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. 2016 Dec 27;61(1):e02101-16. doi: 10.1128/AAC.02101-16

Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance

Xiaolei Wei a, Peiying Chen a,b, Rongsui Gao a, Yeqi Li a, Anxue Zhang a, Feifei Liu a, Ling Lu a,✉
PMCID: PMC5192120  PMID: 27799210

ABSTRACT

The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance.

KEYWORDS: Aspergillus fumigatus, azole resistance, cox10, cyp51A, mutation

INTRODUCTION

The filamentous fungus Aspergillus fumigatus, one of the major opportunistic fungal pathogens, causes invasive fungal infections (IFIs) among immunosuppressed patients, with a high mortality rate (1, 2). Currently, azole compounds are the primary clinical therapy drug for IFIs and have been widely used in agriculture as well (3, 4). However, with the emergence of azole-resistant isolates, the efficacy of azole antifungals has been restricted so that azole resistance is becoming a growing public health menace (5–8). Therefore, the identification of genes whose mutations lead to drug resistance can provide a complete understanding of the molecular mechanisms underlying azole resistance.

Previous studies verified that the most common mechanisms of resistance to azole antifungals observed in A. fumigatus samples are due to Cyp51A mutations, which avoid drug-enzyme interactions (9, 10). To date, more than 30 different Cyp51A point mutations conferring azole resistance have been described in clinical isolates and laboratory-derived mutants (11–14). Moreover, some studies have indicated that there are many unexplored non-cyp51A mutations that contribute to azole resistance in A. fumigatus (15, 16). These findings indicate that many unclear molecular mechanisms related to azole resistance need to be explored further. Most recently, it was found that when fungal pathogens are exposed to antifungal drugs, cellular stress response pathways may be activated via drug stimulation (17). Among these pathways, the calcium signaling pathway is required for the long-term survival of cells undergoing plasma membrane and endoplasmic reticulum stresses induced by azole antifungal drugs (18). Previous studies identified some chemical inhibitors of calcium signaling, such as EGTA and FK506, that could be useful adjuncts for treatment with conventional azole antifungals against the Aspergilli (19). However, knowledge of calcium signaling components related to the azole drug stress response remains limited. In this study, we found that the loss of AlgA, a putative calcium signaling pathway component in A. fumigatus, results in an increased frequency of azole-resistant A. fumigatus isolates. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus, and algA defects make the drug-resistant alleles very easy to isolate. In addition, we identified that a new mutation, R243Q, in the A. fumigatus cox10 (Afcox10) gene (AFUB_065450) contributes to itraconazole resistance.

RESULTS

Loss of algA results in an increased frequency of azole-resistant isolates.

AlgA (AFUB_040560) is a putative homolog of human Alg2 (38% identity and 87% similarity) and Saccharomyces cerevisiae Pef1p (32% identity and 51% similarity) in A. fumigatus. To test whether the predicted AlgA protein participates in a variety of stress response processes, we first generated an algA deletion strain, referred as GRA01. As shown in Fig. S1 in the supplemental material, the ΔalgA mutant displayed colony phenotypes similar to those of the parental wild-type strain in normal minimal medium (MM) or in medium supplemented with selected reagents, including osmotic stressors and cell wall-disruptive reagents, suggesting that algA is not required for colony growth under the tested conditions. Moreover, when cultured in medium supplemented with itraconazole at 37°C for up to 4 days, there were no detectable differences in colony growth and MIC values between the algA deletion mutant and its parental wild-type strain (Fig. 1A to C). However, when cultured for a prolonged time of up to 6 days (Fig. 1D), the inoculated algA deletion strain displayed some visible colonies in medium supplemented with itraconazole antifungals. In contrast, under the same culture conditions with the same amount of inoculated conidia, there were no detectable growth colonies for the parental wild-type or complementation strains, indicating that the deletion of algA may cause an increased frequency of azole-resistant colonies. To verify this phenomenon, we further spread a large amount of conidia (5 × 106) from the parental wild-type and ΔalgA strains onto an above-mentioned media supplemented with itraconazole. After incubation for 6 days at 37°C, about 30 to 50 colonies were formed on the plate inoculated with the ΔalgA mutant, while only about 5 to 20 colonies were found on the plate inoculated with the parental wild-type strain, as shown in Fig. 1E. Thus, these results consistently suggest that the deletion of algA allows more isolated azole-resistant colonies to survive during treatment with the azole antifungal itraconazole.

FIG 1.

FIG 1

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Deletion of algA increases the frequency of azole-resistant isolates. (A) Series of 2-μl 10-fold dilutions of conidia (107 to 105) of parental wild-type strain A1160C (WT) and the ΔalgA mutant and ΔalgA-reconstituted (algAc) strains were spotted onto MM with different concentrations of itraconazole and cultured at 37°C for 2 days (without itraconazole) or 4 days (with itraconazole). (B) Susceptibilities of A1160C (WT) and the ΔalgA mutant to itraconazole determined by Etest. (C) Conidia were inoculated into a multiwell plate containing liquid MM supplemented with the indicated concentrations of itraconazole, and resazurin was used to generate clear-cut endpoints. (D) A total of 2 × 104 conidia of the parental wild-type strain A1160C or the ΔalgA mutant or ΔalgA-reconstituted strains were spotted onto MMPDR for 2 days (without itraconazole) or 6 days (with itraconazole). (E) A total of 5 × 106 conidia were plated onto solid MM with 4 μg/ml itraconazole and incubated at 37°C for 6 days, and quantitative data for the colonies are shown on the right. Error bars represent standard deviations from triplicate experiments (**, P < 0.01).

Independent stable azole-resistant isolates are obtained from the algA deletion strain.

The above-mentioned phenomena led us to speculate whether these itraconazole-resistant isolates belong to the independent and stable mutants. To test our hypothesis, in vitro screening of itraconazole-resistant strains was performed. A total of 105 isolated resistance-stable colonies were obtained from 40 culture plates, and 672 isolated colonies gradually went extinct during the screening process. As shown in Fig. 2, there were varied and independent colony phenotypes on both drug-free and itraconazole-containing media among these isolates. Notably, the majority of isolated colonies had defects in hyphal growth and conidiation.

FIG 2.

FIG 2

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One hundred five itraconazole-resistant isolates classified into nine groups in different color zones based on the colony phenotype on medium with 4 μg/ml itraconazole (for 4 days) or without itraconazole (for 2 days).

