Application of antibacterial peptide with antifungal activity

Abstract

The invention relates to the technical field of antibacterial peptides, in particular to application of an antibacterial peptide with antifungal activity, the antibacterial peptide is Jelleine-Ic, and the antifungal activity, the antibiofilm activity and the action mechanism of the Jelleine-Ic on candida albicans are disclosed. The Jelleine-Ic has a very strong antifungal activity on candida albicans, and the Jelleine-Ic has a very strong antifungal activity on The integrity of cell walls of the candida albicans is destroyed, and the permeability of cell membranes of the candida albicans is enhanced. Besides, Jelleine-Ic interacts with DNA when entering fungal cells, expression of genes participating in DNA replication and repair is affected, generation of active oxygen in the cells is induced, and death of the fungal cells is accelerated. According to the results, the Jelleine-Ic can effectively inhibit the growth of candida albicans and has the potential of treating fungal infection.

Description

Technical Field

[0001] The invention relates to the technical field of antimicrobial peptides, and in particular to the application of an antimicrobial peptide with antifungal activity. Background Art

[0002] Among hospital infections, Candida albicans is one of the most common fungal pathogens. Its incidence in humans is high, and the mortality rate of systemic fungal-related infections is close to 50%, which seriously threatens human health. Currently, the treatment of candidiasis is limited to a limited number of antifungal compounds, including azoles, echinocandins, polyenes, and flucytosine. In addition, drug resistance is a major challenge to the effective treatment of these infections. Cases of Candida albicans resistance to drugs have appeared in hospital settings and are becoming more and more common. Therefore, the discovery of new antimicrobial drugs is an urgent need to combat fungal infections. Antimicrobial peptides (AMPs) have the characteristics of high activity, low resistance rate, and negligible host toxicity, and are expected to become antifungal drugs.

[0003] AMPs, as important innate immune molecules, are widely distributed in nature, such as animals, plants, and microorganisms. AMPs are between 12 and 50 amino acids in length, usually positively charged and amphiphilic. AMPs show a wide range of activities against pathogens such as bacteria, fungi, viruses, and parasites. In addition, many AMPs show activity against drug-resistant microorganisms. Certain AMPs have been studied as alternative methods to control Candida albicans infections. For example, the antimicrobial peptide C16-FengycinA from Bacillus amyloliquefaciens inhibits the growth of Candida albicans by destroying its cell wall and accumulating reactive oxygen species (ROS). The antimicrobial peptide AMP-17 from the housefly Musca domestica exerts its anti-Candida albicans effect through ROS-mediated apoptosis and necrosis. OctomininII is derived from the antimicrobial peptide Octominin from Octopus minor, which inhibits the growth of Candida albicans through multiple mechanisms of action.

[0004] The APD database (https: / / aps.unmc.edu / ) currently contains 890 anti-Candida AMPs, most of which have a large molecular weight and an average length of 28.03 amino acid residues. From an R&D perspective, focusing on the R&D of short peptides is more effective in terms of time and cost-effectiveness. Jelleine-I is a short AMP (PFKLSLHL) found in the royal jelly of bees, with only 8 amino acids. Previous studies have shown that it exerts its anti-Candida activity by targeting cell membranes and intracellular components. Studies have shown that Jelleine-Ic is a newly designed antimicrobial peptide derived from Jelleine-I that has stronger antibacterial activity than Jelleine-I. The antifungal activity and mode of action of Jelleine-Ic against Candida albicans are still unclear.

[0005] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0006] The purpose of the present invention is to solve the problem of how to apply the antimicrobial peptide Jelleine-Ic to the anti-Candida albicans activity, and provide an application of an antimicrobial peptide with antifungal activity.

[0007] In order to achieve the above object, the present invention discloses an application of an antimicrobial peptide with antifungal activity in the preparation of an antifungal infection drug, wherein the antimicrobial peptide is Jelleine-Ic, and has an amino acid sequence of WWKLLRKL-NH2.

[0008] The fungus is Candida albicans.

[0009] The minimum inhibitory concentration (MIC) of the antimicrobial peptide to Candida albicans is 12.5 μg / mL.

