Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia

Abstract

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 (CFΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψmito) 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 Ca2+ and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 minutes and were complete in 1–2 hrs. 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 Δψmito 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 Δψmito and increases in Cacyto 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.

Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia

“…Calu-3 cells routinely had transepithelial resistance (RT) Ͼ400 ⍀·cm 2 and exhibited polarized responses consistent with previous studies (5,33). JME cells, a continuous SV40 large-T antigen-transformed human nasal epithelial cell line homozygous for ⌬F508 CFTR, were used for many imaging experiments, because they exhibit a higher transfection efficiency than Calu-3 cells and have excellent imaging properties (27)(28)(29). JME cells were cultured in DMEM-Ham's F-12 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillinstreptomycin, 10 ng/ml EGF, 1 M hydrocortisone, 5 g/ml insulin, 5 g/ml transferrin, 30 nM triiodothyronine, 180 M adenine, and 5.5 M epinephrine.…”

“…Recent work on fibroblasts has shown that C12-triggered apoptosis, including release of cytochrome c from mitochondria, occurs independent of the proapoptotic mediators Bak and Bax (28). Importantly, P. aeruginosa biofilms elicited lasI-dependent (lasI is the enzyme responsible for synthesis in P. aeruginosa) increases in cytosolic Ca 2ϩ concentration (Ca cyto ) and depolarization of ⌬ mito , indicating that P. aeruginosa biofilms produce sufficient C12 to induce the observed proapoptotic responses (27). It might be expected that biofilm formation and C12-induced host cell apoptosis might be beneficial to the bacteria, in that barrier function may be compromised to permit bacterial access to the body.…”

“…In our previous study (27) of C12 activation of apoptosis in airway epithelial cell lines (Calu-3 and JME cells), 10 M was the C12 threshold concentration and 50 -100 M C12 caused quite similar responses. These responses included 1) activation of caspases 3/7, 8, and 9 within 1-2 h, 2) depolarization of mitochondrial membrane potential (⌬ mito ), beginning within 5 min and reaching a maximum within 30 min, followed by release of cytochrome c into the cytosol after 1 h, and 3) release of endoplasmic reticulum (ER)-targeted, redox-sensitive green fluorescent protein (roGFP) from the lumen of the ER into the cytosol and nucleus beginning within ϳ20 min and becoming complete within 45 min.…”

“…Since C12 caused apoptosis in the airway epithelial cell line Calu-3 (27), we hypothesized that C12-induced breakdown of tight junctions in airway epithelia was part of the C12-induced apoptosis cascade and, furthermore, that Tg might prevent apoptosis-related degradation of tight junctions through its role in altering Ca cyto or mitochondrial Ca 2ϩ concentration (Ca mito ). We first tested whether C12-induced activation of apoptosis included breakdown of barrier function and tight junctions in Calu-3 cells by pretreating Calu-3 cells with the selective pan-caspase blocker carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (ZVAD-fmk) to test whether blockade of caspases would prevent the effects of C12 on barrier function and tight junctions of Calu-3 cells.…”

“…We first tested whether C12-induced activation of apoptosis included breakdown of barrier function and tight junctions in Calu-3 cells by pretreating Calu-3 cells with the selective pan-caspase blocker carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (ZVAD-fmk) to test whether blockade of caspases would prevent the effects of C12 on barrier function and tight junctions of Calu-3 cells. We then tested whether Tg prevented C12's effects on barrier function, as well as other previously identified aspects of the apoptosis cascade in airway epithelia, including activation of caspase 3/7 (the executioner caspase), release of GFP and oxidizing ER contents into the cytosol, and depolarization of ⌬ mito (27). Some experiments were performed in another human airway epithelial cell line (JME cells), which are easier to transfect than Calu-3 cells and are also flatter and better for imaging.…”