Chitin Biosynthesis in Aspergillus Species.

The fungal cell wall (FCW) is a dynamic structure responsible for the maintenance of cellular homeostasis, and is essential for modulating the interaction of the fungus with its environment. It is composed of proteins, lipids, pigments and polysaccharides, including chitin. Chitin synthesis is catalyzed by chitin synthases (CS), and up to eight CS-encoding genes can be found in Aspergillus species. This review discusses in detail the chitin synthesis and regulation in Aspergillus species, and how manipulation of chitin synthesis pathways can modulate fungal growth, enzyme production, virulence and susceptibility to antifungal agents. More specifically, the metabolic steps involved in chitin biosynthesis are described with an emphasis on how the initiation of chitin biosynthesis remains unknown. A description of the classification, localization and transport of CS was also made. Chitin biosynthesis is shown to underlie a complex regulatory network, with extensive cross-talks existing between the different signaling pathways. Furthermore, pathways and recently identified regulators of chitin biosynthesis during the caspofungin paradoxical effect (CPE) are described. The effect of a chitin on the mammalian immune system is also discussed. Lastly, interference with chitin biosynthesis may also be beneficial for biotechnological applications. Even after more than 30 years of research, chitin biosynthesis remains a topic of current interest in mycology.

Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Carbon Catabolite Repression in Filamentous Fungi Is Regulated by Phosphorylation of the Transcription Factor CreA.

Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects.IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence.

G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis.IMPORTANCEA. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Pathogenic Allodiploid Hybrids of Aspergillus Fungi.

Interspecific hybridization substantially alters genotypes and phenotypes and can give rise to new lineages. Hybrid isolates that differ from their parental species in infection-relevant traits have been observed in several human-pathogenic yeasts and plant-pathogenic filamentous fungi but have yet to be found in human-pathogenic filamentous fungi. We discovered 6 clinical isolates from patients with aspergillosis originally identified as Aspergillus nidulans (section Nidulantes) that are actually allodiploid hybrids formed by the fusion of Aspergillus spinulosporus with an unknown close relative of Aspergillus quadrilineatus, both in section Nidulantes. Evolutionary genomic analyses revealed that these isolates belong to Aspergillus latus, an allodiploid hybrid species. Characterization of diverse infection-relevant traits further showed that A. latus hybrid isolates are genomically and phenotypically heterogeneous but also differ from A. nidulans, A. spinulosporus, and A. quadrilineatus. These results suggest that allodiploid hybridization contributes to the genomic and phenotypic diversity of filamentous fungal pathogens of humans.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Mapping the Fungal Battlefield: Using in situ Chemistry and Deletion Mutants to Monitor Interspecific Chemical Interactions Between Fungi.

Fungi grow in competitive environments, and to cope, they have evolved strategies, such as the ability to produce a wide range of secondary metabolites. This begs two related questions. First, how do secondary metabolites influence fungal ecology and interspecific interactions? Second, can these interspecific interactions provide a way to "see" how fungi respond, chemically, within a competitive environment? To evaluate these, and to gain insight into the secondary metabolic arsenal fungi possess, we co-cultured Aspergillus fischeri, a genetically tractable fungus that produces a suite of mycotoxins, with Xylaria cubensis, a fungus that produces the fungistatic compound and FDA-approved drug, griseofulvin. To monitor and characterize fungal chemistry in situ, we used the droplet-liquid microjunction-surface sampling probe (droplet probe). The droplet probe makes a microextraction at defined locations on the surface of the co-culture, followed by analysis of the secondary metabolite profile via liquid chromatography-mass spectrometry. Using this, we mapped and compared the spatial profiles of secondary metabolites from both fungi in monoculture versus co-culture. X. cubensis predominantly biosynthesized griseofulvin and dechlorogriseofulvin in monoculture. In contrast, under co-culture conditions a deadlock was formed between the two fungi, and X. cubensis biosynthesized the same two secondary metabolites, along with dechloro-5'-hydroxygriseofulvin and 5'-hydroxygriseofulvin, all of which have fungistatic properties, as well as mycotoxins like cytochalasin D and cytochalasin C. In contrast, in co-culture, A. fischeri increased the production of the mycotoxins fumitremorgin B and verruculogen, but otherwise remained unchanged relative to its monoculture. To evaluate that secondary metabolites play an important role in defense and territory establishment, we co-cultured A. fischeri lacking the master regulator of secondary metabolism laeA with X. cubensis. We found that the reduced secondary metabolite biosynthesis of the ΔlaeA strain of A. fischeri eliminated the organism's ability to compete in co-culture and led to its displacement by X. cubensis. These results demonstrate the potential of in situ chemical analysis and deletion mutant approaches for shedding light on the ecological roles of secondary metabolites and how they influence fungal ecological strategies; co-culturing may also stimulate the biosynthesis of secondary metabolites that are not produced in monoculture in the laboratory.

