https://orcid.org/0000-0003-0968-6507
Figures
Abstract
Aspergillus flavus is the second most prevalent species of Aspergillus causing invasive aspergillosis, but its treatment efforts had been hindered by the continuous emergence of drug-resistant fungal strains, while the underlying mechanisms remain largely unexplored. In this study, we investigated the role of the chromatin remodeling factor Arp9 in A. flavus drug-resistant. We show that Arp9 up-regulates the chromatin accessibility of the Erg3 and Erg6 promoters, thereby increasing their transcription levels and enhancing ergosterol synthesis. Therefore, the absence of Arp9 enhances A. flavus sensitivity to amphotericin B (AMB). Additionally, by down-regulating chromatin accessibility of Erg11A gene promoter, Arp9 leads to the decrease of its transcription level and subsequently reduces A. flavus resistance to voriconazole (VOR). Co-immunoprecipitation analysis revealed that Arp9 exists in both SWI/SNF and RSC complex. Drug susceptibility test results indicated that the drug sensitivity response induced by Arp9 may be unique to Arp9, as neither SWP82 of the SWI/SNF nor Sth1 of the RSC is required. The role of Arp9 in drug-resistance was also confirmed using the Galleria mellonella model. Furthermore, we found that VOR induces aflatoxin B1 (AFB1) biosynthesis in an Arp9-dependent manner at 35°C and 37°C, and the effect is dramatically magnified in the VOR-resistant A. flavus strain. This study demonstrates that Arp9 plays a critical role in regulating fungal drug-resistance in vitro and in vivo and revealed that Arp9 is an important factor in enhancing AFB1 biosynthesis under Mammalian physiological temperatures. This study provides potential new insights for the control of the infections caused by filamentous pathogenic fungi.
Author summary
As the notorious aflatoxin producing pathogenic fungi in agriculture and medicine, Aspergillus flavus is widely distributed, contaminating various crops, and inducing invasive aspergillosis mainly to immunodeficiency-patients. Since the drug-resistant clinical A. flavus strains is continuously emergent, the underlying mechanisms urgently needs to be explored. This study demonstrates that chromatin remodeling factor Arp9 is a key switch in A. flavus drug-resistance. It reduces the resistance of A. flavus to VOR by down-regulating the expression of Erg11A. Additionally, Arp9 decreases the sensitivity of A. flavus to AMB by up-regulating ergosterol synthesis. And the role of Arp9 in drug-resistance in vivo was further verified by the G. mellonella model. More importantly, our study is the first to discover that Arp9 promotes AFB1 biosynthesis at mammalian physiological temperature (from 35°C to 37°C) under VOR stress, especially in VOR-resistant strain. This study reveals the important role of chromatin remodeling factor Arp9 in fungal drug-resistance, and provides a potential key target for the treatment of A. flavus infections.
Citation: Ma D, Yao Y, Yang C, Lin H, Sun M, Gao Y, et al. (2026) The chromatin remodeling factor Arp9 modulates drug-resistance and plays a key role in aflatoxins biosynthesis under mammalian-physiological-temperature in Aspergillus flavus. PLoS Pathog 22(3): e1014021. https://doi.org/10.1371/journal.ppat.1014021
Editor: Haoping Liu, University of California Irvine, UNITED STATES OF AMERICA
Received: August 25, 2025; Accepted: February 22, 2026; Published: March 2, 2026
Copyright: © 2026 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The transcriptomics data (RNA-seq) is deposited in NCBI’s Gene Expression Omnibus (GEO) and can be accessed through GEO Series accession ID GEO: GSE279121.
Funding: This work was funded by the grants of the National Natural Science Foundation of China (No. 32470207 and No. 32070140 to ZZ), the Nature Science Foundation of Fujian Province (No. 2021J02026 to ZZ), the grants of the State Key Laboratory of Pathogen and Biosecurity (SKLPBS2434 to ZZ), and the Science and Technology Innovation Special Fund Project of Fujian Agriculture and Forestry University (KFB24088 to ZZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Aspergillus flavus, as an opportunistic pathogen, is the second most common species of the genus Aspergillus that causes invasive aspergillosis (IA), surpassed only by A. fumigatus [1]. Despite improvements in the treatment and diagnosis of IA over the past two decades, it remains a devastating fungal disease [2]. IA primarily affects patients with compromised immune systems, underlying hematological diseases, cancer, autoimmune diseases, as well as solid organ transplant (SOT) recipients or critically ill patients [3,4]. Furthermore, A. flavus produces the toxic and carcinogenic aflatoxins (AFs) family, which can generate other toxic derivatives when ingested by animals [5]. Among the AFs, aflatoxin B1 (AFB1) is one of the most toxic and harmful compounds [6]. A recent report on fungal pathogens issued by the World Health Organization (WHO) recognizes invasive fungal diseases as a growing global health concern, and recommends collaboration between governments and researchers to strengthen pathogenic fungal surveillance, antifungal-drug development, and public policy formulation [7]. Currently, the antifungal-drugs used to treat IA include polyenes, azoles, and echinocandins. Among them, azole drugs are the first-line choice for managing and preventing aspergillosis, such as voriconazole (VOR), itraconazole, isavuconazole, and posaconazole, while amphotericin B (AMB) and caspofungin are important antifungals for azole-resistant strains, with caspofungin typically not used alone but rather as part of a multidrug antifungal regimen alongside azole drugs [8,9].
Since its first approval in the 1950s, AMB has been considered a clinically important therapeutic option for a range of invasive fungal diseases, including IA [10]. Recent studies have shown that AMB kills fungi primarily by forming extracellular sponge-like aggregates that extract ergosterol from lipid bilayers [11]. Research indicated that the molecular mechanism of fungal resistance to AMB is associated with specific target genes, primarily including ERG2, ERG3, and ERG6 [12–14]. Additionally, it has also been reported that AMB resistance may be linked to changes in membrane lipid permeability and chromatin remodeling [15]. While azole drugs primarily work by inhibiting the enzyme lanosterol 14-alpha-demethylase Erg11 (also known as Cyp51), leading to the accumulation of toxic intermediates in the ergosterol biosynthetic pathway, ultimately altering cell membrane permeability and finally causing cell death [16]. It is reported that in addition to modifications to the Erg11 enzyme, overexpression of drug-efflux-pumps, such as ABC transporters and the Major Facilitator Superfamily (MFS), can also lead to reduced accumulation of azole drugs within fungal cells, contributing to azole resistance [17–19]. The limited number and variety of antifungal-drugs significantly restrict patients’ treatment options. Long-term use or misuse of antifungal-drugs further exacerbates the problem of drug-resistance [20,21]. Therefore, the development of novel antifungal-drugs, the repurposing of approved drugs, the use of antivirulence drugs, and the combination of antifungal-drugs are currently the most effective and urgent strategies to address this issue [22–25].
Epigenetic changes, such as DNA or chromatin modifications, alter gene expression levels in response to certain stimuli, including interactions with hosts in the case of fungal pathogens, and these changes can confer drug-resistance by altering the expression of target genes or genes encoding drug-efflux-pumps [26,27]. Therefore, targeting epigenetic pathways provides broad outlook to identify candidate drug-targets for the development of broad-spectrum antifungal-drugs [28,29]. As one of the first discovered chromatin remodeling complexes, SWI/SNF (switching defective/sucrose non-fermenting) class of ATP-dependent chromatin modifiers plays a crucial role in gene regulation, environmental stress response, carcinogenesis, and fungal drug-resistance [30–33]. It contains two complexes: SWI/SNF and RSC (remodeling the structure of chromatin) [34]. Arp9 (actin-related protein 9) is a member of both the SWI/SNF and RSC chromatin remodeling complexes in fungi and belongs to the actin-related protein (ARP) module [34]. However, the research on its role in fungal drug-resistance is limited, and no reports addressed on its role in A. flavus drug-resistance.
Our group has long been engaged in the study of the epigenetic regulation of A. flavus. In this work, we screened drug-resistance phenotypes from various epigenetic modification deficient strains of A. flavus prepared in our lab, in which the chromatin remodeling factor Arp9 exhibited two different drug-resistance phenotypes towards AMB and VOR. Simultaneously, we observed that the expression levels of the Arp9 were significantly down-regulated in the laboratory-randomly-screened VOR-resistant A. flavus strains, and the knockout of Arp9 from the VOR-resistant strain also resulted in the same drug susceptibility profile. This suggested that Arp9 plays a crucial role in the development of resistance to VOR in A. flavus. Furthermore, we found that Arp9 dramatically promotes AFB1 biosynthesis at 35°C to 37°C, and the effect is significantly magnified in the VOR-resistant strain CAMS-CCPM-D06242-4. To explore the underlying mechanism, we carried out a serial of experiments at genetic level, transcriptional level, and animal individual level. The results of this study would provide new insights for the development of antifungal-drugs.
Results
Arp9 is involved in the fungal resistance to VOR and AMB
An increasing number of literature reports indicated that epigenetic modifications can affect fungal drug-resistance. In order to explore whether histone methylation modification is involved in the regulation of A. flavus drug-resistance, we conducted a minimum inhibitory concentration (MIC) test with the wild-type (WT) strain of A. flavus using two antifungal-drugs, AMB and VOR, based on the microdilution method specified by European Committee on Antimicrobial Susceptibility Testing (EUCAST). The results showed that the MIC of AMB against the WT strain was 1 μg/mL, while the MIC of VOR against the WT strain was 2 μg/mL. To investigate the underlying mechanism, we conducted a preliminary screening of the histone-methylation-related gene knockout strains previously obtained in our lab using 1/3 MIC (0.33 μg/mL) and 2/3 MIC (0.67 μg/mL) of AMB, or 1/8 MIC (0.25 μg/mL) and 1/4 MIC (0.5 μg/mL) of VOR. An intriguing observation was found that the knockout fungal strain of the chromatin remodeling factor Arp9 (AFLA_009253) exhibited distinct drug-sensitivity responses when exposed to these two antifungal-drugs: compared to the WT, the Arp9 mutant (ΔArp9) showed greater sensitivity to AMB, but increased resistance to VOR. (S1 Fig).
