Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic animal pathogen Aspergillus fumigatus. It is a member of the epipolythiodioxopiperazine (ETP) class of toxins characterised by a disulphide bridged cyclic dipeptide. A putative cluster of 12 genes involved in gliotoxin biosynthesis has been identified in A. fumigatus by a comparative genomics approach based on homology to genes from the sirodesmin (another ETP) biosynthetic gene cluster of Leptosphaeria maculans. The physical limits of the cluster in A. fumigatus have been defined by bioinformatics and by identifying the genes that are co-regulated and whose timing of expression correlates with the production of gliotoxin in culture.     

Fungal Secondary Metabolites and Their Fundamental Roles in Human Mycoses

Understanding which fungal factors allow colonization and infection of a human host is critical to lowering the incidence of human mycoses and related mortalities. In the pathogen Aspergillus fumigatus, secondary metabolites, small bioactive molecules produced by many opportunistic fungal pathogens, have important roles in suppressing and providing protection from host defenses. Deletion of LaeA, a global regulator of secondary metabolism in fungi, significantly decreases A. fumigatus virulence, in part owing to loss of gliotoxin and hydrophobin production. In addition to gliotoxin, dihydroxynaphthalene (DHN) melanin and siderophores are other A. fumigatus virulence factors; all three metabolites are derived from hallmark secondary metabolite gene clusters. Many of the gene clusters producing toxin metabolites have yet to be deciphered, and the study of secondary metabolites and their role in the virulence of human pathogens is a nascent field.     

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.     

Dysregulated gliotoxin biosynthesis attenuates the production of unrelated biosynthetic gene cluster-encoded metabolites in Aspergillus fumigatus

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. The gliotoxin bis-thiomethyltransferase, GtmA, attenuates gliotoxin biosynthesis in A. fumigatus by conversion of dithiol gliotoxin to bis-thiomethylgliotoxin (BmGT). Here we show that disruption of dithiol gliotoxin bis-thiomethylation functionality in A. fumigatus results in significant remodelling of the A. fumigatus secondary metabolome upon extended culture. RP-HPLC and LC–MS/MS analysis revealed the reduced production of a plethora of unrelated biosynthetic gene cluster-encoded metabolites, including pseurotin A, fumagillin, fumitremorgin C and tryprostatin B, occurs in A. fumigatus ΔgtmA upon extended incubation. Parallel quantitative proteomic analysis of A. fumigatus wild-type and ΔgtmA during extended culture revealed cognate abundance alteration of proteins encoded by relevant biosynthetic gene clusters, allied to multiple alterations in hypoxia-related proteins. The data presented herein reveal a previously concealed functionality of GtmA in facilitating the biosynthesis of other BGC-encoded metabolites produced by A. fumigatus.     

Gliotoxin in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>: an example that mycotoxins are potential virulence factors

Moulds produce several different mycotoxins that may improve their chance of survival in particular environments. For example, Aspergillus fumigatus, an important human pathogen, produces several mycotoxins including gliotoxin. This secondary metabolite, a small lipid soluble dipeptide, exerts toxic effects on phagocytic cells and T-lymphocytes at low concentrations in vitro. A. fumigatus also produces high levels of gliotoxin in vivo, and this suggests that host defense mechanisms might be impaired by this metabolite during host infection. In the past few years, the genes responsible for the production of gliotoxin in A. fumigatus have been identified and more recently gliotoxin-minus mutants have been used in animal experiments to ascertain the biological role of this product. Mycotoxins have also been shown to act as virulence factors in some fungal infections of insects and plants.     

Gliotoxin effects on fungal growth: mechanisms and exploitation

Although 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 μg/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 ΔgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Δsod1 and Δyap1) were hypersensitive to exogenous gliotoxin, while Δgsh1 was resistant. Significant gliotoxin-mediated (5 μg/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 μg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus ΔgliT, 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.     

Isolation and identification of Aspergillus fumigatus mycotoxins on growth medium and some building materials

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions.     

Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the non-ribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wild-type strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.     

