Functional characterization of genes encoding cadmium pumping P1B-type ATPases in Aspergillus fumigatus and Aspergillus nidulans.

Several P1B-type ATPases are important Cd2+/Cu2+ pumps in Aspergillus species, and they are tightly associated with the heavy metal stress tolerance of these ascomycetous fungi. To better understand the roles of the two P1B-type ATPases, Aspergillus nidulans CrpA Cd2+/Cu2+ pump (orthologue of the Candida albicans Crp1 Cd2+/Cu2+ pump) and Aspergillus fumigatus PcaA Cd2+ pump (orthologue of the Saccharomyces cerevisiae Pca1 Cd2+ pump), we have generated individual mutants and characterized their heavy metal susceptibilities. The deletion of CrpA in A. nidulans has led to the increased sensitivity of the fungus to stresses induced by Zn2+, Fe2+, or the combination of oxidative-stress-inducing menadione sodium bisulfite and Fe3+. Heterologous expression of A. fumigatus PcaA in the S. cerevisiae pca1 deletion mutant has resulted in enhanced tolerance of the yeast to stresses elicited by Cd2+or Zn2+ but not by Fe2+/Fe3+ or Cu2+. Mammalian host immune defense can attack microbes by secreting Zn2+ or Cu2+, and the oxidative stress induced by host immune systems can also disturb metal (Cu2+, Fe2+, and Zn2+) homeostasis in microbes. In summary, PcaA and CrpA can protect fungal cells from these complex stresses that contribute to the virulence of the pathogenic Aspergillus species. Moreover, due to their presence on the fungal cell surface, these P1B-type ATPases may serve as a novel drug target in the future. IMPORTANCE Mammalian host immune defense disrupts heavy metal homeostasis of fungal pathogens. P1B-type ATPase of Aspergillus fumigatus and Aspergillus nidulans may help to cope with this stress and serve as virulence traits. In our experiments, both A. nidulans Cd2+/Cu2+ pump CrpA and A. fumigatus Cd2+ pump PcaA protected fungal cells from toxic Zn2+, and CrpA also decreased Fe2+ susceptibility most likely indirectly. In addition, CrpA protected cells against the combined stress induced by the oxidative stressor menadione and Fe3+. Since P1B-type ATPases are present on the fungal cell surface, these proteins may serve as a novel drug target in the future.

Species-specific effects of the introduction of Aspergillus nidulans gfdB in osmophilic aspergilli.

Industrial fungi need a strong environmental stress tolerance to ensure acceptable efficiency and yields. Previous studies shed light on the important role that Aspergillus nidulans gfdB, putatively encoding a NAD+-dependent glycerol-3-phosphate dehydrogenase, plays in the oxidative and cell wall integrity stress tolerance of this filamentous fungus model organism. The insertion of A. nidulans gfdB into the genome of Aspergillus glaucus strengthened the environmental stress tolerance of this xerophilic/osmophilic fungus, which may facilitate the involvement of this fungus in various industrial and environmental biotechnological processes. On the other hand, the transfer of A. nidulans gfdB to Aspergillus wentii, another promising industrial xerophilic/osmophilic fungus, resulted only in minor and sporadic improvement in environmental stress tolerance and meanwhile partially reversed osmophily. Because A. glaucus and A. wentii are phylogenetically closely related species and both fungi lack a gfdB ortholog, these results warn us that any disturbance of the stress response system of the aspergilli may elicit rather complex and even unforeseeable, species-specific physiological changes. This should be taken into consideration in any future targeted industrial strain development projects aiming at the fortification of the general stress tolerance of these fungi. KEY POINTS: • A. wentii c' gfdB strains showed minor and sporadic stress tolerance phenotypes. • The osmophily of A. wentii significantly decreased in the c' gfdB strains. • Insertion of gfdB caused species-specific phenotypes in A. wentii and A. glaucus.

NODULIN HOMEOBOX is required for heterochromatin homeostasis in Arabidopsis.

