Mud crabs (S. paramamosain) were obtained from Zhangpu Fish Farm (Fujian, China), and testes from male crabs (body weight 300 ± 10 g) were used for RNA extraction. All commercially available strains used in this study were purchased from China General Microbiological Culture Collection Center (CGMCC), including Pseudomonas fluorescens (CGMCC NO. 1.3202), Pseudomonas stutzeri (CGMCC NO. 1.1803), Pseudomonas aeruginosa (CGMCC NO. 1.2421), Acinetobacter baumannii (CGMCC NO. 1.6769), Escherichia coli (CGMCC NO. 1.2385), Staphylococcus aureus (CGMCC NO. 1.2465), Staphylococcus epidermidis (CGMCC NO. 1.4260), Bacillus cereus (CGMCC NO. 1.3760), Enterococcus faecium (CGMCC NO. 1.131), Enterococcus faecalis (CGMCC NO. 1.2135), C. neoformans (CGMCC NO. 2.1563), Aspergillus niger (CGMCC NO. 3.316), A. fumigatus (CGMCC NO. 3.5835), Fusarium graminearum (CGMCC NO. 3.4521), and F. oxysporum (CGMCC NO. 3.6785). The bacterial strains were cultured in nutrient broth at 37°C, and the fungal strains were grown in potato dextrose agar (PDA) at 28°C. All experiments were carried out in strict accordance with the guidelines of the standard biosecurity and institutional safety procedures established by Xiamen University.

Rapid amplification of cDNA ends (RACE) PCR was performed to obtain the full-length cDNA of Sparamosin. Total RNA from the testes of normal mature crabs was extracted using TRIzol^TM reagent (Invitrogen, United States) and cDNA was synthesized using PrimeScript^TM RT reagent Kit with a gDNA Eraser Kit (Takara, China). In addition, RACE cDNA was prepared using SMARTer^® RACE 5′/3′ Kit (Takara, China) and was used as a template for PCR. Gene-specific primers (Supplementary Table 1) were designed based on the obtained partial cDNA sequence from transcriptome database established by our laboratory to amplify the target gene, and the fragment was recombined into pMD18-T Vector (Takara, China) and sequenced by Bioray biotechnology (Xiamen, China).

The signal peptide of Sparamosin was predicted by SignalP-4.1 Server¹ and the second structure of the mature peptide was predicted by PSIPRED 4.0.² The mature peptide was truncated between residue Ser²⁵ and residue Gly²⁶ to evaluate the antimicrobial activity of different helical regions. The physicochemical parameters of the peptides, such as molecular weight, theoretical isoelectric point, net positive charge and hydropathicity were predicted by ProtParam.³ The total hydrophobicity was calculated through the Antimicrobial Peptide Database.⁴ The peptides used in this study were chemically synthesized by Genscript (Nanjing, China). The purity and molecular weight of the peptides were further confirmed by high performance liquid chromatography and mass spectrometry, respectively. The powdered peptide could be stored for a long time at −80°C. The stock solutions were kept at −20°C for storage.

The antimicrobial activity was determined three times, each time in triplicate, in 96-well microplates according to previous reports with some modifications (Vitale et al., 2002; Wiegand et al., 2008; Castro et al., 2018). Briefly, microorganisms were harvested during their logarithmic growth phase and diluted in Mueller-Hinton broth to approximately 1 × 10⁶ CFU/mL (bacteria) or diluted in RPMI 1640 medium buffered with 0.165 mol/L 3-morpholinopropane-1-sulfonic acid (pH 7.0, referred to as RPMI-MOPS) to approximately 2 × 10⁴ cells/mL (yeasts or conidia of filamentous fungi). Then, the microbial suspension was added to each well and incubated with serial diluted peptides. The microplates were incubated in the dark at 37°C for 24 h (bacteria) or at 28°C for 48 h (yeasts or conidia of filamentous fungi). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) values were determined as previously described (Shan et al., 2016).

