RESUMEN

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.

Asunto(s)

Aspergilosis , Aspergillus fumigatus , Proteínas Fúngicas , Evasión Inmune , Proteoma , Esporas Fúngicas , Aspergillus fumigatus/inmunología , Aspergillus fumigatus/genética , Animales , Esporas Fúngicas/inmunología , Ratones , Proteoma/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/inmunología , Aspergilosis/inmunología , Aspergilosis/microbiología , Humanos , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/metabolismo , Citocinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino

RESUMEN

Trichoderma atroviride and Trichoderma harzianum are widely used as commercial biocontrol agents against plant diseases. Recently, T. harzianum IOC-3844 (Th3844) and T. harzianum CBMAI-0179 (Th0179) demonstrated great potential in the enzymatic conversion of lignocellulose into fermentable sugars. Herein, we performed whole-genome sequencing and assembly of the Th3844 and Th0179 strains. To assess the genetic diversity within the genus Trichoderma, the results of both strains were compared with strains of T. atroviride CBMAI-00020 (Ta0020) and T. reesei CBMAI-0711 (Tr0711). The sequencing coverage value of all genomes evaluated in this study was higher than that of previously reported genomes for the same species of Trichoderma. The resulting assembly revealed total lengths of 40 Mb (Th3844), 39 Mb (Th0179), 36 Mb (Ta0020), and 32 Mb (Tr0711). A genome-wide phylogenetic analysis provided details on the relationships of the newly sequenced species with other Trichoderma species. Structural variants revealed genomic rearrangements among Th3844, Th0179, Ta0020, and Tr0711 relative to the T. reesei QM6a reference genome and showed the functional effects of such variants. In conclusion, the findings presented herein allow the visualization of genetic diversity in the evaluated strains and offer opportunities to explore such fungal genomes in future biotechnological and industrial applications.

Asunto(s)

Trichoderma , Filogenia , Trichoderma/genética , Genómica

RESUMEN

Trichoderma harzianum, whose gene expression is tightly controlled by the transcription factors (TFs) XYR1 and CRE1, is a potential candidate for hydrolytic enzyme production. Here, we performed a network analysis of T. harzianum IOC-3844 and T. harzianum CBMAI-0179 to explore how the regulation of these TFs varies between these strains. In addition, we explored the evolutionary relationships of XYR1 and CRE1 protein sequences among Trichoderma spp. The results of the T. harzianum strains were compared with those of Trichoderma atroviride CBMAI-0020, a mycoparasitic species. Although transcripts encoding carbohydrate-active enzymes (CAZymes), TFs, transporters, and proteins with unknown functions were coexpressed with cre1 or xyr1, other proteins indirectly related to cellulose degradation were identified. The enriched GO terms describing the transcripts of these groups differed across all strains, and several metabolic pathways with high similarity between both regulators but strain-specific differences were identified. In addition, the CRE1 and XYR1 subnetworks presented different topology profiles in each strain, likely indicating differences in the influences of these regulators according to the fungi. The hubs of the cre1 and xyr1 groups included transcripts not yet characterized or described as being related to cellulose degradation. The first-neighbor analyses confirmed the results of the profile of the coexpressed transcripts in cre1 and xyr1. The analyses of the shortest paths revealed that CAZymes upregulated under cellulose degradation conditions are most closely related to both regulators, and new targets between such signaling pathways were discovered. Although the evaluated T. harzianum strains are phylogenetically close and their amino acid sequences related to XYR1 and CRE1 are very similar, the set of transcripts related to xyr1 and cre1 differed, suggesting that each T. harzianum strain used a specific regulation strategy for cellulose degradation. More interestingly, our findings may suggest that XYR1 and CRE1 indirectly regulate genes encoding proteins related to cellulose degradation in the evaluated T. harzianum strains. An improved understanding of the basic biology of fungi during the cellulose degradation process can contribute to the use of their enzymes in several biotechnological applications and pave the way for further studies on the differences across strains of the same species.

RESUMEN

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Asunto(s)

Aspergillus/crecimiento & desarrollo , Gliotoxina/farmacología , Metiltransferasas/genética , Factores de Transcripción/genética , Aspergillus/efectos de los fármacos , Aspergillus/genética , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus oryzae/efectos de los fármacos , Aspergillus oryzae/genética , Aspergillus oryzae/crecimiento & desarrollo , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Gliotoxina/biosíntesis , RNA-Seq

RESUMEN

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation, called invasive pulmonary aspergillosis (IPA), in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over recent decades. Therefore, new strategies are necessary to combat aspergillosis, and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the smiA (sensitive to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and ΔsmiA strains and chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-Seq) of an SmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipid pathway that are directly or indirectly regulated by SmiA. Sphingolipid analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of SmiA represents the first genetic element described and characterized that plays a direct role in miltefosine response in fungi. IMPORTANCE The filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis, and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over recent decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungal agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown, and, aiming to get more insights about it, we identified a transcription factor, SmiA (sensitive to miltefosine), important for miltefosine resistance. Our results suggest that miltefosine displays antifungal activity against A. fumigatus, interfering in sphingolipid biosynthesis.