[0010] The invention also discloses an application of an antimicrobial peptide with antifungal activity in cooperation with amphotericin B in preparing an antifungal infection drug. The antimicrobial peptide is Jelleine-Ic, with an amino acid sequence of WWKLLRKL-NH2; and the fungus is Candida albicans.

[0011] The concentration of the antimicrobial peptide Jelleine-Ic is 0.78-3.13 μg / mL, and the concentration of amphotericin B is 1.25 μg / mL.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention discloses the antifungal activity, antibiofilm activity and mechanism of action of Jelleine-Ic against Candida albicans. Jelleine-Ic has strong antifungal activity against Candida albicans, with an MIC of 12.5 μg / mL. It effectively inhibits the formation of Candida albicans biofilm and destroys the formed biofilm. When it is used in combination with amphotericin B, it exhibits a synergistic effect. When the concentration is lower than 125 μg / mL, it has no hemolytic effect on rabbit red blood cells (rRBCs). Jelleine-Ic damages the integrity of the cell wall and enhances the permeability of the cell membrane of Candida albicans. In addition, Jelleine-Ic interacts with DNA when entering the cell, affects the expression of genes involved in DNA replication and repair, and induces the production of intracellular reactive oxygen species, accelerating the death of fungal cells. Based on the above results, Jelleine-Ic can effectively inhibit the growth of Candida albicans and has the potential to treat fungal infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The antifungal activity kinetics and hemolytic activity of Jelleine-Ic, where (A) is Candida albicans cells (1.0×10 5 cfu / mL) were incubated with 1 MIC of Jelleine-Ic or amphotericin B at 30°C. After different incubation periods, the OD of each culture was measured. 600 Control: sterile water; (B) rRBCs suspension was incubated with different concentrations of Jelleine-I for 1 hour, and then the OD of the supernatant was measured. 414 . TritonX-100 and phosphate buffer saline (PBS) were used as positive and negative controls, respectively. Different lowercase letters indicate significant differences among different groups (P<0.05);

[0014] Figure 2 The inhibitory effects of Jelleine-Ic (A), amphotericin B (B) and their combination (C) on the biofilm formation of Candida albicans. Control: sterile water. Different lowercase letters indicate significant differences among different groups (P<0.05);

[0015] Figure 3 The destructive effects of Jelleine-Ic (A), amphotericin B (B) and their combination (C) on the biofilm of Candida albicans. Control: sterile water. Different lowercase letters indicate significant differences among different groups (P<0.05);

[0016] Figure 4 Jelleine-Ic acts on the cell wall and cell membrane of Candida albicans, where (A) is the cell wall integrity of Candida albicans. The fungal cells were incubated with the peptide for 12 hours, then stained with 5% crystal violet and observed under a microscope. Control: sterile water. Scale bar: 50 μm; (B) is the cell membrane permeability of Candida albicans. The fungal cells were incubated with Jelleine-Ic, and then the membrane permeability was measured, control: PBS; (C) is the cell membrane destruction of Candida albicans cells. Candida albicans cells were incubated with the peptide for 30 minutes, then PI (0.1 μg / mL) was added and fixed, and then the cells were observed under a confocal laser scanning microscope (CLSM), control: sterile water. Scale bar: 10 μm; (D) Expression levels of ergosterol synthesis (ERG1, ERG5 and ERG11), cell wall synthesis (GSL1 and GSL2), and DNA replication and repair-related genes (PSF1, PSF2 and RAD16) in Candida albicans after 12 h of Jelleine-Ic treatment. Control: sterile water, different lowercase letters indicate significant differences among different groups (P<0.05);

[0017] Figure 5 Scanning electron microscopy (AF) and transmission electron microscopy (GL) micrographs of Candida albicans treated with Jelleine-Ic for 12 h, where (A, B, G and H) are controls (Control, sterile water); (CF and IL) are Jelleine-Ic. All tests were repeated 3 times;

