Abstract 1002 Poster Session IV, Tuesday, 5/4 (poster 134)

Many mucosal pathogens cause epithelial damage by stimulating apoptosis. Pseudomonas aeruginosa a lumenal pathogen in cystic fibrosis can elicit a brisk inflammatory response following attachment to asialoGM1 receptors on respiratory epithelial cells and occasionally invades the mucosal barrier particularly when tight junctions are disrupted. We postulated that either interaction might also stimulate an apoptotic response, depending upon the types of bacterial virulence factors expressed. Several well characterized isogenic mutants of P. aeruginosa PAO1 were compared for their ability to stimulate apoptosis. Monolayers of either nasal epithelial cells in primary culture, or 9HTEo- cells were exposed to 1 × 106 PAO1, PAO1exsA (deficient in type III secretion system) PAO/NP (Pil-) or PAONP/fliA (Pil-Fla-) for 1-6 hours. Bacteria were killed by the addition of gentamicin (100mg/ml), monolayers washed and incubated for an additional 6-12 hours. DNA fragmentation was then determined by using the TUNEL reaction to label the free 3′- OH termini with fluorescein and were detected by flow cytometry. Apoptosis in 9HTEo- cells (lacking tight junctions) was stimulated by PAO1 in a time dependent fashion, ranging from 0% at 1 hour, to 6% (+/- 2%) at 3 hrs, and 37% (+/- 2.5%) at 6 hrs. Ligation of the asialoGM1 receptor, which is sufficient to initiate translocation of NFkB, did not stimulate apoptosis at any time point. Exposure to the type III secretion mutant did not trigger apoptosis, nor did the Pil- Fla- strain, suggesting that close apposition of the organism and the epithelial cell is necessary. A Pil-Fla+ mutant, which can bind to GM1 components of the epithelial cell by flagellar adhesins, was able to elicit apoptosis in 7% of the 9HTEo- cells. Respiratory epithelial cells in primary culture, which have tight junctions and are resistant to PAO1 invasion, were significantly less susceptible to apoptosis with only 6.2% apoptosis after 5 hours of exposure to wild type bacteria. Similar levels of resistance to PAO1 induced apoptosis were observed in cell lines with and without abnormalities in CFTR function. Both 9HTEo- cells expressing the pCep vector or pCep-R (overexpressing the CFTR R-domain) had equivalent 3.5-5.5% rates of apoptosis; and IB3 (CF) and C-38 (corrected) cell lines demonstrated equivalent 3% (+/- 0.2%) rates of apoptosis following PAO1 exposure. These experiments suggest that apoptosis is not the typical response of the respiratory epithelium to adherent P.aeruginosa and is not a major factor in cells with CFTR mutations. Organisms, which can induce apoptosis, express type III secretion systems and can access baso-lateral receptors in the absence of tight junctions. Thus, invasion of host cells by P.aeruginosa is more likely to induce apoptosis than the superficial interactions typical of lumenal infection.