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  [...]melanin-deficient M. oryzae mutants produce non-pigmented appressoria that are unable to penetrate epidermal cells of the leaf cuticle [9]. Fungal SM act in different ways and increase the pathogen's ability to counteract adverse conditions in the host environment, irrespective of whether it is an animal or plant host. Since filamentous fungi encode between 30 to 70 secondary metabolism gene clusters (the products of most of these clusters are unknown) and there even exists cross-talk between clusters, resulting in the formation of hybrid molecules, it can be expected that up to 100 SM are produced by a single filamentous fungus.

Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary and systemic model of aspergillosis.

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading.

Key Points Secondary metabolites are structurally heterogenic low-molecular-mass molecules produced by many microorganisms, especially soil-dwelling bacteria and fungi. Unlike primary metabolites, these compounds are not directly required to ensure growth of the organisms that produce them. Most of the fungal secondary metabolites derive from either non-ribosomal peptides or polyketides. A few compounds represent mixed polyketide–non-ribosomal peptide compounds, and some others are derived from different biosynthesis pathways. In general, the biosynthesis genes for fungal secondary metabolites are located in single gene clusters that can span a few tens of kilobases, although there are exceptions for which two gene clusters located on different chromosomes are required for the biosynthesis of a distinct compound. Genome-mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of their biosynthesis gene clusters are silent under standard cultivation conditions, meaning that a plethora of natural products remains to be discovered. Fungal secondary metabolism gene clusters are controlled by a complex regulatory network involving interconnecting subnetworks consisting of multiple proteins and complexes that respond to various environmental stimuli. Global regulation of secondary metabolism gene clusters is achieved by globally acting transcription factors, which are encoded by genes that do not belong to any cluster. Pathway-specific regulation is mediated by transcription factors encoded by genes within the clusters that they regulate. Crosstalk regulation between gene clusters has been shown to occur, adding another level of complexity that could form the basis of combinatorial biosynthesis pathways which result in even more compounds. Chromatin-modifying elements allow specific control of secondary metabolism gene clusters. The modifications mediated by these factors include histone methylation and acetylation. Furthermore, chromatin-modulating complexes appear to be targets for bacterial manipulation of fungi, forming a novel concept for the interaction of organisms at the molecular level. Traditional ways to screen for secondary metabolites produced by microorganisms, based on variations in the growth medium, pH, temperature, aeration, light and so on, are not sufficient if the physiological and/or ecological triggers that activate the silent gene clusters are not known. Cluster activation and metabolite identification has been approached in several novel ways, including genetic engineering, simulation of physiological conditions (such as microbial interactions) that induce clusters, and chemical genomics based on inhibitors of histone acetyltransferases, histone deacetylases or DNA methyltransferases. Fungi produce a diverse array of secondary metabolites that have a range of functions and great pharmacological potential. In this Review, Axel Brakhage describes the regulatory pathways governing the production of these secondary metabolites and discusses how this knowledge provides a new avenue for drug discovery. Fungi produce a multitude of low-molecular-mass compounds known as secondary metabolites, which have roles in a range of cellular processes such as transcription, development and intercellular communication. In addition, many of these compounds now have important applications, for instance, as antibiotics or immunosuppressants. Genome mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of the fungal secondary metabolite biosynthesis gene clusters are silent under standard cultivation conditions. In this Review, I describe our current understanding of the regulatory elements that modulate the transcription of genes involved in secondary metabolism. I also discuss how an improved knowledge of these regulatory elements will ultimately lead to a better understanding of the physiological and ecological functions of these important compounds and will pave the way for a novel avenue to drug discovery through targeted activation of silent gene clusters.

Another important protein on the surface of A. fumigatus conidia is the Mep1p metalloprotease, which is released from spores in the mammalian lung to cleave host complement proteins and enhance infection, similar to the Alp1p serine protease released from mycelia for the same purpose [44]. In particular, detection of fungal conidia from environmental samples might provide an early warning to those suffering from lung conditions like asthma or chronic obstructive pulmonary disease, in which patients show a heightened susceptibility to allergic exacerbations due to fungal sensitization [46]. [...]conidial surface proteins play an integral role at the interface between normal fungal function and pathogenesis while offering a wealth of potential biomarkers and novel therapeutic targets. Levdansky E, Kashi O, Sharon H, Shadkchan Y, Osherov N. The Aspergillus fumigatus cspA gene encoding a repeat-rich cell wall protein is important for normal conidial cell wall architecture and interaction with host cells.

Iron is essential for a wide range of cellular processes. Here we show that the bZIP-type regulator HapX is indispensable for the transcriptional remodeling required for adaption to iron starvation in the opportunistic fungal pathogen Aspergillus fumigatus. HapX represses iron-dependent and mitochondrial-localized activities including respiration, TCA cycle, amino acid metabolism, iron-sulfur-cluster and heme biosynthesis. In agreement with the impact on mitochondrial metabolism, HapX-deficiency decreases resistance to tetracycline and increases mitochondrial DNA content. Pathways positively affected by HapX include production of the ribotoxin AspF1 and siderophores, which are known virulence determinants. Iron starvation causes a massive remodeling of the amino acid pool and HapX is essential for the coordination of the production of siderophores and their precursor ornithine. Consistent with HapX-function being limited to iron depleted conditions and A. fumigatus facing iron starvation in the host, HapX-deficiency causes significant attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrates that HapX-dependent adaption to conditions of iron starvation is crucial for virulence of A. fumigatus.

