In Vitro and In Vivo Characterization of aBordetella bronchisepticaMutant Strain with a Deep Rough Lipopolysaccharide Structure

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Abstract
Bordetella bronchisepticais closely related toBordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research onBordetellahas been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) duringB. bronchisepticainfection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutantB. bronchisepticaLP39 on thewaaCgene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although inB. bronchisepticaLP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS ofB. bronchisepticaLP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, althoughB. bronchisepticaLP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form ofB. bronchisepticaLPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from humanB. bronchisepticachronic infections, the possibility that this phenotype arises as a consequence of selection pressure within the host at a late stage of the infection process is discussed.