Natural-host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C-terminal domain, but not the RGD motif, during infection

Abstract

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human-adapted (e.g. Bordetella pertussis) strains produce a surface-exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg-Gly-Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural-host animal models should apply to B. pertussis FHA as well. We also show that the C-terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage-like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.

Fig. 1. Expression of fhaB_Bb in B. pertussis

A. Schematic of strains used. The B. pertussis fhaB locus is shown in red and the B. bronchiseptica fhaB locus is shown in blue. The regions of integration of pSJ63 and pSJ61 into the chromosomes of Bpe138 and RBX20 to form Bpe138::pSJ63 and RBX20::pSJ61, respectively, are indicated by the black crosses. B. Western blot showing FhaB (~370 kDa) and FHA (~250 kDa) in whole cell lysates (WCLs) and concentrated supernatants (Supe) of the various B. pertussis and B. bronchiseptica strains as indicated below each lane. Blots were probed with the anti-CRD antibody. The position of the 250 kDa molecular mass marker is shown. C. Adherence of wild-type and mutant B. pertussis strains to L2 cells (moi = 200). Asterisks indicate a statistically significant (P < 0.05).

Fig. 2. Expression of fhaB_Bb in B. pertussis does not alter its ability to infect mice

A. The number of cfu recovered from the lungs of BALB/c mice at days 0, 3 or 4, and 11 post inoculation with 50 μl PBS containing 5 × 10⁵ cfu of the indicated strains is shown. Each circle represents the number of cfu recovered from a single animal. The horizontal black bar is the geometric mean for each group. For RBX20 at day 3/4, the means of numbers from moribund animals (~10⁹) and healthy animals (~10⁵) were calculated separately as shown. The dashed line represents the lower limit of detection. Asterisks indicate a statistically significant difference compared with RB50 (P < 0.05). This experiment has been performed at least twice with similar results. The data from one experiment are shown. B. Western blots of whole cell lysates of the strains indicated across the bottom probed with serum from mice inoculated with PBS or the strains indicated across the top. The sera were collected 21 days post inoculation. Molecular mass markers are shown on the right.

Fig. 3. A. Comparison of the FhaB proteins predicted for B. bronchiseptica RB50 (FhaB_Bb, blue) and B. pertussis Tohama 1 (FhaB_Bp, red). Vertical black lines represent the positions of amino acid differences. FhaB_Bb contains six additional 19 aa repeats near the N-terminus (white space in FhaB_Bp). The vertical white dashed line represents the site of SphB1-dependent maturation. The various domains are designated across the bottom. A schematic of mature FHA is shown at the top in purple. The regions used for the production of the anti-CRD and anti-MCD antibodies are indicated

B. Schematic of the various ‘chimeric’ strains constructed and their phenotypes in adherence to L2 cells, tracheal colonization in Wistar rats, and lung inflammation in the lungs of BALB/c mice. Dark blue represents B. bronchiseptica RB50 DNA, red represents B. pertussis Tohama 1 DNA. The yellow ‘star’ in RBFS4 represents the location of the insertion mutation. ND, not determined. The tabular part of part B summarizes many animal experiments. Those involving strains RB50, RBX9, RBX11-T-E, RBFS4 and RBFS10 have been performed many times. Rat and mouse experiments using RB50gap, RB50CRD_Bp and RB50NMCD_Bp were performed once.

