Induction of Aspergillus fumigatus zinc cluster transcription factor OdrA/Mdu2 provides combined cellular responses for oxidative stress protection and multiple antifungal drug resistance.

IMPORTANCE: An overexpression screen of 228 zinc cluster transcription factor encoding genes of A. fumigatus revealed 11 genes conferring increased tolerance to antifungal drugs. Out of these, four oxidative stress and drug tolerance transcription factor encoding odr genes increased tolerance to oxidative stress and antifungal drugs when overexpressed. This supports a correlation between oxidative stress response and antifungal drug tolerance in A. fumigatus. OdrA/Mdu2 is required for the cross-tolerance between azoles, polyenes, and oxidative stress and activates genes for detoxification. Under oxidative stress conditions or when overexpressed, OdrA/Mdu2 accumulates in the nucleus and activates detoxifying genes by direct binding at their promoters, as we describe with the mdr1 gene encoding an itraconazole specific efflux pump. Finally, this work gives new insights about drug and stress resistance in the opportunistic pathogenic fungus A. fumigatus.

Fungal COP9 signalosome assembly requires connection of two trimeric intermediates for integration of intrinsic deneddylase.

The conserved eight-subunit COP9 signalosome (CSN) is required for multicellular fungal development. The CSN deneddylase cooperates with the Cand1 exchange factor to control replacements of E3 ubiquitin cullin RING ligase receptors, providing specificity to eukaryotic protein degradation. Aspergillus nidulans CSN assembles through a heptameric pre-CSN, which is activated by integration of the catalytic CsnE deneddylase. Combined genetic and biochemical approaches provided the assembly choreography within a eukaryotic cell for native fungal CSN. Interactomes of functional GFP-Csn subunit fusions in pre-CSN deficient fungal strains were compared by affinity purifications and mass spectrometry. Two distinct heterotrimeric CSN subcomplexes were identified as pre-CSN assembly intermediates. CsnA-C-H and CsnD-F-G form independently of CsnB, which connects the heterotrimers to a heptamer and enables subsequent integration of CsnE to form the enzymatically active CSN complex. Surveillance mechanisms control accurate Csn subunit amounts and correct cellular localization for sequential assembly since deprivation of Csn subunits changes the abundance and location of remaining Csn subunits.

F-box receptor mediated control of substrate stability and subcellular location organizes cellular development of Aspergillus nidulans.

Fungal growth and development are coordinated with specific secondary metabolism. This coordination requires 8 of 74 F-box proteins of the filamentous fungus Aspergillus nidulans. F-box proteins recognize primed substrates for ubiquitination by Skp1-Cul1-Fbx (SCF) E3 ubiquitin RING ligases and degradation by the 26S proteasome. 24 F-box proteins are found in the nuclear fraction as part of SCFs during vegetative growth. 43 F-box proteins interact with SCF proteins during growth, development or stress. 45 F-box proteins are associated with more than 700 proteins that have mainly regulatory roles. This corroborates that accurate surveillance of protein stability is prerequisite for organizing multicellular fungal development. Fbx23 combines subcellular location and protein stability control, illustrating the complexity of F-box mediated regulation during fungal development. Fbx23 interacts with epigenetic methyltransferase VipC which interacts with fungal NF-κB-like velvet domain regulator VeA that coordinates fungal development with secondary metabolism. Fbx23 prevents nuclear accumulation of methyltransferase VipC during early development. These results suggest that in addition to their role in protein degradation, F-box proteins also control subcellular accumulations of key regulatory proteins for fungal development.

Biosynthesis of Antibacterial Iron-Chelating Tropolones in Aspergillus nidulans as Response to Glycopeptide-Producing Streptomycetes.

