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 ARF001^vec and ARF001^bpsA-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 ARF001^vec or ARF001^bpsA-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 ARF001^vec or ARF001^bpsA-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.