Characterizing the Pathogenic, Genomic, and Chemical Traits of Aspergillus fischeri, a Close Relative of the Major Human Fungal Pathogen Aspergillus fumigatus.

Aspergillus fischeri is closely related to Aspergillus fumigatus, the major cause of invasive mold infections. Even though A. fischeri is commonly found in diverse environments, including hospitals, it rarely causes invasive disease. Why A. fischeri causes less human disease than A. fumigatus is unclear. A comparison of A. fischeri and A. fumigatus for pathogenic, genomic, and secondary metabolic traits revealed multiple differences in pathogenesis-related phenotypes. We observed that A. fischeri NRRL 181 is less virulent than A. fumigatus strain CEA10 in multiple animal models of disease, grows slower in low-oxygen environments, and is more sensitive to oxidative stress. Strikingly, the observed differences for some traits are of the same order of magnitude as those previously reported between A. fumigatus strains. In contrast, similar to what has previously been reported, the two species exhibit high genomic similarity; ∼90% of the A. fumigatus proteome is conserved in A. fischeri, including 48/49 genes known to be involved in A. fumigatus virulence. However, only 10/33 A. fumigatus biosynthetic gene clusters (BGCs) likely involved in secondary metabolite production are conserved in A. fischeri and only 13/48 A. fischeri BGCs are conserved in A. fumigatus Detailed chemical characterization of A. fischeri cultures grown on multiple substrates identified multiple secondary metabolites, including two new compounds and one never before isolated as a natural product. Additionally, an A. fischeri deletion mutant of laeA, a master regulator of secondary metabolism, produced fewer secondary metabolites and in lower quantities, suggesting that regulation of secondary metabolism is at least partially conserved. These results suggest that the nonpathogenic A. fischeri possesses many of the genes important for A. fumigatus pathogenicity but is divergent with respect to its ability to thrive under host-relevant conditions and its secondary metabolism.IMPORTANCEAspergillus fumigatus is the primary cause of aspergillosis, a devastating ensemble of diseases associated with severe morbidity and mortality worldwide. A. fischeri is a close relative of A. fumigatus but is not generally observed to cause human disease. To gain insights into the underlying causes of this remarkable difference in pathogenicity, we compared two representative strains (one from each species) for a range of pathogenesis-relevant biological and chemical characteristics. We found that disease progression in multiple A. fischeri mouse models was slower and caused less mortality than A. fumigatus Remarkably, the observed differences between A. fischeri and A. fumigatus strains examined here closely resembled those previously described for two commonly studied A. fumigatus strains, AF293 and CEA10. A. fischeri and A. fumigatus exhibited different growth profiles when placed in a range of stress-inducing conditions encountered during infection, such as low levels of oxygen and the presence of chemicals that induce the production of reactive oxygen species. We also found that the vast majority of A. fumigatus genes known to be involved in virulence are conserved in A. fischeri, whereas the two species differ significantly in their secondary metabolic pathways. These similarities and differences that we report here are the first step toward understanding the evolutionary origin of a major fungal pathogen.

Genome-wide transcriptome analysis of Aspergillus fumigatus exposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakAHOG1 and MpkC dependent.

