Purpose: RNA triphosphatase catalyses the first step of cap formation. The cap structure is required for efficient pre-mRNA splicing, export, stability and translation initiation in eukaryotes. Structural and functional analyses of the Saccharomcyes cerevisiae RNA triphosphatase suggested the appropriateness of this enzyme as a valuable therapeutic target to treat fungal diseases. Nevertheless, confirmation of the essentiality of the RNA triphosphatase encoding gene in a fungal pathogen, such as Aspergillus fumigatus, is needed to prove the suitability of this potential therapeutic target, especially after demonstrating the dispensability of this gene in Candida albicans. Here, we studied the essentiality of the A. fumigauts triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional expression driven by two regulatable promoters, the alcA and niiA promoters. In addition, we illustrated the feasibility of both conditional expression systems for future applications of gene down-regulation. Methods: A wide range of molecular biology, classical and molecular genetics techniques and phenotypic analyses were utilized in this work. Also, E-tests experiments, using antifungals employed to treat invasive aspergillosis, were performed. Results: The essentiality of the A. fumigatus triA gene, encoding RNA triphosphatase, was firstly analyzed using the conditional expression driven by the A. nidulans alcA promoter (alcAP), a suitable system to validate essential genes in A. fumigatus. TriA depletion mediated by the alcAP causes a very strong growth inhibition, but without showing a strict terminal phenotype. Accordingly, we utilized the heterokaryon rescue technique as an arbiter to decide on the essentiality of the triA gene. Our results undoubtedly demonstrated the essentiality of triA, pointing out the RNA triphosphatase as a valuable therapeutic target in this fungus. In addition, another conditional expression system, based on the A. fumigatus niiA promoter (niiAP), that allows identification of A. fumigatus essential genes was employed. Although the niiAP-mediated repression of triA is less severe than that driven by the alcAP, a strong growth inhibition occurred in niiAP-triA strains. Interestingly, E-tests performed to determine whether triAdown-regulated cells became more sensitive to antifungals indicated a synergic effect betweenamphotericinB and another antifungal inhibiting the A. fumigatus RNA triphosphatase activity. Conclusions: The triA gene, encoding the RNA triphosphatase, is essential in A. fumigatus. The heterokaryon rescue technique is the appropriate arbiter to decide gene essentiality in A. fumigatus. The synergism observed between amphotericin B and TriA depletion might represent further achievements in the treatment of invasive aspergillosis.
Full conference title:
4th Advances Against Aspergillosis
- AAA 4th (2010)