Unless otherwise stated, chemical reagents and plasticware were obtained from Fisher Scientific (St. Louis, MO). PBS used in washing of cryptococcal cells was obtained at a 10X concentration and diluted 1:10 with deionized water, then sterilized before use. The medium used in Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) Assays was RPMI 1640 supplemented with 0.165 M morpholinepropanesulfonic acid (MOPS), pH 6.9–7, filter-sterilized using a 0.22 μm filter. The cell culture medium used in cytotoxicity experiments was DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10% NCTC-109, 1% non-essential amino acids, 100 U penicillin/ml, and 100 μg streptomycin/ml, filter-sterilized using a 0.22 μm filter. All mammalian cell cultures were incubated at 37° C, 5% CO₂ in humidified environments.

Cryptococcus neoformans strains H99 (serotype A, mating type α) (gift of John Perfect, Duke University Medical Center, Durham, NC), Cn145a (serotype A), and Cryptococcus gattii strains R265 (serotype B), R272 (serotype B), R4247 (serotype C), and WSA87 (serotype C) (gift of Brian Wickes, University of Texas Health Science Center, San Antonio, TX), and Cryptococcus deneoformans strain 52D (serotype D) were stored at −80°C in 15% glycerol stocks and were plated on yeast extract peptone-dextrose (YPD) (BD Difco; Franklin Lakes, NJ) agar plates. Prior to experiments, individual cryptococcal strains were incubated with shaking in YPD broth for 18 h at 30°C. Cells were collected through centrifugation and washed three times in sterile phosphate-buffered saline (PBS). The cells were quantified using Trypan blue exclusion in a hemacytometer and were resuspended in required medium at the concentration needed for each experiment.

3,3′-(((5,6-dimethoxy-2-(methoxyxarbonyl)-1-methyl-1H-indole-4,7-diyl)bis(ethyne-2,1-diyl))bis(4,1-phenylene)bis(oxy))bis(N,N,N-trimethylpropan-1-aminium) iodide (EIPE-1) (Figure 1) was synthesized and provided by Dr. Nelson’s laboratory (Adhikari et al., 2022; Reed et al., 2023). EIPE-1 powder was then reconstituted with dimethyl sulfoxide (DMSO) to a stock concentration of 5 mg/mL. Dilutions to working concentrations for experiments were made into the media used for each experiment.

MIC assays were conducted according to CLSI guidelines (CLSI, 2017 #3098). Briefly, either EIPE-1 or Amphotericin B (AmB) was diluted in RPMI-MOPS, pH 7.0 and evaluated in a two-fold dilution in a concentration range of 100 μg/mL to 0.0488 μg/mL. Dilutions were made in RPMI-MOPS, in a 96-well microtiter plate. A single cryptococcal strain was added to all wells at 0.5×10³/ml. Growth controls included the cryptococcal strain grown in media alone. Plates were incubated at 35°C in a humidified incubator for 48 h. The optical densities at 490 nm were measured with a Synergy HTX multi-mode plate reader (BioTek, Winooski, VT), and plates were also visually inspected for turbidity (indicating growth). For MFC assays, dilutions including and above the determined MIC concentration were plated on YPD plates and incubated at 30°C in for 48 h. MFC was defined as the concentration that permitted less than three colony forming units (CFUs), or no growth on the plates, indicating a reduction in 99.9% of the initial inoculum (fungicidal). In other words, the compound has a 99.9% fungicidal activity against the yeast cells (Ernst et al., 1996; Graybill et al., 1997; Espinel-Ingroff et al., 2002; Leite et al., 2014).

