Characterization ofAspergillus fumigatusmutants with reduced susceptibility to caspofungin

Abstract

Caspofungin acetate (CAS) is a member of a new class of clinically-approved echinocandin drugs to treat invasive aspergillosis. CAS inhibits the activity of beta-1,3-D-glucan synthase (GS), thus damaging the fungal cell wall. Although no clinical resistance of Aspergillus to CAS has been reported as yet, the development of in vitro reduced susceptibility is presumed to be inevitable. By contrast, echinocandin resistance in laboratory strains of Candida albicans and Saccharomyces cerevisiae has been well documented. To study the potential for clinical resistance in Aspergillus, two classes of Aspergillus fumigatus mutant strains were isolated that exhibited reduced susceptibility to CAS. In the first class, a site-directed mutation within the target gene (AfFKS1, encoding the putative catalytic subunit of GS) was introduced and shown to confer low-level (16-fold) reduced susceptibility. A second class of spontaneous mutants were sensitive to low levels of drug but displayed nearly normal growth above 0.5 microg/ml, suggesting induction of an unknown resistance mechanism. At higher levels of drug (> or = 16 microg/ml), the mutants displayed partially restored sensitivity. Preliminary studies indicate that neither target site mutations, nor changes in target gene expression are present in these strains, as has been documented for several yeasts. Instead, preliminary results indicate that the molecular mechanism(s) underlying reduced susceptibility of CAS in the A. fumigatus strains is novel, possibly due to remodeling of the cell wall components.

Resistance to echinocandin-class antifungal drugs

“…Biochemical studies have confirmed the conserved behavior of hot-spot mutations in altering the sensitivity of glucan synthase for echinocandin drugs (Kahn et al 2007;. The universality of Fks1 modification as a mechanism for fungal resistance to echinocandin drugs was demonstrated following the engineering of the equivalent C. albicans fks1-S645Y mutation into the mould A. fumigatus, which was found to confer reduced susceptibility (Gardiner et al, 2005); such resistance in Aspergillus has only been observed in laboratory strains.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Caspofungin-resistant isolates of C. albicans and S. cerevisiae have been reported to contain mutations within the FKS1 gene, specifically at two distinct hot-spot regions (157,294,447,455). In A. fumigatus isolates, the introduction of a site-directed mutation within fks1 confers increased resistance to caspofungin (200). Similarly, an A. fumigatus Ser678Pro fks1 mutation, equivalent to a mutation known to confer echinocandin resistance in Candida, confers high levels of cross-resistance to three echinocandins in A. fumigatus (491).…”

“…An analysis of a class of spontaneously generated A. fumigatus mutants with reduced susceptibility to the echinocandins found no mutations in fks1 and no changes in its expression (200), suggesting that other mechanisms of resistance must be involved. In accordance with this, well-characterized stress response pathways have been implicated in A. fumigatus echinocandin resistance and will be discussed here.…”

Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance

“…In one study, two classes of A. fumigatus mutants were isolated that exhibited reduced susceptibility to caspofungin (70). A site-directed mutation in fks1, encoding the catalytic subunit of glucan synthase, conferred a 16-fold reduction in susceptibility.…”