Fig 1.
Bps promotes resistance to antimicrobial peptides.
Survival of WT, ΔbpsA-D, ΔbpsA-Dvec, and ΔbpsA-Dcomp in the presence of .5 μg/ml polymyxin B (a), .5 μg/ml LL-37 (b), 50 μg/ml HNP-1 (c), and 50 μg/ml HNP-2 (d). Bacteria were exposed to the indicated concentrations of AMPs in 10mM Na3PO4 for 2 hours. Each data point represents the mean and s.e.m. of triplicates from one experiment and is representative of at least three independent experiments. Statistical differences were assessed by unpaired two-tailed Student’s t test. *, p<0.05; **, p<0.005; ***, p<0.0005. n.s. not significant.
Fig 2.
Dispersin B hydrolyzes Bps and increases the susceptibility of B. pertussis to PmB and LL-37.
(a) Bps was quantified by ELISA from the WT or ΔbpsA-D strains following incubation with indicated concentrations of Dispersin B. Each data point represents the mean and s.e.m. of triplicates from one experiment and is representative of two independent experiments. Statistical differences were assessed by two-way ANOVA. *, p<0.05; ***, p<0.0005. (b, c) Survival of WT and ΔbpsA-D strains following treatment with 50 μg/ml Dispersin B or buffer in the presence of .5 μg/ml polymyxin B (b) or .5 μg/ml LL-37 (c). Each data point represents the mean and s.e.m. of triplicates from one experiment and is representative of two independent experiments. Statistical differences were assessed by one-way ANOVA. ***, p<0.0005.
Fig 3.
The presence of Bps on the cell surface inhibits LL-37 binding.
(a, b) Bacteria were fixed and exposed to FITC-labeled LL-37 followed by measurement of the fluorescence intensity of FITC-labeled LL-37 bound to bacteria by flow cytometry. (a) Bacterial counts for each strain were normalized to mode. (b) Median fluorescence intensities were quantified and assessed for statistical analysis. Data represent triplicates from one of two independent experiments. Statistical differences were assessed by one-way ANOVA. **, p<0.005.
Fig 4.
B. pertussis releases Bps during laboratory growth, and cell-free Bps provides protection from and binds to PmB and LL-37.
(a) Quantitation of cell-associated and released (supernatant) Bps from the WT and ΔbpsA-D strains by ELISA. Each data point represents the mean and s.e.m. of eight wells from one experiment and is representative of two independent experiments. Statistical differences were assessed by two-way ANOVA. ***, p<0.0005. (b, c) Survival of WT and ΔbpsA-D strains in the presence of PmB (b) or LL-37 (c). Supernatants from WT and the ΔbpsA-D cultures (b) or purified Bps and mock preparation (c) were added as indicated. Each data point represents the mean and s.e.m. of triplicates from one experiment and is representative of two independent experiments. Statistical differences were assessed by one-way ANOVA. ***, p<0.0005. (d, e) Bps binds PmB and LL-37. (d) Bps or mock preparations were spotted on nitrocellulose membranes. To detect PmB binding, the membranes were incubated with PmB, washed and probed with α-polymyxin B antibody conjugated to HRP. (e) Binding of Bps or mock preparations to LL-37 was quantified by ELISA using WGA conjugated to HRP. Asterisks indicate significance compared to mock prep. Each data point represents the mean and s.e.m. from one experiment and is representative of two independent experiments. Statistical differences between Bps and mock preps were assessed by unpaired two-tailed Student’s t test. ***, p<0.0005.
Fig 5.
The presence of the WT strain increases ΔbpsA-D resistance to LL-37 in vitro and enhances its survival in the mouse respiratory tract and Bps is released in mouse lungs during infection.
(a) Survival of WT and ΔbpsA-D strains either in monoculture or in a 1:1 co-culture of both strains against LL-37. Each data point represents the mean and s.e.m. of triplicates from one of five independent experiments. Statistical differences were assessed by two-way ANOVA. **, p<0.005; ***, p<0.0005. n.s. not significant. (b) Bacterial CFUs recovered from the nasal septum and lungs of C57BL/6J mice four days after aerosol infection with WT or ΔbpsA-D in monoculture or in a 1:1 co-culture. Bars indicate the mean and s.e.m. of ten mice. Data from two independent experiments with groups of five mice each are shown. Statistical differences were assessed by two-way ANOVA. *, p<0.05; **, p<0.005; ***, p<0.0005; n.s., not significant. Dotted line represents the lower limit of detection for nasal septum, and dashed line represents the lower limit of detection for lungs. (c) Amounts of Bps in supernatants of lung lysates obtained from mice either instilled with PBS (blank circles) or infected with the indicated strains was quantified by ELISA using WGA conjugated to HRP. For infected mice, bars indicate the mean and s.e.m. of two independent experiments consisting of groups of five mice each (from Fig 5B). For PBS-instilled mice, bars indicate the mean and s.e.m. of one experiment consisting of four mice. Statistical differences were assessed by one-way ANOVA. **, p<0.005; ***, p<0.0005.
Fig 6.
Bps enhances PmB and LL-37 resistance, inhibits AMP binding, and promotes respiratory tract survival when produced in E. coli.
(a, b) Survival of ARF001vec and ARF001bpsA-D strains in the presence of .5 μg/ml polymyxin B (a) or .5 μg/ml of LL-37 (b). Bacteria were exposed to the indicated concentrations of AMPs for 2 hours in 10mM Na3PO4 buffer. Each data point represents the mean and s.e.m. of triplicates from one of two independent experiments. Statistical differences were assessed by unpaired two-tailed Student’s t test. *, p<0.05; ***, p<0.0005. (c, d) Binding of FITC LL-37 to the ARF001vec or ARF001bpsA-D strains was measured by flow cytometry. Bacteria were fixed and exposed to FITC-labeled LL-37 and then the fluorescence intensity of FITC-labeled LL-37 bound to bacteria was measured by flow cytometry. (c) Bacterial counts for each strain were normalized to mode. (d) Median fluorescence intensities were quantified and assessed for statistical analysis. Data represent triplicates from one of two independent experiments. Statistical differences were assessed by one-way ANOVA. ***, p<0.0005. (e, f) Bacterial CFUs recovered from the nasal septum and lungs three days after intranasal challenge (e) or one day after aerosol challenge (f) with either the ARF001vec or ARF001bpsA-D strains. Bars indicate the mean and s.e.m. of groups of five mice each. Data are representative of one of two independent experiments with five mice each. Statistical differences were determined by unpaired two-tailed Student’s t test for each organ. *, p<0.05; **, p<0.005. Dotted line represents the lower limit of detection for nasal septum, and dashed line represents the lower limit of detection for lungs.
Fig 7.
Bps converts E. coli to a respiratory pathogen in C57BL/6 mice.
Pulmonary infection with E. coli expressing bpsA-D results in severe pneumonia (e-h) compared to E. coli expressing the empty vector (a-d). ARF001bpsA-D-infected mice: Neutrophils (filled arrows) admixed with edema (open arrows) at lesion periphery (f) and large numbers of neutrophils (filled arrows), macrophages (open arrows) with bronchiolar epithelial hyperplasia, dysplasia and necrosis (circle) at lesion center (g, h). ARF001vec-infected mice: few mixed leukocytes (arrows) at lesion periphery (b) and multifocal, small numbers of neutrophils (closed arrow) and moderate numbers of macrophages (open arrow) around bronchioles (c, d). Scale bar in a, e = 500 μm, 20x total magnification; Scale bar in b, c, f, g = 100 μm, 100x total magnification; Scale bar in d, h = 50 μm, 200x total magnification.