Fig 1.
Identification of AtrR-family genes in A. oryzae and A. nidulans.
(A) Growth of A. oryzae strains overexpressing the candidate genes on medium plate in the presence of clotrimazole. 102 conidia of the strains were inoculated on CD plate (1% maltose) containing 0.5 μg/ml clotrimazole and incubate at 30°C for 7 days. (B) Growth of the AoatrR deletion strain on medium plate in the presence of miconazole. 102 conidia of A. oryzae LigD (wild type) and the AoatrR deletion mutant (ΔAoatrR) were inoculated on CD plate (1% glucose) containing 0.01 μg/ml miconazole (MCZ) and incubate at 30°C for 5 days. (C) Growth of the AnatrR deletion strain on medium plate in the presence of MCZ. 102 conidia of A. nidulans KU70 (wild type) and the AnatrR deletion mutant (ΔAnatrR) were inoculated on CD plate (1% glucose) containing 0.005 μg/ml miconazole and incubate at 37°C for 5 days. (D) Phylogenetic tree of fungal AtrR ortholog proteins. The protein sequences were retrieved from NCBI database, and the sequence IDs were shown in brackets. The sequences were aligned by Clustal W program and the distances were calculated. The phylogenetic tree was drawn with an NJplot program.
Fig 2.
Drug susceptibility of A. fumigatus strains linked to modifications of the atrR and srbA genes.
(A) Antifungal drug susceptibility test by paper-disc diffusion assay. GMM plates containing conidia of each strain were prepared. A paper-disc was placed on center of the plate, and 10 μl of drug solution indicated was dropped on it (fluconazole (FLCZ): 10 mg/mL; MCZ: 10 mg/mL; amphotericin B (AMPH): 0.25 mg/mL; micafungin (MCFG): 0.02 mg/mL). The plates were incubated for 48 h before photographed. (B) Inhibition test for colony growth by antifungal drugs. The conidia of each strain were center-inoculated onto GMM plates containing indicated concentrations of itraconazole (ITCZ) or MCZ. The plates were incubated for 70 h. The colony diameter was measured in triplicates, and the mean values for plates with drugs were compared with those without drugs. The percentage of growth was plotted in the graphs.
Fig 3.
Expression analysis in atrR and srbA deletion mutants.
(A) Expression levels of the cyp51A and cyp51B as well as the atrR and srbA genes were determined by real-time RT-PCR method. The strains were cultivated in GMM for 24 h. The expression levels of gene of interest were normalized with that of actin gene. Relative expression ratios against Af293 (WT) are shown. Error bars represent the standard deviations based on three independent replicates. (B) MCZ susceptibility test for the Af293, ΔatrR, and ΔsrbA strains carrying a cyp51A gene fused with a thiA promoter. MCZ susceptibility was examined as described above. When thiamine was present, expression of the functional cyp51A mRNA driven by thiA promoter was repressed. The plates were incubated for 48 h before photographed. (C) Expression levels of cyp51A were determined by real-time RT-PCR in the mutant strains of ΔatrR, ΔsrbA, ΔatrR ΔsrbA of Afs35-background. The strains were cultivated in YGMM for 18 h.
Fig 4.
AtrR and SrbA regulate genes associated with the ergosterol biosynthesis pathway.
(A) The AtrR- and SrbA-dependent genes determined by RNA-sequencing analysis. Numbers of gene with more than 4-fold or less than 1/4-fold change in expression level compared to Af293 are shown. The expression level of nine genes in ΔatrR and ΔsrbA strains was more than 4-fold of Af293, and the expression level of eighteen genes in ΔatrR and ΔsrbA strains was less than 1/4-fold of Af293. (B) Heat map of expression of the erg genes. The levels of expression ratio were shown. (C) Expression levels of erg3B, erg24A, erg25A, and cyp51A genes in response to MCZ addition were determined by real-time RT PCR method. The strains were cultivated in GMM for 24 h followed by 2 h culture with MCZ (final concentration, 2 μg/mL) or without MCZ (control). The expression levels of gene of interest were normalized with that of actin gene. Relative expression ratios against Af293 (WT without MCZ) are shown. Error bars represent the standard deviations based on three independent replicates.
Table 1.
RNA-sequencing expression data for the genes regulated by AtrR and SrbA.
Fig 5.
AtrR is involved in adaptation to hypoxia.
(A) Growth test under normoxic and hypoxic conditions. The conidia of each strain were inoculated on GMM plate, and the plate was incubated in anaeropack system (a sealed pack) where initial concentration of oxygen was set at 2% (hypoxia condition). After 96 h, oxygen in the pack appeared to be completely consumed as estimated from no more growth of all colonies on the plate. The plate for normoxia was incubated in a regular incubator (21% oxygen) for 72 h. (B) Growth of Afs35-background strains under normoxic and hypoxic conditions. The 10 to 104 conidia of each strain were inoculated on GMM plate, and the plate was incubated in anaeropack system (a sealed pack) for 48 h, where concentration of oxygen was maintained at 2 to 4% (moderate hypoxia condition). (C) Expression levels of erg3B, erg24A, erg25A, and cyp51A genes in response to oxygen limitation (O2 limit.) were determined by real-time RT PCR method. The strains were cultivated in YGMM for 18 h. A half potion of the culture was transferred into a 50 mL tube with a screwed cap. The tube was closed with a cap carefully eliminating air, and was incubated for another 1 h. The expression levels of gene of interest were normalized with that of actin gene. Relative expression ratios against Af293 (WT before oxygen limitation) are shown. Error bars represent the standard deviations based on three independent replicates.