Candida species fail to produce the immunosuppressive secondary metabolite gliotoxin in vitro

Yeasts of the genus Candida are a major cause of morbidity and mortality in immunocompromised patients. Despite new insights in recent years, the pathogenesis of Candida infection is still incompletely understood. Previous studies have suggested that gliotoxin, a secondary fungal metabolite with well-known immunosuppressive effects, is produced by various species of the genus Candida, and a possible role of gliotoxin as a virulence factor of C. albicans has also been discussed. However, until now, no definitive evidence has been provided that members of the genus Candida are able to produce gliotoxin. To clarify this question, we tested a total of 100 clinical isolates of C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis for gliotoxin production using a highly sensitive HPLC protocol, and, for selected isolates, confirmed our findings by tandem MS. This approach did not detect intracellular or extracellular gliotoxin production by any of the isolates examined, although various culture conditions were applied. Therefore, in contrast to previous studies, our data strongly suggest that at least the Candida species investigated in this study are not able to produce the secondary metabolite gliotoxin.     

Biosynthetic gene clusters for epipolythiodioxopiperazines in filamentous fungi

Epipolythiodioxopiperazines (ETPs) are toxic secondary metabolites made only by fungi. Recent research efforts have provided insight into the molecular mechanisms of the toxicity of these compounds. The availability of complete genome sequences for many fungi has also facilitated the identification of putative ETP biosynthetic gene clusters. Expression and mutational analyses have confirmed the role of such gene clusters in the biosynthesis of two ETPs, sirodesmin PL and gliotoxin. The creation of mutants unable to produce these ETPs has facilitated the assessment of these molecules as virulence factors in interactions between pathogenic fungi and their hosts.     

Cytotoxic Substances from Aspergillus fumigatus in Oxygenated or Poorly Oxygenated Environment

Aspergillus fumigatus often causes serious health problems. The airway of the human body, the most common initial site of damage, is always exposed to an oxygenated condition, and the oxygen concentration may play a critical role in the virulence of A. fumigatus. In this study, oxygen content, fungal growth, the production of cytotoxic substance(s) in the fungal culture, and their relationship were investigated. Two clinical strains of A. fumigatus were cultured under certain oxygen contents (10, 14 and 20%), and cytotoxicity of their culture filtrates on murine macrophages and their fungal growth were evaluated. The components of these filtrates were analyzed by gas chromatography-mass spectrometry. All culture filtrates contained gliotoxin and showed potent cytotoxicity on macrophages at very low concentration. The amount of gliotoxin in the culture filtrate prepared at 10% oxygen was markedly less, but diminutions in fungal growth and cytotoxicity of this culture filtrate were negligible. These results suggest that a well-oxygenated condition is suitable for the production of gliotoxin by A. fumigatus. A significant role of cytotoxic substances(s) other than gliotoxin is also suggested.     

Toll-like receptors and innate antifungal responses

The mammalian Toll-like receptors (TLRs) are homologues of Drosophila Toll and constitute a novel protein family involved in the mediation of innate immunity and the activation of adaptive immunity. Analysis of infection with human pathogenic fungi Candida albicans and Aspergillus fumigatus implicated TLR2 and TLR4 in elicitation of immune responses. Cryptococcus neoformans is recognized by a process that uses TLR4. C. albicans induces immunostimulation through causative agents, such as mannan or its structural derivatives (e.g. phospholipomannan), which are recognized by the immune system as pathogen-associated molecular patterns and are located in the cell wall of fungi. Secreted aspartic proteinases represent a key virulence factor that contributes to the ability of C. albicans to cause mucosal and disseminated infections, and might be a further potential stimulator of TLRs. Simultaneous activation of other pattern recognition receptors collaborating with TLRs illustrates the cooperation of various chains within ligand-specific receptor complexes for the recognition of fungal pathogens and their cell wall components.     

Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.     

Antimicrobial Activities of Selected Cyathus Species

Twelve selected Cyathus species were tested for their abilities to produce antimicrobial metabolites. Most of them were found to produce secondary exo-metabolites that could induce morphological abnormalities of rice pathogenic fungi Pyricularia oryzae. Some extracts from the cultivated liquid obviously inhibited human pathogenic fungi Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Activities against six human pathogenic bacteria were also obtained from some of these extracts.