Cell Microbiol. 2012 May;14(5):698-709. doi: 10.1111/j.1462-5822.2012.01753.x. Epub 2012 Feb 9.
Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia.
Schwarzer C1, Fu Z, Patanwala M, Hum L, Lopez-Guzman M, Illek B, Kong W, Lynch SV, Machen TE.
Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis (CF) patients, a process regulated by quorum-sensing molecules including N-(3-oxododecanoyl)-l-homoserine lactone (C12). C12 (10-100 µM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CF ΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψ(mito)) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca(2+) and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 min and were complete in 1-2 h. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψ(mito) and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψ(mito) and increases in Ca(cyto) like 10-50 µM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed.
JME and CalLu-3 cells were grown on 12-well plates and exposed to C12 or staurosporine (positive control), then processed for activities of caspase 3/7, 8 and 9. JME cells were left untreated or exposed to C12 (50 μM) (A) or staurosporine (50 μM) (B), and activities of caspases 3/7, 8 and 9 were measured after 0.5, 1, 2, and 4 hrs. C. Similar experiments were performed on Calu-3 cells exposed to C12 (50 μM) for 2 hrs. Activities of caspases 3/7, 8 and 9 were expressed relative to untreated controls. Avg +/− SD (n = 2–9 expts for all averages).
A. Confocal image (ex: 488 nm, em: 520–560 nm) of JC-1-stained JME cells shows punctate staining in cytosol characteristic of mitos in control, untreated cells. Nuclei appear as dark, nonfluorescent areas. B. Image of the same field after 10 mins with 50 μM C12. Punctate staining was replaced by diffuse staining, including in the nuclei. C. Time course of JC-1 fluorescence in the nucleus, which tracks loss of JC-1 from the mitos resulting from depolarization of Δψmito. JC-1 signal was normalized by setting initial value in the nucleus as zero and value after FCCP (10 μM) as 100%. D. Summary of effects of C12 (50 μM, 20 min) and FCCP (10 μM, 2 min) on change in nuclear JC-1 fluorescence (ΔJC-1) in JME cells. Avg +/−SD (n = 6 expts, >10 cells in each experiment)..
Immunofluorescence of cytoC of JME (A, B) and Calu-3 (C, D) cells under control conditions (A, C) and after treatment with 50 μM C12 for 1 hr (B, D). Results typical of six similar experiments for each.
Fura-2-loaded JME cells were treated with C12 1, 10 and 100 μM followed by thapsigargin (10 μM, positive control for release from ER) (A) or with thapsigargin followed by 1, 10 and 100 μM C12 (B). Traces show average Cacyto for 10–20 cells, and are typical of four similar experiments.
Cells were transfected with ER-targeted roGFP and observed in the confocal microscope under control conditions (A) and after treatment with C12 (50 μM) for 45 (B) and 90 mins (C). Cells in which roGFP had leaked from the ER and diffused into the cytosol and nucleus are shown by arrows. Results typical of >10 similar.
JME cells were transfected separately with ER-targeted and cytosolic roGFP, and cells were plated onto one coverslip for measurement of redoxer and redoxcyto, as described previously (). As shown by the solid line, C12 (10 μM) caused, after a delay of about 30 mins, redoxer to become reduced and redoxcyto to become oxidized; after about 60 mins, redoxer and redoxcyto reached similar values. Data show the time course of responses to C12 for redoxcyto and redoxer from five cells each. Average values (+/− SD) for redoxer (triangles) and redoxcyto (squares) are shown for control cells in the steady state and then after 1 hr treatment with C12 (10 or 50 μM). Avg +/− SD, n = 6 expts, 47 cellular measurements of redoxcyto and 67 cellular measurements of redoxer.
5JME cells expressing lacZ or CFTR (adenovirus infections) were grown on coverglasses or well plates and exposed to C12 (50 μM) for times to 2 hrs. A. Activation of caspase 3/7 by 50 μM C12 was measured in JME-lacZ and JME-CFTR cells at t = 0, 20, 60 and 120 mins. Avg +/− SD (n = 3) of caspase activity measured in RLU at each time point. * p < 0.05. Other points were insignificantly different from each other. B. Time course of effect of C12 on Δψmito. JME-lacZ and JME-CFTR cells were loaded with JC-1, washed and then exposed to C12 (50 μM, arrow). Nuclear fluorescence (expressed as percentage of maximal JC-1 fluorescence observed during treatment with 10 μM FCCP at the end of the experiment) increased rapidly in both JME-lacZ and JME-CFTR cells. Inset shows average rates (+/− SD, n = 3) of C12-triggered decrease of JC-1 fluorescence in JME-lacZ and JME-CFTR cells. There was no difference in these rates (p > 0.40). C. Time course of effect of C12 on redoxer. JME-lacZ and JME-CFTR cells that had been transfected with ER-roGFP were exposed to C12 (50 μM, arrows). C12 caused the ER redox potential (resulting from reduction of oxidized environment experienced by ER-roGFP) to become reduced at the same rate in JME-lacZ and JME-CFTR cells. The strong reductant DTT (1 mM) was added as shown to induce maximal reduction of the ER-roGFP ratio. Inset shows average rates (+/− SD, n = 3) of C12-triggered decrease of ER-roGFP fluorescence ratio in JME-lacZ and JME-CFTR cells. There was no difference in these rates (p > 0.09).
Biofilms were prepared by vortexing one biofilm-covered mesh into 500 μl of Ringer's, and then adding 10 μL of this suspension to the 100 μl of Ringer's bathing the JC-1-loaded JME cells. Fluorescence (ex: 490 nm, em: 520–560 nm) was measured over nuclei. A. PAO1wt biofilm caused immediate increase of JC-1 fluorescence, and this was increased further by FCCP (10 μM). B. PAO1ΔlasI biofilm did not affect JC-1 fluorescence, but further addition of PAO1wt biofilm caused a large increase in JC-1 fluorescence that was not affected further by FCCP (10 μM). Traces show average JC-1 fluorescence for >10 cells. Results typical of three similar experiments for both PAO1wt and PAO1lasI.
Biofilms were prepared by vortexing one biofilm-covered mesh into 500 μl of Ringer's, and then adding 10 μL of this suspension to the 100 μl of Ringer's bathing the fura-2-loaded JME cells. Fluorescence ratios (ex: 350/380 nm, em: >510 nm) were measured and expressed relative to maximum ratios measured at the end of the experiment. A. PAO1wt biofilm caused immediate increase of fura-2 ratio. B. PAO1ΔlasI biofilm caused no increase of fura-2 ratio. Traces show average fura-2 ratio for >10 cells. Results typical of four similar experiments for both PAO1wt and PAO1lasI.