ABSTRACT
One of the main mechanisms of azole resistance of Aspergillus fumigatus is thought to be a reduction in the drug’s affinity for the target molecule, Cyp51A, due to its amino acid mutation(s). It is known that the azole resistance pattern is closely related to the mutation site(s) of the molecule. In this study, we tried to develop a simple and rapid detection method for cyp51A mutations using the endonuclease Surveyor nuclease. The Surveyor nuclease assay was verified using several azole-resistant strains of A. fumigatus that possess point mutations in Cyp51A. For validation of the Surveyor nuclease assay, blind tests were conducted using 48 strains of A. fumigatus (17 azole-resistant and 31 azole-susceptible strains). The Surveyor nuclease assay could rapidly detect cyp51A mutations with one primer set. Also, all the tested strains harboring different cyp51A single point mutations could be clearly distinguished from the wild type. The Surveyor nuclease assay is a simple method that can detect cyp51A mutations rapidly.
INTRODUCTION
Azole antifungals are widely regarded as first-line therapeutic drugs for invasive aspergillosis. The increase of azole-resistant Aspergillus fumigatus strains in recent years has become a serious problem worldwide (1). The target of azoles is 14α-sterol demethylase, involved in ergosterol biosynthesis, and most azole-resistant A. fumigatus strains have mutations in cyp51A, which encodes the enzyme (2). The Cyp51A molecule with point mutations in hot spots lowered the binding affinity for azole drugs, resulting in azole resistance in the strain (3). Hot spots that confer resistance have been reported, such as mutations at positions glycine 54, glycine 138, proline 216, methionine 220, and glycine 448 (4–9). A point mutation in Cyp51A can occur in some hosts receiving long-term azole therapy. On the other hand, azole-resistant A. fumigatus derived from exposure to azole fungicides in the environment has also been reported (10–12). The cyp51A genes of these environmental strains mostly have a tandem repeat (TR) in the promoter region and a point mutation(s) in the open reading frame. Typically, TR34 (tandem repeat sequence of 34 base pairs) and TR46 are combined with L98H and Y121F/T289A mutations, respectively. Recently, the TR53 azole resistance mechanism has also been reported (13). In addition, five single nucleotide polymorphism (SNP) cyp51A mutations (F46Y, M172V, N248T, D255E, and E427K) in A. fumigatus have been reported worldwide (14–18). These strains are not usually defined as resistant but often have relatively high MICs of azoles compared with the wild type. Furthermore, strains with 3 SNPs (F46Y, M172V, and E427K) that show the same intermediate susceptibility profile for azole drugs have been described (14–18).
In these situations, a rapid detection method for mutations in cyp51A would prove to be a promising tool for the early, adequate treatment of aspergillosis and would be an appropriate epidemiological survey for azole-resistant A. fumigatus. Several methods have been developed to rapidly detect mutations in cyp51A in A. fumigatus. Some methods are based on the LightCycler instrument using a TaqMan assay or melting curve analysis (19–21), and a method using loop-mediated isothermal amplification has also been developed (22).
Surveyor nuclease (SN) (Integrated DNA Technologies, Inc.) is a member of a family of plant endonucleases that cleave heteroduplex DNA with high specificity at sites of base substitution mismatch and DNA distortion. The assay using the enzyme is used to confirm the presence or absence of mutation introduction in genome-editing experiments (TALE nuclease, CRISPR/Cas9, etc.) (23–25). In another study, the SN assay was used to screen for mutations in kidney-related genes. In that study, it was shown that the SN assay could detect a high percentage of DNA variations rapidly and at low cost (26).
In the current study, we show that the SN assay is useful for detecting mutations in cyp51A in A. fumigatus.
RESULTS
cyp51A mutation discrimination by SN assay.As a test sample, genomic DNA of A. fumigatus with a G54W mutation in cyp51A was used, and the SN treatment time was from 20 to 60 min (Fig. 1). Samples treated for 20 and 30 min could clearly confirm the cleavage fragments. Cleavage fragments treated for more than 40 min gradually became less clear. Based on this result, SN treatment was conducted for 30 min in the subsequent experiments. The results of the search for major mutations are shown in Fig. 2. The expected cleavage fragments were confirmed in G54, M220, G448S, and L98H samples. However, in the Y121F/T289A sample, an uncleaved fragment (704 plus 503 bp and 454 plus 503 bp) was also observed. In samples with 5 SNPs, a band pattern different from the others was detected. Samples on which colony direct PCR was performed (strains carrying cyp51A with a G54W [IFM 63345] or M220V [IFM64304] mutation) also provided the same results as the experiment using genomic DNA as a template (see Fig. S1 in the supplemental material).
Efficiency of cleavage by SN. The 1,661-bp amplicons were PCR amplified. The G54W mutant amplicon was annealed in combination with the reference amplicon (wild-type cyp51A). SN treatment was performed at 42°C for 20, 30, 40, 50, and 60 min. The cleavage products were 1,480 and 181 bp. The control was annealed alone with the reference amplicon.
