RESUMEN
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.
Asunto(s)
Aspergillus nidulans , Proteínas F-Box , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Metiltransferasas/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismoRESUMEN
Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.
Asunto(s)
Aspergillus nidulans , Cromatina , Acetiltransferasas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Genes Reguladores , Histona Desacetilasas/metabolismo , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Esterigmatocistina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta.
Asunto(s)
Proteínas Fúngicas/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Esporas Fúngicas , Verticillium/fisiología , Xilema/metabolismo , Proteínas de Unión al ADN , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Interacciones Huésped-Patógeno , Solanum lycopersicum , Modelos Biológicos , Fenotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Metabolismo SecundarioRESUMEN
Gliotoxin (GT) biosynthesis in fungi is encoded by the gli biosynthetic gene cluster. While GT addition autoinduces biosynthesis, Zn2+ has been shown to attenuate cluster activity, and it was speculated that identification of Zn2Cys6 binuclear transcription factor GliZ binding partners might provide insight into this observation. Using the Tet-ON induction system, doxycycline (DOX) presence induced GliZ fusion protein expression in, and recovery of GT biosynthesis by, A. fumigatus ΔgliZ::HA-gliZ and ΔgliZ::TAP-gliZ strains, respectively. Quantitative RT-PCR confirmed that DOX induces gli cluster gene expression (n = 5) in both A. fumigatus HA-GliZ and TAP-GliZ strains. GT biosynthesis was evident in Czapek-Dox and in Sabouraud media, however tagged GliZ protein expression was more readily detected in Sabouraud media. Unexpectedly, Zn2+ was essential for GliZ fusion protein expression in vivo, following 3 h DOX induction. Moreover, HA-GliZ abundance was significantly higher in either DOX/GT or DOX/Zn2+, compared to DOX-only. This suggests that while GT induction is still intact, Zn2+ inhibition of HA-GliZ production in vivo is lost. Co-immunoprecipitation revealed that GT oxidoreductase GliT associates with GliZ in the presence of GT, suggesting a potential protective role. Additional putative HA-GliZ interacting proteins included cystathionine gamma lyase, ribosomal protein L15 and serine hydroxymethyltransferase (SHMT). Total mycelial quantitative proteomic data revealed that GliT and GtmA, as well as several other gli cluster proteins, are increased in abundance or uniquely expressed with GT addition. Proteins involved in sulphur metabolism are also differentially expressed with GT or Zn2+ presence. Overall, we disclose that under DOX induction GliZ functionality is unexpectedly evident in zinc-replete media, subject to GT induction and that GliT appears to associate with GliZ, potentially to prevent dithiol gliotoxin (DTG)-mediated GliZ inactivation by zinc ejection.
Asunto(s)
Aspergillus fumigatus , Gliotoxina , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteómica , Zinc/metabolismoRESUMEN
Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.
Asunto(s)
Malus , Patulina , Penicillium , Virulencia/genética , Patulina/análisis , Patulina/metabolismo , Frutas/química , Frutas/metabolismo , Frutas/microbiología , Penicillium/genética , Penicillium/metabolismoRESUMEN
The filamentous fungus Aspergillus flavus is a plant and human pathogen predominantly found in the soil as spores or sclerotia and is capable of producing various secondary metabolites (SM) such as the carcinogenic mycotoxin aflatoxin. Recently, we have discovered a novel nuclear chromatin binding complex (KERS) that contains the JARID1-type histone demethylase KdmB, a putative cohesion acetyl transferase EcoA, a class I type histone deacetylase RpdA and the PHD ring finger reader protein SntB in the model filamentous fungus Aspergillus nidulans. Here, we show the presence of the KERS complex in A. flavus by immunoprecipitation-coupled mass spectrometry and constructed kdmBΔ and rpdAΔ strains to study their roles in fungal development, SM production and histone post-translational modifications (HPTMs). We found that KdmB and RpdA couple the regulation of SM gene clusters with fungal light-responses and HPTMs. KdmB and RpdA have opposing roles in light-induced asexual conidiation, while both factors are positive regulators of sclerotia development through the nsdC and nsdD pathway. KdmB and RpdA are essential for the productions of aflatoxin (similar to findings for SntB) as well as cyclopiazonic acid, ditryptophenaline and leporin B through controlling the respective SM biosynthetic gene clusters. We further show that both KdmB and RpdA regulate H3K4me3 and H3K9me3 levels, while RpdA also acts on H3K14ac levels in nuclear extracts. Therefore, the chromatin modifiers KdmB and RpdA of the KERS complex are key regulators for fungal development and SM metabolism in A. flavus.