Meanwhile, qRT-PCR on two VOR-resistant strains isolated from the tobacco and from the laboratory selected-clinical-isolations revealed that in both VOR-resistant strains, the expression levels of the Arp9 gene were significantly reduced (Fig 1A and 1B). These findings implied that Arp9 plays a crucial role in fungal resistance to VOR, and related studies have critical clinical significance and important practical value. Furthermore, through phylogenetic tree analysis and domain homology alignment, we found that Arp9 is highly conserved in the genus Aspergillus, but shows significant differences from its homologs in Homo sapiens, Mus musculus, and Arabidopsis thaliana (S2A and S2B Fig). Subsequently, we further conducted agar spotting assays by inoculating various dilutions of WT, ΔArp9, and its complementary strain (Com-Arp9) onto Müller-Hinton medium containing 0.67 μg/mL of AMB and 0.25 μg/mL of VOR. Our results indicated that, compared to WT, ΔArp9 exhibited significantly more sensitivity to AMB, but more resistance to VOR (Fig 1C). The above findings were further corroborated through the Kirby-Bauer test, as shown in Fig 1D-1G. Meanwhile, we found that the inhibition rate of AMB on ΔArp9 was approximately 21.45%, significantly higher than that on WT and Com-Arp9 (14.94% and 14.68%, respectively) (S3A and S3B Fig). In contrast, the inhibition rate of VOR on ΔArp9 was approximately 50.83%, significantly lower than that on WT and Com-Arp9 (62.87% and 62.91%, respectively) (S3C and S3D Fig). This further validates our conclusion that ΔArp9 is more sensitive to AMB and more resistant to VOR compared to WT.
(A) qRT-PCR validation of changes in Arp9 expression levels in clinically isolated strains before and after developing resistance to VOR. (B) qRT-PCR validation of changes in Arp9 expression levels in VOR-resistant strains isolated from tobacco. (C) Spores of WT, ΔArp9, and Com-Arp9 were diluted to 2 × 106, 2 × 105, 2 × 104, and 2 × 103 and inoculated onto MH medium containing 0.67 μg/mL AMB and 0.25 μg/mL VOR. The growth state was observed after 48 h of incubation at 37°C. (D) Antifungal disks containing 10 μg (6 mm in diameter) of AMB were placed on MH agar plates, and observed after 24 h of incubation at 37°C. (E) Statistical analysis of the diameters of the inhibition zones around the AMB disks. (F) Antifungal disks containing 1 μg (6 mm in diameter) of VOR were placed on MH agar plates, and observed after 24 h of incubation at 37°C. (G) Statistical analysis of the inhibition zone diameters of the VOR disks. All experiments were performed at least three biological replicates. Student’s t-test was used for statistical analysis, **, P < 0.01 and ***, P < 0.001.
Arp9 participates in the regulation of fungal biofilm formation and drug-resistance
There is a close relationship between fungal biofilm and drug-resistance. In the aforementioned drug-susceptibility tests, we determined that Arp9 regulates the resistance of A. flavus to AMB and VOR. This prompts the question of whether Arp9 influences the formation of A. flavus biofilm, and whether the drug-resistance of its biofilm is impacted. Herein, the crystal violet staining method was utilized to determine the role of Arp9 in the formation of biofilm, which showed that the biofilm-forming ability of ΔArp9 was significantly reduced compared to WT at both 24 and 48 h (Fig 2A). To analyze the resistance of ΔArp9 biofilm to antifungal-drugs, the XTT assay was employed. The both drugs were added at strain-specific MIC concentrations and sub-MIC concentrations (one-fourth of the previously determined MIC). The drugs were administered at both early and late stages of biofilm maturation (24 and 48 h after incubation). The results indicated that when AMB was added at the MIC concentration at the early stage of fungal biofilm maturation, its inhibitory effect against ΔArp9 was significantly higher than that to WT. Similarly, when AMB was added at sub-MIC concentration at the late stage of biofilm maturation, its inhibitory effect against ΔArp9 was also significantly higher than that to WT. However, the addition of VOR at both 24 and 48 h promoted the formation of ΔArp9 biofilm rather than inhibiting it (Fig 2B-2E). These results suggested that the biofilm formed of ΔArp9 was more sensitive to AMB and more resistant to VOR compared to the WT.
(A) ΔArp9 and WT strains were inoculated onto 96-well polystyrene plates containing RPMI-1640 medium. After 24 and 48 h of incubation, biofilm biomass was determined through the crystal violet assay. (B-E) The above-mentioned fungal strains were inoculated onto 96-well polystyrene plates containing RPMI-1640 medium. After 24 and 48 h of incubation, the fungal strains were treated with AMB and VOR at 1/4 × MIC and MIC concentrations for 24 h. The inhibition rates of AMB and VOR on biofilms formed at 24 and 48 h were determined by the XTT assay. All experiments were performed at least three biological replicates. Student’s t-test was used for statistical analysis: *, P < 0.05, ** P < 0.01, *** P < 0.001.
Arp9 significantly enhances fungal pathogenicity and colonization ability in vivo
To investigate the impact of Arp9 on A. flavus pathogenicity, we established an infection model using Galleria mellonella larvae as hosts. The results showed that, at 72 h post-infection, all larvae injected with WT fungal strain succumbed to the infection, whereas the survival rate of larvae injected with the ΔArp9 remained at 30%. Even at 120 h, the survival rate of ΔArp9-infected larvae still remained 10%. These results demonstrated that ΔArp9 pathogenicity is significantly reduced compared to the WT strain (Fig 3A and 3B). Subsequently, the deceased G. mellonella larvae were incubated at 29°C for 7 d to observe the colonization ability of the ΔArp9. The results indicated that the spore-producing capability of the ΔArp9 on the dead larvae was also significantly reduced compared to the WT strain (Fig 3C and 3D). Further microbial biomass analysis revealed that the hyphal content of ΔArp9 within the larvae was significantly lower (Fig 3E), confirming the reduced colonization ability of ΔArp9. To investigate the role of Arp9 in aflatoxin biosynthesis in A. flavus infected hosts, we extracted AFB1 from the above dead infected larvae, and the results revealed that ΔArp9 strain completely failed to produce AFB1 in the larvae (Fig 3F).
(A) Arp9 enhances the pathogenicity of A. flavus to G. mellonella larvae. 1 × 105 spores of WT, ΔArp9, and Com-Arp9 strains were injected into G. mellonella larvae, respectively, and larval mortality was observed after 120 h. (B) Survival curves of G. mellonella larvae infected with the above fungal strains, and the larvae injected with saline served as the negative control. (C) Sporulation ability of the above strains on the dead G. mellonella larvae. The state was documented after 7 d of incubation at 29°C. (D) The conidium number of the above fungal strains on dead G. mellonella larvae was calculated with hemocytometer according to the result from (C). (E) Content analysis of fungal hyphae through fungal DNA quantitative by qPCR. (F) TLC analysis of the AFB1 yield of the above fungal strains in the infected G. mellonella larvae. (G) Distribution of A. flavus hyphae or spores within G. mellonella larvae and state of the larval body cavity observed by H&E staining. The larvae were injected with the above fungal strains for 48 h, followed by formaldehyde fixation, longitudinal sectioning, and finally H&E staining for observation. The red cycle indicates the mycelium of ΔArp9 in the infected the G.mellonella. (H) The changes of hemocyte number of G. mellonella larvae after infection with the above fungal strains, respectively. All experiments were performed with at least three biological replicates. Log-rank (Mantel-Cox) test was used for (B) statistical analysis. Student’s t-test was used for (D), (E), (H) statistical analysis. *, P < 0.05, ** P < 0.01.
Subsequently, pathological sectioning on the infected larvae were performed (Fig 3G). The results showed that, through hematoxylin and eosin (H&E) staining, the body cavity of larvae infected with ΔArp9 for 48 h remained relatively intact, with fewer germination of spores within the larvae. In contrast, the body cavity of larvae injected by WT spores was nearly completely destroyed, and most of the spores germinated into hyphae, which partially explains the faster mortality rate after WT fungal strain injection. The immune system of G. mellonella larvae is similar to that of mammals, also possessing cellular and humoral immunity, and the hemocyte number, as one of indexes of cellular immunity, have been used to assess larval immune responses to pathogens [35,36]. To further observe Arp9 role in the disruption of host’s immune system, we monitored the hemocyte number of larvae after injecting with the above fungal strains. The results showed that following the injection of ΔArp9, the hemocyte number in larvae decreased by about 36% compared to the control group (CK). Whereas after injecting WT spores, the hemocyte number decreased by about 67%, significantly lower than the ΔArp9 injection group (Figs 3H and S4). Taken together, Arp9 plays an important role in fungal colonization ability and pathogenicity against host immune system.
Arp9 dichotomously regulates fungal drug-sensitivity to VOR and AMB in vivo
The previous results of in vitro experiments have confirmed that, compared to the WT fungal strain, the ΔArp9 is significantly more sensitive to AMB, but more resistant to VOR. To investigate whether Arp9 plays the same role in drug-resistance against VOR and AMB in vivo, the constructed larvae model was used, in which the larvae were treated with 120 ng/larvae of VOR after they are infected with A. flavus for 1 h. The results showed that when larvae infected with WT strain were treated with the same dose of drugs (120 ng/larvae VOR) for 120 h, the survival rate was significantly increased to 50% compared with the untreated WT-infected larvae (0% survival rate) (Fig 4A and 4B). While, when ΔArp9-infected G. mellonella larvae were treated with 120 ng/larvae of VOR for 120 h, no improvement was found in the survival rate, compared with that of the ΔArp9-infected larvae group (Fig 4A and 4C). The results of the above in vivo experiments reflected that the absence of Arp9 significantly enhance fungal drug-resistance against VOR.
