63ª Reunião Anual da SBPC

C. Ciências Biológicas - 3. Bioquímica - 1. Biologia Molecular

TRANSCRIPTIONAL RESPONSE TO ITRACONAZOLE IN THE HUMAN PATHOGENIC FUNGUS Paracoccidioides brasiliensis.

Benedito Rodrigues da Silva Neto 1,2 Nayche Santiago Pacheco de Santana 1 Célia Maria de Almeida Soares 3 Maristela Pereira 4

1. Laboratório de Biologia Molecular, Instituto de Ciências Biológicas Universidade Federal de Goiás. 2. Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás. 3. Profa. Dra. Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás. 4. Profa. Dra/ Orientadora Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás.

INTRODUÇÃO:

The thermally dimorphic fungal pathogen Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This disease is characterized by a granulomatous inflammation with clinical forms ranging from a benign localized infection to a disseminated one, affecting several parts of the body. About 10 million people are infected by the agent cause of this disease. The disease is acquired through of the inhalation of fungal propagules that reach the lungs where occurs the conversion from mycelial to the yeast phase. Itraconazole is a triazole antifungal drug, which is multiringed synthetic compounds containing three nitrogen atoms in the azole ring. The triazole drugs are broad-spectrum antifungal agents and are currently used to treat infections caused by various pathogenic yeast and molds. The mechanism of action of azoles has been elucidated to some fungi. They act by blocking the ergosterol, an essential cell membrane component, biosynthetic pathway through binding to and inhibition of the lanosterol 14-α demethylase enzyme, encoded by the erg11.

METODOLOGIA:

Here we aim to investigate the mechanism of action of itraconazole on P. brasiliensis, by using Representational Difference Analysis (RDA). P. brasiliensis yeast cells were grown on minimum medium in the presence and absence of itraconazole for 1 and 2 hour. ESTs sequences were clustering using the CAP3 program. By using the Blast2GO program ESTs were classified in agreement with the possible functions. The database sequence matches were considered significant at E-values ≤10-10.

RESULTADOS:

The analyses indicated the classification of transcripts in different functional category. Most of the regulated genes were involved in lipid metabolism including the precursors of ergosterol. The genes ERG11, ERG6, ERG3 and ERG5 were up regulated. In addition, were found genes involved in cell stress response, drug efflux, small molecule transport, elongation and transcription factors, cell wall and membrane, and hypothetic proteins. The up and down regulated genes by RDA and bioinformatics analysis were confirmed by real-time PCR.

CONCLUSÃO:

The elucidation of the action mechanism of itraconazole should provide information about the molecular mechanisms of its growth, metabolism, pathogenesis, and drug resistance. Novel genes/protein targets for the development of antifungal agents should be identified.

Palavras-chave: Paracoccidioides brasiliensis, Itraconazole, Transcriptional response.