Antifungal activity of eumelanin-inspired indoylenepheyleneethynylene against Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in >152,000 immunocompromised individuals annually, leading to 112,000 yearly deaths. The four classes of existing antifungal agents target plasma membrane sterols (ergosterol), nucleic acid synthesis, and cell wall synthesis. Existing drugs are not highly effective against Cryptococcus, and antifungal drug resistance is an increasing problem. A novel antimicrobial compound, a eumelanin-inspired indoylenepheyleneethynylene, EIPE-1, was synthesized and has antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MSRA), but not towards Gram-negative organisms. Based on EIPE-1’s antibacterial activity, we hypothesized that EIPE-1 could have antifungal activity. For these studies, we tested EIPE-1 against C. neoformans strain H99 and 6 additional cryptococcal clinical isolates. We examined antifungal activity, cytotoxicity, effects on fungal gene expression, and mechanism of action of EIPE-1. Results showed that EIPE-1 has fungicidal effects on seven cryptococcal strains with MICs ranging from 1.56 to 3.125 μg/mL depending on the strain, and it is non-toxic to mammalian cells. We conducted scanning and transmission electron microscopy on the exposed cells to examine structural changes to the organism following EIPE-1 treatment. Cells exposed displayed structural changes to their cell wall and membranes, with internal contents leaking out of the cells. To understand the effect of EIPE-1 on fungal gene expression, RNA sequencing was conducted. Results showed that EIPE-1 affects several processes involved stress response, ergosterol biosynthesis, capsule biosynthesis, and cell wall attachment and remodeling. Therefore, our studies demonstrate that EIPE-1 has antifungal activity against C. neoformans, which affects both cellular structure and gene expression of multiple fungal pathways involved in cell membrane stability and viability.


