Sigma-Aldrich Inc. (Shanghai, China) provided the hinokitiol powder (CAS 499–44-5), which was then dissolved into dimethyl sulfoxide (DMSO) to a storage concentration of 32 mg/ml and diluted to acceptable working solutions to reach various final concentrations.

The standard A. fumigatus strain (CGMCC 3.14869) was purchased from China General Microbiological Culture Collection Center. Mycelium was obtained following the required number of days (mentioned in the required methods of different experiments) of conidia cultivation on the Sabouraud’s agar medium at 28°C. Conidia on the surface of the medium were collected, counted by a hemocytometer, and adjusted to a final concentration of 1 × 10⁷ /ml.

MIC was determined using the standard microdilution method developed by the American Clinical and Laboratory Standards Institute (CLSI) (Wootton et al., 2020). Conidia suspension (2 × 10⁵ CFU/ml) (CFU means colony-forming unit) was cultured with hinokitiol in various concentrations (2, 4, 8, 16, and 32 μg/ml) and 0.1% DMSO at 28°C in 96-well plates (100 μl per well). A microplate reader was used to read the optical density (OD) at 620 nm at 12 h, 24 h, 48 h, 72 h, and 96 h.

Conidia suspension (2 × 10⁵ CFU/ml) (CFU means colony-forming unit) was cultured with hinokitiol in various concentrations (2, 4, 8, 16, 32,64, and 128 μg/ml) and 0.1% DMSO at 28°C in 96-well plates (100 μl per well). 5 μl of medium from each well was removed and incubated on Sabouraud‘s agar medium for 48 h. When the quantity of CFU (colony forming units) is less than 10, 99% of A. fumigatus conidia are killed, and the medication concentration is the minimal fungicidal concentration.

Conidia suspension (2 × 10⁵ CFU/ml) was cultured for 24 h at 28°C in 96-well plates (100 μl per well) to generate mycelium. 2, 4, 8, 16, and 32 μg/ml of hinokitiol were added with 0.1% DMSO to each well before being incubated for 24 h. A microplate reader was used to read the OD at 620 nm.

HCECs (provided by University of Xiamen, Fujian, China) were incubated according to the previous method by Yin et al. (2022). Cell suspension (3 × 10⁵/mL) to be tested was incubated in a 96-well plate with different concentrations of hinokitiol for 24 h under the original condition in dark. The absorbance values were measured at 450 nm 3 h after the addition of 10 μl of cell counting kit-8 (CCK-8) each well. 6 replicates were set up for each sample.

We seeded the A. fumigatus conidia (2 × 10⁵ CFU/ml) into 6-well plates and cultured there for 24 h at 28°C to generate mycelium. After being washed the mycelium was centrifuged at 12,000 rpm for 10 min before being transferred to a fresh 6-well plate. The mycelium was rinsed with phosphate buffer (PBS), then collected and fixed with 2.5% glutaraldehyde at 4°C overnight after being incubated with 0.1% DMSO or hinokitiol (8 μg/ml) at 28°C for 24 h. The following procedures were followed as per standard procedures to prepare the mycelium sample for TEM observation (Shen et al., 2020). We examined the samples using a JEOL-1200EX transmission electron microscope.

We seeded the A. fumigatus conidia (2 × 10⁵ CFU/ml) into 6-well plates and cultured there for 24 h at 28°C to generate mycelium. The mycelium was washed, centrifuged at 12,000 rpm for 10 min, and then transferred to a new 6-well plate. The mycelium was rinsed with PBS after being incubated with 0.1% DMSO or hinokitiol (8 μg/ml) at 28°C for 24 h. They were then collected and fixed with 2.5% glutaraldehyde at 4°C for 2 h. PBS was used to clean the samples, and they were then mixed for an hour at 4°C with 1% (v/v) osmium tetroxide in PBS. Samples were then gently dehydrated in graded ethanol, critical point dried in CO2, coated with gold, and seen under an SEM (VEGA3; TESCAN Company, Shanghai, China) at magnifications of 2000 and 5,000 (bar =20 or 10 μm).

Aspergillus fumigatus mycelium was prepared and collected as previously indicated and was treated with hinokitiol (4 μg/ml, 8 μg/ml) or without hinokitiol (0.1% DMSO) for 24 h, 36 h, or 48 h. Each group received a 15-min addition of 50 μg/ml propidium iodide (PI) solution before being incubated in the dark at room temperature. Bright-field and fluorescence images were both taken using a fluorescence microscope (Nikon, Tokyo, Japan, 100×) with green excitation light or not. Image J software was used to calculate the fluorescence intensity.

