Search

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. However, an examination of 264 available genomes of A. fumigatus shows up to 20 additional BGCs, with some strains showing considerable variations in BGC number and composition. These new BGCs present a new frontier in the future of secondary metabolism characterization in this important species.

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434KD) were generated for three loci. Data show that the ∆t-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

Aspergillus fumigatus is a ubiquitous opportunistic pathogen. We have previously reported that volatile organic compounds (VOCs) produced by A. fumigatus cause delays in metamorphosis, morphological abnormalities, and death in a Drosophila melanogaster eclosion model. Here, we developed A. fumigatus deletion mutants with blocked oxylipin biosynthesis pathways (∆ppoABC) and then exposed the third instar larvae of D. melanogaster to a shared atmosphere with either A. fumigatus wild-type or oxylipin mutant cultures for 15 days. Fly larvae exposed to VOCs from wild-type A. fumigatus strains exhibited delays in metamorphosis and toxicity, while larvae exposed to VOCs from the ∆ppoABC mutant displayed fewer morphogenic delays and higher eclosion rates than the controls. In general, when fungi were pre-grown at 37 °C, the effects of the VOCs they produced were more pronounced than when they were pre-grown at 25 °C. GC–MS analysis revealed that the wild-type A. fumigatus Af293 produced more abundant VOCs at higher concentrations than the oxylipin-deficient strain Af293∆ppoABC did. The major VOCs detected from wild-type Af293 and its triple mutant included isopentyl alcohol, isobutyl alcohol, 2-methylbutanal, acetoin, and 1-octen-3-ol. Unexpectedly, compared to wild-type flies, the eclosion tests yielded far fewer differences in metamorphosis or viability when flies with immune-deficient genotypes were exposed to VOCs from either wild-type or ∆ppoABC oxylipin mutants. In particular, the toxigenic effects of Aspergillus VOCs were not observed in mutant flies deficient in the Toll (spz6) pathway. These data indicate that the innate immune system of Drosophila mediates the toxicity of fungal volatiles, especially via the Toll pathway.

Mycotoxin contamination in food poses health hazards to humans. Current methods of controlling mycotoxins still have limitations and more effective approaches are needed. During the past decades of years, variable environmental factors have been tested for their influence on mycotoxin production leading to elucidation of a complex regulatory network involved in mycotoxin biosynthesis. These regulators are putative targets for screening molecules that could inhibit mycotoxin synthesis. Here, we summarize the regulatory mechanisms of hierarchical regulators, including pathway-specific regulators, global regulators and epigenetic regulators, on the production of the most critical mycotoxins (aflatoxins, patulin, citrinin, trichothecenes and fumonisins). Future studies on regulation of mycotoxins will provide valuable knowledge for exploring novel methods to inhibit mycotoxin biosynthesis in a more efficient way.

In studying the development of tolerance to common hospital cleaners (Oxivir® and CaviCide™) in clinical isolate stocks of the emerging, multidrug-resistant yeast pathogen Candida auris, we selected for a cleaner-tolerant subpopulation of a more common nosocomial pathogen, Candida glabrata. Through the purification of each species and subsequent competition and other analyses, we determined that C. glabrata is capable of readily dominating mixed populations of C. auris and C. glabrata when exposed to hospital cleaners. This result suggests that exposure to antimicrobial compounds can preferentially select for low-level, stress-tolerant fungal pathogens. These findings indicate that clinical disinfection practices could contribute to the selection of tolerant, pathogenic microbes that persist within healthcare settings.

The activity of fumagillin, a mycotoxin produced by Aspergillus fumigatus, has not been studied in depth. In this study, we used a commercial fumagillin on cultures of two cell types (A549 pneumocytes and RAW 264.7 macrophages). This toxin joins its target, MetAP2 protein, inside cells and, as a result, significantly reduces the electron chain activity, the migration, and the proliferation ability on the A549 cells, or affects the viability and proliferation ability of the RAW 264.7 macrophages. However, the toxin stimulates the germination and double branch hypha production of fungal cultures, pointing out an intrinsic resistant mechanism to fumagillin of fungal strains. In this study, we also used a fumagillin non-producer A. fumigatus strain (∆fmaA) as well as its complemented strain (∆fmaA::fmaA) and we tested the fumagillin secretion of the fungal strains using an Ultra High-Performance Liquid Chromatography (UHPLC) method. Furthermore, fumagillin seems to protect the fungus against phagocytosis in vitro, and during in vivo studies using infection of immunosuppressed mice, a lower fungal burden in the lungs of mice infected with the ∆fmaA mutant was demonstrated.