Introduction
Cryptococcus neoformans is an encapsulated fungal pathogen, transmitted frequently via inhalation of environmental spores found in soil, pigeon excrement, and decomposing wood (Levitz, 1991;Kwon-Chung et al., 2014).Infection by the pathogen can result in cryptococcosis, which manifests initially as a pulmonary disease but can also disseminate to the central nervous system (CNS) leading to cryptococcal meningitis (Chang et al., 2004;Shi et al., 2010;Kwon-Chung et al., 2014).HIV patients are primarily at risk of the development of cryptococcosis.They make up 95% of all cases reported in low-to-middle income countries, such as those in Sub-Saharan Africa, and 80% of all cases in high-income countries (Dhana, 2013;Sloan and Parris, 2014).This is a result of a decrease in their immune system's ability to clear pathogens, due to the HIV suppression of their CD4 + T-cell count (Pappas et al., 2010;Kwon-Chung et al., 2014;Gibson and Johnston, 2015).In an immunocompetent host, a Th-1 type CD4 + T-cell response would typically clear the infection relatively quickly, with little to no symptoms (Pappas et al., 2010;Gibson and Johnston, 2015).However, in an HIV or immunocompromised host, their immune systems are unable to remove the fungal pathogens from their lungs, leading to the development of a cryptococcal infection (Pappas et al., 2010;Gibson and Johnston, 2015;Garelnabi and May, 2018).After initial infection, C. neoformans can traffic from the lungs to the host's central nervous system (Garelnabi and May, 2018).This leads to the development of cryptococcal meningitis, which has a 40% fatality rate, even with the use of antifungal treatments with ideal conditions (Rajasingham et al., 2017;Patel et al., 2018).For many individuals with severely compromised immune systems, complete removal of the cryptococcal infection is impossible, resulting in a lifelong commitment to the use of antifungal therapies to keep the infection at bay (Coelho and Casadevall, 2016).Current estimates show in AIDS patients, roughly 152,000 cases of cryptococcal meningitis occur each year, with an average of 112,000 yearly deaths (Rajasingham et al., 2022).To help prevent fatalities from cryptococcal infections, early diagnosis is crucial.However, the use of potent fungicidal drugs in combination with fungistatic drugs are still important for the treatment of the disease.Without them, cryptococcal meningitis is fatal (Chen et al., 2010;Dhana, 2013;Sloan and Parris, 2014;Guo et al., 2016).The World Health Organization (WHO) recommended treatment regimen for cryptococcal meningitis in AIDS patients involves three phases: (a) Induction (2 weeks of treatment), (b) Consolidation (8 weeks of treatment), and (c) Maintenance (6-12 weeks or until HIV is controlled by Highly Active Antiretroviral Therapy (HAART)) (Sloan and Parris, 2014).This treatment requires a combination of antifungal therapies over a course of 6-12 months depending on the drug availability.Unfortunately, reduced availability of the drugs for treatment of cryptococcal meningitis continues to be an issue in most Asian and African countries where the disease is most prevalent (Sloan and Parris, 2014).
As the prevalence of dangerous fungal infections continues to rise, the importance of antifungal drugs has risen significantly (Perlin et al., 2017;Fuentefria et al., 2018).Despite many advances in antifungal therapies over the past few decades, the antifungal treatment options are currently limited to only four structural classes of drugs -polyenes, azoles, 5-fluorocytosine, and echinocandins (Perlin et al., 2017;Van Daele et al., 2019).These classes are divided into their respective group based on their mechanism of action (Perea and Patterson, 2002;Pappas et al., 2010).Each of the above-mentioned classes of antifungal therapies have limitations in relation to effectiveness, toxicity and/or the development of drug resistance (Fuentefria et al., 2018).Moreover, the emergence of intrinsic resistance and the ongoing evolution of drug resistant strains has put weight on the limited selection of antifungals available and contributes to the challenge of treating these infections (Perfect, 2017;Wiederhold, 2017;Geddes-McAlister and Shapiro, 2019;Bermas and Geddes-McAlister, 2020).Therefore, the discovery of novel antifungal therapies is critical for fighting these deadly infections (Perlin et al., 2017).
Melanins are dark, negatively charged, hydrophobic pigments that are naturally produced by a multitude of microbes, including bacteria and fungi (Casadevall et al., 2000;Nosanchuk and Casadevall, 2006;Eisenman and Casadevall, 2012;Garcia-Rubio et al., 2020).Eumelanin is a black-brown variety of melanin synthesized by phenoloxidases within a select number of microbes from 3,4-dihydroxyphenyalanine (DOPA) (Hogan et al., 1996;Nosanchuk and Casadevall, 2006;Eisenman and Casadevall, 2012).This variety of melanin is comprised of two monomers, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Selvaraju et al., 2015).The unique properties of the pigments are suggested to have a potential application in the field of medicine (Eisenman and Casadevall, 2012).Melanin production has been associated with increased virulence for various pathogenic microorganisms due to its ability to reduce host defenses by means of antimicrobial mechanisms, including protection from oxidative stress (Nosanchuk and Casadevall, 2006).The ability to protect microbes from the defenses of the host could be relevant to the development of antimicrobial therapies since the use of antimicrobials in tandem with the host immune defenses can increase the effectiveness of some antimicrobial therapies (Nosanchuk and Casadevall, 2006;Adhikari et al., 2022).As such, melanin could be a potential target for the discovery of future antimicrobial therapies.A recent study by Adhikari et al. utilized vanillin for the synthesis of a eumelanin-inspired indolyenepheneethylene synthetic compound, EIPE-1 (Adhikari et al., 2022).Through the application of synthetic approaches for derivatization of the methyl-4,7-dibromo-5,6dimethoxy-1-methyl-1H-indole-2-carboxylate (DBI), a eumelanininspired indole core decorated at the 4-and 7-positions, EIPE-1 was prepared as a potential new antimicrobial agent (Selvaraju et al., 2015;Adhikari et al., 2022;Reed et al., 2023).EIPE-1 has two bactericidal moieties ligated to the DBI core that were intended to exhibit similar antibiotic mechanisms to cationic cell-wall disrupting compounds (Baker et al., 1941;Velkov et al., 2013;Adhikari et al., 2022).This compound demonstrated antimicrobial effects against 13 strains of gram-positive bacteria, including two methicillin resistant strains (Adhikari et al., 2022;Reed et al., 2023).Thus, we hypothesized that EIPE-1 may be effective as an antifungal treatment.In this article, we report the antifungal activity of EIPE-1 and its effects on the medically relevant fungal pathogen, Cryptococcus neoformans.

