Zambuzzi-Carvalho Patrícia Fernanda, Fernandes Amanda Gregorim, Valadares Marize Campos, Tavares Patrícia de Mello, Nosanchuk Joshua D, Soares Célia Maria de Almeida, Pereira Maristela
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil.
Laboratório de Farmacologia e Toxicologia Celular, Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brazil.
Med Mycol. 2015 Jun;53(5):477-92. doi: 10.1093/mmy/myv011. Epub 2015 Apr 7.
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.
副球孢子菌病（PCM）是拉丁美洲最常见的真菌病，由副球孢子菌属中的一组真菌引起。该疾病的临床和病理表现范围广泛，从无症状的肺部病变到累及多个器官的播散型。磺胺类药物是最早用于治疗PCM的药物，目前仍用于治疗这种真菌感染。磺胺类药物是对氨基苯甲酸（PABA）的竞争性拮抗剂，PABA的反应由二氢蝶酸合酶（DHPS）催化。然而，磺胺类药物对副球孢子菌属的分子作用尚不清楚。这项工作的目的是研究磺胺甲恶唑对卢氏副球孢子菌的整体作用机制。在有或没有磺胺甲恶唑存在的情况下，将酵母细胞在基本培养基上培养以构建EST文库。代表性差异分析（RDA）技术用于鉴定用磺胺甲恶唑处理后卢氏副球孢子菌上调和下调的基因。大约六个与线粒体功能相关的转录本差异表达。为了证实RDA和生物信息学结果，使用定量实时聚合酶链反应（qRT-PCR）研究了几个相关基因，以评估它们的表达水平。为了证实磺胺甲恶唑对线粒体的影响，我们测量了有或没有接触该药物的卢氏副球孢子菌对四唑盐3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐（MTT）的还原。MTT分析表明，磺胺甲恶唑对卢氏副球孢子菌产生明显的剂量依赖性不良反应。感染巨噬细胞中选定基因的转录活性证实了我们的体外结果。结果表明，磺胺甲恶唑在卢氏副球孢子菌中作为氨基酸、核酸和叶酸辅因子生物合成的竞争者起作用，破坏线粒体功能。
