Application of antibacterial peptide SK-21 in preparation of product for inhibiting drug-resistant candida

Abstract

The invention belongs to the technical field of antibacterial drug research, and particularly relates to application of an antibacterial peptide SK-21 in preparation of a product for inhibiting drug-resistant candida. The amino acid sequence of the antibacterial peptide SK-21 is as shown in SEQ ID NO. 1. Tests prove that the SK-21 has certain in-vitro antifungal activity on drug-resistant candida, and has relatively good biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial drug research technology, specifically relating to the use of an antimicrobial peptide SK-21 in the preparation of products that inhibit drug-resistant Candida. Background Technology

[0002] Opportunistic fungi can cause both surface and invasive infections, leading to significant illness and mortality. Invasive candidiasis is one of the most representative invasive fungal infections, with a mortality rate exceeding 40%. *Candida tropicalis* is one of the most important non-*Candida albicans* genera and is widely considered the second most virulent *Candida* genus after *Candida albicans*.

[0003] Azole antibiotics, especially fluconazole, are widely used to treat and prevent Candida infections. However, with frequent exposure and widespread use of fluconazole, resistance to fluconazole and cross-resistance with other antifungal drugs are increasing. Simultaneously, drug abuse has promoted the emergence of drug-resistant Candida species. *Candida tropicalis* is the most common non-albedococcal Candida isolated from patients with candidiasis, exhibiting increased levels of fluconazole resistance. Studies have reported that the prevalence of fluconazole-resistant *Candida tropicalis* isolates in clinical trials ranges from 20% to 50%, indicating a rising prevalence of fluconazole and multidrug resistance. Data shows that over 21% of *Candida tropicalis* isolates from tropical regions of China are fluconazole-resistant. This resistance may be based on multiple factors, such as altered drug targets, overexpression of drug targets, and overexpression of efflux pumps. However, the number and efficacy of currently available antifungal drugs for treating candidiasis are limited, posing significant challenges to clinical treatment. Current antifungal drug therapies show low efficacy, and achieving a complete cure for the infection remains a difficult task. Therefore, exploring new and potential antifungal drugs and developing new treatment options and therapies are key to solving the current problem of fungal resistance. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides the use of the antimicrobial peptide SK-21 in the preparation of products that inhibit drug-resistant Candida. Tests have shown that SK-21 has certain in vitro antifungal activity against drug-resistant Candida and exhibits good biocompatibility.

[0005] The specific technical solution provided by this invention is as follows:

[0006] This invention provides the use of antimicrobial peptide SK-21 in the preparation of products that inhibit drug-resistant Candida, wherein the amino acid sequence of the antimicrobial peptide SK-21 is shown in SEQ ID NO.1.

[0007] Preferably, the drug-resistant Candida is one or more of FLZ-resistant Candida tropicalis 6984, FLZ-resistant Candida tropicalis 8402, FLZ-resistant Candida tropicalis 4252, FLZ-resistant Candida tropicalis 4171 or drug-resistant Candida albicans ATCC14053.

[0008] Preferably, the minimum inhibitory concentration (MIC) of the antimicrobial peptide SK-21 against FLZ-resistant Candida tropicalis 6984, FLZ-resistant Candida tropicalis 8402 and FLZ-resistant Candida tropicalis 4171 is 128 μg / mL, and the minimum inhibitory concentration (MIC) against FLZ-resistant Candida tropicalis 4252 and drug-resistant Candida albicans ATCC14053 is 64 μg / mL.

[0009] Preferably, the antimicrobial peptide SK-21 is used to prepare products that kill the larvae of the large wax moth.

[0010] Preferably, the antimicrobial peptide SK-21 is used to prepare products that improve fungal resistance.

[0011] Preferably, the fungus is Candida tropicalis or Candida albicans.

[0012] Preferably, the drug resistance is the resistance of fungi to azole drugs.