Checkerboard assays were conducted using a method previously described, to determine the antifungal activity of EIPE-1 in combination with AmB (Bonifácio et al., 2019; Nelson et al., 2021). EIPE-1 or AmB were evaluated in a two-fold dilution as described in the MIC protocol above. Dilutions were made in RPMI-MOPS, in a 96-well microtiter plate. A single cryptococcal strain was added to all wells at 0.5×10³/ml for evaluation of efficacy of combinations. Controls used were EIPE-1 only (row H), AmB only (Column 10), growth control (column 11), and media control (column 12). Plates were incubated at 35°C in a humidified incubator for 48 h. The optical densities at 490 nm were measured with a Synergy HTX multi-mode plate reader (BioTek, Winooski, VT). Results were analyzed using the Fractional Inhibitory Concentration Index (FICI), a non-parametric model based on the Loewe additivity theory to determine the interaction of the combination of EIPE-1 and AmB, where FICI ≤ 0.5 is synergistic, FICI 0.5–4 is indifferent, and FICI ≥ 4 is antagonistic. FICIs were defined as the sum of individual FICs (FICI = FIC_{Am B} + FIC_EIPE-1), with FICs being defined as the MIC derived from the combination therapy divided by their MIC alone (FIC = MIC_Combination/MIC_Alone). Off-scale MICs were considered to be the highest or lowest concentration tested in the microdilution assay (Bonifácio et al., 2019; Nelson et al., 2021).

To test the cytotoxicity of EIPE-1 on mammalian cells, individual cell lines, including the human cervical epithelial cell line HeLa, murine fibroblast cell line McCoy, and human lung epithelial cell line A549 (all acquired from ATCC), were tested using the CyQUANT™ LDH Cytotoxicity Assay, fluorescence (Invitrogen). For this, cells were grown in cell culture medium according to ATTC guidelines at 37° C with 5% CO₂. The Cytotoxicity Kit was used according to the manufacturer’s instructions. Briefly, mammalian cells were added in triplicate to wells of a 96-well plate (1 × 10⁶ cells/ml in 100 μL). EIPE-1 was prepared similarly to the MIC assay (1X MIC, 2X MIC, and 10X MIC) except cell culture media was used for dilutions and was added at 10 μL per well. Negative controls included media alone and untreated cells, and the positive control included fully lysed cells. Plates were incubated for 24 h at 37°C, 5% CO₂. After incubation, 50 μL of reaction mixture was added and incubated for 10 min at room temperature. Following incubation, 50 μL of stop solution was added to each sample. Fluorescence was measured on a Synergy HTX multi-mode plate reader (BioTek) with filters for 560/25 (excitation) and 590/20 (emission). Cytotoxicity of EIPE-1 was conducted in two independent experiments (n = 2) with each cell line, with each condition performed in triplicate. Percent cytotoxicity was defined as the fluorescence of experimental wells (cell line and EIPE-1) divided by negative control untreated cells. Greater than 30% cytotoxicity is considered cytotoxic, whereas lower percentages (<30%) were considered non-toxic (ISO10993-5, 2009).

In order to visualize fungal cells using electron microscopy, a higher quantity of fungal cells (10×10⁶ cells) was used. Prior to conducting electron microscopy experiments, we determined the MIC of EIPE-1 using a higher number of cryptococcal cells (strain H99). We followed the same MIC protocol above and determined the MIC was 6.25 μg/mL for this number of cells. Cryptococcus neoformans cells were resuspended in RPMI-MOPS, pH 7.0, at a concentration of 10 × 10⁶ cells/ml. For compound treated samples, EIPE-1 was added at 6.25 μg/mL. Negative controls included untreated C. neoformans strain H99 cells incubated under the same conditions for each time point. The fungal cells were incubated at 35°C in humidity for 4 h, 6 h, 8 h, or 12 h to detect changes in cell morphology over time. The cells were collected by centrifugation. The pellet was resuspended in 2.0% glutaraldehyde in 0.1 M cacodylate buffer at a volume of 1 mL for a minimum of 2 h and processed for scanning electron microscopy (SEM) or transmission electron microscopy (TEM) at the Oklahoma State University (OSU) Microscopy Laboratory (Stillwater, OK) using their provided protocols.