In order to gain a comprehensive understanding of plant disease in natural and agricultural ecosystems, it is essential to examine plant disease in multi-pathogen–host systems. Ralstonia solanacearum and Fusarium oxysporum f. sp. lycopersici are vascular wilt pathogens that can result in heavy yield losses in susceptible hosts such as tomato. Although both pathogens occupy the xylem, the costs of mixed infections on wilt disease are unknown. Here, we characterize the consequences of co-infection with R. solanacearum and F. oxysporum using tomato as the model host. Our results demonstrate that bacterial wilt severity is reduced in co-infections, that bikaverin synthesis by Fusarium contributes to bacterial wilt reduction, and that the arrival time of each microbe at the infection court is important in driving the severity of wilt disease. Further, analysis of the co-infection root secretome identified previously uncharacterized secreted metabolites that reduce R. solanacearum growth in vitro and provide protection to tomato seedlings against bacterial wilt disease. Taken together, these results highlight the need to understand the consequences of mixed infections in plant disease.

Basic leucine zipper (bZIP) transcription factors play a crucial role in the environmental stress response of eukaryotes. In this work, we studied the effect of gene manipulations, including both deletions and overexpressions, of two selected bZIP transcription factors, NapA and RsmA, in the oxidative stress response and sterigmatocystin production of Aspergillus nidulans. We found that NapA was important in the oxidative stress response by negatively regulating intracellular reactive species production and positively regulating catalase activities, whereas RsmA slightly negatively regulated catalase activities. Concerning sterigmatocystin production, the highest concentration was measured in the ΔrsmAΔnapA double deletion mutant, but elevated sterigmatocystin production was also found in the OErsmA OEnapA strain. Our results indicate that NapA influences sterigmatocystin production via regulating reactive species level whereas RsmA modulates toxin production independently of the redox regulation of the cells.

Fungal secondary metabolites are renowned toxins as well as valuable sources of antibiotics, cholesterol-lowering drugs, and immunosuppressants; hence, great efforts were levied to understand how these compounds are genetically regulated. The genes encoding for the enzymes required for synthesizing secondary metabolites are arranged in biosynthetic gene clusters (BGCs). Often, BGCs contain a pathway specific transcription factor (PSTF), a valuable tool in shutting down or turning up production of the BGC product. In this review, we present an in-depth view of PSTFs by examining over 40 characterized BGCs in the well-studied fungal species Aspergillus nidulans and Aspergillus fumigatus. Herein, we find BGC size is a predictor for presence of PSTFs, consider the number and the relative location of PSTF in regard to the cluster(s) regulated, discuss the function and the evolution of PSTFs, and present application strategies for pathway specific activation of cryptic BGCs.

Fungi serve as a biological scaffold for bacterial attachment. In some specialized interactions, the bacteria will invade the fungal host, which in turn provides protection and nutrients for the bacteria. Mechanisms of the physical interactions between fungi and bacteria have been studied in both clinical and agricultural settings, as discussed in this review. Fungi and bacteria that are a part of these dynamic interactions can have altered growth and development as well as changes in microbial fitness as it pertains to antibiotic resistance, nutrient acquisition, and microbial dispersal. Consequences of these interactions are not just limited to the respective microorganisms, but also have major impacts in the health of humans and plants alike. Examining the mechanisms behind the physical interactions of fungi and bacteria will provide us with an understanding of multi-kingdom community processes and allow for the development of therapeutic approaches for disease in both ecological settings.

Fungal polyketides are a diverse class of natural products, or secondary metabolites (SMs), with a wide range of bioactivities often associated with toxicity. Here, we focus on a group of non-reducing polyketide synthases (NR-PKSs) in the fungal phylum Ascomycota that lack a thioesterase domain for product release, group V. Although widespread in ascomycete taxa, this group of NR-PKSs is notably absent in the mycotoxigenic genus Fusarium and, surprisingly, found in genera not known for their secondary metabolite production (e.g., the mycorrhizal genus Oidiodendron, the powdery mildew genus Blumeria, and the causative agent of white-nose syndrome in bats, Pseudogymnoascus destructans). This group of NR-PKSs, in association with the other enzymes encoded by their gene clusters, produces a variety of different chemical classes including naphthacenediones, anthraquinones, benzophenones, grisandienes, and diphenyl ethers. We discuss the modification of and transitions between these chemical classes, the requisite enzymes, and the evolution of the SM gene clusters that encode them. Integrating this information, we predict the likely products of related but uncharacterized SM clusters, and we speculate upon the utility of these classes of SMs as virulence factors or chemical defenses to various plant, animal, and insect pathogens, as well as mutualistic fungi.

