Figure 1.
Colocalization of the P. aeruginosa exsA mutant with acidified vacuoles in epithelial cells compared to that of wild-type bacteria or a popB (translocon) mutant.
Confocal microscopy images of human corneal epithelial cells at 5 h post-infection with GFP-expressing P. aeruginosa (green). Prior to imaging, infected cultures were infused with LysoTracker (LT) DND-22 (pseudo-colored red). Panels depict (A) Uninfected control, (B) PAO1 exsA mutant (C) PAO1 popB (translocon) mutant and (D) wild-type PAO1. Uninfected cells appeared healthy. The intracellular exsA mutant appeared more frequently in LT (+) (acidified) vacuoles which co-localized yellow (arrows) than either the intracellular popB mutant or wild-type PAO1. PAO1-infected cells which displayed bleb-niche formation (1D inset) showed reduced fluorescence (< 10% fluorescence intensity of PAO1-infected non-blebbing cells, p < 0.001 Welch’s corrected t-Test). Occasional bleb-niches contained LT (+) vacuoles containing bacteria (1D inset, yellow). Representative images are shown. Magnification ~ 600 x.
Figure 2.
Quantification of acidified versus non-acidified vacuole occupation by wild-type P. aeruginosa and its type III secretion mutants.
(A) Confocal microscopy images were used to classify bacteria-occupied vacuoles in human corneal epithelial cells as either LT (+) (acidified) or LT (-) (non-acidic) at 5 h post-infection with P. aeruginosa PAO1 or its type III secretion mutants (exsA or popB). The data are shown as the mean (+/- SEM) number of bacteria-occupied vacuoles per cell. Grey columns denote LT (-) vacuoles, black columns LT (+) vacuoles. The exsA and popB mutants were both associated with increased numbers of acidified LT (+) bacteria-occupied vacuoles per cell compared to wild-type PAO1 (p < 0.001, Welch’s corrected t-Test). The exsA mutant showed more acidified than non-acidified bacteria-occupied vacuoles per cell (p < 0.001, Welch’s corrected t-Test). (B) To normalize differences in internalization and replication, the percentage of LT (+) bacteria-occupied vacuoles was calculated as a function of the total number of bacteria-occupied vacuoles per cell. Mean percentage (+/- SEM) is shown. The exsA mutant was associated with more acidified bacteria-occupied vacuoles per cell than either the popB mutant or wild-type bacteria (p < 0.001, Welch’s corrected t-Test). A representative experiment of 3 independent experiments is shown in both panels (A) and (B). Calculations excluded cells showing bleb-niche formation. Significant differences between all groups were identified using ANOVA analysis (p < 0.0001), and characterized on a pairwise basis using Welch’s correct t-Test [*p < 0.05, **p < 0.001].
Figure 3.
Intracellular survival and replication of P. aeruginosa PAO1 and its type III secretion mutants in corneal epithelial cells in the presence bafilomycin A1 (200 nM) (black boxes) versus control cells treated with vehicle only (grey boxes).
Bafilomycin treatment restored intracellular survival of the exsA mutant to that of the popB mutant and wild-type PAO1. Bafilomycin A1 was added 1 h before infection and continued throughout the assay. Intracellular survival was expressed as the mean percentage increase in viable intracellular bacteria at 8 h versus 4 h post-infection (+/- SEM). A representative experiment of 3 independent experiments in shown above. ANOVA (p = 0.0002) and Welch’s corrected t-test were used for statistical analysis (* p < 0.05).
Figure 4.
Quantification of acidified versus non-acidified vacuole occupation by a triple effector type III secretion mutant of P. aeruginosa complemented with either exoS or exoS without ADPr activity.
(A) Confocal microscopy images were used to classify bacteria-occupied vacuoles as LysoTracker LT (+) (acidified) or LT (-) at 5 h post-infection with a triple effector mutant of P. aeruginosa (PAO1ΔexoSTY) complemented with exoS (pUCPexoS), exoS without ADPr activity (pUCPexoSE381D) or a vector control (pUCP18). Data are shown as the mean (+/- SEM) values of bacteria-occupied vacuoles per cell. Grey columns denote LT (-) vacuoles, black columns denote LT (+) vacuoles. Calculations excluded cells showing bleb-niche formation. Without ExoS ADPr activity (complementation with pUCP18 or pUCPexoSE381D), there were significantly more acidified bacteria-occupied vacuoles per cell (p < 0.05 Welch’s corrected t-Test). (B) The number of LT (+) bacteria-occupied vacuoles per cell was also calculated as a function of the total number of bacteria-occupied vacuoles per cell. Mean percentage (+/- SEM) is shown. Expression of ADPr active exoS was associated with reduced occupation of acidified vacuoles. Calculations also excluded cells showing bleb-niche formation. (C) Mean (+/- SEM) values of intracellular bacteria were determined to account for both the number of bacteria per vacuole and bacteria within blebbing cells in non-vacuolar niches. Complementation of the triple effector mutant PAO1ΔexoSTY with exoS (pUCPexoS) significantly reduced the number of intracellular bacteria per cell within acidified compartments. Grey columns denote LT (-) vacuoles, black columns LT (+) vacuoles. Each panel above is a representative experiment of 3 independent experiments. Significant differences were observed between groups by ANOVA (p < 0.0001). Welch’s corrected t-Test was used in pair-wise comparisons [*p < 0.05, **p < 0.001].
Figure 5.
Colocalization of P. aeruginosa with acidified versus non-acidified vacuoles in relation to exoS transcriptional output.
Confocal and Differential Interference Contrast (DIC) microscopy of human corneal epithelial cells at 5 h post-infection with P. aeruginosa PAO1 complemented with a reporter construct pJNE05 encoding the exoS promoter fused to gfp (green), and p67T1 which constitutively expresses dTomato (red). Bacteria were classified as having a high exoS transcriptional output using a threshold value of 1000 units of GFP fluorescent intensity (green) based on expression levels observed under T3SS-inducing conditions (see Results). Prior to imaging, epithelial cells were infused with LysoTracker DND-22 (blue). ExoS-expressing bacteria (high output, green) [solid arrows] were located primarily outside of acidified (blue) intracellular compartments, which often contained bacteria with low exoS output [dashed arrows]. Blebs are indicated with open arrows. Representative images are shown from two independent experiments. Magnification ~ 600 x.
Figure 6.
Quantification of acidified vacuole occupation by P. aeruginosa in relation to exoS transcriptional output.
Data show the mean (+/- SEM) percentage of bacteria-occupied acidified (LT+) vacuoles at 5 h post-infection for P. aeruginosa PAO1 and a popB (translocon) mutant. Bacteria were transformed with an exoS transcriptional reporter plasmid pJNE05 (exoS-gfp) and plasmid p67T1 (dTomato). Infected cells were also stained with LysoTracker. Bacteria with high exoS expression (grey columns) were significantly less likely to occupy acidified vacuoles than those with a low exoS expression (black columns) (* p < 0.001, Welch’s corrected t-Test). Data is representative of 3 independent experiments.