Reconstitution of the Early Stage of Chetomin Biosynthesis in Aspergillus fumigatus Leads to the Production of Epipolythiodioxopiperazines.

Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.

The evolution of the gliotoxin biosynthetic gene cluster in Penicillium fungi.

Fungi biosynthesize diverse secondary metabolites, small organic bioactive molecules with key roles in fungal ecology. Fungal secondary metabolites are often encoded by physically clustered genes known as biosynthetic gene clusters (BGCs). Fungi in the genus Penicillium produce a cadre of secondary metabolites, some of which are useful (e.g. the antibiotic penicillin and the cholesterol-lowering drug mevastatin) and others harmful (e.g. the mycotoxin patulin and the immunosuppressant gliotoxin) to human affairs. Fungal genomes often also encode resistance genes that confer protection against toxic secondary metabolites. Some Penicillium species, such as Penicillium decumbens, are known to produce gliotoxin, a secondary metabolite with known immunosuppressant activity. To investigate the evolutionary conservation of homologs of the gliotoxin BGC and of genes involved in gliotoxin resistance in Penicillium, we analyzed 35 Penicillium genomes from 23 species. Homologous, lesser fragmented gliotoxin BGCs were found in 12 genomes, mostly fragmented remnants of the gliotoxin BGC were found in 21 genomes, whereas the remaining 2 Penicillium genomes lacked the gliotoxin BGC altogether. In contrast, broad conservation of homologs of resistance genes that reside outside the BGC across Penicillium genomes was observed. Evolutionary rate analysis revealed that BGCs with higher numbers of genes evolve slower than BGCs with few genes, suggestive of constraint and potential functional significance or more recent decay. Gene tree-species tree reconciliation analyses suggested that the history of homologs in the gliotoxin BGC across the genus Penicillium likely involved multiple duplications, losses, and horizontal gene transfers. Our analyses suggest that genes encoded in BGCs can have complex evolutionary histories and be retained in genomes long after the loss of secondary metabolite biosynthesis.

Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

The Toxic Mechanism of Gliotoxins and Biosynthetic Strategies for Toxicity Prevention.

Gliotoxin is a kind of epipolythiodioxopiperazine derived from different fungi that is characterized by a disulfide bridge. Gliotoxins can be biosynthesized by a gli gene cluster and regulated by a positive GliZ regulator. Gliotoxins show cytotoxic effects via the suppression the function of macrophage immune function, inflammation, antiangiogenesis, DNA damage by ROS production, peroxide damage by the inhibition of various enzymes, and apoptosis through different signal pathways. In the other hand, gliotoxins can also be beneficial with different doses. Low doses of gliotoxin can be used as an antioxidant, in the diagnosis and treatment of HIV, and as an anti-tumor agent in the future. Gliotoxins have also been used in the control of plant pathogens, including Pythium ultimum and Sclerotinia sclerotiorum. Thus, it is important to elucidate the toxic mechanism of gliotoxins. The toxic mechanism of gliotoxins and biosynthetic strategies to reduce the toxicity of gliotoxins and their producing strains are summarized in this review.

Characterization of novel gliotoxin biosynthesis-related genes from deep-sea-derived fungus Geosmithia pallida FS140.

Gliotoxins are epipolythiodioxopiperazine toxins produced by the filamentous fungi, which show great potential in the treatment of liver and lung cancer because of its cytotoxicity. In this study, three novel genes related to gliotoxin biosynthesis, gliT, gliM and gliK encoding thioredoxin reductase, O-methyltransferase and gamma-glutamyl cyclotransferase, respectively, from the deep-sea-derived fungus Geosmithia pallida were cloned from G. pallida and expressed in Escherichia coli. The recombinant GliT, GliM and GliK proteins were expressed and purified by Ni affinity column, which was demonstrated by SDS-PAGE and Western blot analysis. The inclusion bodies of GliT were renatured and the corresponding enzymatic properties of the two enzymes were further investigated. Using DTNB as a substrate, GliT showed the highest enzymatic activity of 11041 mU/L at pH 7.0, and the optimal reaction temperature was 40 °C. Using EGCG as a substrate, GliM showed the highest enzymatic activity of 239.19 mU/mg at pH 7.0, the optimum temperature was 35 °C. GliK from G. pallida was firstly reported to show bi-function of glutymal cyclotransferase and acetyltransfearse actvity with highest enzymatic activity of 615.5 U/mg in this study. The results suggested the important enzymatic function of GliT, GliM and GliK in the gliotoxin biosynthesis in G. pallida, which would lay a foundation for the mechanism elucidation of the gliotoxin biosynthesis in G. pallida and the exploitation of novel gliotoxin derivaties.