The cytotoxicity of Sparamosin_26–54 was determined on mouse hepatocytes (AML 12 cells) and human hepatocytes (L02 cells). CellTiter 96^® AQ_ueous one solution Cell Proliferation assay (Promega, United States) was used to assess cell viability. Briefly, 100 μL of AML 12 or L02 cells were seeded in 96-well microplates at 10⁴ cells/well and incubated at 37°C overnight under 5% CO₂. Then, the cells were incubated at 37°C for 24 h in a culture medium supplemented with various concentrations of Sparamosin_26–54 (0, 0.1, 1, 10, 100 μg/mL). After incubation, the cells were treated with 20 μL of MTS-PMS reagent for another 2 h, and then the absorbance value of each well was measured at 492 nm (Tecan, Switzerland).

The biofilm inhibition assays were performed in 96-well microplates according to previous reports with some modifications (Berditsch et al., 2015; Poonam et al., 2017). For the biofilm formation assay, C. neoformans cells were harvested in the logarithmic phase, diluted in RPMI-MOPS medium to a final cell density of approximately 1 × 10⁶ cells/mL, and then aliquoted into microplates. Serially diluted concentrations of Sparamosin_26–54 were added to the wells, and the microplates were incubated at 35°C for 72 h without shaking to allow biofilm formation. The biofilm mass was evaluated by crystal violet (CV) staining as previously described (Berditsch et al., 2015). In experiments with preformed biofilms, C. neoformans cells were incubated at 35°C for 72 h without shaking to allow biofilm formation. Thereafter, RPMI-MOPS medium containing Sparamosin_26–54 and resazurin (final concentration 0.1 mM) was added to the wells, and the microplates were incubated at 35°C for another 24 h. The respiratory activity of cells in biofilm was evaluated by a modified resazurin assay as previously described (Berditsch et al., 2015).

C. neoformans suspensions at approximately 1 × 10⁷ cells/mL in RPMI-MOPS were incubated with different concentrations of Sparamosin_26–54 (6, 12 μM) at 28°C for 1 h. Then, the cells were harvested for RNA extraction. Approximately 200 μL of freeze-dried cells were grinded in liquid nitrogen and the total RNA was isolated using TRIzol reagent. RNA-Seq was performed by Novogene Corporation (Beijing, China) using the Illumina NovaSeq platform, which could generate 150 bp paired-end reads. Reference genome and gene model annotation files of C. neoformans var. neoformans JEC21 were downloaded from GenBank (GCA_000091045.1). An index of the reference genome was constructed using Hisat2 (version 2.0.5), and paired-end clean reads were aligned with the reference genome using Hisat2 (version 2.0.5). FeatureCounts (version 1.5.0-p3) was used to calculate the number of reads mapped to each gene. The expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) for each gene was calculated based on the length of the gene and the count of reads mapped to that gene. Differential expression analysis of the two groups was performed using the DEGSeq2 R package (version 1.16.1). The p-value was adjusted using the Benjamini and Hochberg’s method. The corrected p-value < 0.05 and | log₂(Fold change)| > 1 were set as thresholds for significant differential expression. The gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was implemented by the clusterProfiler R package (version 3.4.4). GO terms with a corrected p-value < 0.05 were considered significantly enriched by DEGs.

To verify the results of RNA-Seq, five up-regulated genes encoding chitin synthase (CNA05300), Rho1 GTPase (CNG02630), catalase (CNL06020), DNA supercoiling (CND02890) and caspase (CNB00130) and six down-regulated genes encoding C-22 sterol desaturase (CNF03720), NADH dehydrogenase (CND01070), succinate dehydrogenase (CNA03530), ubiquinol-cytochrome c reductase complex core protein 2 (CNL04470), cytochrome c oxidase subunit V (CNK03240) and ATP synthase subunit alpha (CNF02280) were selected for quantitative real-time PCR (qRT-PCR). The qRT-PCR reactions were performed on qTOWER 2.2 real time PCR system (Analytik Jena AG, Germany) according to the FastStart Universal SYBR Green Master (ROX) kit (Roche, Switzerland) protocol. The cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. Gene expression levels were calculated using the 2-ΔΔ^Ct method (Livak and Schmittgen, 2001) and normalized to the abundance of the house-keeping gene actin. The primers of target genes were listed in Supplementary Table 2.