(A) The G. mellonella infected by A. flavus strains were treated with VOR. Larvae (n = 10) were injected with WT or ΔArp9 through the penultimate proleg, followed by VOR injection (120 ng/larvae). The fungal free larvae directly injected with VOR (120 ng/larvae) was set as control. Image was taken 120 h after injection. (B) The survival curve of WT-infected G. mellonella larvae after VOR treatment. (C) The survival curve of ΔArp9-infected G. mellonella larvae after VOR treatment. (D) The G. mellonella larvae infected by A. flavus strains were treated with AMB. Larvae (n = 10) were injected with WT or ΔArp9 strain through the penultimate proleg, followed by AMB injection (320 ng/larvae). The AMB (320 ng/larvae) directly injection group was set-up as control. Image was taken 144 h after injection. (E) The survival curve of the WT-injected larvae treated with AMB. (F) The survival curve of ΔArp9-injected larvae treated with AMB. Log-rank (Mantel-Cox) test was used for (B), (C), (E), (F) statistical analysis. *, P < 0.05, ** P < 0.01, ns means no significant.
Similarly, after G. mellonella larvae infected with A. flavus strain for 1 h, these larvae were treated with 320 ng/larvae AMB for 144 h, respectively. The results showed that the survival rate of WT-injected larvae increased from 20% (untreated) to 60% (treated), but no significant difference of statistic was observed between them. When ΔArp9-infected larvae were treated with the same doses of AMB (320 ng/larvae) for 144 h, their survival rate increased significantly from 30% (untreated) to 80% (treated) (Fig 4D and 4F). These in vivo results demonstrated that compared to WT, ΔArp9 exhibits significantly increased resistance to VOR, while significantly more sensitivity to AMB in hosts, which is consistent with the results obtained from in vitro drug-sensitivity tests.
Arp9 mediates VOR-resistance by up-regulating Erg11A
In order to further analyze the underlying mechanism of Arp9-mediated resistance to VOR, we performed RNA-seq analysis with both WT and ΔArp9 fungal strains. Multi-factor principal component (PCA) analysis revealed that the sequencing data was qualified for further analysis (S5A Fig). The analysis identified 1415 up-regulated and 1314 down-regulated genes in ΔArp9 compared to WT strain (S5B Fig). Among the top 20 Gene Ontology (GO) terms (P < 0.05) (Fig 5A), the biological process terms are mainly related to drug-resistance included drug metabolic process (GO:0017144), antibiotic metabolic process (GO:0016999), and drug catabolic process (GO:0042737). KEGG analysis found that differentially expressed genes are most enriched in 20 signaling pathways (P < 0.05) (Fig 5B), among which steroid biosynthesis (afv00100) and ABC transporters (afv02010) were found to be associated with the resistance of A. flavus to VOR and AMB. Compared with the WT, most genes in the ergosterol synthesis pathway of ΔArp9 are significantly down-regulated, such as AFLA_002655 (Erg6), AFLA_003936 (Erg3), AFLA_004897 (Erg25), AFLA_007071 (Erg24), and AFLA_005205 (Erg1). However, the VOR drug-target Erg11A (AFLA_001488) and drug-efflux-genes were significantly up-regulated, including AFLA_000192 (AtrC), AFLA_010719 (Mdr1), AFLA_011311 (AtrF), and AFLA_011408 (dotC) (P < 0.05) (Fig 5C). qRT-PCR analysis was further performed to monitor the expression levels of antifungal drug-related-genes (Erg11A, AtrF, Mdr1, Erg3, and Erg6), and the results verified the transcriptome data and showed that in ΔArp9, Erg11A, AtrF, and Mdr1 were significantly up-regulated, which might explain the fungal resistance to VOR, while Erg3 and Erg6 were dramatically down-regulated, which might underlie the fungal sensitivity to AMB (Fig 5D). ChART-PCR (Chromatin Accessibility by Real-Time PCR) was employed to further investigate whether Arp9 influences the transcription levels of the aforementioned target genes by remodulating chromatin accessibility in their promoter regions. The results indicated that upon Arp9 gene deletion, the relative CAI (chromatin accessibility index) in the promoter regions of Erg11A, AtrF, and Mdr1 genes was significantly increased, whereas it was markedly decreased in the promoter regions of Erg3 and Erg6 genes, and among them, the changes in AtrF and Erg6 were the most pronounced (Figs 5E and S6).
(A) GO analysis of Arp9-regulated differentially expression genes (DEGs). (B) KEGG analysis on Arp9-regulated DEGs. (C) Heatmap analysis of differential expression of drug resistance-related genes between WT and ΔArp9. (D) qRT-PCR was performed to validate the drug resistance-related genes from the transcriptome analysis. (E) ChART-PCR analysis on the changes of the relative CAI (chromatin accessibility index) levels in the promoter regions of drug resistance-related genes (Erg11A, AtrF, Mdr1, Erg3, Erg6) before and after Arp9 deletion. (F) Sensitivity assay of the ΔErg11A, ΔAtrF, ΔMdr1, ΔErg3, ΔErg6 mutant strains, along with the WT strain to the corresponding antifungal drugs. (G) Sensitivity assay of the ΔArp9/ΔErg11A, ΔArp9/ΔAtrF, ΔArp9/ΔMdr1, ΔArp9/ΔErg3, ΔArp9/ΔErg6 mutant strains, along with the WT strain to the corresponding antifungal drugs. The spores of corresponding fungal strains are diluted to a serial of concentrations (2 × 106, 2 × 105, 2 × 104, 2 × 103 spores) and 1 μL inoculated onto Mueller-Hinton (MH) agar plates containing either 0.67 μg/mL AMB or 0.25 μg/mL VOR, while the agar plate without antifungal agent was set as the control. The plates were then incubated at 37°C for 48 h, and the growth status of the strains was recorded. All experiments were performed at least three biological replicates. Student’s t-test: ** P < 0.01, *** P < 0.001.
To further analyze the potential role of these resistance genes on the fungal drug-resistance, these genes were knocked-out by the homologous recombination method. And the double-deletion mutants of Arp9 gene with each of these genes were constructed, respectively. The analysis of the drug resistance revealed that under the treatment of 0.25 μg/mL VOR, the ΔErg11A, ΔAtrF, and ΔMdr1 fungal strains exhibited significantly higher sensitivity than the WT strain (Fig 5F). However, among the double-knockout strains, ΔArp9/ΔAtrF and ΔArp9/ΔMdr1 showed even higher resistance than WT, indicating that the deletion of AtrF or Mdr1 did not reverse fungal resistance caused by Arp9 deletion. In contrast, ΔArp9/ΔErg11A was more sensitive to VOR compared to WT (Fig 5G), which reflected that deletion of Erg11A in ΔArp9 restores fungal sensitivity to VOR despite the deficiency of Arp9.
In summary, these data reflected that Arp9 down-regulates the relative expression levels of the above target genes by decreasing the relative CAI in the promoter regions of the VOR drug target Erg11A, thereby decreases A. flavus’s resistance to VOR.
Arp9-reduced fungal sensitivity to AMB is via the synthesis of ergosterol
As the target of AMB, ergosterol content is crucial in influencing fungal sensitivity to AMB. Through RNA-seq analysis, we found that some genes within the ergosterol synthesis pathway in ΔArp9 was down-regulated compared to the WT strain (S7 Fig). ChART-PCR and qRT-PCR results demonstrated that in ΔArp9, due to the decrease of chromatin accessibility in promoter regions, the gene expression levels of Erg3 and Erg6 were significantly reduced (Fig 5D and 5E). Under 0.67 μg/mL AMB pressure, ΔErg3, ΔErg6, ΔArp9/ΔErg3, and ΔArp9/ΔErg6 all exhibited increased drug resistance compared to WT (Fig 5F and 5G). The aforementioned results demonstrated that Arp9 influences the sensitivity of A. flavus to AMB by adjusting the ergosterol synthesis pathway genes Erg3 and Erg6. To further investigate whether the absence of Arp9 affects ergosterol synthesis, we performed HPLC to detect ergosterol levels in WT and ΔArp9 strains. The results revealed that the WT strain contained about 6000 μg/g of ergosterol, significantly higher than that in the ΔArp9 strain (about 4000 μg/g) (Fig 6A and 6B). In summary, we have revealed that Arp9 up-regulates the relative expression levels of ergosterol biosynthesis pathway genes Erg3 and Erg6 by increasing the relative CAI in their promoter regions, which in turn up-regulates ergosterol synthesis and reduces A. flavus’s sensitivity to AMB.
(A) HPLC analysis of ergosterol production in the ΔArp9 and WT strain. (B) Quantitative analysis of ergosterol in the above fungal strains based on the HPLC results. All experiments were performed with at least three biological replicates. Student’s t-test: ***, P < 0.001.
Arp9 dramatically enhance AFB1 biosynthesis under mammalian-physiological-temperature
Our transcriptome analysis of WT strains with and without VOR treatment also revealed that, under mammalian-physiological-temperature (37°C), the aflatoxin biosynthesis pathway was significantly up-regulated in the WT + VOR group compared to the WT without VOR (Fig 7A). We hypothesized that VOR may enhance aflatoxin biosynthesis in the WT strain at 37°C. Further qRT-PCR results showed that the genes in the aflatoxin-gene-cluster, including aflG, aflL, aflP, aflR, and aflS, were dramatically up-regulated in the WT + VOR group compared to the WT without VOR at 37°C (Fig 7B). In order to investigate the VOR-induced AFB1 production at different temperatures, the experiments were conducted at 35°C, 37°C, and 39°C. The TLC results showed that the addition of VOR at both 35°C and 37°C promoted the production of AFB1 by WT and VOR-resistant strain CAMS-CCPM-D06242-4, but failed to enable the production of AFB1 in ΔArp9, whereas under the high temperature 39°C conditions, there was no toxin production regardless of the presence or absence of VOR (Fig 7C). Our results demonstrated for the first time that Arp9 significantly promotes AFB1 biosynthesis under VOR stress at 35°C and 37°C, and suggested that an appropriate increase in body temperature (about 39°C or above in this study) is beneficial for inhibiting the toxin production of A. flavus, even in the presence of drug-treated or infected by drug-resistant strains.