Copper is an essential micronutrient for the opportunistic human pathogen, Aspergillus fumigatus. Maintaining copper homeostasis is critical for survival and pathogenesis. Copper-responsive transcription factors, AceA and MacA, coordinate a complex network responsible for responding to copper in the environment and determining which response is necessary to maintain homeostasis. For example, A. fumigatus uses copper exporters to mitigate the toxic effects of copper while simultaneously encoding copper importers and small molecules to ensure proper supply of the metal for copper-dependent processes such a nitrogen acquisition and respiration. Small molecules called isocyanides recently found to be produced by A. fumigatus may bind copper and partake in copper homeostasis similarly to isocyanide copper chelators in bacteria. Considering that the host uses copper as a microbial toxin and copper availability fluctuates in various environmental niches, understanding how A. fumigatus maintains copper homeostasis will give insights into mechanisms that facilitate the development of invasive aspergillosis and its survival in nature.

The zebrafish has become a widely accepted model host for studies of infectious disease, including fungal infections. The species is genetically tractable, and the larvae are transparent and amenable to prolonged in vivo imaging and small molecule screening. The aim of this review is to provide a thorough introduction into the published studies of fungal infection in the zebrafish and the specific ways in which this model has benefited the field. In doing so, we hope to provide potential new zebrafish researchers with a snapshot of the current toolbox and prior results, while illustrating how the model has been used well and where the unfulfilled potential of this model can be found.

Aflatoxin biosynthesis is correlated with oxidative stress and is proposed to function as a secondary defense mechanism to redundant intracellular reactive oxygen species (ROS). We find that the antioxidant gallic acid inhibits aflatoxin formation and growth in Aspergillus flavus in a dose-dependent manner. Global expression analysis (RNA-Seq) of gallic acid-treated A. flavus showed that 0.8% (w/v) gallic acid revealed two possible routes of aflatoxin inhibition. Gallic acid significantly inhibited the expression of farB, encoding a transcription factor that participates in peroxisomal fatty acid β-oxidation, a fundamental contributor to aflatoxin production. Secondly, the carbon repression regulator encoding gene, creA, was significantly down regulated by gallic acid treatment. CreA is necessary for aflatoxin synthesis, and aflatoxin biosynthesis genes were significantly downregulated in ∆creA mutants. In addition, the results of antioxidant enzyme activities and the lipid oxidation levels coupled with RNA-Seq data of antioxidant genes indicated that gallic acid may reduce oxidative stress through the glutathione- and thioredoxin-dependent systems in A. flavus.

Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of respiration in the late stationary stage—when sterigmatocystin production occurs—doubled in the overexpressing mutant carrying three aodA gene copies, but decreased to 10% in the deletant. Essentially identical results were obtained regardless whether the cultures were illuminated or protected from light. In contrast, sterigmatocystin yield in the aodA deletant was about half of that in the control when grown in the dark, while aodA overexpression resulted in up to 70% more sterigmatocystin formed, the yield increasing with alternative oxidase activity. Results were quite different when cultures were illuminated: under those conditions, sterigmatocystin volumetric yields were considerably lower, and statistically unvarying, regardless of the presence, absence, or the copy number of aodA. We conclude that the copy number of aodA, and hence, the balance between alternative- and cytochrome C-mediated respiration, appears to correlate with sterigmatocystin production in A. nidulans, albeit only in the absence of light.

Storage of freshly harvested fruit is a key factor in modulating their supply for several months after harvest; however, their quality can be reduced by pathogen attack. Fruit pathogens may infect their host through damaged surfaces, such as mechanical injuries occurring during growing, harvesting, and packing, leading to increased colonization as the fruit ripens. Of particular concern are fungal pathogens that not only macerate the host tissue but also secrete significant amounts of mycotoxins. Many studies have described the importance of physiological factors, including stage of fruit development, biochemical factors (ripening, C and N content), and environmental factors (humidity, temperature, water deficit) on the occurrence of mycotoxins. However, those factors usually show a correlative effect on fungal growth and mycotoxin accumulation. Recent reports have suggested that host factors can induce fungal metabolism, leading to the synthesis and accumulation of mycotoxins. This review describes the new vision of host-factor impact on the regulation of mycotoxin biosynthetic gene clusters underlying the complex regulation of mycotoxin accumulation in ripening fruit.

Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L−1 ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis.