The A. fumigatus standard strain AF293, ITR-resistant clinical isolates (AF1 and AF2), and VRC-resistant clinical strains (AF4 and AF5) were routinely grown on potato dextrose agar (PDA). Before each experiment, the strains used in this study, were cultured on PDA medium for 4 days at 37°C. The drugs, including ITR, VRC, and AmB, were purchased from Macklin Chemicals (Shanghai, China). SNH was obtained from Fengyao Tonghui Chemicals (Wuhan, China). ITR, VRC, and AmB were dissolved in DMSO to prepare a 5.12 mg/mL stock solution. SNH was dissolved in sterile distilled water with 0.05% Tween 80 to prepare a 5 mg/mL stock solution. All drug stock solutions were stored at –20°C.

The minimal inhibitory concentration (MIC) of ITR, VRC, AmB, and SNH inhibiting growth of A. fumigatus were tested according to the reference Clinical and Laboratory Standards Institute M38-A2 document (CLSI, 2008; Espinel-Ingroff et al., 2010). The drug concentrations ranged from 0.06 to 32 μg/mL for both ITR and VRC, from 0.03 to 16 μg/mL for AmB, and from 0.0625 to 2 mg/mL for SNH. The conidia were dispersed in RPMI-1640 medium at a concentration of 0.4 × 10⁴ to 5 × 10⁴ cells/mL. The suspensions were then dispensed into triplicate wells of a 96-well microtiter plate. After incubation at 37°C for 48 h, MIC endpoints were defined as the lowest drug concentrations that caused complete visible inhibition of growth, as compared with the drug-free growth control. The criteria for determining the antifungal susceptibility of molds followed the CLSI M38-A2 document (Espinel-Ingroff et al., 2010). Experiments in each strain were performed in triplicate.

Interactions between SNH and antifungal agents (ITR or VRC) against the test strain were assessed with the MIC method as described above. The final drug concentration SNH ranged from 0.01 to 0.13 mg/mL, and those of antifungal agents (ITR or VRC) ranged from 0.03 to 32 μg/mL. The fractional inhibitory concentration index (FICI) was defined according to the M38-A2 document (Odds, 2003).

The sporulation of A. fumigatus was evaluated by assessment of the initial growth of a droplet of conidia from a serial dilution at final concentrations of 0 × (negative control), 1 ×, 2 ×, and 4 × MIC at two temperatures (37 and 28°C). Spot dilution experiments were performed with 3 μL of a 10-fold dilution series starting at a concentration of 10⁵ cells/mL spotted on PDA solid medium at the two temperatures for 96 h (37°C) and 120 h (28°C). Samples were collected from approximately the same three locations of colonies with an Oxford cup (Beijing Jitai Yuancheng Technology Co., Ltd., Beijing, China) and counted with a hemacytometer to estimate the average sporulation per square centimeter. Meanwhile, the conidial melanin/pigment, isolated from 4-day-old cultures with 2% NaOH, was purified and measured as described previously (Song et al., 2018).

Fresh conidia were prepared with sterile phosphate buffered saline (PBS) and cultured at 37°C in liquid PDA medium at a concentration of 10⁶ cells/mL with various concentrations of SNH as described above. Then conidia were collected after 12, 16, and 20 h incubation. Germination was defined by a germ tube longer than the conidia diameter (Chen et al., 2020). The number of germinated conidia under a microscope (400 ×).

The metabolic activity of biofilms was monitored with tetrazolium salt 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)-carbonyl-2H-tetrazoliumhydroxide (XTT; Macklin, Shanghai, China) reduction assay (Bugli et al., 2013; Bom et al., 2015; González-Ramírez et al., 2016). Briefly, for the biofilm formation assays, A. fumigatus spore suspension was prepared in RPMI-1640 medium supplement at a concentration of 10⁶ cells/mL with the above-described concentration of SNH, and the 96-well polystyrene plates were incubated at 37°C for 24 h. For the preformed biofilms, 200 μL of conidial suspension (10⁶ cells/mL) was added into each well of a microtiter plate and incubated at 37°C for 24 h. Next, the plate was washed with PBS three times. Then 200 μL SNH, at the above-described concentrations, prepared in RPMI-1640 medium was added. After incubation at 37°C for 24 h, the plates were incubated with XTT-menadione solution in the dark at 37°C for 2 h. Finally, the absorbance of the supernatant was measured at 492 nm (Pumeesat et al., 2017).