(A) KEGG analysis of up-regulated DEGs in WT strain upon VOR stress. (B) qRT-PCR analysis on expression levels of candidate genes in the aflatoxin gene cluster (aflG, aflL, aflP, aflR and aflS) in the WT strain under VOR stress. (C) The AFB1 yield in the above A. flavus strains under VOR stress. Purchased AFB1 was used as the standard. The WT, ΔArp9, and CAMS-CCPM-D06242-4 strains were inoculated into PDB, PDB + VOR, and PDB + Dimethyl Sulfoxide (DMSO), respectively. The aflatoxin production was assessed after 7 d of incubation at 35°C, 37°C and 39°C. All experiments were performed at least three biological replicates. Student’s t-test: ***, P < 0.001.
The Arp9 is the key target for the response of A. flavus to AMB and VOR
To further investigate whether the sensitivity of A. flavus to AMB and resistance to VOR caused by the deletion of Arp9 are conserved, we knocked out Arp9 in strains CAMS-CCPM-D06242-4 and YE13. The results showed that both Arp9 deletion mutants (CAMS-CCPM-D06242-4ΔArp9 and YE13ΔArp9), exhibited increased sensitivity to AMB compared to their wild-type counterparts, furthermore, even in VOR-resistant strains, the deletion of Arp9 still exacerbated their resistance to VOR (Fig 8A and 8B). In view of that Arp9 is a key member of the chromatin remodeling complex (CRC), we constructed dual-tagged strains in A. flavus: Arp9–3 × HA + Sfh1-Strep and Arp9–3 × HA + Swp82-Strep. Co-immunoprecipitation (Co-IP) analysis confirmed that Arp9 interacts with both Sfh1 and Swp82 (S8 Fig). Research indicates that, unlike Arp9, which coexists in both the SWI/SNF and RSC complexes, SWP82 is a subunit unique to the SWI/SNF complex, while Sfh1 is exclusive to the RSC complex [34]. By generating sfh1 and swp82 knockout mutants (Δsfh1 and Δswp82), we investigated whether the drug tolerance phenotype induced by Arp9 is associated with other SWI/SNF subunits. Results showed that sfh1, and swp82 deletion mutants exhibit no significant change in resistance to VOR compared to the WT (Fig 8C). However, these mutants displayed the opposite phenotype to AMB compared to the ΔArp9 (Fig 8C). These findings indicated that Arp9-mediated resistance to antifungal drugs (AMB and VOR) in A. flavus is specific to this subunit. Furthermore, we performed an MNase digestion assay. The results revealed that following Arp9 deletion, the release of single nucleosomes was increased than that under WT conditions (S9 Fig).
(A) Sensitivity assay of the CAMS-CCPM-D06242-4 and CAMS-CCPM-D06242-4ΔArp9. The spores of corresponding fungal strains are diluted to a serial of concentrations (2 × 106, 2 × 105, 2 × 104, 2 × 103 spores), and 1 μL spore suspension was inoculated onto Mueller-Hinton (MH) agar plates containing either 0.67 μg/mL AMB or 8 μg/mL VOR, while the agar plate without antifungal agent was set as the control. The plates were then incubated at 37°C for 48 h, and the growth status of the strains was recorded. (B) Sensitivity assay of the YE13 and YE13ΔArp9. The spores were inoculated onto MH agar plates containing either 0.67 μg/mL AMB or 1 μg/mL VOR in the same manner as described above. (C) Sensitivity assay of the Δsfh1, Δswp82 and WT. The spores were inoculated onto MH agar plates containing either 0.67 μg/mL AMB or 0.25 μg/mL VOR in the same manner as described above.
Discussion
A. flavus, the second most invasive Aspergillus pathogen, is distributed worldwide [37,38]. Due to the limited selections and the abuse of antifungal-drugs, clinically resistant strains have gradually increased. However, the pathogenicity and drug-resistance of A. flavus have not been well illuminated. Chromatin remodeling, as epigenetic modifications, is a focus of attention. Several resistance strategies have been linked to the chromatin remodeling complex SWI/SNF. In Candida albicans, Snf2 drives fluconazole resistance in MRR1GOF isolates by remodeling the nucleosomes [31]. Under tebuconazole stress, Fusarium graminearum transcription factor SREBP is phosphorylated by Hog1, which then recruits the FgSWI/SNF proteins Swp73 and Arp9 to the ergosterol biosynthetic gene CYP51A. Consequently, Arp9 mutant becomes sensitive to tebuconazole by inhibiting CYP51A [39]. Moreover, the SWI/SNF of Saccharomyces cerevisiae collaborates with its histone partner Rtt106 to drive the expression of multidrug-resistance-genes [40]. In this study, we discovered that the absence of Arp9 in A. flavus made it more sensitive to AMB, whereas more resistant to VOR (Figs 1C-1G and S3). And the expression levels of Arp9 significantly decreased in the VOR-resistant strains isolated from the tobacco and from the laboratory selected-clinical-isolations (Fig 1A and 1B). Moreover, after knocking out Arp9, they also showed sensitivity to AMB and resistance to VOR (Fig 8A and 8B). To investigate whether the drug resistance phenotype induced by Arp9 is associated with other subunits of the SWI/SNF and RSC complexs, Co-IP analysis was performed and confirmed that Arp9 interacts with both Sfh1 and Swp82 (S8 Fig). Furthermore, we prepared related knockout strains, including Δsfh1 and Δswp82, and the results indicated that the deletion of these genes does not affect A. flavus resistance to VOR. Compared to ΔArp9, they exhibit an opposite phenotype toward AMB, suggesting that Arp9 plays a unique role in VOR resistance and AMB sensitivity relative to other subunits of the SWI/SNF and RSC complexs. (Fig 8C). Additionally, studies indicate that the ARP module within the SWI/SNF and RSC complexs, where Arp9 resides, can influence chromatin remodeling by affecting nucleosome sliding [41,42]. To investigate the chromatin remodeling function of Arp9 in A. flavus, we assessed chromatin accessibility in WT and ΔArp9 strains using MNase digestion assays. Results revealed significantly increased release of mononucleosomes in ΔArp9 compared to WT after treatment with different concentrations of MNase (S9 Fig), suggesting that Arp9 may antagonistically regulate the maintenance of chromatin openness. Analysis of the Arp9 protein sequence revealed that it is relatively conserved among Aspergillus species but exhibits significant divergence from those of H. sapiens and M. musculus (S2 Fig). With significant clinical and practical value, it is crucial to reveal how Arp9 regulates A. flavus drug-resistance.
Biofilm formation not only affects their pathogenicity but also closely relates to their drug-resistance [43,44]. The chromatin-remodeling-factor Snf5 influences biofilm formation and caspofungin sensitivity in C. albicans by affecting the biofilm regulator Ace2 [45]. In S. cerevisiae, biofilm formation modulator Flo11 is regulated by the Swi/Snf complex [46]. Since the clinical treatment of A. flavus is primarily conducted in the context of established infections, where it forms biofilm, we assessed Arp9 role in the drug-resistance of A. flavus biofilm. We found that the absence of Arp9 affected biofilms forming ability of this pathogen. And the mature biofilm of ΔArp9 exhibited the same drug-resistance of hyphae state to the both antifungal-drugs (Fig 2B-2E). These results indicated that Arp9 plays a critical role in the fungal drug-resistance, no matter in hyphae or biofilm state.
To further investigate Arp9 role in drug-resistance in vivo, we conducted a drug-sensitivity test with G. mellonella infection model. The G. mellonella larva is used as an alternative mammalian model to assess fungal virulence and drug-resistance [35,47]. Chromatin remodeling plays a critical role in fungal virulence, and the chromatin remodeling factor RSC4 in C. albicans is essential in its virulence regulation [29,48]. The chromatin remodeling factor sfh1 is also involved in the virulence of Sclerotinia sclerotiorum [49]. In this study, we also found that Arp9 is deeply involved in the AFB1 biosynthesis and virulence of A. flavus in G. mellonella (Fig 3). Compared to the mice model, the G. mellonella model is low-cost, easy to breed, relatively simple to maintain and easy to inoculate [35]. The G. mellonella model has been making increasingly important contributions in the field of antifungal efficacy evaluation in the IA treatment [50,51]. Through G. mellonella model, we revealed that Arp9 plays a key role in the regulation of A. flavus drug-resistance both in vitro and in vivo.
Although both VOR and AMB target the plasma membrane component ergosterol, the drug-resistance against the both antifungal-drugs are developed by different ways. The fungal resistance to VOR is primarily due to mutations or increased expression of the lanosterol 14α-demethylase Erg11A (Cyp51A) site [52]. Additionally, the activation of drug-efflux-pumps, such as ABC transporters, can lead to reduced accumulation of drugs within fungal cells, which is also a cause of azole resistance [16]. The over-expression of the drug-efflux-pump gene Mdr1 is the primary reason for the itraconazole resistance of A. fumigatus mitochondrial-defective strains [53]. The C2H2 transcription factor ZfpA induces a significant up-regulation of the ABC transporter AtrF, thereby enhancing A. fumigatus azole resistance [54]. This study found that Arp9 down-regulate the transcription levels of drug efflux genes AtrF, Mdr1, and the VOR drug target Erg11A by reducing their chromatin accessibility in their promoter regions (Fig 5D and 5E). Further investigation found that ΔAtrF and ΔMdr1 exhibited sensitivity to VOR (Fig 5F). Additionally, when the target of VOR, Erg11A, was deleted, A. flavus failed to grow on plates containing VOR (Fig 5F). Further double-knockout experiments revealed that ΔArp9/ΔAtrF and ΔArp9/ΔMdr1 exhibited VOR resistance similar to ΔArp9, whereas only ΔArp9/ΔErg11A showed sensitivity to VOR (Fig 5G). These results suggested that Arp9 suppresses fungal resistance to VOR by down-regulating the expression of the VOR target gene Erg11A through reducing the CAI of its promoter.
