No 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.

Aspergillus fumigatus is an important cause of life-threatening invasive fungal disease in patients with compromised immune systems. Resistance to itraconazole in A. fumigatus is closely linked to amino acid substitutions in Cyp51A that replace Gly54. In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established. This assay uses molecular beacons corresponding to the wild-type cyp51A gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54. Molecular beacon structure design and real-time PCR conditions were optimized to increase the assay specificity. The multiplex assay was applied to the analysis of chromosomal DNA samples from a collection of 48 A. fumigatus clinical and laboratory-derived isolates, most with reduced susceptibility to itraconazole. The cyp51A allelic identities for codon 54 were established for all of the strains tested, and mutations altering Gly54 in 23 strains were revealed. These mutations included G 54 W (n ‫؍‬ 1), G 54 E (n ‫؍‬ 12), G 54 K (n ‫؍‬ 3), G 54 R (n ‫؍‬ 3), and G 54 V (n ‫؍‬ 4). Molecular beacon assay results were confirmed by DNA sequencing. Multiplex real-time PCR with molecular beacons is a powerful technique for allele differentiation and analysis of resistance mutations that is dynamic and suitable for rapid high-throughput assessment of drug resistance.

Replacement of phenylalanine with leucine at position 391 in squalene epoxidase was identified as being responsible for terbinafine resistance in mutants of Aspergillus nidulans. The equivalent mutation was engineered into the ergA gene of Aspergillus fumigatus, resulting in an F389L substitution that also conferred resistance to this pathogenic mold.

The filamentous fungus Aspergillus nidulans is an obligate aerobe, which is capable of anaerobic survival, but not anaerobic growth. Since cytochrome c forms an essential part of the oxidative respiratory pathway it was expected that mutants lacking this component would be non-viable. Gene replacement of one homologue of the cycA (cytochrome c) gene was carried out in a diploid strain. Benomyl-induced haploidisation of this diploid yielded all cycA+ haploid colonies, initially suggesting that loss of cycA was indeed lethal. However, use of an alternative unbiased method to recover haploids yielded viable, but slow-growing, cycA- mutants. Replacement of the cycA locus in the cycA- mutants was verified by Southern blotting. Spectral analysis confirmed the absence of detectable levels of cytochrome c, and respiratory insensitivity to cyanide suggested the absence of cytochrome c-dependent respiration. Growth parameters were consistent with those expected of a CycA- mutant. Compared to the wild type, the mutants grew slowly on fermentable carbon sources, did not grow on non-fermentable carbon sources, and produced higher levels of ethanol. To our knowledge, this is the first report of a filamentous fungus that remains viable after complete elimination of a functional cytochrome c gene. We propose that the mutants are viable due to their ability to ferment and to use alternative respiratory pathways.