Reagents
Unless otherwise stated, chemical reagents and plasticware were obtained from Fisher Scientific (St. Louis, MO).PBS used in washing Frontiers in Microbiology 03 frontiersin.org 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% CO 2 in humidified environments.

Cryptococcus cultures
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 phosphatebuffered 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.

Minimum inhibitory concentration and minimum fungicidal concentration assays of Cryptococcus
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 0.5×10 3 /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 assay using EIPE-1 in combination with AmB against Cryptococcus neoformans
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 0.5×10 3 /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 Molecular structure of eumelanin-inspired indolyenepheneethylene (EIPE-1).Conn et al. 10.3389/fmicb.2023.1339303Frontiers in Microbiology 04 frontiersin.orginteraction 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).

Cytotoxicity assay with EIPE-1
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% CO 2 .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 × 10 6 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% CO 2 .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).

Electron microscopy
In order to visualize fungal cells using electron microscopy, a higher quantity of fungal cells (10×10 6 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 × 10 6 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.

Examination of Cryptococcus via scanning electron microscopy
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% OsO 4 ) 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.

Examination of Cryptococcus via transmission electron microscopy
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% OsO 4 at room temperature for 1 h.1% OsO 4 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.

RNA purification of Cryptococcus neoformans
Cryptococcus neoformans at a concentration of 10 × 10 6 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

RNA analysis
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).

Statistical analysis
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.

EIPE-1 inhibits cryptococcal growth
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.

EIPE-1 is non-cytotoxic to mammalian cells
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).

Electron microscopy reveals structural changes to cryptococcal cells
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.

EIPE-1 treatment affects cryptococcal gene expression
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 downregulated.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,  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).

EIPE-1 does not clear cryptococcal infection in Galleria mellonella
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).

