Mechanism for glutathione-mediated protection against the Pseudomonas aeruginosa redox toxin, pyocyanin

Abstract

Pseudomonas aeruginosa is an important human pathogen associated with several acute and chronic conditions, including diseases of the airways and wounds. The organism produces pyocyanin, an extracellular redox toxin that induces oxidative stress, depletes intracellular glutathione (GSH) and induces proliferative arrest and apoptosis, thus compromising the ability of tissue to repair itself. GSH is an important intra- and extracellular antioxidant, redox buffer and detoxifies xenobiotics by increasing their polarity, which facilitates their elimination. As previous studies have reported exogenous GSH to be protective against pyocyanin toxicity, this study was undertaken to explore the mechanism by which GSH protects host cells from the deleterious effects of the toxin. Co-incubation of pyocyanin with GSH resulted in a time-dependent diminished recovery of the toxin from the incubation medium. Concurrently, a highly polar green-colored metabolite was recovered that exhibited a UV–visible spectrum similar to pyocyanin and which was determined by mass spectrometry to have a major ion (m/z=516) consistent with a glutathione conjugate. The ability of the conjugate to oxidize NADPH and to reduce molecular oxygen with the production of reactive oxygen species was comparable to pyocyanin yet it no longer demonstrated cytotoxicity towards host cells. These data suggest that GSH forms a cell-impermeant conjugate with pyocyanin and that availability of the thiol may be critical to minimizing the toxicity of this important bacterial virulence factor at infection sites. Our data indicate that for GSH to have a clinically effective role in neutralizing pyocyanin, the thiol needs to be available at millimolar concentrations.