Filamentous hemagglutinin of Bordetella bronchiseptica is required for efficient establishment of tracheal colonization

Abstract

Adherence to ciliated respiratory epithelial cells is considered a critical early step in Bordetella pathogenesis. For Bordetella pertussis, the etiologic agent of whooping cough, several factors have been shown to mediate adherence to cells and cell lines in vitro. These putative adhesins include filamentous hemagglutinin (FHA), fimbriae, pertactin, and pertussis toxin. Determining the precise roles of each of these factors in vivo, however, has been difficult, due in part to the lack of natural-host animal models for use with B. pertussis. Using the closely related species Bordetella bronchiseptica, and by constructing both deletion mutation and ectopic expression mutants, we have shown that FHA is both necessary and sufficient for mediating adherence to a rat lung epithelial (L2) cell line. Using a rat model of respiratory infection, we have shown that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance action of the mucociliary escalator.

FIG. 1

(A) Schematic representation of the two experimental approaches. RB50 alternates between the Bvg⁺ phase, characterized by the expression of FHA (thick lines), fimbriae (thin lines), pertactin (solid ovals), and AC/HLY (solid circles), and the Bvg⁻ phase, characterized by the expression of flagella (wavy lines). Deletion of the FHA structural gene (ΔfhaB) results in a strain, RBX9, which is identical to RB50 except that it lacks FHA. Deletion of BvgS (ΔbvgS) locks RB50 in the Bvg⁻ phase (RB54), and deletion of the flagellin structural gene (ΔflaA) results in lack of flagella (RB57). Addition of Bvg⁻ phase-specific promoters to fhaB and fhaC (fhaB^rfhaC^r) results in ectopic expression of FHA in a Bvg⁻ phase-locked strain (DF8). (B) Construction of the ΔFHA strain, RBX9. bvgAS, fhaB, fimA, fimB, fimC, fimD, and fhaC are contiguous on the Bordetella chromosome. Oligonucleotides containing either a HindIII (H) or a BamHI (B) restriction endonuclease cleavage site at one end were used to amplify DNA fragments by PCR, which were then joined and cloned into our allelic exchange vector to create plasmid pΔfhaB. Antibiotic resistance genes and the sacB and sacR genes encoded on the plasmid are indicated. Two consecutive homologous recombination events occurring on each side of the deletion junction result in deletion of all but the first four and last five codons of fhaB from the B. bronchiseptica chromosome. (C) Construction of the FHA^r strain, DF8. RB57 contains deletion mutations in bvgS, locking it in the Bvg⁻ phase, and flaA, rendering it unable to synthesize flagella. Replacement of the native fhaB promoter (P_fhaB) with the Bvg⁻ phase-specific flagellin promoter (P_flaA; hatched lines) allows expression of fhaB in RB57. Insertion of the flagellin promoter plus the flagellin ribosome binding site (SD_flaA) allows expression of fhaC. Both fhaB and fhaC are required for expression, processing, and localization of FHA.

FIG. 2

Western immunoblot analysis of whole-cell lysates (WCE) and culture supernatants (SUPE) of wild-type B. bronchiseptica strain RB50 grown under Bvg⁺ and Bvg⁻ phase conditions and RBX9, the ΔFHA strain, grown under Bvg⁺ phase conditions. RB57, the ΔbvgS ΔflaA strain, and its derivatives MR8, which contains a flagellin promoter upstream of fhaB, and DF8, which also contains a flagellin promoter upstream of fhaC, are locked in the Bvg⁻ phase. Polypeptides contained in lysates and supernatants were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with either polyclonal anti-FHA antibody (anti-FHA) or monoclonal anti-Fim3 antibody (anti-Fim). Sizes are indicated in kilodaltons on the right.

FIG. 3

Adherence of B. bronchiseptica strains to rat lung epithelial (L2) cells used in a standard adherence assay with RB50 (a), RBX9 (b), RB57 (c), or DF8 (d).

FIG. 4

Colonization of the rat respiratory tract by RB50, RBX9, and DF8. Groups of five or six 3-week-old female Wistar rats were inoculated with approximately 500 CFU of RB50 or RBX9 or with 10⁶ CFU of DF8. Animals were sacrificed on day 10, 26, or 60 postinoculation, and the number of CFU colonizing various sites in the respiratory tract was determined. Each symbol represents log CFU per nasal septum, larynx or centimeter of trachea recovered from a single animal, and short horizontal lines represent the mean for each group. Open circles, RB50; solid circles, RBX9; gray circles, DF8. Statistical significance is designated with asterisks (one asterisk = P < 0.05; two asterisks = P < 0.001; three asterisks = P < 0.0001. Dashed lines represent the lower limit of detection.

FIG. 5

Serum anti-Bordetella antibody titers. Open bars represent antibody titers in sera as determined by ELISA with RB50 used as the antigen; solid bars represent titers determined with RBX9 used as the antigen. Graphs show titers in sera from RB50- and RBX9-infected animals, collected at day 26 or 60 postinoculation, as indicated.

FIG. 6

Colonization of the trachea following intranasal inoculation of anesthetized or unanesthetized rats or intratracheal inoculation of anesthetized rats. Groups of five 3-week-old female Wistar rats were inoculated by delivering 10⁶ CFU of RB50, RBX9, or RB54 in a 50-μl volume to the external nares of unanesthetized (A) or anesthetized (B) rats or by injecting 10⁶ CFU of RB50, RBX9, or RB54 in a 20-μl volume into the trachea of anesthetized rats (C). Animals were sacrificed at 24 h or 5 days postinoculation, as indicated. Each symbol represents the number of CFU per centimeter of trachea from a single animal, and horizontal bars show the mean for each group. Open circles, RB50; solid circles, RBX9; gray circles, RB54. The horizontal dashed lines represent the lower limit of detection. Statistical significance is designated with asterisks (one asterisk = P < 0.05; three asterisks = P < 0.0001.