Inhibition of the neutrophil superoxide-generating NADPH oxidase with fungal gliotoxin

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Reactive oxygen species are a critical weapon in Aspergillus fumigatus killing by neutrophils, as demonstrated by chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins [fumagillin, gliotoxin, and helvolic acid] were examined for their effects on the O_2^--generating NADPH oxidase of human neutrophils. Of these mycotoxins, only gliotoxin greately and stoichiometrically inhibited O_2- generation. The other two toxins were ineffective. The inhibition was dependent on the disulfide bridge of gliotoxin and caused by effects on the activation steps of the oxidase, but not those on the catalysis of the activated oxidase. Specifically, gliotoxin inhibited PMA-stimulated p47^<phox> phosphorylation, its incorporation to p67^<phox> associated with the cytoskeleton, and the translocation of all cytosolic components (p67^<phox>, p47^<phox>, and p40^<phox>) to the membrane, which are crucial steps to assemble the active NADPH oxidase. Thus, it is likely that the primary target of gliotoxin is p47^<phox> phosphorylation. Gliotoxin did not inhibit the translocation of Rac2. Finally, neutrophils showed A. fumigatus-killing activity with an E/T ratio of 3.6. Gliotoxin inhibited this A. fumigatus-killing activity of neutrophils with IC_<50> values at nM levels, in assays performed over at E/T ratios of 15 to 90. These results suggest that A. fumigatus will acquire invasiveness in hosts by inhibiting the activation of the neutrophil NADPH oxidase, a critical defect in their neutrophils to kill A. fumigatus, by means of its hyphal product, gliotoxin.