The analysis of the localization of Sparamosin_26–54 in C. neoformans was carried out based on the previous report with slight modifications (Zhang et al., 2019). Briefly, C. neoformans cells were harvested in the logarithmic phase and washed with 10 mM sodium phosphate buffer (NaPB, pH 7.4). The cells were diluted in the same buffer to a final cell density of approximately 1 × 10⁷ cells/mL, and incubated with 24 μM FITC-labeled Sparamosin_26–54 [synthesized by GL Biochem (Shanghai, China)] at room temperature for 30 min. After incubation, the cells were washed twice with 10 mM NaPB to remove unbound peptide. A confocal laser scanning microscopy (Zeiss, Germany) were used for imaging.

The polyclonal antibody of Sparamosin was prepared by GenScript (Nanjing, China) using the antigen site KVQHSIFSGLGPNPC, which was designed and optimized by OptimumAntigen Design Tool. The Sparamosin_26–54-phospholipid interactions were determined using PIP Strips^TM (Echelon Biosciences, United States) according to the protocol. Briefly, the membrane was blocked with 3% (wt/vol) BSA at room temperature for 1 h, and then incubated with 2 μg/mL Sparamosin_26–54 at room temperature for 1 h. Then, the membrane was incubated with Sparamosin antibody (1:1,000, diluted in 1% [wt/vol] BSA) at room temperature for 2 h. After washing with PBST several times, the membrane was then incubated with HRP conjugated goat anti-rabbit IgG (1:5,000, diluted in 1% [wt/vol] BSA) at room temperature for 40 min. The peptides were visualized on a chemiluminescent imaging system (Tanon Science and Technology, United States) using immobilon^TM western chemiluminescent HRP substrate (Millipore, United States).

The time-killing kinetics assay was carried out as previous described (Shan et al., 2016). Briefly, C. neoformans cells were diluted in RPMI-MOPS medium to a final cell density of approximately 1 × 10⁶ cells/mL. Sparamosin_26–54 was incubated with C. neoformans at a concentration of 1 × or 2 × MIC. The culture was sampled diluted and plated on PDA plates at different time points. After 48 h of incubation, the surviving colonies were counted, and the untreated group was used as a control. The experiments were conducted three times independently.

The effect of Sparamosin_26–54 on C. neoformans was observed using SEM as described earlier (Datta et al., 2016). Briefly, C. neoformans cells were harvested in the logarithmic phase and resuspended at approximately 1 × 10⁷ cells/mL in NaPB, and incubated with 24 μM Sparamosin_26–54 at room temperature for 1 h. After incubation, the cells were fixed with 2.5% (vol/vol) glutaraldehyde at 4°C for 2 h and washed three times before being placed on poly-L-lysine coated glass slides at 4°C for 30 min. The cells were subsequently dehydrated in a graded series of ethanol (30, 50, 70, 90, 95, and 100%), for 15 min each. Thereafter, the samples were dehydrated with tertiary butanol followed by freezing at 4°C and lyophilized using the critical point dryer. Finally, the specimens were coated with gold and observed under a field emission scanning electron microscope (Zeiss SUPRA 55, Germany).

The TEM analysis was carried out based on the previous report with slight modifications (Chen et al., 2003). Briefly, C. neoformans cells were prepared and incubated with Sparamosin_26–54 as described above for SEM analysis. After pre-fixation with 2.5% glutaraldehyde at 4°C overnight, the C. neoformans cells were washed three times with NaPB and then post-fixed with 1% osmium tetroxide at 4°C for 2.5 h. The fixed samples were washed three times with NaPB, dehydrated in a graded ethanol series (30, 50, 70, 90, and 100%), transferred to a graded mixture of absolute acetone and epoxy resin, and finally immersed in pure epoxy resin in a constant-temperature incubator overnight. Finally, the samples were sectioned using an ultramicrotome, stained with uranyl acetate and lead citrate, and observed using a transmission electron microscope (FEI, United States).

