Lycosin-II Exhibits Antifungal Activity and Inhibits Dual-Species Biofilm by Candida albicans and Staphylococcus aureus

The increase and dissemination of antimicrobial resistance is a global public health issue. To address this, new antimicrobial agents have been developed. Antimicrobial peptides (AMPs) exhibit a wide range of antimicrobial activities against pathogens, including bacteria and fungi. Lycosin-II, isolated from the venom of the spider Lycosa singoriensis, has shown antibacterial activity by disrupting membranes. However, the mode of action of Lycosin-II and its antifungal activity have not been clearly described. Therefore, we confirmed that Lycosin-II showed antifungal activity against Candida albicans (C. albicans). To investigate the mode of action, membrane-related assays were performed, including an evaluation of C. albicans membrane depolarization and membrane integrity after exposure to Lycosin-II. Our results indicated that Lycosin-II damaged the C. albicans membrane. Additionally, Lycosin-II induced oxidative stress through the generation of reactive oxygen species (ROS) in C. albicans. Moreover, Lycosin-II exhibited an inhibitory effect on dual-species biofilm formation by C. albicans and Staphylococcus aureus (S. aureus), which are the most co-isolated fungi and bacteria. These results revealed that Lycosin-II can be utilized against C. albicans and dual-species strain infections.


Introduction
Increasing antimicrobial resistance is a severe threat to public health, warranting the development of effective drugs to combat infections that are caused by multidrug-resistant pathogens, such as bacteria and fungi [1]. The abuse of antibiotics to combat infections has increased, resulting in the development of various multidrug-resistant pathogens [2]. Fungal infections, such as candidiasis, are also becoming increasingly problematic. Three classes of antifungal agents-azoles, polyenes, and echinocandins-are only used in clinical settings. Therefore, an increase in resistant fungal pathogens raises concerns regarding fungal diseases and public health [3].
C. albicans is an opportunistic and prevalent fungal pathogen [4]. C. albicans can cause nosocomial bloodstream infections with a mortality rate of 40%, despite the use of antifungal agents [5][6][7]. In particular, C. albicans can cause superficial infections in immunocompromised individuals receiving organ transplants and chemotherapy [8]. Moreover, C. albicans can form biofilms, which are related to C. albicans infections. C. albicans biofilms are a community of yeast-form, pseudohyphal, and hyphal cells encased in extracellular polymeric substances (EPS) that are resistant to the host immune system and existing antifungal agents [9]. Resistance to antifungal agents by C. albicans biofilms and the ability to colonize surfaces, such as implanted medical devices, negatively impacts patient health [10]. C. albicans is the most common fungal pathogen that is regularly found in bacterial-fungal co-infections.
S. aureus is a major pathogen that causes clinical infections, such as bacteremia and osteoarticular, skin, pleuropulmonary, and device-associated infections. Moreover, S. aureus is frequently isolated with C. albicans in bloodstream infections [11]. Importantly, C. albicans and S. aureus co-infections have increased mortality compared to monomicrobial infections [12]. Therefore, a wide range of novel antimicrobials should be developed to treat these polymicrobial infections.
AMPs play a key role in the innate immune system, functioning as a defense against harmful microbes in various organisms. Additionally, AMPs are a promising alternative to conventional antibiotics. In particular, AMPs yielding broad-spectrum antimicrobial activity and decreased rates of resistance have become potential therapies for the control of infections [13,14].
Lycosin-II, a 21-amino-acid peptide isolated from the venom of the spider Lycosa singoriensis, is an α-helix peptide with antibacterial effects against multi-drug resistant nosocomial bacterial pathogens [15]. In a previous study, we confirmed that Lycosin-II, which disrupted the bacterial membrane, was active against oxacillin-resistant S. aureus and meropenemresistant Pseudomonas aeruginosa [16]. However, it is not clear how Lycosin-II has antifungal activity and can inhibit the formation of C. albicans and S. aureus mono-and multispecies biofilms.
In this study, we investigate the antifungal activity and the mechanism of action of Lycosin-II against C. albicans by examining membrane depolarization and integrity after exposure to Lycosin-II. Moreover, we confirm that Lycosin-II can inhibit biofilm formation by C. albicans and multispecies biofilm formation by C. albicans and S. aureus.
C. albicans (Korea Collection for Type Culture [KCTC] 7270) was used as a reference strain, and it was isolated from a human skin lesion of erosion interdigitalis in Uruguay and purchased from the KCTC. C. albicans (Culture Collection of Antibiotics Resistant Microbes [CCARM] 14001, CCARM 14004, and CCARM 14007) isolated in 1999, as well as C. albicans (CCARM 14020) isolated in 2002, were used as representative drug-resistant strains and were purchased from the CCARM [17]. S. aureus (American Type Culture Collection [ATCC] 25923) was purchased from the ATCC.

