Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response

Abstract

Fimbriae are filamentous, cell surface structures which have been proposed to mediate attachment of Bordetella species to respiratory epithelium. Bordetella bronchiseptica has four known fimbrial genes: fim2, fim3, fimX, and fimA. While these genes are unlinked on the chromosome, their protein products are assembled and secreted by a single apparatus encoded by the fimBCD locus. The fimBCD locus is embedded within the fha operon, whose genes encode another putative adhesin, filamentous hemagglutinin (FHA). We have constructed a Fim(-) B. bronchiseptica strain, RB63, by introducing an in-frame deletion extending from fimB through fimD. Western blot analysis showed that RB63 is unable to synthesize fimbriae but is unaffected for FHA expression. Using this mutant, we assessed the role of fimbriae in pathogenesis in vitro and in vivo in natural animal hosts. Although RB63 was not significantly defective in its ability to adhere to various tissue culture cell lines, including human laryngeal HEp-2 cells, it was considerably altered in its ability to cause respiratory tract infections in rats. The number of DeltafimBCD bacteria recovered from the rat trachea at 10 days postinoculation was significantly decreased compared to that of wild-type B. bronchiseptica and was below the limit of detection at 30 and 60 days postinoculation. The number of bacteria recovered from the nasal cavity and larynx was not significantly different between RB63 and the wild-type strain at any time point. The ability of fimbriae to mediate initial attachment to tracheal tissue was tested in an intratracheal inoculation assay. Significantly fewer RB63 than wild-type bacteria were recovered from the tracheas at 24 h after intratracheal inoculation. These results demonstrate that fimbriae are involved in enhancing the ability of B. bronchiseptica to establish tracheal colonization and are essential for persistent colonization at this site. Interestingly, anti-Bordetella serum immunoglobulin M (IgM) levels were significantly lower in animals infected with RB63 than in animals infected with wild-type B. bronchiseptica at 10 days postinoculation. Even at 30 days postinoculation, RB63-infected animals had lower serum anti-Bordetella antibody titers in general. This disparity in antibody profiles suggests that fimbriae are also important for the induction of a humoral immune response.

FIG. 1

(A) Fragments of B. pertussis DNA homologous between B. pertussis and B. bronchiseptica were used to integrate a plasmid into the B. bronchiseptica genome. Flanking regions of B. bronchiseptica DNA were then isolated while the plasmid was excised from the genome. Thick-lined boxes represent the B. pertussis DNA and show the organization of the fim locus; thin-lined boxes represent B. bronchiseptica DNA. Two KpnI-PstI B. pertussis fragments were used to isolate B. bronchiseptica DNA cloned as plasmids pCMA4 and pCMA6. Restriction analysis of B. bronchiseptica DNA revealed differences between B. pertussis and B. bronchiseptica DNA as is represented by the absence of certain restriction sites in B. bronchiseptica. The asterisk represents extra DNA in B. bronchiseptica that does not correspond to B. pertussis sequences in this region. There is evidence that fimA in B. bronchiseptica may be a complete and functional gene (7). A, AlwNI; As, AspI; K, KpnI; N, NspI; P, PstI; S, SmaI; X, XmaI. (B) SalI-AspI and PstI-PstI fragments from B. bronchiseptica were ligated in frame to create the ΔfimBCD mutation. pCMA10 represents the allelic exchange plasmid used to introduce the deletion into the B. bronchiseptica chromosome. RB63 represents the genetic organization of the ΔfimBCD strain. (C) The minimal open reading frame of the fimbrial biogenesis operon (fimBCD) containing the intact fimB promoter was used to complement the Fim⁻ mutant, RB63. pCMA11 represents the complementation plasmid.

