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

如慢性肉芽肿性疾病所证明的那样，活性氧是烟曲霉菌被中性粒细胞杀死的关键武器。在本研究中，研究了烟曲霉菌产生的真菌毒素[烟青素、胶质毒素和贺氏酸]对人中性粒细胞产生O_2^-NADPH氧化酶的影响。在这些真菌毒素中，只有胶质毒素对O_2的产生有强烈的抑制作用。另外两种毒素是无效的。这种抑制作用依赖于胶质毒素的二硫键，并且是通过对酶的激活步骤的影响而引起的，而不是对被激活的酶的催化作用。具体地说，胶质毒素抑制了PMA刺激的p47；；Phox&gt；磷酸化，它与细胞骨架相关的p67；；Phox&gt；的掺入，以及所有细胞液成分(p67；lt；Phox&>t；，p47^&lt；Phox&gt；和p40^&lt；Phox&gt；)的移位，这是组装活性NADPH氧化酶的关键步骤。因此，胶质毒素的主要靶点很可能是p47；Gliotoxx不能抑制rac2的转位。中性粒细胞对烟曲霉菌具有杀伤活性，其E/T比为3.6。在E/T比为15到90的检测中，粘液毒素抑制中性粒细胞对烟曲霉菌的这种杀灭活性，其IC_&lt；50&gt；值在NM水平。这些结果表明，烟曲霉菌将通过其菌丝产物胶质毒素抑制中性粒细胞NADPH氧化酶的激活而获得对宿主的侵袭力。中性粒细胞是杀死烟曲霉的关键缺陷。

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.