Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis

A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elabora ted ATP-dependent and ATP-independent types of cytotoxic factors in the gro wth medium. These cytotoxic factors, active against macrophages, were secre ted during the exponential phase of growth in a complex medium. Commensurat e with the appearance of the cytotoxic activities in the cell-free growth m edium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatas e), were detected in the medium. These ATP-utilizing enzymes are believed t o convert external ATP, presumably effluxed from macrophages, to various ad enine nucleotides, which then activate purinergic receptors such as P2Z, le ading to enhanced macrophage cell death. Pretreatment of macrophages with p eriodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z rec eptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner s uch that it triggered activation of apoptotic processes in macrophages, lea ding to proteolytic conversion of procaspase-3 to active caspase-3. This cy totoxic factor(s) did not appear to act on procaspase-3 present in macropha ge cytosolic extracts. Intact macrophages, when exposed to the cytotoxic fa ctor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of ac tive caspase-3 in their cytosolic extracts. Interestingly, two redox protei ns, azurin and cytochrome c(551), were detected in the cytotoxic preparatio n. When cell-line-derived or peritoneal macrophages or mast cells were incu bated overnight with Q-Sepharose column flow-through fraction or with a mix ture of azurin and cytochrome c(551), they underwent extensive cell death d ue to induction of apoptosis.