A total of 105 isolated itraconazole-resistant colonies were roughly classified into nine groups by different colony phenotypes (colony diameters) on both drug-free and drug-containing (4 μg/ml itraconazole) media, as shown in Fig. 2. However, it is possible that all of the isolates belonged to the independent ones. A clinic-derived reference strain, A1160, was used as a phenotypic control; while A1160 is an itraconazole-susceptible strain, it was unable to form any detectable colony in the drug-containing (4 μg/ml itraconazole) medium. To validate the drug resistance ability of the above-mentioned isolates, we then selected 11 independent resistant representatives (W004, W009, W010, W048, W117, W322, W377, W416, W513, W601, and W770), which were distributed in the different groups and showed different colony phenotypes on both drug-free and drug-containing (4 μg/ml itraconazole) media. For these selected strains, testing of susceptibility to itraconazole was performed according to CLSI (Clinical and Laboratory Standards Institute) document M38-A2 standards. Therefore, we assumed that all selected isolates, the MICs of which were higher than the ECV (epidemiological cutoff value, 1 μg/ml for itraconazole), defined a scope of itraconazole-resistant strains, as shown in Table 1 (20–22). Notably, isolates W377 and W513 showed the highest degree of resistance to itraconazole (MIC = 16 μg/ml). In addition, the majority of selected strains showed phenotypes of cross-resistance to the other antifungals voriconazole, bifonazole, and terbinafine (except W601), but all isolates displayed the same sensitivity as that of the parent strain to caspofungin when spotted onto solid YAG medium (see Materials and Methods) containing the tested antifungals (Fig. 3). To further test whether these isolates were cross-resistant to antifungals, we used a standard broth-based microdilution method to assess responses to azoles, polyenes, and echinocandins. The overall MICs of voriconazole and terbinafine and the minimal effective concentrations (MECs) of caspofungin for 11 isolates are listed in Table 1. Consequently, four isolates (W004, W009, W048, and W377) also showed resistance to voriconazole. Caspofungin MECs were variable and exhibited a wide range among the selected strains. Due to a lack of ECVs for terbinafine and caspofungin in A. fumigatus, cross-resistance to these two antifungals in the tested strains was not identified. Interestingly, as shown in Table 1, all tested isolates had their own antifungal resistance profiles, suggesting that evolved lineages (isolates) may acquire azole resistance independently.

TABLE 1.

MICs or MECs of itraconazole, voriconazole, terbinafine, and caspofungin for selected isolatesa

Strain MIC or MEC (μg/ml)
ITC VRC Terb CAS
A1160 1 0.5 8 0.25
W004 8 2 16 0.25
W009 8 2 16 0.06
W010 4 1 >16 >4
W048 8 2 >16 >4
W117 8 1 8 0.25
W322 4 1 16 0.06
W377 16 2 8 >4
W416 8 1 16 0.125
W513 16 1 16 0.125
W601 8 0.5 8 0.25
W770 4 1 16 0.25
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a

ITC, itraconazole; VRC, voriconazole; Terb, terbinafine; CAS, caspofungin.

FIG 3.

FIG 3

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Testing of cross-resistance and sensitivity to voriconazole (VRC), bifonazole (BFZ), terbinafine (TRB), and caspofungin (CAS) in selected strains. The concentration of itraconazole (ITC) was 16 μg/ml, that of voriconazole was 1 μg/ml, that of bifonazole was 2 μg/ml, that of terbinafine was 0.5 μg/ml, and that of caspofungin was 1 μg/ml.

Identification of mutated genes conferring azole resistance.

To gain insight into the frequency of the Afcyp51A mutation in the above-mentioned 105 itraconazole-resistant isolates, we cloned the Afcyp51A gene from all 105 isolates. After sequencing the entire Afcyp51A gene, including its promoter region, to our surprise, 104 isolates had no mutation in the open reading frame (ORF) or the promoter region of Afcyp51A except for one isolate, referred to as W601, which harbors a single-base change (T726C) that leads to an F219L mutation of AfCyp51A. These data suggest that >99% of itraconazole-resistant isolates retain the wild-type cyp51A gene, indicating that they may have non-cyp51A mutations or other unknown itraconazole-resistant mechanisms in the algA deletion background strain.

To unravel the potential mutations underlying azole resistance, we selected one resistant mutant, W601, which had a cyp51A mutation, and another representative mutant, W004, which contains a wild-type cyp51A gene that likely conferred non-cyp51A-dependent azole resistance. As shown in Fig. 4A and C, the two stable azole-resistant isolates W601 and W004 showed remarkably different colony phenotypes and sensitivities to various antifungal agents. Consistently, MIC assays by the Etest showed that both isolates were resistant to itraconazole, even when the concentration of itraconazole reached 32 μg/ml (Fig. 4B). To identify the mutated gene contributing to azole resistance, next-generation sequencing (NGS) was carried out on isolates W004 and W601. After a BLAST analysis of the whole genomic sequence assembly compared to A1163 genome information, ∼400 single nucleotide polymorphisms (SNPs) were detected in the coding region in both isolates W004 and W601 (see Table S2 in the supplemental material), but most of these SNPs were the same in isolates W004 and W601. Because the two isolates showed entirely different phenotypes and were derived from the same parental strain, we hypothesized that W004 and W601 would have unique mutation sites affecting itraconazole resistance. Therefore, we removed the common SNPs and synonymous mutations shared by both isolates, and we then found that only one unique SNP each was left for W004 and W601 (see Table S3 in the supplemental material). Thus, we successfully narrowed our targets to two individual genes harboring the unique SNP. Consequently, the G728A mutation leading to an amino acid change from arginine (R) to glutamine (Q) at codon 243 in the putative AFUB_065450 protein was found in W004. Based on NCBI information, AFUB_065450 is an uncharacterized gene in A. fumigatus, and this point mutation was located in the predicted active domain of the protein product. However, the ortholog of AFUB_065450 in yeast, cox10, has been identified as a participant in heme biosynthetic processes by catalyzing protoheme (heme B) to heme O via farnesylation (23, 24). Thus, we refer to AFUB_065450 as Afcox10. In the W601 mutant, a T726C nucleotide change at codon 219 that would be responsible for a phenylalanine (F)-to-leucine (L) substitution in Afcyp51A was found. This unique SNP was consistent with our above-described sequence analysis of the cloned Afcyp51A gene from W601. Thus, these data suggest that the R243Q mutation of AfCox10 in W004 and the F219L mutation of AfCyp51A in W601 possibly contribute to itraconazole resistance. These data also suggest that our above-mentioned sequence analysis data for the cloned Afcyp51A gene from all resistance isolates are accurate, indicating that our isolated resistant strains, except for W601, are likely associated with non-cyp51A mutation-mediated itraconazole resistance.

FIG 4.

FIG 4

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Colony phenotypes and sensitivities to various antifungal agents for strains W601 and W004. (A) A series of 2-μl 10-fold dilutions of conidia (107 to 105) of W004, W601, and the parental wild-type strain A1160C (WT) were spotted onto YAG medium containing antifungals (at the concentrations described in the legend Fig. 3). (B) Susceptibilities of A1160C (WT), W004, and W601 to itraconazole determined by Etest. (C) Progenies (from F1 to F11 generations) of the W004 or W601 strain, which had been passaged serially on drug-free medium, were spotted back onto itraconazole-containing medium (with itraconazole at 16 μg/ml).