The confocal laser scanning microscopy (CLSM) was performed to observe biofilm developed on the coverslips (Shanghai Baiyan Bio-Technology Co., Ltd, Shanghai, China) in 12-well plates (Shanghai Huipan Industerial Co., Ltd, Shanghai, China). Method was following the previous investigation with slight modifications (Iwahashi et al., 2020). Briefly, the biofilms were washed three times with PBS and stained with calcofluor white stain (Sigma-Aldrich Trading Co., Ltd, Shanghai, China) supplemented with 20 μL 10% KOH at room temperature for 5 min. Fluorescent images were taken and analyzed by CLSM using OLYMPUS cellSens Dimension 3.2 (Olympus Co., Ltd, Tokyo, Japan).

To investigate the effects of concentration and exposure time on the activity of SNH, we performed time-kill experiments and the methodology referred to previous studies with slight modifications (Bugli et al., 2013). All strains were grown in RPMI-1640 medium with a starting inoculum of 10⁶ cells/mL. The SNH working concentrations were 1 ×, 2 ×, 4 ×, and 8 × MIC. Samples were incubated at 37°C without agitation. At predetermined time points (0, 4, 8, 12, 16, 20, and 24 h) after incubation, the samples were transferred to 96-well plates, and their metabolic activity was determined with XTT reduction assays. The method to detect the metabolic activity was as described above.

After treatment with SNH, propidium iodide (PI) staining assays were used to investigate the A. fumigatus cell membrane integrity (Chen et al., 2020). The inoculum of the AF293 strain was treated with the above-described concentrations of SNH and incubated at 37°C for 12 h. Then the samples were washed with PBS three times. PI (Solarbio, Beijing, China) was incubated with A. fumigatus at a concentration of 20 μg/mL for 30 min in the dark. The specimens were observed under a fluorescence microscope (Leica, Germany). Results represent three independent experiments.

Conidia of the AF293 strain were cultured in RPMI-1640 medium with or without SNH (2 × MIC) for 12 h at 37°C. Then samples were collected, treated with liquid nitrogen, and stored at –80°C for collection of total RNA. Total RNA was prepared with RNAiso plus reagent (TaKaRa, Dalian, China) and reverse-transcribed into cDNA with a PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa). Illumina RNA sequencing was then conducted by Biomarker Technologies (Qingdao, China). To determine time-specific expression patterns of genes in the ergosterol synthesis pathway, we treated the AF293 conidia prepared in RPMI-1640 medium with 2 × MIC SNH and then incubated them at 37°C for 0, 6, and 12 h for RNA extraction. RT-qPCR was performed with a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). Transcripts of the β-tubulin gene were used as an internal control. Transcript ratios were evaluated with the 2^{–Δ Δ CT} method (Vandesompele et al., 2002). The primer sequences are listed in Supplementary Table 1.

Conidia of A. fumigatus AF293 were prepared in RPMI-1640 medium at a concentration of 10⁶ cells/mL with final concentrations of 0, 1/2 ×, and 1 × MIC SNH. Samples were incubated at 37°C for 12 h. Then a 3 μL aliquot containing 10⁵ cells/mL was inoculated onto PDA medium containing 0, 1, 2, or 4 μg/mL AmB. After inoculation at 37°C for 4 days, colony morphology was investigated with a digital camera (60-mm Macro lens, Canon Inc., Japan).

To quantify the concentration of ergosterol, we incubated AF293 conidia in RPMI-1640 medium with different concentrations of SNH (1 ×, 2 ×, and 4 × MIC) at 37°C for 3 days. The mycelia were harvested and washed with PBS twice, as described previously (Pinto et al., 2011). Then 0.500 g samples were dispersed in 100% methanol for ultrasound-assisted extraction. The content of ergosterol was determined by high-performance liquid chromatography (HPLC; 1260 infinity II, Agilent). Ergosterol standard (Macklin, Shanghai, China) was dissolved with 100% methanol to a concentration of 100 μg/mL and used as a reference solution. The concentration of ergosterol was calculated with the following equations: (peak area of experimental group × concentration of ergosterol standard group)/peak area of ergosterol standard product. Experiments for each strain were performed in triplicate.

