Newly Discovered Antimicrobial Peptide Scyampcin44–63 from Scylla paramamosain Exhibits a Multitargeted Candidacidal Mechanism In Vitro and Is Effective in a Murine Model of Vaginal Candidiasis

ABSTRACT The emergence of azole-resistant and biofilm-forming Candida spp. contributes to the constantly increasing incidence of vulvovaginal candidiasis. It is imperative to explore new antifungal drugs or potential substituents, such as antimicrobial peptides, to alleviate the serious crisis caused by resistant fungi. In this study, a novel antimicrobial peptide named Scyampcin44–63 was identified in the mud crab Scylla paramamosain. Scyampcin44–63 exhibited broad-spectrum antimicrobial activity against bacteria and fungi, was particularly effective against planktonic and biofilm cells of Candida albicans, and exhibited no cytotoxicity to mammalian cells (HaCaT and RAW264.7) or mouse erythrocytes. Transcriptomic analysis revealed four potential candidacidal modes of Scyampcin44–63, including promotion of apoptosis and autophagy and inhibition of ergosterol biosynthesis and the cell cycle. Further study showed that Scyampcin44–63 caused damage to the plasma membrane and induced apoptosis and cell cycle arrest at G2/M in C. albicans. Scanning and transmission electron microscopy demonstrated that Scyampcin44–63-treated C. albicans cells were deformed with vacuolar expansion and destruction of organelles. In addition, C. albicans cells pretreated with the autophagy inhibitor 3-methyladenine significantly delayed the candidacidal effect of Scyampcin44–63, suggesting that Scyampcin44–63 might contribute to autophagic cell death. In a murine model of vulvovaginal candidiasis, the fungal burden of vaginal lavage was significantly decreased after treatment with Scyampcin44–63.

endoplasmic reticulum (ER) stress, and apoptosis ( Fig. 1A; Table S4). The downregulated genes mainly related to ergosterol biosynthesis ( Fig. 1A; Table S5). To validate the RNA-seq results, we randomly selected 12 genes, including five upregulated and seven downregulated genes, from the DEG libraries for quantitative reverse-transcription PCR (qRT-PCR) (primers for qRT-PCR are listed in Table S3). The trends of gene expression levels were consistent between qRT-PCR and RNA-seq. Hence, the qRT-PCR results validated the reliability of the RNA-seq data (Fig. 1B).
(ii) KEGG enrichment analysis of DEGs. In response to Scyampcin 44-63 treatment, the KEGG enrichment analysis showed that pathways associated with amino acid metabolism and biosynthesis, biosynthesis of secondary metabolites, 2-oxocarboxylic acid metabolism, autophagy, glycerolipid metabolism, and inositol phosphate metabolism were upregulated, while pathways related to replication and repair, transcription, translation, cell cycle, meiosis, and steroid biosynthesis were downregulated ( Table 2). The DEGs enriched in KEGG pathways (autophagy, cell cycle and steroid biosynthesis, corrected P values , 0.05, and jlog 2 [fold change]j . 1) were individually subjected to hierarchical clustering analysis, and a heat map was constructed (Fig. 1C). Among these DEGs, 19 of the 25 genes participating in the autophagy pathway were upregulated, and 35 of the 39 genes related to the cell cycle and 9 of the 11 genes associated with steroid biosynthesis were downregulated.
Morphological change and extracellular DNA concentration. To explore the anti-candidal mechanism of Scyampcin 44-63 , we used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to observe the morphological and ultrastructural changes of C. albicans induced by Scyampcin 44-63 , respectively. The untreated yeasts were round, while the Scyampcin 44-63 -treated yeasts were deformed and dented (Fig. 5A). TEM images showed that the cell wall was deformed, the membrane was dented and the vacuole was expanded in Scyampcin 44-63 -treated yeasts. There were obvious organelle boundaries in the untreated yeasts, meanwhile, in the Scyampcin 44-63 -treated yeasts, the organelles were barely visible (Fig.  5B). In addition, we measured the extracellular DNA concentration of C. albicans to check whether damaged nuclear and plasma membranes induced the leakage of DNA. The results showed that the concentrations of extracellular DNA in C. albicans were significantly elevated when exposed to 12 to 48 mM Scyampcin 44-63 for 2 h and 4 h (Fig. 5C).