Arabidopsis NODULIN HOMEOBOX (NDX) is a nuclear protein described as a regulator of specific euchromatic genes within transcriptionally active chromosome arms. Here we show that NDX is primarily a heterochromatin regulator that functions in pericentromeric regions to control siRNA production and non-CG methylation. Most NDX binding sites coincide with pericentromeric het-siRNA loci that mediate transposon silencing, and are antagonistic with R-loop structures that are prevalent in euchromatic chromosomal arms. Inactivation of NDX leads to differential siRNA accumulation and DNA methylation, of which CHH/CHG hypomethylation colocalizes with NDX binding sites. Hi-C analysis shows significant chromatin structural changes in the ndx mutant, with decreased intrachromosomal interactions at pericentromeres where NDX is enriched in wild-type plants, and increased interchromosomal contacts between KNOT-forming regions, similar to those observed in DNA methylation mutants. We conclude that NDX is a key regulator of heterochromatin that is functionally coupled to het-siRNA loci and non-CG DNA methylation pathways.

Genome-wide mapping of binding sites of the transposase-derived SETMAR protein in the human genome.

Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these "domesticated", transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a histone-lysine methyltransferase domain and an HsMar1 transposase. To elucidate the biological role of SETMAR, it is crucial to identify genomic targets to which SETMAR specifically binds and link these sites to the regulation of gene expression. Herein, we mapped the genomic landscape of SETMAR binding in a near-haploid human leukemia cell line (HAP1) in order to identify on-target and off-target binding sites at high resolution and to elucidate their role in terms of gene expression. Our analysis revealed a perfect correlation between SETMAR and inverted terminal repeats (ITRs) of HsMar1 transposon remnants, which are considered as natural target sites for SETMAR binding. However, we did not detect any untargeted events at non-ITR sequences, calling into question previously proposed off-target binding sites. We identified sequence fidelity of the ITR motif as a key factor for determining the binding affinity of SETMAR for chromosomes, as higher conservation of ITR sequences resulted in increased affinity for chromatin and stronger repression of SETMAR-bound gene loci. These associations highlight how SETMAR's chromatin binding fine-tune gene regulatory networks in human tumour cells.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

Sequence-based prediction of protein binding mode landscapes.

Interactions between disordered proteins involve a wide range of changes in the structure and dynamics of the partners involved. These changes can be classified in terms of binding modes, which include disorder-to-order (DO) transitions, when proteins fold upon binding, as well as disorder-to-disorder (DD) transitions, when the conformational heterogeneity is maintained in the bound states. Furthermore, systematic studies of these interactions are revealing that proteins may exhibit different binding modes with different partners. Proteins that exhibit this context-dependent binding can be referred to as fuzzy proteins. Here we investigate amino acid code for fuzzy binding in terms of the entropy of the probability distribution of transitions towards decreasing order. We implement these entropy calculations into the FuzPred (http://protdyn-fuzpred.org) algorithm to predict the range of context-dependent binding modes of proteins from their amino acid sequences. As we illustrate through a variety of examples, this method identifies those binding sites that are sensitive to the cellular context or post-translational modifications, and may serve as regulatory points of cellular pathways.

Sequence-Based Prediction of Fuzzy Protein Interactions.

It is becoming increasingly recognised that disordered proteins may be fuzzy, in that they can exhibit a wide variety of binding modes. In addition to the well-known process of folding upon binding (disorder-to-order transition), many examples are emerging of interacting proteins that remain disordered in their bound states (disorder-to-disorder transitions). Furthermore, disordered proteins may populate ordered and disordered states to different extents depending on their partners (context-dependent binding). Here we assemble three datasets comprising disorder-to-order, context-dependent, and disorder-to-disorder transitions of 828 protein regions represented in 2157 complexes and elucidate the sequence-determinants of the different interaction modes. We found that fuzzy interactions originate from local sequence compositions that promote the sampling of a wide range of different structures. Based on this observation, we developed the FuzPred method (http://protdyn-fuzpred.org) of predicting the binding modes of disordered proteins based on their amino acid sequences, without specifying their partners. We thus illustrate how the amino acid sequences of proteins can encode a wide range of conformational changes upon binding, including transitions from disordered to ordered and from disordered to disordered states.

Two Targets, One Hit: new Anticancer Therapeutics to Prevent Tumorigenesis Without Cardiotoxicity.

A serious adverse effect of cancer therapies is cardiovascular toxicity, which significantly limits the widespread use of antineoplastic agents. The promising new field of cardio-oncology offers the identification of potent anti-cancer therapeutics that effectively inhibit cancer cell proliferation without causing cardiotoxicity. Future introduction of recently identified cardio-safe compounds into clinical practice (including ERK dimerization inhibitors or BAX allosteric inhibitors) is expected to help oncologists avoid unwanted cardiological complications associated with therapeutic interventions.