Fungi produce numerous low molecular weight molecules endowed with a multitude of biological activities. However, mining the full-genome sequences of fungi indicates that their potential to produce secondary metabolites is greatly underestimated. Because most of the biosynthesis gene clusters are silent under laboratory conditions, one of the major challenges is to understand the physiological conditions under which these genes are activated. Thus, we cocultivated the important model fungus Aspergillus nidulans with a collection of 58 soil-dwelling actinomycetes. By microarray analyses of both Aspergillus secondary metabolism and full-genome arrays and Northern blot and quantitative RT-PCR analyses, we demonstrate at the molecular level that a distinct fungal-bacterial interaction leads to the specific activation of fungal secondary metabolism genes. Most surprisingly, dialysis experiments and electron microscopy indicated that an intimate physical interaction of the bacterial and fungal mycelia is required to elicit the specific response. Gene knockout experiments provided evidence that one induced gene cluster codes for the long-sought after polyketide synthase (PKS) required for the biosynthesis of the archetypal polyketide orsellinic acid, the typical lichen metabolite lecanoric acid, and the cathepsin K inhibitors F-9775A and F-9775B. A phylogenetic analysis demonstrates that orthologs of this PKS are widespread in nature in all major fungal groups, including mycobionts of lichens. These results provide evidence of specific interaction among microorganisms belonging to different domains and support the hypothesis that not only diffusible signals but intimate physical interactions contribute to the communication among microorganisms and induction of otherwise silent biosynthesis genes.

Aspergillus fumigatus , an opportunistic human pathogen, frequently infects the lungs of people with cystic fibrosis and is one of the most common causes of infectious-disease death in immunocompromised patients. Here, we construct 252 strain-specific, genome-scale metabolic models of this important fungal pathogen to study and better understand the metabolic component of its pathogenic versatility. The models show that 23.1% of A. fumigatus metabolic reactions are not conserved across strains and are mainly associated with amino acid, nucleotide, and nitrogen metabolism. Profiles of non-conserved reactions and growth-supporting reaction fluxes are sufficient to differentiate strains, for example by environmental or clinical origin. In addition, shotgun metagenomics analysis of sputum from 40 cystic fibrosis patients (15 females, 25 males) before and after diagnosis with an A. fumigatus colonization suggests that the fungus shapes the lung microbiome towards a more beneficial fungal growth environment associated with aromatic amino acid availability and the shikimate pathway. Our findings are starting points for the development of drugs or microbiome intervention strategies targeting fungal metabolic needs for survival and colonization in the non-native environment of the human lung. Here, the authors generate strain-specific genome-scale metabolic models of Aspergillus fumigatus and analyze fungal metabolism of infection of the lung of cystic fibrosis patients, finding that the fungus shapes the lung microbiome to promote its own growth.

Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

Breathe easy: why inhaled fungal spores don't provoke an immune reaction Every day we inhale thousands of tiny fungal spores (conidia), originating from many different fungal species. Yet although these spores are packed with antigens and allergens, their inhalation does not continuously activate our innate immune cells or provoke inflammatory responses. A series of immunological, biochemical and genetic experiments shows why: immune recognition of these spores is prevented by a hydrophobic layer of rodlet proteins covering the conidial surface. If this layer is removed, spores activate the immune system. A pathogenic spore equipped with this defensive layer might lie dormant beyond host defences until conditions are suitable for germination. Therapeutically the robust nature of the rodlet proteins might be exploited to generate nanoparticles containing embedded molecules targeted to a specific location in the body, or optimized for sustained delivery. Fungal spores are ubiquitous in the air we breathe and contain many antigens and allergens, and yet they neither continuously activate the host innate immune cells nor induce detrimental inflammatory responses after their inhalation. Here, the surface layer on dormant spores is shown to mask their recognition by the immune system and hence prevent an immune response. The air we breathe is filled with thousands of fungal spores (conidia) per cubic metre, which in certain composting environments can easily exceed 10 9 per cubic metre. They originate from more than a hundred fungal species belonging mainly to the genera Cladosporium , Penicillium , Alternaria and Aspergillus 1 , 2 , 3 , 4 . Although these conidia contain many antigens and allergens 5 , 6 , 7 , it is not known why airborne fungal microflora do not activate the host innate immune cells continuously and do not induce detrimental inflammatory responses following their inhalation. Here we show that the surface layer on the dormant conidia masks their recognition by the immune system and hence prevents immune response. To explore this, we used several fungal members of the airborne microflora, including the human opportunistic fungal pathogen Aspergillus fumigatus , in in vitro assays with dendritic cells and alveolar macrophages and in in vivo murine experiments. In A. fumigatus , this surface ‘rodlet layer’ is composed of hydrophobic RodA protein covalently bound to the conidial cell wall through glycosylphosphatidylinositol-remnants. RodA extracted from conidia of A. fumigatus was immunologically inert and did not induce dendritic cell or alveolar macrophage maturation and activation, and failed to activate helper T-cell immune responses in vivo . The removal of this surface ‘rodlet/hydrophobin layer’ either chemically (using hydrofluoric acid), genetically (Δ rodA mutant) or biologically (germination) resulted in conidial morphotypes inducing immune activation. All these observations show that the hydrophobic rodlet layer on the conidial cell surface immunologically silences airborne moulds.