DISCUSSION
C. albicans is responsible for 90% of VVC cases, afflicting 75% of women globally (1). With the increasing incidence of azole-resistant and biofilm-forming Candida species, there is an urgent need to explore new antifungal drugs (3,14). In the present study, we identified a novel AMP with broad-spectrum antimicrobial activity named Scyampcin 44-63 from the mud crab S. paramamosain. Scyampcin 44-63 inhibited C. albicans planktonic growth and biofilm formation and effectively inhibited preformed biofilms, without cytotoxicity toward mammalian cells (HaCaT and RAW264.7) or mouse erythrocytes. Thus, we performed RNA-seq to analyze the potential candidacidal mechanism of Scyampcin 44-63 ; the results suggest that Scyampcin 44-63 might inhibit ergosterol biosynthesis, trigger autophagic cell death and apoptosis, and interfere with the cell cycle. Following these clues, we designed experiments to verify the antifungal features and mechanism and further evaluated the efficacy of Scyampcin 44-63 in vivo.
Several reports have shown that apoptosis is closely associated with cell cycle arrest; for example, antifungal natural products and their derivatives cause cellular apoptosis and dysregulate the cell cycle (29). The plant defensin HsAFP1 also causes S. cerevisiae cell cycle arrest at the G 2 /M phase (30). AMB (4 to 8 mg/mL) (31) and an antifungal compound citral derivative (29) cause cell cycle arrest and apoptosis in C. albicans. The antimicrobial peptides APP and BUF (5-21) cause C. albicans cell cycle arrest in the S phase and subsequently inhibit normal processes (32). However, the precise link between the cell cycle and apoptosis in C. albicans remains unclear. In the present study, several cell cycle-and mitosisrelated genes of C. albicans were downregulated after Scyampcin 44-63 treatment, and we demonstrated that Scyampcin 44-63 induced cell cycle arrest at the G 2 /M phase in C. albicans,  suggesting that one of the candidacidal mechanisms of Scyampcin 44-63 is interference with cell proliferation and division.
The murine model of VVC has been well established, and it is helpful to evaluate the efficacy of antifungal agents, which is an indispensable step for future applications. Cytokines, antibodies, probiotics, and AMPs play vital roles in the immune defense of vaginal tissues against the invasion of Candida species (33). Several AMPs, such as gomesin (34), Hst-5 (35), and NFAP2 (3) significantly reduce the number of C. albicans cells in a murine model of VVC, even though the vaginal fungal burden is still present. Similarly, in this study, intravaginal administration of Scyampcin [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] was effective in reducing the number of C. albicans cells, and fewer pseudohyphae were observed in the murine vagina, which could not eliminate the vaginal fungal burden to undetectable levels. Although AMPs are considered promising new antibiotic candidates, there are still some issues (low stability and bioavailability, potential toxicity, and high cost) that need to be solved for their application in the clinic (36). Some AMPs (such as LL-37 and b-defensins) are severely dysfunctional under physiological salt concentrations and are affected by host factors (37). These findings may partially explain why AMPs cannot fully eradicate the vaginal fungal burden in the murine model of VVC. Furthermore, C. albicans is a polymorphic fungus that grows as yeast, pseudohyphae, and true hyphae (38). C. albicans has the chance to convert yeast to hyphae once in contact with host cells, and the hyphae are more resistant to antifungal drugs, which greatly hampers treatment in vivo (39). Seeking methods to improve antimicrobial activity and safety in vivo requires further research. Previous studies have shown that some AMPs act synergistically with AMPs, surface-active agents, and traditional antibiotics, and these synergistic effects enhance their antimicrobial effect (36). For example, NFAP2 combined with fluconazole indeed enhances the efficacy in vivo (3). Loading AMPs into systems based on nanoparticles or microparticles with a targeted delivery capacity is another strategy to overcome the shortcomings of AMPs (36). In the present study, we provide an option for topical treatment of murine VVC with Scyampcin 44-63 , which needs further optimization. In the future, we will attempt to explore the synergistic effect of Scyampcin 44-63 with AMPs or antibiotics and optimize the delivery approach to improve the efficacy of AMP in vivo.

