RESUMO
Lipases comprise the third most commercialized group of enzymes worldwide and those of microbial origin are sought for their multiple advantages. Agro-industrial waste can be an alternative culture medium for producing lipases, reducing production costs and the improper disposal of waste frying oil (WFO). This study aimed to produce yeast lipases through submerged fermentation (SF) using domestic edible oil waste as inducer and alternative culture medium. The optimal culture conditions, most effective inducer, and purification method for a new lipase from Moesziomyces aphidis BRT57 were identified. Yeast was cultured in medium containing green coconut pulp and WFO waste for 72 h. The maximum production of lipases in SF occurred in a culture medium containing WFO and yeast extract at 48 and 72 h of incubation, with enzyme activities of 8.88 and 11.39 U mL-1, respectively. The lipase was isolated through ultrafiltration followed by size exclusion chromatography, achieving a 50.46 % recovery rate. To the best of our knowledge, this is the first study to report the production and purification of lipases from M. aphidis, demonstrating the value of frying oil as inducer and alternative medium for SF, contributing to the production of fatty acids for biodiesel from food waste.
Assuntos
Cocos , Lipase , Lipase/isolamento & purificação , Lipase/química , Lipase/biossíntese , Lipase/metabolismo , Cocos/química , Óleos de Plantas/química , Fermentação , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genéticaRESUMO
Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some ß-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.
Assuntos
Celulase , Cryptococcus gattii , Escherichia coli , Cryptococcus gattii/genética , Cryptococcus gattii/enzimologia , Cryptococcus gattii/química , Celulase/genética , Celulase/química , Celulase/isolamento & purificação , Celulase/metabolismo , Celulase/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/biossíntese , Expressão Gênica , Clonagem Molecular , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Introduction. Adherence is a major virulence trait in Candida glabrata that, in many strains, depends on the EPA (epithelial adhesin) genes, which confer the ability to adhere to epithelial and endothelial cells of the host. The EPA genes are generally found at subtelomeric regions, which makes them subject to subtelomeric silencing. In C. glabrata, subtelomeric silencing depends on different protein complexes, such as silent information regulator and yKu complexes, and other proteins, such as Repressor/activator protein 1 (Rap1) and Abf1. At the EPA1 locus, which encodes the main adhesin Epa1, we previously found at least two cis-acting elements, the protosilencer Sil2126 and the negative element, that contribute to the propagation of silencing from the telomere to the subtelomeric region.Hypothesis. Abf1 binds to the regulatory regions of EPA1 and other regions at the telomere E-R, thereby negatively regulating EPA1 transcription.Aim. To determine whether Abf1 and Rap1 silencing proteins bind to previously identified cis-acting elements on the right telomere of chromosome E (E-R subtelomeric region), resulting in negative regulation of EPA1 transcription and infer Abf1 and Rap1 recognition sites in C. glabrata.Methodology. We used chromatin immunoprecipitation (ChIP) followed by quantitative PCR to determine the binding sites for Abf1 and Rap1 in the intergenic regions between EPA1 and EPA2 and HYR1 and EPA1, and mutants were used to determine the silencing level of the EPA1 promoter region.Results. We found that Abf1 predominantly binds to the EPA1 promoter region, leading to negative regulation of EPA1 expression. Furthermore, the mutant abf1-43, which lacks the last 43 amino acids at its C-terminal end and is defective for subtelomeric silencing, exhibits hyperadherence to epithelial cells in vitro compared to the parental strain, suggesting that EPA1 is derepressed. We also determined the motif-binding sequences for Abf1 and Rap1 in C. glabrata using data from the ChIP assays.Conclusion. Together these data indicate that Abf1 negatively regulates EPA1 expression, leading to decreased adhesion of C. glabrata to epithelial cells.
Assuntos
Candida glabrata , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Candida glabrata/genética , Candida glabrata/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Adesão Celular , Telômero/metabolismo , Telômero/genética , Humanos , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Regiões Promotoras Genéticas , LectinasRESUMO
BACKGROUND: The incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains. RESULTS: Fluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman's correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models. CONCLUSIONS: The fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance.
