Mutation of an NADH-Ubiquinone Oxidoreductase Subunit in aspergillus fumigatus Leads to Azole Resistance.

J. MOSQUERA, M.J. ANDERSON, H.E. ROSS, M. BIRCH, J.L. BROOKMAN, D.W. DENNING

Author address: 

University of Manchester, Manchester, United Kingdom, F2G, Manchester, United Kingdom, F2g, Manchester, United Kingdom.

Abstract: 

Background: We employed a random insertional mutagenesis screen in order to find genes involved in azole resistance in A. fumigatus. Methods: aspergillus fumigatus pyrG- protoplasts were transformed using a restriction-enzyme-mediated integration (REMI) system with a linearised plasmid containing the A. nidulans pyrG gene and the restriction enzyme XhoI. After 48h, itraconazole was added at a subinhibitory concentration and plates incubated further. Colonies that showed reduced or increased growth in relation to the majority (after the addition of itraconazole) were subcultured and tested for their susceptibility to various azoles. The disrupted genes were identified by plasmid rescue and the association of these genes with the altered phenotype confirmed by reintroduction of the plasmid into the parental strain. Results: One transformant was more resistant to azoles with anti- aspergillus activity: MICs=1.0, 0.50 and 4.0 µg/ml for itraconazole, posaconazole and ravuconazole, respectively, versus 0.25, 0.12 and 1.0 µg/ml for the parental clinical isolate. The insertional mutation had occurred at an XhoI site 534 bp downstream from the start of a 841 bp ORF that encodes a 242 amino acid protein. The best BLAST hit to a characterised fungal protein was to the NADH-ubiquinone oxidoreductase 29.9 kDa subunit from Neurospora crassa. This protein is an essential mitochondrial precursor of Complex I, which provides the input to the respiratory chain from the NAD-linked dehydrogenases of the citric-acid cycle. The complex couples the oxidation of NADH and the reduction of ubiquinone to the generation of a proton gradient, which is then used for ATP synthesis. Conclusions: Resistance to azoles due to an alteration in mitochondrial respiration has been described in Candida. Nevertheless this is the first report of such a mutation leading to azole resistance in aspergillus.
2003

abstract No: 

M-391

Full conference title: 

43rd Interscience Conference on Antimicrobial Agents
    • ICAAC 43rd