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.

Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.

Correction: Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

[This corrects the article DOI: 10.1371/journal.pgen.1007511.].

Genome sequencing of evolved aspergilli populations reveals robust genomes, transversions in A. flavus, and sexual aberrancy in non-homologous end-joining mutants.

BACKGROUND: Aspergillus spp. comprises a very diverse group of lower eukaryotes with a high relevance for industrial applications and clinical implications. These multinucleate species are often cultured for many generations in the laboratory, which can unknowingly propagate hidden genetic mutations. To assess the likelihood of such events, we studied the genome stability of aspergilli by using a combination of mutation accumulation (MA) lines and whole genome sequencing. RESULTS: We sequenced the whole genomes of 30 asexual and 10 sexual MA lines of three Aspergillus species (A. flavus, A. fumigatus and A. nidulans) and estimated that each MA line accumulated mutations for over 4000 mitoses during asexual cycles. We estimated mutation rates of 4.2 × 10-11 (A. flavus), 1.1 × 10-11 (A. fumigatus) and 4.1 × 10-11 (A. nidulans) per site per mitosis, suggesting that the genomes are very robust. Unexpectedly, we found a very high rate of GC → TA transversions only in A. flavus. In parallel, 30 asexual lines of the non-homologous end-joining (NHEJ) mutants of the three species were also allowed to accumulate mutations for the same number of mitoses. Sequencing of these NHEJ MA lines gave an estimated mutation rate of 5.1 × 10-11 (A. flavus), 2.2 × 10-11 (A. fumigatus) and 4.5 × 10-11 (A. nidulans) per base per mitosis, which is slightly higher than in the wild-type strains and some ~ 5-6 times lower than in the yeasts. Additionally, in A. nidulans, we found a NHEJ-dependent interference of the sexual cycle that is independent of the accumulation of mutations. CONCLUSIONS: We present for the first time direct counts of the mutation rate of filamentous fungal species and find that Aspergillus genomes are very robust. Deletion of the NHEJ machinery results in a slight increase in the mutation rate, but at a rate we suggest is still safe to use for biotechnology purposes. Unexpectedly, we found GC→TA transversions predominated only in the species A. flavus, which could be generated by the hepatocarcinogen secondary metabolite aflatoxin. Lastly, a strong effect of the NHEJ mutation in self-crossing was observed and an increase in the mutations of the asexual lines was quantified.

Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species.

The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen Aspergillus fumigatus.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

Proteomic profiling of the antifungal drug response of Aspergillus fumigatus to voriconazole.

Antifungal resistance is an emerging problem and one of the reasons for treatment failure of invasive aspergillosis (IA). Voriconazole has become a standard therapeutic for the treatment of this often fatal infection. We studied the differentially expressed proteins as a response of Aspergillus fumigatus to voriconazole by employing the two-dimensional difference gel electrophoresis (DIGE) technique. Due to addition of drug, a total of 135 differentially synthesized proteins were identified by MALDI-TOF/TOF-mass spectrometry. In particular, the level of proteins involved in the general stress response and cell detoxification increased prominently. In contrast, cell metabolism and energy proteins were down-regulated, which suggests the cellular effort to maintain balance in energy utilization while trying to combat the cellular stress exerted by the drug. We detected several so-far uncharacterized proteins which may play a role in stress response and drug metabolism and which could be future targets for antifungal treatment. A mutant strain, which is deleted in the cross-pathway control gene cpcA, was treated with voriconazole to investigate the contribution of the general control of amino acid biosynthesis to drug resistance. We compared the mutant strain's protein expression profile with the wild-type strain. The absence of CpcA led to an increased resistance to voriconazole and a reduced activation of some general stress response proteins, while the transcript level of the triazole target gene erg11A (cyp51A) remained unchanged. In contrast, the sensitivity of strain ΔcpcA to terbinafine and amphotericin B was slightly increased. These findings imply a role of CpcA in the cellular stress response to azole drugs at the post transcriptional level.

RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior.

BACKGROUND: Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. RESULTS: We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. CONCLUSIONS: We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.

SAGA/ADA complex subunit Ada2 is required for Cap1- but not Mrr1-mediated upregulation of the Candida albicans multidrug efflux pump MDR1.

Overexpression of the multidrug efflux pump MDR1 is one mechanism by which the pathogenic yeast Candida albicans develops resistance to the antifungal drug fluconazole. The constitutive upregulation of MDR1 in fluconazole-resistant, clinical C. albicans isolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activates MDR1 transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However, MDR1 expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response in C. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotes MDR1 overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 caused MDR1 overexpression in a wild-type strain but only weakly in mutants lacking ADA2. In the presence of benomyl or H2O2, compounds that induce MDR1 expression in an Mrr1- and Cap1-dependent fashion, MDR1 was upregulated with the same efficiency in wild-type and ada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to the MDR1 promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required for MDR1 overexpression in fluconazole-resistant C. albicans strains containing gain-of-function mutations in Mrr1.

White-opaque switching of Candida albicans allows immune evasion in an environment-dependent fashion.

Candida albicans strains that are homozygous at the mating type locus can spontaneously and reversibly switch from the normal yeast morphology (white) to an elongated cell type (opaque), which is the mating-competent form of the fungus. White-opaque switching also influences the ability of C. albicans to colonize and proliferate in specific host niches and its susceptibility to host defense mechanisms. We used live imaging to observe the interaction of white and opaque cells with host phagocytic cells. For this purpose, we generated derivatives of the switching-competent strain WO-1 that express green fluorescent protein from a white-specific promoter and red fluorescent protein from an opaque-specific promoter or vice versa. When mixed populations of these differentially labeled white and opaque cells were incubated with human polymorphonuclear neutrophils (PMNs) on a glass slide, the neutrophils selectively phagocytosed and killed white cells, despite frequent physical interaction with opaque cells. White cells were attacked only after they started to form a germ tube, indicating that the suppression of filamentation in opaque cells saved them from recognition by the PMNs. In contrast to neutrophils, dendritic cells internalized white as well as opaque cells. However, when embedded in a collagen matrix, the PMNs also phagocytosed both white and opaque cells with similar efficiency. These results suggest that, depending on the environment, white-opaque switching enables C. albicans to escape from specific host defense mechanisms.

Roles of different peptide transporters in nutrient acquisition in Candida albicans.

Fungi possess two distinct proton-coupled peptide transport systems, the dipeptide/tripeptide transporters (PTR) and the oligopeptide transporters (OPT), which enable them to utilize peptides as nutrients. In the pathogenic yeast Candida albicans, peptide transporters are encoded by gene families consisting of two PTR genes and eight OPT genes. To gain insight into the functions and importance of specific peptide transporters, we generated mutants lacking the two dipeptide/tripeptide transporters Ptr2 and Ptr22, as well as the five major oligopeptide transporters Opt1 to Opt5. These mutants were unable to grow in media containing peptides as the sole nitrogen source. Forced expression of individual peptide transporters in the septuple mutants showed that Ptr2 and Ptr22 could utilize all tested dipeptides as substrates but differed in their abilities to transport specific tripeptides. Interestingly, several oligopeptide transporters, which are thought to transport peptides consisting of more than three amino acids, also mediated the uptake of tripeptides. Opt1 especially turned out to be a highly flexible transporter that enabled growth on all tripeptides tested and could even utilize a dipeptide, a function that has never been ascribed to this family of peptide transporters. Despite their inability to grow on proteins or peptides, the opt1Δ opt2Δ opt3Δ opt4Δ opt5Δ ptr2Δ ptr22Δ septuple mutants had no in vivo fitness defect in a mouse model of gastrointestinal colonization. Therefore, the nutritional versatility of C. albicans enables it to utilize alternative nitrogen sources in this host niche, which probably contributes to its success as a commensal and pathogen in mammalian hosts.

