Marcio José Poças-Fonseca

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature‐dependent cell morphology change from mycelium (22° C) to yeast (36° C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3938 (Y = 1654 and M = 2274) ESTs were sequenced and clustered into 597 contigs and 1563 singlets, making up a total of 2160 genes, which possibly represent one‐quarter of the complete gene repertoire in P. brasiliensis. From this total, 1040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes—cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage‐specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis. All EST sequences have been deposited in GenBank under Accession Nos CA580326–CA584263. Copyright

A truncated form of the Carbon catabolite repressor 1 increases cellulase production in Trichoderma reesei

BackgroundRut-C30 is a cellulase-hyperproducing Trichoderma reesei strain and, consequently, became the ancestor of most industry strains used in the production of plant cell wall-degrading enzymes, in particular cellulases. Due to three rounds of undirected mutagenesis its genetic background differs from the wild-type QM6a in many ways, of which two are the lack of a 83 kb large sequence in scaffold 15 and the partial lack of the gene encoding the Carbon catabolite repressor 1 (CREI). However, it is still unclear, what exactly enhances cellulase production in Rut-C30.ResultsThe investigation of the expression of two genes encoding cellulases (cbh1 and cbh2) and the gene encoding their main transactivator (xyr1) revealed that the presence of the truncated form of CREI (CREI-96) contributes more to the Rut-C30 phenotype than a general loss of CREI-mediated carbon catabolite repression (cre1 deletion strain) or the deletion of 29 genes encoded in the scaffold 15 (83 kb deletion strain). We found that the remaining cre1 in Rut-C30 (cre1-96) is transcribed into mRNA, that its putative gene product (Cre1-96) is still able to bind DNA, and that the CREI-binding sites in the upstream regulatory regions of the chosen CREI-target genes are still protected in Rut-C30. As it was previously reported that CREI acts on the nucleosome positioning, we also analyzed chromatin accessibility of the core promoters of CREI-target genes and found them open even on D-glucose in the presence of CREI-96.ConclusionsThe lack of the full version of CREI in Rut-C30 corresponds with a partial release from carbon catabolite repression but is not completely explained by the lack of CREI. In contrast, the truncated CREI-96 of Rut-C30 exerts a positive regulatory influence on the expression of target genes. Mechanistically this might be explained at least partially by a CREI-96-mediated opening of chromatin.

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.

Thermophilic molds: Biology and applications

Abstract Thermophilic molds thrive in a variety of natural habitats including soils, composts, wood chip piles, nesting materials of birds and other animals, municipal refuse and others, and ubiquitous in their distribution. These molds grow in simple media containing carbon and nitrogen sources and mineral salts. Polyamines are synthesized in these molds and the composition of lipids varies considerably, predominantly containing palmitic, oleic and linoleic acids with low levels of lauric, palmiotoleic and stearic acids. Thermophilic molds are capable of efficiently degrading organic materials by secreting thermostable enzymes, which are useful in the bioremediation of industrial wastes and effluents that are rich in oil, heavy metals, anti-nutritional factors such as phytic acid and polysaccharides. Thermophilic molds synthesize several antimicrobial substances and biotechnologically useful miscellaneous enzymes. The analysis of genomes of thermophilic molds reveals high G:C contents, shorter introns and intergenic regions with lesser repetitive sequences, and further confirms their ability to degrade agro-residues efficiently. Genetic engineering has aided in ameliorating the characteristics of the enzymes of thermophilic molds. This review is aimed at focusing on the biology of thermophilic molds with emphasis on recent developments in the analysis of genomes, genetic engineering and potential applications.

The Relation Between Promoter Chromatin Status, Xyr1 and Cellulase Ex-pression in Trichoderma reesei

The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes in industrial scale. The interplay of the transactivator Xyr1 and the repressor Cre1 mainly regulates the expression of these enzymes. During induc-ing conditions, such as in the presence of sophorose, the transcription of the two major cellulase-encoding genes, cbh1 and cbh2, is activated as well as the expression of xyr1. In the presence of D-glucose carbon catabolite repression mediated by Cre1 takes place and the expression of Xyr1 and the plant cell wall-degrading enzymes is down-regulated. In this study we compare the chromatin status of xyr1, cbh1, and cbh2 promoters in the wild-type strain and the Cre1-deficient strain Rut-C30. Chromatin rearrangement occurs in the xyr1 promoter during induction on sophorose. Chromatin opening and protein-DNA interactions in the xyr1 promoter were detected especially in a region located 0.9 kb upstream the translation start co-don, which bears several putative Cre1-binding sites and a CCAAT-box. Moreover, the xyr1 promoter is overall more acces-sible in a cre1-truncated background, no matter which carbon source is present. This makes the xyr1 regulatory sequence a good target for promoter engineering aiming at the enhancement of cellulase production.