Discussion
Despite advances in antifungal therapies over the decades, antifungals are limited to only 14 individual agents (in 4 classes) that have been approved by the U.S. Federal Drug and Food Administration (FDA) for use in the treatment of fungal infections (Dismukes, 2000;Nett and Andes, 2016).The goal of this study is to explore the potential of EIPE-1 as an effective and non-toxic antifungal for the purpose of increasing the current therapies on the market for the treatment of fungal infections, in particular the infection caused by C. neoformans (Perlin et al., 2017;Van Daele et al., 2019).While its original purpose was as a building block for organic semiconductors, it was later discovered that this core has the interesting capability of serving as a foundation for the integration of antibactericidal moieties and possesses intrinsic antimicrobial activity that causes cytoplasmic membrane disruption in gram positive bacteria.Additionally, it was revealed that thirteen different strains from eight gram-positive bacteria, including two methicillin resistant strains were found to be susceptible to EIPE-1 (Adhikari et al., 2022;Reed et al., 2023).While not previously studied in fungal organisms, we found enough evidence to support studying this compound as an antifungal against Cryptococcus isolates.Therefore, we decided to study this compound to determine its use as a novel antifungal drug against Cryptococcus.
Cells come into contact with environmental stressors (Cowen and Steinbach, 2008).A microbe's ability to adapt to these stresses present in its surrounding environment is crucial for survival in their biological niches.One key attribute of Cryptococcus is its ability to survive in harsh environments via sensing, responding, and adapting to changes for its survival and proliferation.During antimicrobial treatments, the fungal organism senses and initiates stress signal pathways which allows them to adapt (Dismukes, 2000;Cowen and Steinbach, 2008;Fuchs and Mylonakis, 2009;Nett and Andes, 2016).A stress response can be seen during treatment with EIPE-1, as several stress-associated genes recognized in previous literature have been  identified in the RNAseq analysis.These genes include but are not limited to the Ricin Beta Lectin superfamily, ATPases associated with diverse cellular activities (AAA+) superfamily, Bcl-2-associated athanogen (BAG)-family proteins and SLC2A (GLUT) family (Doong et al., 2002;Ishikawa et al., 2011;Mueckler and Thorens, 2013;Gallegos et al., 2014;Khan et al., 2022).In addition, we saw a reduction in ribosomal protein translation in our treated populations.Regulation of translation is crucial for C. neoformans to adapt to the environmental stressors (Knowles et al., 2021).This capability of C. neoformans to adapt to stressors demonstrates not only its cellular mechanisms, but also its plasticity of its cell wall, which plays a key role in the defense of the cell from environmental stress and maintains integrity of the cell (Rodrigues et al., 2008;Garcia-Rubio et al., 2020;Upadhya et al., 2023).Disruption of the fungal cellular wall by interfering with glucosidases and chitinase may be an important mechanism by which EIPE-1 exerts its antifungal effects.Specifically, during fungal growth, chitinase is involved in the breakdown of chitin and chitosan by hydrolyzing polymers of chitin at the beta-(1-4) linkages.Chitin and chitosan are vital components of the fungal cell wall and have been shown to contribute to the general stability of the cellular wall (Banks et al., 2005;Baker et al., 2009).All this is an expected response of the cell when exposed to stress and/or apoptotic stimuli of an agent with antifungal capabilities.However, while we believe these genes and transcriptional pathways are the most important for the morphological changes and cellular death observed in the EIPE-1 treated population, we must note that there may be important genes involved that were not identified during initial analyses.Additionally, whereas the C. neoformans genome for strain H99 has been previously sequenced, not all the genes have been annotated to determine the function.Moreover, many of the annotated genes of fungal species are generated by comparison of genomes and by automatic sequence analysis pipelines.Therefore, it is possible that important genes were excluded from our analyses, due to these limitations (Janbon et al., 2014).In the future, follow-up studies need to be done to validate the gene expression data.
Combining all the data from the RNA sequencing and the electron microscopy, we composed a putative model based on the effects of EIPE-1 against C. neoformans as displayed in Figure 6.As found in all living eukaryotic cells, beneath the fungal cell wall, C. neoformans possesses a plasma membrane that consists of a phospholipid bilayer (Rodrigues et al., 2008;Agustinho et al., 2018;Upadhya et al., 2023;Zhukov and Popov, 2023).This membrane maintains the viability of a cell and prevents the free exchange of molecules from the cytoplasm to the cell's environment and vice versa (Upadhya et al., 2023).Previously, microorganism membrane permeability to hydrophobic molecules was identified as being pertinent for susceptibility to the molecule (Reed et al., 2023).Since C. neoformans has a high cellular surface hydrophobicity due to the presence of mannoproteins, lipids, glucan, and chitin molecules, it could allow the hydrophobic EIPE-1 molecule to passively diffuse across the membrane into the cell (van der Rest et al., 1995;Danchik and Casadevall, 2020;Vij et al., 2020).Once within the cell, it interacts with the cell's ability to synthesize the cell wall and the membrane.During this process, the cell responds to the presence of EIPE-1 by the up-regulation of genes involved in stress response, including efflux pumps, heat shock proteins, etc. (Doong et al., 2002;Cowen and Steinbach, 2008;Ishikawa et al., 2011;Mueckler and Thorens, 2013;Gallegos et al., 2014;Holmes et al., 2016;Kurop et al., 2021;Khan et al., 2022).Specifically, efflux pumps allow the organism to regulate its internal environment by the removal of antimicrobial substances (Holmes et al., 2016).While the overall function of EIPE-1 is still relatively unknown, it has been shown to interfere with several pathways involved in the biosynthesis of ergosterol, GPI-anchored proteins, GXM/GalXM, chitin, and chitosan.The result of this interference, the cell's membrane and wall are damaged leading to the formation of a pore and/or breakdown of the cellular membrane.When this occurs, the cell is no longer able to maintain the internal environment and may allow leakage of cellular organelles, as seen in Figure 4C, leading to cellular death.
This model provides perhaps the clearest illustration of the dynamics of EIPE-1 against C. neoformans.However, we must recognize that the true mechanism of the synthetic molecule's antifungal activity against Cryptococcus is still largely unknown.It is possible that the damage we are observing in the treated populations could be a downstream effect of the true target of EIPE-1.Future investigations involving the use of C. neoformans mutant libraries to pinpoint the molecular target of EIPE-1 are currently underway in our laboratory.While these data suggest that EIPE-1 may have EIPE-1 does not provide antifungal protection during in-vivo infection of Galleria mellonella.G. mellonella larvae were inoculated with either PBS alone or EIPE-1 at the concentration of 15 μg/mL, 20 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 150 μg/mL, or 200 μg/mL diluted in PBS.Infected G. mellonella were incubated at 37°C and were examined every 12 h for mortality for 10 days.Larvae were considered dead following full-body melanism and immobility.Data are representative of three independent experiments.potential as a novel antifungal against C. neoformans, we are aware that we do not know the true efficiency of the synthetic compound within an in vivo model.There are various factors that can impact the efficiency of a therapeutic treatment in a living model over time from host-pathogen interactions to the distribution, metabolism, and elimination of EIPE-1 from the host's body.All these can impact the bioavailability and efficiency of the administered drug since less of our drug may remain active or as potent at the target sites of infection (Gillette, 1971;Ekins et al., 2000;Adepu and Ramakrishna, 2021).Additionally, during pathogenesis, C. neoformans cells typically interfere with immune cell recognition and phagocytosis with its virulence factors, including melanin production and a capsule composed of galactoxylomannan (GalXM) and glucuronoxylomannan (GXM).These factors add protective features to the fungal cells.For example, the components of the capsule have an anti-phagocytic influence on immune phagocytes, allowing the pathogen to evade phagocytosis (Kozel and Gotschlich, 1982;Kozel et al., 1988;Yauch et al., 2006;Zaragoza et al., 2009;Vecchiarelli et al., 2013;Conn and Wozniak, 2023).Interestingly, our EIPE-1 treated C. neoformans cells have a reduction in genes that regulate capsule biosynthesis.While this pathway is not typically required for viability of the yeast since acapsular mutants can survive and replicate in vitro (Grijpstra et al., 2009;Tefsen et al., 2014), it is required for virulence in vivo (Chang and Kwon-Chung, 1994;Chang et al., 1996), and the absence of capsule results in a reduction in virulence.This indicates that during treatment our C. neoformans may remain in a less virulent state, which may also aid our immune cells during pathogen clearance when the correct bioavailability and potency is maintained (Gillette, 1971;Ekins et al., 2000;Adepu and Ramakrishna, 2021).It is important to learn the role our innate immune cells will play during pathogen clearance of C. neoformans during EIPE-1 treatment.Studies are ongoing in our lab to understand how treatment with EIPE-1 may affect immune-mediated clearance of C. neoformans.
Finally, C. neoformans is capable of producing its own melanin in the host in the presence of L-DOPA.This plays an important role in protecting C. neoformans from host induced damage due to reactive oxygen species.Melanin is also capable of binding and impacting the effect of antifungal treatments on the fungal cells (Wang and Casadevall, 1994;Zaragoza et al., 2009;Lee et al., 2019).This leads to the question of whether EIPE-1 will be effective against cryptococcal cells when in a melanized form.As previously mentioned, the structure of the EIPE-1 indole core is inspired by eumelanin molecular structure (Adhikari et al., 2022).We do not know if structural similarities will contribute to stronger binding or decrease the susceptibility of C. neoformans to the novel compound.Future studies are being conducted on the potential of EIPE-1 on melanized C. neoformans.A putative model for the mechanism of antifungal activity of EIPE-1.C. neoformans H99 have a complex cell wall comprised of chitin, chitosan, a-1,3 glucan, B-1,3 glucan, B-1,6 glucan, mannoproteins and GPI-anchored proteins.Additionally, they possess a capsule that is consistently maintained on the outer cell wall and composed of GXM and GalXM.EIPE-1, a hydrophobic eumelanin inspired molecule, may enter the cell through a passive method and alters components of the cell by a downregulation in genes involved with capsule biosynthesis, cell wall attachment and remodeling, and ergosterol biosynthesis.

