A. fumigatus is a human pathogenic fungus capable of inducing a range of disease states in immunocompromised patients. Fungal respiration exhibits peculiar features and involves an alternative oxidase pathway not yet fully elucidated. In this sense, we have silenced an aox gene in A. fumigatus (Afaox) by RNA interference. Inverted repeats of Afaox coding sequence were cloned in the pALB1 plasmid, which contain an alb1 gene cloned. In xylose medium, the Afaox/alb1 and alb1 transformants had the same phenotype, a green color, as the wild strain, indicating that the interference was not induced. However, in maltose medium the Afaox/alb1 transformant grew in white color indicating an entire silence and a Real-time PCR revealed a decrease of 95% in RNAm levels. It has been suggested that AOX may be important for fungal survival in macrophages, based on accumulating evidence for its antioxidant defense. ROS production was monitored using fluorescent probe CM-H2DCFDA. In Afaox/alb1 transformants, ROS production increased 12.5 and 2-fold when compared with wild-strain and alb1 transformants, respectively, suggesting that AOX can prevent ROS formation. The survival rate of silenced strain in macrophages was measured by MTT assay. Afaox, double silenced strain, revealed a highest survival when compared with the other strains. Conidia were incubated with different ROS donors and conidia viability was measured by flow cytometria with FUN-1 and PI uptakes. These results indicate that the alternative oxidase has an important role in oxidative defense in Aspergillus fumigatus.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)