Pertussis toxin and adenylate cyclase toxin provide a one-two punch for establishment of Bordetella pertussis infection of the respiratory tract

Abstract

Previously we found that pertussis toxin (PT), an exotoxin virulence factor produced by Bordetella pertussis, plays an important early role in colonization of the respiratory tract by this pathogen, using a mouse intranasal infection model. In this study, we examined the early role played by another exotoxin produced by this pathogen, adenylate cyclase toxin (ACT). By comparing a wild-type strain to a mutant strain (DeltaCYA) with an in-frame deletion of the cyaA gene encoding ACT, we found that the lack of ACT confers a significant peak (day 7) colonization defect (1 to 2 log(10)). In mixed-infection experiments, the DeltaCYA strain was significantly outcompeted by the wild-type strain, and intranasal administration of purified ACT did not increase colonization by DeltaCYA. These data suggest that ACT benefits the bacterial cells that produce it and, unlike PT, does not act as a soluble factor benefiting the entire infecting bacterial population. Comparison of lower respiratory tract infections over the first 4 days after inoculation revealed that the colonization defect of the PT deletion strain was apparent earlier than that of DeltaCYA, suggesting that PT plays an earlier role than ACT in the establishment of B. pertussis infection. Examination of cells in the bronchoalveolar lavage fluid of infected mice revealed that, unlike PT, ACT does not appear to inhibit neutrophil influx to the respiratory tract early after infection but may combat neutrophil activity once influx has occurred.

FIG. 1.

(A) Groups of four to five BALB/c mice were inoculated with the indicated doses of either WT (darker bars) or ΔCYA (lighter bars), and colonization levels were assessed after 7 days. Results show a significant defect in colonization by ΔCYA at both doses. (B) Comparison of the time courses of respiratory tract colonization by WT (squares) and ΔCYA (open circles) after inoculation of BALB/c mice with 2.5 × 10⁵ CFU of either strain. Each value is the mean ± standard deviation (SD) of results for groups of four to five mice. *, P < 0.05.

FIG. 2.

Groups of four BALB/c mice were inoculated with 5 × 10⁵ CFU of a mixture of WT and ΔCYA at the indicated ratios. (A) Colonization levels from each infection at 7 days postinoculation. Bars indicate means + SDs. (B) Ratios of the two strains in the inoculum (Inoc) or the CFU recovered from individual mice infected with each mixture, expressed as percentages.

FIG. 3.

BALB/c mice (four per group) were inoculated with approximately 3 × 10⁵ CFU of WT (dark bar) or an equivalent dose of ΔCYA, with administration of 200 ng purified ACT or an equivalent volume of buffer on days 0, 1, and 2 postinoculation. Colonization levels at day 4 postinoculation are shown. Bars indicate means + SDs.

FIG. 4.

(A) BALB/c mice were inoculated with approximately 2 × 10⁵ CFU of WT, ΔPT, or ΔCYA, and colonization levels were assessed at the indicated times after inoculation (points indicate means + SDs of values from four mice). (B) BALB/c mice were inoculated with approximately 4 × 10⁵ CFU of WT, ΔPT, ΔCYA, or ΔPT ΔCYA, and colonization levels were assessed at the indicated times after inoculation (points indicate means + SDs of values from four mice). (C) Neutrophil numbers in bronchoalveolar lavage fluid from mice in the experiment shown in panel B. *, P < 0.05.