Antifungal Activity
To determine the antifungal activity of Lycosin-II, melittin, and the antifungal agents, the minimum inhibitory concentrations (MICs) were determined using a broth microdilution method with some modification [18,19]. Lycosin-II, melittin, fluconazole, and amphotericin B were serially diluted two-fold in 10 mM sodium phosphate buffer in 96-well plates. C. albicans was cultured overnight at 28 • C in YPD media and then seeded into 96-well plates (2 × 10 4 colony-forming units (CFUs)/mL; 50 µL/well). After incubation for 16-24 h at 28 • C, the growth of the C. albicans strains was determined by measuring the absorbance at 600 nm using a Spectra Max M3 microplate reader. The MICs were defined as the lowest peptide or antifungal agent concentration that could inhibit 90% of the growth of C. albicans compared to the negative control.

Time-Kill Kinetics Assay
The time-kill kinetics of Lycosin-II against C. albicans (KCTC 7270) were determined. C. albicans was grown to mid-log phase in YPD medium at 28 • C. Then, C. albicans was diluted in the YPD medium to a final concentration of 2 × 10 4 CFU/mL. The suspensions were incubated with Lycosin-II at 1× and 2× MICs and with 10 mM sodium phosphate buffer as a control. Next, aliquots were plated on the YPD agar plates and incubated for 16-24 h at 28 • C. After incubation, the CFUs were counted. The survival rate was calculated using the following equation [20]: Percentage survival rate = (CFUs of C. albicans in the Lycosin-II solution-CFUs of C. albicans in the control solution) × 100%

PI Uptake Assay
The membrane integrity of C. albicans (KCTC 7270) after exposure to Lycosin-II was determined using a PI uptake assay. Propidium iodide is an impermeable dye that is used to determine the permeability of cells by agents [22,23]. C. albicans (2 × 10 7 CFU/mL) in PBS was incubated with Lycosin-II at 0.5×, 1×, 2×, and 4× MICs at 28 • C for 10 min. Then, C. albicans was harvested by centrifugation and resuspended in PBS. Next, PI (10 µg/mL) was added to the cells, and these were incubated for 10 min. To remove unbound PI, the cells were washed with PBS. The fluorescence intensity of the PI was measured using flow cytometry (Beckman). Additionally, the PI uptake was visualized using fluorescence microscopy and an EVOS FL Auto 2 imaging system (Invitrogen, Waltham, MA, USA).

SYTOX Green Uptake Assay
To further investigate C. albicans membrane damage by Lycosin-II, we used SYTOX green, which is an impermeable dye that interacts with nucleic acids, resulting in enhanced fluorescent intensity. C. albicans (KCTC 7270) was washed and resuspended in PBS. Then, C. albicans (2 × 10 7 CFU/mL) in PBS loaded with SYTOX green (1 µM) was treated with Lycosin-II, and the fluorescence was measured using excitation and emission wavelengths of 485 nm and 520 nm, respectively [24]. Additionally, the SYTOX green uptake was visualized using fluorescence microscopy and an EVOS FL Auto 2 imaging system (Invitrogen).

Measurement of ROS
The accumulation of reactive oxygen species (ROS) in C. albicans (KCTC 7270) due to Lycosin-II was detected using DCFH-DA [25]. DCFH-DA is oxidized by ROS to dichlorofluorescein (DCF), which emits green fluorescence [26]. C. albicans was washed with YPD medium and resuspended at a final concentration of 1 × 10 7 CFU/mL in YPD medium. Next, suspensions of C. albicans were exposed to Lycosin-II at 0.5×, 1×, 2×, and 4× MICs for 1 h at 28 • C. After centrifugation, C. albicans was incubated with 10 µM DCFH-DA for 1 h and then washed with PBS. The fluorescence intensity of DCFH-DA was measured using flow cytometry (Beckman).