Aspergillus fumigatus mycelium was prepared in sabouraud medium for 5 days and was treated with hinokitiol (4 μg/ml, 8 μg/ml) or without hinokitiol (0.1% DMSO) for 48 h. Mycelium was washed in sterile distilled water, filtered, and fully dried. Total ergosterol contents of A. fumigatus mycelium with different hinokitiol treatments were determined by the method described previously (OuYang et al., 2019) with a slight modification. 48 h were spent vacuum-freezing the samples before 0.1 g of each group was saponified in 4 ml of freshly made 30% (w/v) methanolic KOH and 8 ml of 100% ethanol at 90°C. 3 ml of petroleum ether was used three times to extract the mixtures containing 10 ml of saturated sodium chloride solution. After placing the samples in the extraction flask, the supernatants were collected. At 60 to 90°C, the petroleum ether solution was then distilled. With the help of 10 ml anhydrous ethanol, the solute crystallized and was retrieved. High-Performance Liquid Chromatography (HPLC) analysis was carried out using a HITACHI high-performance liquid chromatograph (Chromaster, HITA-CHI, Japan). At a constant temperature, the sample extracts were separated and examined on a C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase was made up of Solvent A (methanol). A flow of 1 ml per minute was present. Chromatographic peaks were discovered by measuring retention times and spectra against the specified standard. The detection wavelength was set at 282 nm. For the analysis, 20 μl aliquots were directly injected into the HPLC. Ergosterol standard curve is provided in Supplementary Table S1.

Aspergillus fumigatus mycelium was prepared and collected as previously indicated and the experimental groups and the control groups were classified using the same approach as in the cell membrane permeability assay. We utilized calcofluor white (Sigma, St. Louis, MO, United States) and fluorescence microscopy to observe. The collected mycelia were stained with 10 μl KOH (10%) followed by 10 μl of calcofluor white stain as the manufacturer’s instructions.

The experimental groups and the control groups were classified using the same approach as in the cell membrane permeability assay. We assayed chitin content according to the previous method described with slight modifications (Ke et al., 2022). The mycelium samples were cultured in a 96-well plate. They were twice rinsed with distilled water before being incubated for 5 min in the dark with Congo red (0.025%). The materials were examined using a microplate reader (Tecan, Switzerland) with excitation and emission wavelengths of 540 and 600–850 nm, respectively.

Aspergillus fumigatus mycelium was prepared in sabouraud medium for 5 days and was treated with hinokitiol (4 μg/ml, 8 μg/ml) or without hinokitiol (0.1% DMSO) for 24 and 48 h. Mycelium was washed in sterile distilled water, filtered, and fully dried. We ground the mycelium into a paste with the chitinase extraction reagent in an ice bath and then centrifuged them at 8000 rpm at 4°C for 25 min. The supernatant was collected to get the extracted enzyme solution, and a commercial BC0820 kit was used to measure chitinase activity (Solarbio Technology, Beijing, China).

Conidia suspension (2 × 10⁵/mL) containing 0.1% DMSO or hinokitiol (4 μg/ml) was incubated on a 6-well plate at 28°C. The Control (DMSO treated) and Test (HK treated) samples were harvested after incubation for 36 h and then stored at −80°C for RNA-Seq analysis. All the experiments were carried out in 3 independent biological replicates. Total RNA was extracted using the TRIzol reagent according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, United States). RNA integrity was assessed using the Agilent 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). Then the libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the as manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The Sequence Read Archive has been released in NCBI¹ and the 6 runs can be searched by accession numbers SRR22582946, SRR22582945, SRR22582944, SRR22582943, SRR22582942, and SRR22582941.

Hub genes were screened from the differently expressed genes (DEGs) in the RNA-seq result. We utilized the STRING database (version 11.5²) to analyze the protein–protein interactions (PPI) of Hub DEGs. Cytoscape software (version 3.8.0) was used to visualize the PPI network. The Molecular Complex Detection (MCODE) plugin of Cytoscape was then used to determine the important gene modules in the PPI networks. Finally, the cytoHubba plugin was used to rank the genes and highlight Hub genes. Overall, the features we got from different plugins were represented in the PPI network.