Fixed C. neoformans cells were collected by centrifugation and transferred to a 12-well plate. Cells were rinsed three times in a buffered wash (30% cacodylate buffer, and 6.15% sucrose) at fixed intervals of 15 min. C. neoformans were incubated in osmium tetroxide (1% OsO₄) for 1 h in a 36-well plate with a clear coverslip. Following incubation, C. neoformans was rinsed three times in a buffered wash at fixed intervals of 15 min. Cryptococcus neoformans were dehydrated in ethanol (50, 70, 90, 95, and 100%) in increasing percentages three times at fixed intervals of 15 min. Cryptococcus neoformans were washed two times with hexamethyldisilane at a time interval of 5 min. Coverslips were removed and placed on a clear sheet for 12 h until dried. Coverslips were mounted on stubs using silver paint. Sample mounts were covered in an Au-Pd coat by the OSU Microscopy Laboratory. Images were examined with a FEI Quanta 600 field-emission gun Environmental Scanning Electron Microscope with a Bruker EDS X-ray microanalysis system and HKL EBSD system. Images were examined at 10000X and 20,000x for SEM. At least 8 fields per condition were examined.

Fixed C. neoformans cells were collected by centrifugation. Media was removed and C. neoformans was rinsed 3 times in a buffered wash at fixed intervals of 15 min. Rinsed cells were resuspended in 1% OsO₄ at room temperature for 1 h. 1% OsO₄ was removed. Cryptococcus neoformans was rinsed 3 times in a buffered wash at fixed intervals of 15 min. Cryptococcus neoformans were dehydrated in ethanol (50, 70, 90, 95, and 100%) in increasing concentrations three times at fixed intervals of 15 min. Cryptococcus neoformans were washed three times in propylene oxide for fixed intervals of 15 min. Cells were placed in 1:1 propylene oxide and Poly/Bed for 12 h. Cryptococcus neoformans cells were embedded (100% embedding medium) and sliced to 80 nm in thickness by the staff of the OSU Microscopy Laboratory. Images were examined with a JEOL JEM-2100 with Bruker EDS at 8000X for TEM. At least 8 fields per condition per time point were examined.

Cryptococcus neoformans at a concentration of 10 × 10⁶ cells/ml was incubated with EIPE-1 and RPMI MOPS using the minimum inhibitory concentration from the previously described MIC assays for this number of cells. Cells were incubated at 35°C in a humid incubator for 6 h, which correlated to the time point we observed morphological changes in the fungal cells by SEM and TEM. Untreated cryptococcal cells incubated under the same conditions were used as controls. RNA was purified using AllPrep© Fungal DNA/RNA/Protein kit (Qiagen) and quantified using the Take3 plate on a Synergy HTX multi-mode plate reader (BioTek). RNA was determined pure at a 260/280 ratio of 2.0 (ISO20395, 2019). RNA experiments were conducted in triplicate.

RNA was sent for sequencing to Novogene Corp (Sacramento, CA). Fungal RNA-sequencing was conducted using SMARTer Stranded V2 library prep and samples were sequenced on the Illumina Platform (PE150 Q30 ≥ 80%) (Novogene Corp). Gene expression was compared between each untreated C. neoformans strain H99 incubated for 6 h compared to H99 treated with EIPE-1 for 6 h. This time point was chosen because the fungal cells were still alive, but initial microsocopy studies showed changes in cell wall/membrane were observed starting at 4 h incubation. Statistics were performed by Novogene, and statistically significant differentially-expressed genes (DEG) in the treated vs. untreated cells were reported. Differentially-regulated genes and their reported functions were examined using FungiDB – Fungal & Oomycete Informatics Resources (Amos et al., 2022).

Galleria mellonella larvae (Carolina Biological Supply, Burlington, NC) were briefly examined for melanization before storage in groups of ten. Prior to experiments, G. mellonella were removed from food for 24 h. Larvae were washed in 70% ethanol and ampicillin (1 mg/mL) or Rifampicin (1 mg/mL). Galleria mellonella larvae were given an injection into the last proleg with 10 μL of C. neoformans H99 (1×10⁴ cells/ml), heat-killed C. neoformans H99 (1×10⁴ cells/ml), or PBS (Mylonakis et al., 2005; Fuchs et al., 2010; Kay et al., 2019). Following a 2 h incubation period at room temperature, the larvae were injected with 10 μL of EIPE-1 at 15 μg/mL, 20 μg/mL, or 25 μg/mLdiluted in PBS (treatment) or with 10 μL PBS (control) in the second to last proleg (Mylonakis et al., 2005; Tsai et al., 2016; Kay et al., 2019). Galleria mellonella were incubated at 37°C and were examined every 12 h for 10 days. Every 12 h, survival was checked and cocoons were removed to arrest the G. mellonella in their larval stage (Sprynski et al., 2014). Galleria mellonella larvae were considered dead following full-body melanism (turning brown/black) and immobility (Kay et al., 2019).

