Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity

Pseudomonas aeruginosa is a persistent pathogen in the airways of patients with cystic fibrosis or bronchiectasis from other causes and appears to hav e evolved strategies to survive the inflammatory response of the host. We h ypothesized that the secreted hemolytic phospholipase C (PLC) of P. aerugin osa (PlcHR) would decrease neutrophil respiratory burst activity. We found that while intact wild-type P, aeruginosa cells stimulated moderate respira tory burst activity from human neutrophils, an isogenic mutant pseudomonas (Delta HR strain) containing a targeted deletion of the plcHR operon induce d a much more robust oxidative burst from neutrophils. In contrast, a secon d pseudomonas mutant (Delta N) containing a disruption in the gene encoding the nonhemolytic PLC (PlcN) was not different from the wild type in stimul ating neutrophil O-2(-) production. Readdition of purified PlcHR to the Del ta HR strain suppressed neutrophil O-2(-) production to levels stimulated b y wild-type bacteria. Interestingly, purified PlcHR decreased phorbol myris tate acetate (PMA)- but not formyl methionyl-leucyl-proline (fMLP)-induced respiratory burst activity, suggesting interference by PlcHR with a protein kinase C (PKC)-specific signaling pathway. Accordingly, the PKC inhibitor bisindolylmaleimide inhibited the oxidative burst induced by either PMA or intact pseudomonas, but not by fMLP, whereas the p38 kinase inhibitor SB-20 3580 fully inhibited the respiratory burst induced by fMLP or the PlcHR-rep lete wild-type bacteria, but not PMA or the PLHR-deficient Delta HR bacteri al mutant. We conclude that expression of PlcHR by P. aeruginosa suppresses bacterium-induced neutrophil respiratory burst by interfering with a PKC-d ependent, non-p38 kinase-dependent pathway.