Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism

Abstract

The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213^* in svf1 and 167^* in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167^* in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.

Keywords: Aspergillus fumigatus; Azole resistance; CRISPR-Cas9; Ergosterol; In-host adaptation.

Fig. 1

Verification of SNP generation via Sanger sequencing. (A) Verification of A → G in cyp51A (AFUA_4G06890). (B) Verification of C → T in svf1 (AFUA_5G11820) (C). Verification of T → A in uncharacterised protein (AFUA_7G01960).

Fig. 2

Mycelial growth of A. fumigatus strains V130-15, V130-15svf1^213* and V157-62 with or without H₂O₂. Solid lines indicate growth in the absence of H₂O₂; dotted lines indicate growth in the presence of 2.5 mM H₂O_2. Sabouraud dextrose agar plates were spot inoculated with 5x10²Aspergillus conidia in 5 µl PBS and cultured at 37 °C. Colony diameter was measured every 24 h. Data represents two independent experiments with 3 repeats per experiment. Error bars represent ± SD.

Fig. 3

Mycelial growth of A. fumigatus strains V130-15, V130-15unc^167* and V157-62 at different temperatures. Sabouraud dextrose agar plates were spot inoculated with 5x10²Aspergillus conidia in 5 µl PBS and cultured at different temperatures. Colony diameter was measured after 96 h. Data were obtained in two experiments each comprising triplicate repeats. Mean values ± SD are shown (^*p < 0.01; ^***p < 0.0001; two-tailed Students T-test).

Fig. 4

Morphology and growth differences of A. fumigatus strains V130-15, V130-15unc^167* and V157-62 at different temperatures. Sabouraud dextrose agar plates were spot inoculated with 5 × 10²Aspergillus conidia in 5 µl PBS and cultured. Images were taken after 96 h.

Fig. 5

Quantification of total ergosterol content of A. fumigatus strain V130-15, V130-15unc^167* and V157-62. Strains were cultured overnight in liquid glucose minimal media at 37 °C shaking at 200 rpm. Fungal mass was dried and ground in liquid nitrogen. Samples were subjected to alkaline hydrolysis and ergosterol was solvent extracted and derivatised. Ergosterol was then quantified using gas chromatography-mass spectrometry (GC–MS). Data represents two independent extraction experiments, comprising 6 biological repeats per strain and 3 technical repeats per sample. Error bars represent ± SD (^*p < 0.01; ^***p < 0.0001; two-tailed Students T-test).

Fig. 6

Quantification of phosphatidylinositol content of A. fumigatus strain V130-15, V130-15unc^167* and V157-62. Strains were cultured overnight in liquid glucose minimal media at 37 °C shaking at 200 rpm. Fungal mass was dried and ground in liquid nitrogen. Samples were subjected to chloroform-methanol extraction. Phosphatidylinositol (32:4) was then quantified using liquid chromatography-mass spectrometry (LC–MS). Data represents two independent extraction experiments, comprising 6 biological repeats per strain and 3 technical repeats per sample. Error bars represent ± SD (^*p < 0.05; two-tailed Students T-test).

Fig. 7

Susceptibility of A. fumigatus strains V130-15, V130-15unc^167* and V157-62 to lovastatin and nystatin. Metabolic activity of the strains was assessed using the XTT spectrophotometric assay after 48 h incubation with 16 µg/ml nystatin and 256 µg/ml lovastatin at 37 °C. Data represents two independent experiments with 3 repeats per experiment. Error bars represent ± SD (^*p < 0.05; ^**p < 0.01; two-tailed Students T-test).

Fig. A.1

Schematic showing the protospacer adjacent motif (PAM), protospacer and Cas9 cut site for each target. Colour scheme is as follows: red text represents 3 bp PAM site, orange text represents 20 bp protospacer, scissors represent Cas9 cut site and underlining indicates the location of the target nucleotide for modification.