[0018] Figure 6 Jelleine-Ic enters cells, interacts with DNA and induces intracellular ROS generation. Among them, (A) is the localization of Jelleine-Ic in Candida albicans cells. The cells were stained with FITC-Jelleine-Ic and DAPI at the same time, and then observed under CLSM, scale: 10μm; (B) is the DNA binding activity of Jelleine-Ic. Different concentrations of Jelleine-Ic were incubated with Candida albicans genomic DNA. The mixture was electrophoresed and observed. (C) is the effect of Jelleine-Ic on intracellular ROS. Candida albicans cells were incubated with Jelleine-Ic for 12h, and then the ROS level was determined. Control (Control): sterile water. Different lowercase letters indicate significant differences between different groups (P<0.05). DETAILED DESCRIPTION

[0019] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0020] The experimental data were analyzed by one-way analysis of variance (ANOVA) using DPS data processing software, and the significance was determined by Duncan's new multiple range test, with the significant difference level analyzed according to P < 0.05. Graphpadprism 5 was used for drawing.

[0021] Example 1: Test fungi and their cultivation

[0022] Candida albicans was provided by China Academy of Chinese Medical Sciences (Beijing, China). Candida albicans cells were cultured in Sabouraud dextrose broth (1% tryptone, 2% glucose) at 30°C and 160 rpm.

[0023] Example 2: Peptides and Chemicals

[0024] Jelleine-I (PFKLSLHL) and Jelleine-Ic (WWKLLRKL-NH2) were synthesized by Synpeptide (Shanghai, China) using a solid phase method. All chemicals were of analytical grade. Amphotericin B was purchased from Solarbio (Beijing, China).

[0025] Example 3: MIC and MFC determination

[0026] Thaw the C.albicans glycerol stock stored at -80℃ on ice, take 100μL of glycerol stock to 2mL of fresh SDB medium, and culture overnight at 30℃, 160r / min. Transfer the next day, take 100μL of C.albicans bacterial liquid from the overnight bacteria to 2mL of fresh SDB medium, and culture at 30℃, 160r / min until the logarithmic growth phase. Use fresh SDB medium to prepare C.albicans bacterial suspension (concentration of 1.0×10 5 cfu / mL). 10 μL of different concentrations of antimicrobial peptides diluted with sterile water were added to a 96-well ELISA plate using the two-fold dilution method, and then 90 μL of C.albicans bacterial suspension was added to the wells corresponding to the different concentrations of antimicrobial peptides. The bacterial suspension plus sterile water was used as a positive control, and the SDB medium plus sterile water was used as a negative control. The plates were placed in a 30°C incubator for 24 hours, and the OD values ​​of the 96-well ELISA plate were measured using an ELISA reader. 600 value.

[0027] The method for determining the minimum fungicidal concentration (MFC) refers to the MIC determination method. After the 96-well ELISA plate in the MIC determination experiment was incubated at 30°C for 24 hours, 50 μL of bacterial solution was taken from each well (mixed before taking) to the corresponding SDA culture medium and evenly spread with coating beads. After culturing in a 30°C constant temperature incubator for 24 hours, the colonies of C. albicans on the SDA plate were counted, and the lowest concentration without colony growth was taken as the MFC of the antimicrobial peptide against C. albicans.

[0028] Jelleine-Ic showed strong antifungal activity against Candida albicans with a MIC of 12.5 μg / mL, which was superior to the parent peptide Jelleine-I with a MIC of 25 μg / mL. The MIC of amphotericin B against Candida albicans was 5 μg / mL. The MFCs of Jelleine-Ic, Jelleine-I and amphotericin B against Candida albicans were 25 μg / mL, 50 μg / mL and 10 μg / mL, respectively.

[0029] Example 4: Dynamic Antibacterial Activity and Hemolytic Activity Assays

[0030] Dynamic antibacterial activity assay: C.albicans was cultured to the logarithmic growth phase, and a C.albicans suspension (concentration of 1.0×10 5 cfu / mL). The diluted bacterial solution was incubated with Jelleine-Ic or amphotericin B at a final concentration of 1 MIC. Sterile water was used as a negative control, and the mixture was incubated at 30°C and 160 rpm for 24 h. The OD of the culture was measured using a microplate reader. 600 value.