Point mutations in Afcyp51A and Afcox10 confer azole resistance in an algA-independent manner.

To further confirm whether these two missense mutations analyzed by next-generation sequencing could confer resistance to itraconazole, the ORF regions of Afcox10 and Afcyp51A were amplified from W004 and W601, respectively. Sequence analysis showed that the mutations in Afcox10 and Afcyp51A were consistent with those detected by next-generation sequencing. Moreover, to verify whether the itraconazole-resistant phenotypes in W004 and W601 were dependent on the deletion of algA, mutation strains WX01 (Afcox10MtR), for the R243Q mutation in AfCox10, and WX03 (Afcyp51AMtR), for the F219L mutation in AfCyp51A, were re-created by reverse-genetics approaches in a wild-type A1160 background. As expected, a phenotype of hyperresistance to itraconazole was also present in our reconstructed strains WX01 and WX03 (Fig. 5). These data collectively indicate that either the R243Q mutation in AfCox10 or the F219L mutation in AfCyp51A was able to induce azole resistance in an algA-independent manner. In addition, Afcox10MtR displayed phenotypes of cross-resistance to voriconazole and terbinafine similar to that of W004, suggesting that AfCox10R243Q is able to confer multidrug resistance in A. fumigatus.

FIG 5.

FIG 5

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Itraconazole drug resistance induced by a point mutation in Afcox10 and Afcyp51A in an algA-independent manner. Series of 2-μl 10-fold dilutions of conidia (107 to 105) of AfCox10R243Q (W004 [Afcox10MtF] and WX01 [Afcox10MtR]), AfCyp51AF219L (W601 [Afcyp51AMtF] and WX03 [Afcyp51AMtR]) and their parental wild-type strains (ΔalgA [W004 and W601 parental strain ΔalgA mutant] and the wild type [A1160C]) were spotted onto YAG medium containing antifungals.

We then wondered whether the AfCox10 orthologs in fungi are functionally conserved. Three putative orthologs from different fungal species were selected to test functional complementation: Aspergillus nidulans, a model fungus for the Aspergilli, and Candida albicans and Cryptococcus neoformans, two leading human pathogens. As shown in Fig. S2 in the supplemental material, all colonies transformed by ectopically expressed Afcox10 or its orthologs from A. nidulans, referred to as WX04 and WX05, almost completely rescued the colony growth defects in W004, accompanied by normal susceptibility to itraconazole, compared to the parental strain. In contrast, the colonies transformed by ectopically expressed Afcox10 orthologs from C. albicans and C. neoformans, referred to as WX06 and WX07, showed no significant difference from the recipient strain W004. These findings suggest that Cox10 orthologs in Aspergilli are conserved and that A. nidulans Cox10 (AnCox10) virtually functionally substitutes for AfCox10 in growth and itraconazole susceptibility. Notably, as shown in Fig. S2 in the supplemental material, Afcox10 was able to completely rescue the growth defects in W004 under the control of the AngpdA promoter, suggesting that phenotypic defects of W004 are due to Afcox10 dysfunction. The slight phenotypic difference between WX01 (Afcox10MtR) and W004 (Afcox10MtF) may be attributed to the different genetic background.

Validation of the azole resistance mechanism in Afcox10 and Afcyp51A mutants.

Previous studies have verified that azoles are capable of interfering with the activity of 14-α-lanosterol demethylase to inhibit ergosterol biosynthesis (25). To further analyze the resistance mechanism of two mutations, AfCox10R243Q and AfCyp51AF219L, we tested and compared the contents of ergosterol and the sterol intermediates in itraconazole-resistant isolates and the parental wild-type strain. Total sterol was extracted as previously described and measured via high-performance liquid chromatography (HPLC). Unexpectedly, there was no significant difference in total ergosterol levels between the mutant (WX01 or WX03) and the parental wild-type strain (see Fig. S3 in the supplemental material). In addition, no extra sterol derivatives were found in WX01 and WX03 compared to the parental wild-type strain. These data indicate that there may be other mechanisms underlying azole resistance in these mutants.

Another well-known azole resistance mechanism is the overexpression of efflux pumps, which may cause drug resistance by reducing cellular drug accumulation by pumping the drugs out of the cell (26). We therefore examined the relationship between the efflux transporters and the itraconazole resistance of Afcox10-related mutants and Afcyp51A-related mutants. Rhodamine 6G (R6G) (a fluorescent compound) is used as a mimicking substrate of ATP-binding cassette (ABC) transporters (27). As shown in Fig. 6A, significantly greater absorption of R6G was observed in the parental wild-type strain than in the Afcox10-related mutant strains W004 and WX01. The relative mean fluorescence intensity of R6G that accumulated in the parental wild-type strain was almost 10-fold higher than that in the Afcox10-related mutants. Moreover, no significant difference in R6G uptake was detected in the Afcyp51A-related mutant isolates W601 and WX03 compared to their parental wild-type strains (Fig. 6B). In comparison, the results for the activity of ATP-driven efflux pumps among all tested strains in Fig. 6C and D show that no significant difference in R6G efflux was observed after the addition of glucose. These data suggest that the AfCox10R243Q mutants (W004 and WX01) had a decrease in R6G absorption, but the ability to efflux R6G was not significantly different from that of the parent strain.

FIG 6.

FIG 6

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Flow cytometry analysis showing decreased R6G absorption in the AfCox10G243Q mutant. The uptake (A and B) and efflux (C and D) abilities of R6G were assessed in AfCox10G243Q mutants (W004 [Afcox10MtF] and WX01 [Afcox10MtR]), AfCyp51AF219L mutants (W601 [Afcyp51AMtF] and WX03 [Afcyp51AMtR]), and parental wild-type strain A1160C (WT). FL1-A at the x axis represents the relative fluorescence intensity value. The fluorescence intensity was normalized to that of the parental strain under the same conditions. Error bars represent standard deviations from triplicate experiments (**, P < 0.01).

To further directly detect the intracellular retention of itraconazole in Afcox10-related mutants compared to the parental wild-type strain, conidia were inoculated in liquid medium supplemented with itraconazole for 1 h, and the content of itraconazole extracted from the mycelia was then detected by HPLC. As shown in Fig. 7, Afcox10 mutant isolates W004 and WX01 had a remarkably and significantly low (almost 2.5-fold lower) intracellular retention of itraconazole compared to that of the parental wild-type strain. These results suggest that the decreased absorption of itraconazole in the Afcox10 mutant isolates W004 and WX01 was likely the main reason for their itraconazole resistance.

FIG 7.

FIG 7

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HPLC chromatograms showing the decreased intracellular retention of itraconazole in the AfCox10G243Q mutant. (A and B) Samples were extracted from parental wild-type strain A1160C (WT) and the W004 (Afcox10MtF) and WX01 (Afcox10MtR) strains treated with 1 μg/ml itraconazole (A) or 4 μg/ml itraconazole (B) for 1 h compared with the commercial itraconazole (ITC) standard. (C) Normalized quantification and comparison of the itraconazole contents in mutants and the parental wild-type strain under the same cultural conditions. Error bars represent standard deviations from triplicate experiments (**, P < 0.01). The single peak at the ∼4-min retention time marked as I represents itraconazole.

