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.

A single-copy gene, designated TruMDR2, encoding an ATP-binding cassette (ABC) transporter was cloned and sequenced from the dermatophyte Trichophyton rubrum. The ORF of TruMDR2 was 4048 nt and the deduced amino acid sequence showed high homology with ABC transporters involved in drug efflux in other fungi. The encoded ABC protein predicted 12 transmembrane segments (TMSs) and two almost identical nucleotide-binding domains (NBDs) arranged in two halves in a (TMS 6 -NBD) 2 configuration and could be classified as a member of the multidrug-resistance (MDR) class of ABC transporters. Northern blot analyses revealed an increased level of transcription of the TruMDR2 gene when mycelium was exposed to acriflavine, benomyl, ethidium bromide, ketoconazole, chloramphenicol, griseofulvin, fluconazole, imazalil, itraconazole, methotrexate, 4-nitroquinoline N-oxide (4NQO) or tioconazole. Disruption of the TruMDR2 gene rendered the mutant more sensitive to terbinafine, 4NQO and ethidium bromide than the control strain, suggesting that this transporter plays a role in modulating drug susceptibility in T. rubrum. INTRODUCTIONTrichophyton rubrum is a cosmopolitan filamentous fungus that can infect human keratinized tissue (skin, nails and, rarely, hair) and is the causal agent of 80-90 % of all chronic and recurrent dermatophytoses (Fernández-Torres et al., 2000). This pathogen, which normally causes wellcharacterized superficial infections, can also cause deep dermal invasion in immunocompromised patients (Smith et al., 2001;Squeo et al., 1998). Griseofulvin, terbinafine and itraconazole are widely used to treat dermatophytosis. Although in routine clinical practice the response to these antifungals is usually satisfactory, the occurrence of strains that are insensitive to antifungal agents (Mukherjee et al., 2003;Osborne et al., 2005) could play a relevant role in the failure of antifungal treatments. Thus, studies of the mechanisms of antifungal resistance are crucial for a more rational use of drugs, to minimize or overcome resistance.A clinical isolate of T. rubrum submitted to a mutagenic treatment presented simultaneous resistance to griseofulvin and tioconazole in vitro, suggesting the existence of a multidrug-resistance (MDR) mechanism based on cellular efflux involved in this event (Fachin et al., 1996). The T. rubrum TruMDR1 gene, which encodes an ATP-binding cassette (ABC) transporter, is also differentially expressed in the presence of unrelated toxic compounds, including the antifungals griseofulvin and itraconazole (Cervelatti et al., 2006). ABC transporters are highly conserved ATPases, ubiquitous from bacteria to humans, and this mechanism protects cells against the cytotoxic effects of compounds by reducing the accumulation of toxic compounds in the cell. In eukaryotes, most ABC transporters are composed of two similar halves, each consisting of a cytoplasmic nucleotidebinding domain (NBD) and six transmembrane segments (TMSs). The NBDs of ABC transporters contain conserved amino acid sequences, called th...

We report here the isolation, molecular cloning and initial characterization of the Trichophytonrubrum pacC gene, which encodes a putative protein that is homologous to the PacC/Rim101p family of pH signaling transcription regulators. The promoter region of the T. rubrumpacC gene contains four recognition sites 5'-GCCAAG-3' for the PacC protein, suggesting that the transcription of this gene itself could be induced under alkaline growth conditions. The enhanced expression profile of the T. rubrumpacC gene in an alkaline environment was confirmed by Northern blotting analysis. We also report that the disruption of pacC gene decreased both the secretion of keratinolytic proteases and the ability of the mutant pacC-1 to grow on human nail fragments as the sole source of nutrition, i.e., growth of the dermatophyte T. rubrum appear to be related to molecular events which depend on the action of protein PacC.