The integrity of the cell membrane after Sparamosin_26–54 treatment was determined by live-dead staining according to the protocol of a LIVE/DEAD^® FungaLight^TM Yeast Viability Kit (Invitrogen, United States). Briefly, C. neoformans cells were prepared and incubated with Sparamosin_26–54 as described above for SEM analysis. After incubation, the cells were washed twice with PBS and placed on poly-L-lysine slides at room temperature for 15 min. The cells were washed twice and then stained with SYTO^® 9 and PI for 15 min in the dark. Fluorescent images were obtained with a confocal laser scanning microscopy (Zeiss, Germany).

The calcein leakage assay was carried out according to a previous report with the following modifications (Vylkova et al., 2007). Briefly, C. neoformans cells were harvested in the logarithmic phase and washed with 10 mM NaPB. The cells were diluted in 10 mM NaPB to a final cell density of approximately 1 × 10⁷ cells/mL and loaded with Calcein-AM (Thermo Fisher Scientific, United States) at a final concentration of 5 μM at room temperature for 2 h. Then, the cells were washed three times with 10 mM NaPB to remove unincorporated dye. Different concentrations of Sparamosin_26–54 (from 6 to 48 μM) were incubated with calcein-loaded cells in a 96-well microplate for analysis. In a microplate reader (Tecan, Switzerland), the fluorescence intensity of the induced calcein release was recorded every 15 min at the excitation and emission wavelengths of 485 nm and 530 nm, respectively. Percent leakage was calculated using the formula: percentage leakage (%) = (F_sample − F₀)/(F_T − F₀) × 100. Where F_sample is the fluorescence intensity after adding Sparamosin_26–54, and F₀ is the fluorescence intensity determined by measuring the amount of calcein released from loaded cells without peptide treatment in 240 min, and F_T is the maximum fluorescence intensity measured after the cells were boiled for 10 min. After boiling, the fluorescence intensity of calcein-loaded C. neoformans was assumed to be equal to the total potentially available intracellular calcein.

C. neoformans suspensions at 1 × 10⁷ cells/mL in RPMI-MOPS were incubated with different concentrations of Sparamosin_26–54 (6, 12, and 24 μM) at 28°C for 0.5, 1, and 2 h. Then, the yeasts were collected by centrifugation, and the supernatant was subjected to DNA quantitative analysis in a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States), or an ATP determination kit (Invitrogen, United States) for quantitative analysis of ATP, as described previously (Castro et al., 2018; Liu et al., 2020).

The production of intracellular reactive oxygen species (ROS) was measured by fluorometric assay using 2,7-dichlorofuorescin diacetate (DCFH-DA), as described previously (Hwang et al., 2011b). Briefly, C. neoformans suspensions at 1 × 10⁷ cells/mL in RPMI-MOPS were incubated with Sparamosin_26–54 (6, 12 μM) or 20 mM H₂O₂ at 28°C for 1 h. After incubation, the cells were washed with PBS and then stained with 10 μM of DCFH-DA (Nanjing Jiancheng Bioengineering Institute, China). The cells were then harvested and observed under fluorescence microscopy (Zeiss, Germany).

The mitochondrial membrane potential (MMP) was determined by fluorometric assay using 3,3′-dihexyloxacarbocyanine iodide DiOC₆(3), as mentioned earlier (Hwang et al., 2011b). Briefly, C. neoformans suspensions at 1 × 10⁷ cells/mL in RPMI-MOPS were incubated with Sparamosin_26–54 (6, 12 μM) or 20 mM H₂O₂ at 28°C for 3 h. After incubation, the cells were washed with PBS, and incubated with 2 ng of DiOC₆(3) (Sigma, United States) at 28°C for 30 min. The cells were then harvested and analyzed by flow cytometer (Becton Dickinson, United States).

Terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay was carried out as described previously (Castro et al., 2018). Briefly, C. neoformans suspensions at 1 × 10⁷ cells/mL in RPMI-MOPS were incubated with Sparamosin_26–54 (6, 12 μM) or amphotericin B (4 μg/mL) at 28°C for 24 h. After incubation, the cells were washed with PBS and fixed with 2% formaldehyde in PBS at room temperature for 30 min. Then, the cells were permeabilized with 0.1% Triton X-100 for 30 min and subjected to TUNEL reaction using one step TUNEL apoptosis assay kit (Beyotime, China), according to the manufacturer’s instructions. Finally, all samples were examined by fluorescence microscopy (Zeiss, Germany).

All data were represented as mean ± standard error of mean. Statistical analysis was performed with Student’s t-test using GraphPad Prism 6 (GraphPad Software Inc., United States) and SPSS 25 (IBM Corp., United States). Two-tailed p-values were used for all analyses, and a p-value < 0.05 was considered statistically significant. Transcriptome analysis was performed using Novomagic (Novogene, China). Pearson analysis was used to correlate gene expression determined by RNA-seq and qRT-PCR.

The full-length cDNA sequence of Sparamosin was obtained (Genbank accession number MH423837). This gene consists of a 5′ untranslated region (UTR) of 181 bp, an open reading frame (ORF) of 231 bp and a 3′ UTR of 161 bp. The ORF of Sparamosin encodes a 76-amino acid protein, which contains a putative 22-amino acid signal peptide and a 54-amino acid mature peptide (Figure 1A). No similar nucleotide or amino acid sequence was found to match this sequence in the existing online databases, indicating that it is an uncharacterized protein. Two truncated peptides, Sparamosin_1–25 and Sparamosin_26–54 (which located in the 1st to 25th and 26th to 54th amino acid of the mature peptide, respectively), were designed and synthesized based on the secondary structure of Sparamosin mature peptide, which has two predicted α-helices located at residues Ile¹³-Phe²⁴ and residues Pro³¹-Arg³⁹, respectively (Figures 1B,C). The key physicochemical parameters of Sparamosin and its truncated peptides were shown in Figure 1D. In addition, the peptide sequences of Cecropin A (Steiner et al., 1981), Magainin II (Zasloff, 1987), and LL-37 (Gudmundsson et al., 1996) were also analyzed as comparative controls. The measured molecular weights of Sparamosin and its truncated peptides were consistent with their theoretical values, indicating that these peptides were successfully synthesized. The hydrophobic residue ratio of six peptides ranged from 32 to 45%, the net charge ranged from −3 to + 6, and the hydrophobic moment ranged from 0.105 to 0.521. The results showed that unlike the other two synthetic peptides (Sparamosin and Sparamosin_1–25), the physicochemical parameters of Sparamosin_26–54 have values closer to those defined for AMP.

Our preliminary study on six commercially available CGMCC strains showed that Sparamosin_26–54 displayed stronger antimicrobial activity than Sparamosin, while no antimicrobial activity of Sparamosin_1–25 was observed in the tested strains (Supplementary Table 3). Then, we further evaluated the antimicrobial efficacy of Sparamosin_26–54 by determining its MIC, MBC and MFC against a series of strains. As shown in Table 1, Sparamosin_26–54 displayed a broad-spectrum antibacterial activity against several Gram-negative (P. fluorescens, P. stutzeri, P. fluorescens, A. baumannii, E. coli) and Gram-positive (S. aureus, S. epidermidis, B. cereus, E. faecium, E. faecalis) bacteria with MIC values in the range of 6–48 μM, as well as the MBC values lower than 48 μM. In addition, Sparamosin_26–54 showed a profound inhibitory effect against a pathogenic fungus C. neoformans, and could inhibit the conidial germination of several filamentous fungi, such as A. niger, A. fumigatus, F. graminearum, and F. oxysporum with MIC values in the range of 6–24 μM. However, it was not cytotoxic to mammalian cells tested (AML12 and L02 cells) (Supplementary Figures 1A,B), indicating that Sparamosin_26–54 had good biocompatibility.