The soil microbiome comprises numerous filamentous fungi and bacteria that mutually react and challenge each other by the production of bioactive secondary metabolites. Herein, we show in liquid co-cultures that the presence of filamentous Streptomycetes producing antifungal glycopeptide antibiotics induces the production of the antibacterial and iron-chelating tropolones anhydrosepedonin (1) and antibiotic C (2) in the mold Aspergillus nidulans. Additionally, the biosynthesis of the related polyketide tripyrnidone (5) was induced, whose novel tricyclic scaffold we elucidated by NMR and HRESIMS data. The corresponding biosynthetic polyketide synthase-encoding gene cluster responsible for the production of these compounds was identified. The tropolones as well as tripyrnidone (5) are produced by genes that belong to the broad reservoir of the fungal genome for the synthesis of different secondary metabolites, which are usually silenced under standard laboratory conditions. These molecules might be part of the bacterium-fungus competition in the complex soil environment, with the bacterial glycopeptide antibiotic as specific environmental trigger for fungal induction of this cluster.

Hülle Cells of Aspergillus nidulans with Nuclear Storage and Developmental Backup Functions Are Reminiscent of Multipotent Stem Cells.

Some aspergilli are among the most cosmopolitan and ecologically dominant fungal species. One pillar of their success is their complex life cycle, which creates specialized cell types for versatile dispersal and regenesis. One of these cell types is unique to aspergilli-the Hülle cells. Despite being known for over a century, the biological and ecological roles of Hülle cells remain largely speculative. Previously reported data on in vivo Hülle cell formation and localization have been conflicting. Our quantification reveals that Hülle cells can occur at all locations on hyphae and that they show cellular activity similar to that seen with adjacent hyphae, indicating that they develop as intricate parts of hyphal tissue. In addition, we show that during sexual development associated with two parental strains, the typically multinucleate Hülle cells can inherit nuclei from both parents, indicating that they may serve as genetic backups. We provide an easy, reproducible method to study Hülle cell biology and germination with which we investigate the 90-year-old puzzle of whether and how Hülle cells germinate. We present clear evidence for the germination of Hülle cells, and we show that Hülle cells grow hyphae that develop into a spore-producing colony. Finally, we show that Hülle cell-derived colonies produce conidiospores faster than spore-derived colonies, providing evidence for an as-yet-undescribed developmental shortcut program in Aspergillus nidulans We propose that Hülle cells represent a unique cell type as specialized hypha-derived sexual tissue with a nucleus storage function and may act as fungal backup stem cells under highly destructive conditions.IMPORTANCE The in vivo identification of Hülle cells in cases of aspergillosis infections in animals and humans illustrates their biological relevance and suggests that they might be involved in pathogenicity. It is striking that aspergilli have developed and maintained a multinucleate nurse cell that is presumably energy-intensive to produce and is usually found only in higher eukaryotes. Our findings shed light on how the understudied Hülle cells might contribute to the success of aspergilli by acting not only as nurse cells under detrimental conditions (sexual development) but also as fungal backup stem cells with the capacity to produce genetically diverse spores in an accelerated manner, thereby substantially contributing to survival in response to predator attack or under otherwise severely destructive conditions. Our study solved the 90-year-old puzzle of Hülle cell germination and provides easy, reproducible methods that will facilitate future studies on biological and ecological roles of Hülle cells in aspergilli.

Integration of Fungus-Specific CandA-C1 into a Trimeric CandA Complex Allowed Splitting of the Gene for the Conserved Receptor Exchange Factor of CullinA E3 Ubiquitin Ligases in Aspergilli.