Asunto(s)
Aflatoxinas , Aspergillus flavus , Humanos , Cromatina/metabolismo , Metabolismo Secundario/genética , Virulencia , Proteínas Fúngicas/metabolismo , Aflatoxinas/genética , Regulación Fúngica de la Expresión GénicaRESUMEN
Filamentous fungal cells, unlike yeasts, fuse during vegetative growth. The orthologs of mitogen-activated protein (MAP) kinase Fus3 and transcription factor Ste12 are commonly involved in the regulation of cell fusion. However, the specific regulatory mechanisms underlying cell fusion in filamentous fungi have not been revealed. In the present study, we identified the novel protein FsiA as an AoFus3- and AoSte12-interacting protein in the filamentous fungus Aspergillus oryzae. The expression of AonosA and cell fusion-related genes decreased upon fsiA deletion and increased with fsiA overexpression, indicating that FsiA is a positive regulator of cell fusion. In addition, the induction of cell fusion-related genes by fsiA overexpression was also observed in the Aoste12 deletion mutant, indicating that FsiA can induce the cell fusion-related genes in an AoSte12-independent manner. Surprisingly, the fsiA and Aoste12 double deletion mutant exhibited higher cell fusion efficiency and increased mRNA levels of the cell fusion-related genes as compared to the fsiA single deletion mutant, which revealed that AoSte12 represses the cell fusion-related genes in the fsiA deletion mutant. Taken together, our data demonstrate that FsiA activates the cell fusion-related genes by suppressing the negative function of AoSte12 as well as by an AoSte12-independent mechanism.
Asunto(s)
Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Transcripción/metabolismo , Fusión Celular , ADN de Hongos , Genes Fúngicos , Mapas de Interacción de Proteínas , Eliminación de SecuenciaRESUMEN
Eukaryotic striatin forms striatin-interacting phosphatase and kinase (STRIPAK) complexes that control many cellular processes including development, cellular transport, signal transduction, stem cell differentiation and cardiac functions. However, detailed knowledge of complex assembly and its roles in stress responses are currently poorly understood. Here, we discovered six striatin (StrA) interacting proteins (Sips), which form a heptameric complex in the filamentous fungus Aspergillus nidulans. The complex consists of the striatin scaffold StrA, the Mob3-type kinase coactivator SipA, the SIKE-like protein SipB, the STRIP1/2 homolog SipC, the SLMAP-related protein SipD and the catalytic and regulatory phosphatase 2A subunits SipE (PpgA), and SipF, respectively. Single and double deletions of the complex components result in loss of multicellular light-dependent fungal development, secondary metabolite production (e.g. mycotoxin Sterigmatocystin) and reduced stress responses. sipA (Mob3) deletion is epistatic to strA deletion by supressing all the defects caused by the lack of striatin. The STRIPAK complex, which is established during vegetative growth and maintained during the early hours of light and dark development, is mainly formed on the nuclear envelope in the presence of the scaffold StrA. The loss of the scaffold revealed three STRIPAK subcomplexes: (I) SipA only interacts with StrA, (II) SipB-SipD is found as a heterodimer, (III) SipC, SipE and SipF exist as a heterotrimeric complex. The STRIPAK complex is required for proper expression of the heterotrimeric VeA-VelB-LaeA complex which coordinates fungal development and secondary metabolism. Furthermore, the STRIPAK complex modulates two important MAPK pathways by promoting phosphorylation of MpkB and restricting nuclear shuttling of MpkC in the absence of stress conditions. SipB in A. nidulans is similar to human suppressor of IKK-ε(SIKE) protein which supresses antiviral responses in mammals, while velvet family proteins show strong similarity to mammalian proinflammatory NF-KB proteins. The presence of these proteins in A. nidulans further strengthens the hypothesis that mammals and fungi use similar proteins for their immune response and secondary metabolite production, respectively.
