Up-regulation of Fas expression by Pseudomonas aeruginosa-infected endothelial cells depends on modulation of iNOS and enhanced production of NO induced by bacterial type III secreted proteins.
To obtain a better understanding of the mechanisms involved in the up-regulation of the Fas apoptotic signaling cascade induced by P. aeruginosa type III secretion system (TTSS), human umbilical vein endothelial cells (HUVEC) 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 annexin V reactivity) were more prominent in cultures infected with the wild-type bacteria. PAO-1 induced the up-regulation of Fas and the release of soluble FasL (sFasL) from infected cells but cell treatment with antagonist anti-Fas did not completely abrogate apoptosis suggesting that, besides the activated Fas-FasL pathway, other mechanisms are likely to be associated with the induction of apoptosis. LNMMA, a potent inhibitor of NO synthesis, completely inhibited apoptosis in both PAO-1 and PAO-1::exsA infected cultures. Moreover, PAO-1 was shown to up-regulate both the expression of iNOS and NO production by HUVEC. Treatment of cells with LNMMA completely inhibited cell expression of mFas. Based on these results we speculate that P. aeruginosa TTSS not only accounts for HUVEC higher expression of Fas and release of sFasL but also leads to overproduction of NO and to a NO-dependent up-regulation of the Fas-FasL proteins.
Assis
Maria-Cristina
MC
Department of Microbiology and Immunology, Universidade do Estado do Rio de Janeiro, 20 551-030 Rio de Janeiro, Brazil.
Freitas
Carla
C
Saliba
Alessandra M
AM
D'A Carvalho
Ana Paula
AP
Simao
Tatiana A
TA
Albano
Rodolpho M
RM
Plotkowski
Maria-Cristina
MC
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D004791:Enzyme Inhibitors / Q000378:metabolism; D053222:Fas Ligand Protein / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000378:metabolism* / Q000472:pathogenicity; D015854:Up-Regulation; D019014:fas Receptor / Q000378:metabolism*; D019323:omega-N-Methylarginine / Q000378:metabolism
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D053222:Fas Ligand Protein; D019014:fas Receptor; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II