MATERIALS AND METHODS
Scyampcin gene cloning. The full-length cDNA of Scyampcin was amplified by rapid amplification of cDNA ends (RACE) PCR. The primers for RACE PCR were listed in Table S1. Mud crabs (S. paramamosain, weight: 300 6 10 g) were obtained from Zhangpu FishFarm (Fujian, China), and tissues or organs were collected for further RNA extraction, respectively. TRIzol reagent (Invitrogen, USA), PrimeScript RT reagent kit with a genomic DNA (gDNA) eraser kit (TaKaRa, China), SMARTer RACE 59/39 kit were used to extract total RNA, synthesize cDNA, and RACE cDNA, respectively. The obtained fragment was cloned into pMD18-T Vector (TaKaRa, China) and sequenced by Bioray Biotechnology (Xiamen, China).
Strains and cultivation. The strains used in antimicrobial activity assay were purchased from the China General Microbiological Culture Collection Center (CGMCC). Bacterial strains were cultivated in nutrient broth (NB) at 37°C, and fungal strains were cultured in yeast extract peptone dextrose (YPD) or potato dextrose agar (PDA) at 28°C. All experiments were strictly executed according to the guidelines of the standard biosecurity and institutional safety procedures established by Xiamen University.
Cell line and cultivation. Murine macrophage RAW264.7 was cultured in Dulbecco's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS). Human immortalized keratinocytes HaCaT were maintained in minimal essential medium (MEM) with 10% FBS. The aforementioned cell lines were cultivated at 37°C with 5% CO 2 atmosphere.
Cytotoxicity assay. The CellTiter 96 AQ ueous assay (Promega, USA) was used to determine cell viability (8). The CellTiter 96 AQ ueous Assay is composed of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS). RAW264.7 or HaCaT were seeded to 96-well plates (1 Â 10 4 cells/well) for 18 to 24 h. Fresh media with or without different concentrations of the test peptide were added slightly to the wells and incubated for another 24 h. The vehicle group was treated with fresh medium containing 5% water. After incubation with tested peptide, 20 mL of MTS-PMS reagent was added to each well and incubated for 1 to 4 h. The absorbance of each well was measured at 492 nm in a multimode microplate reader (Tecan, Switzerland). The experiments were repeated three times independently.
Antimicrobial activity assay. The broth dilution method was used to determine the antimicrobial activity of the peptide based on a previous report with some modifications (9). Briefly, microorganisms were cultured to midlogarithmic phase and then harvested. The bacteria were diluted to approximately 5 Â 10 5 CFU/mL with Mueller-Hinton broth, and yeasts or conidia of filamentous fungi were diluted to approximately 5 Â 10 4 cells/mL in RPMI 1640 medium buffered with 0.165 mol/liter 3-morpholinopropane-1-sulfonic acid (pH of 7.0, referred to as RPMI-MOPS). Finally, the obtained microbial suspensions were mixed with serially diluted peptides in 96-well plates and then incubated under the corresponding conditions for 24 or 48 h. The final concentration contains 50% RPMI-MOPS (vehicle control). The MIC, MBC, and MFC values were recorded as previously described (9). The experiments were performed three times independently.
Transcriptomic analysis. C. albicans (3.75 Â 10 7 cells/mL in RPMI-MOPS) were incubated with an equal volume of Scyampcin 44-63 maintained in shaking at 28°C for 1 h. The yeast cells were collected and ground in liquid nitrogen. Total RNA was extracted with TRIzol reagent. RNA integrity was assessed, and qualified RNA was used to construct a library. Transcriptomic sequencing was performed by Novogene Corporation (Beijing, China) using the Illumine NovaSeq platform. The reference genome and annotation files of C. albicans SC5314 were downloaded from NCBI (RefSeq accession number GCF_000182965.3). Hisat2 version 2.0.5 was applied to construct an index of the reference genome, and sequence alignment was performed between paired-end clean reads and the reference genome. The reads mapped to each gene were counted using FeatureCounts version 1.5.0-p3. The DESeq2 R package (1.20.0) was used to analyze differential gene expression, with thresholds of corrected P values , 0.05 and jlog 2 (fold change)j . 1 (P values were corrected based on Benjamini and Hochberg's approach). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was performed using the clusterProfiler R package (3.4.4).