[0031] Hemolytic activity assay: Take fresh rabbit erythrocytes, centrifuge at 2000r / min for 5min, discard the supernatant, wash the erythrocytes 3 times with PBS and resuspend. Incubate at 37℃ with shaking for 1h, centrifuge at 500×g for 10min, take 80μL of the obtained supernatant and detect the absorbance at 414nm. Calculate the hemolytic activity.

[0032] The antifungal activity kinetics assay showed that Jelleine-Ic almost completely killed 1MIC of Candida albicans within 24 hours ( Figure 1 A). Amphotericin B showed a similar effect. No significant increase in the hemolytic activity of Jelleine-Ic was observed at tested concentrations below 125 μg / mL compared to the control group (7.63%). Figure 1B). Even at the highest concentration (10 MIC, 125 μg / mL), Jelleine-Ic only exhibited 10.65% hemolytic activity.

[0033] Example Five: Detection of Synergistic Effect

[0034] Detect the effect of the combined action of Jelleine-Ic and amphotericin B on the growth of C. albicans. The tested C. albicans was cultured to the logarithmic growth phase, and a C. albicans bacterial suspension (concentration of 1.0×10 5 cfu / mL) was prepared with fresh SDB medium. Using the two-fold dilution method, 5 μL of antibacterial peptides at different concentrations (diluted with sterile water) and 5 μL of amphotericin B at different concentrations were added to a 96-well microplate, and then 90 μL of the bacterial suspension was added to the wells corresponding to the mixed solution of different concentrations of the compounds. It was placed in an incubator at 30 °C for constant temperature culture for 24 h, and the OD 600 value was detected with a microplate reader. The positive control was the bacterial solution plus sterile water, and the negative control was SDB medium plus sterile water. The synergistic effect was evaluated by the Fractional Inhibitory Concentration Index (FICI). The comprehensive effect was defined as follows:

[0035] The FICI values obtained from different combinations of antibacterial compounds were interpreted as synergistic effect (FICI ≤ 0.5), additive effect (0.5 < FICI ≤ 1.0), irrelevant effect (1.0 < FICI ≤ 2.0), and antagonistic effect (FICI > 2.0).

[0036] Interactions of synergistic, additive, and irrelevant effects were observed between Jelleine-Ic and amphotericin B, and the FICI values were between 0.31 - 2. As shown in Table 1, Jelleine-Ic at a concentration of 0.78 - 3.13 μg / mL showed a synergistic effect when used in combination with 1.25 μg / mL of amphotericin B.

[0037] Table 1 Combined action of Jelleine-Ic and amphotericin B on Candida albicans

[0038]

[0039] Example Six: Antibiofilm Activity

[0040] The antibiofilm activities of Jelleine-Ic, amphotericin B, and their combination were determined. To inhibit biofilm formation, 20 μL of serially diluted antibacterial agents were introduced into a 96-well microplate. Then 180 μL of logarithmic-phase Candida albicans cells (1.0×10 5cfu / mL) solution and incubated at 30°C. Sterile water was used as a control. After 24 h of incubation, the biofilm formation inhibition effect was evaluated by crystal violet staining.

[0041] For the disruption of preformed biofilms, 200 μL of Candida albicans cells (1.0 × 10 4 cfu / mL) were added to a 96-well microplate and incubated at 30°C for 24 h to form a biofilm. The biofilm was washed with PBS and treated with different concentrations of antimicrobial agents. Sterile water was used as a control. The plate was incubated again at 30°C for 24 h. The preformed biofilm disruption effect was quantified by crystal violet staining.

[0042] It was found that Jelleine-Ic and amphotericin B had excellent biofilm inhibition activity. At 1 MIC, Jelleine-Ic and amphotericin B almost completely inhibited the formation of biofilm ( Figure 2 A and 2B). When used in combination with 0.25 MIC of amphotericin B, all tested concentrations of Jelleine-Ic inhibited biofilm formation by >90% ( Figure 2 C).

