Background: CAS is an echinocandin antifungal that inhibits β -1,3-glucan synthase, an enzyme essential for fungal cell wall biogenesis. In yeasts, resistance to CAS results from mutations in the target enzyme-encoding gene. We have identified and characterized CAS-resistant Aspergillus mutants, in the genetically tractable mold A. nidulans. Materials and methods: Conidia (105 - 106) of A. nidulans mutagenized with 4-nitroquinoline N-oxide were placed as streaks or spots on to plates of MAG containing 5μg/ml CAS and incubated 7 days at 37ºC. A total of 32 CAS resistant mutants were isolated and genetic analysis determined that all were allelic. The fksA gene was amplified using the Expand Long PCR system from 18 of these strains, cloned and sequenced to determine the site of mutations in this target gene. Results: We found that CAS resistance in A. nidulans was the result of mutations in fksA, the gene for the enzyme β -1,3-glucan synthase. Seven mutants had a single base pair difference compared to the wild type. Six of these mutations were at Arg 1385 of the polypeptide chain, changing it to either Gly or Ser. The seventh mutant had a single base change resulting in substitution of Asp 677 to Tyr. This mutation is near Ser 645 of the Candida albicans FKS1 gene that has been described as a hot spot for mutations in this fungus. Mutation of Arg 1385 was also seen in nine other strains but these mutants had additional base changes that could have contributed to the resistance phenotype. Thus 15 of 18 CAS resistant strains had mutation at Arg 1385. Conclusions: Arg 1385 of FksA of A. nidulans is a hot spot for mutations that is associated with resistance to CAS. We are currently isolating CAS resistant mutant in A. fumigatus and will sequence these to see if this conserved amino acid is also a hot spot for mutations resulting in CAS resistance.
Full conference title:
47th Interscience Conference on Antimicrobial agents and Chemotherapy
- ICAAC 47th