Male 6–8-week-old Balb/c mice (Chongqing Tengxin Biotechnology Co., Ltd., Chongqing, China) with a weight of 20–25 g was given food and water ad libitum. Mice were immunosuppressed by injection of cyclophosphamide at 200 mg/kg intravenously for 3 days (Denning et al., 1995, 1997). Then the mice were infected by injection of a 100 μL inoculum of A. fumigatus AF293 into the tail vein at a final concentration of 10⁷ cells/mL. After the successful establishment of an IA mouse model, treatment was started 2 h after inoculation and was continued for 7 days by gastric gavage with SNH at 10 mg/kg/d and 30 mg/kg/d. The same volume of normal saline and ITR with a concentration of 75 mg/kg/d were used in the control group.

Fungal burden and histopathology were used to evaluate the efficacy of SNH against IA. The liver, kidney, lung, and serum were collected after the mice were treated for 1, 4, and 7 days. One half of the collected liver, kidney, and lung samples was used to prepared dilutions of the homogenates and plated on PDA. The number of colony formation units per gram of tissue was determined after at least 48 h of incubation at 37°C. The other tissues were fixed in 10% methanol and embedded in paraffin. Thin sections were cut and stained with periodic acid-Schiff stain (PAS) for microscopic observations.

Cytokines were detected with a BD Cytometric Bead Array Mouse Inflammation Kit (Univ, Shanghai, China) in serum treated for 1 and 4 days. IL-6, IL-17A, IFN-γ, and TNF-α were detected according to the manufacturer’s protocol. Then samples were measured on a BD FACSCalibur Flow Cytometer and analyzed in FCAP Array Software Version 3.0 (BD Bioscience).

Each experiment was performed three independent times. Graphing and statistical analyses were performed in GraphPad Prism, version 7.0 (GraphPad Software Inc., La Jolla, CA, United States) with Student’s t-test. Cytokine data were compared with unpaired two-tailed Mann-Whitney (non-parametric) tests. Results represent the average of three independent experiments ± standard deviation (SD), and the level of statistical significance was set at *P < 0.05.

To evaluate the antifungal potential of SNH against A. fumigatus, we tested the MIC. The MIC of SNH against the AF293, AF1, and AF2 strains was 100 μg/mL, whereas that against AF4 and AF5 strains was 50 μg/mL. For AmB, the MIC for the AF293, AF1, and AF2 strains was 4 μg/mL, and was 0.5–1 μg/mL for AF4 and AF5. However, the FICI indicated that the combination ITR or VRC with SNH produced no synergistic effects against ITR- or VRC-resistant strains (data not shown). Furthermore, 1 ×, 2 ×, and 4 × MIC SNH against A. fumigatus was used to assess sporulation at 37 and 28°C. As shown in Figure 1 and Supplementary Figure 2, strains treated with SNH showed a significant decrease in conidial yield to 40–50% that in the untreated group (P < 0.05). Additionally, the A. fumigatus had a whitish color in the SNH treatment group. Further investigations confirmed that the conidia of SNH-treated strains accumulated less melanin than those of the control group (Figure 1G).

The anti-biofilm activity of SNH was evaluated with XTT reduction analysis. The metabolic activity of biofilm was significantly decreased by treatment with even low concentrations of SNH (Figure 2A; P < 0.05). In the mature biofilms pretreated with 1 × MIC, XTT reduction assays demonstrated that the inhibition rate was approximately 20%. However, compared with the control treatment, 4 × MIC treatment resulted in an 80% lower inhibition rate (Figure 2B). Next, the fluorescent filamentous biomass of biofilms was examined by the CLSM. Compared with control group, as shown in Figures 2C,D, A. fumigatus mycelium length was shortened and the mature biofilm was thinner after SNH treatment. Hyphal growth, the basis of biofilm formation, was modest at 1 × and 2 × MIC; however, hyphal growth was absent at 4 × MIC (Figure 3). These results correlated with the results of the XXT reduction assays.

Time-kill assays were used to investigate the kinetics of SNH against A. fumigatus (Figures 4A–E). Similar to amphotericin B (AmB), SNH exhibited fungistatic activity against all tested A. fumigatus strains at a concentration of 0.4 mg/mL. Further investigations were evaluated with PI staining. Statistical analysis indicated that the percentage of PI-stained cells rose to 42.37, 66.23, and 79.72% after treatment with SNH at concentrations of 0.1, 0.2, and 0.4 mg/mL, respectively (Figure 4F). In particular, the percentage of PI-stained cells treated with the 4 × MIC approached that in the AmB treatment group. These results revealed that SNH exhibited fungicidal activity against A. fumigatus.