[0043] Jelleine-Ic can also destroy the formed biofilm. After 24h treatment with 1MIC of Jelleine-Ic and amphotericin, the formed biofilm was reduced from 100% to 41.90% and 26.93% ( Figure 3 A and 3B). When combined with 0.25 MIC of amphotericin B, Jelleine-Ic reduced biofilm to 24.71% at 0.5 MIC ( Figure 3 C).

[0044] Example 7: Cell Wall Integrity

[0045] Take C.albicans in the logarithmic growth phase and prepare a C.albicans suspension (concentration of 2.0×10 7 cfu / mL), treated the C.albicans suspension with Jelleine-Ic and amphotericin B at a final concentration of 2MIC, and sterilized water was used as a control. Each group was placed in a 30°C incubator for 10 hours, centrifuged at 3000r / min for 10 minutes to collect the C.albicans bacteria, and washed 3 times with PBS. Add 5% crystal violet to stain for 10 minutes, add PBS to suspend the bacterial solution and centrifuge until the bacterial solution is colorless, take 5μL of the bacterial solution and precipitate it on a glass slide, then add 10% tannic acid to fix it, cover it with a coverslip and observe it under an inverted microscope, randomly observe 100 cells and calculate the integrity rate of the C.albicans cell wall. Each group of experiments was repeated three times.

[0046] like Figure 4 As shown in A, the control group (Control) Candida albicans cells were round or oval, their intact cell walls were stained light purple or remained colorless, and the cell wall integrity rate was 92.83%. In contrast, the cell walls and cytoplasm of the Candida albicans cells treated with Jelleine-Ic were dark purple, and the cell integrity rate was significantly reduced to 57.19%, which was significantly lower than the control.

[0047] Example 8: Cell membrane permeability

[0048] Take out C.albicans from -80℃ ultra-low temperature freezer, add to fresh SDB medium, and place in constant temperature shaker at 30℃, 160r / min for overnight culture. Centrifuge at 3000r / min for 10min, keep the precipitate, and resuspend C.albicans with PBS to a final concentration of 2.0×107cfu / mL. Treat the Candida albicans suspension with Jelleine-Ic and amphotericin B at a final concentration of 1MIC. Treat the suspended C.albicans suspension with 10% Triton X-100 as a positive control, and PBS as a negative control. Measure the OD of the filtrate with a UV spectrophotometer. 260 The cell membrane permeability was calculated.

[0049] Treatment with Jelleine-Ic rapidly enhanced the membrane permeability of Candida albicans cells ( Figure 4 B). After Jelleine-Ic treatment, cell membrane permeability increased from 5.45% to 15.11% at 0.5 h and further increased to 27.37% at 12 h. This increase was significantly greater than that observed in the control. These findings suggest that Jelleine-Ic specifically targets and permeabilizes fungal cell membranes.

[0050] Example 9: Propidium iodide uptake assay

[0051] The disruption of the cell membrane of Candida albicans cells was assessed by measuring the uptake of propidium iodide (PI). 7 cfu / mL) were cultured with different concentrations of Jelleine-Ic at 30°C, and sterile water was used as a control. After incubation for 30 min, PI (0.1 μg / mL) was used and stored in the dark. The cells were then observed using a Carl Zeiss LSM800 confocal laser scanning microscope (CLSM).

[0052] Disruption of the C. albicans cell membrane was further assessed by PI uptake ( Figure 4C). Control C. albicans cells showed no red fluorescence. In contrast, fungal cells treated with Jelleine-Ic exhibited red fluorescence of PI. In addition, the number of red fluorescent cells increased with increasing peptide concentrations, indicating that Jelleine-Ic enhanced cell membrane disruption of C. albicans.

[0053] Jelleine-Ic treatment affected the expression levels of genes related to ergosterol (ERG1, ERG5, and ERG11) and cell wall (GSL1 and GSL2) synthesis. Figure 4 D). In C. albicans treated with Jelleine-Ic, the expression levels of ERG1, ERG5, and ERG11 were significantly increased by 6.89-fold, 5.91-fold, and 11.22-fold, respectively, compared to the control. In addition, Jelleine-Ic treatment resulted in a significant upregulation of GSL1 and GSL2 expression by 8.27-fold and 23-52-fold, respectively, compared to the control group.

