Antibacterial peptide LR-24 and application thereof in preparation of antibacterial drugs

Abstract

The application discloses an antibacterial peptide LR-24 and application thereof in preparation of antibacterial drugs, and belongs to the technical field of biological medicine. The amino acid sequence of the antibacterial peptide LR-24 is shown as SEQ ID NO. 2. The antibacterial peptide LR-24 provided by the application has high efficient killing capacity on various gram-negative / positive bacteria, has broad-spectrum stability, still has strong antibacterial activity under salt ions, 50% serum, protease and high-temperature environment, and improves the problem that the activity of the antibacterial peptide is reduced in some complex environments. Moreover, the antibacterial peptide LR-24 greatly reduces hemolytic activity and cytotoxicity while maintaining antibacterial activity, and is a new antibacterial drug candidate with great clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antimicrobial peptide LR-24 and its application in the preparation of antimicrobial drugs. Background Technology

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides or antimicrobial peptides, are a class of small protein molecules transcribed and translated from specific genes. As key components of innate immunity, they possess broad-spectrum antimicrobial activity, high biostability, and are less prone to inducing drug resistance. In addition to directly killing pathogenic microorganisms, antimicrobial peptides also have various biological functions such as immunomodulation, promoting wound healing, and neutralizing endotoxins. Unlike traditional antibiotics that act on specific molecular targets, antimicrobial peptides primarily work by disrupting the integrity of microbial cell membranes, causing leakage of cell contents and leading to target cell death. Some antimicrobial peptides can also enter the cell interior and act on biomolecules such as DNA, RNA, or enzymes, interfering with cellular metabolism. Currently, there are no reports of large-scale bacterial resistance to antimicrobial peptides; resistance has only been occasionally observed after long-term passage at subinhibitory concentrations.

[0003] The Cathelicidins family is an important class of antimicrobial peptides in mammals, and their C-terminal structure has a crucial influence on antibacterial activity. BMAP-28 is a member of the Cathelicidins family derived from bovine bone marrow, composed of 28 amino acids with a net positive charge of +7, exhibiting an α-helical conformation and strong hydrophobicity. This hydrophobic property, while conferring highly efficient bactericidal activity, also introduces potential toxicity to eukaryotic cells. However, this toxicity typically occurs at doses far above the minimum inhibitory concentration (MIC) and is further attenuated in the physiological environment of serum.

[0004] Despite their wide availability and diverse functions, natural antimicrobial peptides face numerous challenges in clinical translation. For example, their long amino acid sequences lead to high synthesis costs and hinder large-scale production. More critically, their cell selectivity is generally poor, potentially damaging normal human cells (e.g., causing hemolysis) while killing pathogens, thus limiting their systemic drug application. Therefore, the field urgently needs to develop novel design strategies to overcome the limitations of traditional modification methods, precisely controlling the cationic and hydrophobic properties of antimicrobial peptides while maintaining or even enhancing their antibacterial efficacy, in order to achieve high therapeutic index antimicrobial peptide molecular design. Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide LR-24 and its application in the preparation of antimicrobial drugs, thereby addressing the problems existing in the prior art. The antimicrobial peptide LR-24 provided by this invention exhibits highly efficient killing ability against a variety of Gram-negative / positive bacteria and possesses broad-spectrum stability. It maintains potent antimicrobial activity even under conditions of salt ions, 50% serum, proteases, and high temperatures, thus improving the problem of reduced activity of antimicrobial peptides in some complex environments. Furthermore, while maintaining antimicrobial activity, this antimicrobial peptide LR-24 shows significantly reduced hemolytic activity and cytotoxicity, making it a promising novel antimicrobial drug candidate for clinical application.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides an antimicrobial peptide LR-24, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides the application of the above-mentioned antimicrobial peptide LR-24 in the preparation of antimicrobial drugs.

[0008] Furthermore, the antibacterial drug has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria.

[0009] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis; The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

[0010] The present invention also provides an antibacterial drug, wherein the antibacterial drug uses the above-mentioned antimicrobial peptide LR-24 as an active ingredient.