Time-killing kinetics. Time-killing kinetics of different concentrations of peptide were conducted in the same way as the antimicrobial activity assay. The cell density of C. albicans was adjusted to approximately 5 Â 10 4 cells/mL. The suspensions of C. albicans were mixed with equal volumes of testing peptide (1Â, 2Â MBC) and incubated for 0, 1, 2, 4, 6, 8, or 10 h. The mixed suspensions were diluted and spread onto yeast extract peptone dextrose agar plates. After incubation for 24 h, the surviving colonies were counted. The experiments were repeated three times independently. When evaluating the effect of 3-MA on the candidacidal activity of Scyampcin 44-63 , C. albicans cells were pretreated with or without 10 mM 3-MA for 1 h. Different concentrations of Scyampcin 44-63 were added and incubated for 30 min, 1 h, and 2 h. The final concentration contains 50% RPMI-MOPS (vehicle control). The mixed suspensions were diluted and spread onto plates. After incubation for 24 h, the surviving colonies were counted. The experiments were repeated twice independently.
Antibiofilm activity assay. Antibiofilm activity assay was performed as described previously, with some modifications (41). Briefly, 200 mL of a 2.5 Â 10 6 CFU/mL suspension of C. albicans in RPMI-MOPS was seeded in 96-well plates for 4 or 24 h, followed by washing with RPMI-MOPS to remove planktonic C. albicans. Fresh RPMI-MOPS with or without Scyampcin 44-63 was added to the plate and further incubated for 24 h. The final concentration contains 50% RPMI-MOPS (vehicle control). After incubation, the planktonic C. albicans were removed by washing three times with sterile water. The biofilm was then stained with 100 mL of 0.1% crystal violet. Excessive stains were removed by washing the plate three times, 95% ethanol was used to dissolve the stain, and the absorbance was measured at 595 nm. The experiments were repeated three times independently.
TUNEL assay. Apoptosis in C. albicans was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays as previously reported with some modifications (9). Briefly, C. albicans (2.5 Â 10 6 cells/mL) in RPMI-MOPS were treated with or without Scyampcin 44-63 for 2 h at 28°C. The final concentration contains 50% RPMI-MOPS (vehicle control). The samples were washed twice with PBS and then fixed in 4% paraformaldehyde for more than 30 min at 4°C. The traces of paraformaldehyde were washed with PBS, and the cells were permeabilized with 0.3% Triton X-100 on ice for 30 min. TUNEL staining was performed according to the manufacturer's instructions of the one-step TUNEL apoptosis assay kit (Beyotime, China). After staining, the samples were imaged with a confocal laser microscope. The experiments were repeated three times independently.
Transmission electron microscopy (TEM). TEM samples were prepared following the protocol reported by Liu and colleagues (43) with some modifications. Briefly, C. albicans (1.25 Â 10 7 cells/mL) were suspended in RPMI-MOPS, and an equal volume of peptide or sterile water (vehicle control) was added and followed by incubated for 1 h (28°C, 230 rpm). After treatment, the fungal cells were fixed with 2.5% glutaraldehyde and 0.5% paraformaldehyde. Glutaraldehyde and paraformaldehyde were diluted in 0.2 M phosphate buffer (pH of 6.8) and PIPES buffer (pH of 6.8), respectively. PIPES buffer contained 1 mM MgCl 2 , 1 mM CaCl 2 , and 0.1 M sorbitol. After fixation for 2 h at 4°C, the fungal cells were washed three times and pre-embedded in 2% agar. Then the specimens were postfixed with 1.5% KMnO 4 for 2 h at 4°C and washed three times with sterile water. All samples were dehydrated in a graded acetone series (50%, 70%, 85%, 95%, 100%, 100%, and 100%) for 5 min each. After dehydration, all samples were transferred to graded ratios between epoxy resin and acetone (1:3, 1:1, and 3:1) for 1.5 h each, and all samples were incubated in pure epoxy resin overnight. Finally, all samples were sectioned into ultrathin sections, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (Hitachi HT-7800, Japan). The experiments were repeated two times independently.