Analysis of the Effect of NAC on the Antifungal Activity of Lycosin-II
To determine the effect of ROS production on the mode of action of Lycosin-II, we used the ROS scavenger NAC [27]. C. albicans (KCTC 7270) was washed and resuspended with PBS at 1 × 10 7 CFU/mL, and SYTOX green at 1 µM was added to the suspension. Then, NAC was added to the suspension at 5, 7, 7.5, and 10 mM, which was then incubated with Lycosin-II at 1× MIC for 1 h. The fluorescence intensity of the SYTOX green was measured at excitation and emission wavelengths of 485 nm and 520 nm using a Spectramax M3 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

Biofilm Inhibition Assays
To analyze the effect of Lycosin-II on C. albicans single-species biofilms and polymicrobial biofilms, we used C. albicans (CCARM 14020), S. aureus (ATCC 25923), and S. aureus (ATCC 25923) expressing green fluorescent protein (GFP). We demonstrated in a previous study that the strain CCARM 14020 of C. albicans could form a large biofilm [23,28]. C. albicans (CCARM 14020) and S. aureus (ATCC 25923) were incubated in YPD medium and LB medium. The two strains were washed with RPMI medium supplemented with 2% glucose and adjusted to 1 × 10 6 CFU/mL for the single-species biofilm evaluation. A total of 180 µL aliquots of C. albicans or S. aureus and the two mixed strains was added to 96-well plates and incubated with 20 µL Lycosin-II at 0.5-8 µM for 24 h at 37 • C. The supernatant was discarded, and 100% methanol was added to fix the biofilm for 20 min. Then, the methanol was discarded, and the sample was dried completely. The biofilm formation was stained with 0.1% crystal violet for 20 min and then washed three times with distilled water. Next, ethanol (95%) was added to the stained biofilms for dissolution. Biofilm quantification was determined by measuring absorbance at 595 nm using a Versa-Max microplate enzyme-linked immunosorbent assay reader (Molecular Devices) [29,30]. The percentage of biofilm formation was determined using the following equation: Biofilm formation (%) = (A595 of treated biofilm/A595 of untreated biofilm) × 100

Determination of C. albicans and S. aureus Viability in a Dual-Species Biofilm
The biofilms that were formed by C. albicans and S. aureus were suspended by pipetting. The biofilm suspension was transferred to a 1.5 mL tube, diluted in 1 mL PBS, and vortexed for 5 min [31,32]. Then, YPD and LB agar plates containing vancomycin and amphotericin B were used to distinguish between C. albicans and S. aureus, respectively. The aliquots were spread on agar plates and incubated at 37 • C for 16 h. The numbers of C. albicans and S. aureus were determined by counting the CFUs.

Visualization of the Biofilm
To visualize the biofilm after treatment with Lycosin-II, we used calcofluor white (10 µg/mL) to stain C. albicans in the biofilm. Moreover, the S. aureus (ATCC 25923) strain expressing GFP allowed for the visualization of this bacterium. The images were visualized using EVOS FL Auto 2 fluorescence (Invitrogen).

Statistics
All the data were expressed as three independent experiments using the mean ± standard deviation. Two-tailed Student's t-tests were used to determine the significance of the differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant compared to the control).

Antifungal Activity against C. albicans
The antifungal activities of Lycosin-II, melittin, fluconazole, and amphotericin B against the C. albicans strains are summarized in Table 1. Melittin is a lytic peptide with antimicrobial activity that was isolated from Iranian honeybee (Apis mellifera meda) venom and was used as a control in this study. Lycosin-II showed antifungal activity against C. albicans with a MIC of 2 µM. Melittin showed antifungal activity against C. albicans at concentrations ranging from 2 to 4 µM. We also found that Lycosin-II showed antifungal activity against fluconazole-resistant C. albicans strains. A time-kill kinetics assay was performed to determine the time that was required to kill C. albicans cells, and Lycosin-II showed antifungal activity in dose-and time-dependent manners. Within 60 min, Lycosin-II at 1× MIC reduced C. albicans to >10% viability. In addition, Lycosin-II at 2× MIC was able to completely kill C. albicans in 60 min ( Figure 1). ized using EVOS FL Auto 2 fluorescence (Invitrogen).

