Mining for novel natural compounds is of eminent importance owing to the continuous need for new pharmaceuticals. Filamentous fungi are historically known to harbor the genetic capacity for an arsenal of natural compounds, both beneficial and detrimental to humans. The majority of these metabolites are still cryptic or silent under standard laboratory culture conditions. Mining for these cryptic natural products can be an excellent source for identifying new compound classes. Capitalizing on the current knowledge on how secondary metabolite gene clusters are regulated has allowed the research community to unlock many hidden fungal treasures, as described in this chapter.

Summary The Fenton-chemistry generating properties of copper ions are considered a potent phagolysosome defense against pathogenic microbes, yet our understanding of underlying host/microbe dynamics remains unclear. We address this issue in invasive aspergillosis and demonstrate that host and fungal responses inextricably connect copper and reactive oxygen intermediate (ROI) mechanisms. Loss of the copper-binding transcription factor AceA yields an A. fumigatus strain displaying increased sensitivity to copper and ROI in vitro, increased intracellular copper concentrations, decreased survival in challenge with murine alveolar macrophages and reduced virulence in a non-neutropenic murine model. ΔaceA survival is remediated by dampening of host ROI (chemically or genetically) or enhancement of copper-exporting activity (CrpA) in A. fumigatus. Our study exposes a complex host/microbe multifactorial interplay that highlights the importance of host immune status and reveals key targetable A. fumigatus counter-defenses.

SUMMARY Aerial spores, crucial for propagation and dispersal of the Kingdom Fungi, are commonly the initial inoculum of pathogenic fungi. Natural products (secondary metabolites) have been correlated with fungal spore development and enhanced virulence in the human pathogen Aspergillus fumigatus but mechanisms for metabolite deposition in the spore are unknown. Metabolomic profiling of A. fumigatus deletion mutants of fumiquinazoline (Fq) cluster genes reveal that the first two products of the Fq cluster, FqF and FqA, are produced to comparable levels in all fungal tissues but the final enzymatically-derived product, FqC, predominantly accumulates in the fungal spore. Loss of the sporulation-specific transcription factor, BrlA, yields a strain unable to produce FqA or FqC. Fluorescence microscopy showed FmqD, the oxidoreductase required to generate FqC, was secreted via the Golgi apparatus to the cell wall in an actin-dependent manner. In contrast, all other members of the Fq pathway including the putative transporter, FmqE – which had no effect on Fq biosynthesis – were internal to the hyphae. The coordination of BrlA-mediated tissue specificity with FmqD secretion to the cell wall presents a previously undescribed mechanism to direct localization of specific secondary metabolites to spores of the differentiating fungus.

Summary Filamentous fungi are renowned for the production of bioactive secondary metabolites. Typically, one distinct metabolite is generated from a specific secondary metabolite cluster. Here, we characterize the newly described trypacidin (tpc) cluster in the opportunistic human pathogen Aspergillus fumigatus. We find that this cluster as well as the previously characterized endocrocin (enc) cluster both contribute to the production of the spore metabolite endocrocin. Whereas trypacidin is eliminated when only tpc cluster genes are deleted, endocrocin production is only eliminated when both the tpc and enc non-reducing polyketide synthase-encoding genes, tpcC and encA, respectively, are deleted. EncC, an anthrone oxidase, converts the product released from EncA to endocrocin as a final product. In contrast, endocrocin synthesis by the tpc cluster likely results from incomplete catalysis by TpcK (a putative decarboxylase), as its deletion results in a nearly 10-fold increase in endocrocin production. We suggest endocrocin is likely a shunt product in all related non-reducing polyketide synthase clusters containing homologues of TpcK and TpcL (a putative anthrone oxidase), e.g. geodin and monodictyphenone. This finding represents an unusual example of two physically discrete secondary metabolite clusters generating the same natural product in one fungal species by distinct routes.

ABSTRACTEndocrocin is a simple anthraquinone frequently identified in extracts of numerous fungi. Several biosynthetic schemes for endocrocin synthesis have been hypothesized, but to date, no dedicated secondary metabolite gene cluster that produces this polyketide as its major metabolite has been identified. Here we describe our biosynthetic and regulatory characterization of the endocrocin gene cluster inAspergillus fumigatus. This is the first report of this anthraquinone in this species. The biosynthetic genes required for endocrocin production are regulated by the global regulator of secondary metabolism, LaeA, and encode an iterative nonreducing polyketide synthase (encA), a physically discrete metallo-β-lactamase type thioesterase (encB), and a monooxygenase (encC). Interestingly, the deletion of a gene immediately adjacent toencC, termedencDand encoding a putative 2-oxoglutarate-Fe(II) type oxidoreductase, resulted in higher levels of endocrocin production than in the wild-type strain, whereas overexpression ofencDeliminated endocrocin accumulation. We found that overexpression of theencAtranscript resulted in higher transcript levels ofencA-Dand higher production of endocrocin. We discuss a model of theenccluster as one evolutionary origin of fungal anthraquinones derived from a nonreducing polyketide synthase and a discrete metallo-β-lactamase-type thioesterase.

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The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new “function-omic” avenues downstream of the metabolomics, identification, and purification phases.

Microbial secondary metabolites, including isocyanide moieties, have been extensively mined for their repertoire of bioactive properties. Although the first naturally occurring isocyanide (xanthocillin) was isolated from the fungus Penicillium notatum over half a century ago, the biosynthetic origins of fungal isocyanides remain unknown. Here we report the identification of a family of isocyanide synthases (ICSs) from the opportunistic human pathogen Aspergillus fumigatus. Comparative metabolomics of overexpression or knockout mutants of ICS candidate genes led to the discovery of a fungal biosynthetic gene cluster (BGC) that produces xanthocillin (xan). Detailed analysis of xanthocillin biosynthesis in A. fumigatus revealed several previously undescribed compounds produced by the xan BGC, including two novel members of the melanocin family of compounds. We found both the xan BGC and a second ICS-containing cluster, named the copper-responsive metabolite (crm) BGC, to be transcriptionally responsive to external copper levels and further demonstrated that production of metabolites from the xan BGC is increased during copper starvation. The crm BGC includes a novel type of fungus-specific ICS-nonribosomal peptide synthase (NRPS) hybrid enzyme, CrmA. This family of ICS-NRPS hybrid enzymes is highly enriched in fungal pathogens of humans, insects, and plants. Phylogenetic assessment of all ICSs spanning the tree of life shows not only high prevalence throughout the fungal kingdom but also distribution in species not previously known to harbor BGCs, indicating an untapped resource of fungal secondary metabolism.