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.

Degradation mechanism of AtrA mediated by ClpXP and its application in daptomycin production in Streptomyces roseosporus.

The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A-factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull-down assays, bacterial two-hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA-QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.

Urease of Aspergillus fumigatus Is Required for Survival in Macrophages and Virulence.

The number of patients suffering from fungal diseases has constantly increased during the last decade. Among the fungal pathogens, the airborne filamentous fungus Aspergillus fumigatus can cause chronic and fatal invasive mold infections. So far, only three major classes of drugs (polyenes, azoles, and echinocandins) are available for the treatment of life-threatening fungal infections, and all present pharmacological drawbacks (e.g., low solubility or toxicity). Meanwhile, clinical antifungal-resistant isolates are continuously emerging. Therefore, there is a high demand for novel antifungal drugs, preferentially those that act on new targets. We studied urease and the accessory proteins in A. fumigatus to determine their biochemical roles and their influence on virulence. Urease is crucial for the growth on urea as the sole nitrogen source, and the transcript and protein levels are elevated on urea media. The urease deficient mutant displays attenuated virulence, and its spores are more susceptible to macrophage-mediated killing. We demonstrated that this observation is associated with an inability to prevent the acidification of the phagosome. Furthermore, we could show that a nickel-chelator inhibits growth on urea. The nickel chelator is also able to reverse the effects of urease on macrophage killing and phagosome acidification, thereby reducing virulence in systemic and trachea infection models. IMPORTANCE The development of antifungal drugs is an urgent task, but it has proven to be difficult due to many similarities between fungal and animal cells. Here, we characterized the urease system in A. fumigatus, which depends on nickel for activity. Notably, nickel is not a crucial element for humans. Therefore, we went further to explore the role of nickel-dependent urease in host-pathogen interactions. We were able to show that urease is important in preventing the acidification of the phagosome and therefore reduces the killing of conidia by macrophages. Furthermore, the deletion of urease shows reduced virulence in murine infection models. Taken together, we identified urease as an essential virulence factor of A. fumigatus. We were able to show that the application of the nickel-chelator dimethylglyoxime is effective in both in vitro and in vivo infection models. This suggests that nickel chelators or urease inhibitors are potential candidates for the development of novel antifungal drugs.

Redirection of acyl donor metabolic flux for lipopeptide A40926B0 biosynthesis.

The metabolic flux of fatty acyl-CoAs determines lipopeptide biosynthesis efficiency, because acyl donor competition often occurs from polyketide biosynthesis and homologous pathways. We used A40926B0 as a model to investigate this mechanism. The lipopeptide A40926B0 with a fatty acyl group is the active precursor of dalbavancin, which is considered as a new lipoglycopeptide antibiotic. The biosynthetic pathway of fatty acyl-CoAs in the A40926B0 producer Nonomuraea gerenzanensis L70 was efficiently engineered using endogenous replicon CRISPR (erCRISPR). A polyketide pathway and straight-chain fatty acid biosynthesis were identified as major competitors in the malonyl-CoA pool. Therefore, we modified both pathways to concentrate acyl donors for the production of the desired compound. Combined with multiple engineering approaches, including blockage of an acetylation side reaction, overexpression of acetyl-CoA carboxylase, duplication of the dbv gene cluster and optimization of the fermentation parameters, the final strain produced 702.4 mg l-1 of A40926B0, a 2.66-fold increase, and the ratio was increased from 36.2% to 81.5%. Additionally, an efficient erCRISPR-Cas9 editing system based on an endogenous replicon was specifically developed for L70, which increased conjugation efficiency by 660% and gene-editing efficiency was up to 90%. Our strategy of redirecting acyl donor metabolic flux can be widely adopted for the metabolic engineering of lipopeptide biosynthesis.

Structural insights into ligand recognition and activation of the melanocortin-4 receptor.

Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning MC4R-specific activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric Gs protein stimulated by the endogenous peptide ligand α-MSH, FDA-approved drugs afamelanotide (Scenesse™) and bremelanotide (Vyleesi™), and a selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and a distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.

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.

Reconstitution of Enzymatic Carbon-Sulfur Bond Formation Reveals Detoxification-Like Strategy in Fungal Toxin Biosynthesis.

