Enhancing gliotoxins production in deep-sea derived fungus Dichotomocyes cejpii by engineering the biosynthetic pathway.

Gliotoxin can be developed as potent biopesticide. In this study, the positive transcriptional factor gliZ, glutathione-S transferase encoding gene gliG and gliN were firstly deleted by CRISPR/Cas9 system, which abolished the production of gliotoxin-like compounds in Dichotomomyces cejpii. CRISPR/dCas9 system targeting promoter of gliG was used to activate the biosynthetic genes in gli cluster. The overexpression of gliZ, gliN and gliG can significantly improve the yield of gliotoxin-like compunds. The gliotoxin yields was improved by 16.38 ± 1.36 fold, 18.98 ± 1.28 fold through gliZ overexpression and gliM deletion in D. cejpii FS110. In addtion, gliN was heterologously expressed in E. coli, the purified GliN can catalyze gliotoxin into methyl-gliotoxin. Furthermore, the binding sequences of GliZ in the promoters of gliG was determined by Dnase footprinting. This study firstly illustrated the transcriptional regulatory mechanism of DcGliZ for the gliotoxin biosynthesis in D. cejpii, and improved the yields of gliotoxins significantly in D. cejpii via biosynthetic approaches.

Structural and Mechanistic Insights into C-S Bond Formation in Gliotoxin.

Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.

N-Heterocyclization in Gliotoxin Biosynthesis is Catalyzed by a Distinct Cytochrome P450 Monooxygenase.

Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human-pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways.

Julichrome derivatives and gliotoxin from a soil derived Streptomyces sp.

Six julichrome derivatives including a new monomeric julichrome named as julichrome Q10 (1), and previous reported julichrome Q6 (2), julichrome Q6.6 (4), julichrome Q3.5 (5), julichrome Q5.6 (6), julichrome Q2.3 (7), along with a diketopiperazine gliotoxin (3) were isolated from a soil derived strain Streptomyces sp. The structures of these compounds were identified by HR-ESI-MS, UV, IR and NMR methods. The isolated compounds were tested for their in vitro cytotoxicity against human hepatocarcinoma HepG-2 and SMMC-7721 cell lines, human breast cancer MCF-7 and MDA-MB-231 cell lines, and human normal heptical LO2 cell line. Gliotoxin (3) showed the most cytotoxic activity against the tested tumor cell lines, with IC50 values ranging from 0.11 to 1.45 µM. Julichrome Q6.6 (4) displayed selective cytotoxic activity against SMMC-7721, MCF-7 and MDA-MB-231 cell lines.

Dechdigliotoxins A-C, Three Novel Disulfide-Bridged Gliotoxin Dimers from Deep-Sea Sediment Derived Fungus Dichotomomyces cejpii.

Dechdigliotoxins A-C (1-3), which represented the first examples of gliotoxin dimers with an unprecedented exocyclic disulfide linkage, were obtained from a deep-sea derived fungus Dichotomomyces cejpii FS110. The structures of these compounds were elucidated on the basis of spectroscopic analysis and the absolute configurations were unambiguously determined through quantum chemical calculations, as well as DP4+ probability simulations. The proposed biosynthetic pathway suggested 1-3 were generated from unusual L-Phe and D-Ser. All the isolates were evaluated for their cytotoxicity against four tumor cell lines.

Diketopiperazine Formation in Fungi Requires Dedicated Cyclization and Thiolation Domains.

Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5-diketopiperazines (DKPs) is a crucial step in the biosynthesis of a large number of bioactive natural products. However, the mechanism of DKP formation in fungi has remained unclear, despite extensive studies of their biosyntheses. Here we show that DKP formation en route to the fungal virulence factor gliotoxin requires a seemingly extraneous couplet of condensation (C) and thiolation (T) domains in the NRPS GliP. In vivo truncation of GliP to remove the CT couplet or just the T domain abrogated production of gliotoxin and all other gli pathway metabolites. Point mutation of conserved active sites in the C and T domains diminished cyclization activity of GliP in vitro and abolished gliotoxin biosynthesis in vivo. Verified NRPSs of other fungal DKPs terminate with similar CT domain couplets, suggesting a conserved strategy for DKP biosynthesis by fungal NRPSs.