After verifying SNH’s potent antifungal activity, we proceeded to investigate the antifungal mechanism of SNH. Transcriptomic changes in A. fumigatus treated with SNH were analyzed with Illumina sequencing, and 2.83 Gb clean data were obtained from each sample, with differential expression of 175 annotated genes. Raw sequence data have been deposited in the Beijing Institute of Genomics Genome Sequence Archive (accession number PRJCA007831). Genes with fold change ≥ 2 and false discovery rate (FDR) < 0.01 were considered significantly differentially expressed. The results showed that 66 genes were up-regulated and 109 genes were down-regulated (Supplementary Figure 3A). These differentially expressed genes (DEGs) were functionally grouped into gene ontology classes including 39 functional categories (Supplementary Figure 3B) and 16 clusters of orthologous groups (Supplementary Figure 3C). The DEGs were also assigned to 50 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The main KEGG enrichment pathway was the steroid biosynthesis pathway (Figure 5A).

To verify the transcriptomic results described above, we investigated genes associated with the ergosterol biosynthetic gene pathway through qRT-PCR analysis. In accordance with the transcriptomic data, the expression of 14 genes, including Erg2 (C-8 sterol isomerase, putative), Erg3 (Sterol desaturase), Erg4 [C-24(28) sterol reductase], Erg5 (Cytochrome P450 sterol C-22 desaturase, putative), Erg6 (Sterol 24-c-methyltransferase, putative), Erg7 (Oxidosqualene: lanosterol cyclase), Erg26 (C-3 sterol dehydrogenase/C-4 decarboxylase), and Erg27 (3-ketosteroid reductase), were down-regulated after SNH treatment. Furthermore, four other genes, Erg1 (Squalene monooxygenase), Erg24 (C-14 sterol reductase), Erg25 (C-4 methyl sterol oxidase), and Cyp51A (14-alpha sterol demethylase), were up-regulated (Figure 5B). These data suggested that the antifungal activity of SNH against A. fumigatus involved interference with the steroid synthesis pathway. We further verified the antifungal mechanism on ergosterol biosynthesis by performing cell membrane stress assays. The strain treated with SNH showed significantly diminished sensitivity to AmB (Figure 5C). HPLC results (Figure 5D) were determined after treatment with SNH by 1 ×, 2 ×, and 4 × MIC, and the concentration of ergosterol decreased by 31.31, 53.25, and 62.42%, respectively. In summary, these data indicated that SNH inhibited the growth of A. fumigatus by inhibiting ergosterol synthesis.

The IA murine model was used to assess the in vivo efficacy of antifungal activity. Compared with treatment with normal saline, treatment with SNH at doses of 10 mg/kg/day and 30 mg/kg/day significantly decreased the fungal burden in the liver, kidney, and lung (P < 0.05), and had effects equal to those of ITR from day 4 to day 7 (Figures 6A,C). Histopathology investigations using PAS-staining were consistent with the fungal burden results. Moreover, sections of PAS-stained kidney and lung were analyzed microscopically for abnormalities. The results revealed that the SNH or ITR treatment groups, compared with the treatment with normal saline, resulted in significantly fewer inflammatory cell infiltrates (Figure 6D), thus suggesting that repeated treatment with SNH was efficacious in IA mice.

To determine the effect of SNH on murine cellular immunity, we analyzed the serum of sacrificed mice 1 and 4 days after A. fumigatus challenge and drug treatment. Overall, the levels of IL-6 and IL-17A in mice with a treatment dose of SNH of 10 mg/kg/day or 30 mg/kg/day decreased (Figures 6E,F). On day 1, compared with the levels in untreated IA mice, the level of the pro-inflammatory cytokine IL-6 after treatment with SNH at a dose of 10 mg/kg/day was 3.26-fold lower, and that of IL-17A was 8-fold lower. After completion of 1 day of therapy in the SNH 30 mg/kg/day group, the level of IL-6 was decreased by 4.53-fold, and the level of IL-17A was undetectable. In addition, the levels of IFN-γ, and TNF-α were unchanged with respect to those in the sham control (Supplementary Figures 4B,C).