Gabriela F. Persinoti

Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses

BackgroundThe filamentous fungus Trichoderma reesei is a major producer of lignocellulolytic enzymes utilized by bioethanol industries. However, to achieve low cost second generation bioethanol production on an industrial scale an efficient mix of hydrolytic enzymes is required for the deconstruction of plant biomass. In this study, we investigated the molecular basis for lignocellulose-degrading enzyme production T. reesei during growth in cellulose, sophorose, and glucose.ResultsWe examined and compared the transcriptome and differential secretome (2D-DIGE) of T. reesei grown in cellulose, sophorose, or glucose as the sole carbon sources. By applying a stringent cut-off threshold 2,060 genes were identified as being differentially expressed in at least one of the respective carbon source comparisons. Hierarchical clustering of the differentially expressed genes identified three possible regulons, representing 123 genes controlled by cellulose, 154 genes controlled by sophorose and 402 genes controlled by glucose. Gene regulatory network analyses of the 692 genes differentially expressed between cellulose and sophorose, identified only 75 and 107 genes as being specific to growth in sophorose and cellulose, respectively. 2D-DIGE analyses identified 30 proteins exclusive to sophorose and 37 exclusive to cellulose. A correlation of 70.17% was obtained between transcription and secreted protein profiles.ConclusionsOur data revealed new players in cellulose degradation such as accessory proteins with non-catalytic functions secreted in different carbon sources, transporters, transcription factors, and CAZymes, that specifically respond in response to either cellulose or sophorose.

Expression pattern of cellulolytic and xylanolytic genes regulated by transcriptional factors XYR1 and CRE1 are affected by carbon source in Trichoderma reesei.

Trichoderma reesei is the most important fungus for the industrial production of enzymes to biomass deconstruction. Most of the genes encoding cellulases and hemicellulases are regulated by the transcription factors CRE1 and XYR1. In this work, the regulation of 22 genes of cellulases and xylanases by these transcription factors was investigated under three different carbon sources. Analysis of gene expression and enzymatic profiles of CMCase, β-glucosidase, and xylanases showed different regulation that was depended of the carbon source in both Δxyr1 and Δcre1 mutants. In the presence of glucose, the majority of genes evaluated (82%) showed increased expression levels in the Δcre1 mutant compared to the parental QM9414 strain. In the Δxyr1 mutant, it was observed that expression of cellulase and xylanase genes was reduced compared to the parental QM9414 strain, when cultured in the presence of cellulose or sophorose. Interesting, in the presence of glucose, approximately 60% of the analyzed genes had increased expression in the Δxyr1 mutant compared to parental strain. Furthermore, no correlation between gene expression and the number of putative binding sites of XYR1 and CRE1 to promoter region of cellulolytic and xylanolytic studied genes was observed. Therefore, these results demonstrated that the regulation of cellulase and xylanase by the transcription factors CRE1 and XYR1 is influenced by different carbon sources.

Defining the genome-wide role of CRE1 during carbon catabolite repression in Trichoderma reesei using RNA-Seq analysis.

The ascomycete Trichoderma reesei is one of the most well-studied cellulolytic fungi and is widely used by the biotechnology industry in the production of second generation bioethanol. The carbon catabolite repression (CCR) mechanism adopted by T. reesei is mediated by the transcription factor CRE1. CCR represses genes related to cellulase production when a carbon source is readily available in the medium. Using RNA sequencing, we investigated CCR during the synthesis of cellulases, comparing the T. reesei Δcre1 mutant strain with its parental strain, QM9414. Of 9129 genes in the T. reesei genome, 268 genes were upregulated and 85 were downregulated in the presence of cellulose (Avicel). In addition, 251 genes were upregulated and 230 were downregulated in the presence of a high concentration of glucose. Genes encoding cellulolytic enzymes and transcription factors and genes related to the transport of nutrients and oxidative metabolism were also targets of CCR, mediated by CRE1 in a carbon source-dependent manner. Our results also suggested that CRE1 regulates the expression of genes related to the use of copper and iron as final electron acceptors or as cofactors of enzymes that participate in biomass degradation. As a result, the final effect of CRE1-mediated transcriptional regulation is to modulate the access of cellulolytic enzymes to cellulose polymers or blocks the entry of cellulase inducers into the cell, depending on the glucose content in the medium. These results will contribute to a better understanding of the mechanism of carbon catabolite repression in T. reesei, thereby enhancing its application in several biotechnology fields.