Asunto(s)
Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Depuradores de Radicales Libres/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luz , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Membrana Nuclear/metabolismo , Estructura Cuaternaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estrés FisiológicoRESUMEN
For eukaryotes like fungi to regulate biological responses to environmental stimuli, various signalling cascades are utilized, like the highly conserved mitogen-activated protein kinase (MAPK) pathways. In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module regulates development and the production of secondary metabolites (SMs). This pathway consists five proteins, the three kinases SteC, MkkB and MpkB, the adaptor SteD and the scaffold HamE. In this study, homologs of these five pheromone module proteins have been identified in the plant and human pathogenic fungus Aspergillus flavus. We have shown that a tetrameric complex consisting of the three kinases and the SteD adaptor is assembled in this species. It was observed that this complex assembles in the cytoplasm and that MpkB translocates into the nucleus. Deletion of steC, mkkB, mpkB or steD results in abolishment of both asexual sporulation and sclerotia production. This complex is required for the positive regulation of aflatoxin production and negative regulation of various SMs, including leporin B and cyclopiazonic acid (CPA), likely via MpkB interactions in the nucleus. These data highlight the conservation of the pheromone module in Aspergillus species, signifying the importance of this pathway in regulating fungal development and secondary metabolism.
Asunto(s)
Aflatoxinas/metabolismo , Aspergillus flavus/crecimiento & desarrollo , Aspergillus flavus/metabolismo , Proteínas Fúngicas/metabolismo , Feromonas/metabolismo , Aflatoxina B1 , Aspergillus flavus/genética , Aspergillus nidulans , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fenotipo , Metabolismo SecundarioRESUMEN
The ubiquitin proteasome system is critical for the regulation of protein turnover, which is implicated in the modulation of a wide array of biological processes in eukaryotes, ranging from cell senescence to virulence in plant and human hosts. Proteins to be marked for ubiquitination and subsequent degradation are bound by F-box proteins, which are interchangeable substrate-recognising receptors. These F-box proteins bind a wide range of substrates and associate with the adaptor protein Skp1 and the scaffold Cul1 to form Skp1-Cul1-F-box (SCF) complexes. SCF complex components are highly conserved in eukaryotes, ranging from yeast to humans. However, information regarding the composition of these complexes and the biological roles of F-box proteins is limited, specifically in filamentous fungal species like the genus Aspergillus. In this study, we have identified 51 and 55 fbx-encoding genes in the genomes of two pathogenic fungi, A. fumigatus and A. flavus, respectively. Immunoprecipitations of the HA-tagged SkpA adaptor protein revealed that 26 F-box proteins in A. fumigatus and 30 F-box proteins in A. flavus are involved in SCF complex formation during vegetative growth. These interactome data also revealed that a diverse array of SCF complex conformations exist in response to various exogenous stressors. Lastly, we have provided evidence that the F-box protein Fbx45 interacts with SkpA in both species in response to Amphotericin B. Orthologs of the fbx45 gene are highly conserved in Aspergillus species, but are not present within the genomes of organisms such as yeast, plants or humans. This suggests that Fbx45 could potentially be a novel F-box protein that is unique to specific filamentous fungi such as Aspergillus species.
Asunto(s)
Aspergilosis/genética , Aspergillus fumigatus/ultraestructura , Proteínas Cullin/genética , Proteínas F-Box/genética , Secuencia de Aminoácidos/genética , Aspergilosis/microbiología , Aspergilosis/patología , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidad , Proteínas Cullin/ultraestructura , Proteínas F-Box/ultraestructura , Humanos , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/ultraestructura , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
Mitogen-activated protein kinase (MAPK) pathways are highly conserved from yeast to human and are required for the regulation of a multitude of biological processes in eukaryotes. A pentameric MAPK pathway known as the Fus3 pheromone module was initially characterised in Saccharomyces cerevisiae and was shown to regulate cell fusion and sexual development. Individual orthologous pheromone module genes have since been found to be highly conserved in fungal genomes and have been shown to regulate a diverse array of cellular responses, such as cell growth, asexual and sexual development, secondary metabolite production and pathogenicity. However, information regarding the assembly and structure of orthologous pheromone modules, as well as the mechanisms of signalling and their biological significance is limited, specifically in filamentous fungal species. Recent studies have provided insight on the utilization of the pheromone module as a central signalling hub for the co-ordinated regulation of fungal development and secondary metabolite production. Various proteins of this pathway are also known to regulate reproduction and virulence in a range of plant pathogenic fungi. In this review, we discuss recent findings that help elucidate the structure of the pheromone module pathway in a myriad of fungal species and its implications in the control of fungal growth, development, secondary metabolism and pathogenicity.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/genética , Feromonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Metabolismo Secundario/genética , Hongos/genética , Hongos/patogenicidad , Genoma Fúngico/genética , Humanos , Fosforilación/genética , Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Virulencia/genéticaRESUMEN
Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA-sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane-associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.
