Pertussis toxin plays an early role in respiratory tract colonization by Bordetella pertussis

Abstract

In this study, we sought to determine whether pertussis toxin (PT), an exotoxin virulence factor produced exclusively by Bordetella pertussis, is important for colonization of the respiratory tract by this pathogen by using a mouse intranasal infection model. By comparing a wild-type Tohama I strain to a mutant strain with an in-frame deletion of the ptx genes encoding PT (deltaPT), we found that the lack of PT confers a significant peak (day 7) colonization defect (1 to 2 log(10) units) over a range of bacterial inoculum doses and that this defect was apparent within 1 to 2 days postinoculation. In mixed-strain infection experiments, the deltaPT strain showed no competitive disadvantage versus the wild-type strain and colonized at higher levels than in the single-strain infection experiments. To test the hypothesis that soluble PT produced by the wild-type strain in mixed infections enhanced respiratory tract colonization by deltaPT, we coadministered purified PT with the deltaPT inoculum and found that colonization was increased to wild-type levels. This effect was not observed when PT was coadministered via a systemic route. Intranasal administration of purified PT up to 14 days prior to inoculation with deltaPT significantly increased bacterial colonization, but PT administration 1 day after bacterial inoculation did not enhance colonization versus a phosphate-buffered saline control. Analysis of bronchoalveolar lavage fluid samples from mice infected with either wild-type or deltaPT strains at early times after infection revealed that neutrophil influx to the lungs 48 h postinfection was significantly greater in response to deltaPT infection, implicating neutrophil chemotaxis as a possible target of PT activity promoting B. pertussis colonization of the respiratory tract.

FIG. 1.

(A) The ptx-ptl gene region. The intact ptx genes in B. pertussis strain WT encode the subunits (S1 to S5) of PT, and the downstream ptl genes are in the same operon (the promoter is indicated by the arrow upstream of the ptx genes). The B. pertussis ΔPT strain was constructed by introducing an in-frame deletion of the ptx genes (indicated by the Δ symbol and broken line) into the chromosome of B. pertussis strain Tohama I. (B) Western blot showing PtlF expression in strains WT, ΔPT, and ΔBVG.

FIG. 2.

(A) Groups of four or five BALB/c mice were inoculated with the indicated doses of either B. pertussis WT (squares) or ΔPT (triangles), and colonization levels were assessed after 7 days (values from individual mice are shown, with the bar indicating the mean). Results show a significant defect (P values by the t test are shown) in colonization by ΔPT at all doses. (B) Comparison of the time course of respiratory tract colonization by WT or ΔPT after inoculation of BALB/c mice with 5 × 10⁵ CFU of either strain. Each value is the mean ± standard deviation (error bar) for a group of four or five mice. Values that are significantly different from the values for the WT strain are indicated as follows: **, P < 0.05; *, P < 0.07. (C) Comparison of the time course of respiratory tract colonization by WT or ΔPT after inoculation of C57BL6 mice with 5 × 10⁵ CFU of either strain. Each value is the mean ± standard deviation of a group of four or five mice. Values that are significantly different (P < 0.05) from the values for the WT strain are indicated (**).

FIG. 3.

Groups of four BALB/c mice were inoculated with 5 × 10⁵ CFU of a mixture of B. pertussis strains WT and ΔPT at the indicated ratios. (A) Colonization levels from each infection at days 2, 8, and 15 postinoculation. The means ± standard deviations (error bars) are shown. (B) Ratios of the two strains in the inoculum (Inoc) or the CFU recovered from the infections at days 2, 8, and 15 expressed as a percentage. Ratios were determined by colony hybridization.

FIG. 4.

(A) BALB/c mice (three or four per group) were inoculated with approximately 5 × 10⁵ CFU of B. pertussis strain ΔPT coadministered with 200 ng of purified PT or PT-9K/129G (PT*) or PBS, and colonization levels at day 4 postinoculation are shown. The means ± standard deviations (error bars) are shown in all four panels. Values that are significantly different (P < 0.05) from the values for the control (PBS) or PT* strain are indicated by asterisks. (B) BALB/c mice (three or four per group) were inoculated with approximately 5 × 10⁵ CFU of ΔPT with coadministration of the indicated amount of purified PT, and colonization levels at day 4 postinoculation (results from two different experiments) are shown. Values that are significantly different (P < 0.05) from the values when no PT was added to the inoculum are indicated by asterisks. (C) BALB/c mice (three per group) were inoculated with approximately 5 × 10⁶ CFU of B. pertussis strain ΔBVG with coadministration of the indicated amount of purified PT, and colonization levels at day 4 postinoculation are shown. (D) BALB/c mice (four per group) were inoculated intranasally with approximately 5 × 10⁵ CFU of ΔPT, with either intranasal (i.n.), intramuscular (i.m.), intraperitoneal (i.p.), or subcutaneous (s.c.) coadministration of 2 ng of PT (or intranasal coadministration of PBS), and colonization levels at day 4 postinoculation are shown. The value that is significantly different (P < 0.05) from the value when no PT was added to the inoculum is indicated by an asterisk.

FIG. 5.

BALB/c mice (three or four per group) were inoculated with approximately 5 × 10⁵ CFU of B. pertussis strain ΔPT. Mice were given either 2 ng of PT or PBS intranasally at the indicated time relative to bacterial inoculation (from 28 days before [−28] to 1 day after), and colonization levels at day 4 postinoculation are shown. Means ± standard deviations (error bars) are shown. Values that are significantly different (P < 0.05) from the values for the control (PBS given intranasally [i.n.]) are indicated (**).

FIG. 6.

BALB/c mice were inoculated with approximately 5 × 10⁵ CFU of B. pertussis Tohama I or ΔPT (or PBS), and at the indicated times after inoculation, groups of mice were sacrificed and assayed. (A) Colonization levels of B. pertussis strains. Means ± standard deviations (error bars) of the values for four mice are shown. (B) Neutrophil numbers in BAL fluid samples (expressed as a percentage of total white blood cells). Means ± standard deviations (error bars) for three mice are shown. Values that are significantly different from the values for Tohama I strain are indicated as follows: #, P = 0.08; *, P < 0.05.

FIG. 7.

SCID mice (BALB/c background) were inoculated with approximately 5 × 10⁵ CFU of B. pertussis strain WT or ΔPT, and colonization levels were assessed at days 2, 7, and 14 postinoculation. Means ± standard deviations (error bars) of the values for four mice are shown. Values that are significantly different from the values for the WT strain are indicated as follows: *, P < 0.05; **, P < 0.001.