FIG. 2

Western immunoblot analysis of whole-cell lysates of RB50 (+ phase), RB50 (− phase), RB53, RB54, RB63, RB63(pBBR1MCS), and complementation strain RB63(pCMA11). Approximately 25 OD₆₀₀ units of lysates were loaded per lane and probed with a 1:4,000 dilution of anti-Fim3 antibody. The cluster of bands running at approximately 21 to 24 kDa represents fimbriae. The position of the 32-kDa molecular weight marker is shown on the left.

FIG. 3

Western immunoblot analysis of whole-cell lysates of RB50 (+ phase), RB50 (− phase), RB53, RB54, RB63, and RBX9 probed with anti-FHA antibody. The cluster of bands running at approximately 220 kDa represents FHA. Positions of molecular weight markers (220 and 195 kDa) are shown on the right.

FIG. 4

In vitro analysis of adhesive functions of different strains of B. bronchiseptica. HEp-2 cells infected with RB50, RB63, RB54, or RBX9 at an MOI of 10, 20, 100, 200, 400, or 500 were washed, and the bacteria adhering to 30 representative epithelial cells were counted and averaged after staining with Giemsa stain. The asterisk represents significantly different attachment levels between RB50 and RB63 with a P value of ≤0.0002. Error bars represent the standard error from the mean. Bordetella strains used for this assay were the wild-type strain RB50 (open squares), Fim⁻ strain RB63 (open diamonds), FHA⁻ strain RBX9 (open triangles), and constitutively Bvg⁻ strain RB54 (open circle).

FIG. 5

Histograms showing mean colonization levels in the noses, larynxes, and tracheas of 4-week-old, female Wistar rats inoculated intranasally with B. bronchiseptica. Rats were inoculated with the wild-type strain RB50 (hatched bars) and the ΔfimBCD strain RB63 (solid bars) and sacrificed after 10, 30, and 60 days. The lower limit of detection was four bacteria and is represented by the dashed line. Significantly different colonization levels are designated by ∗ for P values of ≤0.005. Error bars represent the standard error from the mean (SEM).

FIG. 6

Histograms showing mean colonization levels in the tracheas of 4-week-old, female Wistar rats inoculated intranasally with 10⁶ CFU (A) and intratracheally with 10⁵ CFU (B) of B. bronchiseptica. Rats were inoculated with wild-type strain RB50 (hatched bars), the Bvg⁻ phase-locked strain RB54 (open bars), and the ΔfimBCD strain RB63 (solid bars) and sacrificed after 24 h and 5 days. The lower limit of detection was four bacteria and is represented by the dashed line; ∗ indicates that colonization levels for RB63 were significantly lower than those of RB50 with a P value of ≤0.05. Error bars represent the standard error from the mean (SEM).

FIG. 7

Anti-Bordetella antibody titers in sera collected from RB50- and RB63-infected animals were measured by ELISA using RB50 (A) or RB63 (B) whole cells as the antigen. Specifically, total serum antibody, IgM, and IgG titers were determined. Sera collected from Lewis rats sacrificed at 10 and 30 days postinoculation were tested. Hatched bars represent RB50-infected animals; solid bars represent RB63-infected animals; ∗ indicates that antibody titers for RB63-infected animals were significantly lower than those for RB50-infected animals, with a P value of ≤0.02. Error bars represent 1 standard error from the mean. The lower limit of detection of antibody titer is 10 U. Mock (PBS)-infected animals had antibody titers below the level of detection.

FIG. 8

Anti-Bordetella IgG2b titers in sera collected from RB50-, RB63-, and mock-infected Lewis rats were measured by ELISA using RB50 whole cells as antigen. Sera collected from animals sacrificed at 30 days postinoculation were tested. Open bars represent PBS-treated (mock-infected) animals; hatched bars represent RB50-infected animals; solid bars represent RB63-infected animals; ∗ indicates that antibody titers for RB63-infected animals were significantly lower than those for RB50-infected animals, with a P value of ≤0.05. The dashed line represents the lower limit of detection, i.e., 10 U. Error bars indicate standard error from the mean (SEM).