[0011] The present invention also provides the use of the above-mentioned antimicrobial peptide LR-24 in the combined preparation of an antimicrobial composition with an antibiotic, wherein the antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

[0012] Furthermore, the antibacterial composition has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria.

[0013] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis; The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

[0014] The present invention also provides an antibacterial composition, wherein the antibacterial composition comprises the above-mentioned antimicrobial peptide LR-24 and an antibiotic as active ingredients.

[0015] Furthermore, the antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

[0016] The present invention discloses the following technical effects: This invention uses BMAP-28 as the parent peptide and, through simultaneous N- / C-terminal truncation and selective C-terminal truncation, obtains a novel antimicrobial peptide LR-24, whose amino acid sequence is shown in SEQ ID NO.2. This antimicrobial peptide LR-24 exhibits broad-spectrum stability, maintaining potent antimicrobial activity under various harsh environments. Its minimum inhibitory concentration (MIC) remained largely unchanged under different salt ion environments, environments containing 50% serum, environments containing multiple proteases, and high temperatures, thus addressing the issue of reduced activity of antimicrobial peptides in some complex environments. This lays a solid foundation for its in vivo application and development as a food preservative or topical formulation. Most importantly, antimicrobial peptide LR-24 successfully resolves the inherent contradiction between high activity and high toxicity of antimicrobial peptides. Through truncation design, while maintaining highly effective killing ability against various Gram-negative and Gram-positive bacteria, its hemolytic activity against human erythrocytes and toxicity to mammalian cells are significantly reduced, ensuring high therapeutic potential and achieving an excellent balance between activity and safety. Furthermore, LR-24 exhibits rapid bactericidal kinetics, acting within minutes of bacterial contact and demonstrating an inhibition rate of nearly 50% against biofilm formation, which is significant for controlling chronic and refractory infections. In summary, the antimicrobial peptide LR-24 provided by this invention possesses excellent stability, high cell selectivity, and rapid and efficient antimicrobial activity, offering a promising candidate molecule for the development of next-generation, highly effective, and low-toxicity antimicrobial drugs, with broad application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The growth curve of Cronobacter sakazakii ATCC 12868 at an antimicrobial peptide concentration of 1×MIC; Figure 2 Growth curve of Staphylococcus aureus CGMCC 1.490 at an antimicrobial peptide concentration of 1×MIC; Figure 3 The inhibitory effect of different concentrations of antimicrobial peptides on biofilm formation of Cronobacter sakazakii ATCC 12868 was measured; P < 0.05. Figure 4The images are high-resolution fluorescence micrographs of Cronobacter sakazakii ATCC 12868 after treatment with FITC-labeled peptides; where a1 is a Bright Field image, a2 is a FITC image, a3 is a PI image, and a4 is a Merge image; the scale bar is 50 µM. Figure 5 The results show the effect of different concentrations of the antimicrobial peptide LR-24 on the leakage of β-galactosidase in Cronobacter sakazakii ATCC 12868. Figure 6 Scanning electron microscopy (SEM) images of Cronobacter sakazakii ATCC 12868 before and after treatment with the antimicrobial peptide LR-24; where a1-a3 are growth images of untreated Cronobacter sakazakii ATCC 12868; b1-b3 are growth images of Cronobacter sakazakii ATCC 12868 after treatment with the antimicrobial peptide LR-24; scale bar is 2 µM. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0025] The sequence of the antimicrobial peptide BMAP-28 in the following embodiments of the present invention is as follows: GGLRSLGRKILRAWKKYGPIIVPIIRIG (SEQ ID NO. 1).

[0026] The antimicrobial peptides in the following embodiments of the present invention were all synthesized by Fmoc-solid phase synthesis and purified by high performance liquid chromatography (HPLC) and verified by mass spectrometry (MS) to obtain products with a purity of over 95%.

[0027] The test strains involved in the following embodiments of the present invention are shown in Table 1.

