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Induction of inflammatory mediator release (12-hydroxyeicosatetraenoic acid) from human platelets by Pseudomonas
1AG Infektabwehr, Ruhr-Universität Bochum, BRD, FRG.
Abstract:
The role of platelets in acute and chronic infection has been widely discussed in various disease processes. We studied the effects of two lipolytic enzymes (phospholipase C, lipase) secreted by Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients with regard to 12-hydroxyeicosatetraenoic acid (12-HETE) generation from human platelets. Both phospholipase C (PLC) and lipase were secreted into the culture supernatant at the end of the logarithmic growth phase. Indeed, only culture supernatants obtained from the late logarithmic/early stationary phase of CF strains induced the generation of 12-hydroxyeicosatetraenoic acid (12-HETE) (from 15 +/- 9 to 370 +/- 98 ng, n = 7). Purified P. aeruginosa lipase itself generated only small amounts of 12-HETE from human platelets with a maximum of 30 +/- 7 ng/10(8) platelets at the highest concentration tested (20 nkat). A partially purified culture supernatant from P. aeruginosa strain PAO1 containing phospholipase C and lipase, but lacking glycolipid and protease activities, induced time- and dose-dependently a significant 12-HETE generation from human platelets. Maximal 12-HETE generation was observed at the highest enzyme concentrations tested (PLC: 1.35 nkat, lipase: 3.7 nkat/10(8) platelets). To analyze whether lipase exhibits a modulatory role on PLC-induced 12-HETE generation from human platelets we inhibited lipase activity in the P. aeruginosa partially purified culture supernatant by treatment with the lipase-specific inhibitor hexadecylsulfonylfluoride (AMSF) leaving the activity of PLC unaffected (lipase-free culture supernatant). The capacity of lipase-free culture supernatant to induce 12-HETE generation was diminished by up to 100% depending on the PLC activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Pseudomonas aeruginosa enzymes, phospholipase C (PLC) and lipase, from cystic fibrosis patients induce 12-hydroxyeicosatetraenoic acid (12-HETE) generation in human platelets. Lipase activity modulates PLC
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Platelets play a critical role in both acute and chronic infections.
- Pseudomonas aeruginosa is a significant opportunistic pathogen, particularly in cystic fibrosis (CF) patients.
- Lipolytic enzymes secreted by bacteria can influence host immune responses.
Purpose of the Study:
- To investigate the role of Pseudomonas aeruginosa lipolytic enzymes (phospholipase C and lipase) in generating 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets.
- To determine the specific contribution of phospholipase C (PLC) and lipase to 12-HETE production.
- To explore the modulatory effect of lipase on PLC-induced 12-HETE generation.
Main Methods:
- Isolation and culture of Pseudomonas aeruginosa strains from cystic fibrosis patients.
- Collection of bacterial culture supernatants at the late logarithmic/early stationary growth phase.
- Assay of 12-HETE generation from human platelets induced by bacterial culture supernatants and purified enzymes.
- Inhibition of lipase activity using a specific inhibitor (AMSF) to assess its role.
Main Results:
- Culture supernatants from CF strains, particularly during the late logarithmic/early stationary phase, significantly induced 12-HETE generation.
- Purified Pseudomonas aeruginosa lipase alone generated only small amounts of 12-HETE.
- A partially purified supernatant containing both PLC and lipase induced dose- and time-dependent 12-HETE generation.
- Inhibition of lipase activity diminished the capacity of the supernatant to induce 12-HETE generation, indicating a modulatory role.
Conclusions:
- Pseudomonas aeruginosa phospholipase C is a primary inducer of 12-HETE generation from human platelets.
- Bacterial lipase plays a significant modulatory role in this process.
- These findings highlight a novel mechanism by which P. aeruginosa may influence platelet function during infection, particularly in CF patients.