Table 1.
HBE culture characteristics upon Bp infection.
CBF was quantified from slow-motion capture of 240 frames-per-second. TEER was measured using an epithelial voltohmeter, with 300 Ohms·cm2 as the cut-off for acceptable epithelium integrity. Percent LDH was quantified from basal supernatants. Percent acetylated-α-tubulin was quantified from Fig 4B. Mean values are represented, with standard error of the mean values in parentheses. Data represent at least two independent experiments. Significance was calculated by using one-way ANOVA. *, p < 0.05 compared to uninfected HBE cells; #, p < 0.05 compared to HBE cells infected with WT.
Fig 1.
Bps and FHA promote infection of HBE cells.
(A) CFUs of WT, ΔbpsA-D, and ΔfhaB strains after 1 and 24 h of infection. Mean values of four biological replicates in technical duplicate are presented with standard error of the mean. Statistical differences were assessed by two-way ANOVA. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. (B and C) Bacterial strains were added to empty wells (without Transwells) of a 24-well plate in either SS, DMEM, or apical HBE washes. After 1 h (B) or 24 h (C), CFUs were enumerated on BG agar. Mean and standard error of the mean of three biological replicates in technical duplicate are presented. Statistical differences were assessed by two-way ANOVA. **, p < 0.005; n.s., not significant.
Fig 2.
Bp induces mucin secretion in HBE cells.
HBE cells were infected as described in the ‘Materials and Methods’ at an MOI of 10, for 24 h with different Bp strains. Fixed cells were, sectioned and stained with (A) Hematoxylin & Eosin, or (B) PAS/AB. (C) Semi-quantification of mucin production from PAS/AB-stained images. Images are representative of two biological replicates. Statistical differences were assessed by One-way ANOVA. *, p≤0.05; n.s., not significant.
Fig 3.
Bp forms aggregates and clusters on HBE cells.
(A) Representative live cell images of GFP-labeled bacteria on apical surface of HBE cells after 24 and 48 h (10X objective). (B) Immunofluorescent staining of α-tubulin (cilia) and DAPI (nuclei) on fixed HBE cells infected with GFP-labeled bacteria after 48 h (10X objective). Zoomed in image of HBE cells infected with the WT strain showing colocalization of bacterial aggregates with punctate acetylated-α-tubulin staining (arrow). (C-E) Scanning electron microscopy of Bp strains 24 h after infection of HBE cells. All images are representative of three biological replicates.
Fig 4.
Visualization of Bp biofilms and ECM components on HBE cells.
HBE cells infected with GFP-expressing WT (A-D), ΔbpsA-D (E-H), or ΔfhaB (I-L) strains for 24 h (A-B, E-F, I-J) or 48 h (C-D, G-H, K-L) were fixed and stained for Bps (α-PNAG, red) and eDNA (α-dsDNA, blue), then imaged using an Olympus FV3000 confocal microscope (20X objective). B, D, F, H, J, and L are zoomed in images of A, C, E, G, I, and K, respectively. Z-stacks were acquired at 1-μm intervals. IMARIS software was used to produce a shading picture of the biofilms.
Table 2.
COMSTAT analysis of CLSM images.
Values correspond to Fig 4A–4L, 5C, and 5D. Mean values are represented, with standard error of the mean values in parentheses. Data represent at least two independent experiments. To compare mutants to WT, significance was calculated by using one-way ANOVA and Dunnett’s post hoc: *, p < 0.05; **, p < 0.01, ***p < 0.001; ****, p < 0.0001. To compare 24 h to 48 h, significance was calculated using unpaired Student’s t-test: #, p < 0.05.
Fig 5.
The role of adenylate cyclase toxin in Bp biofilm formation on HBE cells.
(A) Biofilm formation after 96 h was evaluated by GFP-labeled bacteria in microtiter plates by crystal violet method. Bars indicate the mean and standard error of the mean of two independent experiments performed by triplicate. Significant differences were calculated by using one-way ANOVA and Bonferroni post hoc. ***, p < 0.0005. (B-E) HBE cells were apically infected with bacterial strains in DMEM for 1 h at MOI 10. (B) CFUs were enumerated after 24 h on BG agar. Mean and standard error of the mean of two biological replicates in technical duplicate are presented. Statistical differences were assessed by unpaired Student’s t-test. n.s., not significant. (C-D) HBE cells infected with GFP-expressing WT (C) or ΔcyaA (D) strains for 48 h were fixed and imaged using an Olympus FV3000 confocal microscope (20X objective). Z-stacks were acquired at 1-μm intervals. IMARIS software was used to produce a shading picture of the biofilms. (E) Representative live cell images of GFP-labeled bacteria on apical surface of HBE cells after 48 h (10X objective). (F) Scanning electron microscopy of bacterial strains 24 h after infection of HBE cells. For C-F, images are representative of at least two biological replicates.
Table 3.
Strains and Plasmids.