Invasive aspergillosis is predominantly caused by Aspergillus fumigatus, and adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. The central signal transduction pathway operating during hyperosmotic stress is the high osmolarity glycerol mitogen-activated protein kinase cascade. A. fumigatus MpkC and SakA, orthologues of the Saccharomyces cerevisiae Hog1p, constitute the primary regulator of the hyperosmotic stress response. We compared A. fumigatus wild-type transcriptional response to osmotic stress with the ΔmpkC, ΔsakA, and ΔmpkC ΔsakA strains. Our results strongly indicate that MpkC and SakA have independent and collaborative functions during the transcriptional response to transient osmotic stress. We have identified and characterized null mutants for four A. fumigatus basic leucine zipper proteins transcription factors. The atfA and atfB have comparable expression levels with the wild-type in ΔmpkC but are repressed in ΔsakA and ΔmpkC ΔsakA post-osmotic stress. The atfC and atfD have reduced expression levels in all mutants post-osmotic stress. The atfA-D null mutants displayed several phenotypes related to osmotic, oxidative, and cell wall stresses. The ΔatfA and ΔatfB were shown to be avirulent and to have attenuated virulence, respectively, in both Galleria mellonella and a neutropenic murine model of invasive pulmonary aspergillosis.

RNAseq reveals hydrophobins that are involved in the adaptation of Aspergillus nidulans to lignocellulose.

BACKGROUND: Sugarcane is one of the world's most profitable crops. Waste steam-exploded sugarcane bagasse (SEB) is a cheap, abundant, and renewable lignocellulosic feedstock for the next-generation biofuels. In nature, fungi seldom exist as planktonic cells, similar to those found in the nutrient-rich environment created within an industrial fermenter. Instead, fungi predominantly form biofilms that allow them to thrive in hostile environments. RESULTS: In turn, we adopted an RNA-sequencing approach to interrogate how the model fungus, Aspergillus nidulans, adapts to SEB, revealing the induction of carbon starvation responses and the lignocellulolytic machinery, in addition to morphological adaptations. Genetic analyses showed the importance of hydrophobins for growth on SEB. The major hydrophobin, RodA, was retained within the fungal biofilm on SEB fibres. The StuA transcription factor that regulates fungal morphology was up-regulated during growth on SEB and controlled hydrophobin gene induction. The absence of the RodA or DewC hydrophobins reduced biofilm formation. The loss of a RodA or a functional StuA reduced the retention of the hydrolytic enzymes within the vicinity of the fungus. Hence, hydrophobins promote biofilm formation on SEB, and may enhance lignocellulose utilisation via promoting a compact substrate-enzyme-fungus structure. CONCLUSION: This novel study highlights the importance of hydrophobins to the formation of biofilms and the efficient deconstruction of lignocellulose.

Diverse Regulation of the CreA Carbon Catabolite Repressor in Aspergillus nidulans.

Carbon catabolite repression (CCR) is a process that selects the energetically most favorable carbon source in an environment. CCR represses the use of less favorable carbon sources when a better source is available. Glucose is the preferential carbon source for most microorganisms because it is rapidly metabolized, generating quick energy for growth. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, a C2H2 finger domain DNA-binding protein. The aim of this work was to investigate the regulation of CreA and characterize its functionally distinct protein domains. CreA depends in part on de novo protein synthesis and is regulated in part by ubiquitination. CreC, the scaffold protein in the CreB-CreC deubiquitination (DUB) complex, is essential for CreA function and stability. Deletion of select protein domains in CreA resulted in persistent nuclear localization and target gene repression. A region in CreA conserved between Aspergillus spp. and Trichoderma reesei was identified as essential for growth on various carbon, nitrogen, and lipid sources. In addition, a role of CreA in amino acid transport and nitrogen assimilation was observed. Taken together, these results indicate previously unidentified functions of this important transcription factor. These novel functions serve as a basis for additional research in fungal carbon metabolism with the potential aim to improve fungal industrial applications.