Biology subjects

Horta, M. A. C.

Publications and source records attributed to Horta, M. A. C..

10 recordsLinked to original sources

bioRxiv2025

Genetic response to light and carbon source variations in Trichoderma harzianum: The key regulatory roles of env1, cre1, and blr2

In filamentous fungi, light plays a key role in regulating physiological processes such as growth, conidiation, secondary metabolism, and the expression of hydrolytic enzymes. The processes that depend on light are controlled by photoreceptors, including BLR1, BLR2, and ENV1, as well as by signaling pathways involving heterotrimeric G-proteins and cyclic adenosine monophosphate (cAMP). Trichoderma harzianum is a promising candidate for biotechnological use and is able to promote hydrolytic reactions under biomass degradation conditions. However, the genetic mechanisms underlying its response to light remain poorly understood, especially under degradative conditions. This study aimed to assess the expression of carbohydrate-active enzymes (CAZymes), transcription factors (TFs), and signaling pathway proteins under different light conditions and carbon sources. The results revealed distinct patterns of relative gene expression influenced by these environmental factors, highlighting the complex regulatory mechanisms at play in T. harzianum. Moreover, our results suggested that env1, cre1, and blr2 are critical for adjusting to different light conditions and carbon sources. This highlights the importance of both factors in regulating gene expression and supporting metabolic adaptation in T. harzianum. To our knowledge, such findings have not been previously reported in the context of cellulose degradation for this species. Overall, these results offer valuable insights into how T. harzianum responds to environmental changes, revealing a complex regulatory network that is not only crucial for optimizing fungal growth in industrial applications but also deepens our understanding of its biology and ecological interactions. Highlights{blacksquare} Trichoderma harzianum is a key candidate for biotechnology applications. {blacksquare}Light and carbon control CAZymes, photoreceptors, key regulators, and signaling. {blacksquare}T. harzianum has distinct gene expression patterns under different light conditions and carbon sources. {blacksquare}env1, cre1, and blr2 regulate metabolic adaptation in T. harzianum. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/657067v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@8167c7org.highwire.dtl.DTLVardef@1c46f6borg.highwire.dtl.DTLVardef@16eddc8org.highwire.dtl.DTLVardef@1a2afa6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis

Aspergillus fumigatus, an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores or conidia for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, and the cryptic pathogen Aspergillus lentulus. After identifying 62 proteins uniquely expressed on the A. fumigatus conidial surface, we deleted 42 genes encoding conidial proteins. We found deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1{beta}, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection.

microbiology↗

bioRxiv2022

Examination of genome-wide ortholog variation in clinical and environmental isolates of the fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus is both an environmental saprobe and an opportunistic human fungal pathogen. Knowledge of genomic variation across A. fumigatus isolates is essential for understanding the evolution of pathogenicity, virulence, and resistance to antifungal drugs. Here, we investigated 206 A. fumigatus isolates (133 clinical and 73 environmental isolates) aiming to identify genes with variable presence across isolates and test whether this variation was related to the clinical or environmental origin of isolates. The PanCore genome of A. fumigatus constitutes 13,085 ortholog groups, of which 7,773 (59.4%) are shared by all isolates (CORE) and 5,312 (40.6%) vary in their gene presence across isolates (ACCESSORY). Despite differences in the distribution of orthologs across all isolates, no significant differences were observed among clinical vs. environmental isolates when accounting for phylogeny. Orthologs that differ in their distribution across isolates tend to occur in low frequency and/or be restricted to specific isolates; thus, the degree of genomic conservation between orthologs of A. fumigatus is high. These results suggest that differences in the distribution of orthologs within A. fumigatus cannot be associated with the clinical or environmental origin of isolates. ImportanceAspergillus fumigatus is a cosmopolitan species of fungi responsible for thousands of cases of invasive disease. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. A comprehensive survey of the genomic diversity present in A. fumigatus and its relationship to the clinical or environmental origin of isolates can contribute to the prediction of the mechanisms of evolution and infection of the species. Our results suggest that there is no significant variation in ortholog distribution between clinical and environmental isolates when accounting for evolutionary history. The work supports the hypothesis that environmental and clinical isolates of A. fumigatus do not differ in their gene contents.

bioinformatics↗

bioRxiv2022

Whole-Genome Sequencing and Comparative Genomic Analysis of Potential Biotechnological Strains from Trichoderma harzianum, Trichoderma atroviride, and Trichoderma reesei

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.

genomics↗

bioRxiv2021

Regulation of gliotoxin biosynthesis and protection in Aspergillus species

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. Author SummaryA. fumigatus secretes mycotoxins that are essential for its virulence and pathogenicity. Gliotoxin (GT) is a sulfur-containing mycotoxin, which is known to impair several aspects of the human immune response. GT is also toxic to different fungal species, which have evolved several GT protection strategies. To further decipher these responses, we used transcriptional profiling aiming to compare the response to GT in the GT producer A. fumigatus and the GT non-producer A. nidulans. This analysis allowed us to identify additional genes with a potential role in GT protection. We also identified 15 transcription factors (TFs) encoded in the A. fumigatus genome that are important for conferring resistance to exogenous gliotoxin. One of these TFs, KojR, which is essential for A. oryzae kojic acid production, is also important for virulence and GT protection in A. fumigatus, A. nidulans and A. oryzae. KojR regulates the expression of another TF, an oxidoreductase, previously shown to be essential for GT protection, and sulfur metabolism. Together, this work identified conserved components required for gliotoxin protection in Aspergillus species.