Gliotoxin is a virulence factor of the human pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. The activity of this metabolite is mediated by a transannular disulfide bond, a hallmark of the epipolythiodiketopiperazine (ETP) family. Through the creation of fungal gene deletion mutants and heterologous protein expression, we unveiled the critical role of the cytochrome P450 monooxygenase (CYP450) GliC for the stepwise bishydroxylation of the diketopiperazine (DKP) core. We show for the first time the formation of the C-S bond from the DKP in a combined assay of GliC and the glutathione- S-transferase (GST) GliG in vitro. Furthermore, we present experimental evidence for an intermediary imine species. The flexible substrate scope of GliC and GliG in combination parallels P450/GST pairs used in eukaryotic phase I/II detoxification pathways.

A Nonredundant Phosphopantetheinyl Transferase, PptA, Is a Novel Antifungal Target That Directs Secondary Metabolite, Siderophore, and Lysine Biosynthesis in Aspergillus fumigatus and Is Critical for Pathogenicity.

Secondary metabolites are key mediators of virulence for many pathogens. Aspergillus fumigatus produces a vast array of these bioactive molecules, the biosynthesis of which is catalyzed by nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs). Both NRPSs and PKSs harbor carrier domains that are primed for acceptance of secondary metabolic building blocks by a phosphopantetheinyl transferase (P-pant). The A. fumigatus P-pant PptA has been shown to prime the putative NRPS Pes1 in vitro and has an independent role in lysine biosynthesis; however, its role in global secondary metabolism and its impact on virulence has not been described. Here, we demonstrate that PptA has a nonredundant role in the generation of the vast majority of detectable secondary metabolites in A. fumigatus, including the immunomodulator gliotoxin, the siderophores triacetylfusarinine C (TAFC) and ferricrocin (FC), and dihydroxy naphthalene (DHN)-melanin. We show that both the lysine and iron requirements of a pptA null strain exceed those freely available in mammalian tissues and that loss of PptA renders A. fumigatus avirulent in both insect and murine infection models. Since PptA lacks similarity to its mammalian orthologue, we assert that the combined role of this enzyme in both primary and secondary metabolism, encompassing multiple virulence determinants makes it a very promising antifungal drug target candidate. We further exemplify this point with a high-throughput fluorescence polarization assay that we developed to identify chemical inhibitors of PptA function that have antifungal activity.IMPORTANCE Fungal diseases are estimated to kill between 1.5 and 2 million people each year, which exceeds the global mortality estimates for either tuberculosis or malaria. Only four classes of antifungal agents are available to treat invasive fungal infections, and all suffer pharmacological shortcomings, including toxicity, drug-drug interactions, and poor bioavailability. There is an urgent need to develop a new class of drugs that operate via a novel mechanism of action. We have identified a potential drug target, PptA, in the fungal pathogen Aspergillus fumigatus PptA is required to synthesize the immunotoxic compound gliotoxin, DHN-melanin, which A. fumigatus employs to evade detection by host cells, the amino acid lysine, and the siderophores TAFC and FC, which A. fumigatus uses to scavenge iron. We show that strains lacking the PptA enzyme are unable to establish an infection, and we present a method which we use to identify novel antifungal drugs that inactivate PptA.

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.

Enzymatic Carbon-Sulfur Bond Formation in Natural Product Biosynthesis.

Sulfur plays a critical role for the development and maintenance of life on earth, which is reflected by the wealth of primary metabolites, macromolecules, and cofactors bearing this element. Whereas a large body of knowledge has existed for sulfur trafficking in primary metabolism, the secondary metabolism involving sulfur has long been neglected. Yet, diverse sulfur functionalities have a major impact on the biological activities of natural products. Recent research at the genetic, biochemical, and chemical levels has unearthed a broad range of enzymes, sulfur shuttles, and chemical mechanisms for generating carbon-sulfur bonds. This Review will give the first systematic overview on enzymes catalyzing the formation of organosulfur natural products.

Gliotoxin--bane or boon?

Gliotoxin (GT) is the most important epidithiodioxopiperazine (ETP)-type fungal toxin. GT was originally isolated from Trichoderma species as an antibiotic substance involved in biological control of plant pathogenic fungi. A few isolates of GT-producing Trichoderma virens are commercially marketed for biological control and widely used in agriculture. Furthermore, GT is long known as an immunosuppressive agent and also reported to have anti-tumour properties. However, recent publications suggest that GT is a virulence determinant of the human pathogen Aspergillus fumigatus. This compound is thus important on several counts - it has medicinal properties, is a pathogenicity determinant, is a potential diagnostic marker and is important in biological crop protection. The present article addresses this paradox and the ecological role of GT. We discuss the function of GT as defence molecule, the role in aspergillosis and suggest solutions for safe application of Trichoderma-based biofungicides.