Fig. 4. Assessment of the FHA RGD triplet in B. bronchiseptica pathogenesis

A. Wistar rats were inoculated intranasally with 10 μl PBS containing 1000 cfu of B. bronchiseptica (strains indicated across the bottom) and the number of cfu recovered from the nasal cavity (NC) and 1 cm trachea determined at days 14 and 28 post inoculation. Each circle represents the number of cfu recovered from a single animal. The horizontal line shows the geometric mean for each group. The dashed line represents the lower limit of detection. y-axis is log scale. Asterisks indicate a statistically significant difference compared with RB50 (P < 0.05). B. BALB/c mice were inoculated intranasally with 50 μl PBS containing 5 × 10⁵ cfu of B. bronchiseptica (strains indicated across the bottom) and the number of cfu in the right lung lobes was determined 60 min (day 0), 3 and 11 days post inoculation. Each circle represents the number of cfu recovered from a single animal. The horizontal line shows the geometric mean for each group. The dashed line represents the lower limit of detection. For RBX9, the means at day 3 for moribund (~10⁹) and healthy (~10⁶) animals were calculated separately as indicated. y-axis is log scale. Asterisks indicate a statistically significant difference compared with RB50 (P < 0.05). C and D. SCID/Bg mice were inoculated intranasally with 50 μl PBS containing 5 × 10⁵ (for C) or 10 μl containing 1000 (for D) cfu of RB50 (solid line) or RB50RAE (dashed line) and the number of survivors plotted over time. The mean time to death was not significantly different between RB50 and RB50RAE for either experiment, as determined by the Log Rank (Mantel-Cox) test. RB50 and RBX9 have been compared in these models many times with similar results. Each type of animal experiment that included the RB50RAE strain was performed once and not repeated in the interest of minimizing the number of animals used because no difference between RB50RAE and RB50 was detected in any of the experiments.

Fig. 5

ICAM-1 induction. BEAS-2B cells were incubated with the indicated strains of B. bronchiseptica or B. pertussis at a moi of 100 and the amount of surface ICAM-1 was determined by flow cytometry. (Bp536 = wild-type B. pertussis, Bpe138 = B. pertussis ΔfhaB, Bpe129 = B. pertussis RAD mutant, RB50 = wild-type B. bronchiseptica, RBX9 = RB50ΔfhaB, RB50RAE = RB50 RAE mutant, RBX11 = RB50ΔfhaS, RBX11Δ28 = RB50 containing the FHA ‘Δ28’ mutation.) Fluorescence of cells incubated with PBS alone was set as a value of one and relative levels of fluorescence in samples incubated with bacteria are shown. Asterisks indicate a statistically significant difference compared with the wild-type parental strain (P < 0.05).

Fig. 6. Antibodies to the MCD, but not the CRD, block adherence to L2 and J774A.1 cells

A. B. pertussis and B. bronchiseptica strains (indicated) were incubated with anti-MCD (αM) or anti-CRD (αC) antibodies then tested for adherence to L2 or J774A.1 cells. The number of bacteria per cell is shown. Asterisks indicate statistically significant differences in adherence compared with wild-type bacteria that were not incubated with antibody. *P < 0.05 and **P < 0.01. B. Aliquots of the bacteria used in the adherence assays were incubated with goat anti-chicken antibody conjugated to IRdye 680 (for anti-CRD) or goat anti-rabbit conjugated to IRdye 800 (for anti-MCD), washed, then spotted into nitrocellulose and fluorescence was visualized using an Odyssey imager.

Fig. 7. The MCD is required for FHA function in vivo

A. Western blot showing FHA (~250 kDa) in whole cell lysates (WCLs) and concentrated supernatants (Supe) of the indicated B. bronchiseptica strains. Blots were probed with the anti-CRD antibody. B. Adherence of RB50 and RBX11Δ28 to L2 cells is shown. Moi = 200. The difference between the values was not statistically significant. C. Wistar rats were inoculated intranasally with 10 μl PBS containing 1000 cfu of B. bronchiseptica (strains indicated across the bottom) and the number of cfu recovered from the nasal cavity and 1 cm trachea determined at days 14 and 28 post inoculation. Each circle represents the number of cfu recovered from a single animal. The horizontal line shows the geometric mean for each group. The dashed line represents the lower limit of detection. y-axis is log scale. *P < 0.05. D. BALB/c mice were inoculated intranasally with 50 μl PBS containing 5 × 10⁵ cfu of B. bronchiseptica (strains indicated across the bottom) and the number of cfu in the right lung lobes was determined 60 min (day 0), 3 and 11 days post inoculation. Each circle represents the number of cfu recovered from a single animal. The horizontal line shows the geometric mean for each group. The dashed line represents the lower limit of detection. For RBX9 and RBX11Δ28, the means at day 3 for moribund (~10⁸) and healthy (~10^4–5) animals were calculated separately as indicated. y-axis is log scale. *P < 0.05. The rat and mouse experiments using RBX11Δ28 were performed at least twice with similar results and the data from one of each type of experiment are shown.