Data analyses were conducted using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Depending on the data collected and interaction observed between cryptococcal cells and the compound, the one-way ANOVA with the Tukey’s multiple comparison test was used to compare the data. For G. mellonella studies, the log-rank test was used to compare survival rates.

To determine the antifungal activity of the compound EIPE-1 against cryptococcal strains (H99, Cn145a, R272, R2625, R4247, 52D, and WSA87), we conducted minimum inhibitory concentration (MIC) assays. AmB is an established antifungal drug used against C. neoformans in immunocompromised patients, therefore it was used as a control compound for MIC value comparison against EIPE-1 (Perfect et al., 2010; Sloan and Parris, 2014). Statistical analysis showed a significant difference (p < 0.05) in antifungal activity following incubation with EIPE-1 compared to C. neoformans alone or AmB in RMPI-MOPS (Figure 2). The AmB MIC value had high variation between cryptococcal strains ranging from 0.39 to 6.25 μg/ml. The MIC of EIPE-1 in our assay was 3.125 μg/mL against C. neoformans strain H99, C. gattii strains R272, R625, and R4247. The MIC value of EIPE-1 was 1.56 μg/mL against C. neoformans strain Cn145a, the C. deneoformans strain 52D, and the C. gattii strainWSA87 (Figure 2). However, despite the variation in the EIPE-1 MIC data, it demonstrates that EIPE-1 can inhibit cryptococcal growth of multiple cryptococcal strains at low concentrations.

To determine whether the antifungal activity demonstrated by EIPE-1 is fungistatic or fungicidal, we conducted minimum fungicidal concentration (MFC) assays. The results of our YPD plates displayed no visible CFUs present after 48 h incubation. This indicates that EIPE-1 is fungicidal at the MIC concentration for each cryptococcal strain.

The in vitro interaction of antifungal therapy combinations can have a greater efficacy than the sum of their individual actions, such as seen in the current cryptococcal meningitis treatment guidelines that advises treatment via combination drug therapy (Perfect et al., 2010; Sloan and Parris, 2014; Nelson et al., 2021). Therefore, we tested the synthetic compound EIPE-1 in combination with AmB against C. neoformans H99 using a checkerboard assay and categorized the results by the FICI. Both drugs maintained their individual MICs as determined above. Each EIPE-1/AmB combination had an average FICI between 1.17–1.19, placing them in the indifferent category (0.5–4.0). This result is not dependent on the concentration of EIPE-1 used.

In order to determine the relative cytotoxicity of EIPE-1 to mammalian cells, the CyQUANT™ Cytotoxicity Assay Kit was used with three different mammalian cell lines, including the murine fibroblast cell line McCoy, human lung epithelial cell line A549, and the human cervical epithelial cell line HeLa. EIPE-1 was shown to have a cytotoxicity of <30% (non-toxic) at the MIC concentration tested in all cell lines (Figures 3A–C). At 2X concentration, EIPE-1 was non-toxic for two of the three cell lines, but at 10X concentration, EIPE-1 was toxic (>30%) for all three cell lines. However, since the compound cytotoxicity was less than 30% at the 1X and 2X MIC concentration in most cell lines, the compound was determined to be non-toxic to mammalian cells (ISO10993-5, 2009).