[0028] Table 1 Test strains Example 1: Obtaining Antimicrobial Peptides It is generally believed that antimicrobial properties are related to cationic and hydrophobic residues. Cationic residues induce antimicrobial peptides to target the cell membrane surface, while hydrophobic residues help them insert into the bacterial cell membrane, thereby causing damage. Low hydrophobicity reduces the bactericidal activity of antimicrobial peptides, but high hydrophobicity can damage mammalian cell membranes and is a factor inducing cytotoxicity. Therefore, appropriately reducing hydrophobicity can enhance the antimicrobial activity and biocompatibility of antimicrobial peptides. Computer simulations are used to calculate the changes in charge number and hydrophobicity during amino acid truncation. Gravy is commonly used to represent the hydrophobicity of antimicrobial peptides; a higher value indicates greater hydrophobicity. The net positive charge and GRAVY value of antimicrobial peptides also affect their antibacterial properties. Generally, antimicrobial peptides with a positive charge between 5 and 7 exhibit the highest cell selectivity. A higher GRAVY value indicates stronger bacterial killing activity.

[0029] In this embodiment, the antimicrobial peptide BMAP-28 (hereinafter referred to as GG-28) was used as the parent peptide. Its amino acid sequence was truncated by simultaneously shortening the N- and C-termini and selectively shortening the C-terminus to explore the influence of the BMAP-28 structure on the properties of the antimicrobial peptide and to identify antimicrobial peptides with better antimicrobial and physiological / biochemical properties. The sequences and physicochemical parameters of the obtained truncated peptides are shown in Table 2.

[0030] Table 2. Amino acid composition and parameters of antimicrobial peptides Example 2: Determination of the bioactivity of antimicrobial peptides I. Experimental Methods 1. Antibacterial activity assay The minimal inhibitory concentration (MIC) method recommended by the Clinical and Laboratory Standards Institute (CLSI) was adopted, and the specific steps are as follows: (1) Cell culture: The -20℃ frozen bacterial solution was inoculated into MHB medium at 2% (bacterial solution volume / medium medium volume) and cultured overnight in a shaker at 168 rpm and 37℃ until it reached the logarithmic growth phase. Then, a second-generation strain was inoculated and cultured again, and the second-generation strain was adjusted to OD using MHB. 600 =0.4, diluted 100 times as the bacterial solution to be tested.

[0031] (2) Peptide dilution: Add 95 μL and 50 μL of BSA solution to row A and other rows respectively. Then, add 5 μL of antimicrobial peptide solution to row A. Gradually dilute 50 μL from top to bottom. After gradient dilution in row G, discard 50 μL of the mixture.

[0032] (3) Inoculation of bacterial culture: 50 μL of diluted second-generation bacterial culture was inoculated into the AG row and the first 6 wells of the H row, and 50 μL of sterile MHB medium was added to the last 6 wells of the H row. The first 6 wells and the last 6 wells of the H row were used as positive and negative control groups, respectively. After 24 h of incubation, the negative control wells served as the contamination group of the 96-well plate. If no turbidity was observed, the absorbance was measured at 600 nm using a microplate reader. The concentration of antimicrobial peptide at which no obvious turbidity was observed in the solution in the wells was taken as the minimum inhibitory concentration. This experiment was independently repeated three times.

[0033] 2. Stability testing of antimicrobial peptides The stability of antimicrobial peptides was evaluated using Cronobacter sakazakii ATCC 12868 and Staphylococcus albus CGMCC 1.490 as test strains.

[0034] 2.1 Stability under different salt ion environments Prepare BSA solutions with final concentrations of 150 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 6 mM NH4Cl, and 8 mM ZnCl2. Replace the BSA solutions in "1. Antibacterial Activity Assay" with BSA solutions containing the different salt ions to dilute the peptide solution, and then inoculate the bacterial culture.

[0035] 2.2 Stability under different temperature environments The antimicrobial peptides were incubated at 40℃, 60℃, 80℃, and 100℃ for 1 h. The peptide solutions incubated at different temperatures were used to replace the peptide solution added in row A of "1. Antimicrobial Activity Assay" to dilute the peptide solutions, which were then inoculated with bacterial culture.