AMB mainly kills fungi by forming extracellular spongy aggregates to extract ergosterol from the lipid bilayer, so ergosterol amount is one of the indexes for AMB resistance [11,55]. It was shown that disruption of GCN5 impairs ergosterol synthesis and compromises the integrity of fungal cell membranes, which in turn causes the gcn5Δ mutant to exhibit increased sensitivity to amphotericin B [56]. In the same way, we confirmed by HPLC analysis that ergosterol levels in ΔArp9 were significantly lower than those in WT and showing greater sensitivity to AMB (Figs 1C, 6A and 6B). In addition, in ΔArp9, the CAI of both Erg3 and Erg6 promoters significantly decreased, which lead to the reduction of their expression levels (Fig 5D and 5E). However, the further exploration indicated that ΔErg3, ΔErg6, ΔArp9ΔErg3 and ΔArp9ΔErg6 acquired higher resistance to AMB than WT (Fig 5F and 5G). It is reported that the absence of ergosterol caused by mutations in the ERG gene resulted in the reduction of AMB sensitivity in Candida [57–60]. It was shown that ergosterol was not detected in a smooth Candida glabrata Erg6 nonsense mutant strain with reduced AMB sensitivity [12]. Moreover, Neurospora crassa strains with Erg3 deletion also showed resistance to AMB [61]. We hypothesized that Erg3 and Erg6, as two key genes downstream of the ergosterol synthesis pathway, are extremely important for ergosterol synthesis, and that complete deletion of Erg3 and Erg6 may lead to ergosterol severe deficiency and consequent resistance to AMB. In our study, ΔArp9 showed sensitivity to AMB, while ΔErg3, ΔErg6, ΔArp9ΔErg3 and ΔArp9ΔErg6 showed resistance to AMB. All the above results indicated that the chromatin remodeling factor Arp9 up-regulates the synthesis of ergosterol in A. flavus by increasing the expression of Erg3 and Erg6 genes, thereby reducing the sensitivity of A. flavus to AMB.
As the most toxic mycotoxin, AFB1 is the most significant and abundant Aflatoxin [6]. Most A. flavus strains isolated clinically produce AFB1 at temperatures around 28°C, few A. flavus strains can produce toxins at 37°C [62]. Our experiments have also found that when simulating the human body’s ambient temperature of 37°C in vitro, all fungal strains (WT, ΔArp9 and CAMS-CCPM-D06242-4 strains) could not produce AFB1 (Fig 7C). However, after treatment with 1/8 × MIC of VOR, the AFB1 biosynthesis capacity of both the WT and CAMS-CCPM-D06242-4 strains was significantly enhanced (Fig 7C). Furthermore, VOR-resistant CAMS-CCPM-D06242-4 produced at least five times of AFB1 more than that of the WT strain at 37°C under VOR stress. The above finding may explain that why the aspergillosis caused by A. flavus is more toxic than other pathogenic fungi [63]. This study also found that ΔArp9, which is also a VOR-resistant strain, did not produce AFB1, regardless of the presence or absence of VOR, which reflected that Arp9 is the key regulator to promote the biosynthesis of AFB1 at mammalian-physiological-temperature under the stress of anti-fungus antibiotics, and it would be made an ideal target to suppress the virulence of A. flavus in the clinical treatment of invade aspergillosis. A series of temperature experiments (including 35°C, 37°C and 39°C) in this study were tested, and the results reflected that an appropriate increase in body temperature is important in controlling the harm of fungal infection (Fig 7C).
In summary, Arp9 serves as a crucial regulator that up-regulates the chromatin accessibility of the Erg3 and Erg6 gene promoters, thereby increasing their transcription levels and enhancing ergosterol synthesis, reducing A. flavus sensitivity to AMB. And Arp9 down-regulates the chromatin accessibility of the Erg11A gene promoters, leading to decreased transcription levels of it and subsequently reducing A. flavus resistance to VOR. Meanwhile, Arp9 is also a crucial virulence factor that regulates the pathogenicity and toxin production of A. flavus, especially to regulates the AFB1 biosynthesis at mammalian-physiological-temperature under antibiotics treatment (Fig 9). Our study is the first to uncover the critical role of the chromatin remodeling factor Arp9 in drug-resistance and virulence of A. flavus, providing novel insights for antifungal-drug development.
Arp9 reduces the resistance of A. flavus to VOR by inhibiting the expression of Erg11A, and it promotes the synthesis of ergosterol by enhancing the expression of Erg3 and Erg6, thereby reducing the sensitivity of A. flavus to AMB. Additionally, Arp9 promotes biofilm formation in A. flavus, enhances its virulence towards G. mellonella larvae, and augments the ability of A. flavus to produce AFB1 at mammalian-physiological-temperature under antibiotics treatment.
Methods
Strains and growth conditions
The fungal strains used in this study were displayed in S1 Table. Potato dextrose agar (PDA, Franklin, NJ, USA) were used for mycelial growth, sporulation, and sclerotia formation.
Antifungal susceptibility testing
According to EUCAST E.DEF 9.4, the broth microdilution method was used to determine the sensitivity to antifungal-drugs [64]. Briefly, amphotericin B (AMB, Aladdin, Shanghai, China) and voriconazole (VOR, Aladdin, Shanghai, China) were gradient-diluted into a 96-well plate (100 μL per well) using RPMI 1640 (Sigma-Aldrich, France). Then, the spores of WT were added to the 96-well plate containing the above gradient-diluted antifungal-drugs, then incubated at 37°C for 48 h. The Kirby-Bauer test was performed as previously described, WT and ΔArp9 spores were spread onto Müller-Hinton agar plates, then antifungal discs for voriconazole (Liofilchem’s 9168) and amphotericin B (Liofilchem’s 9137) were spread on the plates. The diameters of the zones of inhibition were measured after incubated at 37°C for 24 h [65].
Agar spotting assays
Müller-Hinton medium was supplemented with 0.67 μg/mL AMB and 0.25 μg/mL VOR, respectively. Then, 1 μL conidial suspensions (2 × 106, 2 × 105, 2 × 104, 2 × 103 conidia/mL) from WT, ΔArp9, and Com- Arp9 grown at 37°C for 48 h, then observed and documented.
qRT-PCR analysis
Following previously described protocol [66], qRT-PCR was performed to analyze gene expression levels using the QuantStudio 1 Plus real-time PCR system (Thermo Fisher Scientific, USA). Primers were listed in S2 Table. The relative gene expression level was calculated through 2−ΔΔCT and normalized by tublin.
Biofilm formation assays
All experiments were conducted with RPMI 1640 medium. The crystal violet (CV) assay was carried out as previously described [67], and the microplate reader (Varioskan Flash 4.00.53, Thermo) was used to read the optical density (OD570). The metabolic activity of biofilm after antifungal treatment was assessed through XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) [43,68], After inoculation and cultivation, fresh medium containing 1/4 × MIC and 1 × MIC of antifungal-drugs (AMB, VOR) was added and kept for another 24 h, respectively. The OD490 was read using microplate reader (Varioskan Flash 4.00.53, Thermo). Finally, the inhibition rate of the antifungal-drugs to the biofilm was calculated using the following formula: Inhibition Rate = (OD of biofilm without antifungal-drugs - OD of biofilm with antifungal drugs)/ OD of biofilm without antifungal-drugs × 100%.
Galleria mellonella virulence assay
The G. mellonella virulence assay was performed with slight modification [69]. The WT, ΔArp9, and Com-Arp9 strains were diluted in saline to 1 × 107 conidia/mL. G. mellonella larvae (Keyun Biopesticide Co., Ltd, Henan, China) were injected with 10 μL of spore suspension per larva, with 10 larvae in each group. The control group received an equal volume of saline injection. The larvae were observed under dark at 37°C for 5 d, and mortality was recorded every 24 h.
Fungal colonization in G. mellonella
Experimental procedure of this test refers to our previous methods [70]. After the aforementioned G. mellonella larvae died, they were incubated at 29°C for 7 d. From each group, three larvae were randomly selected to detect AFB1 yield in the infected larvae.
Determination of fungal load in infected G. mellonella
The infected G. mellonella larvae (n = 3) were ground in liquid nitrogen. DNA was extracted following previous descriptions [71,72], and the relative fungal load in the infected larvae was determined by qPCR targeting the fungal ribosomal 28S subunit gene.
Observation of histopathological sections of G. mellonella
The G. mellonella larvae infected with A. flavus for 48 h were fixed overnight in 4% paraformaldehyde fixative solution (Solarbio, Beijing, China), then sent to Aisen Biotechnology Co., Ltd. (Fuzhou, China) for embedding, sectioning, and subsequent hematoxylin-eosin staining. Following staining, the sections were scanned and digitized for observation (Microscopy digital camera MD60, MSHOT).
Determination of G. mellonella hemocyte density
As described above, after 36 h of injection, the tails of the G. mellonella larvae were cut off using a scalpel, and 20 μL of hemolymph was aspirated from the larvae. The hemolymph was then diluted in 80 μL of pre-cooled anticoagulant buffer [73]. The number of hemocytes were counted and documented.