Measurement of extracellular DNA concentration. C. albicans (2.5 Â 10 8 cells/mL) in RPMI-MOPS were exposed to different concentrations of peptide or sterile water (vehicle control) and incubated for 2 h or 4 h. After incubation, the centrifuged supernatants were collected and measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The experiments were repeated three times independently.
Cell cycle assay. The DNA-specific fluorescent dye Sytox Green was applied to measure the DNA content. C. albicans (10 6 cells) were collected after exposure to different concentrations of Scyampcin 44-63 for 1 h. The final concentration contains 50% RPMI-MOPS (vehicle control). The samples were washed twice with 50 mM sodium citrate buffer and then fixed in cold 70% ethanol at 4°C for 12 h. The washing process was repeated for twice, and the cells were resuspended in buffer containing RNase A and incubated for 1 h at 37°C. SYTOX Green was then added and stained for 30 min (44). The DNA contents were measured with flow cytometer (Becton, Dickinson, USA). The data were analyzed using ModFit LT software (Verity Software House, USA). The experiments were repeated three times independently.
Murine model of VVC. Healthy C57BL/6J female mice (6 to 8 weeks old, 16 to 18 g) were purchased from GemPharmatech and acclimated for 1 week before the experiment. The mice were housed in individually ventilated cages (IVCs) on a 12-h light:12-h dark cycle at room temperature and provided access to sterile food and water ad libitum. The murine model of Candida vaginitis was constructed following a previously described study with some modifications (35,45). Briefly, each mouse was injected subcutaneously with 100 mL of sesame oil containing 200 mg of estradiol valerate (Sigma-Aldrich, Unite States). Three days later, each mouse was inoculated intravaginally with 10 mL of C. albicans (2.5 Â 10 7 CFU/mL). Twenty-four hours postinfection, mice received vaginal perfusion of 10 mL phosphate-buffered saline or different concentrations of Scyampcin 44-63 (12 or 48 mM/group) or 100 mg/mL fluconazole every 12 h. After 3 consecutive days, the vagina of each mouse was rinsed with 200 mL of PBS. The lavage fluid was diluted with PBS and then spread on Sabouraud's dextrose agar plates containing 1.25 mg/mL chloramphenicol (Solarbio, China) for fungal quantification. The aforementioned animal experiments were performed according to national guidelines and approved by the Laboratory Animal Management and Ethics Committee of Xiamen University (XMULAC20220048). The experiments were performed twice independently.
For histology, the vaginal tissues were fixed with 4% formaldehyde, and then paraffin-embedded vaginal tissues were obtained. Sections were cut from the embedded tissues and subjected to hematoxylin and eosin staining (3).
Statistical analysis. All data are represented as the means 6 standard error of the mean. Two-tailed Student's t test, one-way analysis of variance (ANOVA) or two-way ANOVA was used to analyze the differences between different groups. Figure 2 and 7 used one-way ANOVA, Fig. 3 used two-way ANOVA, and the other figures used Student's t test. P values , 0.05 were considered significant.
The DESeq2 R package (1.20.0) was used to analyze transcriptomic data for differential gene expression with thresholds of corrected P values , 0.05 and jlog 2 (fold change)j . 1 (P values corrected base on Benjamini and Hochberg's approach).
Data availability. All relevant data are within the article and its supporting information files. The full-length cDNA sequence of Scyampcin and transcriptomic data of C. albicans are both available on NCBI database (GenBank accession number MW388710; BioProject number PRJNA906206).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, DOCX file, 11.1 MB.