DISCUSSION

Scientists and clinicians have found that there are complicated and varied mechanisms underlying azole drug resistance in A. fumigatus (28). The most prevalent resistance comes from the mutational modification of the azole target protein Cyp51A (29). Therefore, over the last few decades, most investigators have focused their attention on cyp51A-mediated azole resistance and largely neglected other resistance mechanisms. It is noteworthy that the genomic sequence survey analysis for cyp51A in azole-resistant isolates collected from surveillance sites (Manchester, United Kingdom) in 2008 and 2009 revealed that 43% of the tested A. fumigatus isolates harbored the sequence of the wild-type cyp51A gene (12). Similarly, another survey in Germany in 2013 reported that 47.1% of resistant isolates had the normal wild-type cyp51A gene sequence without any mutation (30). Thus, repeated evidence of non-cyp51A mutations conferring azole resistance led us to believe that these unknown resistance mechanisms need to be explored.

Previous studies have shown that fungal pathogens such as A. fumigatus and C. albicans are able to respond to drug-induced stress by modifying intracellular signaling pathways (31, 32). In this study, we found that the deletion of algA, a putative calcium signaling pathway member in A. fumigatus, resulted in a colony phenotype similar to that of the parental wild-type strain under calcium-limited conditions or other tested stress treatments. However, the loss of algA significantly increased the frequency of azole resistance compared to those of its parental wild-type and reconstituted strains. Furthermore, some isolated resistant colonies showed stable drug resistance and may belong to independent resistance isolates. In contrast to previously identified azole resistance isolates related to the cyp51A mutation in A. fumigatus, only one resistance isolate carried a point mutation in cyp51A in the background of the algA deletion strain. This phenomenon indicates that non-cyp51A mutation-mediated azole resistance mechanisms play a dominant role in adapting the azole environment in the algA deletion strain. Through next-generation sequencing, we successfully identified the mutation sites in two selected isolates. Moreover, the reconstitutive strains harboring the mutation in the background of a wild-type strain, A1160, confirmed that mutations AfCox10R243Q and AfCyp51AF219L contribute to itraconazole resistance in an algA-independent manner. These data suggest that the loss of algA results in an increased frequency of azole-resistant isolates rather than directly affecting drug resistance. These resistance isolates had their own antifungal resistance profiles, indicating that these mutations may have originally existed in the A. fumigatus strain and also suggesting that evolved lineages (isolates) may acquire azole resistance independently, while algA defects made the drug-resistant alleles much easier to isolate. Since the algA homolog in humans has a role in apoptotic cell death, this suggests that the loss of algA in fungal cells may allow them to more easily escape from cell apoptosis induced by itraconazole and result in survival mutants conferring drug resistance (33). In addition, of 11 selected independent itraconazole-resistant representatives, we found that they all had various MEC values of caspofungin, suggesting that the cell wall synthesis-related drug target of caspofungin in some isolates may be changed. However, the underlying mechanism of this phenomenon is not clear.

In addition, more lines of evidence combined with our data collectively indicate the existence of non-cyp51A mutation-mediated azole resistance (16). Through next-generation sequencing and reverse-genetics approaches, two point mutations were found to confer azole resistance in our isolated strains. One of these mutations was an F219L mutation in Cyp51A. F219 substitutions have already been shown to be associated with azole resistance in resistant clinical strains (34). Another R243Q substitution mutation in AfCox10 (AFUB_065450) conferring azole resistance has not yet been reported in A. fumigatus or in any fungal pathogens. Putative AfCox10 orthologs in S. cerevisiae are nucleus-encoded accessory proteins needed for cytochrome c oxidase (COX) assembly. Cox10 is a farnesyltransferase that catalyzes the conversion of protoheme to heme O in the biosynthesis of heme A. Heme A is required to maintain the stability and prevent the degradation of MTCO1, which forms both the catalytic and the structural cores of COX (35, 36). In addition, missense mutations of Cox10 in human and mouse are associated with mitochondrial disorders (37–40). Our data suggest that an amino acid substitution, R243Q, in AfCox10 causes azole resistance in A. fumigatus. Nevertheless, further studies are required to determine whether this mutation is related to the clinical itraconazole resistance isolates of A. fumigatus. Moreover, a homolog complementation experiment demonstrated that a Cox10 ortholog in A. nidulans almost completely rescued the phenotype of the Afcox10 mutant in terms of growth and itraconazole susceptibility, suggesting that Cox10 of the Aspergilli possesses common functions in hyphal growth and itraconazole resistance.

Regarding the resistance mechanism in the AfCox10R243Q mutant, the measurements of intracellular R6G and itraconazole levels indicated that azole resistance is likely due to the reduced intracellular retention of itraconazole. Given that AfCox10R243Q mutants are cross-resistant to voriconazole, bifonazole, and terbinafine, we hypothesize that the R243Q mutation of AfCox10 may influence the activity of COX due to the lack of heme A. In addition, COX defects further lead to mitochondrial disorders and thus may explain why the AfCox10R243Q mutants displayed very severe growth defects compared to the parental wild-type strain. However, the specific relationship between AfCox10 and drug resistance has to be addressed in further detailed investigations.

Taken together, our study indicates that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus. Moreover, our data suggest that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to find unique SNPs contributing to drug resistance.

MATERIALS AND METHODS

Strains, media, and cultural conditions.

All A. fumigatus strains used in this study are listed in Table 2 and were grown on the following media, as previously described (41, 42). Briefly, YAG medium contains 2% glucose, 0.5% yeast extract, 2% agar, and trace elements. For YG liquid medium, agar is omitted from YAG medium. YUU medium contains YAG plus 5 mM uridine and 10 mM uracil. MM (minimal medium) includes 1% glucose, trace elements, and 50 ml/liter of a salt solution (pH 6.5). MMPDR medium represents MM plus 0.5 mg/liter pyridoxine and 2.5 mg/liter riboflavin (pH 6.5). RPMI 1640 medium is composed of 10.4 g/liter RPMI 1640 (catalog no. 31800-014; Invitrogen Gibco) and 34.53 g/liter MOPS [3-(N-morpholino)propanesulfonic acid]. All strains were cultured at 37°C or 35°C (according to M38-A2 guidelines).

TABLE 2.