FIGURE 2
FIGURE 2Minimum Inhibition Concentration of EIPE-1 and Amphotericin B against Cryptococcal Strains.Cryptococcal yeast cells (strains H99, Cn145a, R265, R272, R4247, WSA87, or 52D) were incubated in RMPI-MOPs alone, in RPMI-MOPS with either EIPE-1, or Amphotericin B in a two-fold dilution for 48 h at 35°C, with humidity.Optical Densities were determined using a multi-mode plate reader.Data shown are from two independent experiments with each cryptococcal strain and means ± SEM are shown.Statistical significance (p < 0.05) is shown with an asterisk *.Some strains had no variation between experiments and do not have an error bar.
FIGURE 3EIPE-1 is not cytotoxic to mammalian cells.(A) Human cervical epithelial cell line HeLa, (B) Murine fibroblast cell line McCoy, and (C) human lung epithelial cell line A549 were incubated in RPMI-MOPS, in RPMI-MOPS with either 6.25 µg/ml EIPE-1, 12.5 µg/ml EIPE-1, or 62.5 µg/ml EIPE-1 for 24 h at 37°C, 5% CO 2 .Cytotoxicity was defined as greater than 30%.Fluorescence was measured on a Synergy HTX multi-mode plate reader.Data displayed are the mean ± SEM of results of 2 independent experiments.

