Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up-or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

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Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.

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Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes a human systemic mycosis with high incidence in Latin America. Owing to their participation in the control of pathogen morphogenesis, differentiation and virulence, it was decided to characterize ras genes in P. brasiliensis. ras1 and ras2 were identified to be coding for two different proteins with high identity. The ras transcriptional pattern was investigated by reverse transcription PCR (RT-PCR) during mycelium-to-yeast (M-->Y) transition, heat shock at 42 degrees C and after internalization of yeast cells by murine macrophages. Both genes were downregulated inside macrophages and ras1, at 42 degrees C. In contrast, ras genes did not show any transcriptional variation during the M-->Y transition. The fact that Ras proteins are attached to the membrane via farnesylation prompted the use of a farnesyltransferase inhibitor to investigate the importance of this process for vegetative growth and dimorphic transition. Farnesylation blockage interfered with the vegetative growth of yeast cells and stimulated germinative tube production even at 37 degrees C. During Y-->M transition, the inhibitor increased filamentation in a dose-dependent manner, indicating that impaired farnesylation favours the mycelium form of P. brasiliensis. The results suggest that ras genes might have a role in dimorphism, heat shock response and in host-pathogen interaction.