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

Abstract

A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c 551 , were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c 551 , they underwent extensive cell death due to induction of apoptosis.

Comprehensive Analysis of the Secreted Proteins of the Parasite Haemonchus contortus Reveals Extensive Sequence Variation and Differential Immune Recognition

“…In addition, the protein is predicted to be glycosylphosphatidylinositol-anchored and the observed molecular mass of ϳ62 kDa (C-terminal half, spots 40, 41, 42,) and ϳ52 kDa (N-terminal half, spots 60,63,65,69) fit to a predicted 127.6 kDa for the full-length protein (including signal peptide and anchor peptide). Based on sequence comparisons, both halves may very well encode an active serine protease, thereby classifying the full-length protein as a polyprotein.…”

“…Nucleoside diphosphate kinase (NDK) (spot 157) is responsible for maintaining the levels of intracellular nucleotide pools but is also involved in cell growth, differentiation, and tumor metastasis (66). A secreted form has been found only in some bacteria (67)(68)(69) and in the nematode Trichinella spiralis (70). A function is suggested by observed ATP-dependent cytotoxicity for macrophages and mast cells and prevention of cells from apoptosis (67,71).…”

Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages

“…Chakrabarty and others [4,[8][9][10][11] have demonstrated that many pathogenic microorganisms release enzymes that modulate levels of NTPs. Of particular interest is extracellular ATP (eATP), which is expected to accumulate at the site of inflammation [12][13][14], important in immunity against microbial infection.…”

“…ATP-binding to P2X 7 receptors of macrophages results in their cytolysis in vitro via necrotic [23,24] or apoptotic pathways [25,26], dependent presumably on the duration of exposure to eATP [27,28]. Extracellular pathogenic bacteria, such as Pseudomonas aeruginosa [10,18], have evolved strategies to avoid phagocytic activity of macrophages by releasing enzymes that increases the level of eATP, thereby triggering apoptosis of these and other professional phagocytes. In contrast, intracellular parasites, like Leishmania spp., are expected to develop strategies in the opposite direction by releasing eATP-utilizing enzymes, thereby prolonging the survival of their host cells.…”

“…The released enzymes include adenylate kinase, 5′-nucleotidase (and/or ATPase/phosphatase), ecto-ATPase [15], ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) [16] and NdK. The last enzyme utilizes eATP as its principal substrate [9,10,[17][18][19][20]. eATP is known to trigger the death of immune cells, including macrophages, by its binding to purinergic receptor of the P2X family, i. e. P2X 7 [12,13,21,22].…”