Asunto(s)
Aspergillus fumigatus/genética , Esporas Fúngicas/genética , Aspergilosis/microbiología , Aspergillus fumigatus/metabolismo , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/genética , Humanos , Proteínas de la Membrana/metabolismo , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Virulencia/genéticaRESUMEN
Aspergillus fumigatus is an opportunistic human pathogen that causes various complications in patients with a weakened immune system functions. Asexual spores of A. fumigatus are responsible for initiation of aspergillosis. Long-term viability and proper germination of dormant conidia depend on trehalose accumulation, which protect the spores against thermal and oxidative stress. A putative Myb transcription factor, MybA has been recently found to be responsible for a variety of physiological and molecular roles ranging from conidiation, spore viability, trehalose accumulation, cell wall integrity and protection against reactive oxygen species. In this perspective review, we discuss the recent findings of MybA and its overlapping functions with the other regulators of conidia viability and trehalose accumulation. Therefore, the aim of this perspective is to raise interesting and stimulating questions on the molecular functions of MybA in conidiation and trehalose biogenesis and to question its genetic and physical interactions with the other regulators of conidial viability.
Asunto(s)
Aspergillus fumigatus/fisiología , Viabilidad Microbiana/genética , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Factores de Transcripción/genética , Ambiente , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Interacción Gen-Ambiente , Interacciones Huésped-Patógeno , Humanos , Factores de Transcripción/metabolismoRESUMEN
F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus.
Asunto(s)
Aspergilosis/microbiología , Aspergillus fumigatus/enzimología , Regulación Fúngica de la Expresión Génica , Proteínas Ligasas SKP Cullina F-box/metabolismo , Secuencia de Aminoácidos , Animales , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/patogenicidad , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Modelos Animales de Enfermedad , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Gliotoxina/metabolismo , Humanos , Ratones , Mutación , Estrés Oxidativo , Fosforilación , Transporte de Proteínas , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitinas/metabolismo , VirulenciaRESUMEN
Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.
Asunto(s)
Aspergillus fumigatus/enzimología , Aspergillus fumigatus/patogenicidad , Hierro/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Metabolismo Secundario , Animales , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Cromatografía Liquida , Modelos Animales de Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Aspergilosis Pulmonar Invasiva/microbiología , Aspergilosis Pulmonar Invasiva/patología , Espectrometría de Masas , Metabolómica , Ratones , Fosfoproteínas Fosfatasas/genética , Proteoma/análisis , VirulenciaRESUMEN
Fungal development and secondary metabolite production are coordinated by regulatory complexes as the trimeric velvet complex. Light accelerates asexual but decreases sexual development of the filamentous fungus Aspergillus nidulans. Changes in gene expression and secondary metabolite accumulation in response to environmental stimuli have been the focus of many studies, but a comprehensive comparison during entire development is lacking. We compared snapshots of transcript and metabolite profiles during fungal development in dark or light. Overall 2.014 genes corresponding to 19% of the genome were differentially expressed when submerged vegetative hyphae were compared to surface development. Differentiation was preferentially asexual in light or preferentially sexual connected to delayed asexual development in dark. Light induces significantly gene expression within the first 24-48h after the transfer to surfaces. Many light induced genes are also expressed in dark after a delay of up to two days, which might be required for preparation of enhanced sexual development. Darkness results in a massive transcriptional reprogramming causing a peak of lipid-derived fungal pheromone synthesis (psi factors) during early sexual development and the expression of genes for cell-wall degradation presumably to mobilize the energy for sexual differentiation. Accumulation of secondary metabolites like antitumoral terrequinone A or like emericellamide start under light conditions, whereas the mycotoxin sterigmatocystin or asperthecin and emodin appear under dark conditions during sexual development. Amino acid synthesis and pool rapidly drop after 72-96h in dark. Subsequent initiation of apoptotic cell-death pathways in darkness happens significantly later than in light. This illustrates that fungal adaptation in differentiation and secondary metabolite production to light conditions requires the reprogramming of one fifth of the potential of its genome.