The stepwise acquisition of fluconazole resistance mutations causes a gradual loss of fitness in Candida albicans.

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1, Tac1 and Upc2, which result in constitutive overexpression of multidrug efflux pumps and ergosterol biosynthesis genes respectively. It is not known how the permanently changed gene expression program in resistant strains affects their fitness in the absence of drug selection pressure. We have systematically investigated the effects of activating mutations in Mrr1, Tac1 and Upc2, individually and in all possible combinations, on the degree of fluconazole resistance and on the fitness of C. albicans in an isogenic strain background. All combinations of different resistance mechanisms resulted in a stepwise increase in drug resistance, culminating in 500-fold increased fluconazole resistance in strains possessing mutations in the three transcription factors and an additional resistance mutation in the drug target enzyme Erg11. The acquisition of resistance mutations was associated with reduced fitness under non-selective conditions in vitro as well as in vivo during colonization of a mammalian host. Therefore, without compensatory mutations, the inability to appropriately regulate gene expression results in a loss of competitive fitness of drug-resistant C. albicans strains.

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.

Inducible and constitutive activation of two polymorphic promoter alleles of the Candida albicans multidrug efflux pump MDR1.

Overexpression of the multidrug efflux pump MDR1 confers resistance to the antifungal drug fluconazole on Candida albicans. It has been reported that two types of MDR1 promoters exist in C. albicans and that homozygosity for the allele with higher activity may promote fluconazole resistance. We found that the two MDR1 promoter alleles in strain SC5314 were equally well activated by inducing chemicals or hyperactive forms of the transcription factors Mrr1 and Cap1, which control MDR1 expression. In addition, no loss of heterozygosity at the MDR1 locus was observed in MDR1-overexpressing clinical C. albicans strains that developed fluconazole resistance during therapy.

Shaping the fungal adaptome--stress responses of Aspergillus fumigatus.

Aspergillus fumigatus as prime pathogen to cause aspergillosis has evolved as a saprophyte, but is also able to infect and colonise immunocompromised hosts. Based on the 'dual use' hypothesis of fungal pathogenicity, general characteristics have to be considered as unspecific virulence determinants, among them stress adaptation capacities. The susceptible, warm-blooded mammalian host represents a specific ecological niche that poses several kinds of stress conditions to the fungus during the course of infection. Detailed knowledge about the cellular pathways and adaptive traits that have evolved in A. fumigatus to counteract situations of stress and varying environmental conditions is crucial for the identification of novel and specific antifungal targets. Comprehensive profiling data accompanied by mutant analyses have shed light on such stressors, and nutritional deprivation, oxidative stress, hypoxia, elevated temperature, alkaline pH, extensive secretion, and, in particular during treatment with antifungals, cell membrane perturbations appear to represent the major hazards A. fumigatus has to cope with during infection. Further efforts employing innovative approaches and advanced technologies will have to be made to expand our knowledge about the scope of the A. fumigatus adaptome that is relevant for disease.

Secondary metabolites of Hülle cells mediate protection of fungal reproductive and overwintering structures against fungivorous animals.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones accumulate emodins and are delayed in maturation and growth of fruiting bodies. Emodin represses fruiting body and resting structure formation in other fungi. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. Our findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

Basal expression of the Aspergillus fumigatus transcriptional activator CpcA is sufficient to support pulmonary aspergillosis.

Aspergillosis is a disease determined by various factors that influence fungal growth and fitness. A conserved signal transduction cascade linking environmental stress to amino acid homeostasis is the Cross-Pathway Control (CPC) system that acts via phosphorylation of the translation initiation factor eIF2 by a sensor kinase to elevate expression of a transcription factor. Ingestion of Aspergillus fumigatus conidia by macrophages does not trigger this stress response, suggesting that their phagosomal microenvironment is not deficient in amino acids. The cpcC gene encodes the CPC eIF2alpha kinase, and deletion mutants show increased sensitivity towards amino acid starvation. CpcC is specifically required for the CPC response but has limited influence on the amount of phosphorylated eIF2alpha. Strains deleted for the cpcC locus are not impaired in virulence in a murine model of pulmonary aspergillosis. Accordingly, basal expression of the Cross-Pathway Control transcriptional activator appears sufficient to support aspergillosis in this disease model.