Anion receptors based on halogen bonding with halo-1,2,3-triazoliums.

A systematic series of anion receptors based on bidentate halogen bonding by halo-triazoles and -triazoliums is presented. The influence of the halogen bond donor atom, the electron-withdrawing group, and the linker group that bridges the two donor moieties is investigated. Additionally, a comparison with hydrogen bond-based analogues is provided. A new, efficient synthetic approach to introduce different halogens into the heterocycles is established using silver(I)-triazolylidenes, which are converted to the corresponding halo-1,2,3-triazoliums with different halogens. Comprehensive nuclear magnetic resonance binding studies supported by isothermal titration calorimetry studies were performed with different halides and oxo-anions to evaluate the influence of key parameters of the halogen bond donor, namely, polarization of the halogen and the bond angle to the anion. The results show a larger anion affinity in the case of more charge-dense halides as well as a general preference of the receptors to bind oxo-anions, in particular sulfate, over halides.

Draft Genome Sequence and Gene Annotation of the Entomopathogenic Fungus Verticillium hemipterigenum.

Verticillium hemipterigenum (anamorph Torrubiella hemipterigena) is an entomopathogenic fungus and produces a broad range of secondary metabolites. Here, we present the draft genome sequence of the fungus, including gene structure and functional annotation. Genes were predicted incorporating RNA-Seq data and functionally annotated to provide the basis for further genome studies.

Genetic engineering activates biosynthesis of aromatic fumaric acid amides in the human pathogen Aspergillus fumigatus.

The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

Identification of immunogenic antigens from Aspergillus fumigatus by direct multiparameter characterization of specific conventional and regulatory CD4+ T cells.

CD4(+) T cells orchestrate immune responses against fungi, such as Aspergillus fumigatus, a major fungal pathogen in humans. The complexity of the fungal genome and lifestyle questions the existence of one or a few immune-dominant Ags and complicates systematic screening for immunogenic Ags useful for immunotherapy or diagnostics. In this study, we used a recently developed flow cytometric assay for the direct ex vivo characterization of A. fumigatus-specific CD4(+) T cells for rapid identification of physiological T cell targets in healthy donors. We show that the T cell response is primarily directed against metabolically active A. fumigatus morphotypes and is stronger against membrane protein fractions compared with cell wall or cytosolic proteins. Further analysis of 15 selected single A. fumigatus proteins revealed a highly diverse reactivity pattern that was donor and protein dependent. Importantly, the parallel assessment of T cell frequency, phenotype, and function allowed us to differentiate between proteins that elicit strong memory T cell responses in vivo versus Ags that induce T cell exhaustion or no reactivity in vivo. The regulatory T cell (Treg) response mirrors the conventional T cell response in terms of numbers and target specificity. Thus, our data reveal that the fungal T cell immunome is complex, but the ex vivo characterization of reactive T cells allows us to classify Ags and to predict potential immunogenic targets. A. fumigatus-specific conventional T cell responses are counterbalanced by a strong Treg response, suggesting that Treg-depletion strategies may be helpful in improving antifungal immunity.

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.

Flavoenzyme-catalyzed formation of disulfide bonds in natural products.

Nature provides a rich source of compounds with diverse chemical structures and biological activities, among them, sulfur-containing metabolites from bacteria and fungi. Some of these compounds bear a disulfide moiety that is indispensable for their bioactivity. Specialized oxidoreductases such as GliT, HlmI, and DepH catalyze the formation of this disulfide bridge in the virulence factor gliotoxin, the antibiotic holomycin, and the anticancer drug romidepsin, respectively. We have examined all three enzymes by X-ray crystallography and activity assays. Despite their differently sized substrate binding clefts and hence, their diverse substrate preferences, a unifying reaction mechanism is proposed based on the obtained crystal structures and further supported by mutagenesis experiments.

Epidithiodiketopiperazine biosynthesis: a four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges.

Enzyme quartet: Isolation of the first sulfur-bearing intermediate of the gliotoxin pathway in Aspergillus fumigatus and successful in vitro conversion of the bisglutathione adduct into an intact epidithiodiketopiperazine by a four-enzyme cascade (including glutamyltransferase GliK and dipeptidase GliJ) revealed an outstanding adaptation of a primary metabolic pathway into natural product biosynthesis that is widespread in fungi.