Assuntos
Antifúngicos , Biofilmes , Candida , Candidíase , Reposicionamento de Medicamentos , Farmacorresistência Fúngica , Fluconazol , Testes de Sensibilidade Microbiana , Infecções Urinárias , Fluconazol/farmacologia , Egito , Humanos , Farmacorresistência Fúngica/genética , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candida/genética , Candida/isolamento & purificação , Candida/classificação , Candidíase/microbiologia , Candidíase/tratamento farmacológico , Candidíase/urina , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Animais , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Camundongos , Virulência/genética , Virulência/efeitos dos fármacos , Feminino , Masculino , Fosfolipases/genética , Fosfolipases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Background: In the face of increasing antifungal resistance among Candida albicans biofilms, this study explores the efficacy of a combined treatment using Kangbainian lotion (KBN) and miconazole nitrate (MN) to address this challenge. Methods: Using UPLC-Q-TOF/MS Analysis for Identification of Active Compounds in KBN Lotion; FICI for synergy evaluation, XTT and ROS assays for biofilm viability and oxidative stress, fluorescence and confocal laser scanning microscopy (CLSM) for structural and viability analysis, and real-time fluorescence for gene expression. Conclusion: Our study indicates that the combined application of KBN and MN somewhat impacts the structural integrity of Candida albicans biofilms and affects the expression of several key genes involved in biofilm formation, including ALS1, ALS3, HWP1, HSP90, and CSH1. These preliminary findings suggest that there may be a synergistic effect between KBN and MN, potentially influencing not only the structural aspects of fungal biofilms but also involving the modulation of genetic pathways during their formation.
Assuntos
Antifúngicos , Biofilmes , Candida albicans , Farmacorresistência Fúngica , Sinergismo Farmacológico , Miconazol , Testes de Sensibilidade Microbiana , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Antifúngicos/farmacologia , Miconazol/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
The glycoside hydrolase family 3 (GH3) ß-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 ß-glucosidases. Two distinct configurations were identified in fungal GH3 ß-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in ß-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger ß-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric ß-glucosidases enzymes for industrial application.
Assuntos
Engenharia de Proteínas , Streptomyces , beta-Glucosidase , Streptomyces/enzimologia , Streptomyces/genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , beta-Glucosidase/química , Engenharia de Proteínas/métodos , Biotecnologia/métodos , Aspergillus niger/enzimologia , Aspergillus niger/genética , Domínios Proteicos , Aspergillus/enzimologia , Aspergillus/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Domínio Catalítico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/químicaRESUMO
BACKGROUND: ââThe genus Fusarium poses significant threats to food security and safety worldwide because numerous species of the fungus cause destructive diseases and/or mycotoxin contamination in crops. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets for control strategies. DESCRIPTION: In response to these challenges, we developed the Fusarium Protein Toolkit (FPT), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum and F. verticillioides. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome based on 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework. CONCLUSION: FPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium. FPT has the potential to deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets for control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats resulting from climate change. Thus, FPT has the potential to contribute to improving food security and safety worldwide.
Assuntos
Proteínas Fúngicas , Fusarium , Internet , Fusarium/genética , Fusarium/metabolismo , Fusarium/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genoma Fúngico/genética , Variação Genética , Modelos Moleculares , Software , Conformação ProteicaRESUMO
Thermophily is a trait scattered across the fungal tree of life, with its highest prevalence within three fungal families (Chaetomiaceae, Thermoascaceae, and Trichocomaceae), as well as some members of the phylum Mucoromycota. We examined 37 thermophilic and thermotolerant species and 42 mesophilic species for this study and identified thermophily as the ancestral state of all three prominent families of thermophilic fungi. Thermophilic fungal genomes were found to encode various thermostable enzymes, including carbohydrate-active enzymes such as endoxylanases, which are useful for many industrial applications. At the same time, the overall gene counts, especially in gene families responsible for microbial defense such as secondary metabolism, are reduced in thermophiles compared to mesophiles. We also found a reduction in the core genome size of thermophiles in both the Chaetomiaceae family and the Eurotiomycetes class. The Gene Ontology terms lost in thermophilic fungi include primary metabolism, transporters, UV response, and O-methyltransferases. Comparative genomics analysis also revealed higher GC content in the third base of codons (GC3) and a lower effective number of codons in fungal thermophiles than in both thermotolerant and mesophilic fungi. Furthermore, using the Support Vector Machine classifier, we identified several Pfam domains capable of discriminating between genomes of thermophiles and mesophiles with 94% accuracy. Using AlphaFold2 to predict protein structures of endoxylanases (GH10), we built a similarity network based on the structures. We found that the number of disulfide bonds appears important for protein structure, and the network clusters based on protein structures correlate with the optimal activity temperature. Thus, comparative genomics offers new insights into the biology, adaptation, and evolutionary history of thermophilic fungi while providing a parts list for bioengineering applications.