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.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Gliotoxin, a Known Virulence Factor in the Major Human Pathogen Aspergillus fumigatus, Is Also Biosynthesized by Its Nonpathogenic Relative Aspergillus fischeri.

Aspergillus fumigatus is a major opportunistic human pathogen. Multiple traits contribute to A. fumigatus pathogenicity, including its ability to produce specific secondary metabolites, such as gliotoxin. Gliotoxin is known to inhibit the host immune response, and genetic mutants that inactivate gliotoxin biosynthesis (or secondary metabolism in general) attenuate A. fumigatus virulence. The genome of Aspergillus fischeri, a very close nonpathogenic relative of A. fumigatus, contains a biosynthetic gene cluster that is homologous to the A. fumigatus gliotoxin cluster. However, A. fischeri is not known to produce gliotoxin. To gain further insight into the similarities and differences between the major pathogen A. fumigatus and the nonpathogen A. fischeri, we examined whether A. fischeri strain NRRL 181 biosynthesizes gliotoxin and whether the production of secondary metabolites influences the virulence profile of A. fischeri We found that A. fischeri biosynthesizes gliotoxin under the same conditions as A. fumigatus However, whereas loss of laeA, a master regulator of secondary metabolite production (including gliotoxin biosynthesis), has previously been shown to reduce A. fumigatus virulence, we found that laeA loss (and loss of secondary metabolite production) in A. fischeri does not influence its virulence. These results suggest that LaeA-regulated secondary metabolites are virulence factors in the genomic and phenotypic background of the major pathogen A. fumigatus but are much less important in the background of the nonpathogen A. fischeri Understanding the observed spectrum of pathogenicity across closely related pathogenic and nonpathogenic Aspergillus species will require detailed characterization of their biological, chemical, and genomic similarities and differences.IMPORTANCEAspergillus fumigatus is a major opportunistic fungal pathogen of humans, but most of its close relatives are nonpathogenic. Why is that so? This important, yet largely unanswered, question can be addressed by examining how A. fumigatus and its close nonpathogenic relatives are similar or different with respect to virulence-associated traits. We investigated whether Aspergillus fischeri, a nonpathogenic close relative of A. fumigatus, can produce gliotoxin, a mycotoxin known to contribute to A. fumigatus virulence. We discovered that the nonpathogenic A. fischeri produces gliotoxin under the same conditions as those of the major pathogen A. fumigatus However, we also discovered that, in contrast to what has previously been observed in A. fumigatus, the loss of secondary metabolite production in A. fischeri does not alter its virulence. Our results are consistent with the "cards of virulence" model of opportunistic fungal disease, in which the ability to cause disease stems from the combination ("hand") of virulence factors ("cards") but not from individual factors per se.

The Role of Zinc in Gliotoxin Biosynthesis of Aspergillus fumigatus.