Statistics
All the data were expressed as three independent experiments using the mean ± standard deviation. Two-tailed Student's t-tests were used to determine the significance of the differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant compared to the control).

Antifungal Activity against C. albicans
The antifungal activities of Lycosin-II, melittin, fluconazole, and amphotericin B against the C. albicans strains are summarized in Table 1. Melittin is a lytic peptide with antimicrobial activity that was isolated from Iranian honeybee (Apis mellifera meda) venom and was used as a control in this study. Lycosin-II showed antifungal activity against C. albicans with a MIC of 2 μM. Melittin showed antifungal activity against C. albicans at concentrations ranging from 2 to 4 μM. We also found that Lycosin-II showed antifungal activity against fluconazole-resistant C. albicans strains. A time-kill kinetics assay was performed to determine the time that was required to kill C. albicans cells, and Lycosin-II showed antifungal activity in dose-and time-dependent manners. Within 60 min, Lycosin-II at 1ⅹ MIC reduced C. albicans to >10% viability. In addition, Lycosin-II at 2ⅹ MIC was able to completely kill C. albicans in 60 min ( Figure 1).

Membrane Potential Levels of C. albicans
DiBAC4(3) was used to analyze the effect of the membrane potential in C. albicans after exposure to Lycosin-II. C. albicans was treated with Lycosin-II at 0.5×, 1×, 2×, and 4× MICs. As shown in Figure 2A,B, 1.58% of the C. albicans control group stained positive for DiBAC4(3). However, in C. albicans exposed to Lycosin-II at 0.5×, 1×, 2×, and 4× MICs, the fluorescence intensity increased by 5.06%, 24.82%, 90.44%, and 99.04%, respectively, compared to those of the control groups (Figure 2A,B). These results suggested that Lycosin-II has membrane-depolarizing activity against C. albicans. The survival rates were determined by counting the number of colony-forming units at each time point. Abbreviation: MIC, minimum inhibitory concentration.

Effect of Lycosin-II on Membrane Integrity
We analyzed whether the integrity of the fungal membrane was damaged by Lycosin-II using PI and SYTOX green. These dyes cannot penetrate intact membranes. However, they interact with nucleic acids after membranes are compromised with antimicrobial agents, thereby increasing the fluorescence intensity. We demonstrated the disruption of C. albicans membrane integrity by flow cytometric analysis using PI. Among the control groups, the proportion of C. albicans stained with PI was 0.6%, indicating an intact cell membrane. After treatment with Lycosin-II at 0.5×, 1×, 2×, and 4× MICs, the proportions of PI-positive C. albicans increased to 2.92%, 30.50%, 89.48%, and 99.92%, respectively (Figure 3A). We further confirmed the integrity of the C. albicans membrane using SYTOX green. We also demonstrated that treatment with Lycosin-II induced an increase in the fluorescence intensity of SYTOX in a dose-dependent manner ( Figure 3B). Moreover, PI and SYTOX green stained the nucleic acid DNA in C. albicans after treatment with Lycosin-II in a dose-dependent manner. Additionally, we confirmed that Lycosin-II damaged the C. albicans membranes, causing the uptake of PI and SYTOX green, as demonstrated by the increase in the red and green fluorescence intensities, respectively ( Figure S1).

Effect of Lycosin-II on Membrane Integrity
We analyzed whether the integrity of the fungal membrane was damaged by Lycosin-II using PI and SYTOX green. These dyes cannot penetrate intact membranes. However, they interact with nucleic acids after membranes are compromised with antimicrobial agents, thereby increasing the fluorescence intensity. We demonstrated the disruption of C. albicans membrane integrity by flow cytometric analysis using PI. Among the control groups, the proportion of C. albicans stained with PI was 0.6%, indicating an intact cell membrane. After treatment with Lycosin-II at 0.5×, 1×, 2×, and 4× MICs, the proportions of PI-positive C. albicans increased to 2.92%, 30.50%, 89.48%, and 99.92%, respectively ( Figure 3A). We further confirmed the integrity of the C. albicans membrane using SYTOX green. We also demonstrated that treatment with Lycosin-II induced an increase in the fluorescence intensity of SYTOX in a dose-dependent manner ( Figure 3B). Moreover, PI and SYTOX green stained the nucleic acid DNA in C. albicans after treatment with Lycosin-II in a dose-dependent manner. Additionally, we confirmed that Lycosin-II damaged the C. albicans membranes, causing the uptake of PI and SYTOX green, as demonstrated by the increase in the red and green fluorescence intensities, respectively ( Figure S1).