A. fumigatus strains used in this studya

Strain Genotype Source or reference
A1160 Δku80 pyrG1 FGSC
A1160C Δku80 A1160::pyrG1 53
ATCC MYA-3626 NA ATCC
GRA01 Δku80 pyrG1 ΔalgA::pyr4 This study
GRA02 Δku80 pyrG1 ΔalgA::pyr4 algA(p)::algA::hph This study
WX01 Δku80 pyrG1 cox10(p)::cox10R243Q::pyr4 This study
WX02 Δku80 pyrG1 Δcyp51A::pyr4 This study
WX03 Δku80 pyrG1 Δcyp51A::pyr4 cyp51A(p)::cyp51AF219L::hph This study
WX04 Δku80 pyrG1 ΔalgA::pyr4 cox10R243Q gpdA(p)::Afcox10::hph This study
WX05 Δku80 pyrG1 ΔalgA::pyr4 cox10R243Q gpdA(p)::Ancox10::hph This study
WX06 Δku80 pyrG1 ΔalgA::pyr4 cox10R243Q gpdA(p)::Cacox10::hph This study
WX07 Δku80 pyrG1 ΔalgA::pyr4 cox10R243Q gpdA(p)::Cncox10::hph This study
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a

FGSC, Fungal Genetics Stock Center (http://www.fgsc.net/); NA, not applicable; ATCC, American Type Culture Collection.

Construction of mutant strains and cyp51A sequence analysis.

To construct the ΔalgA mutant (GRA01), the Neurospora crassa pyr4 gene, as a selectable nutritional marker, was amplified from plasmid pLB01 with the primers pyr4-up and pyr4-down. The 5′-flanking and 3′-flanking fragments of the algA gene were amplified from A. fumigatus A1160 genomic DNA by using primers algA-P1/algA-P3 and algA-P4/algA-P6, respectively. These three PCR products were then fused with the nested primer pair algA-P2/algA-P5. The fusion PCR product was transformed into the recipient strain A1160 to generate the ΔalgA mutant. The transformants were confirmed by diagnostic PCR with primers algA-s-up/algA-s-down and algA-P1/algA-P5. The algA-reconstituted strain GRA02 was generated as follows: the algA gene was amplified from A1160 genomic DNA with primers algA-F/algA-R and then cloned into the pEASY-Blunt cloning vector (TransGen Biotech) to generate plasmid G01. The hygromycin resistance gene amplified from plasmid pAN7-1 with primers hyg-up-SpeI and hyg-down-SpeI was then subcloned into plasmid G01, creating plasmid G02. Plasmid G02 was transformed with GRA01 to generate GRA02. The transformants were confirmed by PCR with primers algA-F and algA-R.

To construct strain WX01 (cox10MtR), the 5′-flanking region and ORF of cox10R243Q from strain W004 were amplified with primers cox10-P1 and cox10-P3. The 3′-flanking region of the cox10R243Q gene was amplified from strain W004 with primer pair cox10-P4/cox10-P6, and a pyr4 selectable marker was amplified as described above. The three PCR products were fused with the nested primer pair cox10-P2/cox10-P5 and then transformed into recipient strain A1160 to generate mutant strain WX01 (cox10MtR).

For the Δcyp51A mutant (WX02), the 5′-flanking and 3′-flanking fragments of the cyp51A gene were amplified from A. fumigatus A1160 genomic DNA by using the primers cyp51A-P1/cyp51A-P3 and cyp51A-P4/cyp51A-R. The N. crassa pyr4 selectable marker was amplified as described above. These three PCR products were then fused with the nested primer pair cyp51A-P2/cyp51A-P5. The fusion PCR product was transformed into receipt strain A1160 to generate the Δcyp51A mutant. The transformants were confirmed by diagnostic PCR with primers cyp51A-s-up/cyp51A-s-down, cyp51A-P1/pyr4-R, and cyp51A-R/pyr4-F. To construct WX03 (cyp51AMtR), the 5′-flanking region, ORF, and 3′-flanking region of W601 (cyp51AMtF) were amplified from strain W601 with primer pair cyp51A-p2/cyp51A-P5. This fragment was cloned into the pEASY-Blunt cloning vector to generate plasmid W1. The hygromycin resistance gene was amplified from plasmid pAN7-1 with primers hyg-up-SpeI and hyg-down-SpeI. This fragment and plasmid W1 digested by SpeI were ligated, yielding plasmid W2. Plasmid W2 was further transformed into strain WX02 to obtain WX03 (cyp51AMtR) on selected medium containing 200 μg/ml hygromycin. All constructed strains were confirmed by diagnostic PCR and sequencing.

For the construction of Afcox10 and its putative orthologs from Aspergillus nidulans, Candida albicans, and Cryptococcus neoformans under the control of the gpdA promoter, the ORF of each ortholog was amplified from the corresponding cDNA with the primers Afcox10-s-up/Afcox10-s-down for Afcox10, Ancox10-s-up/Ancox10-s-down for Ancox10, Cacox10-s-up/Cacox10-s-down for C. albicans cox10 (Cacox10), and Cncox10-s-up/Cncox10-s-down for C. neoformans cox10 (Cncox10). Using the One Step cloning kit, these fragments were cloned into reconstituted plasmid pBARGPE, which contains a gpdA promoter, a trpC terminator, and a hygromycin resistance gene, to generate plasmids W3, W4, W5, and W6 (43). These four plasmids were transformed into mutant strain W004 to construct strains WX04, WX05, WX06, and WX07, respectively.

The cyp51A coding region and its promoter were amplified by PCR with primers cyp51A-F and cyp51A-R. Each fragment was sequenced by using cyp51A-F. Primers used in this study are listed in Table S1 in the supplemental material.

In vitro screening of itraconazole-resistant strains.

First, conidia of parental wild-type strain A1160C or the ΔalgA mutant or ΔalgA-reconstituted strains were spotted or spread onto medium supplemented with 4 μg/ml itraconazole antifungals for 6 days. Subsequently, 5 million freshly harvested spores from the ΔalgA mutant were spread with 40 replicas to isolate drug-resistant strains. The surviving isolates from culture plates were serially streaked and cultured on itraconazole-containing medium (4 or 16 μg/ml) continuously for four generations with an incubation period of 6 days at 37°C. The surviving strains on the final-step plate amended with 16 μg/ml itraconazole were kept for further studies.

Antifungal susceptibility testing.

Testing for susceptibility to itraconazole was carried out by the Etest and broth-based microdilution methods. The Etest was performed in accordance with the manufacturer's instructions, as described previously (44). The broth-based microdilution method for susceptibility testing was performed according to CLSI document M38-A2 guidelines (45). Briefly, conidial suspensions were diluted in RPMI 1640 liquid medium and then transferred into a multiwell plate containing itraconazole dilutions. Plates were incubated at 35°C for 48 h. The MIC was defined visually as the lowest concentration that prevents any discernible growth. A. fumigatus ATCC MYA-3626 and A1160, as reference strains, were included in related tests as references for quality controls.

Cross-resistance testing.