Drug-sensitivity test of A. flavus in G. mellonella
Based on previous literature [74,75], G. mellonella larvae with similar sizes were randomly divided into 5 groups, with 10 larvae in each group. They were treated as follows: VOR (120 ng/larvae), WT, WT + VOR (120 ng/larvae), ΔArp9 and ΔArp9 + VOR (120 ng/larvae). The spore suspension was diluted in the same manner and 5 μL was injected into larvae. The above antibiotic solutions (120 ng/larvae for VOR, 320 ng/larvae for AMB) were injected according to the group assignments at 1 h after the spore injection. The larvae were observed and documented over a period of 5 d or 6 d.
RNA-seq analysis
The WT and ΔArp9 strains were cultured in RPMI 1640 for 48 h. The hyphae were then collected and frozen in liquid nitrogen. RNA isolation, mRNA purification, cDNA synthesis and sequencing were performed by Wuhan IGENEBOOK Biotechnology Co., Ltd. Data was analyzed according to the previous study [72].
Chromatin accessibility real-time PCR (CHART-PCR) analysis
The experiments were performed based on the previous method with slight modifications [76,77]. WT and ΔArp9 strains were inoculated separately into PDB at 37°C, shaking at 180 rpm for 48 h. Then, the mycelium was collected and lyzed with DNaseⅠ (Vazyme, Nanjing, China). DNase I hypersensitive sites (DHSs) are regions of chromatin highly sensitive to DNase I enzyme, typically located within open chromatin domains. This open chromatin structure permits binding of transcription factors and other regulatory proteins, thereby governing gene expression. Consequently, DNase I-treated chromatin samples were extracted using phenol-chloroform. Finally, chromatin accessibility levels of promoter regions were analyzed using the QuantStudio 1 Plus real-time PCR system (Thermo Fisher Scientific, USA). In this experiment, the promoter regions of genes (Erg11A, AtrF, Mdr1, Erg3, Erg6) was predicted by BDGP (https://fruitfly.org/seq_tools/promoter.html) website, and corresponding primer pairs for these promoter regions were designed using NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi). All relative primers were listed in S2 Table. The relative chromatin accessibility index (CAI) was calculated through 2−ΔΔCT and normalized by tublin.
Co-immunoprecipitation (Co-IP)
To detect the interaction between Arp9 and Sfh1 in A. flavus, we grew the Arp9–3 × HA + Sfh1-Strep strain in PDB medium at 180 rpm for 48 h. After ground in liquid nitrogen, IP lysis buffer (Beyotime, Shanghai, China) was added to lyse proteins, then centrifuged at 4°C to collect supernatant. The supernatant was incubated overnight at 4°C with Mouse IgG Magnetic Beads (Beyotime, Shanghai, China) and Streptavidin Magnetic Beads (MedChemExpress, Shanghai, China). Target proteins were eluted from beads, with residual supernatants serving as the Input group. Samples underwent SDS-PAGE, transferred to PVDF membranes, and finally subjected to Western blotting using rabbit Anti-HA-tag Antibody (Beyotime, Shanghai, China) and rabbit Anti-Strep-tag Ⅱ antibody (Abcam, Cambridge, UK). For Arp9–3 × HA + Swp82-Strep, the same procedure was performed as the above.
MNase assay
The experiments were performed based on the previous method with slight modifications [76]. WT and ΔArp9 strains were inoculated separately with PDB at 37°C, shaking at 180 rpm for 48 h. Then, mycelium was ground into powder via liquid nitrogen, 1 mL nuclease buffer for each 0.1 g mycelium was added, mixed thoroughly, and divided into three equal portions to serve as the untreated control group (0 U MNase ((Takara, Tokyo, Japan)), the 10 U MNase-treated group, and the 40 U MNase-treated group, respectively. After Incubated at 37°C for 5 minutes, the reaction was immediately terminated and DNA was extracted. Finally, the digestion productions were analyzed by gel electrophoresis using 1.5% agarose.
The construction of mutant strains
All mutant strains, including Erg3, Erg6, Erg11A, AtrF and Mdr1 gene knock-out strains (ΔErg3, ΔErg6, ΔErg11A, ΔAtrF and ΔMdr1) and their double knockout strains with Arp9 (ΔArp9/ΔErg3, ΔArp9/ΔErg6, ΔArp9/ΔErg11A, ΔArp9/ΔAtrF and ΔArp9/ΔMdr1), were constructed following the protocol of homologous recombination [78] and the detail protocol was as described in our previous study [79]. The related primers were listed in S3 Table.
Ergosterol quantitative analysis
WT and ΔArp9 were inoculated into liquid GMM and cultured at 37°C shaking at 180 r/min for 48 h. Then, the hyphae were collected and ground in liquid nitrogen, and finally the ergosterol content was determined by HPLC in Keming Biotechnology Co., Ltd. (Suzhou, China).
Aflatoxin analysis
The spores (104/mL) of WT, ΔArp9, and CAMS-CCPM-D06242-4 strains were inoculated into 10 mL of PDB, PDB + 0.25 μg/mL VOR or added with an equal volume of DMSO, and cultured in dark at 35°C, 37°C, 39°C for 7 d. The extraction and detection methods are performed following the previous experiments [79].
Supporting information
S1 Fig. Drug-resistance analysis of gene deletion mutants related to the histone methylation modification in A. flavus.
The spores of above fungal strains were diluted to 2 × 106, 2 × 105, 2 × 104, and 2 × 103 conidia/mL, respectively, and 1 μL of the above spore suspension was inoculated onto MH medium containing corresponding antifungal drugs (0.33 or 0.67 μg/mL AMB, 0.25 or 0.5 μg/mL VOR). The growth status was observed after 48 h incubation at 37°C.
https://doi.org/10.1371/journal.ppat.1014021.s001
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S2 Fig. Bioinformatics analysis of Arp9.
(A) Evolutionary relationship between Arp9 from A. flavus NRRL 3357 and other 10 species (Aspergillus oryzae RIB40, Aspergillus fumigatus Af293, Aspergillus parasiticus, Homo sapiens, Mus musculus, Saccharomyces cerevisiae S288C, Arabidopsis thaliana, Candidozyma auris, Aspergillus niger, Aspergillus turcosus). (B) Analysis of the domain of homologous Arp9 from A. flavus and other species.
https://doi.org/10.1371/journal.ppat.1014021.s002
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S3 Fig. Arp9 plays an important role in the fungal resistance to AMB and VOR.
(A) Spores of WT, ΔArp9, and Com-Arp9 were diluted to 1 × 107 and 1 μL spore suspension was inoculated onto MH medium containing 0.67 μg/mL AMB. The growth state was observed after 96 h of incubation at 37°C. (B) The inhibition rate of 0.67 μg/mL AMB against the above-mentioned strains. (C) Spores of the above-mentioned strains were diluted to 1 × 107 and 1 μL suspension was inoculated onto MH medium containing 0.25 μg/mL VOR. The growth state was observed after 96 h of incubation at 37°C. (D) The inhibition rate of 0.25 μg/mL VOR against the above-mentioned strains.
https://doi.org/10.1371/journal.ppat.1014021.s003
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S4 Fig. The hemocytes in the hemolymph of G. mellonella larvae after injection with related A. flavus strains.
The spores of WT, ΔArp9, and Com-Arp9 strains were diluted to 1 × 107 conidia/mL with physiological saline and 5 μL was injected into G. mellonella larvae. Hemocytes were collected at 36 h post-injection and observed under a microscope with a scale of 100 μm.
https://doi.org/10.1371/journal.ppat.1014021.s004
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S5 Fig. RNA-seq analysis of the role of Arp9 in A. flavus drug-resistance.
(A) Two-dimensional principal component analysis (PCA) of fungal samples, including two ΔArp9 strains (Arp9_CK, the red dots) and two WT strains (WT_CK, the blue dots). (B) A volcano plot reflecting the distribution of differentially expressed genes (DEGs). Red is up-regulated, blue is down-regulated, and gray is unchanged ones.
https://doi.org/10.1371/journal.ppat.1014021.s005
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S6 Fig. Schematic representation of the promoter position of drug resistance-related genes (Erg11A, AtrF, Mdr1, Erg3, Erg6).
Promoter prediction of the related genes was performed using the BDGP (https://fruitfly.org/seq_tools/promoter.html) website, where the red boxes located represents the position of predicted promoter.
https://doi.org/10.1371/journal.ppat.1014021.s006
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S7 Fig. Ergosterol synthesis pathway showing the key nodes adjusted by Arp9.
In this pathway, solid red color boxes indicate up-regulated genes, while solid blue color boxes indicate down-regulated genes. Ergosterol is indicated by the red hollow box.
https://doi.org/10.1371/journal.ppat.1014021.s007
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S8 Fig. Co-IP analysis was performed to identify the interactions between Arp9 and its complex subunits, Sfh1 and Swp82.
https://doi.org/10.1371/journal.ppat.1014021.s008
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S9 Fig. Arp9 is essential for maintaining chromatin structural accessibility.
The gel electrophoregram with 1.5% gel showed the distribution of nucleosomes from WT and ∆Arp9 after MNase digestion for 5 minutes. M: DNA Marker (5,000 bp), T: trinucleosome, D: dinucleosome, M: mononucleosome.
https://doi.org/10.1371/journal.ppat.1014021.s009
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S3 Table. Primers used in the construction of fungal strains.
https://doi.org/10.1371/journal.ppat.1014021.s012
(PDF)
Acknowledgments
We especially thank Professor Jun Yuan, Xiuna Wang, Yu Wang and Xinyi Nie for their support in instrument maintenance and reagent ordering. And all authors would like to express their deepest gratitude to Professor Zhengyu Shu of Fujian Normal University for his generous donation of the YE13 strains used in this study. We are truly grateful for their continued support and encouragement throughout this project.