[0013] Preferably, the azole drug is fluconazole.

[0014] Preferably, the product is an aqueous solution containing the antimicrobial peptide SK-21, wherein the content of the antimicrobial peptide SK-21 in the aqueous solution is 64-256 μg / mL.

[0015] Preferably, the product includes pharmaceutically acceptable excipients.

[0016] The dosage forms of the products described in this invention can be selected from clinically acceptable formulations, such as conventional oral, topical, or injectable formulations. Oral formulations include, but are not limited to, capsules, granules, tablets, and liquid formulations; topical formulations include, but are not limited to, gels, patches, ointments, sprays, liniments, and lotions; and injectable formulations include, but are not limited to, intravenous or intramuscular injection formulations. Appropriate excipients may also be added during the specific preparation process, such as preservatives, suspending agents, pH adjusters, flavoring agents, surfactants, suspending agents, isotonic agents, dispersants, emulsifiers, and antifreeze agents. The specific type of excipient is selected according to the specific type of formulation. For example, gel formulations may include pH adjusters, humectants, preservatives, stabilizers, solubilizers, hydrosolvents, and transdermal absorption enhancers; sprays may include suspending agents, wetting agents, and surfactants; and intravenous or intramuscular injection formulations may include solubilizers, hydrosolvents, isotonic agents, pH adjusters, emulsifiers, and antioxidants. Different formulation types can be applied to different scenarios, such as gels for external use in skin inflammation related to Candida infection, sprays for preventing Candida biofilm infection in medical devices, and intravenous or intramuscular injections for systemic candidiasis, etc.

[0017] This invention utilizes a novel multi-task adaptive modeling theory to model antifungal peptide drugs. Using this model, a novel antimicrobial peptide, SK-21, with good antibacterial activity was identified from over three million unknown functional sequences in the Uniport database. SK-21 exhibits certain in vitro antifungal activity against drug-resistant Candida and demonstrates good biocompatibility. These findings suggest that SK-21 could be used as a potential candidate drug for treating infections caused by drug-resistant Candida. Attached Figure Description

[0018] Figure 1 The relevant physicochemical properties of SK-21 include: A) the helical ring structure of the peptide, showing amino acid polarity; B) the peptide sequence and model secondary helical structure; and C) the relevant physicochemical parameters of the peptide.

[0019] Figure 2 The time-growth kinetics and time-fungicide kinetics of SK-21 against azole-resistant Candida albicans are shown; A. Growth curve; B. Fungicide kinetic curve.

[0020] Figure 3 This describes the transition from yeast phase to mycelial phase; scale bar: 10 μm.

[0021] Figure 4 This study investigated the effect of SK-21 on the formation of azole-resistant Candida biofilms. The inhibitory effect of different concentrations of SK-21 on Candida biofilm formation was observed using laser confocal microscopy. Images obtained from live / dead staining (SYTO 9, green; PI, red) are shown, with a scale bar of 20 μm.

[0022] Figure 5 This study investigated the effect of SK-21 on the removal of mature biofilms from azole-resistant Candida albicans. The removal efficiency of different concentrations of SK-21 was observed using laser confocal microscopy. Images obtained from live / dead staining (SYTO 9, green; PI, red) are shown, with a scale bar of 20 μm.

[0023] Figure 6 The effect of SK-21 on azole-resistant Candida albicans biofilm was determined by the XTT method; the biofilm activity level under different concentrations of SK-21 was determined by the XTT reduction method, and the absorbance was measured at OD490 nm. Error bars represent the standard deviations of the three independent experiments. ***P<0.001 compared with the control group; A) Inhibitory effect on biofilm formation; B) Removal effect on mature biofilm;

[0024] Figure 7 The images show the effects of SK-21 treatment on the morphology of azole-resistant Candida albicans observed using scanning electron microscopy. A) Untreated cells with SK-21; B) Candida albicans cells treated with SK-21 for 24 hours; C) A magnified view of the area within the white dashed line in image A; D) A magnified view of the area within the white dashed line in image B.