Antifungal Activity against C. albicans
The induction of ROS production by antimicrobial agents is acknowledged as an antifungal mechanism that damages membrane integrity [33]. To analyze intracellular ROS generation by C. albicans after Lycosin-II exposure, we used a fluorescent dye, DCFH-DA, which is widely used as an indicator of ROS. We confirmed that Lycosin-II induced ROS production by using flow cytometry to quantify DCF fluorescence. A total of 0.56% of C. albicans exhibited ROS-positive staining within the control group. However, dosedependent ROS generation was observed, with 1.24%, 1.82%, 6.60%, and 27.48% exhibiting ROS-positive staining after treatment with 0.5×, 1×, 2×, and 4× MICs of Lycosin-II, respectively ( Figure 4A,B). We used NAC as an ROS scavenger to demonstrate the association between ROS generation due to Lycosin-II and antifungal activity. We confirmed that pretreatment with NAC reduced the fluorescence intensity of SYTOX green ( Figure 4C). These results suggested that Lycosin-II induces ROS generation, leading to oxidative and fungal membrane damage.

Antifungal Activity against C. albicans
The induction of ROS production by antimicrobial agents is acknowledged as an antifungal mechanism that damages membrane integrity [33]. To analyze intracellular ROS generation by C. albicans after Lycosin-II exposure, we used a fluorescent dye, DCFH-DA, which is widely used as an indicator of ROS. We confirmed that Lycosin-II induced ROS production by using flow cytometry to quantify DCF fluorescence. A total of 0.56% of C. albicans exhibited ROS-positive staining within the control group. However, dose-dependent ROS generation was observed, with 1.24%, 1.82%, 6.60%, and 27.48% exhibiting ROS-positive staining after treatment with 0.5×, 1×, 2×, and 4× MICs of Lycosin-II, respectively ( Figure 4A,B). We used NAC as an ROS scavenger to demonstrate the association between ROS generation due to Lycosin-II and antifungal activity. We confirmed that pretreatment with NAC reduced the fluorescence intensity of SYTOX green ( Figure 4C). These results suggested that Lycosin-II induces ROS generation, leading to oxidative and fungal membrane damage.

Inhibitory Effects of Lycosin-II on Biofilm
Biofilms produced by pathogens such as bacteria and fungi worsen disease progression and increase drug resistance [34]. C. albicans and S. aureus were tested to assess the biofilm inhibitory effect of Lycosin-II. The results show that Lycosin-II at 8 and 2 μM inhibited more than 90% of the biofilm formation by a single species (C. albicans or S. aureus; Figure 5A,B). Lycosin-II also inhibited the formation of dual-species biofilms containing C. albicans and S. aureus ( Figure 5C). Furthermore, to investigate the viability of C. albicans and S. aureus in dual-species biofilms, a colony count assay was performed. Lycosin-II

Inhibitory Effects of Lycosin-II on Biofilm
Biofilms produced by pathogens such as bacteria and fungi worsen disease progression and increase drug resistance [34]. C. albicans and S. aureus were tested to assess the biofilm inhibitory effect of Lycosin-II. The results show that Lycosin-II at 8 and 2 µM inhibited more than 90% of the biofilm formation by a single species (C. albicans or S. aureus; Figure 5A,B). Lycosin-II also inhibited the formation of dual-species biofilms containing C. albicans and S. aureus ( Figure 5C). Furthermore, to investigate the viability of C. albicans and S. aureus in dual-species biofilms, a colony count assay was performed. Lycosin-II resulted in the inhibition of C. albicans and S. aureus within the biofilm (Figure 5D,E). To visualize biofilm inhibition by Lycosin-II, we used calcofluor white to distinguish the fungi and GFP-expressing S. aureus. Treatment with 4 µM Lycosin-II decreased the number of C. albicans in the biofilm. Moreover, 2 µM Lycosin-II markedly reduced S. aureus in the biofilms ( Figure 5F). These results indicate that Lycosin-II effectively inhibited biofilms that contained C. albicans and S. aureus. with Lycosin-II. The biofilm mass was quantified by using crystal violet and measuring the optical density at 595 nm. The colony-forming unit (CFU) numbers of (D) C. albicans and (E) S. aureus in the dual-species biofilms were analyzed using a plating method on yeast extract peptone dextrose and Luria-Bertani medium supplemented with vancomycin and amphotericin B, respectively. Black dot indicated number of C. albicans and S. aureus. (F) The viability of the dual-species biofilm was analyzed using fluorescence microscopy. C. albicans was stained using calcofluor white (blue), and S. aureus expressing green fluorescent protein (GFP; green) was used to detect the bacterial cells. The graph is expressed as the mean ± standard deviation. Significance: *, p < 0.05; ***, p < 0.001; ns, not significant compared with control.

