Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase

Abstract

Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O 2 ؊ generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47 phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67 phox , p47 phox , and p40 phox , which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47 phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47 phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) ␤II rather than an effect of GT on PKC ␤II activity, suggesting a failure of PKC ␤II to associate with the substrate, p47 phox , on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.The best-known NADPH oxidase occurs in phagocytes and provides large quantities of reactive oxygen species (ROS) to oxidatively modify microbes as a microbicidal mechanism. The phagocyte NADPH oxidase is a highly regulated multisubunit enzyme composed of p67 phox , p47 phox , p40 phox , and Rac2 in the cytosol, as well as flavocytochrome b 558 , which consists of gp91 phox and p22 phox , in the membrane (4,9,15,29,36,39,57). The enzyme is dormant in resting cells, with these phox (for phagocyte oxidase) components being distributed to their cellular compartments. However, once phagocytes encounter invading microbes or soluble stimulants, such as phorbol myristate acetate (PMA), cytosolic phox components and Rac2 migrate to the membrane to assemble with flavocytochrome b 558 , forming the active enzyme. The activated flavocytochrome b 558 works as the catalytic redox center, where electrons are transferred from NADPH to molecular oxygen to generate O 2 Ϫ , a precursor of ROS. Recently, increasing attention has been directed toward the Nox/Duox family in nonphagocytes (33), which is composed of homologues of gp91 phox . Although the rates of ROS production are quite low in nonphagocytes, their ROS are believed to play important roles in cell signaling, hypoxic response, the immune system, etc.Over the past decade, the biochemistry of phagocyte NADPH oxidase has been intensively studied, particularly concerning the roles of Src homology 3 (SH3) domains and proline-rich regions (PRR), as well as those of other domains recently identified in...

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

“…GT is an epipolythiodioxopiperazine metabolite of pathogenic fungi which is capable of altering function and inducing apoptosis in macrophages [63]. GT has been shown to inhibit assembly of the NADPH oxidase of neutrophils via inhibition of PKC and the subsequent membrane translocation of p67 phox and p47 phox [40]. In this study we investigated the effect of GT on insect hemocytes and showed that GT inhibits both translocation of the hemocyte 67 and 47 kDa proteins and O 2 À generation upon PMA activation.…”

“…This is of tremendous interest as an immunologically related protein was detected in insect hemocytes [6], possibly suggesting that this antibody recognizes a region of gp91 phox that has remained conserved, consisting of cysteine residues against which GT exerts its effect. Compared to other investigators [40], throughout this investigation low concentrations of GT were employed to inhibit membrane translocation (0.06-0.25 mg/ml compared to 1-3 mg/ml). To address this, it has been shown that gliotoxin is actively concentrated in cells in a glutathione-dependent manner and intracellular levels of the toxin can be up to 1500-fold greater than the applied concentration [64].…”

“…Reports of O 2 À production by PMA activated hemocytes [6] and the ability of GT to inhibit O 2 À production by neutrophils [40] prompted an investigation into the effects of GT on O 2 À production by insect hemocytes. O 2 À was measured by determining the superoxide dismutase (SOD) (50 mg/ml)-inhibitable reduction of cytochrome c [43] and the characteristics of O 2 À production and cytochrome c reduction by hemocytes are summarized in Fig.…”

“…In addition this toxin has been associated with cases of clinical aspergillosis [38,39]. Reports have suggested that A. fumigatus may retard the action of neutrophils by inhibiting protein kinase C bII-related responses: p47 phox phosphorylation, its incorporation to the cytoskeleton, and the subsequent membrane translocation of p67 phox and p47 phox [40]. In a recent study it was shown that GT directly harms sites crucial for electron transport in flavocytochrome b 558 [41].…”

A Polysaccharide Virulence Factor of a Human Fungal Pathogen Induces Neutrophil Apoptosis via NK Cells

“…Some of the toxins and enzymes it produces may facilitate its growth within the host and impair the host's immune response. In particular, toxins, such as gliotoxin and fumagillin, disrupt the formation of a functional NADPH oxidase complex in PMNs and inhibit both PMN degranulation and F-actin formation (13,14). In addition, the rodlet layer of the conidial surface silences the PMN response (15).…”