[0025] Figure 8 The flow cytometry plots show the staining of fungal cells after incubation with SK-21 for 1 hour at different concentrations of antimicrobial peptides using two dyes.

[0026] Figure 9 The effects of SK-21 on the accumulation of reactive oxygen species (ROS) and cell membrane depolarization in azole-resistant Candida albicans; A. Degree of cell membrane depolarization; B. Effects on cellular ROS;

[0027] Figure 10 The study included the in vivo toxicity and therapeutic activity of SK-21 in the *Candida albicans* model; A) toxicity of SK-21 to *Candida albicans* larvae; B) larval survival rate after SK-21 treatment; and C) fungal load of larvae in each SK-21-treated group. Compared with the azole-resistant *Candida albicans* + PBS group, *** P<0.001. Detailed Implementation

[0028] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0029] It should be understood that in various embodiments of the present invention, the order of the above-mentioned processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0030] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0031] The weights of the relevant components mentioned in the embodiments of this invention can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this invention is within the scope disclosed in the embodiments of this invention. Specifically, the mass described in the embodiments of this invention can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0032] 1. Materials and Methods

[0033] 1.1 Materials

[0034] 1.1.1, Microbial strains

[0035] All azole-resistant Candida tropicalis strains were clinical isolates from the blood of patients with invasive infections, and were obtained from the Laboratory Center of the Affiliated Hospital of Guizhou Medical University. The drug-resistant Candida albicans strain was Candida albicans ATCC14053.

[0036] 1.1.2 Main Reagents

[0037] Yeast Extract Peptone Dextrose Medium (YPD) and agar powder were purchased from Solarbio; Fluconazole (FLZ) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; XTT cell proliferation and cytotoxicity assay kit was purchased from Shanghai BeatBio Biotechnology Co., Ltd.; RPMI 1640 was purchased from Gibco, USA; SYTO 9 was purchased from Invitrogen; and Propidium lodide was purchased from Sigma-Aldrich.

[0038] 1.1.3 Main Instruments

[0039] The ultra-clean workbench was purchased from Suzhou Purification Co., Ltd.; the multifunctional microplate reader was purchased from Thermo Scientific, USA; the YS100 ordinary optical microscope was purchased from Nikon Corporation, Japan; the Nikon ti-u-ds-ri2 inverted microscope was purchased from Japan; the FV1000 laser confocal microscope was purchased from Olympus Corporation, Japan; and the flow cytometer (FACSCanton II) was purchased from BD Corporation, USA.

[0040] 1.2 Methods

[0041] 1.2.1 Synthesis of Peptide SK-21

[0042] The designed antimicrobial peptide SK-21 (SPGKKKKKKKKKKTKKKKKK, as shown in SEQ ID NO.1) sequence was synthesized by Shanghai Jier Biochemical Co., Ltd. using a solid-phase chemical synthesis method, with a purity (HPLC) >95%. It was dissolved in sterile ddH2O to a concentration of 5 mg / mL and stored at -80℃ for later use. The physicochemical properties of peptide SK-21 were analyzed using the Ex-PASy Prot-Param software (http: / / web.expasy.org / protparam / ); a helical diagram was plotted using Heliquest software (https: / / heliquest.ipmc.cnrs.fr / ).

[0043] 1.2.2 Determination of Minimum Inhibitory Concentration

[0044] The minimum inhibitory concentration of peptide SK-21 against drug-resistant Candida strains was determined using the microdilution method, in accordance with the standards of the Clinical and Laboratory Standards Institute (CLSI).