References
- 1. Rudramurthy SM, Paul RA, Chakrabarti A, Mouton JW, Meis JF. Invasive Aspergillosis by Aspergillus flavus: Epidemiology, Diagnosis, Antifungal Resistance, and Management. J Fungi (Basel). 2019;5(3):55. pmid:31266196
- 2. Boyer J, Feys S, Zsifkovits I, Hoenigl M, Egger M. Treatment of Invasive Aspergillosis: How It’s Going, Where It’s Heading. Mycopathologia. 2023;188(5):667–81. pmid:37100963
- 3. Arastehfar A, Carvalho A, Houbraken J, Lombardi L, Garcia-Rubio R, Jenks JD, et al. Aspergillus fumigatus and aspergillosis: From basics to clinics. Stud Mycol. 2021;100:100115. pmid:34035866
- 4. Hoenigl M, Seidel D, Sprute R, Cunha C, Oliverio M, Goldman GH, et al. COVID-19-associated fungal infections. Nat Microbiol. 2022;7(8):1127–40. pmid:35918423
- 5. Nesbitt BF, O’Kelly J, Sargeant K, Sheridan A. Aspergillus flavus and turkey X disease. Toxic metabolites of Aspergillus flavus. Nature. 1962;195:1062–3. pmid:14479064
- 6. Zhao Y, Zeng R, Chen P, Huang C, Xu K, Huang X, et al. Transcriptomic and Proteomic Insights into the Effect of Sterigmatocystin on Aspergillus flavus. J Fungi (Basel). 2023;9(12):1193. pmid:38132793
- 7. Parums DV. Editorial: The World Health Organization (WHO) Fungal Priority Pathogens List in Response to Emerging Fungal Pathogens During the COVID-19 Pandemic. Med Sci Monit. 2022;28:e939088. pmid:36453055
- 8. Patterson TF, Thompson GR 3rd, Denning DW, Fishman JA, Hadley S, Herbrecht R, et al. Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis. 2016;63(4):e1–60. pmid:27365388
- 9. Verweij PE, Chowdhary A, Melchers WJG, Meis JF. Azole Resistance in Aspergillus fumigatus: Can We Retain the Clinical Use of Mold-Active Antifungal Azoles?. Clin Infect Dis. 2016;62(3):362–8. pmid:26486705
- 10. Cavassin FB, Baú-Carneiro JL, Vilas-Boas RR, Queiroz-Telles F. Sixty years of Amphotericin B: An Overview of the Main Antifungal Agent Used to Treat Invasive Fungal Infections. Infect Dis Ther. 2021;10(1):115–47. pmid:33523419
- 11. Maji A, Soutar CP, Zhang J, Lewandowska A, Uno BE, Yan S, et al. Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal. Nature. 2023;623(7989):1079–85. pmid:37938782
- 12. Vandeputte P, Tronchin G, Larcher G, Ernoult E, Bergès T, Chabasse D, et al. A nonsense mutation in the ERG6 gene leads to reduced susceptibility to polyenes in a clinical isolate of Candida glabrata. Antimicrob Agents Chemother. 2008;52(10):3701–9. pmid:18694952
- 13. Carolus H, Pierson S, Muñoz JF, Subotić A, Cruz RB, Cuomo CA, et al. Genome-Wide Analysis of Experimentally Evolved Candida auris Reveals Multiple Novel Mechanisms of Multidrug Resistance. mBio. 2021;12(2):e03333-20. pmid:33820824
- 14. Tian S, Rong C, Li H, Wu Y, Wu N, Chu Y, et al. Genetic microevolution of clinical Candida auris with reduced Amphotericin B sensitivity in China. Emerg Microbes Infect. 2024;13(1):2398596. pmid:39234778
- 15. Shivarathri R, Jenull S, Chauhan M, Singh A, Mazumdar R, Chowdhary A, et al. Comparative Transcriptomics Reveal Possible Mechanisms of Amphotericin B Resistance in Candida auris. Antimicrob Agents Chemother. 2022;66(6):e0227621. pmid:35652307
- 16. Pérez-Cantero A, López-Fernández L, Guarro J, Capilla J. Azole resistance mechanisms in Aspergillus: update and recent advances. Int J Antimicrob Agents. 2020;55(1):105807. pmid:31542320
- 17. Tobin MB, Peery RB, Skatrud PL. Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus. Gene. 1997;200(1–2):11–23. pmid:9373135
- 18. Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, et al. Efflux-mediated antifungal drug resistance. Clin Microbiol Rev. 2009;22(2):291–321, Table of Contents. pmid:19366916
- 19. Fraczek MG, Bromley M, Buied A, Moore CB, Rajendran R, Rautemaa R, et al. The cdr1B efflux transporter is associated with non-cyp51a-mediated itraconazole resistance in Aspergillus fumigatus. J Antimicrob Chemother. 2013;68(7):1486–96. pmid:23580559
- 20. Fisher MC, Hawkins NJ, Sanglard D, Gurr SJ. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science. 2018;360(6390):739–42. pmid:29773744
- 21. Zakaria A, Osman M, Dabboussi F, Rafei R, Mallat H, Papon N, et al. Recent trends in the epidemiology, diagnosis, treatment, and mechanisms of resistance in clinical Aspergillus species: A general review with a special focus on the Middle Eastern and North African region. J Infect Public Health. 2020;13(1):1–10. pmid:31672427
- 22. Vila T, Romo JA, Pierce CG, McHardy SF, Saville SP, Lopez-Ribot JL. Targeting Candida albicans filamentation for antifungal drug development. Virulence. 2017;8(2):150–8. pmid:27268130
- 23. Wall G, Lopez-Ribot JL. Screening Repurposing Libraries for Identification of Drugs with Novel Antifungal Activity. Antimicrob Agents Chemother. 2020;64(9):e00924-20. pmid:32660991
- 24. Hoenigl M, Sprute R, Egger M, Arastehfar A, Cornely OA, Krause R, et al. The Antifungal Pipeline: Fosmanogepix, Ibrexafungerp, Olorofim, Opelconazole, and Rezafungin. Drugs. 2021;81(15):1703–29. pmid:34626339
- 25. Donlin MJ, Meyers MJ. Repurposing and optimization of drugs for discovery of novel antifungals. Drug Discov Today. 2022;27(7):2008–14. pmid:35489676
- 26. Lee Y, Robbins N, Cowen LE. Molecular mechanisms governing antifungal drug resistance. NPJ Antimicrob Resist. 2023;1(1):5. pmid:38686214
- 27. Patra S, Raney M, Pareek A, Kaur R. Epigenetic Regulation of Antifungal Drug Resistance. J Fungi (Basel). 2022;8(8):875. pmid:36012862
- 28. Chang Z, Yadav V, Lee SC, Heitman J. Epigenetic mechanisms of drug resistance in fungi. Fungal Genet Biol. 2019;132:103253. pmid:31325489
- 29. Balachandra VK, Ghosh SK. Emerging roles of SWI/SNF remodelers in fungal pathogens. Curr Genet. 2022;68(2):195–206. pmid:35001152
- 30. Meng Y, Ni Y, Li Z, Jiang T, Sun T, Li Y, et al. Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans. Elife. 2024;13:e85728. pmid:38251723
- 31. Liu Z, Myers LC. Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance. Antimicrob Agents Chemother. 2017;61(11):e01344-17. pmid:28807921
- 32. Song Z-T, Liu J-X, Han J-J. Chromatin remodeling factors regulate environmental stress responses in plants. J Integr Plant Biol. 2021;63(3):438–50. pmid:33421288
- 33. Wanior M, Krämer A, Knapp S, Joerger AC. Exploiting vulnerabilities of SWI/SNF chromatin remodelling complexes for cancer therapy. Oncogene. 2021;40(21):3637–54. pmid:33941852
- 34. Zhu L, Liu Q, Zhao C, Zhu M, Yang X, Yang J. Structural and functional characterisation and regulatory mechanisms of SWI/SNF and RSC chromatin remodelling complexes in fungi. Mycology. 2025;16(3):988–1010. pmid:40937128
- 35. Borman AM. Of mice and men and larvae: Galleria mellonella to model the early host-pathogen interactions after fungal infection. Virulence. 2018;9(1):9–12. pmid:28933671
- 36. Sheehan G, Kavanagh K. Analysis of the early cellular and humoral responses of Galleria mellonella larvae to infection by Candida albicans. Virulence. 2018;9(1):163–72. pmid:28872999
- 37. De Francesco MA. Drug-Resistant Aspergillus spp.: A Literature Review of Its Resistance Mechanisms and Its Prevalence in Europe. Pathogens. 2023;12(11):1305. pmid:38003770
- 38. Fakhim H, Badali H, Dannaoui E, Nasirian M, Jahangiri F, Raei M, et al. Trends in the Prevalence of Amphotericin B-Resistance (AmBR) among Clinical Isolates of Aspergillus Species. J Mycol Med. 2022;32(4):101310. pmid:35907396
- 39. Liu Z, Jian Y, Chen Y, Kistler HC, He P, Ma Z, et al. A phosphorylated transcription factor regulates sterol biosynthesis in Fusarium graminearum. Nat Commun. 2019;10(1):1228. pmid:30874562
- 40. Nikolov VN, Malavia D, Kubota T. SWI/SNF and the histone chaperone Rtt106 drive expression of the Pleiotropic Drug Resistance network genes. Nat Commun. 2022;13(1):1968. pmid:35413952
- 41. Clapier CR, Kasten MM, Parnell TJ, Viswanathan R, Szerlong H, Sirinakis G, et al. Regulation of DNA Translocation Efficiency within the Chromatin Remodeler RSC/Sth1 Potentiates Nucleosome Sliding and Ejection. Mol Cell. 2016;62(3):453–61. pmid:27153540
- 42. Schubert HL, Wittmeyer J, Kasten MM, Hinata K, Rawling DC, Héroux A, et al. Structure of an actin-related subcomplex of the SWI/SNF chromatin remodeler. Proc Natl Acad Sci U S A. 2013;110(9):3345–50. pmid:23401505