Exposure to N-chlorotaurine induces oxidative stress responses in Aspergillus fumigatus.

PURPOSE: The neutrophil-derived oxidant N-chlorotaurine (NCT) displays remarkable in vivo tolerability and efficacy against a range of pathogens. The aim of this study was to characterize the response of the pulmonary pathogen Aspergillus fumigatus to NCT. METHODOLOGY: The effect of NCT on the growth and viability of A. fumigatus was characterized. NCT-induced alteration of amino acids and gliotoxin from A. fumigatus mycelium was assessed. Label-free shotgun quantitative proteomic analysis was performed on A. fumigatus exposed to NCT for 24 h. RESULTS: Incubation of A. fumigatus with NCT at concentrations ranging from 6.8 to 55 mM decreased conidial growth and viability, and mycelium biomass relative to the controls. Exposure to NCT (13.77 mM) resulted in increased amino acids and gliotoxin levels from A. fumigatus mycelium. Exposure of A. fumigatus mycelium to NCT (6.8 mM) revealed an enrichment in proteins associated with the ribosome, transcription and translation and non-ribosomal peptide biosynthesis (e.g. Pes1, Pes3), which play an essential role in oxidative stress resistance in A. fumigatus. A decrease in the abundance of proteins associated with fumagillin and pseurotin biosynthesis highlighted the anti-virulence activity of NCT. CONCLUSION: These results indicate that NCT induces an oxidative stress response in A. fumigatus as evidenced by alterations in the proteome and inhibits conidial and mycelial growth. Clinical investigations of topical application of NCT to treat Aspergillus infections are encouraged.

Interaction of a Novel Zn2Cys6 Transcription Factor DcGliZ with Promoters in the Gliotoxin Biosynthetic Gene Cluster of the Deep-Sea-Derived Fungus Dichotomomyces cejpii.

Gliotoxin is an important epipolythiodioxopiperazine, which was biosynthesized by the gli gene cluster in Aspergillus genus. However, the regulatory mechanism of gliotoxin biosynthesis remains unclear. In this study, a novel Zn2Cys6 transcription factor DcGliZ that is responsible for the regulation of gliotoxin biosynthesis from the deep-sea-derived fungus Dichotomomyces cejpii was identified. DcGliZ was expressed in Escherichia coli and effectively purified from inclusion bodies by refolding. Using electrophoretic mobility shift assay, we demonstrated that purified DcGliZ can bind to gliG, gliM, and gliN promoter regions in the gli cluster. Furthermore, the binding kinetics and affinity of DcGliZ protein with different promoters were measured by surface plasmon resonance assays, and the results demonstrated the significant interaction of DcGliZ with the gliG, gliM, and gliN promoters. These new findings would lay the foundation for the elucidation of future gliotoxin biosynthetic regulation mechanisms in D. cejpii.

Reduced-gliotoxin induces ROS-mediated anoikis in human colorectal cancer cells.

Reduced-gliotoxin is a small molecule derived from the secondary metabolites of marine fungi; compared to other gliotoxin analogues, it exhibits potent anticancer effects. However, the molecular basis of the death of colorectal cancer (CRC) cells induced by reduced-gliotoxin is unclear. Thus, the aim of this study was to investigate the potency of reduced-gliotoxin against CRC cells and to elucidate the underlying mechanisms. Cell morphology, flow cytometric analysis and western bolt analysis were performed to examine the functions and mechanisms of cell death induced by reduced-gliotoxin. Our findings demonstrated that reduced-gliotoxin triggered rapid cell detachment and induced anoikis in CRC cells. Mechanistically, our data indicated that the anoikis induced by reduced-gliotoxin was associated with the disruption of integrin-associated cell detachment and multiple signaling pathways. Furthermore, reduced-gliotoxin induced the excessive production of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP), resulting in the activation of both endogenous and exogenous apoptotic pathways and eventually, in the apoptosis of CRC cells. The blockage of ROS generation with N-acetylcysteine (NAC) attenuated the anoikis induced by reduced-gliotoxin. Taken together, these results suggest that reduced-gliotoxin may prove to be a potential candidate in the treatment of CRC.

