Molecular cloning, expression and insulin reduction activity of a thioredoxin 1 homologue (TRX1) from the pathogenic fungus Paracoccidioides lutzii.

The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. The Paracoccidioides lutzii response to oxidative stress is largely unexplored. Thioredoxins (TRX) are involved in the regulation of the redox environment in the cell, responding to oxidative stress in several organisms. In this study, we describe the isolation and characterization of a cDNA encoding a thioredoxin 1 from yeast cells from P. lutzii. The cDNA codes for a 12kDa protein containing the characteristic thioredoxin active site. The thioredoxin 1 gene was expressed in Escherichia coli and the isolated thioredoxin 1 recombinant protein as the native PlTRX1 from yeast cells showed insulin reduction activity in vitro. We also showed by semi-quantitative RT-PCR analysis that the expression of thioredoxin 1 gene was induced in response to H2O2 and may exert an antioxidant activity in vivo. Our results suggest that the thioredoxin 1 may play an important role in controlling the redox status in P. lutzii which may contribute to this organism's virulence.

Isolation of osteosarcoma-associated human antibodies from a combinatorial Fab phage display library.

Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain) and Vkappa (kappa chain variable domain) regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction.

Paracoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http://www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53%) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75% of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47% of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.

The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process.

BACKGROUND: Paracoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The fungus is thermally dimorphic with two distinct forms corresponding to completely different lifestyles. Upon elevation of the temperature to that of the mammalian body, the fungus adopts a yeast-like form that is exclusively associated with its pathogenic lifestyle. We describe expressed sequence tags (ESTs) analysis to assess the expression profile of the mycelium to yeast transition. To identify P. brasiliensis differentially expressed sequences during conversion we performed a large-scale comparative analysis between P. brasiliensis ESTs identified in the transition transcriptome and databases. RESULTS: Our analysis was based on 1107 ESTs from a transition cDNA library of P. brasiliensis. A total of 639 consensus sequences were assembled. Genes of primary metabolism, energy, protein synthesis and fate, cellular transport, biogenesis of cellular components were represented in the transition cDNA library. A considerable number of genes (7.51%) had not been previously reported for P. brasiliensis in public databases. Gene expression analysis using in silico EST subtraction revealed that numerous genes were more expressed during the transition phase when compared to the mycelial ESTs 1. Classes of differentially expressed sequences were selected for further analysis including: genes related to the synthesis/remodeling of the cell wall/membrane. Thirty four genes from this family were induced. Ten genes related to signal transduction were increased. Twelve genes encoding putative virulence factors manifested increased expression. The in silico approach was validated by northern blot and semi-quantitative RT-PCR. CONCLUSION: The developmental program of P. brasiliensis is characterized by significant differential positive modulation of the cell wall/membrane related transcripts, and signal transduction proteins, suggesting the related processes important contributors to dimorphism. Also, putative virulence factors are more expressed in the transition process suggesting adaptation to the host of the yeast incoming parasitic phase. Those genes provide ideal candidates for further studies directed at understanding fungal morphogenesis and its regulation.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells.

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Enzymatic bleaching of organosolv sugarcane bagasse pulps with recombinant xylanase of the fungus Humicola grisea and with commercial Cartazyme HS xylanase.

Organosolv (ethanol/water and acetosolv) pulps were treated with Humicola grisea var. thermoidea and compared with Cartazyme HS xylanase-treated pulp. The ethanol/water pulps treated with H. grisea had the same viscosity as unbleached pulps (8 cP). Ethanol/water pulps treated with Cartazyme had higher viscosity than H. grisea-treated pulps (12 cP). Acetosolv pulps treated with H. grisea and Cartazyme presented a reduction in viscosity; however, the pulps treated with H. grisea had a lower reduction in viscosity than Cartazyme-treated pulps. Ethanol/water pulps treated with H. grisea had a 23% reduction in kappa number in 4 and 8 h of treatment, compared with the unbleached pulps. Cartazyme-treated pulps had a kappa number similar to that of the control pulps for 4 h of treatment. Extending the treatment time to 12 h resulted in a reduction of 33%. The acetosolv pulp treated with H. grisea had a kappa number reduced to 23% in 4 h. Cartazyme treatment resulted in a reduction of 55 and 44% in kappa number for 4 and 8 h of treatment, respectively, when compared with control pulp. Extending the treatment time to 12 h decreased the kappa number 72%. Fourier transform infrared spectra and principal component analysis showed differences among unbleached, H. grisea-treated, and Cartazyme-treated pulps.

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein.

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.

Paracoccidioides brasiliensis presents two different cDNAs encoding homologues of the fructose 1,6-biphosphate aldolase: protein isolation, cloning of the cDNAs and genes, structural, phylogenetic, and expression analysis.

A proteomic approach was used to identify a 39 kDa antigen of Paracoccidioides brasiliensis. Amino acid sequences of the N-terminal and of endoproteinase Lys-C digested peptides revealed the protein to be a fructose 1,6-biphosphate aldolase (FBA) Class II of P. brasiliensis. Two cDNA homologues, Pbfba1 and Pbfba2, were cloned and characterized. Pbfba1 encoded a predicted polypeptide of 360 amino acids that was highly homologous in the primary structure to the same enzyme from fungi and bacteria. The other DNA, Pbfba2, encoded a polypeptide predicted to be 363 amino acids. The sequence of Pbfba2 differed significantly from Pbfba1. Phylogenetic and molecular analysis supports the concept of gene duplication for FBAs in P. brasiliensis, constituting a two-member family. Expression analysis demonstrated differential expression for both fbas genes in P. brasiliensis cells.

Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts.

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.