Role of pH in the pathogenesis of dermatophytoses

The secretion of proteolytic enzymes by dermatophytes is a key factor in their invasion and subsequent dissemination through the stratum corneum of the host. During the first stages of infection, dermatophytes respond to the skin by de‐repressing a number of genes coding for proteins and enzymes such as adhesins, lipases, phosphatases, DNAses, non‐specific proteases, and keratinases. These proteins have their optimal activity at acidic pH values, which matches the acidic pH of human skin, allowing the pathogen to adhere and penetrate the host tissue, scavenge nutrients and overcome host defence mechanisms. The conserved PacC/Rim101p signal transduction pathway mediates diverse metabolic events involved in ambient pH sensing and in the virulence of pathogenic microorganisms. The seven dermatophyte genomes analysed here revealed the presence of the PacC/Rim101p pH‐responsive signal transduction pathway, which consists of the six pal genes (palA, B, C, F, H and I) and the transcription factor PacC. The PacC binding site was present in the promoter regions of pacC, palB, palI and palH genes of all dermatophytes, suggesting functional equivalency with the signalling cascade of other fungi. Moreover, the promoter region of pacC gene of the seven dermatophytes had multiple PacC DNA‐binding sites, suggesting that these genes, like their homologues in model fungi, are auto‐regulated.

The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

BackgroundTrichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.ResultsComparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.ConclusionsThe regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis

We defined the role of the transcriptional factor—XYR1—in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Deciphering the Cis-Regulatory Elements for XYR1 and CRE1 Regulators in Trichoderma reesei

In this work, we report the in silico identification of the cis-regulatory elements for XYR1 and CRE1 proteins in the filamentous fungus Trichoderma reesei, two regulators that play a central role in the expression of cellulase genes. Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative cis-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from cel7a and xyn1 promoters, and we also mapped the potential target sequence for this regulator at the cel6a promoter that has been suggested but not identified previously. Additionally, seven other promoters (for cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 and swo genes) presented a putative XYR1-binding site, and strong sites for CRE1 were found at the xyr1 and cel7b promoters. Using the cis-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in T. reesei mutant strains lacking xyr1 or cre1 and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.

Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway.

The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. In a previous work, we identified a number of genes modulated by NUC-2 protein, including the mak-2 gene, which codes for a mitogen-activated protein kinase (MAPK), suggesting its participation in the phosphate signaling pathway. Thus, to identify other genes involved in metabolic responses to exogenous phosphate sensing and the functioning of the MAPK MAK-2, we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. Quantitative Real-time PCR gene expression analysis of 18 selected genes, using independent RNA samples, validated our microarray results. A high Pearson correlation between microarray and quantitative Real-time PCR data was observed. The analysis of these differentially expressed genes in the Δmak-2 strain provide evidence that the mak-2 gene participates in the hierarchical phosphate-signaling pathway in N. crassa in addition to its involvement in other metabolic routes such as the isoprenylation pathway, thus revealing novel aspects of the N. crassa phosphorus-sensing network.

In vitro and ex vivo infection models help assess the molecular aspects of the interaction of Trichophyton rubrum with the host milieu

Dermatophytes are fungal pathogens that cause cutaneous infections such as onychomycosis and athletes foot in both healthy and immunocompromised patients.Trichophyton rubrum is the most prevalent dermatophyte causing human nail and skin infections worldwide, and because of its anthropophilic nature, animal infection models are limited. The purpose of this work was to compare the expression profile of T. rubrum genes encoding putative virulence factors during growth in ex vivo and in vitro infection models. The efficiency of the ex vivo skin infection model was confirmed by scanning electron microscopy (SEM), which showed that the conidia had produced hyphae that penetrated into the epidermis. Quantitative RT-PCR (qRT-PCR) analysis showed that the expression of some genes is modulated in response to the infection model used, as compared to that observed in cells grown in glucose-containing media. We concluded that ex vivo infection models help assess the molecular aspects of the interaction of T. rubrum with the host milieu, and thus provide insights into the modulation of genes during infection.