Assuntos
Evolução Molecular , Genoma Fúngico , Genômica/métodos , Filogenia , Fungos/genética , Fungos/classificação , Adaptação Fisiológica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
The regulation of virulence in plant-pathogenic fungi has emerged as a key area of importance underlying host infections. Recent work has highlighted individual transcription factors (TFs) that serve important roles. A prominent example is PnPf2, a member of the Zn2Cys6 family of fungal TFs, which controls the expression of effectors and other virulence-associated genes in Parastagonospora nodorum during infection of wheat. PnPf2 orthologues are similarly important for other major fungal pathogens during infection of their respective host plants, and have also been shown to control polysaccharide metabolism in model saprophytes. In each case, the direct genomic targets and associated regulatory mechanisms were unknown. Significant insight was made here by investigating PnPf2 through chromatin-immunoprecipitation (ChIP) and mutagenesis approaches in P. nodorum. Two distinct binding motifs were characterised as positive regulatory elements and direct PnPf2 targets identified. These encompass known effectors and other components associated with the P. nodorum pathogenic lifestyle, such as carbohydrate-active enzymes and nutrient assimilators. The results support a direct involvement of PnPf2 in coordinating virulence on wheat. Other prominent PnPf2 targets included TF-encoding genes. While novel functions were observed for the TFs PnPro1, PnAda1, PnEbr1 and the carbon-catabolite repressor PnCreA, our investigation upheld PnPf2 as the predominant transcriptional regulator characterised in terms of direct and specific coordination of virulence on wheat, and provides important mechanistic insights that may be conserved for homologous TFs in other fungi.
Assuntos
Ascomicetos , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Doenças das Plantas , Fatores de Transcrição , Triticum , Triticum/microbiologia , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Virulência , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ascomicetos/patogenicidade , Ascomicetos/genética , Ascomicetos/metabolismoRESUMO
Invasive fungal infections are associated with high mortality, which is exacerbated by the limited antifungal drug armamentarium and increasing antifungal drug resistance. Echinocandins are a frontline antifungal drug class targeting ß-glucan synthase (GS), a fungal cell wall biosynthetic enzyme. Echinocandin resistance is generally low but increasing in species like Candida glabrata, an opportunistic yeast pathogen colonizing human mucosal surfaces. Mutations in GS-encoding genes (FKS1 and FKS2 in C. glabrata) are strongly associated with clinical echinocandin failure, but epidemiological studies show that other, as yet unidentified factors also influence echinocandin susceptibility. Furthermore, although the gut is known to be an important reservoir for emergence of drug-resistant strains, the evolution of resistance is not well understood. Here, we studied the evolutionary dynamics of C. glabrata colonizing the gut of immunocompetent mice during treatment with caspofungin, a widely-used echinocandin. Whole genome and amplicon sequencing revealed rapid genetic diversification of this C. glabrata population during treatment and the emergence of both drug target (FKS2) and non-drug target mutations, the latter predominantly in the FEN1 gene encoding a fatty acid elongase functioning in sphingolipid biosynthesis. The fen1 mutants displayed high fitness in the gut specifically during caspofungin treatment and contained high levels of phytosphingosine, whereas genetic depletion of phytosphingosine by deletion of YPC1 gene hypersensitized the wild type strain to caspofungin and was epistatic to fen1Δ. Furthermore, high resolution imaging and mass spectrometry showed that reduced caspofungin susceptibility in fen1Δ cells was associated with reduced caspofungin binding to the plasma membrane. Finally, we identified several different fen1 mutations in clinical C. glabrata isolates, which phenocopied the fen1Δ mutant, causing reduced caspofungin susceptibility. These studies reveal new genetic and molecular determinants of clinical caspofungin susceptibility and illuminate the dynamic evolution of drug target and non-drug target mutations reducing echinocandin efficacy in patients colonized with C. glabrata.