Detection of major mutations by SN. Six major mutations were tested by SN assay. (a) Detection patterns of the G54 mutation. Lane 1, control; lane 2, G54W (cleavage fragments, 181 and 1,480 bp); lane 3, G54R (181 and 1,480 bp); lane 4 G54V (182 and 1,479 bp). (b) Detection patterns of the M220 mutation. Lane 5, control; lane 6, M220K (fragments, 751 and 910 bp); lane 7, M220I (752 and 909 bp); lane 8, M220V (750 and 911 bp). (c) Detection patterns of Y120F/T289A (lane 10; the fragments were 454, 503, and 704 bp), G448S (lane 11; 227 and 1,434 bp), and L98H (lane 12; 385 and 1,276 bp) mutations. In the Y120F/T289A lane, incomplete cleavage fragments (957 and 1,207 bp) can be observed. Lane 9, control. (d) Lane 14, 5SNP (Af293) detection pattern (many cleavage fragments were detected); lane 13, control.
Validation of the SN assay.Blind tests were conducted to verify the SN assay for the detection of cyp51A mutations. The test was performed on 17 azole-resistant and 31 azole-susceptible strains. All the resistant strains used in the test had mutations in cyp51A and could be distinguished from susceptible strains (see Table S1 in the supplemental material). Mutations were detected in 4 sensitive strains in which the sequence of cyp51A was not analyzed. Of these, 3 strains had N248K and 1 strain had M39I mutations.
DISCUSSION
The amino acid mutations in Cyp51A have been reported to be a major cause of the reduction of drug affinity for the molecule, which is the leading cause of azole resistance in A. fumigatus (3). It is also known that azole resistance shows different patterns correlated with the mutated amino acid sites (4–9). Therefore, rapid determination of the mutation pattern in the Cyp51A molecule should be useful for selecting the appropriate treatment for aspergillosis. For example, G54 and P216 mutant strains are susceptible to voriconazole (VRCZ), and TR34/L98H strains are multi-azole resistant (4–9). In addition, such a method would be suitable for performing epidemiological studies, such as evaluating the rate of azole-resistant A. fumigatus or extracting strains potentially having various or unknown mutations in cyp51A from among a large number of environmental or clinical isolates for screening.
In this study, we developed a novel method for detecting point mutations in the cyp51A gene using the SN assay. The assay could clearly distinguish wild-type cyp51A from point-mutated genes. The assay is able to reduce the time needed to determine the presence or absence of mutations. In addition, various mutations in the gene can be detected with only one primer set, and moreover, unknown mutations in the target region can also be detected. In fact, an unreported mutation, M39I, was detected in this study, although it is not related to azole resistance. Based on the above, the SN assay is suitable for primary screening analysis to extract isolates possessing point mutations from a large number of A. fumigatus strains.
Several methods have previously been developed for the detection of mutations that confer azole resistance (19–22). Most of them require different assays for specific mutations. On the other hand, the SN assay is characterized as being able to detect different mutations with only one primer set that amplifies the cyp51A gene region. In addition, mutations can be detected directly from a fungal colony because the SN assay can be performed when the target region of the sample is amplified using colony direct PCR. Therefore, the time required for extracting DNA can be shortened considerably. With the SN assay, sequence analysis by the Sanger method is necessary to determine the detailed mutation site. However, known mutations, such as G54, M220, and G448, can be assumed from the size of the cleaved fragment. On the other hand, many SNPs in the cyp51A gene that do not confer azole resistance have been reported (19). If these sites are close to mutations that cause azole resistance, it is difficult to differentiate restriction fragments in an electrophoresis gel. Recently, A. fumigatus strains having cyp51A with 5 amino acid mutations (F46Y, M172V, N248T, D255E, and E427K), termed 5SNPs, have been reported (14–18). The strains with cyp51A with 5SNPs usually have higher azole MICs. Since the 5SNPs have 3 silent mutations (G89G, L358L, and C454C), the gene encoding the 5SNPs has many mutations. In the SN assay, the 5SNPs can be identified by the pattern of characteristic cleavage fragments.
In conclusion, this is the first study using the SN assay for detection of cyp51A changes in A. fumigatus. The assay is a simple method by which mutations in the cyp51A gene of A. fumigatus can be rapidly detected with only one primer set. Using this method, the time required to obtain information regarding the mutations in azole-resistant strains can be shortened. The assay can be performed if one target gene of the specimen is amplified by PCR. Therefore, the SN assay is indeed a promising method that can be readily applied to, e.g., the detection of mutations that confer resistance in other fungal species. In future work, it will be necessary to develop technology to detect cyp51A mutations directly from clinical samples using the SN assay.
