title: Transcriptional profile of the human pathogenic fungus Paracoccidioides lutzii in response to sulfamethoxazole.
abstract: 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.
authors: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors: Patrícia Fernanda
authors: PF
authors: Zambuzzi-Carvalho
authors: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors: Amanda Gregorim
authors: AG
authors: Fernandes
authors: Laboratório de Farmacologia e Toxicologia Celular, Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors: Marize Campos
authors: MC
authors: Valadares
authors: Departments of Medicine and Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY, USA.
authors: Patrícia de Mello
authors: Pde M
authors: Tavares
authors: Departments of Medicine and Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY, USA.
authors: Joshua D
authors: JD
authors: Nosanchuk
authors: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors: Célia Maria de Almeida
authors: CM
authors: Soares
authors: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil maristelaufg@gmail.com.
authors: Maristela
authors: M
authors: Pereira
mesh_terms: D000890:Anti-Infective Agents / Q000378:metabolism*; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D050296:Microbial Viability / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D010084:Oxidation-Reduction; D010228:Paracoccidioides / Q000187:drug effects*; D060888:Real-Time Polymerase Chain Reaction; D013420:Sulfamethoxazole / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue; D013420:Sulfamethoxazole
keywords: Paracoccidioides lutzii; antifungal; representational difference analysis; sulfamethoxazole; transcriptome