Insight into the Significance of Aspergillus fumigatus cyp51A Polymorphisms

Abstract

Triazole antifungal compounds are the first treatment choice for invasive aspergillosis. However, in the last decade the rate of azole resistance among Aspergillus fumigatus strains has increased notoriously. The main resistance mechanisms are well defined and mostly related to point mutations of the azole target, 14-α sterol demethylase (cyp51A), with or without tandem repeat integrations in the cyp51A promoter. Furthermore, different combinations of five Cyp51A mutations (F46Y, M172V, N248T, D255E, and E427K) have been reported worldwide in about 10% of all A. fumigatus isolates tested. The azole susceptibility profile of these strains shows elevated azole MICs, although on the basis of the azole susceptibility breakpoints, these strains are not considered azole resistant. The purpose of the study was to determine whether these cyp51A polymorphisms (single nucleotide polymorphisms [SNPs]) are responsible for the azole susceptibility profile and whether they are reflected in a poorer azole treatment response in vivo that could compromise patient treatment and outcome. A mutant with a cyp51A deletion was generated and became fully susceptible to all azoles tested. Also, three cyp51A gene constructions with different combinations of SNPs were generated and reintroduced into an azole-susceptible wild-type (WT) strain (the ΔakuB^KU80 strain). The alternative model host Galleria mellonella was used to compare the virulence and voriconazole response of G. mellonella larvae infected with A. fumigatus strains with WT cyp51A or cyp51A with SNPs. All strains were pathogenic in G. mellonella larvae, although they did not respond similarly to voriconazole therapeutic doses. Finally, the full genomes of these strains were sequenced and analyzed in comparison with those of A. fumigatus WT strains, revealing that they belong to different strain clusters or lineages.

Keywords: Aspergillus fumigatus; Galleria mellonella; azole resistance; cyp51A polymorphisms; whole-genome sequencing.

FIG 1

Overall view of the 3D model of the A. fumigatus Cyp51A protein in complex with VRC. Cyp51A-WT (A) and Cyp51A-F46Y (B) protein structures. (Panels 1 and 2) different protein views; (panels 3) structural formulas of the amino acid placed in position 46 showing the 2D structure (left) and the 3D conformer (right) of each amino acid. The location of the F46Y amino acid change is circled.

FIG 2

Construction of the A. fumigatus Δcyp51A-SNP fusion cassette, confirmation of the PCR mutant, and determination of VRC susceptibility by Etest. (A) Map of the parental strain with cyp51A-5SNPs (TP11) (map 1), map of the cyp51A WT strain (map 2), and design of the fusion vector for A. fumigatus cyp51A gene deletion (map 3). The deleted cyp51A coding fragment is indicated in light green. Primers (p) are indicated by arrows. 3′ T, 3′ cyp51A terminator. (B) PCR analysis of the TP11 parental strain and the T1.15 Δcyp51A mutant. PCR verification of integration of the fragment cassette at the 5′ end (p7 to p9) (lanes a) and the 3′ end (p10 to p8) (lanes b) and verification of the absence of the cyp51A fragment (p11 to p12) (lanes c). Expected fragment sizes are indicated at the bottom. Lanes M, 1-kb DNA molecular ladder. (C) VRC susceptibility of the ΔakuB^KU80 strain (panel 1), strain TP11 (panel 2), and Δcyp51A-hypersusceptible mutant T1.15 (panel 3) by Etest.

FIG 3

Construction of the fusion cassettes for heterologous expression of the A. fumigatus cyp51A SNPs in the ΔakuB^KU80 strain with WT cyp51A and PCR confirmation of the mutants. (A) Design of the fusion cassettes for heterologous expression of cyp51A-5SNPs (map 1), cyp51A-3SNPs (map 2), and cyp51A F46Y (map 3). Primers are indicated by arrows. Blue, the homologous sequence outside cyp51A (5′ region, including the promoter, and 3′ region); dark green, the 3′ cyp51A terminator. (B) PCR analysis for verification of vector integration in the parental strain with WT cyp51A (the ΔakuB^KU80 strain) and the strains with cyp51A-5SNPs (T1.5), cyp51A-3SNPs (T2.3), and cyp51A-F46Y (T3.1). Lanes: a, full vector (A to H2) cassette integration; b, 5′ fragment (A1 to p9) cassette integration; c, 3′ fragment (H1 to p10) cassette integration; and d, hyg fragment (HYGp1 to HYGp2) integration. Expected fragment sizes are indicated at the bottom. Lanes M, 1-kb DNA molecular ladder.

FIG 4

Comparison of killing rates and VRC responses of G. mellonella larvae infected with A. fumigatus strains and their expression mutants. (A) Percent survival of G. mellonella larvae infected with the strain with the cyp51A WT (the ΔakuB^KU80 strain) and the strain with cyp51A-5SNPs (CM7632) with or without VRC treatment. (B) Percent survival of G. mellonella larvae infected with the strain with the cyp51A WT (ΔakuB^KU80) and the strain with cyp51A-3SNPs (CM3249) with or without VRC treatment. (C) Percent survival of G. mellonella larvae infected with the strain with the cyp51A WT (ΔakuB^KU80) and the expression mutant with cyp51A-5SNP (T1.5) with or without VRC treatment. (D) Percent survival of G. mellonella larvae infected with the strain with the cyp51A WT (ΔakuB^KU80) and the expression mutant with cyp51A-3SNPs (T2.3) with or without VRC treatment. NS, nonsignificant (P ≥ 0.01); *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

FIG 5

Phylogenetic analysis of A. fumigatus strains by whole-genome sequencing. Dendrograms were formed using the genome of strain A1163 (CBS144.89) (A) or AF293 (B) as a reference. Indigo dots, strains with the cyp51A WT; orange dots, strains with cyp51A-5SNPs; green dots, strains with cyp51A-3SNPs.