Background: A collection of highly itraconazole resistant (RIT) A. fumigatus mutants was isolated and used to identify novel drug transporter genes in order to better understand the potential contribution of multi-drug resistance (MDR) transporters to clinical itraconazole (ITRA) resistance. Methods: Twenty-three A. fumigatus mutants were selected in vitro following ultraviolet irradiation and grew in the presence of 100 mg/ml ITRA. Primers designed to highly conserved regions of MDR pumps were used in RT-PCR amplification reactions to identify six novel genes encoding potential MDR efflux pumps. Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail. Analysis of the deduced amino acid sequence encoded by AfuMDR3 gene revealed high similarity to MFS (Major Facilitator Superfamily) transporters, while AfuMDR4 was a typical member of the ABC (ATP-Binding Cassette) superfamily. Real-time quantitative PCR with molecular beacon probes was used to measure the expression levels of AfuMDR3 and AfuMDR4 genes. Results: Many RIT mutants showed either constitutive high level expression of one or both genes or induction of expression upon exposure to itraconazole. Two RIT strains RIT11 and RIT13, which over express both AfuMDR3 and AfuMDR4, retained virulence in a mouse aspergillosis model. Conclusions: Our results suggest that these newly identified drug efflux pump genes of A. fumigatus contribute to the itraconazole resistance phenotypes displayed by the RIT mutant strains, and may have clinical significance.
Full conference title:
42nd Interscience Conference on Antimicrobial Agents and Chemotherapy
- ICAAC 42nd