Assuntos
Antifúngicos , Candida glabrata , Candidíase , Caspofungina , Farmacorresistência Fúngica , Mutação , Esfingolipídeos , Candida glabrata/genética , Candida glabrata/efeitos dos fármacos , Candida glabrata/metabolismo , Caspofungina/farmacologia , Camundongos , Antifúngicos/farmacologia , Animais , Esfingolipídeos/biossíntese , Esfingolipídeos/metabolismo , Farmacorresistência Fúngica/genética , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Equinocandinas/farmacologia , HumanosRESUMO
New molecular technologies have helped unveil previously unexplored facets of the genome beyond the canonical proteome, including microproteins and short ORFs, products of alternative splicing, regulatory non-coding RNAs, as well as transposable elements, cis-regulatory DNA, and other highly repetitive regions of DNA. In this Review, we highlight what is known about this 'hidden genome' within the fungal kingdom. Using well-established model systems as a contextual framework, we describe key elements of this hidden genome in diverse fungal species, and explore how these factors perform critical functions in regulating fungal metabolism, stress tolerance, and pathogenesis. Finally, we discuss new technologies that may be adapted to further characterize the hidden genome in fungi.
Assuntos
Fungos , Genoma Fúngico , Genoma Fúngico/genética , Fungos/genética , Elementos de DNA Transponíveis/genética , Fases de Leitura Aberta/genética , Processamento Alternativo/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
BACKGROUND: Terbinafine is widely used to treat onychomycosis caused by dermatophyte fungi. Terbinafine resistance in recent years is causing concern. Resistance has so far been associated with single-nucleotide substitutions in the DNA sequence of the enzyme squalene epoxidase (SQLE) but how this affects SQLE functionality is not understood. OBJECTIVES: The aim of this study was to understand newly discovered resistance in two Australian strains of Trichophyton interdigitale. PATIENTS/METHODS: Resistance to terbinafine was tested in four newly isolated strains. Three-dimensional SQLE models were prepared to investigate how the structure of their SQLE affected the binding of terbinafine. RESULTS: This study found the first Australian occurrences of terbinafine resistance in two T. interdigitale strains. Both strains had novel deletion mutations in erg1 and frameshifts during translation. Three-dimensional models had smaller SQLE proteins and open reading frames as well as fewer C-terminal α-helices than susceptible strains. In susceptible strains, the lipophilic tail of terbinafine was predicted to dock stably into a hydrophobic pocket in SQLE lined by over 20 hydrophobic amino acids. In resistant strains, molecular dynamics simulations showed that terbinafine docking was unstable and so terbinafine did not block squalene metabolism and ultimately ergosterol production. The resistant reference strain ATCC MYA-4438 T. rubrum showed a single erg1 mutation that resulted in frameshift during translation, leading to C-terminal helix deletion. CONCLUSIONS: Modelling their effects on their SQLE proteins will aid in the design of potential new treatments for these novel resistant strains, which pose clinical problems in treating dermatophyte infections with terbinafine.
Assuntos
Antifúngicos , Arthrodermataceae , Farmacorresistência Fúngica , Esqualeno Mono-Oxigenase , Terbinafina , Terbinafina/farmacologia , Esqualeno Mono-Oxigenase/genética , Esqualeno Mono-Oxigenase/metabolismo , Farmacorresistência Fúngica/genética , Austrália , Antifúngicos/farmacologia , Humanos , Arthrodermataceae/efeitos dos fármacos , Arthrodermataceae/genética , Arthrodermataceae/enzimologia , Testes de Sensibilidade Microbiana , Onicomicose/microbiologia , Onicomicose/tratamento farmacológico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos MolecularesRESUMO
Filamentous fungi with their diverse inventory of carbohydrate-active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE-1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real-time evaluation of CreA/CRE-1-mediated CCR at the transcriptional level, we developed a luciferase-based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE-1-dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE-1-mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours.
Assuntos
Repressão Catabólica , Genes Reporter , Luciferases , Neurospora crassa , Luciferases/genética , Luciferases/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Regulação Fúngica da Expressão Gênica , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Fungos/metabolismo , Carbono/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas RepressorasRESUMO
Background: Aspergillus cristatus was a filamentous fungus that produced sexual spores under hypotonic stress and asexual spores under hypertonic stress. It could be useful for understanding filamentous fungi's sporulation mechanism. Previously, we conducted functional studies on Achog1, which regulated the hyperosmotic glycerol signaling (HOG) pathway and found that SI65_02513 was significantly downregulated in the transcriptomics data of ΔAchog1 knockout strain. This gene was located at multiple locations in the HOG pathway, indicating that it might play an important role in the HOG pathway of A. cristatus. Furthermore, the function of this gene had not been identified in Aspergillus fungi, necessitating further investigation. This gene's conserved domain study revealed that it has the same protein tyrosine phosphatases (PTPs) functional domain as Saccharomyces cerevisiae, hence SI65_02513 was named Acptp2,3. Methods: The function of this gene was mostly validated using gene knockout and gene complementation approaches. Knockout strains exhibited sexual and asexual development, as well as pigments synthesis. Morphological observations of the knockout strain were carried out under several stress conditions (osmotic stress, oxidative stress, Congo Red, and sodium dodecyl sulfate (SDS). Real-time fluorescence polymerase chain reaction (PCR) identified the expression of genes involved in sporulation, stress response, and pigments synthesis. Results: The deletion of Acptp2,3 reduced sexual and asexual spore production by 4.4 and 4.6 times, demonstrating that Acptp2,3 positively regulated the sporulation of A. cristatus. The sensitivity tests to osmotic stress revealed that ΔAcptp2,3 strains did not respond to sorbitol-induced osmotic stress. However, ΔAcptp2.3 strains grew considerably slower than the wild type in high concentration sucrose medium. The ΔAcptp2,3 strains grew slower than the wild type on media containing hydrogen peroxide, Congo red, and SDS. These findings showed that Acptp2,3 favorably controlled osmotic stress, oxidative stress, and cell wall-damaging chemical stress in A. cristatus. Deleting Acptp2,3 resulted in a deeper colony color, demonstrating that Apctp2,3 regulated pigment synthesis in A. cistatus. The expression levels of numerous stress-and pigments-related genes matched the phenotypic data. Conclusion: According to our findings, Acptp2,3 played an important role in the regulation of sporulation, stress response, and pigments synthesis in A. cristatus. This was the first study on the function of PTPs in Aspergillus fungi.
Assuntos
Aspergillus , Proteínas Fúngicas , Pressão Osmótica , Esporos Fúngicos , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Aspergillus/metabolismo , Aspergillus/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pigmentos Biológicos/metabolismo , Pigmentos Biológicos/biossíntese , Estresse Fisiológico , Regulação Fúngica da Expressão Gênica , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/genética , Técnicas de Inativação de Genes , Estresse Oxidativo , Vermelho Congo/farmacologiaRESUMO
Zn(II)2Cys6 proteins constitute the largest group of fungal-specific transcription factors. However, little is known about their functions in the crop killer Botrytis cinerea. In this work, a T-DNA insertion strain M13448 was identified which was inserted into the Zn(II)2Cys6 TF-encoding gene BcTBS1. Knockout of BcTBS1 did not affect mycelia growth, appressorium formation, and sclerotium germination, but impaired fungal conidiation, conidial morphogenesis, conidial germination, infection cushion development, and sclerotial formation. Accordingly, ΔBctbs1 mutants showed reduced virulence in its host plants. Further study proved that BcTBS1, BCIN_15g03870, and BCIN_12g06630 were induced by cellulose. Subsequent cellulase activity assays revealed that the loss of BcTBS1 significantly decreased cellulase activity. In addition, we verified that the BCIN_15g03870 and BCIN_12g06630 genes were positive regulated by BcTBS1 by quantitative real-time reverse-transcription-polymerase chain reaction (qRT-PCR). Taken together, these results suggested that BcTBS1 can promote pathogenicity by modulating cellulase-encoding genes that participate in host cellulose degradation.
