Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter

Abstract

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

Fig. 1.

Colony morphology of three of the isolates on SDA medium after incubation at 35°C for 96 h. The colony diameter of F12776 was 30 to 35 mm and was 60 to 70 mm for the other two isolates. F13535 is an example of the morphology of the majority of the isolates.

Fig. 2.

Germination of conidia from all eight isolates. Counts were measured at hourly intervals in SDB at 35°C. Each point represents the means from eight replicates ± standard deviations. Typical apical hyphae grow singly or with a bifurcate structure, lateral branches that tend to grow out perpendicularly, and hyphae that grow relatively straight. F122776, however, showed atypical swollen hyphae that did not grow in straight lines.

Fig. 3.

Relative expression levels of the five cyp51A, cyp51B, AfuMDR1, AfuMDR3, and AfuMDR4 genes in three of the isolates. Expression was normalized to β-tubulin and is the means from four experiments.

Fig. 4.

Effect of itraconazole treatment on the expression levels of five genes in three of the isolates and the susceptible isolate AF41. Expression was determined at 1, 2, and 4 h after the addition of 4 mg/liter ITR and is compared to that at 0 h. Values are expressed relative to those for a cyclodextrin control. The results represent the results from two independent experiments performed in duplicate. Significant differences are indicated by asterisks (P < 0.05).

Fig. 5.

Southern hybridizations of restriction digestions of genomic DNA from five of the isolates. (A) A BglI digestion hybridized with a 282-bp Aft1 probe derived from PCR carried out on F12776 DNA. A band (indicated by the arrow) of approximately 4.9 kb is present only in the F12776 lane; the expected size is 4,892 bp. (B) An EcoRI digestion hybridized with a 282-bp Aft1 probe derived from a PCR carried out on F12776 DNA. A band (indicated by the arrow) of approximately 7.5 kb is present only in the F12776 lane; the expected size is 7,522 bp. In addition, a band (indicated by an oval) of approximately 12 kb is missing from the F12776 lane, and two bands (indicated by asterisks) have not hybridized with the same intensity as in the other four lanes. The positions of the size markers are indicated on the left side of the gels. Hyperladder I (Bioline) and λ DNA monocut mix (New England BioLabs) were used.