MATERIALS AND METHODS
Strains.Forty-eight characterized A. fumigatus clinical strains, A1163 (= IFM 53842), and Af293 (= IFM 54229) were provided through the National Bio-Resource Project (NBRP) Japan (http://www.nbrp.jp/). A1163 was used as a reference strain of Cyp51A (AFUB_063960), and Af293 was used as a strain having 5 SNPs of Cyp51A (AFUA_4G06890). Antifungal susceptibility testing had already been performed according to the Clinical and Laboratory Standards Institute reference broth microdilution method, document M38, with partial modifications (27). Briefly, tests were performed in triplicate with itraconazole (ITCZ) and VRCZ in RPMI 1640 medium (pH 7.0) at 35°C using a dried plate for antifungal susceptibility testing (Eiken Chemicals, Tokyo, Japan). In some strains, susceptibility test results were read on the 3rd day because of their slow growth. Among the 48 clinical strains, 17 were azole resistant and 31 were azole susceptible. The cyp51A sequences of all the azole-resistant strains had been analyzed previously, and all of them were confirmed to possess a mutation(s) in cyp51A (see Table S1). Also, five azole-susceptible strains had been analyzed previously, and no mutations were found in cyp51A.
DNA extraction.Genome DNA was extracted from overnight-cultured mycelia by the urea-phenol method. Mycelia were mixed with 0.5-mm glass beads, 0.5 ml phenol-chloroform-isoamyl alcohol (PCI), and 0.5 ml DNA extraction buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.3 M NaCl, 0.5% SDS, 5 M urea) and disrupted with a beads musher (FastPrep FP100A; MP-Biomedicals, Santa Ana, CA). After centrifugation, the upper layer was transferred to a new tube and subjected to ethanol precipitation. The resulting DNA pellet was suspended in 100 μl Tris-EDTA (TE) buffer.
Amplification of the cyp51A region.The cyp51A gene region was amplified from genome DNA by PCR using the primer set cyp51A_AF2P_F (5′-CCCTCCCTGTGTCTCCTCGAA-3′) and cyp51A_AF4P_R (5′-TCCTCGATGGTTACAACAGTCTCAC-3′). Amplification was carried out using KOD FX Neo (Toyobo Co., Ltd., Osaka, Japan). The amplification was carried out with an initial denaturation at 94°C for 2 min, followed by 30 cycles of 98°C for 10 s, 57°C for 30 s, and 68°C for 1 min, with a final extension at 68°C for 1 min. PCR products were purified with a Fast Gene gel/PCR extraction kit (Nippon Genetics Co., Ltd., Tokyo, Japan). The purification step was added in this study to determine the exact concentration of PCR amplicon. As a reference DNA, cyp51A of Fungal Genetics Stock Center (FGSC) strain A1163 (= IFM 53842) incorporated in the plasmid pUC119 was used. Colony direct PCR was performed using fungal cells cultured for 48 h at 37°C on a peptone-dextrose agar (PDA) plate. A few mycelia were added to the reaction mixture, and the cyp51A region was amplified with KOD FX Neo.
DNA hybridization.In a total volume of 10 μl, 250 ng each of the test and reference PCR amplicons, 1 μl 10× buffer (100 mM Tris-HCl, pH 8.0, 15 mM MgCl2, 500 mM KCl), and distilled water were mixed. As a control, 500 ng of the reference PCR amplicon was used. The mixture was heated to 95°C for 10 min for denaturing, followed by a slow reduction in temperature until a temperature of 25°C was reached. This reaction was performed using a thermal cycler (95°C for 10 min, 85°C for 90 s, 75°C for 90 s, 65°C for 90 s, 55°C for 90 s, 45°C for 90 s, 35°C for 90 s, and 25°C for 90 s). Then, the sample was stored at 4°C until the next step.
Treatment with SN.A Surveyor mutation detection kit was purchased from Integrated DNA Technologies (Coralville, IA, USA); 10 μl hybridized sample, 1 μl Surveyor nuclease S, 1 μl Surveyor enhancer S, and 1.3 μl 0.15 mM MgCl2 were mixed. The reaction mixtures were incubated at 42°C for 20, 30, 40, 50, or 60 min. After incubation, 1.5 μl of stop solution was added.
Analysis of DNA fragments.Analysis of DNA fragments was performed by agarose gel electrophoresis using 2% agarose and 1× Tris-acetate-EDTA (TAE) and then by staining with ethidium bromide.
Blind test for validation of the SN assay.A blind test was conducted with the 48 strains (see Table S1). Among the strains used, the cyp51A sequences of 5 susceptible strains and 17 resistant strains had been analyzed previously. The remaining 26 susceptible strains used were those whose cyp51A sequences had not been analyzed before the blind test. After determining the presence or absence of mutations by SN assay, the cyp51A sequences of these strains were analyzed.
Sequence analysis.The nucleotide sequences of DNA fragments were determined using BigDye Terminator v1.1/3.1 (Applied Biosystems, Tokyo, Japan) and an ABI automatic sequencer (PerkinElmer, Tokyo, Japan). The primers for DNA sequencing are shown in Table S2 in the supplemental material.
Data availability.All the data analyzed during this study are included here and in the supplemental material.
ACKNOWLEDGMENTS
This research was supported by AMED under grants no. JP19jm0110015, 19fm0208024, and JP19fk0108094.
We have no conflict of interest to declare.
FOOTNOTES
- Received 12 November 2019.
- Returned for modification 15 December 2019.
- Accepted 22 January 2020.
- Accepted manuscript posted online 3 February 2020.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