[0054] Example 10: Scanning Electron Microscope and Transmission Electron Microscope

[0055] Fungal cells (2.0×10 7 cfu / mL) and Jelleine-Ic (1MIC) were cultured at 30°C, and the control was sterile water. After incubation for 12 hours, cell samples for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were prepared according to the previously established method (K. Ding, P. Shen, Z. Xie, L. Wang, X. Dang, In vitro and in vivo antifungal activity of two peptides with the same composition and different distribution, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 252 (2022) 109243). The samples were examined under a Hitachi SU8100 scanning electron microscope and a Hitachi HT-7700 transmission electron microscope, respectively.

[0056] The surface morphology and structural changes of C.albicans after Jelleine-Ic treatment for 10 hours were analyzed by scanning electron microscopy (SEM). As can be seen from the figure, the C.albicans cells in the control group were round or oval, the cells were plump, and the cell surface was smooth ( Figure 5A and 5B). In contrast, treatment with Jelleine-Ic for 12 h resulted in significant damage and morphological changes ( Figure 5 C), such as cell surface depressions ( Figure 5 D) Perforation Figure 5 E) and deformation ( Figure 5 F).

[0057] Transmission electron microscopy (TEM) was used to analyze the changes in the internal morphology and structure of C.albicans cells after Jelleine-Ic treatment. The C.albicans cells in the control group were round or oval in morphology, and the cell structure was intact ( Figure 5 G and 5H). On the other hand, cells treated with Jelleine-Ic showed many ultrastructural changes ( Figure 5 I), including deformation of the external structure of the cell ( Figure 5 Red arrows in J), loosely arranged cell walls ( Figure 5 blue arrows in K and 5L), uneven electron density, and formation of intracellular vacuoles ( Figure 5 K and red arrows in 5L).

[0058] Example 11: Fluorescence Microscope

[0059] Evaluation of peptide localization in Candida albicans cells. Fungal cells (2.0 × 10 7 cfu / mL) and fluorescein isothiocyanate (FITC)-Jelleine-Ic (1MIC) were incubated at 30°C for 1 h, and sterile water was used as a control. The cells were then harvested and washed with PBS. Subsequently, they were treated with 1 μg / mL of 4',6-diamidino-2-phenylindole (DAPI) and examined under Carl Zeiss LSM800CLSM.

[0060] Compared with control cells, green fluorescence was found in C. albicans cells exposed to FITC-Jelleine-Ic ( Figure 6 A), indicating that Jelleine-Ic successfully enters cells by penetrating the cell wall and cell membrane. When cells were stained with both dyes, colocalization of FITC-Jelleine-Ic and DAPI was observed, indicating that the peptide interacts with fungal DNA.

[0061] Example 12: Electrophoretic mobility shift assay

[0062] Genomic DNA was isolated from Candida albicans cells. 40 ng of genomic DNA was incubated with different concentrations of Jelleine-Ic (15.63-1000 μg / mL) at 25°C. The control was sterile water. After 1.5 hours of incubation, the DNA binding activity of Jelleine-Ic was detected by evaluating DNA migration on 0.8% agarose gel electrophoresis.

[0063] The results showed that 125 μg / mL of Jelleine-Ic almost inhibited the migration of Candida albicans genomic DNA ( Figure 6 B). This finding suggests that Jelleine-Ic interacts with the genomic DNA of C. albicans. In addition, the expression levels of the genes PSF1, PSF2, and RAD16 involved in DNA replication and repair in C. albicans treated with Jelleine-Ic were 2.13-fold, 2.82-fold, and 1.56-fold higher, respectively, compared to the control ( Figure 4 D), indicating that Jelleine-Ic disrupts DNA structure and affects its function.