E3 cullin-RING ubiquitin ligase (CRL) complexes recognize specific substrates and are activated by covalent modification with ubiquitin-like Nedd8. Deneddylation inactivates CRLs and allows Cand1/A to bind and exchange substrate recognition subunits. Human as well as most fungi possess a single gene for the receptor exchange factor Cand1, which is split and rearranged in aspergilli into two genes for separate proteins. Aspergillus nidulans CandA-N blocks the neddylation site, and CandA-C inhibits the interaction to the adaptor/substrate receptor subunits similar to the respective N-terminal and C-terminal parts of single Cand1. The pathogen Aspergillus fumigatus and related species express a CandA-C with a 190-amino-acid N-terminal extension domain encoded by an additional exon. This extension corresponds in most aspergilli, including A. nidulans, to a gene directly upstream of candA-C encoding a 20-kDa protein without human counterpart. This protein was named CandA-C1, because it is also required for the cellular deneddylation/neddylation cycle and can form a trimeric nuclear complex with CandA-C and CandA-N. CandA-C and CandA-N are required for asexual and sexual development and control a distinct secondary metabolism. CandA-C1 and the corresponding domain of A. fumigatus control spore germination, vegetative growth, and the repression of additional secondary metabolites. This suggests that the dissection of the conserved Cand1-encoding gene within the genome of aspergilli was possible because it allowed the integration of a fungus-specific protein required for growth into the CandA complex in two different gene set versions, which might provide an advantage in evolution.IMPORTANCEAspergillus species are important for biotechnological applications, like the production of citric acid or antibacterial agents. Aspergilli can cause food contamination or invasive aspergillosis to immunocompromised humans or animals. Specific treatment is difficult due to limited drug targets and emerging resistances. The CandA complex regulates, as a receptor exchange factor, the activity and substrate variability of the ubiquitin labeling machinery for 26S proteasome-mediated protein degradation. Only Aspergillus species encode at least two proteins that form a CandA complex. This study shows that Aspergillus species had to integrate a third component into the CandA receptor exchange factor complex that is unique to aspergilli and required for vegetative growth, sexual reproduction, and activation of the ubiquitin labeling machinery. These features have interesting implications for the evolution of protein complexes and could make CandA-C1 an interesting candidate for target-specific drug design to control fungal growth without affecting the human ubiquitin-proteasome system.

COP9 Signalosome Interaction with UspA/Usp15 Deubiquitinase Controls VeA-Mediated Fungal Multicellular Development.

COP9 signalosome (CSN) and Den1/A deneddylases physically interact and promote multicellular development in fungi. CSN recognizes Skp1/cullin-1/Fbx E3 cullin-RING ligases (CRLs) without substrate and removes their posttranslational Nedd8 modification from the cullin scaffold. This results in CRL complex disassembly and allows Skp1 adaptor/Fbx receptor exchange for altered substrate specificity. We characterized the novel ubiquitin-specific protease UspA of the mold Aspergillusnidulans, which corresponds to CSN-associated human Usp15 and interacts with six CSN subunits. UspA reduces amounts of ubiquitinated proteins during fungal development, and the uspA gene expression is repressed by an intact CSN. UspA is localized in proximity to nuclei and recruits proteins related to nuclear transport and transcriptional processing, suggesting functions in nuclear entry control. UspA accelerates the formation of asexual conidiospores, sexual development, and supports the repression of secondary metabolite clusters as the derivative of benzaldehyde (dba) genes. UspA reduces protein levels of the fungal NF-kappa B-like velvet domain protein VeA, which coordinates differentiation and secondary metabolism. VeA stability depends on the Fbx23 receptor, which is required for light controlled development. Our data suggest that the interplay between CSN deneddylase, UspA deubiquitinase, and SCF-Fbx23 ensures accurate levels of VeA to support fungal development and an appropriate secondary metabolism.

The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin­proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

Integration of the catalytic subunit activates deneddylase activity in vivo as final step in fungal COP9 signalosome assembly.

The eight-subunit COP9 signalosome (CSN) is conserved from filamentous fungi to humans and functions at the interface between cellular signalling and protein half-life control. CSN consists of six PCI and two MPN domain proteins and forms a scaffold for additional interacting proteins. CSN controls protein stability in the ubiquitin-proteasome system where the MPN domain CSN5/CsnE subunit inactivates cullin-RING ligases. The CSN5/CsnE isopeptidase functions as deneddylase and removes the ubiquitin-like protein Nedd8. The six PCI domain proteins of human CSN form a horseshoe-like ring and all eight subunits are connected by a bundle of C-terminal α-helices. We show that single deletions of any csn subunit of Aspergillus nidulans resulted in the lack of deneddylase activity and identical defects in the coordination of development and secondary metabolism. The CSN1/CsnA N-terminus is dispensable for deneddylase activity but required for asexual spore formation. Complex analyses in mutant strains revealed the presence of a seven-subunit pre-CSN without catalytic activity. Reconstitution experiments with crude extracts of deletion strains and recombinant proteins allowed the integration of CSN5/CsnE into pre-CSN resulting in an active deneddylase. This supports a stable seven subunit pre-CSN intermediate where deneddylase activation in vivo can be controlled by CSN5/CsnE integration as final assembly step.