Fig 6.
AtrR is involved in A. fumigatus virulence.
(A) Mouse infection test with the Af293-background strains. Outbred ICR mice (n = 12) were immune-compromised by intraperitoneally injected cyclophosphamide (200 mg/kg) at Day -4, -2, 1, 3, 5, and 7. Mice were infected intratracheally with 2.5×107 conidia in a volume of 25 μL of each strain (Af293, ΔatrR, and Co-atrR). Statistical significance was examined by log-rank test. P value for comparison between ΔatrR and Af293 was lower than 0.01. (B) Mouse infection test with the Afs35-background strains. Outbred ICR mice (n = 9 to 11) were immune-compromised by intraperitoneally injected cyclophosphamide (150 mg/kg) at Day -4, -1, 3, 6, 9, and 12. Cortisone acetate was also administrated subcutaneously at a concentration of 200 mg per kg of body weight on day -1. Mice were infected intratracheally with 3×105 conidia in a volume of 30 μL of each strain (Afs35, ΔatrR, ΔsrbA, and ΔatrR ΔsrbA) on Day 0.
Fig 7.
FLCZ- and MCZ-responsive and AtrR-dependent genes determined by RNA-sequencing analysis.
(A) The ΔatrR and Af293 strains were cultivated in PDB for 20 h followed by FLCZ (final concentration, 64 μg/mL), MCZ (2 μg/mL), or DMSO (as a control) treatment for 2 h. Number of gene with less than 1/3-fold change in expression level in ΔatrR (DMSO) compared to Af293 (DMSO) was shown. Numbers of gene with more than 3-fold upregulation upon FLCZ or MCZ treatment (2 h) in Af293 were shown. Expression of six genes was induced upon FLCZ and MCZ treatment and these genes were dependent on AtrR, and expression of thirteen genes was induced upon FLCZ and MCZ treatment and these genes were independent on AtrR. (B) Expression level of atrR and srbA genes in response to MCZ addition was determined by real-time RT PCR method. The Af293 strain was cultivated in GMM for 24 h followed by 2 h culture with MCZ (final concentration, 2 μg/mL) or without MCZ (control). The expression levels of gene of interest were normalized with that of actin gene. Relative expression ratios against control (Af293 without MCZ) are shown. Error bars represent the standard deviations based on three independent replicates.
Table 2.
RNA-sequencing analysis for Af293 and ΔatrR in response to azole drugs.
Fig 8.
Expression levels of the cdr1B gene in response to azoles were determined by real-time RT PCR method.
(A) AtrR is involved in regulating cdr1B expression. Af293, ΔatrR, and ΔsrbA strains were cultivated in GMM for 24 h followed by 2 h culture with or without MCZ (final concentration, 2 μg/mL). (B) Af293 and ΔatrR strains were cultivated in GMM for 24 h followed by 2h culture with ITCZ, MCZ, or propiconazole (PRCZ) (final concentrations, 2 μg/mL). The expression level of cdr1B was normalized with that of actin gene. Relative expression ratios against Af293 (WT without drug) are shown. Error bars represent the standard deviations based on three independent replicates.
Fig 9.
Chromatin immunoprecipitation assay showing binding of AtrR protein to the promoter regions of cyp51A and cdr1B.
(A) Chromatin immunoprecipitation (ChIP) was done using a mouse monoclonal antibody directed against the HA epitope. Crosslinked chromatin was prepared from a WT strain or an isogenic strain expressing a 3x HA-tagged form of atrR (shown as atrR-3× HA). Immunoprecipitated DNA was examined by semiquantitative RT PCR analysis using primers that amplified the cyp51A and cdr1B promoters. The relative enrichment of the act1 promoter was also assessed as a control for a promoter not responsive to AtrR. (B) Quantitative analysis for enrichment of the cyp51A and cdr1B promoters by real-time RT PCR method. Chromatin immunoprecipitated from the atrR-3× HA strains exhibited a 20-fold enrichment of cyp51A promoter DNA along with a ~33-fold enrichment of cdr1B promoter sequence. No detectable enrichment of act1 was found. Data are presented as the mean and standard error of two separate ChIP experiments. % input represent signals obtained from the ChIP that are divided by signals obtained from the input sample.
Table 3.
Minimal inhibitory concentrations of antifungals in A. fumigatus mutant strains.
Fig 10.
The clinical azole resistant strain with an atrR gene deleted shows azole hyper-susceptibility.
(A) Drug susceptibility test by paper-disc diffusion assay. A paper-disc was placed on center of the GMM plate, and 10 μl of drug solution indicated was dropped on it (FLCZ: 10 mg/mL; MCZ: 10 mg/mL; ITCZ: 10 mg/mL). An azole resistant strain IFM 61567 and the independently obtained atrR deleted strains (ΔatrR No. 2–4) were streaked outward from the center. The plates were incubated for 48 h before photographed. The IFM 61567 fully grew along the streaked line regardless of the presence of FLCZ and ITCZ, whereas the ΔatrR mutants showed growth inhibition. (B) Expression levels of cdr1B, cyp51A, and cyp51B genes in IFM 61567 and the atrR-deleted strain (ΔatrR No.2) were determined by real-time RT PCR method. IFM 61567 and the ΔatrR were cultivated in YPG for 18 h followed by 1 h culture with or without FLCZ, MCZ, and ITCZ (final concentrations, 2 μg/mL). The expression levels of genes of interest were normalized with that of actin gene. Relative expression ratios against IFM 61567 without drug (control) are shown. Error bars represent the standard deviations based on three independent replicates.
Table 4.
Strains used in this study.