[0045] Activated single colonies were picked from the plate and inoculated into YPD liquid medium. The culture was incubated at 35°C until the logarithmic growth phase. The bacterial suspension was then adjusted to 0.5 × 10⁻⁶ using a hemocytometer. 3 ~2.5×10 3 Different concentrations of SK-21 were co-cultured with the above bacterial suspension in 96-well plates at 35°C for 24 h. The blank group was sterile YPD medium, and the negative control group was YPD medium supplemented with bacterial suspension. The drug concentration corresponding to the well with no visible fungal growth was defined as the minimum inhibitory concentration (MIC).

[0046] 1.2.3 Growth kinetics and bactericidal kinetics

[0047] Following the method in 1.2.2, the bacterial concentration was adjusted to 2 × 10⁻⁶. 6 CFU / mL, the diluted bacterial culture was incubated with the peptide at 35℃ for 48 h to achieve final peptide concentrations of 64, 128, and 256 μg / mL. OD was recorded every 2 h during co-culture. 630nm Cultures were collected at specific time intervals (0, 2, 4, 6, 8, 10, 12 h), serially diluted, and then subjected to agar plate experiments. Fungal colonies were counted after incubation at 35°C for 24 h.

[0048] 1.2.4 Effect of SK-21 on mycelial formation

[0049] Different concentrations of SK-21 were thoroughly mixed with fungal suspensions diluted in RPMI 1640 medium (containing 15% fetal bovine serum) to achieve final drug concentrations of 64, 128, and 256 μg / mL. After incubation at 37°C for 3, 6, 9, 12, and 24 hours, the fungal suspensions were removed and their hyphal formation was observed and photographed under an upright fluorescence microscope.

[0050] 1.2.5. In vitro biofilm resistance assay

[0051] The fungal cells cultured to the logarithmic growth phase were adjusted to a concentration of 2.0 × 10⁻⁶ using RPMI-1640 liquid medium. 6 CFU / mL. Incubate in 96-well flat-bottomed polypropylene plates at 37°C for 90 min (early biofilm formation) or 48 h (mature biofilm), followed by incubation with different concentrations of SK-21 for 24 h. Add 100 μl of freshly prepared XTT solution to each well and incubate at 37°C for 2 h. Measure absorbance at OD490 nm using a microplate reader. Place sterile polylysine-containing cell slides at the bottom of 24-well plates, add 500 μL of the above-mentioned bacterial suspension, and prepare early and mature biofilms according to the XTT method. Add 500 μL of SYTO9 and PI staining solution to a final concentration of 10 μM, incubate at 37°C for 20 min, and then mount with nail polish. Observe and obtain images using a laser confocal microscope.

[0052] 1.2.6 Scanning Electron Microscope

[0053] Following the method described in 1.2.2 above, the bacterial concentration was adjusted to 2.0 × 10⁻⁶. 6 Cells were incubated at CFU / mL in SK-21 at 35°C for 4 h with culture medium. After centrifugation at 5000 rpm for 10 min, the cells were fixed overnight at 4°C with 2.5% glutaraldehyde and dehydrated for 10 min with a series of tert-butanol solutions (50%, 75%, 95%, and 100%). The cells were then dried in a vacuum evaporator and coated with a thin layer of gold-palladium. The samples were observed and images obtained using a scanning electron microscope.

[0054] 1.2.7 Flow cytometry

[0055] Following the method in 1.2.2, the bacterial concentration was adjusted to 2 × 10⁻⁶. 6 After incubating the cells with CFU / mL of SK-21 at 35°C for 1 h, the suspension was then incubated with SYTO 9 and PI staining solution at a final concentration of 10 μM at 35°C for 15 min. Flow cytometry was used to perform quantitative analysis of the stained cells.

[0056] 1.2.8 Membrane Potential Detection

[0057] Following the method described in 1.2.2 above, the bacterial concentration was adjusted to 2.0 × 10⁻⁶. 6 CFU / mL, the membrane potential DiSC3(5) probe was added to the bacterial suspension to be tested, followed by different final concentrations of SK-21. 10 mM PBS was used as a negative control. Fluorescence intensity changes were continuously monitored for 1 h using a multifunctional fluorescent microplate reader.

