Apoptosis of infected mammalial cells has been shown to depend on bacterial type III secretion system (TTSS) and host cell overexpression of membrane-bound Fas (mFas) and Fas ligand (FasL). We examined the role of TTSS proteins, the Fas/FasL system in apoptosis of P. aeruginosa-infected endothelial cells and the mechanism accounting for upregulation of mFas expression in infected endothelial cells.

To obtain a better understanding of the mechanisms accounting for the up-regulation of the Fas apoptotic signaling cascade by P. aeruginosa type III secretion system (TTSS), human endothelial cells (HEC) were infected with P. aeruginosa PAO-1 or its TTSS-negative mutant PAO-1::exsA.

). PAO-1 was significantly more cytotoxic than the mutant and features of apoptosis (DNA fragmentation and reactivity with annexin V) were much more prominent in cultures infected with the wild type bacteria. The up-regulation of mFas was measured by cytometry, PAO-1 induced significant levels of mFAS (38.7% ± 6.8) when compared with the mutant (19.3±5.0) and non-infected cells (15.2± 1.4). The release of soluble FasL (sFasL) from infected cells with PAO-1 (2.1 pg/mL± 0.1) was significantly higher than cells infeted with mutant (0.4 pg/mL± 0.1) and non-infected cells (0.0 pg/mL± 0.0). The treatment with antagonist anti-Fas IgG did not completely abrogate apoptosis suggesting that, besides activated Fas-FasL pathway, other mechanisms are likely associated with P. aeruginosa-induced apoptosis. LNMMA completely inhibited apoptosis in both PAO-1 and PAO-1::exsA infected cultures. Moreover, the detection of iNOS expression by cytometry showed that PAO-1 induced significant increase of median of intensity fluorescence (11.4±1.3) when compared with the mutant and non-infected cells (2.7±0.2 and 2.7±1.1, respectivately). PAO-1 induced higher amount of nitrite detected by Griess reaction. Finally, cell treatment with LNMMA completely inhibited cell expression of mFas.

P. aeruginosa TTSS induced higher expression of mFas and release of sFasL and overproduction of NO.The upregulation of the mFas-FasL proteins was NO-dependent. Therefore, the major mechanism leading to apoptosis of infected HUVEC is likely to be the bacterial induction of NO production, rather than activation of the Fas-FasL pathway.

Instituição de fomento: CNPQ

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