Mapeamento de proteínas alvo para novos antifúngicos na fração microssomal e citosólica do patógeno humano Aspergillus fumigatus

• Autor(a) principal: Silva, Ivy Ortega Medeiros Zanon da
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/12747
• topic: Aspergillus fumigatus; Microsome; Cytosol; OFFGEL electrophoresis; Sterol-c-24-methyltransferase; Tioredoxin reductase; Aspergillus fumigatus; Microssoma; Citosol; Eletroforese OFFGEL; Esterol-c-24-metiltransferase; Tioredoxina redutase; Aspergillus fumigatus; Frações Subcelulares; Eletroforese; Proteínas Fúngicas; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS::MICOLOGIA

The invasive fungal infections incidence has increased in recent years. These infections usually presents high mortality rates. Antifungal prophylaxis remains the most common clinical strategy to decrease mortality and prevent invasive fungal infections, however, it has low efficiency and drug resistance reports. Furthermore, antifungal therapy is limited to a small group of drugs such as polyenes, azoles, and echinocandins. Thus, the search for new drug targets is imperative for the new antifungal agents development. In silico studies have indicated four genes as potential drug target in pathogenic fungi. In this context, our aim was to investigate the expression of two proteins encoded by two putative target genes, erg6 in the microsomal fraction, and trr1 in the cytosolic fraction of A. fumigatus hyphae. To achieve this goal, we first standardized all steps of cell fractionation to isolate these two fractions of A. fumigatus hyphae. Subsequently, was optimized the protein extraction and rehidratation protocols of these two subfractions, such as cytosolic proteins rehidratation. These extracts were submitted to different protocols for protein fractionation in an OFFGEL electrophoresis system (OGE). The Western immunoblot results showed that these two proteins, erg6 and trr1, are expressed in filamentous phase of A. fumigatus. The microsomal protein extract submitted to the OGE in twelve fractions, showed three erg6 protein subunits recognized by monoclonal antibody, with distincts molecular weight and pI: a subunit with approximately 79 kDa, with pI in the range of 5,91 and 6,49, and others two subunits with 35 kDa and 32 kDa, both with pI between 6,49 and 7,08. The enzyme erg6 was described as a homotetramer in other fungi, however, our results suggest that in A. fumigatus the erg6 has a heterotetrameric structure. Regarding trr1 protein, in both, total and fractionated (OGE) extracts, a single band of approximately 40 kDa, with pI in the range of 4.79 and 5.33, was recognized by the polyclonal antibody, suggesting that this protein appears to have a homodimeric structure, as described in other microorganisms.