Fig. 6: Influx of Ca²⁺ promoted release of vacuolar bacteria.

A Image analysis of timelapse images measuring vacuole numbers for P. aeruginosa PAO1 wild type (WT) in corneal epithelial cells (hTCEpi) at MOI 100 when treated with different compounds. B Area Under the Curve bar plots for the percent infected cells of wild-type PAO1 in human corneal epithelial cells (hTCEpi) at MOI 100 when treated with different compounds. C Growth of PAO1 wild type measured at OD₆₀₀ in the presence of different compounds. D Representative widefield microscopy images of human corneal epithelial cells (hTCEpi) infected with different T3SS mutant strains of PAO1 (green) at MOI 100 and increases in Ca²⁺ levels are detected using Fluo-5F AM (magenta). Images were taken 5 h post-infection at ×40 magnification. P. aeruginosa (green), Hoechst (gray), Fluo-5F AM (magenta). Scale bar equals 50 µm. E Bar plot of ratio of hTCEpi cells with positive Ca²⁺ signal (Fluo-5F AM) over number of infected cells of the respective strain 5 hours post infection. F Ratio of hTCEpi cells with positive Ca²⁺ signal (Fluo-5F AM) 5 hours post-infection over number of infected cells of the respective strain when treated with 10 µM Nifedipine for 20 h prior to infection, compared to DMSO (vehicle control). G Number of vacuoles per field of view of the respective strains 5 hours post infection in hTCEpi cells when treated with 10 µM Nifedipine for 20 h prior to infection, compared to DMSO (vehicle control). H Growth of PAO1 ∆exsE and ∆exsE∆exoS/T/Y measured at OD₆₀₀ in the presence of 10 µM Nifedipine or vehicle control (DMSO). Data of biological replicates represented as mean ± SEM (A) or SD (B, C, E–H), N = 4 (A, B, E) N = 3 (C, F–H). For statistical analysis, a Two-way ANOVA with multiple comparisons (A, C, F–H) or One-way ANOVA with multiple comparisons (B, E) was performed. Exact P values—A: No treatment vs. Calcimycin = 0.0179, DMSO vs. Caclimycin = 0.0473; E: WT vs. ∆exsE = >0.0001, WT vs. ∆exsE∆exoSTY = 0.0075, ∆exsE vs. ∆exsE∆popBD = >0.0001, ∆exsE vs. ∆exsE∆pscC = >0.0001, ∆exsE vs. ∆exsE∆exoSTY = 0.0001, ∆exsE∆popBD vs. ∆exsE∆exoSTY = 0.0029, ∆exsE∆pscC vs. ∆exsE∆exoSTY = 0.0072; F = 0.0042; H = 0.0391. P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = ***, P ≤ 0.0001 = ****. Source data are provided as a Source Data file.