Fungal Gliotoxin Targets the Onset of Superoxide-Generating NADPH Oxidase of Human Neutrophils

Abstract

Gliotoxin from Aspergillus, bearing a SS bond in its structure, prevented the onset of O−2 generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA). Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O−2 generation persisted even if cells treated with gliotoxin were subsequently washed, with half-inhibition concentrations (IC50) of 5.3, and 3.5 μM for treatments of 15 and 30 min, respectively. In addition, gliotoxin made neutrophils reduce cytochrome c regardless of absence of PMA, through its reaction with intracellular reductants in an oxygen-dependent process, named redox cycling. Thus, we next tested whether preincubation of neutrophils with gliotoxin under hypoxic conditions would relieve the inhibition of NADPH oxidase. Instead, this prevention of redox cycling significantly favored damage to the NADPH oxidase with an IC50 of 0.009 μM. Moreover, conversion of gliotoxin to its dithiol derivative by addition of reduced dithiothreitol during incubation protected cells from losing oxidase activity. These findings support that the disulfide form of gliotoxin targets NADPH oxidase activation.