Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans

• Author(s): Ricardo, Elisabete Travassos Araújo
• Date: 2011
• Persistent ID: http://hdl.handle.net/10216/22064
• Origin: Repositório Aberto da Universidade do Porto
• Subject(s): Medicina e Oncologia Molecular; Porto
• Document Type: Master thesis
• Language: Portuguese

Description

2. Abstract Several resistance mechanisms are associated to antifungal resistance in pathogenic fungi, namely C. albicans: over-expression of CDR1, CDR2 and MDR1 genes encoding for eflux pumps; alterations in gene expression levels or point mutations in ERG11 gene, encoding for the azoles target enzyme lanosterol 14 - demethylase, associated to ergosterol biosynthesis. The aim of the present work was to uncover the resistance mechanisms in a large group of clinical C. albicans strains, performing phenotypic and gene expression studies. Five control strains and 62 clinical strains of C. albicans were used. Standard protocol from CLSI M27-A2 was used to determine the susceptibility phenotype calculating minimal inhibitory concentration for the antifungals fluconazole, itraconazole and voriconazole, with and without ibuprofen (100µg/ml). Also, reversion by efflux hypothesis was tested by flow cytometry, using as fluorescent marker FUN-1, known as an efflux marker. Next, real time PCR protocols for detection and quantification of the target genes CDR1, CDR2, MDR1 e ERG11, were optimized. Different reaction parameters were tested, to achieve values of efficiency and error obtained from standard curves, according to reference values, to ensure accuracy. ACT1 gene, which encodes for actin was used as normalizing gene for the gene expression levels. Most clinical strains resistant to all tested azoles showed a significant over-expression of CDR1 and specially CDR2 when compared to ERG11; in these strains ibuprofen induced the reversion of resistance and an increase in FUN-1 fluorescence. The 2 strains that did not revert their phenotype, ibuprofen did not increase the fluorescence of cells stained with FUN-1 and gene over-expression was not only for CDR1 and CDR2 but mostly for ERG11 gene. It should be stressed that C. albicans control strain 12-99 with known multiple resistance mechanisms did not revert the resistance phenotype in the presence of ibuprofen. MDR1 expression was not detected in none of the C. albicans clinical strains. Strains with multiple resistance mechanisms did not revert, wich might indicate that ibuprofen could be a potential antifungal efflux blocker. Real Time PCR is easily available and a sensitive method for gene expression studies, and also SYBR Green is acceptable, although being an unspecific marker, when a melting curve is added in each assay.