Fuzziness enables context dependence of protein interactions.

Proteins may undergo adaptive structural transitions to accommodate to their cellular milieu and respond to external signals. Modulation of conformational ensembles can rewire the intra- or intermolecular interaction networks and shift between different functional states. Adaptive conformational transitions are associated with protein fuzziness, which enables (a) rewiring interaction networks via alternative motifs, (b) new functional features via allosteric motifs, (c) functional switches upon post-translational modifications, or (d) regulation of higher-order organizations. We propose that all these context-dependent functional changes are intertwined with structural multiplicity or dynamic disorder in protein assemblies and can only be described by stochastic structure-function relationships.

Study on the glutathione metabolism of the filamentous fungus Aspergillus nidulans.

Yeast protein sequence-based homology search for glutathione (GSH) metabolic enzymes and GSH transporters demonstrated that Aspergillus nidulans has a robust GSH uptake and metabolic system with several paralogous genes. In wet laboratory experiments, two key genes of GSH metabolism, gcsA, and glrA, encoding γ-l-glutamyl-l-cysteine synthetase and glutathione reductase, respectively, were deleted. The gene gcsA was essential, and the ΔgcsA mutant required GSH supplementation at considerably higher concentration than the Saccharomyces cerevisiae gsh1 mutant (8-10 mmol l-1 vs. 0.5 µmol l-1). In addition to some functions known previously, both genes were important in the germination of conidiospores, and both gene deletion strains required the addition of extra GSH to reach wild-type germination rates in liquid cultures. Nevertheless, the supplementation of cultures with 10 mmol l-1 GSH was toxic for the control and ΔglrA strains especially during vegetative growth, which should be considered in future development of high GSH-producer fungal strains. Importantly, the ΔglrA strain was characterized by increased sensitivity toward a wide spectrum of osmotic, cell wall integrity and antimycotic stress conditions in addition to previously reported temperature and oxidative stress sensitivities. These novel phenotypes underline the distinguished functions of GSH and GSH metabolic enzymes in the stress responses of fungi.

FuzDB: database of fuzzy complexes, a tool to develop stochastic structure-function relationships for protein complexes and higher-order assemblies.

FuzDB (http://protdyn-database.org) compiles experimentally observed fuzzy protein complexes, where intrinsic disorder (ID) is maintained upon interacting with a partner (protein, nucleic acid or small molecule) and directly impacts biological function. Entries in the database have both (i) structural evidence demonstrating the structural multiplicity or dynamic disorder of the ID region(s) in the partner bound form of the protein and (ii) in vitro or in vivo biological evidence that indicates the significance of the fuzzy region(s) in the formation, function or regulation of the assembly. Unlike the other intrinsically disordered or unfolded protein databases, FuzDB focuses on ID regions within a biological context, including higher-order assemblies and presents a detailed analysis of the structural and functional data. FuzDB also provides interpretation of experimental results to elucidate the molecular mechanisms by which fuzzy regions-classified on the basis of topology and mechanism-interfere with the structural ensembles and activity of protein assemblies. Regulatory sites generated by alternative splicing (AS) or post-translational modifications (PTMs) are also collected. By assembling all this information, FuzDB could be utilized to develop stochastic structure-function relationships for proteins and could contribute to the emergence of a new paradigm.

Insights into Adaptations to a Near-Obligate Nematode Endoparasitic Lifestyle from the Finished Genome of Drechmeria coniospora.

Nematophagous fungi employ three distinct predatory strategies: nematode trapping, parasitism of females and eggs, and endoparasitism. While endoparasites play key roles in controlling nematode populations in nature, their application for integrated pest management is hindered by the limited understanding of their biology. We present a comparative analysis of a high quality finished genome assembly of Drechmeria coniospora, a model endoparasitic nematophagous fungus, integrated with a transcriptomic study. Adaptation of D. coniospora to its almost completely obligate endoparasitic lifestyle led to the simplification of many orthologous gene families involved in the saprophytic trophic mode, while maintaining orthologs of most known fungal pathogen-host interaction proteins, stress response circuits and putative effectors of the small secreted protein type. The need to adhere to and penetrate the host cuticle led to a selective radiation of surface proteins and hydrolytic enzymes. Although the endoparasite has a simplified secondary metabolome, it produces a novel peptaibiotic family that shows antibacterial, antifungal and nematicidal activities. Our analyses emphasize the basic malleability of the D. coniospora genome: loss of genes advantageous for the saprophytic lifestyle; modulation of elements that its cohort species utilize for entomopathogenesis; and expansion of protein families necessary for the nematode endoparasitic lifestyle.