[0064] Example 13: RT-qPCR

[0065] Candida albicans cells (2.0×10 7 cfu / mL) and Jelleine-Ic (1MIC) were cultured at 30°C. After incubation for 12 h, the cells were harvested and total RNA was extracted. Sterile water was used as a control. The expression levels of Candida albicans cell wall synthesis genes (GSL1 and GSL2), ergosterol synthesis genes (ERG1, ERG5 and ERG11), and genes involved in DNA replication and repair (PSF1, PSF2 and RAD16) were analyzed. The internal reference gene was the 18S gene. The primer sequences are listed in Table 2.

[0066] Table 2 Primer sequences

[0067]

[0068] Example 14: Detection of intracellular ROS in Candida albicans

[0069] C.albicans in the logarithmic growth phase was taken, and a C.albicans suspension with a concentration of 2.0×107 cfu / mL was prepared with fresh SDB medium. It was treated with Jelleine-Ic and amphotericin B with final concentrations of 1 / 2MIC, 1MIC and 2MIC, and sterile water was used as a control. After culturing in a 30℃ constant temperature incubator for 12 hours, the cells were taken out and centrifuged at 3500r / min for 10 minutes to collect the cells, washed twice with extracellular fluid, and each group of samples was resuspended with PBS to a bacterial suspension concentration of 2.0×10 7cfu / mL, and add 100 μL of 2',7'-dichlorodi-hydrofluorescein diacetate (DCFH-DA) solution with a final concentration of 10 μM, and incubate at 30°C in the dark for 30 minutes. Take 200 μL of each group of bacterial solution in each well of a 96-well plate black board, and use a multifunctional microplate reader to detect the fluorescence intensity of 2',7'-dichlorofluorescein (2',7'-Dichlorofluorescein, DCF) (excitation wavelength / emission wavelength: 488 / 525 nm).

[0070] It was found that Jelleine-Ic enhanced the fluorescence intensity of DCF ( Figure 6 C). Compared with control cells, 5.30-fold, 3.43-fold, and 1.71-fold higher fluorescence intensities were detected in cells exposed to 2MIC, 1MIC, and 0.5MIC peptides, indicating that Jelleine-Ic promoted the generation of intracellular ROS.

[0071] Example 15: Effect of ROS scavengers on peptide activity

[0072] 10 mM glutathione (GSH), N-acetylcysteine ​​(NAC) and vitamin C (VC) solutions were preloaded into 96-well microplates, and fungal cells at a concentration of 1.0×105 cfu / mL were added and incubated at 30°C for 30 minutes. Dilutions of peptides (6.25 to 400 μg / mL) were then added, and the plates were incubated at 30°C for another 24 hours. After incubation, the MIC value of each group was evaluated. Sterile water was the control.

[0073] The test results showed that the presence of ROS scavengers significantly reduced the activity of the peptide. After adding 10mM Vc, GSH or NAC, the MIC value increased from 12.5μg / mL to 50μg / mL, indicating that the antifungal activity of Jelleine-Ic may be mediated by inducing ROS accumulation.

[0074] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. Use of an antimicrobial peptide having antifungal activity in the preparation of an antifungal infection drug, characterized in that: The antimicrobial peptide is Jelleine-Ic, with an amino acid sequence of WWKLLRKL-NH2.

2. The use of an antimicrobial peptide with antifungal activity as claimed in claim 1 in the preparation of an antifungal infection drug, characterized in that: The fungus is Candida albicans.

3. The use of an antimicrobial peptide with antifungal activity as claimed in claim 2 in the preparation of an antifungal infection drug, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide to Candida albicans is 12.5 μg / mL.

4. Use of an antimicrobial peptide with antifungal activity in combination with amphotericin B in the preparation of an antifungal infection drug, characterized in that: The antimicrobial peptide is Jelleine-Ic, with an amino acid sequence of WWKLLRKL-NH2.

5. The use of an antimicrobial peptide with antifungal activity in combination with amphotericin B in the preparation of an antifungal infection drug according to claim 1, characterized in that: The fungus is Candida albicans.

6. The use of an antimicrobial peptide with antifungal activity in combination with amphotericin B in the preparation of an antifungal infection drug according to claim 1, characterized in that: The concentration of the antimicrobial peptide Jelleine-Ic is 0.78-3.13 μg / mL, and the concentration of amphotericin B is 1.25 μg / mL.

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