To understand the mechanism of action of EIPE-1, SEM and TEM analyses were conducted. SEM and TEM can provide vital information about the surface and internal structures of cells (Nixon, 1964; Koga et al., 2021). Cryptococcus neoformans strain H99 cells were incubated with EIPE-1 at 4 h, 8 h, or 12 h, following which cells were prepared for electron microscopy. SEM images displayed cell wall/membrane damage as early as 4 h post-incubation (Figure 4A). Damage was indicated by c-shaped cells, mis-shaped cells, etc. (see arrows Figure 4A), indicative of dead/dying cryptococcal cells (Hole et al., 2012). To determine further the effects of EIPE-1 on the cell wall and membrane of the cryptococcal cells, TEM was conducted. TEM allows the internal structures of the cell to be imaged by sectioning of the sample (Winey et al., 2014). Cryptococcus neoformans cells were incubated with EIPE-1 at 4 h, 8 h, or 12 h. Time points remained the same as with the SEM to provide a comparison between the SEM and TEM images. The TEM images confirmed that the compound is affecting the cell wall and cell membrane of the fungal cells. Four morphologies were identified within the images (Figure 4C). We observed damage to the cell wall and membrane at 8 h and 12 h (Figure 4B). In addition, it appears that the membrane damage results in a leakage of internal contents into the surrounding media (Figures 4B,C). The TEM results confirmed that the compound is affecting these specific cellular structures on the fungal cells. Additionally, TEM showed two other cell morphologies that represented dying or dead cells, including c-shaped cells. The images also include cells with degraded membranes and a black smudge. TEM incorporates the use of heavy metals to prevent electrons from passing through the prepared sample. These metals bind to regions concentrated with DNA and proteins, or components of the cell that are rich in lipids. In a bright-field TEM, these regions, and regions high in mass density tend to appear dark in the imaging to allow contrast. Thus, the black region (smudge) observed in the TEM images could be a representation of regions rich in DNA, protein or lipids (Dempster, 1960; Belazi et al., 2009; Klein et al., 2015; Lange et al., 2021). Nucleic acid or DNA, protein and lipids make up a majority of the internal macromolecules of a living eukaryotic cell. Due to the composition of the internal cellular components of C. neoformans, it is likely the black region observed in the TEM images was comprised of internal structures leaking into the media from a pore present in the cellular wall or membrane of the fungal cell (Schie et al., 2016). As assessed by both the SEM and TEM images, the compound appears to affect the cell wall and membrane of the C. neoformans cells, leading to cell lysis.

To understand the effect of EIPE-1 on fungal gene expression, we were interested in identifying differential gene expression between C. neoformans incubated with EIPE-1 compared to control. Purified RNA was sent for sequencing at Novogene (Novogene Corp, Sacramento, CA). The analyses identified 4,936 statistically significantly differentially expressed genes (DEG) between untreated and EIPE-1 treated cryptococcal cells. Of these genes, 2,486 were significantly upregulated and 2,450 genes were significantly down-regulated. Due to a limitation on the information available for C. neoformans strain H99, one third of the greatest DEGs listed (Table 1) have unknown functions. However, our analyses of FungiDB showed that the genes with available information have roles in metabolic processes, stress response, and virulence of the cells (Amos et al., 2022). Descriptions of the top thirty differentially-regulated genes and their putative functions are shown (Table 2). Furthermore, analyses identified 91 enriched pathways through the KEGG online database. Of these 91 pathways, none were statistically significant. The top five active pathways and their p-values are shown (Table 2). These pathways are involved in amino acid biosynthesis, carbon metabolism, ribosome formation, and the replication of DNA (Amos et al., 2022). We further filtered through the list of genes and identified several DEGs involved in C. neoformans viability, capsule biosynthesis, capsule attachment and remodeling and ergosterol biosynthesis (Table 3).

To determine the efficacy of EIPE-1 in a living infection model, G. mellonella larvae were infected with C. neoformans H99 and treated with various concentrations of EIPE-1 as mentioned in the methods. As shown in Figure 5, larvae of the G. mellonella inoculated with C. neoformans H99 experienced rapid death by day five of infection. Additionally, larvae inoculated with C. neoformans and treated with EIPE-1 experienced death at similar time points to H99 alone, regardless of the concentration of EIPE-1 (Figure 5).