Transcriptional profile of the human pathogenic fungus Paracoccidioides lutzii in response to sulfamethoxazole.

Paracoccidioidomycosis (PCM) is the most prevalent mycosis in Latin America and is caused by a group of fungi within the Paracoccidioides genus. The disease may present clinical and pathological manifestations ranging from asymptomatic pneumonia pulmonary lesions, to disseminated forms involving multiple organs. Sulfonamides were the first drugs used to treat PCM and are still used against this fungal infection. Sulfa drugs are competitive antagonists of ρ-aminobenzoic acid (PABA), a reaction catalyzed by dihydropteroate synthase (DHPS). However, the molecular effects of sulfonamides against the Paracoccidioides genus are unknown. The aim of this work was to investigate the global mechanism of action of sulfamethoxazole on Paracoccidioides lutzii. Yeast cells were grown on minimum medium in the presence or absence of sulfamethoxazole to construct EST libraries. The representational difference analysis (RDA) technique was used to identify up- and down-regulated P. lutzii genes after treatment with sulfamethoxazole. Approximately six transcripts related to mitochondrial function were differentially expressed. To confirm the RDA and bioinformatics results, several relevant genes were studied with quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate their levels of expression. To confirm the impact of sulfamethoxazole on mitochondria, we measured the reduction of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by P. lutzii with or without exposure to the drug. MTT assays reveal that sulfamethoxazole produces a marked dose-dependent adverse effect on P. lutzii. The transcriptional activity of selected genes in infected macrophages corroborated our in vitro results. The results indicated that sulfamethoxazole acts in P. lutzii as a competitor for amino acid, nucleic acids and folate cofactor biosynthesis, disrupting mitochondrial functions.

Transcriptional profile of Paracoccidioides induced by oenothein B, a potential antifungal agent from the Brazilian Cerrado plant Eugenia uniflora.

BACKGROUND: The compound oenothein B (OenB), which is isolated from the leaves of Eugenia uniflora, a Brazilian Cerrado plant, interferes with Paracoccidioides yeast cell morphology and inhibits 1,3-ß-D-glucan synthase (PbFKS1) transcript accumulation, which is involved in cell wall synthesis. In this work we examined the gene expression changes in Paracoccidioides yeast cells following OenB treatment in order to investigate the adaptive cellular responses to drug stress. RESULTS: We constructed differential gene expression libraries using Representational Difference Analysis (RDA) of Paracoccidioides yeast cells treated with OenB for 90 and 180 min. Treatment for 90 min resulted in the identification of 463 up-regulated expressed sequences tags (ESTs) and 104 down-regulated ESTs. For the 180 min treatment 301 up-regulated ESTs and 143 down-regulated were identified. Genes involved in the cell wall biosynthesis, such as GLN1, KRE6 and FKS1, were found to be regulated by OenB. Infection experiments in macrophages corroborated the in vitro results. Fluorescence microscopy showed increased levels of chitin in cells treated with OenB. The carbohydrate polymer content of the cell wall of the fungus was also evaluated, and the results corroborated with the transcriptional data. Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment. CONCLUSIONS: The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile. Some of the changes may represent specific adaptive responses to this compound in this important pathogenic fungus.

The malate synthase of Paracoccidioides brasiliensis Pb01 is required in the glyoxylate cycle and in the allantoin degradation pathway.

In the present study, we examined the characteristics of cDNA, the regulation of the gene expression of Paracoccidioides brasiliensis MLS (Pbmls), and the enzymatic activity of the protein P. brasiliensis MLS (PbMLS) from the P. brasiliensis Pb01 isolate. Pbmls cDNA contains 1617 bp, encoding a protein of 539 amino acids with a predicted molecular mass of 60 kDa. The protein presents the MLSs family signature, the catalytic residues essential for enzymatic activity and the peroxisomal/glyoxysomal targeting signal PTS1. The high level of Pbmls transcript observed in the presence of two-carbon (2C) sources suggests that in P. brasiliensis, the primary regulation of carbon flux into the glyoxylate cycle (GC) was at the level of the Pbmls transcript. The gene expression, protein level, and enzymatic activity of Pbmls were highly induced by oxalurate in the presence of glucose and by proline in the presence of acetate. In the presence of glucose, the gene expression, protein level, and enzymatic activity of Pbmls were mildly stimulated by proline. Our results suggested that PbMLS condenses acetyl-CoA from both 2C sources (GC) and nitrogen sources (from proline and purine metabolism) to produce malate. The regulation of Pbmls by carbon and nitrogen sources was reinforced by the presence of regulatory motifs CREA and UIS found in the promoter region of the gene.