Stress tolerances of nullmutants of function-unknown genes encoding menadione stress-responsive proteins in Aspergillus nidulans.

A group of menadione stress-responsive function-unkown genes of Aspergillus nidulans (Locus IDs ANID_03987.1, ANID_06058.1, ANID_10219.1, and ANID_10260.1) was deleted and phenotypically characterized. Importantly, comparative and phylogenetic analyses of the tested A. nidulans genes and their orthologs shed light only on the presence of a TANGO2 domain with NRDE protein motif in the translated ANID_06058.1 gene but did not reveal any recognizable protein-encoding domains in other protein sequences. The gene deletion strains were subjected to oxidative, osmotic, and metal ion stress and, surprisingly, only the ΔANID_10219.1 mutant showed an increased sensitivity to 0.12 mmol l(-1) menadione sodium bisulfite. The gene deletions affected the stress sensitivities (tolerances) irregularly, for example, some strains grew more slowly when exposed to various oxidants and/or osmotic stress generating agents, meanwhile the ΔANID_10260.1 mutant possessed a wild-type tolerance to all stressors tested. Our results are in line with earlier studies demonstrating that the deletions of stress-responsive genes do not confer necessarily any stress-sensitivity phenotypes, which can be attributed to compensatory mechanisms based on other elements of the stress response system with overlapping functions.

Fuzziness endows viral motif-mimicry.

Motif-mimicry is exploited by viruses to interfere with host regulatory networks and has also been suggested as a prevalent strategy for eukaryotic and prokaryotic pathogens. Using the same peptide motif however does not guarantee more effective interactions with the host. Motif-mediated interactions require a flexible or disordered environment, with structural and dynamic features that should differ between the competing host and viral proteins. Using the eukaryotic linear motif (ELM) database we analyzed the protein regions which contained the eukaryotic and viral motifs, including human and human virus ELMs with common target sites. We found that although the eukaryotic motifs are associated with a lack of structure, they are more stable than their flanking regions and can serve as molecular recognition elements. In contrast, eukaryotic viral motifs are often located in more ordered regions, but have increased local flexibility or disorder compared to their embedding environment. Most viral ELMs are devoid of stable binding elements and remain fuzzy after binding. Fuzziness reduces the entropic cost of binding and imparts versatile interaction modes to increase binding promiscuity and to compete with multiple host peptides. Fuzzy interactions confer further functional benefits such as the combinatorial usage of motifs, and a fine-tuning affinity via post-translational modifications.

Fuzzy complexes: Specific binding without complete folding.

Specific molecular recognition is assumed to require a well-defined set of contacts and devoid of conformational and interaction ambiguities. Growing experimental evidence demonstrates however, that structural multiplicity or dynamic disorder can be retained in protein complexes, termed as fuzziness. Fuzzy regions establish alternative contacts between specific partners usually via transient interactions. Nature often tailors the dynamic properties of these segments via post-translational modifications or alternative splicing to fine-tune affinity. Most experimentally characterized fuzzy complexes are involved in regulation of gene-expression, signal transduction and cell-cycle regulation. Fuzziness is also characteristic to viral protein complexes, cytoskeleton structure, and surprisingly in a few metabolic enzymes. A plausible role of fuzzy complexes in increasing half-life of intrinsically disordered proteins is also discussed.

Toxicity of abiotic stressors to Fusarium species: differences in hydrogen peroxide and fungicide tolerance.

Stress sensitivity of three related phytopathogenic Fusarium species (Fusarium graminearum, Fusarium oxysporum and Fusarium verticillioides) to different oxidative, osmotic, cell wall, membrane, fungicide stressors and an antifungal protein (PAF) were studied in vitro. The most prominent and significant differences were found in oxidative stress tolerance: all the three F. graminearum strains showed much higher sensitivity to hydrogen peroxide and, to a lesser extent, to menadione than the other two species. High sensitivity of F. verticillioides strains was also detectable to an azole drug, Ketoconazole. Surprisingly, no or limited differences were observed in response to other oxidative, osmotic and cell wall stressors. These results indicate that fungal oxidative stress response and especially the response to hydrogen peroxide (this compound is involved in a wide range of plant-fungus interactions) might be modified on niche-specific manner in these phylogenetically related Fusarium species depending on their pathogenic strategy. Supporting the increased hydrogen peroxide sensitivity of F. graminearum, genome-wide analysis of stress signal transduction pathways revealed the absence one CatC-type catalase gene in F. graminearum in comparison to the other two species.