Zinc performs diverse physiological functions, and virtually all living organisms require zinc as an essential trace element. To identify the detailed function of zinc in fungal pathogenicity, we carried out cDNA microarray analysis using the model system of Aspergillus fumigatus, a fungal pathogen. From microarray analysis, we found that the genes involved in gliotoxin biosynthesis were upregulated when zinc was depleted, and the microarray data were confirmed by northern blot analysis. In particular, zinc deficiency upregulated the expression of GliZ, which encodes a Zn2-Cys6 binuclear transcription factor that regulates the expression of the genes required for gliotoxin biosynthesis. The production of gliotoxin was decreased in a manner inversely proportional to the zinc concentration, and the same result was investigated in the absence of ZafA, which is a zinc-dependent transcription activator. Interestingly, we found two conserved ZafA-binding motifs, 5'-CAAGGT-3', in the upstream region of GliZ on the genome and discovered that deletion of the ZafA-binding motifs resulted in loss of ZafA-binding activity; gliotoxin production was decreased dramatically, as demonstrated with a GliZ deletion mutant. Furthermore, mutation of the ZafA-binding motifs resulted in an increase in the conidial killing activity of human macrophage and neutrophil cells, and virulence was decreased in a murine model. Finally, transcriptomic analysis revealed that the expression of ZafA and GliZ was upregulated during phagocytosis by macrophages. Taken together, these results suggest that zinc plays an important role in the pathogenicity of A. fumigatus by regulating gliotoxin production during the phagocytosis pathway to overcome the host defense system.

RgsA Attenuates the PKA Signaling, Stress Response, and Virulence in the Human Opportunistic Pathogen Aspergillus fumigatus.

The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, six RGS protein-encoding genes are present. To characterize the rgsA gene predicted to encode a protein with an RGS domain, we generated an rgsA null mutant and observed the phenotypes of the mutant. The deletion (Δ) of rgsA resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators abaA, brlA, and wetA are elevated in the ΔrgsA mutant. Moreover, ΔrgsA resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by ΔrgsA. In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of rgsA, and the ΔrgsA spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the ΔrgsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the rgsA null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by ΔrgsA, and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation-likely by attenuating PKA signaling.

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.

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.

Heterogeneity in Pathogenicity-related Properties and Stress Tolerance in Aspergillus fumigatus Clinical Isolates.

Stress responses and pathogenicity have been extensively studied in Aspergillus fumigatus, the main causative pathogen of life-threatening aspergillosis. The heterogeneity in this pathogen's biology has recently attracted increasing attention. In the present work, we used 16 clinically isolated strains to investigate several properties relevant to the pathogenicity of A. fumigatus, namely, gliotoxin production, elastase activity, hypoxia growth, adaptation to iron-limiting conditions, and growth upon nitrosative, oxidative, and high osmotic stresses. The range of phenotypes was diverse across the strains, with gliotoxin production and elastase activity being negatively correlated at an intermediate index (R=-0.4717). Notably, there were strains that showed extraordinary high production of gliotoxin or elastase activity and hypersensitivity to nitrosative or oxidative stresses. Clustering analysis showed that the 7 potentially pathogenicity-related phenotypes were not correlated with the genetic sub-group or pathotype. These results contribute to the growing awareness of the genetic and phenotypic diversity in A. fumigatus isolates.

Ferricrocin, the intracellular siderophore of Trichoderma virens, is involved in growth, conidiation, gliotoxin biosynthesis and induction of systemic resistance in maize.

Fungal siderophores are known to be involved in iron acquisition and storage, as well as pathogenicity of mammals and plants. As avirulent plant symbionts, Trichoderma spp. colonize roots and induce resistance responses both locally and systemically. To study the role of intracellular siderophore(s) in Trichoderma-plant interactions, we have obtained mutants in a non-ribosomal peptide synthetase, TvTex10, that was predicted to be involved in intracellular siderophore(s) biosynthesis. This gene has a detectable basal level of expression and is also upregulated under iron-deplete conditions. This is unlike two other siderophore-encoding genes, which are tightly regulated by iron. Disruption of tex10 gene using homologous recombination resulted in mutants with enhanced growth rate, reduced conidiation and hyper-sensitivity to oxidative stress as compared to wildtype strain. The mutants also produced reduced levels of gliotoxin and dimethyl gliotoxin but have enhanced ability to colonize maize seedling roots. The mutants were also impaired in induction of induced systemic resistance (ISR) in maize against the foliar pathogen Cochliobolus heterostrophus.

De Novo Transcriptome Sequencing of the Deep-Sea-Derived Fungus Dichotomomyces cejpii and Analysis of Gliotoxin Biosynthesis Genes.

