Biosynthesis and function of gliotoxin in Aspergillus fumigatus.

Gliotoxin (GT) is the prototype of the epidithiodioxopiperazine (ETP)-type fungal toxins. GT plays a critical role in the pathobiology of Aspergillus fumigatus. It modulates the immune response and induces apoptosis in different cell types. The toxicity has been attributed to the unusual intramolecular disulfide bridge, which is the functional motif of all ETPs. Because of the extraordinary structure and activity of GT, this fungal metabolite has been the subject of many investigations. The biosynthesis of GT involves unprecedented reactions catalysed by recently discovered enzymes. Here, we summarize the recent progress in elucidating the GT biosynthetic pathway and its role in virulence.

The MAP kinase MpkA controls cell wall integrity, oxidative stress response, gliotoxin production and iron adaptation in Aspergillus fumigatus.

The saprophytic fungus Aspergillus fumigatus is the most important air-borne fungal pathogen. The cell wall of A. fumigatus has been studied intensively as a potential target for development of effective antifungal agents. A major role in maintaining cell wall integrity is played by the mitogen-activated protein kinase (MAPK) MpkA. To gain a comprehensive insight into this central signal transduction pathway, we performed a transcriptome analysis of the ΔmpkA mutant under standard and cell wall stress conditions. Besides genes involved in cell wall remodelling, protection against ROS and secondary metabolism such as gliotoxin, pyomelanin and pseurotin A, also genes involved in siderophore biosynthesis were regulated by MpkA. Consistently, northern and western blot analyses indicated that iron starvation triggers phosphorylation and thus activation of MpkA. Furthermore, localization studies indicated that MpkA accumulates in the nucleus under iron depletion. Hence, we report the first connection between a MAPK pathway and siderophore biosynthesis. The measurement of amino acid pools and of the pools of polyamines indicated that arginine was continuously converted into ornithine to fuel the siderophore pool in the ΔmpkA mutant strain. Based on our data, we propose that MpkA fine-tunes the balance between stress response and energy consuming cellular processes.

A dedicated glutathione S-transferase mediates carbon-sulfur bond formation in gliotoxin biosynthesis.

Gliotoxin is a virulence factor of the human pathogen Aspergillus fumigatus , the leading cause of invasive aspergillosis. Its toxicity is mediated by the unusual transannular disulfide bridge of the epidithiodiketopiperazine (ETP) scaffold. Here we disclose the critical role of a specialized glutathione S-transferase (GST), GliG, in enzymatic sulfurization. Furthermore, we show that bishydroxylation of the diketopiperazine by the oxygenase GliC is a prerequisite for glutathione adduct formation. This is the first report of the involvement of a GST in enzymatic C-S bond formation in microbial secondary metabolism.

Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin.

The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and in part immunocompetent patients. A. fumigatus can grow in multicellular communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic- and biofilm-grown A. fumigatus mycelium after 24 and 48 h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24 h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes, which code for hydrophobins, and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by real-time PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may also play a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma.

Transannular disulfide formation in gliotoxin biosynthesis and its role in self-resistance of the human pathogen Aspergillus fumigatus.

Gliotoxin (1), the infamous representative of the group of epipolythiodioxopiperazines (ETPs), is a virulence factor of the human pathogenic fungus Aspergillus fumigatus. The unique redox-sensitive transannular disulfide bridge is critical for deleterious effects caused by redox cycling and protein conjugation in the host. Through a combination of genetic, biochemical, and chemical analyses, we found that 1 results from GliT-mediated oxidation of the corresponding dithiol. In vitro studies using purified GliT demonstrate that the FAD-dependent, homodimeric enzyme utilizes molecular oxygen as terminal electron acceptor with concomitant formation of H(2)O(2). In analogy to the thiol-disulfide oxidoreductase superfamily, a model for dithiol-disulfide exchange involving the conserved CxxC motif is proposed. Notably, while all studied disulfide oxidases invariably form intra- or interchenar disulfide bonds in peptides, GliT is the first studied enzyme producing an epidithio bond. Furthermore, through sensitivity assays using wild type, Delta gliT mutant, and complemented strain, we found that GliT confers resistance to the producing organism. A phylogenetic study revealed that GliT falls into a clade of yet fully uncharacterized fungal gene products deduced from putative ETP biosynthesis gene loci. GliT thus not only represents the prototype of ETP-forming enzymes in eukaryotes but also delineates a novel mechanism for self-resistance.