Conidia suspension (2 × 10⁵/mL) containing 0.1% DMSO or hinokitiol (4 μg/ml) was incubated on a 6-well plate at 28°C. The Control (DMSO treated) and Test (HK treated) samples were harvested after incubation for 36 h. The total RNA was prepared using the Fungal Total RNA198 Isolation Kit (B518629, Sangon Biotech, Shanghai, China), and reverse transcribed with HiScript III RT SuperMix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The PCR method was based on previous studies (Sun et al., 2019). Primers used for the qRT-PCR are listed in Supplementary Table S2.

For comparisons with data from the control group or HK treatment group, we performed an unpaired, two-tailed Student’s t-test or one-way ANOVA with post hoc analysis. GraphPadPrism 8.0 (San Diego, California, United States) and ImageJ1.44p (National Institutes of Mental Health, USA) were used for statistical analyzes, and values were presented as the mean ± standard deviation (SD). p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001) was considered statistically significant (ns = no significance). All the experiments were carried out in 3 independent biological replicates.

According to MIC data, the germination of A. fumigatus conidia and growth of A. fumigatus mycelium were inhibited by hinokitiol at 2 μg/ml, with the extent varying proportionally to concentrations from 2 to 32 μg/ml (Figures 1A,B). The 0.1% DMSO treatment group showed no meaningful effect on the growth of A. fumigatus compared to the control group. The time-kill curve in Figure 1C showed that as the hinokitiol dose increased, the absorbance values at the same time point gradually decreased, with a higher difference at 48 h between 4 μg/ml and 8 μg/ml treated groups. MFC assay results indicated that A. fumigatus conidia were 100% killed by hinokitiol at 16 μg/ml (Figure 1D).

Fungal keratitis is a serious blinding eye disease caused by a fungal infection, which has an acute onset, rapid progression, and poor prognosis. The common causative fungi are Aspergillus spp., Candida spp. and Fusarium spp. (Brown et al., 2021; Sharma et al., 2022).The effect of hinokitiol on the proliferative capacity of HCECs was investigated, and the results are shown in Figure 1E. The proliferative capacity of HCECs experienced a significant rise at 2 and 4 μg/ml of hinokitiol, and then saw a gradual drop thereafter. With the exposure of 8 and 10 μg/ml of hinokitiol, no differences in cell proliferation degree were seen compared to the control group. It was not until 12 μg/ml of hinokitiol that HCECs were inhibited to grow. Therefore, hibokitiol with concentrations being below 10 μg/ml is considered to be high-safe and low-toxic.

We used TEM and SEM to examine the micromorphological alterations in hinokitiol-treated A. fumigatus, focusing on the cell wall and cell membrane. SEM revealed that hinokitiol-treated mycelium grew in a chaotic and uneven manner (Figures 1F2,F3) compared to the control (Figure 1F1). They atrophied and deformed, collapsed or flattened and hollowed. The mycelium in Figure 1F2 had twisted and virtually shattered shapes. Figure 1F3 also showed branching and odd shape. TEM results of mycelium ultrastructure displayed that the cell membrane was coiled, discontinuous, and inconspicuous under the action of hinokitiol. The cell membrane separated from the cell wall became ruptured (Figures 1F5,F6). The hinokitiol-treated group likewise exhibited a fuzzy structure of the cell wall that dissolved into fragments and was noticeably shallower than in the control group (Figures 1F5,F6). The findings suggested that hinokitiol interfered with cell wall integrity and cell membrane continuity in some way.

We used a propidium iodide dye that is only taken up by cells with impaired cell integrity to evaluate cell membrane permeability. Hinokitiol (4 g/ml, 8 g/ml) was applied to the mycelium for 24 h, 36 h, and 48 h. In bright fields, A. fumigatus mycelium width and density decreased with medication concentrations and length of time, while the fluorescence intensity in fluorescence images rose gradually (Figures 2A–C). Besides that, the percentage of mycelium stained by PI solution also increased in the same manner. The fluorescence was extensively distributed in the field of vision in the 8 g/ml, 48-h treatment group, and the brilliant spots representing exudation were extremely dense. We utilized the software ImageJ to quantitatively clarified the fluorescence intensity in each PI staining field which showed a clear rising trend over time and across concentration (Figure 2D). Overall, hinokitiol was shown to have a detrimental impact on the pattern of mycelium growth, cell integrity; and increased cell membrane permeability depending on the duration of action and drug concentration proporationally.