Gliotoxin Biosynthesis: Structure, Mechanism, and Metal Promiscuity of Carboxypeptidase GliJ.

The formation of glutathione (GSH) conjugates, best known from the detoxification of xenobiotics, is a widespread strategy to incorporate sulfur into biomolecules. The biosynthesis of gliotoxin, a virulence factor of the human pathogenic fungus Aspergillus fumigatus, involves attachment of two GSH molecules and their sequential decomposition to yield two reactive thiol groups. The degradation of the GSH moieties requires the activity of the Cys-Gly carboxypeptidase GliJ, for which we describe the X-ray structure here. The enzyme forms a homodimer with each monomer comprising one active site. Two metal ions are present per proteolytic center, thus assigning GliJ to the diverse family of dinuclear metallohydrolases. Depending on availability, Zn2+, Fe2+, Fe3+, Mn2+, Cu2+, Co2+, or Ni2+ ions are accepted as cofactors. Despite this high metal promiscuity, a preference for zinc versus iron and manganese was noted. Mutagenesis experiments revealed details of metal coordination, and molecular modeling delivered insights into substrate recognition and processing by GliJ. The latter results suggest a reaction mechanism in which the two scissile peptide bonds of one gliotoxin precursor molecule are hydrolyzed sequentially and in a given order.

Antituberculosis compounds from a deep-sea-derived fungus Aspergillus sp. SCSIO Ind09F01.

Eleven diketopiperazine and fumiquinazoline alkaloids (1-11) together with a tetracyclic triterpenoid helvolic acid (12) were obtained from the cultures of a deep-sea derived fungus Aspergillus sp. SCSIO Ind09F01. The structures of these compounds (1-12) were determined mainly by the extensive NMR, ESIMS spectra data and by comparison with previously described compounds. Besides, anti-tuberculosis, cytotoxic, antibacterial, COX-2 inhibitory and antiviral activities of these compounds were evaluated. Gliotoxin (3), 12,13-dihydroxy-fumitremorgin C (11) and helvolic acid (12) exhibited very strong anti-tuberculosis activity towards Mycobacterium tuberculosis with the prominent MIC50 values of <0.03, 2.41 and 0.894 µM, respectively, which was here reported for the first time. Meanwhile gliotoxin also displayed significant selective cytotoxicities against K562, A549 and Huh-7 cell lines with the IC50 values of 0.191, 0.015 and 95.4 µM, respectively.

Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus.

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and ß-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.

Resistance is not futile: gliotoxin biosynthesis, functionality and utility.

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 self-protection 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.

Bioprospection of cytotoxic compounds in fungal strains recovered from sediments of the Brazilian coast.

The cytotoxic activities of extracts (50 µg/ml) from 48 fungal strains, recovered from sediments of Pecém's offshore port terminal (Northeast coast of Brazil), against HCT-116 colon cancer cell lines were investigated. The most promising extract was obtained from strain BRF082, identified as Dichotomomyces cejpii by phylogenetic analyses of partial RPB2 gene sequence. Thus, it was selected for bioassay-guided isolation of the cytotoxic compounds. Large-scale fermentation of BRF082 in potato dextrose broth, followed by chromatographic purification of the bioactive fractions from the liquid medium, yielded gliotoxin (4) and its derivatives acetylgliotoxin G (3), bis(dethio)bis(methylsulfanyl)gliotoxin (1), acetylgliotoxin (5), 6-acetylbis(dethio)bis(methylsulfanyl)gliotoxin (2), besides the quinazolinone alkaloid fiscalin B. All isolated compounds were tested for their cytotoxicities against the tumor cell lines HCT-116, revealing 4 and 3 as the most cytotoxic ones (IC50 0.41 and 1.06 µg/ml, resp.).

Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC).

A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb(3+)-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.

Exploring the chemodiversity and biological activities of the secondary metabolites from the marine fungus Neosartorya pseudofischeri.