All tested antifungal drugs were purchased from Sigma, except that voriconazole was obtained from Shanghai Yuanye Bio-Technology. Cross-resistance testing was performed with assays using agar plates and the broth-based microdilution method. For assays using agar plates, each drug stock solution was mixed with melted YAG medium and then poured into plates. Freshly harvested spores were spotted onto antifungal-drug-containing medium, and the inoculated plate was then cultured at 37°C for observation. The broth-based microdilution method for the cross-resistance assay was performed according to CLSI document M38-A2 protocols (45). The MICs were defined as described above in the section on antifungal susceptibility testing. For caspofungin, the MEC was used instead of the MIC. The MEC is the lowest concentration of caspofungin that led to the growth of small, rounded, compact hyphal forms compared to the hyphal growth seen in the growth control wells (46).

Next-generation sequencing.

Conidial spores from A. fumigatus strains W004 and W601 were inoculated into liquid medium and shaken for 18 h at 37°C at 200 rpm, and the harvested mycelia were then dried and extracted to obtain genome DNA. The NGS experiment was performed at Shanghai OE Biotechnology Co., Ltd., as a commercial service. Strains W004 and W601 had been sequenced in separate lanes by using the Illumina HiSeq 2000 platform with the PE100 method (100-bp paired-end reads in a high-output mode). 5 GB raw data (raw bases) were generated from each run, and an average 140× depth of each nucleotide (genome size of about 30 Mb) was gained. Sequence assembly and mapping were based on the A. fumigatus A1163 genome (GenBank assembly accession no. GCA_000150145.1 [http://www.ncbi.nlm.nih.gov/assembly/GCA_000150145.1]). Analysis of mapping quality and SNPs was carried out by using SHORE software, which is a next-generation sequencing data analysis suite.

Total sterol extraction.

Total sterol in the A. fumigatus strains was extracted as described previously, with slight modifications (47–49). Briefly, mycelia were harvested by filtration, and 200 mg of dry mycelia was then treated with 3 ml of a 25% alcohol potassium hydroxide solution (3:2 dilution of methanol-ethanol) and mixed by vortexing for 1 min. After incubation at 85°C for 1 h, the samples were treated with 1 ml of distilled water and 3 ml of hexane and vortexed for 3 min. The upper hexane layer was transferred to another tube and evaporated in a fume hood at room temperature. Before analysis, the dried samples were dissolved in 1 ml of methanol and syringe filtered through 0.2-μm-pore-size filters. Total sterol was analyzed by using HPLC (Agilent Technologies) and detected at 280 nm on an AQ-C18 column (250 mm by 4.6 mm; 5 μm).

Measurement of R6G uptake and glucose-induced efflux abilities.

The intracellular concentration of R6G (Sigma) in A. fumigatus was evaluated as described previously, with slight modifications (27, 50). Approximately 5 × 106 fresh conidia were grown in YG medium at 37°C until conidia began to germinate. The samples in culture dishes were washed and then suspended in phosphate-buffered saline (PBS), to which R6G was added at a final concentration of 10 μM, and the mixtures were incubated at 37°C for 1 h. The samples were then washed with PBS, and the fluorescence of R6G in samples was measured by fluorescence-activated cell sorter (FACS) analysis (Accuri C6; BD). To assess energy-dependent R6G efflux, the samples were suspended in PBS containing 1 M glucose and incubated for 0.5 h at 37°C. After the treated conidia had been washed twice with PBS, the fluorescence intensity of samples was measured by FACS analysis.

Cellular itraconazole extraction.

Total cellular itraconazole in the tested A. fumigatus strains was extracted as previously described (51, 52). Briefly, 107 conidia were inoculated into 100 ml of liquid medium (MM) and cultured at 37°C until they grew into mycelia, and itraconazole was then added to the medium at a final concentration of 1 or 4 μg/ml. After incubation for 1 h, the mycelia were washed with distilled water and then harvested via vacuum filtration, lyophilized, and weighed. Thirty milligrams of dry fungal mycelia was homogenized, mixed with ceramic beads in 2 ml of methanol, and then centrifuged at 16,000 × g. The supernatant from the cellular itraconazole extraction step was transferred to another tube for analysis by HPLC. Commercial itraconazole was used as a standard.

Supplementary Material

Supplemental material
supp_61_1_e02101-16__index.html (885B, html)

ACKNOWLEDGMENTS

This work was financially supported by grants from the National Natural Science Foundation of China (NSFC81330035) to L.L., from the National Natural Science Foundation of China (NSFC31500122) to P.C., and from the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and the Research.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02101-16.