Quantitative measurement of the main component of A. fumigatus cell membrane, ergosterol, was assayed and the ratio of dry weight (DW) of mycelium was calculated. We demonstrated that there was a substantial difference in ergosterol levels between each group with or without hinokitiol treatment. After being exposed to hinokitiol with 4 or 8 μg/ml for 48 h, the HPLC result showed that ergosterol content decreased compared with the control group (Figure 2E). The ratios of DW in the control, 4 μg/ml, 8 μg/ml group were 5.160734 mg/g, 0.664238 mg/g, and 0.317686 mg/g, respectively. Therefore, we found that hinokitiol may compromise cell membrane integrity by losing ergosterol content in cell membranes.

We tested calcofluor white staining to explore how hinokitol damage A. fumigatus cell wall. Control mycelium witnessed a strong fluorescence intensity in its chitin-rich cell wall and septa (Figures 3A1–A3). After being treated with hinokitiol, the overall fluorescence intensity of the cell wall and septum fell in a hinokitiol concentration and time-dependent manner (Figures 3A4–A9). In addition, we found that in the 8 μg/ml treatment group especially, the fluorescence profile became blurred and the background fluorescence was greatly increased as indicated by arrows (Figures 3A7–A9). It suggested that the cell wall was disrupted by hinokitiol, where the chitin content leaked out and dispersed. Besides that, the fungus cultured with hinokitiol became narrower in diameter, more branched, and shorter in mycelium length.

Then the chitin content of A. fumigatus in the control and treatment groups was further measured (Figures 3B,C). Both graphs displayed that hinokitiol reduced the chitin content compared to the group without hinokitiol. In the comparison between the different exposure period groups, after being treated with hinokitiol, the increase degrees in chitin content levels were smaller in the 4 and 8 μg/ml hinokitiol-treated groups than that in the control group, for the difference in chitin content levels between the control and treated groups increased in the same time point. The results indicated that hinokitiol disrupted the cell wall components, and chitin, and inhibited the synthesis of cell wall components.

Subsequently, we examined the activity of chitinase in A. fumigatus after treatment with different concentrations of hinokitiol for 24 and 48 h (Figure 3D). Overall, chitinase activity proved to increase in hinokitiol-treated groups in both 24 h and 48 h group (p < 0.0001), despite seeing a drop in chitinase activity at 8 μg/ml (p<0.0001). With the same concentration of hinokitol, there was no statistically significant difference between the chitinase activities of the two groups at two treatment periods (p>0.05). The figure suggested that hinokitiol influenced the biological activity of chitinase, which might explain why the chitin content of the A. fumigatus cell wall decreased after hinokitiol exposure. And the higher concentration of hinokitiol could be able to damage the whole bioactivity, responsible for the fall of chitinase activity at 8 μg/ml.

To explore the effects at the gene level, we used RNA-seq analysis to investigate the transcriptional characteristics of A. fumigatus treated with (4 μg/ml, 36 h) or without hinokitiol treatment. RNA-seq results and derived DEGs related to the cell wall or cell membrane remodeling were subsequently analyzed and divided into two heat maps (Figures 4A,B). There are 67 genes in Figure 4A and 197 genes in Figure 4B. The heat maps provide a global view of specific DEGs, with red representing up-regulation trends and blue representing down-regulation trends. Figure 4A displays genes associated with the fungal cell wall, most of which are involved in the synthesis and degradation of mannans, glucan, and chitin, in addition to some genes related to the activities of the fungal cell wall such as germination, septogenesis, and extracellular sensing. Figure 4B shows genes related to fungal cell membranes, which are involved in the transmembrane transport of various substances in fungi, the formation of cell membranes, regulation of ion channel activity, and mediating the membrane structure with the endoplasmic reticulum, Golgi apparatus, and other organelles to form an integral membrane structure.

Nineteen hub genes associated with the cell wall (Figure 4C) and 10 hub genes related to the cell membrane (Supplementary Figure S3) were obtained and were considered to play an important function in A. fumigatus cultured with hinokitiol (Figure 4C). EglC, pgxB, plyA, gel3, gel1, chiA1, crf1, and ftrA from the PPI network performed qRT-PCR experiments. Among them, eglC, which encodes an endoglucanase, is thought to play the most indispensable role. PCR results validated the reliability of RNA-seq, as the expression trends of these genes were the same with those of heat maps, albeit to a different extent (Figure 4D).