Assuntos
Botrytis , Celulose , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Doenças das Plantas , Fatores de Transcrição , Botrytis/genética , Botrytis/patogenicidade , Botrytis/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismoRESUMO
Trichoderma longibrachiatum is a filamentous fungus used as a biological control agent against different plant diseases. The multifunctional secondary metabolites synthesized by Trichoderma, called peptaibols, have emerged as key elicitors in plant innate immunity. This study obtained a high-quality genome sequence for the T. longibrachiatum strain 40418 and identified two peptaibol biosynthetic gene clusters using knockout techniques. The two gene cluster products were confirmed as trilongin AIV a (11-residue) and trilongin BI (20-residue) using liquid chromatography coupled with tandem mass spectrometry. Further investigations revealed that these peptaibols induce plant resistance to Pseudomonas syringae pv tomato (Pst) DC3000 infection while triggering plant immunity and cell death. Notably, the two peptaibols exhibit synergistic effects in plant-microbe signaling interactions, with trilongin BI having a predominant role. Moreover, the induction of tomato resistance against Meloidogyne incognita showed similarly promising results.
Assuntos
Resistência à Doença , Peptaibols , Doenças das Plantas , Pseudomonas syringae , Solanum lycopersicum , Trichoderma , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Solanum lycopersicum/microbiologia , Solanum lycopersicum/imunologia , Trichoderma/química , Trichoderma/metabolismo , Trichoderma/genética , Peptaibols/farmacologia , Peptaibols/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Tylenchoidea/efeitos dos fármacos , Imunidade Vegetal , AnimaisRESUMO
This study describes the first genome sequence and analysis of Coniella granati, a fungal pathogen with a broad host range, which is responsible for postharvest crown rot, shoot blight, and canker diseases in pomegranates. C. granati is a geographically widespread pathogen which has been reported across Europe, Asia, the Americas, and Africa. Our analysis revealed a 46.8 Mb genome with features characteristic of hemibiotrophic fungi. Approximately one third of its genome was compartmentalised within 'AT-rich' regions exhibiting a low GC content (30 to 45%). These regions primarily comprised transposable elements that are repeated at a high frequency and interspersed throughout the genome. Transcriptome-supported gene annotation of the C. granati genome revealed a streamlined proteome, mirroring similar observations in other pathogens with a latent phase. The genome encoded a relatively compact set of 9568 protein-coding genes with a remarkable 95% having assigned functional annotations. Despite this streamlined nature, a set of 40 cysteine-rich candidate secreted effector-like proteins (CSEPs) was predicted as well as a gene cluster involved in the synthesis of a pomegranate-associated toxin. These potential virulence factors were predominantly located near repeat-rich and AT-rich regions, suggesting that the pathogen evades host defences through Repeat-Induced Point mutation (RIP)-mediated pseudogenisation. Furthermore, 23 of these CSEPs exhibited homology to known effector and pathogenicity genes found in other hemibiotrophic pathogens. The study establishes a foundational resource for the study of the genetic makeup of C. granati, paving the way for future research on its pathogenicity mechanisms and the development of targeted control strategies to safeguard pomegranate production.
Assuntos
Proteínas Fúngicas , Genoma Fúngico , Doenças das Plantas , Punica granatum , Proteoma , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Punica granatum/genética , Punica granatum/microbiologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Anotação de Sequência Molecular , Frutas/microbiologia , Frutas/genética , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
Two-component systems (TCSs) are diverse cell signaling pathways that play a significant role in coping with a wide range of environmental cues in both prokaryotic and eukaryotic organisms. These transduction circuitries are primarily governed by histidine kinases (HKs), which act as sensing proteins of a broad variety of stressors. To date, nineteen HK groups have been previously described in the fungal kingdom. However, the structure and distribution of these prominent sensing proteins were hitherto investigated in a limited number of fungal species. In this study, we took advantage of recent genomic resources in fungi to refine the fungal HK classification by deciphering the structural diversity and phylogenetic distribution of HKs across a large number of fungal clades. To this end, we browsed the genome of 91 species representative of different fungal clades, which yielded 726 predicted HK sequences. A domain organization analysis, coupled with a robust phylogenomic approach, led to an improved categorization of fungal HKs. While most of the compiled sequences were categorized into previously described fungal HK groups, some new groups were also defined. Overall, this study provides an improved overview of the structure, distribution, and evolution of HKs in the fungal kingdom.
