Gliotoxin is a redox-active nonribosomal peptide produced by Aspergillus fumigatus. Like many other disulfide-containing epipolythiodioxopiperazines, a bis-thiomethylated form is also produced. In the case of gliotoxin, bisdethiobis(methylthio)gliotoxin (BmGT) is formed for unknown reasons by a cryptic enzyme. Here, we identify the S-adenosylmethionine-dependent gliotoxin bis-thiomethyltransferase (GtmA), which converts dithiogliotoxin to BmGT. This activity, which is induced by exogenous gliotoxin, is only detectable in protein lysates of A. fumigatus deficient in the gliotoxin oxidoreductase, gliT. Thus, GtmA is capable of substrate bis-thiomethylation. Deletion of gtmA completely abrogates BmGT formation and we now propose that the purpose of BmGT formation is primarily to attenuate gliotoxin biosynthesis. Phylogenetic analysis reveals 124 GtmA homologs within the Ascomycota phylum. GtmA is encoded outside the gliotoxin biosynthetic cluster and primarily serves to negatively regulate gliotoxin biosynthesis. This mechanism of postbiosynthetic regulation of nonribosomal peptide synthesis appears to be quite unusual.

Gliotoxin biosynthesis is encoded by the gli gene cluster in Aspergillus fumigatus. The biosynthesis of gliotoxin is influenced by a suite of transcriptionally-active regulatory proteins and a bis-thiomethyltransferase. A selfprotection system against gliotoxin is present in A. fumigatus. Several additional metabolites are also produced via the gliotoxin biosynthetic pathway. Moreover, the biosynthesis of unrelated natural products appears to be influenced either by gliotoxin or by the activity of specific reactions within the biosynthetic pathway. The activity of gliotoxin against animal cells and fungi, often mediated by interference with redox homeostasis or protein modification, is revealing new metabolic interactions within eukaryotic systems. Nature has provided a most useful natural product with which to reveal some of its many molecular secrets. Contextualizing and rethinking gliotoxinAspergillus fumigatus is an opportunistic fungal pathogen and primarily infects immunocompromised individuals where it can cause fatal invasive aspergillosis (IA) [1]. A. fumigatus exposure can also induce debilitating aspergillosis and allergy in immunocompetent individuals [2,3]. Selected secondary metabolites produced by A. fumigatus, in particular siderophores and the non-ribosomal peptide gliotoxin, are generally considered to be front-line virulence factors [4,5]. Gliotoxin is an epipolythiodioxopiperazine (ETP) of molecular mass 326 Da, and contains a disulfide bridge which can undergo repeating cleavage and reformation, thereby resulting in a potent intracellular redox activity (Figure 1) [6]. Indeed, the dithiol form of gliotoxin has also been posited to be responsible for the observed biological activities of gliotoxin [7]. Bisdethiobis(methylthio)gliotoxin (BmGT) and related gliotoxin metabolites (Figure 1) are also biosynthesized by A. fumigatus [8,9]. Incredibly, the gliotoxin biosynthetic pathway had remained elusive since the discovery of gliotoxin in 1936; however, recent studies have not only dissected this unusual molecular assembly system but have revealed the necessity for gliotoxin-producing fungi to possess an endogenous resistance system against gliotoxin [10,11]. Moreover, because gliotoxin can be considered as the prototype ETP, studies on gliotoxin can be instrumental in revealing the biosynthetic mechanisms of related ETPs which are biosynthesized by a range of fungi [12]. Amongst others, these include sirodesmin A, sporidesmin A, chaetocin, aranotin, and chetomin [13]. Studies on the biosynthetic mechanism of ETPs, particularly gliotoxin, are also serving to inspire new synthetic chemistry approaches for ETP synthesis and desulfurization, which are somewhat beyond the scope of the present review [13,14].In addition to studying how gliotoxin contributes to organismal virulence, it has also been deployed to explore and reveal novel biochemistry within both fungal and animal cells [15,16]. Thus, we contend that it is the ability of gliotoxin to interfere with so many cellular processes that makes it...

Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the A. mellea genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (n = 629 unique) and secreted (n = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (n = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the A. mellea proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that A. mellea exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive A. mellea proteins (n = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms.

Mechanistic studies on gliotoxin biosynthesis and self-protection in Aspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an A. fumigatus ⌬gliA strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the gli cluster, as disabling gli cluster activation, via gliZ deletion, attenuates the sensitivity of an A. fumigatus ⌬gliT strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin bis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ⌬gliT strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant S-adenosylmethionine (SAM) depletion and S-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of gliT-deficient A. fumigatus to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the A. fumigatus ⌬gliK strain, but not the ⌬gliG strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence. Biosynthesis, self-protection mechanisms, and functionality of gliotoxin and related epidithiodiketopiperazine (ETP) molecular species, such as chaetocin and acetylaranotin, are attracting ever-increasing attention as a consequence of findings from highthroughput genome sequencing projects, application of gene deletion technologies, and mass spectrometric analytical methodologies (1-5). Indeed, existing paradigms of gliotoxin (Fig. 1) as a toxin and the perspective of the disulfide bridge-containing (oxidized) form as the final, or only, product are undergoing significant reconsideration (6-11).Self-protection against disulfide-containing metabolites appears to be essential in both fungi and bacteria. It has been demonstrated that the gliotoxin oxidoreductase GliT (12), encoded within the gli cluster, protects Aspergillus fumigatus against exogenous gliotoxin and is essential for gliotoxin biosynthesis (12, 13). A similar mechanism for self-protection against holomycin in Streptomyces clavulige...

a b s t r a c tAlthough initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 lg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up-and down-regulated >1.9-fold; p < 0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p < 0.05), along with concomitant resistance to diamide, were evident in A. fumigatus DgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Dsod1 and Dyap1) were hypersensitive to exogenous gliotoxin, while Dgsh1 was resistant. Significant gliotoxin-mediated (5 lg/ml) growth inhibition (p < 0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p < 0.001) at gliotoxin (10 lg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus DgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.