Heavy Metal-Induced Expression of PcaA Provides Cadmium Tolerance to Aspergillus fumigatus and Supports Its Virulence in the Galleria mellonella Model.

Most of the metal transporters in Aspergillus fumigatus are yet uncharacterized. Their role in fungal metabolism and virulence remains unclear. This paper describes the novel PIB-type cation ATPase PcaA, which links metal homeostasis and heavy metal tolerance in the opportunistic human pathogen A. fumigatus. The protein possesses conserved ATPase motif and shares 51% amino acid sequence identity with the Saccharomyces cerevisiae cadmium exporter Pca1p. A pcaA deletion, an overexpression and a gfp-pcaA complementation strain of A. fumigatus were constructed and their heavy metal susceptibilities were studied. The pcaA knock out strain showed drastically decreased cadmium tolerance, however, its growth was not affected by the exposure to high concentrations of copper, iron, zinc, or silver ions. Although the lack of PcaA had no effect on copper adaption, we demonstrated that not only cadmium but also copper ions are able to induce the transcription of pcaA in A. fumigatus wild type Af293. Similarly, cadmium and copper ions could induce the copper exporting ATPase crpA. These data imply a general response on the transcriptomic level to heavy metals in A. fumigatus through the induction of detoxification systems. Confocal microscopy of the gfp-pcaA complementation strain expressing functional GFP-PcaA supports the predicted membrane localization of PcaA. The GFP-PcaA fusion protein is located in the plasma membrane of A. fumigatus in the presence of cadmium ions. Virulence assays support a function of PcaA for virulence of A. fumigatus in the Galleria mellonella wax moth larvae model, which might be linked to the elimination of reactive oxygen species.

Amino acid acquisition, cross-pathway control, and virulence in Aspergillus.

Supply of all amino acids required for translation is crucial for the synthesis of new proteins. Fungal amino acid biosynthesis has to be coordinated with amino acid uptake as well as protein degradation. A global regulator that connects amino acid biosynthesis and developmental programs is the transcription factor CpcA/Gcn4p. This transcriptional activator is conserved within the fungal kingdom and the cellular levels of this protein are carefully regulated. Deletion of the encoding cpcA gene in the opportunistic pathogen Aspergillus fumigatus results in impaired virulence in immuno-compromised mice, suggesting a role of the cross-pathway control system in fungal pathogenicity.

Gene deletion in Candida albicans wild-type strains using the SAT1-flipping strategy.

Targeted gene inactivation is an important method to investigate gene function. In the diploid yeast Candida albicans, the generation of homozygous knock-out mutants requires the sequential replacement of both alleles of a gene by a selection marker. Targeted gene deletion is often performed in auxotrophic host strains, which are rendered prototrophic after the insertion of appropriate nutritional marker genes into the target locus. The SAT1-flipping strategy described in this chapter allows gene deletion in prototrophic C. albicans wild-type strains with the help of a recyclable dominant selection marker. The SAT1 flipper cassette used for this purpose consists of the caSAT1 marker, which confers resistance to the antibiotic nourseothricin, and the caFLP gene, which encodes the site-specific recombinase FLP. The addition of flanking sequences of the target gene allows specific genomic insertion of the SAT1 flipper cassette by homologous recombination and selection of nourseothricin-resistant transformants. Expression of the FLP recombinase results in subsequent excision of the cassette, which is bordered by direct repeats of the FLP recognition sequence FRT, from the genome. The homozygous mutants obtained after two rounds of insertion and recycling of the SAT1 flipper cassette differ from the wild-type parental strain only by the absence of the target gene and can be used for the inactivation of additional genes and the generation of complemented strains using the same strategy.