[0058] 1.2.9 Detection of reactive oxygen species levels

[0059] Following the method described in 1.2.2 above, the bacterial concentration was adjusted to 2.0 × 10⁻⁶. 6 CFU / mL was added to the bacterial suspension to which the test sample was to be administered, followed by the addition of different final concentrations of SK-21. 10 mM PBS was used as a negative control. Fluorescence intensity changes were continuously monitored over 1 hour using a multi-functional fluorescent microplate reader.

[0060] 1.2.10. Infection experiment with the large wax moth

[0061] The larvae of the large wax moth used in the experiment were purchased from Tianjin Huiyude Biotechnology Co., Ltd., each weighing 250-300 mg and approximately 2-3 cm in length. Before the experiment, all larvae were placed overnight in a dark incubator at 35°C. Ten larvae (10 μL each) were randomly selected from each group and injected with SK-21 at a concentration of 16-32 mg / kg to assess peptide toxicity. An equal volume of sterile PBS was used as a negative control.

[0062] To evaluate the efficacy of the peptide therapy, 12 larvae were randomly selected from each group and fed a 5×10⁻⁶ dose. 710 μL of a CFU / mL bacterial suspension was injected into the left distal leg of each larva. After 1 hour in an incubator, the same amount of peptide was injected into the right distal leg using the same method. The larvae were incubated at 35°C for 5 days, with live and dead counts performed every 24 hours. Larvae were considered dead when they turned black or soft and showed no obvious tactile response. 24 hours after peptide injection (SK-21), three larvae from each group were randomly selected, homogenized in 3 mL of sterile PBS solution, serially diluted, and 10 μL was spotted onto sterile solid YPD plates and incubated for 24 hours. The amount of fungal growth was recorded, and the bacterial load for each larva was calculated.

[0063] 1.2.11 Statistical Analysis

[0064] All experiments in this study were repeated three times. Graphpad Prism 8.0 software was used for statistical graphing and data analysis. Quantitative data were analyzed using... The results indicate that one-way ANOVA was used to compare differences among multiple groups, and log-rank test was used to analyze differences in survival curves. The significance level was set at α = 0.05, and P < 0.05 was considered statistically significant.

[0065] 2. Results and Analysis

[0066] 2.1 Analysis of the Physicochemical Properties of SK-21

[0067] The antimicrobial peptide SK-21 contains 21 amino acids, and the model predicts that its helical diagram is an α-helix structure. Figure 1 (A~B). Its theoretical molecular weight was predicted to be 2539.33 Da, and mass spectrometry analysis confirmed the actual molecular weight to be 2539.35. Furthermore, the peptide's net charge and hydrophobicity were +17 and -0.757, respectively. Figure 1 C) indicates that the polypeptide is a hydrophilic cationic peptide.

[0068] 2.2 Antifungal activity

[0069] Antifungal susceptibility testing was performed on four azole-resistant Candida strains using the broth dilution method. The results (Table 1) showed that SK-21 exhibited effective antifungal activity against all five strains, with MIC values ​​ranging from 64 to 128 μg / mL. Further experiments were conducted using the azole-resistant Candida tropicalis 4252, which showed the best antifungal activity. The MIC values ​​of these strains against fluconazole were all greater than 1024 μg / mL, indicating that SK-21 has good antifungal activity against azole-resistant Candida.

[0070] Table 1. Determination of antibacterial activity of SK-21

[0071]

[0072]

[0073] 2.3 Growth kinetics and bactericidal kinetics

[0074] The growth curve of azole-resistant Candida albicans shows that ( Figure 2 A) Without SK-21, the bacteria entered the logarithmic phase within 6 hours of incubation and the stationary phase after 18 hours. However, SK-21-treated azole-resistant Candida albicans only entered the logarithmic phase after 12 hours of incubation at a concentration of 128 μg / mL, and could not grow and reproduce normally at a peptide concentration of 256 μg / mL, indicating that SK-21 has a concentration-dependent inhibitory effect on drug-resistant Candida albicans.

