Abstract
Pseudomonas aeruginosa is a gram-negative pathogen, which causes life-threatening infections in immunocompromized patients. These bacteria express a secreted lipoxygenase (PA-LOX), which oxygenates free arachidonic acid to 15S-hydro(pero)xyeicosatetraenoic acid. It binds phospholipids at its active site and physically interacts with lipid vesicles. When incubated with red blood cells membrane lipids are oxidized and hemolysis is induced but the structures of the oxygenated membrane lipids have not been determined. Using a lipidomic approach, we analyzed the formation of oxidized phospholipids generated during the in vitro incubation of recombinant PA-LOX with human erythrocytes and cultured human lung epithelial cells. Precursor scanning of lipid extracts prepared from these cells followed by multiple reaction monitoring and MS/MS analysis revealed a complex mixture of oxidation products. For human red blood cells this mixture comprised forty different phosphatidylethanolamine and phosphatidylcholine species carrying oxidized fatty acid residues, such as hydroxy-octadecadienoic acids, hydroxy- and keto-eicosatetraenoic acid, hydroxy-docosahexaenoic acid as well as oxygenated derivatives of less frequently occurring polyenoic fatty acids. Similar oxygenation products were also detected when cultured lung epithelial cells were employed but here the amounts of oxygenated lipids were smaller and under identical experimental conditions we did not detect major signs of cell lysis. However, live imaging indicated an impaired capacity for trypan blue exclusion and an augmented mitosis rate. Taken together these data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type.
Highlights
Lipoxygenases (LOX) catalyze oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives
Under our experimental conditions (70 μg/ml PA-LOX with 100 μl packed red blood cells dissolved in 1 ml PBS), 2.1 ± 0.3 % of the erythrocytes were disrupted as determined by hemoglobin release during 12 h of incubation
To explore the molecular basis of PA-LOX-induced hemolysis, we analyzed by RP-HPLC the red cell membrane lipid extracts for oxygenated polyenoic fatty acids, following alkaline hydrolysis of the ester lipids (Fig. 1A-I)
Summary
Lipoxygenases (LOX) catalyze oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives. In plants and non-mammalian animals downstream products of these hydroperoxides are important for wound healing and defense against pests [1] In mammals, they play extensive roles in inflammation [2,3]. Ii) Virulence factor: In vitro studies employing PA-LOX-expressing versus PA-LOX-deficient pathogens and cultured lung epithelial cells have suggested that the invasive capacity of the pathogen improves when PA-LOX is expressed [11] These data suggest a role for PA-LOX as a virulence factor and recent studies of PA-LOX-erythrocyte interactions support this hypothesis [13]. If formed in vivo these anti-inflammatory and pro-resolving mediators might downregulate the immune response of the host. The formation of such products augments the likelihood of pathogen survival and lipoxin synthase activity might be considered part of a
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Schedule a callMore From: Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
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