In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipopolysaccharide structure

Abstract

Bordetella bronchiseptica is closely related to Bordetella 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 on Bordetella has been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) during B. bronchiseptica infection 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 mutant B. bronchiseptica LP39 on the waaC gene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although in B. bronchiseptica LP39 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 of B. bronchiseptica LP39 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, although B. bronchiseptica LP39 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 of B. bronchiseptica LPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from human B. bronchiseptica chronic 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.

FIG. 1.

Construction of a waaC defective mutant in B. bronchiseptica 9.73. Panel A shows the mutagenesis strategy used, based on site-specific recombination of the nonreplicative vector pK18mob. The internal 200-bp DNA fragment of the waaC coding region used in this strategy was obtained by PCR using primers waaC_pf and waaC_pr, as indicated in Materials and Methods. Panel B shows the Southern blot analysis that confirmed the genetic structure of the mutant B. bronchiseptica LP39. Total DNA from B. bronchiseptica 9.73 and B. bronchiseptica LP39 was digested with EcoRI and probed with the 200-bp waaC PCR product labeled with digoxigenin.

FIG. 2.

SDS-PAGE profiles and immunoblot analyses of phenol-water-extracted LPS from wild-type B. bronchiseptica and B. bronchiseptica waaC mutant LP39. (A) Silver-stained SDS-PAGE (8 to 25% [wt/vol]) of LPS extracted from wild-type B. bronchiseptica in virulent phase (lane 1) and from a waaC mutant in virulent culture conditions (lane 2). (B) Immunoblots of the SDS-PAGE gel shown in panel A. The gel blot was exposed to mouse antiserum obtained 10 months after infection with wild-type B. bronchiseptica as the primary antibody. LPS samples corresponded in all cases to material extracted from approximately 1 mg (wet weight) of bacterial cells. KDO, 3-deoxy-d-manno-octulosonic acid.

FIG. 3.

Semiquantitative analysis of virulence determinant expression in wild-type B. bronchiseptica and in the B. bronchiseptica waaC mutant B. bronchiseptica LP39 by immunoblotting. (A) SDS-PAGE (8 to 25% [wt/vol]) of bacterial lysates corresponding to 10⁸ CFU of wild-type B. bronchiseptica in avirulent phase (lane 1), B. bronchiseptica in virulent phase (lane 2), and B. bronchiseptica waaC mutant LP39 in virulent culture conditions (lane 3). Proteins were stained overnight in an aqueous solution of Coomassie brilliant blue R250 (0.2% [wt/vol]). (B to D) Western blots of the SDS-PAGE gel shown in panel A. Antisera against adenylate cyclase (B), filamentous hemagglutinin (C), and pertactin (D) were used.

FIG. 4.

SDS-PAGE (18% [wt/vol]) analysis showing the genetic complementation of the waaC mutant B. bronchiseptica LP39 with the genomic library of the parental strain. Each well contained LPS extracted from the wild-type B. bronchiseptica strain (lane 1), waaC mutant B. bronchiseptica LP39 carrying the pJB3FS recombinant plasmid (lane 2), and waaC mutant B. bronchiseptica LP39 (lane 3). The samples were obtained from around 1 to 2 mg (wet weight) of bacterial cells using EDTA and polymyxin B as described in Materials and Methods. The positions of the main LPS components in the gel are indicated to the left. KDO, 3-deoxy-d-manno-octulosonic acid.

FIG. 5.

In vivo persistence of wild-type B. bronchiseptica 9.73 and B. bronchiseptica waaC mutant LP39 in a murine respiratory model. Lungs were extracted at different times, and the number of viable bacteria per lung was determined. The results represent the means ± standard deviation of three independent experiments.

FIG. 6.

Phase-contrast micrographs of the adherence of B. bronchiseptica strains to human A549 alveolar epithelial cells, used in a standard adherence assay with (A) the parental strain and (B) the waaC mutant B. bronchiseptica LP39. Magnification, 1,000×. Panels are representative of one to three independent experiments.

FIG. 7.

Intracellular survival of B. bronchiseptica wild-type strain and B. bronchiseptica waaC mutant LP39 in the human alveolar epithelial cell line A549. At selected time periods, the number of CFU per alveolar cell was determined. The data represent the means ± standard deviation of three independent experiments.