From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach.

BackgroundHypocrea jecorina (anamorph Trichoderma reesei) is a filamentous ascomycete of industrial importance due to its hydrolases (e.g., xylanases and cellulases). The regulation of gene expression can influence the composition of the hydrolase cocktail, and thus, transcription factors are a major target of current research. Here, we design an approach for identifying a repressor of a xylanase-encoding gene.ResultsWe used streptavidin affinity chromatography to isolate the Xylanase promoter-binding protein 1 (Xpp1). The optimal conditions and templates for the chromatography step were chosen according to the results of an electrophoretic mobility shift assay performed under repressing conditions, which yielded a DNA-protein complex specific to the AGAA-box (the previously identified, tetranucleotide cis-acting element). After isolating AGAA-box binding proteins, the eluted proteins were identified with Nano-HPLC/tandem MS-coupled detection. We compared the identified peptides to sequences in the H. jecorina genome and predicted in silico the function and DNA-binding ability of the identified proteins. With the results from these analyses, we eliminated all but three candidate proteins. We verified the transcription of these candidates and tested their ability to specifically bind the AGAA-box. In the end, only one candidate protein remained. We generated this protein with in vitro translation and used an EMSA to demonstrate the existence of an AGAA-box-specific protein-DNA complex. We found that the expression of this gene is elevated under repressing conditions relative to de-repressing or inducing conditions.ConclusionsWe identified a putative transcription factor that is potentially involved in repressing xylanase 2 expression. We also identified two additional potential regulatory proteins that bind to the xyn2 promoter. Thus, we succeeded in identifying novel, putative transcription factors for the regulation of xylanase expression in H. jecorina.

Carbon source and pH-dependent transcriptional regulation of cellulase genes of Humicola grisea var. thermoidea grown on sugarcane bagasse.

Time-course expression profiles of one xylanase and eight cellulase encoding genes, as well as of two transcription factor encoding genes of Humicola grisea var. thermoidea were established in different culture media pHs and carbon sources (glucose and sugarcane bagasse). Quantitative real-time RT-PCR analysis revealed a remarkable and parallel increase in mRNA accumulation for cbh1.1, cbh1.2, egl1, egl2, egl3, bgl4 and xyn1 at alkaline pH and with sugarcane bagasse employed as the sole carbon source. Glucose utilization led to a higher creA mRNA accumulation compared to the other genes. A distinct pattern was observed for egl4, whose mRNA preferably accumulated in acidic conditions. The transcriptional profile data combined with the analysis of the in vitro binding of PacC and CreA transcription factors to the promoters support the CreA-mediated carbon repression and the PacC-related pH regulation of H. grisea cellulase and xylanase encoding genes. Moreover, EMSA analyses suggest a role for CreA on pacC transcriptional regulation. These data will be useful to H. grisea hydrolytic enzymes production improvement, as well as to the design of optimized promoters aiming industrial heterologous proteins production.

Interaction of bracken-fern extract with vitamin C in human submandibular gland and oral epithelium cell lines

The consumption of bracken-fern (Pteridium aquilinum) as food is associated with a high incidence of cancer in humans and animals. Thus far, the carcinogenic effects of bracken-fern consumption could be related to chromosome aberrations verified in animal and in human peripheral lymphocytes. We tested the in vitro effects of vitamin C (10 and 100 microg/ml) on the reversibility of DNA damage caused by bracken-fern on human submandibular gland (HSG) cells and on oral epithelium cells (OSCC-3) previously exposed to bracken-fern extract. DNA damage (i.e. nuclei with increased levels of DNA migration) was determined by comet assay, cell morphology was evaluated by light microscopy and cellular degeneration was assessed by the acridine orange/ethidium bromide fluorescent-dyeing test. Results showed that vitamin C alone did not reduce DNA damage caused by bracken-fern in HSG and OSSC-3 cells. However, at a higher concentration (100 microg/ml), vitamin C induced DNA damage in both cell lines. Moreover, vitamin C (10 and 100 microg/ml) together with bracken-fern extract showed synergistic effects on the frequency of DNA damage in HSG cells. In addition, cells treated with bracken-fern extract or vitamin C alone, or with their association, showed apoptosis morphological features, such as chromatin condensation, cytoplasmic volume loss, changes in membrane symmetry and the appearance of vacuoles; these alterations were observed in both cell lines. These results demonstrate that bracken-fern extract was cytotoxic to HSG and OSCC-3 cells, causing cell death by apoptosis, and that vitamin was not able to revert these effects.

Overview and perspectives on the transcriptome of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.

Histone deacetylases inhibitors effects on Cryptococcus neoformans major virulence phenotypes

Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects.