Year

2002

Degree Name

Doctor of Philosophy

Department

Department of Biological Sciences

Abstract

Bordetella bronchiseptica is a Gram-negative respiratory pathogen of a variety of animals. This bacterium causes kennel cough in dogs, bronchopneumonia in rabbits and guinea pigs, and has been associated with the disease atrophic rhinitis in swine. To investigate whether B. bronchiseptica could be attenuated through disruption of aromatic compound biosynthesis, the aroA gene of B. bronchiseptica was PCR amplified and cloned. DNA sequence analysis of the area surrounding the aroA gene revealed it to be part of a mixedfunction superoperon in the order of gyrA, serC, pheA, tyrA, aroA, cmk, rpsA and ihfB that has been shown to be well conserved through wide phylogenetic distances. The aroA O RF was disrupted by inserting a kanamycin resistance cassette within it. This construct was used to produce an insertion mutation in aroA of B. bronchiseptica via homologous recombination. This mutation resulted in strain CMJ25 that displayed a slower growth rate in minimal medium when compared to the parental strain but was not completely auxotrophic for aromatic compounds. Growth rate comparable to that of the parental strain was exhibited when aromatic supplements were added to the medium or when CMJ25 was subjected to growth conditions resulting in the strain displaying a bvg-repressed phase. CMJ25 was not attenuated in a murine model of respiratory infection. To obtain an aromatic amino acid auxotrophic mutant of B. bronchiseptica that was attenuated in vivo, CMJ25 was subjected to rounds of mutagenesis using the gentamycin resistant minitransposon, mini-Tn5/Gm. The resulting strain CMJ60, was unable to grow in minimal SSX medium in the absence of aromatic amino acid (aamix) supplementation, specifically when tryptophan was not present. The region of the chromosome containing the transposon was cloned and D N A sequence analysis revealed that the mini-transposon had inserted into an O R F encoding trpE. Analysis of the sequence surrounding B. bronchiseptica trpE was performed using the genome sequence currently being assembled at the Sanger Center (http://www.sanger.ac.uk/projects/B_bronchiseptica). This revealed trpE to be organised into an operon with genes that code for other tryptophan biosynthetic enzymes, trpG, trpD and trpC. The presence of a putative trpL leader sequence upstream of trpE may indicate that B. bronchiseptica uses an attenuation mechanism to regulate expression of this operon. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with CMJ60 displayed significant dose-dependent increases in B. bronchiseptica-STpecific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain and the humoral immune response in vaccinated and protected mice was directed primarily against B. bronchiseptica lipopolysaccharide. These results demonstrate that B. bronchiseptica, when made auxotrophic for aromatic compounds, is attenuated in mice and can be used as a live intranasal vaccine that elicits strong and protective humoral and cellmediated immune responses. Such a strain m a y not only be utilised for the prevention of B. bronchiseptica associated disease, but could also be used to deliver heterologous antigens to a variety of mammalian species.

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