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.

In 1999, the first biosynthetic gene cluster (BGC), synthesizing the virulence factor DHN melanin, was characterized in Aspergillus fumigatus. Since then, 19 additional BGCs have been linked to specific secondary metabolites (SMs) in this species. Here, we provide a comprehensive timeline of A. fumigatus BGC discovery and find that initial advances centered around the commonly expressed SMs where chemical structure informed rationale identification of the producing BGC (e.g., gliotoxin, fumigaclavine, fumitremorgin, pseurotin A, helvolic acid, fumiquinazoline). Further advances followed the transcriptional profiling of a ΔlaeA mutant, which aided in the identification of endocrocin, fumagillin, hexadehydroastechrome, trypacidin, and fumisoquin BGCs. These SMs and their precursors are the commonly produced metabolites in most A. fumigatus studies. Characterization of other BGC/SM pairs required additional efforts, such as induction treatments, including co-culture with bacteria (fumicycline/neosartoricin, fumigermin) or growth under copper starvation (fumivaline, fumicicolin). Finally, four BGC/SM pairs were discovered via overexpression technologies, including the use of heterologous hosts (fumicycline/neosartoricin, fumihopaside, sphingofungin, and sartorypyrone). Initial analysis of the two most studied A. fumigatus isolates, Af293 and A1160, suggested that both harbored ca. 34–36 BGCs. An examination of 264 available genomes of A. fumigatus located only four additional new BGCs, suggesting the secondary metabolome across A. fumigatus isolates is remarkably conserved. Based on our analysis, around 20 of the genetically characterized BGCs within the A. fumigatus species complex still lack a known chemical product. Such BGCs remain the final hurdle in fully understanding the secondary metabolism in this important species.

Pleurotus eryngii produces various functional molecules that mediate physiological functions in humans. Recently, we observed that P. eryngii produces molecules that have antidepressant functions. An ethanol extract of the fruiting body of P. eryngii was obtained, and the extract was purified by XAD-16 resin using an open column system. The ethanol eluate was separated by HPLC, and the fraction with an antidepressant function was identified. Using LC-MS, the molecular structure of the HPLC fraction with antidepressant function was identified as that of tryptamine, a functional molecule that is a tryptophan derivative. The antidepressant effect was identified from the ethanol extract, XAD-16 column eluate, and HPLC fraction by a serotonin receptor binding assay and a cell-based binding assay. Furthermore, a forced swimming test (FST) showed that the mice treated with purified fractions of P. eryngii exhibited decreased immobility time compared with nontreated mice. From these results, we suggest that the extract of P. eryngii has an antidepressant function and that it may be employed as an antidepressant health supplement.

Copper is an essential metal ion that performs many physiological functions in living organisms. Deletion of Afmac1, which is a copper-responsive transcriptional activator in A. fumigatus, results in a growth defect on aspergillus minimal medium (AMM). Interestingly, we found that zinc starvation suppressed the growth defect of the Δafmac1 strain on AMM. In addition, the growth defect of the Δafmac1 strain was recovered by copper supplementation or introduction of the CtrC gene into the Δafmac1 strain. However, chelation of copper by addition of BCS to AMM failed to recover the growth defect of the Δafmac1 strain. Through Northern blot analysis, we found that zinc starvation upregulated CtrC and CtrA2, which encode membrane copper transporters. Interestingly, we found that the conserved ZafA binding motif 5′-CAA(G)GGT-3′ was present in the upstream region of CtrC and CtrA2 and that mutation of the binding motif led to failure of ZafA binding to the upstream region of CtrC and upregulation of CtrC expression under zinc starvation. Furthermore, the binding activity of ZafA to the upstream region of CtrC was inversely proportional to the zinc concentration, and copper inhibited the binding of ZafA to the upstream region of CtrC under a low zinc concentration. Taken together, these results suggest that ZafA upregulates copper metabolism by binding to the ZafA binding motif in the CtrC promoter region under low zinc concentration, thus regulating copper homeostasis. Furthermore, we found that copper and zinc interact in cells to maintain metal homeostasis.

The field of secondary metabolism has greatly benefitted from computational advances in recent years. This has been particularly true for fungal natural product studies. Strides in genome mining have led to the identification of an extraordinary number of secondary metabolite biosynthetic gene clusters (BGCs) across the fungal Kingdom and metabologenomic platforms can group BGCs into gene cluster families and link them to initial chemical structures. Missing are computational applications focused on identifying BGC regulatory networks. Here, we applied the new online gene regulatory network resource, GRAsp ( G ene R egulation of Asp ergillus fumigatus ), to identify unknown and unpredictable BGC trans-acting transcriptional/metabolite production modules. GRAsp correctly predicted a two-component regulatory module composed of the transcription factors (TFs), RogA ( r egulation o f g liotoxin) and HsfA, which negatively regulate the gliotoxin BGC and are also involved in gliotoxin self-protection. RogA functions through the repression of gliZ, the pathway-specific gliotoxin TF, and HsfA functions by activating rogA expression. Furthermore, GRAsp identified TFs that regulate the production of two BGCs lacking pathway-specific TFs, the helvolic acid and fumitremorgin BGCs, respectively. Finally, the known TF, NsdD, was predicted and found to regulate the hexadehydroastechrome BGC. These advances highlight the power of inference algorithms to uncover unpredictable networks in specialized metabolite synthesis. IMPORTANCE: Toxic secondary metabolites are virulence factors of the opportunistic fungal pathogen Aspergillus fumigatus, yet the transcriptional networks regulating secondary metabolite production remain elusive. Uncovering novel regulators without any prior information is challenging. Computational programs have gained prominence in the field of secondary metabolite research due to their accuracy and ability to handle vast amounts of data, including DNA, RNA, and protein data. In this study, a newly developed online computer platform, Gene Regulation of A. fumigatus , was used to identify five regulators involved in the production of several A. fumigatus toxins, including gliotoxin, helvolic acid, fumitremorgin, and hexadehydroastechrome. This work illustrates the potential for discovering new trans-acting regulators and mechanisms of secondary metabolite regulation through the examination of computational gene regulatory networks.

Aspergillus fumigatus is a notorious pathogenic fungus responsible for various harmful, sometimes lethal, diseases known as aspergilloses. Understanding the gene regulatory networks that specify the expression programs underlying this fungus’ diverse phenotypes can shed mechanistic insight into its growth, development, and determinants of pathogenicity. We used eighteen RNA-seq datasets (seventeen publicly available and one previously unpublished) of Aspergillus fumigatus to construct a comprehensive gene regulatory network resource. Our resource, named GRAsp ( G ene R egulation of Asp ergillus fumigatus ), was able to recapitulate known regulatory pathways such as response to hypoxia, iron and zinc homeostasis, and secondary metabolite synthesis. Further, GRAsp was experimentally validated in two cases: one in which GRAsp accurately identified an uncharacterized transcription factor negatively regulating the production of the virulence factor gliotoxin and another where GRAsp revealed the bZip protein, AtfA, as required for fungal responses to microbial signals known as lipo-chitooligosaccharides. Our work showcases the strength of using network-based approaches to generate new hypotheses about regulatory relationships in Aspergillus fumigatus . We also unveil an online, user-friendly version of GRAsp available to the Aspergillus research community.