Asunto(s)
Aspergillus nidulans/metabolismo , Aspergillus nidulans/efectos de la radiación , Perfilación de la Expresión Génica , Luz , Metaboloma , Metabolismo Secundario , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Factores de TiempoRESUMEN
Morphological development of fungi and their combined production of secondary metabolites are both acting in defence and protection. These processes are mainly coordinated by velvet regulators, which contain a yet functionally and structurally uncharacterized velvet domain. Here we demonstrate that the velvet domain of VosA is a novel DNA-binding motif that specifically recognizes an 11-nucleotide consensus sequence consisting of two motifs in the promoters of key developmental regulatory genes. The crystal structure analysis of the VosA velvet domain revealed an unforeseen structural similarity with the Rel homology domain (RHD) of the mammalian transcription factor NF-κB. Based on this structural similarity several conserved amino acid residues present in all velvet domains have been identified and shown to be essential for the DNA binding ability of VosA. The velvet domain is also involved in dimer formation as seen in the solved crystal structures of the VosA homodimer and the VosA-VelB heterodimer. These findings suggest that defence mechanisms of both fungi and animals might be governed by structurally related DNA-binding transcription factors.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica/fisiología , FN-kappa B/genética , Aspergillus nidulans/genética , Aspergillus nidulans/fisiología , Secuencia de Consenso/genética , Secuencia de Consenso/fisiología , ADN de Hongos/genética , ADN de Hongos/fisiología , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Genes rel/genética , Genes rel/fisiología , FN-kappa B/fisiologíaRESUMEN
Fungi and many other eukaryotes use specialized mitogen-activated protein kinases (MAPK) of the Hog1/p38 family to transduce environmental stress signals. In Aspergillus nidulans, the MAPK SakA and the transcription factor AtfA are components of a central multiple stress-signaling pathway that also regulates development. Here we characterize SrkA, a putative MAPK-activated protein kinase, as a novel component of this pathway. ΔsrkA and ΔsakA mutants share a derepressed sexual development phenotype. However, ΔsrkA mutants are not sensitive to oxidative stress, and in fact, srkA inactivation partially suppresses the sensitivity of ΔsakA mutant conidia to H2O2, tert-butyl-hydroperoxide (t-BOOH), and menadione. In the absence of stress, SrkA shows physical interaction with nonphosphorylated SakA in the cytosol. We show that H2O2 induces a drastic change in mitochondrial morphology consistent with a fission process and the relocalization of SrkA to nuclei and mitochondria, depending on the presence of SakA. SakA-SrkA nuclear interaction is also observed during normal asexual development in dormant spores. Using SakA and SrkA S-tag pulldown and purification studies coupled to mass spectrometry, we found that SakA interacts with SrkA, the stress MAPK MpkC, the PPT1-type phosphatase AN6892, and other proteins involved in cell cycle regulation, DNA damage response, mRNA stability and protein synthesis, mitochondrial function, and other stress-related responses. We propose that oxidative stress induces DNA damage and mitochondrial fission and that SakA and SrkA mediate cell cycle arrest and regulate mitochondrial function during stress. Our results provide new insights into the mechanisms by which SakA and SrkA regulate the remodelling of cell physiology during oxidative stress and development.
Asunto(s)
Aspergillus nidulans/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/genética , Transducción de Señal/efectos de los fármacos , Aspergillus fumigatus/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Transducción de Señal/genética , Esporas Fúngicas/genéticaRESUMEN
Deneddylases remove the ubiquitin-like protein Nedd8 from modified proteins. An increased deneddylase activity has been associated with various human cancers. In contrast, we show here that a mutant strain of the model fungus Aspergillus nidulans deficient in two deneddylases is viable but can only grow as a filament and is highly impaired for multicellular development. The DEN1/DenA and the COP9 signalosome (CSN) deneddylases physically interact in A. nidulans as well as in human cells, and CSN targets DEN1/DenA for protein degradation. Fungal development responds to light and requires both deneddylases for an appropriate light reaction. In contrast to CSN, which is necessary for sexual development, DEN1/DenA is required for asexual development. The CSN-DEN1/DenA interaction that affects DEN1/DenA protein levels presumably balances cellular deneddylase activity. A deneddylase disequilibrium impairs multicellular development and suggests that control of deneddylase activity is important for multicellular development.