title: Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters.
abstract: The identification of essential genes represents a critical step in the discovery of novel therapeutic targets in Aspergillus fumigatus. Structural analyses of the Saccharomyces cerevisiae RNA triphosphatase pointed out this enzyme as an attractive therapeutic target for fungal infections. In addition, demonstration of the essentiality of the S. cerevisiae RNA triphosphatase encoding gene enhanced the value of this potential therapeutic target. Nevertheless, consideration of a fungal RNA triphosphatase as an ideal therapeutic target needs confirmation of the essentiality of the respective gene in a fungal pathogen. In this work, we analyzed the essentiality of the A. fumigatus triA gene, encoding RNA triphosphatase, by conditional gene expression and heterokaryon deletion. Using the conditional gene expression driven by the alcA promoter (alcA(P)), we found that TriA depletion causes morphological abnormalities that result in a very strong growth inhibition. Nevertheless, since a strict terminal phenotype was not observed, the essentiality of the triA gene could not be ensured. Accordingly, the essentiality of this gene was analyzed by the heterokaryon rescue technique. Results obtained unequivocally demonstrated the essentiality of the A. fumigatus triA gene, indicating the suitability of the RNA triphosphatase as an ideal therapeutic target to treat A. fumigatus infections. Besides, a second conditional gene expression system, based on the niiA promoter (niiA(P)), was utilized in this work. Although the niiA(P)-mediated repression of triA was less severe than that driven by the alcA(P), a strong growth inhibition was also found in niiA(P)-triA strains. Finally, E-tests performed to determine whether triA down-regulated cells became more sensitive to antifungals suggest a synergic effect between amphotericin B and another antifungal inhibiting the A. fumigatus RNA triphosphatase activity.
authors.affiliation: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Campus Universitario, Universidad de Alcalá, Alcalá de Henares, ES-28871 Madrid, Spain.
authors.forename: M Cândida
authors.initials: MC
authors.lastname: Monteiro
authors.forename: J Ramón
authors.initials: JR
authors.lastname: De Lucas
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology / Q000627:therapeutic use; D001228:Aspergillosis / Q000188:drug therapy / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015536:Down-Regulation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020043:Genes, Essential; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase