Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H ₂O ₂), and NaNO ₂ generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ~7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H ₂O ₂/NO ₂̄ systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN ̄ (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl ̄ (MPO), which with Cl ̄ yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO ₂̄ in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.