Discussion
Antibiotics are highly useful for the treatment of infections. However, antimicrobial resistance is increasing globally and is considered a serious threat to public health. The development of antimicrobial agents is a key factor in the treatment of infectious diseases. The increasing incidence of infections by pathogens, such as bacteria and fungi, that are , and (C) dual-species biofilms containing C. albicans and S. aureus were incubated with Lycosin-II. The biofilm mass was quantified by using crystal violet and measuring the optical density at 595 nm. The colony-forming unit (CFU) numbers of (D) C. albicans and (E) S. aureus in the dual-species biofilms were analyzed using a plating method on yeast extract peptone dextrose and Luria-Bertani medium supplemented with vancomycin and amphotericin B, respectively. Black dot indicated number of C. albicans and S. aureus. (F) The viability of the dual-species biofilm was analyzed using fluorescence microscopy. C. albicans was stained using calcofluor white (blue), and S. aureus expressing green fluorescent protein (GFP; green) was used to detect the bacterial cells. The graph is expressed as the mean ± standard deviation. Significance: *, p < 0.05; ***, p < 0.001; ns, not significant compared with control.

Discussion
Antibiotics are highly useful for the treatment of infections. However, antimicrobial resistance is increasing globally and is considered a serious threat to public health. The development of antimicrobial agents is a key factor in the treatment of infectious diseases. The increasing incidence of infections by pathogens, such as bacteria and fungi, that are resistant to conventional antimicrobial agents warrants the development of novel antimicrobial agents [35].
Azoles are commonly used to treat Candida spp. infections. Azoles have antifungal activity due to inhibiting the ergosterol biosynthesis pathway and increasing membrane permeability. Recently, many studies have reported that Candida species can be resistant to antifungal agents [36]. Amphotericin B is commonly used to treat fungal infections. However, amphotericin B has side effects, including chills, headaches, kidney damage, and inflammation [37]. Therefore, new antifungal agents should be developed to prevent resistance and toxicity.
Therefore, AMPs offer an effective therapeutic strategy against a wide range of pathogens. The mechanism of action of AMPs is associated with membrane disruption and the targeting of intracellular components, including nucleic acids [38]. Moreover, resistance to AMPs develops slower than it does to conventional antibiotics [39]. Thus, AMPs may be used to overcome antimicrobial resistance.
We previously reported that Lycosin-II, which is derived from the venom of the spider L. singoriensis, has antibacterial activity and can act on Gram-negative and Grampositive bacteria by disrupting the membrane within a concentration range of 1-2 µM. Lycosin-II also exhibited antimicrobial activity against multidrug-resistant (MDR)-S. aureus and MDR-P. aeruginosa. Moreover, Lycosin-II showed lower cytotoxicity than melittin, which is a known lytic peptide [16]. Lycosin-II also has anti-inflammatory activity by reducing pro-inflammatory cytokines, such as IL-6, IL-8, TNF-α, and IL-1β, in mammalian cells during S. aureus and P. aeruginosa infections. However, the antifungal activity and mechanism of action of Lycosin-II are not known. Thus, this study aimed to investigate the Lycosin-II antifungal activity, mechanism, and prevention of biofilm formation by C. albicans and S. aureus.
The fungal cell wall is an important target in the development of antifungal agents [40]. Therefore, we investigated whether Lycosin-II affects the membrane of C. albicans. The mechanisms of action of AMPs include depolarization of the cytoplasmic membrane, which induces membrane permeation [41]. In a previous study, Bac8c, the peptide analog of Ba2A, induced a change in the membrane potential in C. albicans [42]. To investigate the mechanism of action of Lycosin-II, we verified if Lycosin-II could depolarize the membrane using DiBAC 4 (3). This dye is a potential sensing probe that can enter a depolarized membrane and bind to intracellular components, increasing the