Since Sparamosin_26–54 showed good activity against planktonic C. neoformans cells, we further evaluated whether Sparamosin_26–54 could inhibit C. neoformans biofilm formation. The biofilm mass of C. neoformans was quantified by CV staining, and the results were shown in Figures 2A,B. The concentration of Sparamosin_26–54 required to inhibit C. neoformans biofilm formation was 12 μM. At a concentration of 48 μM, the inhibition rate of biofilm formation was more than 90%. In addition, we used the redox indicator resazurin to monitor the respiratory activity of the preformed C. neoformans biofilms treated with Sparamosin_26–54. The results showed that Sparamosin_26–54 could inhibit the respiration of C. neoformans in preformed biofilms. At a concentration of 48 μM, the inhibition rate of respiratory activity exceeded 50% (Figures 2C,D). These results suggested that Sparamosin_26–54 exhibited potent anti-biofilm activity against C. neoformans.

To build a comprehensive model of C. neoformans response to Sparamosin_26–54, RNA sequencing was used to determine DEGs in response to treatment with a low dose of Sparamosin_26–54. We obtained 2,573 genes (up-regulated, 1,474; down-regulated, 1,099) and 3,157 genes (up-regulated, 1,602; down-regulated, 1,555) that were differentially expressed after 0.25 × and 0.5 × MIC Sparamosin_26–54 treatment, respectively (Supplementary Tables 4, 5). Overlapping analysis showed that 1,330 genes were commonly up-regulated under both Sparamosin_26–54 concentrations tested, while 1,023 genes were commonly down-regulated under the same conditions (Figure 3A and Supplementary Tables 6, 7). To further investigate the biological processes involving these up-regulated or down-regulated genes, two DEG lists were separately analyzed for enrichment of GO terms. Genes up-regulated after Sparamosin_26–54 treatment were associated with several significantly enriched biological processes, including cell wall organization or biogenesis, transmembrane transport, lipid oxidation, DNA repair, etc. (Supplementary Table 8). In the biological process category of the down-regulated genes, translation and metabolic processes were significantly enriched (Supplementary Table 8). Figure 3B is a heatmap showing the up-regulated genes involved in the biosynthesis of cell wall component, CWI signaling pathway, anti-oxidative stress, apoptosis and DNA repair, as well as the downregulated genes involved in the ergosterol biosynthesis pathway and mitochondrial oxidative phosphorylation in C. neoformans cells after Sparamosin_26–54 treatment. To verify the RNA-Seq results, a total of 11 genes were selected including 5 up-regulated and 6 down-regulated from DGEs for qRT-PCR analysis. As shown in Figures 3C,D, the qRT-PCR results showed that the expression patterns of selected genes were consistent to those obtained by RNA-seq and the Pearson correlation analysis revealed that these genes were significantly correlated between the RT-qPCR and RNA-seq (Pearson correlation coefficient > 0.98). Thus, the qRT-PCR results confirmed the reliability of the RNA-Seq data.

In many cases, binding to the surface of fungal cell is the first step for AMPs to kill fungi. Therefore, we hypothesized that Sparamosin_26–54 might directly bind to the cell surface of C. neoformans. To test this hypothesis, the distribution of Sparamosin_26–54 in C. neoformans was investigated by FITC-labeled Sparamosin_26–54. As shown in Figure 4A, the fluorescence of FITC-labeled Sparamosin_26–54 was mainly located on the cell surface, indicating that Sparamosin_26–54 might interact with the cell walls/membranes. To further determine the binding properties of Sparamosin_26–54 to cell wall or cell membrane components, a modified ELISA assay and chitin-binding assay were performed. The results showed that Sparamosin_26–54 could not bind glucan or chitin (data not shown). Next, we assessed the ability of Sparamosin_26–54 to bind different bioactive membrane phospholipids using the protein-phospholipid interaction assay. As shown in Figures 4B,C, Sparamosin_26–54 strongly bound to phosphoinositides (PIPs) and phosphatidic acid (PA), but weakly bound to a variety of other phospholipids, including lysophosphatidic acid (LPA), lysophosphocholine (LPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine 1-phosphate (S1P), and phosphatidylserine (PS). These results suggest that Sparamosin_26–54 might bind to fungal cell membrane rather than cell wall.