[0075] From the bactericidal kinetic curve, it can be seen that ( Figure 2 B) Compared with the control, SK-21 showed good antibacterial effects against drug-resistant Candida at concentrations ranging from 64 μg / mL to 128 μg / mL. SK-21 at a concentration of 256 μg / mL could kill azole-resistant Candida within 2 hours.

[0076] 2.4. Transformation from yeast phase to mycelial phase

[0077] Morphological changes in azole-resistant Candida hyphae show ( Figure 3 In the control group, the length and density of hyphae increased with prolonged incubation time. After 9 hours of incubation, the hyphae of the drug-resistant Candida cells grew very long and could cross-connect to form a network. When the concentration of SK-21 was 128 μg / mL and 256 μg / mL, the transformation of azole-resistant Candida from spores to hyphae was completely inhibited, and only single, small numbers of yeast-like cells could be observed.

[0078] 2.5. Anti-biofilm effect of SK-21

[0079] In the experimental results ( Figures 4-5 First, using laser confocal microscopy for visualization, a large number of densely aggregated biofilms were observed in the negative control, mainly emitting green fluorescence. After SK-21 treatment, the dense biofilm structure became looser, and the entangled cross-linked structures decreased and became sparser. The number of cells also decreased, and the number of dead fungi increased, mainly emitting red fluorescence. Figures 4-5 Further quantitative analysis using the XTT assay revealed that SK-21 significantly reduced the percentage of biofilm activity in a concentration-dependent manner. Compared with the control group, SK-21 at concentrations of 128, 256, and 512 μg / mL inhibited biofilm formation by 49.75%, 71.28%, and 88.32%, respectively. Figure 6 A) reduced the amount of mature biofilm by 17.53%, 42.07%, and 58.28%, respectively. Figure 6B). The above results indicate that SK-21 can inhibit the formation of azole-resistant Candida biofilms and eliminate a certain amount of mature biofilms.

[0080] 2.6 Scanning electron microscopy examination

[0081] To visualize the damaging effect of SK-21 on the cell membrane of azole-resistant Candida, the morphological changes of bacteria treated with SK-21 for 2 hours were directly observed using scanning electron microscopy. Untreated bacteria (SK-21 cells) Figure 7 A) The cells were morphologically intact, with a smooth and undamaged cell surface. Zolidine-resistant Candida cells treated with 256 μg / mL SK-21 for 24 h showed significant damage. Figure 7 B) The surface of the bacteria is irregularly shaped, and some bacteria are pitted, indicating that the integrity of the azole-resistant Candida bacteria has been damaged.

[0082] 2.7 Flow cytometry detection of membrane permeability

[0083] Cell membrane is one of the main pathways through which drugs exert their antibacterial effects. Flow cytometry analysis showed that after treatment with 64, 128, and 256 μg / mL SK-21, approximately 45.02%–85.02% of cells were positive for PI staining. Figure 8 Furthermore, the positive rate showed a dose-dependent change, indicating that the peptide SK-21 can increase cell membrane permeability, disrupt the cell membrane integrity of drug-resistant Candida, and thus kill the fungus.

[0084] 2.8 Effect of SK-21 on cell membrane potential

[0085] Changes in cell membrane permeability can be reflected by the degree of cell membrane depolarization. We used the membrane potential-sensitive fluorescent probe DiSC3(5) to assess the degree of fungal cell membrane depolarization. Figure 9 A) Compared with the control group, after treatment with different concentrations of antimicrobial peptide SK-21, the fluorescence intensity of the samples increased rapidly, and the fluorescence intensity gradually increased with the increase of SK-21 concentration, showing a concentration-dependent effect.