Asunto(s)

Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Fosforilcolina/análogos & derivados , Bibliotecas de Moléculas Pequeñas/farmacología , Esfingolípidos/metabolismo , Animales , Antifúngicos/uso terapéutico , Aspergilosis/tratamiento farmacológico , Aspergilosis/microbiología , Aspergillus fumigatus/química , Aspergillus fumigatus/patogenicidad , Farmacorresistencia Fúngica , Larva/efectos de los fármacos , Larva/microbiología , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/microbiología , Fenotipo , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Virulencia

RESUMEN

Aspergillus fumigatus is an opportunistic fungus, capable of causing Invasive Aspergillosis in immunocompromised patients, recently transplanted or undergoing chemotherapy. In the present work, we continued the investigation on A. fumigatus AtfA-D transcription factors (TFs) characterizing possible genetic and physical interactions between them after normal growth and stressing conditions. We constructed double null mutants for all the possible combinations of ΔatfA-, -B, -C, and -D, and look into their susceptibility to different stressing conditions. Our results indicate complex genetic interactions among these TFs that could impact the response to different kinds of stressful conditions. AtfA-D interactions also affect the A. fumigatus virulence in Galleria mellonella. AtfA:GFP is ~97% located in the nucleus while about 20-30% of AtfB, -C, and -D:GFP locate into the nucleus in the absence of any stress. Under stressing conditions, AtfB, -C, and -D:GFP translocate to the nucleus about 60-80% upon the addition of sorbitol or H2O2. These four TFs are also interacting physically forming all the possible combinations of heterodimers. We also identified that AtfA-D physically interact with the MAPK SakA in the absence of any stress and upon osmotic and cell wall stresses. They are involved in the accumulation of trehalose, glycogen and metabolic assimilation of different carbon sources.

RESUMEN

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RESUMEN

Understanding relationships between genes responsible for enzymatic hydrolysis of cellulose and synergistic reactions is fundamental for improving biomass biodegradation technologies. To reveal synergistic reactions, the transcriptome, exoproteome, and enzymatic activities of extracts from Trichoderma harzianum, Trichoderma reesei and Trichoderma atroviride under biodegradation conditions were examined. This work revealed co-regulatory networks across carbohydrate-active enzyme (CAZy) genes and secreted proteins in extracts. A set of 80 proteins and respective genes that might correspond to a common system for biodegradation from the studied species were evaluated to elucidate new co-regulated genes. Differences such as one unique base pair between fungal genomes might influence enzyme-substrate binding sites and alter fungal gene expression responses, explaining the enzymatic activities specific to each species observed in the corresponding extracts. These differences are also responsible for the different architectures observed in the co-expression networks.

Asunto(s)

Enzimas/metabolismo , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Proteómica/métodos , Trichoderma/fisiología , Productos Biológicos/análisis , Biomasa , Celulosa/química , Enzimas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Hidrólisis , Filogenia , Análisis de Secuencia de ARN/métodos

RESUMEN

BACKGROUND: Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. RESULTS: In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. CONCLUSIONS: Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

Asunto(s)

Biocombustibles , Metabolismo de los Hidratos de Carbono , Biología Computacional , Trichoderma/enzimología , Celulosa/metabolismo , Perfilación de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Anotación de Secuencia Molecular , Polisacáridos/metabolismo

RESUMEN

Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes.

Asunto(s)

Trichoderma/genética , Trichoderma/metabolismo , Biodegradación Ambiental , Biocombustibles , Biomasa , Biotecnología , Celulasa/genética , Celulasa/metabolismo , Cromosomas Artificiales Bacterianos/genética , Etanol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Genoma Fúngico , Hidrólisis

RESUMEN

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. In the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. The sequencing generated 14.7 Gbp for downstream analyses. De novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. The present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.

Asunto(s)

Celulosa/metabolismo , Perfilación de la Expresión Génica , Saccharum/química , Trichoderma/genética , Trichoderma/metabolismo , Celulasa/genética , Celulasa/metabolismo , Bases de Datos Genéticas , Genes Fúngicos/genética , Anotación de Secuencia Molecular , Análisis de Secuencia de ARN , Trichoderma/enzimología