FIGURE 4 Electron
FIGURE 4Electron Microscopy of C. neoformans with EIPE-1 shows structural changes.C. neoformans H99 were grown in the presence of EIPE-1 at the calculated MIC of 1.749 μg/mL, for 4, 6, 8, and 12 h, fixed with 2.0% glutaraldehyde in 0.1 M cacodylate buffer, prepared for electron microscopy, and examined for TEM or SEM.(A) SEM of C. neoformans displayed structural changes (as indicated by the arrows) to the cells incubated with EIPE-1 for 4, 8, and 12 h, but not in the untreated cells.(B) TEM of C. neoformans displayed structural changes (arrows) to cells treated with EIPE-1 for 4, 8 and 12 h, but not in the untreated cells.(C) TEM of C. neoformans cells treated with EIPE-1 for 12 h cells displayed four variations in structure -rounded cell (undamaged), degraded membrane, black smudge present over the cell, and c-shape.Magnification is 10,000X for SEM and 8,000X for TEM.Images are representative of at least 8 fields per condition per time point examined.

TABLE 1
Gene descriptions of the top 30 differentially expressed genes.
Top 30 most differentially expressed genes in order of p-value greatest to least.General gene descriptions were obtained from Novogene RNA analysis.Adjusted p-values followed by * are below the lowest reliable number from R (p > 2.2 × 10 −308 ).

TABLE 3
Differently regulated genes associated with C. neoformans capsule and cellular wall biosynthesis.

TABLE 3 (
Continued) Genes are in order of p-value greatest to least.General gene descriptions were obtained from Novogene RNA analysis and FungiDB: Fungal & Oomycete Informatics Resources.