[0086] 2.9 Effects of SK-21 on Reactive Oxygen Species in Cells

[0087] The results show ( Figure 9 (B) Exposure of azole-resistant Candida albicans to SK-21 led to an increasing trend in fungal ROS levels. Compared to the 64 μg / mL group, 256 μg / mL of SK-21 induced a higher concentration of ROS accumulation in azole-resistant Candida albicans, and the ROS level in azole-resistant Candida albicans increased with time. These results suggest that the presence of SK-21 can induce ROS production.

[0088] 2.8. Efficacy of SK-21 against azole-resistant Candida albicans infections in vivo.

[0089] The larvae of the large wax moth are a widely accepted model for studying fungal pathogen-host interactions. In peptide toxicity experiments ( Figure 10 A) By day 5, all larvae were alive, indicating that the peptide exhibited low toxicity in the experimental model. Furthermore, the results showed that the survival rates of the SK-21 treatment groups at 16 mg / kg and 32 mg / kg were 60% and 70% respectively 5 days after infection. Figure 10 B). Meanwhile, bacterial load reflects these results ( Figure 10 C) After 24 hours of SK-21 treatment, the number of larval colonies decreased in all peptide-treated groups.

[0090] In summary, the antimicrobial peptide SK-21 exhibits good antibacterial activity, anti-biofilm formation ability, and clearance ability against azole-resistant Candida, demonstrating a certain therapeutic effect against its infection. Furthermore, it exerts its antibacterial effect by disrupting cell membrane structure. Therefore, the antimicrobial peptide SK-21 is a promising antifungal drug for future development and has the potential to address fungal resistance. However, due to the significant structural differences between the larvae of the giant wax moth and the human body, it cannot completely replace mammalian experiments; its efficacy in mammalian vivo requires further investigation in subsequent experiments.

[0091] Currently, due to the widespread overuse of antibiotics, antifungal drugs are rapidly developing resistance, and the incidence and mortality rates of invasive fungal infections are also increasing significantly. The isolation rates of Candida species, such as Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, and Candida parapsilosis, in hospitals are gradually rising. It has been reported that the proportion of azole-resistant Candida isolates in China has been continuously increasing in recent years. Therefore, the development of new antibacterial drugs to address the current resistance problem is urgent. This invention screened an antimicrobial peptide, SK-21, with good antimicrobial activity from an antimicrobial peptide database. Experiments verified that peptide SK-21 exhibits strong antimicrobial activity against azole-resistant Candida, low resistance, and good in vivo efficacy, and exerts its antimicrobial effect through a membrane damage mechanism.

[0092] This invention reveals that SK-21 exhibits certain antibacterial activity against azole-resistant Candidae. SK-21 effectively kills azole-resistant Candidae within 2 hours and also shows some efficacy against infected larvae of the large wax moth. Recent studies have shown that morphological transformation during biofilm formation is considered an important virulence factor in Candida tropicalis and may be related to the pathogenesis of Candida. Hyphae developing during morphological transformation can penetrate cells and invade the bloodstream to express many virulence factors, thus being considered a more virulent phenotype than yeast. Furthermore, hyphal development is considered a key stage in biofilm formation and maturation. In this invention, SK-21 inhibited the transformation from yeast to hyphal morphology. As a crucial step in the maturation of Candida tropicalis biofilms, we observed a reduction in hyphae, a decrease in the number of embryospores, and a reduction in entanglement structures. Compared to other Candidae species, the yeast stage of Candida tropicalis is characterized by a strong ability to form biofilms, which may be related to the increased biomass in the membrane and extracellular matrix, leading to a more compact structure. However, in this study, SK-21 at concentrations several times higher than the MIC was able to inhibit biofilm formation and showed a certain degree of scavenging effect on mature biofilms, exhibiting a concentration-dependent effect. Therefore, SK-21 demonstrated good biological activity in inhibiting morphological transformation and resisting biofilm formation.

[0093] It has been reported that most antimicrobial peptides exert their antimicrobial effects primarily by targeting the cell wall and cell membrane. In this invention, scanning electron microscopy results showed that SK-21 acts on the cell wall or cell membrane to disrupt the morphological structure of azole-resistant Candida tropicalis. However, the specific cause of morphological damage and eventual cell death is unknown. We speculate that it is related to the electrostatic interaction between the positively charged peptide SK-21 and the negatively charged components in the fungal cell membrane, leading to increased membrane permeability. Experimental results verified this, showing a significant increase in the number of PI-positive fungal cells after SK-21 treatment. This indicates that SK-21 treatment increased cell membrane permeability and disrupted the integrity of the membrane structure. Changes in cell membrane permeability usually trigger changes in membrane potential, and cell membrane potential is closely related to cell function. DiSC3(5), as a membrane potential-sensitive probe, can aggregate within the phospholipid bilayer, causing dye self-quenching. When membrane-modifying compounds (such as peptides) depolarize the membrane and cause loss of potential, DiSC3(5) is released into the solution, resulting in enhanced fluorescence. After treatment with SK-21, the cytoplasmic membrane undergoes a concentration-dependent depolarization change, indicating that the dissipation of membrane potential may participate in the formation of channels or pores, thereby allowing ions or macromolecules to pass through, leading to cytoplasmic membrane dysfunction. Studies have found that ROS produced by aerobic metabolism are usually present in cells in balance with antioxidant enzymes, while excessive ROS has various harmful effects on the basic structure of fungi, such as damage to nucleic acids, DNA, amino acid residues, and cell membranes. In this invention, it was found that SK-21 also induces the accumulation of reactive oxygen species in a dose-dependent manner. In summary, we speculate that the cationic peptide SK-21 can interact with certain negatively charged molecules on the cell membrane through electrostatic interaction, leading to increased membrane permeability, changes in membrane potential depolarization, loss of membrane integrity structure, and accumulation of reactive oxygen species, further resulting in leakage of cellular contents and other consequences, ultimately leading to cytoplasmic membrane dysfunction and cell death.

[0094] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. Use of an antibacterial peptide SK-21 in the manufacture of a product for inhibiting drug-resistant Candida, characterized in that, The amino acid sequence of the antibacterial peptide SK-21 is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The drug-resistant Candida is one or more of FLZ-resistant Candida tropicalis 6984, FLZ-resistant Candida tropicalis 8402, FLZ-resistant Candida tropicalis 4252, FLZ-resistant Candida tropicalis 4171, or drug-resistant Candida albicans ATCC14053.

3. Use according to claim 2, characterized in that, The minimum inhibitory concentration of the antibacterial peptide SK-21 to FLZ-resistant Candida tropicalis 6984, FLZ-resistant Candida tropicalis 8402, and FLZ-resistant Candida tropicalis 4171 is 128 μg / mL, and the minimum inhibitory concentration to FLZ-resistant Candida tropicalis 4252 and drug-resistant Candida albicans ATCC14053 is 64 μg / mL.

4. Use according to claim 1, characterized in that, The antibacterial peptide SK-21 is used for preparing a product for killing Galleria mellonella larvae.

5. Use according to claim 1, characterized in that, The antibacterial peptide SK-21 is used for preparing a product for improving fungal drug resistance.

6. Use according to claim 5, characterized in that, The fungus is Candida tropicalis or Candida albicans.

7. Use according to claim 6, characterized in that, The drug resistance is the drug resistance of the fungus to azole drugs.

8. Use according to claim 6, characterized in that, The azole drug is fluconazole.

9. Use according to claim 4 or 5, characterized in that, The product is an aqueous solution containing the antibacterial peptide SK-21, and the content of the antibacterial peptide SK-21 in the aqueous solution is 64-256 μg / mL.

10. Use according to claim 4 or 5, characterized in that, The product comprises a pharmaceutically acceptable excipient.

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