Rosália Santos Amorim Jesuíno

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

New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen

By means of genealogical concordance phylogenetic species recognition (GCPSR), we have investigated coding and non-coding regions from various genes and the ITS sequences of 7 new and 14 known isolates of Paracoccidioides brasiliensis. Such isolates grouped within the three phylogenetic groups recently reported in the genus Paracoccidioides, with one single exception, i.e., Pb01, a strain that has been the subject of intense molecular studies for many years. This isolate clearly separates from all other Paracoccidioides isolates in phylogenetic analyses and greatly increases the genomic variation known in this genus.

Two-dimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a fungal pathogen of humans. To identify antigens from P. brasiliensis we fractionated a crude preparation of proteins from the fungus and detected the IgG reactive proteins by immunoblot assays of yeast cellular extracts with sera of patients with paracoccidioidomycosis (PCM). We identified and characterized six new antigens by amino acid sequencing and homology search analyses with other proteins deposited in a database. The newly characterized antigens were highly homologous to catalase, fructose-1,6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and triosephosphate isomerase from several sources. The characterized antigens presented preferential synthesis in yeast cells, the host fungus phase.

Avaliação do cetoconazol em camundongos inoculados com Paracoccidioides brasiliensis pela histopatologia de fígado e baço e pela intradermorreação de paracoccidioidina

Male albino mice were inoculated intravenously with 0.5 x 10(7) viable yeast forms of P. brasiliensis (strain 2052). These animals were treated with two doses of ketoconazole (50 and 100 mg/kg) during fifty days and the sacrificed. We studied the presence of P. brasiliensis, the inflammatory granulomatous response of liver and spleen and the anti P. brasiliensis delayed hypersensitivity response measured by the footpad test after 48 hours. It was observed that: 1. animals infected and treated with ketoconazole showed reduction in the number of fungi in the organs studied; 2. there was no difference in the number of granulomas among animals treated and non-treated; 3. the cutaneous delayed tests intensity was similar in all animals.

Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis.

Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus–host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome‐PTS‐1‐targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen‐dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis. Copyright

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.

Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production

Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.

Characterization of a chaperone ClpB homologue of Paracoccidioides brasiliensis

We report the cloning and sequence analysis of a genomic clone encoding a Paracoccidioides brasiliensis ClpB chaperone homologue (PbClpB). The clpb gene was identified in a λ Dash II library. Sequencing of Pbclpb revealed a long open reading frame capable of encoding a 792 amino acid, 87.9 kDa protein, pI of 5.34. The predicted polypeptide contains several consensus motifs of the ClpB proteins. Canonical sequences such as two putative nucleotide‐binding sites, chaperonins ClpA/B signatures and highly conserved casein kinase phosphorylation domains are present. ClpB is 69% to 49% identical to members of the ClpB family from several organisms from prokaryotes to eukaryotes. The transcript of PbclpB was detected as a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phase of the fungus. A 89 kDa protein was also detected in yeast cells of P. brasiliensis. The sequence of the clpb gene and the deduced ClpB protein have been submitted to GenBank under Accession No. AF449501. Copyright

Identification, N-terminal region sequencing and similarity analysis of differentially expressed proteins in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America. This human pathogen is a dimorphic fungus existing as mycelium (26 degrees C) and in infected tissues as a yeast form (36 degrees C). The in vitro differentiation process is reversible and dependent on temperature shift. In the present study, the total proteins from both forms of P. brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis. Differentially expressed proteins were identified. Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions. The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program. The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum. This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P. brasiliensis.

Effects of Antifungal Agents in Sap Activity of Candida albicans Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV+) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.