DIFFERENTIAL EFFECTS ON CASPASE MEDIATED APOPTOSIS OF HELA CELLS INDUCED BY DIFFERENT PSEUDOMONAS AERUGINOSA CULTURE FRACTIONS

The aim of this work was to investigate intracellular survival of Pseudomonas aeruginosa clinical strains in non-phagocytic, epithelial cells and the capacity to induce apoptosis of the host cell using qualitative (evaluation of virulence factors expression, adherence to HeLa cells) and quantitative assays (invasion assay, qRT-PCR). Taken together the results demonstrated that all tested strains adhered to HeLa cells, exhibiting a diffuse, aggregative or mixed diffuse-aggregative pattern with 20-70% adherence rates. The assays confirmed that P. aeruginosa had the ability to enter and invade the epithelial, non-phagoytic cells, probably due to the pores forming enzymes (hemolysins, lecithinase, lipase) released by the analyzed strains. Two strains proved the ability to induce apoptosis of HeLa cells, inducing an increased expression of caspase 3 and Bax genes, correlated with a decreased level of the anti-apoptotic factor Bcl-2. The apoptotic gene expression levels were also analyzed for HeLa cell treated with cell free culture supernatants, responsible for decreased expression levels of caspase 3 and caspase 9 genes. The results corroborate well with other reported data, proving the ability of these bacteria to penetrate non-phagocytic cells and to induce changes at molecular level, including apoptosis. The cell free culture supernatants did not demonstrate the ability to induce apoptosis by caspase – mediated pathways, leading us to the hypothesis that the host cells apoptosis is requiring cell to cell contact, probably implicating the activation of a type III secretion system responsible for the intracellular release of pro-apoptotic factors.