- 43. Kowalski CH, Morelli KA, Schultz D, Nadell CD, Cramer RA. Fungal biofilm architecture produces hypoxic microenvironments that drive antifungal resistance. Proc Natl Acad Sci U S A. 2020;117(36):22473–83. pmid:32848055
- 44. Ciofu O, Moser C, Jensen PØ, Høiby N. Tolerance and resistance of microbial biofilms. Nat Rev Microbiol. 2022;20(10):621–35. pmid:35115704
- 45. Finkel JS, Xu W, Huang D, Hill EM, Desai JV, Woolford CA, et al. Portrait of Candida albicans adherence regulators. PLoS Pathog. 2012;8(2):e1002525. pmid:22359502
- 46. Barrales RR, Korber P, Jimenez J, Ibeas JI. Chromatin modulation at the FLO11 promoter of Saccharomyces cerevisiae by HDAC and Swi/Snf complexes. Genetics. 2012;191(3):791–803. pmid:22542969
- 47. Jemel S, Guillot J, Kallel K, Botterel F, Dannaoui E. Galleria mellonella for the Evaluation of Antifungal Efficacy against Medically Important Fungi, a Narrative Review. Microorganisms. 2020;8(3):390. pmid:32168839
- 48. Balachandra VK, Verma J, Shankar M, Tucey TM, Traven A, Schittenhelm RB, et al. The RSC (Remodels the Structure of Chromatin) complex of Candida albicans shows compositional divergence with distinct roles in regulating pathogenic traits. PLoS Genet. 2020;16(11):e1009071. pmid:33151931
- 49. Liu L, Wang Q, Sun Y, Zhang Y, Zhang X, Liu J, et al. Sssfh1, a Gene Encoding a Putative Component of the RSC Chromatin Remodeling Complex, Is Involved in Hyphal Growth, Reactive Oxygen Species Accumulation, and Pathogenicity in Sclerotinia sclerotiorum. Front Microbiol. 2018;9:1828. pmid:30131794
- 50. Jemel S, Guillot J, Kallel K, Jouvion G, Brisebard E, Billaud E, et al. In Vivo Efficacy of Voriconazole in a Galleria mellonella Model of Invasive Infection Due to Azole-Susceptible or Resistant Aspergillus fumigatus Isolates. J Fungi (Basel). 2021;7(12):1012. pmid:34946994
- 51. Forastiero A, Bernal-Martínez L, Mellado E, Cendejas E, Gomez-Lopez A. In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection. Int J Antimicrob Agents. 2015;46(5):511–7. pmid:26358971
- 52. Snelders E, Karawajczyk A, Schaftenaar G, Verweij PE, Melchers WJG. Azole resistance profile of amino acid changes in Aspergillus fumigatus CYP51A based on protein homology modeling. Antimicrob Agents Chemother. 2010;54(6):2425–30. pmid:20385860
- 53. Li Y, Zhang Y, Zhang C, Wang H, Wei X, Chen P, et al. Mitochondrial dysfunctions trigger the calcium signaling-dependent fungal multidrug resistance. Proc Natl Acad Sci U S A. 2020;117(3):1711–21. pmid:31811023
- 54. Li Y, Dai M, Lu L, Zhang Y. The C2H2-Type Transcription Factor ZfpA, Coordinately with CrzA, Affects Azole Susceptibility by Regulating the Multidrug Transporter Gene atrF in Aspergillus fumigatus. Microbiol Spectr. 2023;11(4):e0032523. pmid:37318356
- 55. Silva LN, Oliveira SSC, Magalhães LB, Andrade Neto VV, Torres-Santos EC, Carvalho MDC, et al. Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex. ACS Infect Dis. 2020;6(5):1273–82. pmid:32239912
- 56. Zhang Y, Zeng L, Huang X, Wang Y, Chen G, Moses M, et al. Targeting epigenetic regulators to overcome drug resistance in the emerging human fungal pathogen Candida auris. Nat Commun. 2025;16(1):4668. pmid:40394068
- 57. Kannan A, Asner SA, Trachsel E, Kelly S, Parker J, Sanglard D. Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae. mBio. 2019;10(6):e02512-19. pmid:31874914
- 58. Sanglard D, Ischer F, Parkinson T, Falconer D, Bille J. Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents Chemother. 2003;47(8):2404–12. pmid:12878497
- 59. Young LY, Hull CM, Heitman J. Disruption of ergosterol biosynthesis confers resistance to amphotericin B in Candida lusitaniae. Antimicrob Agents Chemother. 2003;47(9):2717–24. pmid:12936965
- 60. Ahmad S, Joseph L, Parker JE, Asadzadeh M, Kelly SL, Meis JF, et al. ERG6 and ERG2 Are Major Targets Conferring Reduced Susceptibility to Amphotericin B in Clinical Candida glabrata Isolates in Kuwait. Antimicrob Agents Chemother. 2019;63(2):e01900-18. pmid:30455247
- 61. Yu P, Zhou M, Yu D, Zhang Z, Ye S, Yu Y, et al. Targeted regulation of sterol biosynthesis genes according to perturbations in ergosterol biosynthesis in fungi. J Adv Res. 2025;77:341–56. pmid:39892608
- 62. Zhou S, Ismail MAI, Aimanianda V, de Hoog GS, Kang Y, Ahmed SA. Aflatoxin profiles of Aspergillus flavus isolates in Sudanese fungal rhinosinusitis. Med Mycol. 2024;62(4):myae034. pmid:38578660
- 63. Mosquera J, Warn PA, Morrissey J, Moore CB, Gil-Lamaignere C, Denning DW. Susceptibility testing of Aspergillus flavus: inoculum dependence with itraconazole and lack of correlation between susceptibility to amphotericin B in vitro and outcome in vivo. Antimicrob Agents Chemother. 2001;45(5):1456–62. pmid:11302810
- 64. Guinea J MJ A-AS, Muehlethaler K, Kahlmeter G, Arendrup MC. EUCAST Definitive Document E.Def 9.4. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds 2022. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_EDef_9.4_method_for_susceptibility_testing_of_moulds.pdf
- 65. Espinel-Ingroff A, Cantón E, Pemán J. Antifungal Susceptibility Testing of Filamentous Fungi. Curr Fungal Infect Rep. 2012;6(1):41–50.
- 66. Yang C, Wu D, Lin H, Ma D, Fu W, Yao Y, et al. Role of RNA Modifications, Especially m6A, in Aflatoxin Biosynthesis of Aspergillus flavus. J Agric Food Chem. 2024;72(1):726–41. pmid:38112282
- 67. Chen Y, Le Mauff F, Wang Y, Lu R, Sheppard DC, Lu L, et al. The Transcription Factor SomA Synchronously Regulates Biofilm Formation and Cell Wall Homeostasis in Aspergillus fumigatus. mBio. 2020;11(6):e02329-20. pmid:33173002
- 68. Kirchhoff L, Dittmer S, Furnica D-T, Buer J, Steinmann E, Rath P-M, et al. Inhibition of azole-resistant Aspergillus fumigatus biofilm at various formation stages by antifungal drugs, including olorofim. J Antimicrob Chemother. 2022;77(6):1645–54. pmid:35289361
- 69. Long N, Orasch T, Zhang S, Gao L, Xu X, Hortschansky P, et al. The Zn2Cys6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus. PLoS Genet. 2018;14(10):e1007762. pmid:30365497
- 70. Huang Z, Wu D, Yang S, Fu W, Ma D, Yao Y, et al. Regulation of Fungal Morphogenesis and Pathogenicity of Aspergillus flavus by Hexokinase AfHxk1 through Its Domain Hexokinase_2. J Fungi (Basel). 2023;9(11):1077. pmid:37998882
- 71. Chen X, Wu L, Lan H, Sun R, Wen M, Ruan D, et al. Histone acetyltransferases MystA and MystB contribute to morphogenesis and aflatoxin biosynthesis by regulating acetylation in fungus Aspergillus flavus. Environ Microbiol. 2022;24(3):1340–61. pmid:34863014
- 72. Hao L, Zhang M, Yang C, Pan X, Wu D, Lin H, et al. The epigenetic regulator Set9 harmonizes fungal development, secondary metabolism, and colonization capacity of Aspergillus flavus. Int J Food Microbiol. 2023;403:110298. pmid:37392609
- 73. Shrestha S, Kim Y. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2). J Invertebr Pathol. 2007;96(1):64–70. pmid:17395196
- 74. Gu W, Yu Q, Yu C, Sun S. In vivo activity of fluconazole/tetracycline combinations in Galleria mellonella with resistant Candida albicans infection. J Glob Antimicrob Resist. 2018;13:74–80. pmid:29191612
- 75. Passos JS, de Martino LC, Dartora VFC, de Araujo GLB, Ishida K, Lopes LB. Development, skin targeting and antifungal efficacy of topical lipid nanoparticles containing itraconazole. Eur J Pharm Sci. 2020;149:105296. pmid:32151706
- 76. Gonzalez R, Scazzocchio C. A rapid method for chromatin structure analysis in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 1997;25(19):3955–6. pmid:9380523
- 77. Mello-de-Sousa TM, Rassinger A, Pucher ME, dos Santos Castro L, Persinoti GF, Silva-Rocha R, et al. The impact of chromatin remodelling on cellulase expression in Trichoderma reesei. BMC Genomics. 2015;16(1):588. pmid:26248555
- 78. Zhuang Z, Pan X, Zhang M, Liu Y, Huang C, Li Y, et al. Set2 family regulates mycotoxin metabolism and virulence via H3K36 methylation in pathogenic fungus Aspergillus flavus. Virulence. 2022;13(1):1358–78. pmid:35943142
- 79. Pan X, Hao L, Yang C, Lin H, Wu D, Chen X, et al. SWD1 epigenetically chords fungal morphogenesis, aflatoxin biosynthesis, metabolism, and virulence of Aspergillus flavus. J Hazard Mater. 2023;455:131542. pmid:37172387