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

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

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this record. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (65) partner repository with the data set identifier PXD018967. ChIP-seq results were submitted to NCBI under accession number SUB7547091.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 C_2 H_2 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.Medical Research Council (MRC)Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Technical University of Munich-Institute for Advanced Study (TUMIAS

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

Chitin Biosynthesis in <i>Aspergillus</i> 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

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

Aspergillus 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.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

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