The production of fungal metabolites can be remarkably influenced by various cultivation parameters. To explore the biosynthetic potentials of the marine fungus, Neosartorya pseudofischeri, which was isolated from the inner tissue of starfish Acanthaster planci, glycerol-peptone-yeast extract (GlyPY) and glucose-peptone-yeast extract (GluPY) media were used to culture this fungus. When cultured in GlyPY medium, this fungus produced two novel diketopiperazines, neosartins A and B (1 and 2), together with six biogenetically-related known diketopiperazines,1,2,3,4-tetrahydro-2, 3-dimethyl-1,4-dioxopyrazino[1,2-a]indole (3), 1,2,3,4-tetrahydro-2-methyl-3-methylen e-1,4-dioxopyrazino[1,2-a]indole (4), 1,2,3,4-tetrahydro-2-methyl-1,3,4-trioxopyrazino[1,2-a] indole (5), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio)gliotoxin (11), didehydrobisdethiobis(methylthio)gliotoxin (12) and N-methyl-1H-indole-2-carboxamide (6). However, a novel tetracyclic-fused alkaloid, neosartin C (14), a meroterpenoid, pyripyropene A (15), gliotoxin (7) and five known gliotoxin analogues, acetylgliotoxin (8), reduced gliotoxin (9), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio) gliotoxin (11) and bis-N-norgliovictin (13), were obtained when grown in glucose-containing medium (GluPY medium). This is the first report of compounds 3, 4, 6, 9, 10 and 12 as naturally occurring. Their structures were determined mainly by MS, 1D and 2D NMR data. The possible biosynthetic pathways of gliotoxin-related analogues and neosartin C were proposed. The antibacterial activity of compounds 2-14 and the cytotoxic activity of compounds 4, 5 and 7-13 were evaluated. Their structure-activity relationships are also preliminarily discussed.

Regulation of nonribosomal peptide synthesis: bis-thiomethylation attenuates gliotoxin biosynthesis in Aspergillus fumigatus.

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.

Opposed effects of enzymatic gliotoxin N- and S-methylations.

Gliotoxin (1), a virulence factor of the human pathogenic fungus Aspergillus fumigatus, is the prototype of epipoly(thiodioxopiperazine) (ETP) toxins. Here we report the discovery and functional analysis of two methyl transferases (MTs) that play crucial roles for ETP toxicity. Genome comparisons, knockouts, and in vitro enzyme studies identified a new S-adenosyl-l-methionine-dependent S-MT (TmtA) that is, surprisingly, encoded outside the gli gene cluster. We found that TmtA irreversibly inactivates ETP by S-alkylation and that this detoxification strategy appears to be not only limited to ETP producers. Furthermore, we unveiled that GliN functions as a freestanding amide N-MT. GliN-mediated amide methylation confers stability to ETP, damping the spontaneous formation of tri- and tetrasulfides. In addition, enzymatic N-alkylation constitutes the last step in gliotoxin biosynthesis and is a prerequisite for the cytotoxicity of the molecule. Thus, these specialized alkylating enzymes have dramatic and fully opposed effects: complete activation or inactivation of the toxin.

Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis.

Invasive aspergillosis is an opportunistic infection that is mainly caused by Aspergillus fumigatus, which is known to produce several secondary metabolites, including gliotoxin, the most abundant metabolite produced during hyphal growth. The diagnosis of invasive aspergillosis is often made late in the infection because of the lack of reliable and feasible diagnostic techniques; therefore, early detection is critical to begin treatment and avoid more serious complications. The present work reports the development and validation of an HPLC-MS/MS method for the detection of gliotoxin in the serum of patients with suspected aspergillosis. Chromatographic separation was achieved using an XBridge C18 column (150 × 2.1 mm id; 5 mm particle size) maintained at 25 °C with the corresponding guard column (XBridge C18, 10 × 2.1 mm id, 5 mm particle size). The mobile phase was composed of a gradient of water and acetonitrile/water (95:5 v/v), both containing 1 mM ammonium formate with a flow rate of 0.45 mL min(-1). Data from the validation studies demonstrate that this new method is highly sensitive, selective, linear, precise, accurate and free from matrix interference. The developed method was successfully applied to samples from patients suspected of having aspergillosis. Therefore, the developed method has considerable potential as a diagnostic technique for aspergillosis.

Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation.

The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement.