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MAGNANI, T. Cloning, functional expression, and biochemical characterization of an alternative oxidase mitochondrial gene from A. fumigatus. 2008. 199f. Tese (Doutorado). Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, 2008. The saprophytic species Aspergillus fumigatus is a deuteromycete fungus found worldwide, which has an essential role in recycling carbon and nitrogen. Following inhalation of conidia by the immunocompetent host, the innate cellular immune system is responsible for killing the conidia, exposing them to reactive oxygen. However, A. fumigatus is capable of surviving and replicating within the phagolysosomal compartment of immunocompromised macrophages. It was previously demonstrated that A. fumigatus mitochondria possess an alternative oxidase (aoxAf) wich is a cyanideresistant protein. A partial genomic DNA library was screened to cloning an aoxAf gene. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 352 amino acid sequence with a calculated molecular mass of 40 kDa and a theoretical pI of 9.51. The deduced amino acid sequence revealed four regions completely conserved among the AOXs sequences (LET, NERMHL, LEEA and RADE-H), where six conserved amino acids residues are proposed to be a metal ligand site. To characterize the AOX protein, a cDNA of aoxAf gene was cloned into pYES2 plasmid and transformed in S. cerevisiae INVSc1. After the incubation of the cells in a nonfermentable medium in the presence of KCN, S. cerevisiae expressing AOX was able to grow, while it was lethal for the control yeast. These results suggest that the recombinant AOXAf is properly targeted to the S. cerevisiae mitochondria where it has functional activity. Studies with different species demonstrated that AOX is induced by a variety of treatments usually labeled as stresses. To verify the function of AOX in A. fumigatus under oxidative stress conditions, conidia were treated with different donors of ROS. These treatments caused an increase in aoxAf activity and transcription levels. To identify genetically attributes of virulence and oxidative defense in A. fumigatus, we construct a RNA interference plasmid. Two inverted repeated sequences of conserved region of an interest gene were amplified and cloned in pALB1 plasmid. In maltose medium pALB1 and pALB1/aoxAf transformants demonstrated white colonies, attributable to the reduction of alb1 gene expression. The aoxAf mRNA levels were analyzed by Real time RT-PCR, showing an efficient alternative oxidase gene silencing in pALB1 plasmid construction. It was previously demonstrated that ROS can stimulate the AOXs activity, so, we used the dye CM-H2DCFDA to measure ROS production in RNAi transformants, showing that the decrease in aoxAf gene expression caused an increase in ROS production. After incubation with ROS donors the viability of these strains was determined by flow cytometry analysis. The pALB1/aoxAf strain showed higher lethality, when compared with CEA and pALB1, suggesting the involvement of AOX in antioxidant defense in A. fumigatus. Besides, ROS produced by alveolar macrophages play an essential role in the killing of A. fumigatus conidia. In the same way, phagocytosis assay revealed that pALB1/aoxAf strain was more lethal than CEA and pALB1. With these results, we concluded that alternative oxidase is an efficient antioxidant system and might contribute with defense mechanism of A. fumigatus.

Clonagem, expressão e caracterização do gene da oxidase alternativa mitocrondial de Aspergilus fumigatus