FSRD: fungal stress response database.

Adaptation to different types of environmental stress is a common part of life for today's fungi. A deeper understanding of the organization, regulation and evolution of fungal stress response systems may lead to the development of novel antifungal drugs and technologies or the engineering of industrial strains with elevated stress tolerance. Here we present the Fungal Stress Response Database (http://internal.med.unideb.hu/fsrd) aimed to stimulate further research on stress biology of fungi. The database incorporates 1985 fungal stress response proteins with verified physiological function(s) and their orthologs identified and annotated in 28 species including human and plant pathogens, as well as important industrial fungi. The database will be extended continuously to cover other fully sequenced fungal species. Our database, as a starting point for future stress research, facilitates the analysis of literature data on stress and the identification of ortholog groups of stress response proteins in newly sequenced fungal genomes. Database URL: http://internal.med.unideb.hu/fsrd

Transcriptome changes initiated by carbon starvation in Aspergillus nidulans.

Carbon starvation is a common stress for micro-organisms both in nature and in industry. The carbon starvation stress response (CSSR) involves the regulation of several important processes including programmed cell death and reproduction of fungi, secondary metabolite production and extracellular hydrolase formation. To gain insight into the physiological events of CSSR, DNA microarray analyses supplemented with real-time RT-PCR (rRT-PCR) experiments on 99 selected genes were performed. These data demonstrated that carbon starvation induced very complex changes in the transcriptome. Several genes contributing to protein synthesis were upregulated together with genes involved in the unfolded protein stress response. The balance between biosynthesis and degradation moved towards degradation in the case of cell wall, carbohydrate, lipid and nitrogen metabolism, which was accompanied by the production of several hydrolytic enzymes and the induction of macroautophagy. These processes provide the cultures with long-term survival by liberating nutrients through degradation of the cell constituents. The induced synthesis of secondary metabolites, antifungal enzymes and proteins as well as bacterial cell wall-degrading enzymes demonstrated that carbon-starving fungi should have marked effects on the micro-organisms in their surroundings. Due to the increased production of extracellular and vacuolar enzymes during carbon starvation, the importance of the endoplasmic reticulum increased considerably.

Protein phosphatase Z modulates oxidative stress response in fungi.

The genome of the filamentous fungus Aspergillus nidulans harbors the gene ppzA that codes for the catalytic subunit of protein phosphatase Z (PPZ), and the closely related opportunistic pathogen Aspergillus fumigatus encompasses a highly similar PPZ gene (phzA). When PpzA and PhzA were expressed in Saccharomyces cerevisiae or Schizosaccharomyces pombe they partially complemented the deleted phosphatases in the ppz1 or the pzh1 mutants, and they also mimicked the effect of Ppz1 overexpression in slt2 MAP kinase deficient S. cerevisiae cells. Although ppzA acted as the functional equivalent of the known PPZ enzymes its disruption in A. nidulans did not result in the expected phenotypes since it failed to affect salt tolerance or cell wall integrity. However, the inactivation of ppzA resulted in increased sensitivity to oxidizing agents like tert-butylhydroperoxide, menadione, and diamide. To demonstrate the general validity of our observations we showed that the deletion of the orthologous PPZ genes in other model organisms, such as S. cerevisiae (PPZ1) or Candida albicans (CaPPZ1) also caused oxidative stress sensitivity. Thus, our work reveals a novel function of the PPZ enzyme in A. nidulans that is conserved in very distantly related fungi.

Polyphasic characterization of "Aspergillus nidulans var. roseus" ATCC 58397.

Polyphasic characterization of the echinocandin B producer Aspergillus nidulans var. roseus ATCC 58397 strain was carried out to elucidate its taxonomical status. According to its carbon source utilization and secondary metabolite spectrum as well as the partial ß-tubulin, calmodulin, and γ-actin gene sequences, A. nidulans var. roseus belongs to the Emericella rugulosa species. Auxotroph mutants of A. nidulans var. roseus ATCC 58397 and E. rugulosa CBS 171.71 and CBS 133.60 formed stable heterokaryons on minimal medium with several A. nidulans strains, and in the case of A. nidulans var. roseus, even cleistothecia were developed.