄ciently than B. pertussis, a B. bronchiseptica aroA mutant was as highly attenuated for lung colonisation in mice as its B. pertussis aroA counterpart ( Fig. 1) [34,36]. However, the immune response of mice i/n immunised with a B. bronchiseptica aroA strain di¡ered considerably from that of mice immunised with the B. pertussis aroA strain.…”