The time-killing curves for Sparamosin_26–54 on C. neoformans was determined at the concentrations of 1 × and 2 × MIC. As shown in Figure 5A, Sparamosin_26–54 showed rapid killing activity against C. neoformans and completely killed the fungus within 10 min. This rapid fungicidal rate indicated that Sparamosin_26–54 might kill C. neoformans by disrupting the integrity of cell membrane. To further evaluate the effect of Sparamosin_26–54 on the permeability of the C. neoformans cell membrane, the release of calcein, DNA and ATP were measured. As shown in Figure 5B, when exposed to Sparamosin_26–54, calcein leaked from calcein-loaded C. neoformans in a time- and concentration-dependent manner. In addition, Sparamosin_26–54 increased the amount of DNA and ATP detected in the supernatant of the C. neoformans suspensions (Figures 5C,D). Then, we employed SEM and TEM to observe the changes in the morphology and ultrastructure of C. neoformans cells after Sparamosin_26–54 treatment (Figures 6A,B). The SEM images of the peptide-treated cells showed that Sparamosin_26–54 had a morbid effect on C. neoformans cells surface. After treatment with 24 μM Sparamosin_26–54 for 1 h, the surface of the fungal cells became rough and corrugated. In contrast, the control cells that were not treated with Sparamosin_26–54 exhibited a bright and smooth surface. The TEM image of the peptide-treated cells clearly showed the disruption of cell wall and cell membrane and the leakage of intracellular contents, while the fungal cells in the control group showed an intact cell wall and cell membrane and a homogeneous cytoplasm. SYTO 9 can enter all yeasts regardless of their membrane integrity, while PI can only enter yeasts with damaged membranes. Therefore, the integrity of the C. neoformans cell membrane was observed by staining with SYTO 9/PI. As shown in Figure 6C, the untreated cells were stained only with SYTO 9 instead of PI, while cells treated with Sparamosin_26–54 were stained with SYTO 9 and PI. Taken together, these results suggested that Sparamosin_26–54 disrupts the integrity of the cell wall and cell membrane of C. neoformans.

Among the DEGs identified, a gene coding for a metacaspase was found to be up-regulated under Sparamosin_26–54 treatment, indicating that Sparamosin_26–54 may induce apoptosis in C. neoformans. Thus, we first used DCFH-DA probe to measure intracellular ROS in C. neoformans during exposure to Sparamosin_26–54. As shown in Figure 7A, cells treated with 20 mM H₂O₂ as a positive control showed increased DCH staining compared to untreated cells. In accordance with this, cells exposed to Sparamosin_26–54 (6 and 12 μM) displayed a significant increase in intracellular ROS levels compared to untreated cells. These data indicated that Sparamosin_26–54 treatment resulted in the accumulation of ROS in C. neoformans. The dissipation of MMP is considered to be an early and key cellular event in the occurrence of apoptosis (Barroso et al., 2006). To investigate mitochondria-mediated pathway during apoptosis in C. neoformans induced by Sparamosin_26–54, we next examined MMP of C. neoformans by using DiOC₆(3) which is a membrane potential-sensitive dye that aggregating in healthy mitochondria. After treatment with 6 and 12 μM Sparamosin_26–54 for 3 h, MMP was observed by the decrease of the fluorescence intensity of DiOC₆(3), indicating that Sparamosin_26–54 disrupted the MMP of C. neoformans (Figure 7B). To obtain more evidence of apoptosis in C. neoformans induced by Sparamosin_26–54, the TUNEL assay was conducted, which is one of the most reliable methods for identifying the late stage of yeast cell apoptosis, and is used to visualize the amount of DNA fragmentation in individual cells. We used amphotericin B as a positive control because it has been proven to induce apoptosis in C. neoformans cells (Castro et al., 2018). The microscopic images showed the presence of green fluorescence in the cells treated with 6 and 12 μM Sparamosin_26–54, which was consistent with the positive control cells treated with 4 μg/mL amphotericin B (Figure 7C). Since accumulation, MMP dissipation and DNA fragmentation are all hallmarks of apoptosis (Munoz et al., 2012), our data collectively confirmed that C. neoformans treated with Sparamosin_26–54 exhibited apoptosis mechanism.