Assuntos
Fungos , Histidina Quinase , Filogenia , Histidina Quinase/genética , Histidina Quinase/metabolismo , Histidina Quinase/química , Fungos/genética , Fungos/enzimologia , Fungos/classificação , Genoma Fúngico , Transdução de Sinais , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Evolução Molecular , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Quinases/químicaRESUMO
Histidine kinases (HKs) are important sensor proteins in fungi and play an essential role in environmental adaptation. However, the mechanisms by which fungi sense and respond to fungivores attack via HKs are not fully understood. In this study, we utilized Neurospora crassa to investigate the involvement of HKs in responding to fungivores attack. We found that the 11 HKs in N. crassa not only affected the growth and development, but also led to fluctuations in antioxidant production. Ten mutants in the genes encoding HKs (except ∆phy1) showed increased production of reactive oxygen species (ROS), especially upon Sinella curviseta attack. The ROS burst triggered changes in conidia and perithecial beaks formation, as well as accumulation of ß-glucan, ergothioneine, ergosterol, and carotenoids. ß-glucan was increased in ∆hk9, ∆os1, ∆hcp1, ∆nik2, ∆sln1, ∆phy1 and ∆phy2 mutants compared to the wild-type strain. In parallel, ergothioneine accumulation was improved in ∆phy1 and ∆hk16 mutants and further increased upon attack, except in ∆os1 and ∆hk16 mutants. Additionally, fungivores attack stimulated ergosterol and dehydroergosterol production in ∆hk9 and ∆os1 mutants. Furthermore, deletion of these genes altered carotenoid accumulation, with wild-type strain, ∆hk9, ∆os1, ∆hcp1, ∆sln1, ∆phy2, and ∆dcc1mutants showing an increase in carotenoids upon attack. Taken together, HKs are involved in regulating the production of conidia and antioxidants. Thus, HKs may act as sensors of fungivores attack and effectively improve the adaptive capacity of fungi to environmental stimuli.
Assuntos
Histidina Quinase , Neurospora crassa , Espécies Reativas de Oxigênio , Neurospora crassa/genética , Neurospora crassa/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Regulação Fúngica da Expressão Gênica , Artrópodes/genética , Artrópodes/microbiologia , Mutação , Adaptação Fisiológica/genética , Ergosterol/metabolismo , beta-Glucanas/metabolismo , Antioxidantes/metabolismo , Carotenoides/metabolismo , ErgotioneínaRESUMO
The important role of dihydroxynaphthalene-(DHN) melanin in enhancing fungal stress resistance and its importance in fungal development and pathogenicity are well-established. This melanin also aids biocontrol fungi in surviving in the environment and effectively infecting insects. However, the biosynthetic origin of melanin in the biocontrol agents, Metarhizium spp., has remained elusive due to the complexity resulting from the divergence of two DHN-like biosynthetic pathways. Through the heterologous expression of biosynthetic enzymes from these two pathways in baker's yeast Saccharomyces cerevisiae, we have confirmed the presence of DHN biosynthesis in M. roberstii, and discovered a novel naphthopyrone intermediate, 8, that can produce a different type of pigment. These two pigment biosynthetic pathways differ in terms of polyketide intermediate structures and subsequent modification steps. Stress resistance studies using recombinant yeast cells have demonstrated that both DHN and its intermediates confer resistance against UV light prior to polymerization; a similar result was observed for its naphthopyrone counterpart. This study contributes to the understanding of the intricate and diverse biosynthetic mechanisms of fungal melanin and has the potential to enhance the application efficiency of biocontrol fungi such as Metarhizium spp. in agriculture.