REFERENCES

  • 1.Shao PL, Huang LM, Hsueh PR. 2007. Recent advances and challenges in the treatment of invasive fungal infections. Int J Antimicrob Agents 30:1–14. doi: 10.1016/j.ijantimicag.2007.07.019. [DOI] [PubMed] [Google Scholar]
  • 2.van der Linden JW, Snelders E, Kampinga GA, Rijnders BJ, Mattsson E, Debets-Ossenkopp YJ, Kuijper EJ, Van Tiel FH, Melchers WJ, Verweij PE. 2011. Clinical implications of azole resistance in Aspergillus fumigatus, The Netherlands, 2007-2009. Emerg Infect Dis 17:1846–1854. doi: 10.3201/eid1710.110226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Verweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ. 2009. Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use? Lancet Infect Dis 9:789–795. doi: 10.1016/S1473-3099(09)70265-8. [DOI] [PubMed] [Google Scholar]
  • 4.Zavrel M, White TC. 2015. Medically important fungi respond to azole drugs: an update. Future Microbiol 10:1355–1373. doi: 10.2217/FMB.15.47. [DOI] [PubMed] [Google Scholar]
  • 5.Howard SJ, Cerar D, Anderson MJ, Albarrag A, Fisher MC, Pasqualotto AC, Laverdiere M, Arendrup MC, Perlin DS, Denning DW. 2009. Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis 15:1068–1076. doi: 10.3201/eid1507.090043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Denning DW, Perlin DS. 2011. Azole resistance in Aspergillus: a growing public health menace. Future Microbiol 6:1229–1232. doi: 10.2217/fmb.11.118. [DOI] [PubMed] [Google Scholar]
  • 7.Chowdhary A, Kathuria S, Xu J, Meis JF. 2013. Emergence of azole-resistant Aspergillus fumigatus strains due to agricultural azole use creates an increasing threat to human health. PLoS Pathog 9:e1003633. doi: 10.1371/journal.ppat.1003633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Bader O, Tunnermann J, Dudakova A, Tangwattanachuleeporn M, Weig M, Gross U. 2015. Environmental isolates of azole-resistant Aspergillus fumigatus in Germany. Antimicrob Agents Chemother 59:4356–4359. doi: 10.1128/AAC.00100-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Snelders E, Karawajczyk A, Schaftenaar G, Verweij PE, Melchers WJ. 2010. Azole resistance profile of amino acid changes in Aspergillus fumigatus CYP51A based on protein homology modeling. Antimicrob Agents Chemother 54:2425–2430. doi: 10.1128/AAC.01599-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Becher R, Wirsel SG. 2012. Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) and azole resistance in plant and human pathogens. Appl Microbiol Biotechnol 95:825–840. doi: 10.1007/s00253-012-4195-9. [DOI] [PubMed] [Google Scholar]
  • 11.Cowen LE, Sanglard D, Howard SJ, Rogers PD, Perlin DS. 2015. Mechanisms of antifungal drug resistance. Cold Spring Harb Perspect Med 5:a019752. doi: 10.1101/cshperspect.a019752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Bowyer P, Moore CB, Rautemaa R, Denning DW, Richardson MD. 2011. Azole antifungal resistance today: focus on Aspergillus. Curr Infect Dis Rep 13:485–491. doi: 10.1007/s11908-011-0218-4. [DOI] [PubMed] [Google Scholar]
  • 13.Arendrup MC. 2014. Update on antifungal resistance in Aspergillus and Candida. Clin Microbiol Infect 20(Suppl 6):42–48. doi: 10.1111/1469-0691.12513. [DOI] [PubMed] [Google Scholar]
  • 14.Sharma C, Hagen F, Moroti R, Meis JF, Chowdhary A. 2015. Triazole-resistant Aspergillus fumigatus harbouring G54 mutation: is it de novo or environmentally acquired? J Glob Antimicrob Resist 3:69–74. doi: 10.1016/j.jgar.2015.01.005. [DOI] [PubMed] [Google Scholar]
  • 15.Chowdhary A, Sharma C, Hagen F, Meis JF. 2014. Exploring azole antifungal drug resistance in Aspergillus fumigatus with special reference to resistance mechanisms. Future Microbiol 9:697–711. doi: 10.2217/fmb.14.27. [DOI] [PubMed] [Google Scholar]
  • 16.Moye-Rowley WS. 2015. Multiple mechanisms contribute to the development of clinically significant azole resistance in Aspergillus fumigatus. Front Microbiol 6:70. doi: 10.3389/fmicb.2015.00070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Wei X, Zhang Y, Lu L. 2015. The molecular mechanism of azole resistance in Aspergillus fumigatus: from bedside to bench and back. J Microbiol 53:91–99. doi: 10.1007/s12275-015-5014-7. [DOI] [PubMed] [Google Scholar]
  • 18.Wang S, Cao J, Liu X, Hu H, Shi J, Zhang S, Keller NP, Lu L. 2012. Putative calcium channels CchA and MidA play the important roles in conidiation, hyphal polarity and cell wall components in Aspergillus nidulans. PLoS One 7:e46564. doi: 10.1371/journal.pone.0046564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Liu FF, Pu L, Zheng QQ, Zhang YW, Gao RS, Xu XS, Zhang SZ, Lu L. 2015. Calcium signaling mediates antifungal activity of triazole drugs in the Aspergilli. Fungal Genet Biol 81:182–190. doi: 10.1016/j.fgb.2014.12.005. [DOI] [PubMed] [Google Scholar]
  • 20.Arikan-Akdagli S. 2012. Azole resistance in Aspergillus: global status in Europe and Asia. Ann N Y Acad Sci 1272:9–14. doi: 10.1111/j.1749-6632.2012.06815.x. [DOI] [PubMed] [Google Scholar]
  • 21.Espinel-Ingroff A, Diekema DJ, Fothergill A, Johnson E, Pelaez T, Pfaller MA, Rinaldi MG, Canton E, Turnidge J. 2010. Wild-type MIC distributions and epidemiological cutoff values for the triazoles and six Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document). J Clin Microbiol 48:3251–3257. doi: 10.1128/JCM.00536-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Pfaller M, Boyken L, Hollis R, Kroeger J, Messer S, Tendolkar S, Diekema D. 2011. Use of epidemiological cutoff values to examine 9-year trends in susceptibility of Aspergillus species to the triazoles. J Clin Microbiol 49:586–590. doi: 10.1128/JCM.02136-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kim HJ, Khalimonchuk O, Smith PM, Winge DR. 2012. Structure, function, and assembly of heme centers in mitochondrial respiratory complexes. Biochim Biophys Acta 1823:1604–1616. doi: 10.1016/j.bbamcr.2012.04.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Barros MH, Tzagoloff A. 2002. Regulation of the heme A biosynthetic pathway in Saccharomyces cerevisiae. FEBS Lett 516:119–123. doi: 10.1016/S0014-5793(02)02514-0. [DOI] [PubMed] [Google Scholar]
  • 25.Warris A. 2015. Azole-resistant aspergillosis. J Infect 71(Suppl 1):S121–S125. doi: 10.1016/j.jinf.2015.04.023. [DOI] [PubMed] [Google Scholar]
  • 26.Lamping E, Baret PV, Holmes AR, Monk BC, Goffeau A, Cannon RD. 2010. Fungal PDR transporters: phylogeny, topology, motifs and function. Fungal Genet Biol 47:127–142. doi: 10.1016/j.fgb.2009.10.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Maesaki S, Marichal P, Vanden Bossche H, Sanglard D, Kohno S. 1999. Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candida albicans strains. J Antimicrob Chemother 44:27–31. doi: 10.1093/jac/44.1.27. [DOI] [PubMed] [Google Scholar]