fluorescence intensity. C. albicans treated with Lycosin-II showed a greater increase in DiBAC 4 (3) fluorescence intensity than untreated C. albicans. These results showed that Lycosin-II induced the depolarization of the plasma membrane of C. albicans (Figure 2). Previous studies reported that the membrane of C. albicans was damaged by antifungal peptides, such as protonectin isolated from Agelaia pallipes pallipes [33]. In contrast, peptides consisting of lysine and tryptophan 4 repeats showed antifungal activity against C. albicans but did not affect the membrane integrity [23]. To explore the antifungal mechanism of Lycosin-II in the C. albicans membrane, the integrity of the fungal membranes was analyzed using PI and SYTOX green. Both dyes are nucleic acid affinity red and green dyes that enter the fungal cell after membrane integrity is compromised by antimicrobial agents [43]. Therefore, increases in PI and SYTOX green fluorescence intensities indicate a loss in fungal membrane integrity. We confirmed the increased uptake of PI and SYTOX green after Lycosin-II treatment compared to untreated C. albicans using flow cytometry and fluorescence spectrophotometry ( Figure 3). Moreover, the fluorescence microscopy data showed that Lycosin-II treatment led to increased PI and SYTOX green fluorescence in dose-and time-dependent manners. These data were consistent with the results of the flow cytometry and spectrophotometry (Supplement S1). The natural production of ROS plays a role in homeostasis and cellular metabolism. Previous studies have reported the mechanisms of conventional antifungal agents associated with ROS production. The excessive production of ROS induced by antifungal agents affects lipid retention and induces membrane permeability [22,44]. For example, the antifungal agent fluconazole can increase oxidative stress in fungal strains by inducing ROS production [45]. Therefore, we used the ROS probes DCFH-DA and NAC as general scavengers of ROS. Our results showed that Lycosin-II induced ROS generation in C. albicans in a dose-dependent manner. In addition, NAC indicated reduced membrane permeability of C. albicans, which was caused by Lycosin-II. These results indicated that enhanced ROS production after exposure to Lycosin-II affected membrane permeability in C. albicans (Figure 4).
Biofilms are communities of microorganisms that are trapped by EPS and are protected from the host immune system and antibiotics. Microbes in multispecies infections can have altered virulence, proliferation, and antioxidant tolerance. Because microorganisms within mixed fungal-bacterial biofilms have high levels of resistance to antimicrobial agents, fungal-bacterial infections are complex and challenging to treat. C. albicans and S. aureus are major opportunistic fungi and bacterial pathogens, respectively [46]. Approximately 27% of C. albicans infections are polymicrobial with S. aureus [47]. S. aureus and C. albicans exhibit synergistic pathogenicity. For example, C. albicans can enhance the resistance of S. aureus to vancomycin [48]. Therefore, approaches to combat both bacteria and fungi are urgently needed. In this study, we explored whether Lycosin-II inhibited single-species biofilm formation using a crystal violet assay for biofilm quantification. Additionally, we used calcofluor white and S. aureus expressing GFP for biofilm visualization. Lycosin-II prevented biofilm formation by C. albicans and S. aureus in a dose-dependent manner. Moreover, we confirmed that Lycosin-II inhibited the formation of dual-species biofilms. Additionally, the numbers of live C. albicans and S. aureus in the biofilms were significantly reduced by Lycosin-II ( Figure 5). These results indicated that Lycosin-II had inhibitory activity against dual-species biofilms.

Conclusions
In conclusion, Lycosin-II had antifungal activity against C. albicans. Lycosin-II increased membrane depolarization and permeabilization in C. albicans, and Lycosin-II-induced ROS generation was associated with antifungal activity against C. albicans. Lycosin-II effectively inhibited the formation of single-and dual-species biofilms. Our results suggested that Lycosin-II may be a useful therapeutic alternative against polymicrobial infections.