Perfil transcricional do fundo patogênico Paracoccidioides brasiliensis em resposta a sulfametoxazol

Abstract

Paracoccidioidomycosis (PCM) is a systemic fungal infection caused by thermodimorphic fungus Paracoccidioides brasiliensis. Mycelia and conidia growing in vitro at 23ºC-26ºC and 18ºC ± 4ºC, respectively, and as saprophytes in soil, water and plants to room temperature are considered the infective forms of the fungus. The yeast phase occurs at 35º-37ºC in vitro and host tissues. Sulfonamides were the first drugs used for treatment of PCM and continue to be quite active medications nowadays against this fungal infection. It is known that sulfa drugs are competitive antagonist of ρ-aminobenzoic acid (PABA), which condenses with 2-amino-4- hydroxy-6-hydroxymethyl-7,8 dihydropteridine pyrophosphate (DHPPP) to form dihydropteroate (DHP), a reaction catalyzed by dihydropteroate synthase (DHPS). However, no study was realized to P. brasiliensis yet. The aim of this work was investigating the global mechanism of action of sulfamethoxazole on P. brasiliensis. Yeast cells were grown on minimum medium in the presence and absence of sulfamethoxazole for 1 and 2 h, at 36C. After extraction of total RNA, the cDNA was obtained and used to representational difference analysis (RDA) experiments to identify the genes up and down regulated. ESTs sequences were clustering using the CAP3 program and classified in agreement with the functions by using BLAT2GO program. Several transcripts related to mitochondrial function were differentially expressed. Among them could be cited transmembrane GTPase, succinate/fumarate mitochondrial transporter, 3-demethylubiquinone-9-methyltransferase, ATP synthase subunit beta, NADPH dehydrogenase and carnitine/acyl carnitine carrier. Other transcripts related to metabolism, and to unknown functions were up or down regulated. Aiming to validate the RDA and bioinformatics results, we identified genes 7 potentially relevant and then validated by quantitative real-time PCR (qRT-PCR). Furthermore, to confirm the mitochondrial alteration, the method of reduction of tetrazolium salt 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The results indicated that sufamethoxazole acts in P. brasiliensis as a competitor for the synthesis of amino acids, nucleic acids and precursors of biosynthesis of folate cofactors, and then destabilizing mitochondrial functions.