Gliotoxin, produced by fungi, is an epipolythiodioxopiperazine (ETP) toxin with bioactivities such as anti-liver fibrosis, antitumor, antifungus, antivirus, antioxidation, and immunoregulation. Recently, cytotoxic gliotoxins were isolated from a deep-sea-derived fungus, Dichotomomyces cejpii. However, the biosynthetic pathway for gliotoxins in D. cejpii remains unclear. In this study, the transcriptome of D. cejpii was sequenced using an Illumina Hiseq 2000. A total of 19,125 unigenes for D. cejpii were obtained from 9.73 GB of clean reads. Ten genes related to gliotoxin biosynthesis were annotated. The expression levels of gliotoxin-related genes were detected through quantitative real-time polymerase chain reaction (qRT-PCR). The GliG gene, encoding a glutathione S-transferase (DC-GST); GliI, encoding an aminotransferase (DC-AI); and GliO, encoding an aldehyde reductase (DC-AR), were cloned and expressed, purified, and characterized. The results suggested the important roles of DC-GST, DC-AT, and DC-AR in the biosynthesis of gliotoxins. Our study on the genes related to gliotoxin biosynthesis establishes a molecular foundation for the wider application of gliotoxins from D. cejpii in the biomedical industry in the future.

Selenate sensitivity of a laeA mutant is restored by overexpression of the bZIP protein MetR in Aspergillus fumigatus.

LaeA is a conserved global regulator of secondary metabolism and development in filamentous fungi. Examination of Aspergillus fumigatus transcriptome data of laeA deletion mutants have been fruitful in identifying genes and molecules contributing to the laeA mutant phenotype. One of the genes significantly down regulated in A. fumigatus ΔlaeA is metR, encoding a bZIP DNA binding protein required for sulfur and methionine metabolism in fungi. LaeA and MetR deletion mutants exhibit several similarities including down regulation of sulfur assimilation and methionine metabolism genes and ability to grow on the toxic sulfur analog, sodium selenate. However, unlike ΔmetR, ΔlaeA strains are able to grow on sulfur, sulfite, and cysteine. To examine if any parameter of the ΔlaeA phenotype is due to decreased metR expression, an over-expression allele (OE::metR) was placed in a ΔlaeA background. The OE::metR allele could not significantly restore expression of MetR regulated genes in ΔlaeA but did restore sensitivity to sodium selenate. In A. nidulans a second bZIP protein, MetZ, also regulates sulfur and methionine metabolism genes. However, addition of an OE::metZ construct to the A. fumigatus ΔlaeA OE::metR strain still was unable to rescue the ΔlaeA phenotype to wildtype with regards gliotoxin synthesis and virulence in a zebrafish aspergillosis model.

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.

Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice.

Delayed diagnosis in invasive aspergillosis (IA) contributes to its high mortality. Gliotoxin (GT) and bis-methyl-gliotoxin (bmGT) are secondary metabolites produced by Aspergillus during invasive, hyphal growth and may prove diagnostically useful. Because IA pathophysiology and GT's role in virulence vary depending on the underlying host immune status, we hypothesized that GT and bmGT production in vivo may differ in three mouse models of IA that mimic human disease. We defined temporal kinetics of GT and bmGT in serum, bronchoalveolar lavage fluid (BALF) and lungs of A. fumigatus-infected chronic granulomatous disease (CGD), hydrocortisone-treated, and neutropenic mice. We harvested lungs for assessment of fungal burden, histology and GT/bmGT biosynthetic genes' mRNA induction. GT levels were higher in neutropenic versus CGD or steroid-treated lungs. bmGT was persistently detected only in CGD lungs. GT, but not bmGT, was detected in 71% of sera and 50% of BALF of neutropenic mice; neither was detected in serum/BALF of CGD or steroid-treated mice. Enrichment of GT in Aspergillus-infected neutropenic lung correlated with fungal burden and hyphal length but not induction of GT biosynthetic genes. In summary, GT is detectable in mouse lungs, serum and BALF during neutropenic IA, suggesting that GT may be useful to diagnose IA in neutropenic patients.

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.