Effect of EDE on <i>P.</i><i>aeruginosa</i> corneal colonization in SP-D knockout mice.

<p>Corneal colonization by <i>P. aeruginosa</i> in normal Black Swiss mice (A) or SP-D deficient age/sex-matched Black Swiss mice (B) under normal (NC) and experimental dry eye (EDE) conditions. After 5 days EDE induction, otherwise uninjured corneas were challenged with 10⁹ cfu of <i>P. aeruginosa</i> strain PAO1 (T = 0). EDE did not affect bacterial colonization in wild-type mice after 6 h. However, EDE in SP-D knockout mice (<i>sp-d</i> −/−) resulted in a ∼5-fold increase in corneal colonization after 6 h. Data shown is representative of two independent experiments with SP-D-deficient Black Swiss mice (n≥5 animals per group). P values were obtained using the Mann-Whitney Test. Data for each sample are shown as the median (black square) with upper and lower quartiles (boxed area), and range of the data (error bars).</p

Induction of experimental dry eye.

<p>Tear volumes (A) and fluorescein staining (B) in the eyes of C57BL/6 mice under experimental dry eye (EDE) conditions versus normal controls (NC). (A) EDE resulted in significant decreases in tear volume after 2 days. Tears were collected from the lateral canthus using cotton thread and reported as millimeters of wetted thread. Data are expressed as the mean +/− standard deviation per group from three independent experiments (≥3 mice per group for each experiment). * Denotes significance differences between treatment groups (determined with a parametric repeated measures ANOVA and Bonferroni post-test), p<0.001 in each instance). (B) Corneal integrity was assessed by fluorescein staining in EDE mice or normal controls after 5 or 10 days. Eyes were examined under blue light illumination at 20-x magnification. Photographs are representative of three independent experiments (≥3 mice per group for each experiment). Control mouse eyes are shown in the upper panels (a, b), EDE mouse eyes are shown in the lower panels (c, d). Arrows denote regions of fluorescein staining on the ocular surface.</p

Ocular clearance of <i>P.</i><i>aeruginosa</i> in EDE.

<p>Levels of viable <i>P. aeruginosa</i> (cfu) in corneal homogenates (A) or ocular surface washes (B) of C57BL/6 EDE mice compared to normal controls (NC) at 6 h post-inoculation with 10⁹ cfu of <i>P. aeruginosa</i> strain PAO1 (T = 0). EDE was induced for 5 days prior to bacterial inoculation. Bacteria were rapidly cleared from the murine ocular surface of both groups of mice after 6 h. Similar bacterial levels were found in corneal homogenates (A), but fewer bacteria were recovered from the ocular surface washes of EDE mice compared to controls (p = 0.049, Mann-Whitney test) (B). Data are representative of three independent experiments (≥5 animals per group in each experiment). Data for each sample are shown as the median (black square) with upper and lower quartiles (boxed area), and range of the data (error bars).</p

Incidence and severity of <i>P. aeruginosa</i> infections in EDE mice.

<p>C57BL/6 mice were exposed to EDE or control conditions for 10 d prior to topical challenge with 10⁹ cfu of <i>P. aeruginosa</i> strain PAO1. Mice were monitored for corneal infiltrates, opacities, and changes in epithelial surface regularity. Pathology was graded at 96 h post-inoculation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065797#s2" target="_blank">Materials and Methods</a>). Incidences of pathology in EDE and control groups were not significantly different (Chi-square analysis). Data is representative of two independent experiments.</p

SP-D expression in EDE before and after <i>P.</i><i>aeruginosa</i> challenge.

<p>Western immunoblot blot analysis of SP-D expression in pooled ocular surface washes from EDE and control mice (10 mice per group) after 5 days EDE induction, and before and 6 h after inoculation with <i>P. aeruginosa</i> strain PAO1 (10⁹ cfu). To normalize for differences in tear volume, equivalent amounts of protein (2 µg) were used in the analysis (BCA protein assay). Purified recombinant SP-D (rSP-D, ∼43 kDa monomer), and a relevant number of bacteria suspended in PBS (5×10³ cfu, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065797#pone-0065797-g002" target="_blank">Fig. 2B</a>), were included as positive and negative controls, respectively. SP-D expression in ocular surface washes was increased under EDE conditions before bacterial inoculation. The experiment was repeated once.</p

Colocalization of the <i>P. aeruginosa exsA</i> mutant with acidified vacuoles in epithelial cells compared to that of wild-type bacteria or a <i>popB</i> (translocon) mutant.

<p>Confocal microscopy images of human corneal epithelial cells at 5 h post-infection with GFP-expressing <i>P. aeruginosa</i> (green). Prior to imaging, infected cultures were infused with LysoTracker (LT) DND-22 (pseudo-colored red). Panels depict (A) Uninfected control, (B) PAO1 <i>exsA</i> mutant (C) PAO1 <i>popB</i> (translocon) mutant and (D) wild-type PAO1. Uninfected cells appeared healthy. The intracellular <i>exsA</i> mutant appeared more frequently in LT (+) (acidified) vacuoles which co-localized yellow (arrows) than either the intracellular <i>popB</i> 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.</p

Colocalization of <i>P. aeruginosa</i> with acidified versus non-acidified vacuoles in relation to <i>exoS</i> transcriptional output.

<p>Confocal and Differential Interference Contrast (DIC) microscopy of human corneal epithelial cells at 5 h post-infection with <i>P. aeruginosa</i> PAO1 complemented with a reporter construct pJNE05 encoding the <i>exoS</i> promoter fused to <i>gfp</i> (green), and p67T1 which constitutively expresses dTomato (red). Bacteria were classified as having a high <i>exoS</i> 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 <i>exoS</i> output [dashed arrows]. Blebs are indicated with open arrows. Representative images are shown from two independent experiments. Magnification ~ 600 x.</p

Quantification of acidified vacuole occupation by <i>P. aeruginosa</i> in relation to <i>exoS</i> transcriptional output.

<p>Data show the mean (+/- SEM) percentage of bacteria-occupied acidified (LT+) vacuoles at 5 h post-infection for <i>P. aeruginosa</i> PAO1 and a <i>popB</i> (translocon) mutant. Bacteria were transformed with an <i>exoS</i> transcriptional reporter plasmid pJNE05 (<i>exoS</i>-<i>gfp</i>) and plasmid p67T1 (<i>dTomato</i>). Infected cells were also stained with LysoTracker. Bacteria with high <i>exoS</i> expression (grey columns) were significantly less likely to occupy acidified vacuoles than those with a low <i>exoS</i> expression (black columns) (* p < 0.001, Welch’s corrected t-Test). Data is representative of 3 independent experiments.</p

Intracellular survival and replication of <i>P. aeruginosa</i> 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).

<p>Bafilomycin treatment restored intracellular survival of the <i>exsA</i> mutant to that of the <i>popB</i> 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).</p

Quantification of acidified versus non-acidified vacuole occupation by wild-type <i>P. aeruginosa</i> and its type III secretion mutants.

<p>(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 <i>P. aeruginosa</i> PAO1 or its type III secretion mutants (<i>exsA</i> or <i>popB</i>). 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 <i>exsA</i> and <i>popB</i> 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 <i>exsA</i> 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 <i>exsA</i> mutant was associated with more acidified bacteria-occupied vacuoles per cell than either the <i>popB</i> 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].</p