  • 28.Verweij PE, Chowdhary A, Melchers WJ, Meis JF. 2016. Azole resistance in Aspergillus fumigatus: can we retain the clinical use of mold-active antifungal azoles? Clin Infect Dis 62:362–368. doi: 10.1093/cid/civ885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Spiess B, Postina P, Reinwald M, Cornely OA, Hamprecht A, Hoenigl M, Lass-Florl C, Rath PM, Steinmann J, Miethke T, Lauten M, Will S, Merker N, Hofmann WK, Buchheidt D. 2014. Incidence of Cyp51 A key mutations in Aspergillus fumigatus—a study on primary clinical samples of immunocompromised patients in the period of 1995-2013. PLoS One 9:e103113. doi: 10.1371/journal.pone.0103113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Bader O, Weig M, Reichard U, Lugert R, Kuhns M, Christner M, Held J, Peter S, Schumacher U, Buchheidt D, Tintelnot K, Gross U, MykoLabNet-D Partners. 2013. cyp51A-based mechanisms of Aspergillus fumigatus azole drug resistance present in clinical samples from Germany. Antimicrob Agents Chemother 57:3513–3517. doi: 10.1128/AAC.00167-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Cannon RD, Lamping E, Holmes AR, Niimi K, Tanabe K, Niimi M, Monk BC. 2007. Candida albicans drug resistance another way to cope with stress. Microbiology 153:3211–3217. doi: 10.1099/mic.0.2007/010405-0. [DOI] [PubMed] [Google Scholar]
  • 32.Hartmann T, Sasse C, Schedler A, Hasenberg M, Gunzer M, Krappmann S. 2011. Shaping the fungal adaptome—stress responses of Aspergillus fumigatus. Int J Med Microbiol 301:408–416. doi: 10.1016/j.ijmm.2011.04.008. [DOI] [PubMed] [Google Scholar]
  • 33.Yoon JH, Choi YJ, Kim SG, Nam SW, Lee JY, Park WS. 2012. Programmed cell death 6 (PDCD6) as a prognostic marker for gastric cancers. Tumour Biol 33:485–494. doi: 10.1007/s13277-011-0280-4. [DOI] [PubMed] [Google Scholar]
  • 34.Camps SM, van der Linden JW, Li Y, Kuijper EJ, van Dissel JT, Verweij PE, Melchers WJ. 2012. Rapid induction of multiple resistance mechanisms in Aspergillus fumigatus during azole therapy: a case study and review of the literature. Antimicrob Agents Chemother 56:10–16. doi: 10.1128/AAC.05088-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Williams SL, Valnot I, Rustin P, Taanman JW. 2004. Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1, or SURF1. J Biol Chem 279:7462–7469. doi: 10.1074/jbc.M309232200. [DOI] [PubMed] [Google Scholar]
  • 36.Pitceathly RD, Taanman JW, Rahman S, Meunier B, Sadowski M, Cirak S, Hargreaves I, Land JM, Nanji T, Polke JM, Woodward CE, Sweeney MG, Solanki S, Foley AR, Hurles ME, Stalker J, Blake J, Holton JL, Phadke R, Muntoni F, Reilly MM, Hanna MG, UK10K Consortium. 2013. COX10 mutations resulting in complex multisystem mitochondrial disease that remains stable into adulthood. JAMA Neurol 70:1556–1561. doi: 10.1001/jamaneurol.2013.3242. [DOI] [PubMed] [Google Scholar]
  • 37.Valnot I, von Kleist-Retzow JC, Barrientos A, Gorbatyuk M, Taanman JW, Mehaye B, Rustin P, Tzagoloff A, Munnich A, Rotig A. 2000. A mutation in the human heme A:farnesyltransferase gene (COX10) causes cytochrome c oxidase deficiency. Hum Mol Genet 9:1245–1249. doi: 10.1093/hmg/9.8.1245. [DOI] [PubMed] [Google Scholar]
  • 38.Antonicka H, Leary SC, Guercin GH, Agar JN, Horvath R, Kennaway NG, Harding CO, Jaksch M, Shoubridge EA. 2003. Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency. Hum Mol Genet 12:2693–2702. doi: 10.1093/hmg/ddg284. [DOI] [PubMed] [Google Scholar]
  • 39.Moraes CT, Diaz F, Barrientos A. 2004. Defects in the biosynthesis of mitochondrial heme c and heme a in yeast and mammals. Biochim Biophys Acta 1659:153–159. doi: 10.1016/j.bbabio.2004.09.002. [DOI] [PubMed] [Google Scholar]
  • 40.Diaz F, Thomas CK, Garcia S, Hernandez D, Moraes CT. 2005. Mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency. Hum Mol Genet 14:2737–2748. doi: 10.1093/hmg/ddi307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kafer E. 1977. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet 19:33–131. [DOI] [PubMed] [Google Scholar]
  • 42.Zhong G, Wei W, Guan Q, Ma Z, Wei H, Xu X, Zhang S, Lu L. 2012. Phosphoribosyl pyrophosphate synthetase, as a suppressor of the sepH mutation in Aspergillus nidulans, is required for the proper timing of septation. Mol Microbiol 86:894–907. doi: 10.1111/mmi.12026. [DOI] [PubMed] [Google Scholar]
  • 43.Zhang C, Meng X, Wei X, Lu L. 2016. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet Biol 86:47–57. doi: 10.1016/j.fgb.2015.12.007. [DOI] [PubMed] [Google Scholar]
  • 44.Pfaller JB, Messer SA, Hollis RJ, Diekema DJ, Pfaller MA. 2003. In vitro susceptibility testing of Aspergillus spp.: comparison of Etest and reference microdilution methods for determining voriconazole and itraconazole MICs. J Clin Microbiol 41:1126–1129. doi: 10.1128/JCM.41.3.1126-1129.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard—2nd ed CLSI document M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
  • 46.Espinel-Ingroff A, Fothergill A, Fuller J, Johnson E, Pelaez T, Turnidge J. 2011. Wild-type MIC distributions and epidemiological cutoff values for caspofungin and Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document). Antimicrob Agents Chemother 55:2855–2859. doi: 10.1128/AAC.01730-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Mallory JC, Crudden G, Johnson BL, Mo C, Pierson CA, Bard M, Craven RJ. 2005. Dap1p, a heme-binding protein that regulates the cytochrome P450 protein Erg11p/Cyp51p in Saccharomyces cerevisiae. Mol Cell Biol 25:1669–1679. doi: 10.1128/MCB.25.5.1669-1679.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Alcazar-Fuoli L, Mellado E, Garcia-Effron G, Lopez JF, Grimalt JO, Cuenca-Estrella JM, Rodriguez-Tudela JL. 2008. Ergosterol biosynthesis pathway in Aspergillus fumigatus. Steroids 73:339–347. doi: 10.1016/j.steroids.2007.11.005. [DOI] [PubMed] [Google Scholar]
  • 49.Song J, Zhai P, Zhang Y, Zhang C, Sang H, Han G, Keller NP, Lu L. 2016. The Aspergillus fumigatus damage resistance protein family coordinately regulates ergosterol biosynthesis and azole susceptibility. mBio 7:e01919–15. doi: 10.1128/mBio.01919-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Jia C, Zhang K, Yu Q, Zhang B, Xiao C, Dong Y, Chen Y, Zhang B, Xing L, Li M. 2015. Tfp1 is required for ion homeostasis, fluconazole resistance and N-acetylglucosamine utilization in Candida albicans. Biochim Biophys Acta 1853:2731–2744. doi: 10.1016/j.bbamcr.2015.08.005. [DOI] [PubMed] [Google Scholar]
  • 51.Cendejas-Bueno E, Cuenca-Estrella M, Gomez-Lopez A. 2013. A simple, sensitive HPLC-PDA method for the quantification of itraconazole and hydroxy itraconazole in human serum: a reference laboratory experience. Diagn Microbiol Infect Dis 76:314–320. doi: 10.1016/j.diagmicrobio.2013.03.012. [DOI] [PubMed] [Google Scholar]
  • 52.Long N, Xu X, Qian H, Zhang S, Lu L. 2016. A putative mitochondrial iron transporter MrsA in Aspergillus fumigatus plays important roles in azole-, oxidative stress responses and virulence. Front Microbiol 7:716. doi: 10.3389/fmicb.2016.00716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Jiang H, Shen Y, Liu W, Lu L. 2014. Deletion of the putative stretch-activated ion channel Mid1 is hypervirulent in Aspergillus fumigatus. Fungal Genet Biol 62:62–70. doi: 10.1016/j.fgb.2013.11.003. [DOI] [PubMed] [Google Scholar]

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