microbiology↗

bioRxiv2021

Screening of chemical libraries for new antifungal drugs against Aspergillus fumigatus reveals the potential mechanism of action of miltefosine

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 the last 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 more 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 rmiA (resistant to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and the {Delta}rmiA strains and the Chromatin Immunoprecipitation coupled to next generation sequencing (ChIP-Seq) of a RmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipids pathway that are directly or indirectly regulated by RmiA. Sphingolipids analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of RmiA represents the first genetic element described and characterized which plays a direct role in miltefosine response in fungi. Author summaryThe 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 the last decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungals 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 RmiA (Resistant to miltefosine) important for miltefosine resistance. Our results suggest that miltefosine plays antifungal activity against A. fumigatus interfering in the sphingolipids biosynthesis.

microbiology↗

bioRxiv2020

Comparative gene coexpression networks analysis reveals different strategies of Trichoderma spp. associated with XYR1 and CRE1 during cellulose degradation

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.

bioinformatics↗

bioRxiv2020

The synergistic actions of hydrolytic genes in coexpression networks reveal the potential of Trichoderma harzianum for cellulose degradation

Bioprospecting genes and proteins related to plant biomass degradation is an attractive approach for the identification of target genes for biotechnological purposes, especially genes with potential applications in the biorefinery industry that can enhance second-generation ethanol production technology. Trichoderma harzianum is a potential candidate for cellulolytic enzyme prospection and production. Herein, the enzymatic activities, transcriptome, exoproteome, and coexpression networks of the T. harzianum strain CBMAI-0179 were examined under biomass degradation conditions. We used RNA-Seq to identify differentially expressed genes (DEGs) and carbohydrate-active enzyme (CAZyme) genes related to plant biomass degradation and compared them with the genes of strains from congeneric species (T. harzianum IOC-3844 and T. atroviride CBMAI-0020). T. harzianum CBMAI-0179 harbors strain- and treatment-specific CAZyme genes and transcription factors. We detected important proteins related to biomass degradation, including {beta}-glucosidases, endoglucanases, cellobiohydrolases, lytic polysaccharide monooxygenases, endo-1,4-{beta}-xylanases and {beta}-mannanases, in the exoproteome under cellulose growth conditions. Coexpression networks were constructed to explore the relationships among the genes and corresponding secreted proteins that act synergistically for cellulose degradation. An enriched cluster with degradative enzymes was described, and the subnetwork of CAZymes revealed strong correlations among the secreted proteins (AA9, GH6, GH10, GH11 and CBM1) and differentially expressed CAZyme genes (GH45, GH7, AA7 and GH1). Our results provide valuable information for future studies on the genetic regulation of plant cell wall-degrading enzymes. This knowledge can be exploited for the improvement of enzymatic reactions in biomass degradation for bioethanol production. Key pointsDifferent biotechnological approaches were used to understand the mechanism of cellulose degradation of Trichoderma spp. T. harzianum CBMAI-0179 is a potential candidate for the production of cellulolytic enzymes. Coexpression networks revealed genes and proteins acting synergistically for cellulose hydrolysis.

bioinformatics↗

bioRxiv2019

Integrative Genomic Analysis for the Bioprospection of Regulators and Accessory Enzymes Associated with Cellulose Degradation in a Filamentous Fungus (Trichoderma harzianum)

BackgroundUnveiling fungal genome structure and function reveals the potential biotechnological use of fungi. Trichoderma harzianum is a powerful CAZyme-producing fungus. We studied the genomic regions in T. harzianum IOC3844 containing CAZyme genes, transcription factors and transporters.\n\nResultsWe used bioinformatics tools to mine the T. harzianum genome for potential genomics, transcriptomics, and exoproteomics data and coexpression networks. The DNA was sequenced by PacBio SMRT technology for multi-omics data analysis and integration. In total, 1676 genes were annotated in the genomic regions analyzed; 222 were identified as CAZymes in T. harzianum IOC3844. When comparing transcriptome data under cellulose or glucose conditions, 114 genes were differentially expressed in cellulose, with 51 CAZymes. CLR2, a transcription factor physically and phylogenetically conserved in T. harzianum spp., was differentially expressed under cellulose conditions. The genes induced/repressed under cellulose conditions included those important for plant biomass degradation, including CIP2 of the CE15 family and a copper-dependent LPMO of the AA9 family.\n\nConclusionsOur results provide new insights into the relationship between genomic organization and hydrolytic enzyme expression and regulation in T. harzianum IOC3844. Our results can improve plant biomass degradation, which is fundamental for developing more efficient strains and/or enzymatic cocktails for the production of hydrolytic enzymes.

genomics↗

bioRxiv2019

Broad substrate-specific phosphorylation events are associated with the initial stage of plant cell wall recognition in Neurospora crassa

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unkown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 minutes) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

molecular biology↗