[0036] 2.3 Stability under different serum conditions Prepare BSA solutions with final serum concentrations of 50%, 25%, and 12.5%. Replace the BSA solution in "1. Antibacterial Activity Assay" with BSA solutions containing different serum concentrations to dilute the peptide solution, and then inoculate the bacterial culture.

[0037] 2.4 Stability under different enzyme environments The antimicrobial peptide was incubated with 1 mg / mL of pepsin, trypsin, chymotrypsin, and papain at 37°C for 2 h. The peptide solution was diluted using a peptide solution co-incubated with different enzymes instead of the peptide solution added in row A of section "1. Antimicrobial Activity Assay," and then inoculated with bacterial culture.

[0038] 3. Hemolytic activity assay Healthy human blood was centrifuged at 1000×g at 4℃ for 10 min, and the supernatant was discarded to obtain healthy human red blood cells. The cells were washed three times with PBS, and then resuspended in PBS solution. An equal volume of human red blood cells and different concentrations of peptides were mixed in 96-well plates and incubated at 37℃ for 4 h. The supernatant was then collected by centrifugation, and its absorbance was measured at 570 nm using a microplate reader. Untreated human red blood cells served as a negative control, and the 0.1% Triton X-100 treatment group served as a positive control. The minimum peptide concentration required to induce 10% hemolytic activity was defined as the minimum hemolytic concentration (MHC). This experiment was independently repeated three times. The formula for calculating hemolytic activity is as follows: In the formula, OD （样品测定值） The absorbance of human red blood cells treated with peptide solution; OD （阴性对照） Absorbance of untreated human erythrocytes without added peptide solution and Triton X-100; OD （阳性对照） The absorbance of human red blood cells treated with Triton X-100.

[0039] 4. Cytotoxicity test For specific operating procedures, refer to "1. Antibacterial Activity Assay," simply replace the bacterial culture with cell suspension. After incubating the treated 96-well plates in a CO2 incubator for 24 h, add 10 µL of CCK-8 solution and incubate for another 2 h. Use wells without cells as a blank control group and melitoxin as a positive control. Measure the absorbance at 450 nm using a microplate reader. Repeat each experiment independently three times. Calculate cell viability using the following formula: In the formula, OD (样品测定值) The absorbance of the wells containing cells, culture medium, CCK-8 solution, and drug solution; OD (阴性对照) The absorbance of the pores containing culture medium, CCK-8 solution, and no cells; OD (阳性对照) The absorbance is the value of the well containing cells, culture medium, and CCK-8 solution, but no drug solution.

[0040] 5. Sterilization time-kinetic curve The bacterial culture method follows the steps (1) of "1. Determination of Antimicrobial Activity". Equal volumes of different concentrations of antimicrobial peptides were added to the bacterial suspension, and the mixture was incubated at 37°C. The absorbance at 600 nm was measured at 0, 5, 10, 15, 30, 60, 90, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, and 720 min. The group without added antimicrobial peptides served as a positive control.

[0041] 6. Inhibits biofilm formation The bacterial culture method follows the steps (1) of "1. Antibacterial Activity Assay". The bacterial culture was incubated in a 96-well plate for 24 h. The MHB liquid was slowly aspirated using a pipette, and the plate was gently rinsed with PBS to remove any non-adherent bacteria. The biofilm at the bottom of the wells was then stained with 0.1% crystal violet. Excess dye was removed by washing with PBS, and the dye was dissolved in 70% ethanol. The absorbance at 595 nm was measured.

[0042] II. Test Results 1. Hemolytic activity A hemolytic activity of less than 10% was considered a good blood safety standard. The results are shown in Table 3. It can be seen that the hemolytic activity of the peptide decreased after truncation. At a concentration of 8 µM, LR-24 exhibited good safety, with a hemolytic activity of 8.23 ​​± 2.16%. In contrast, GG-28, the parent peptide, and the positive control meliofemort peptide showed extremely high hemolytic activity, exhibiting hemolytic activities of 10.95 ± 1.30% and 11.89 ± 3.57% respectively at the lowest tested concentration (4 µM).

[0043] Table 3 Hemolytic activity of antimicrobial peptides 2. Cytotoxicity The results are shown in Table 4. GG-28, LR-24, and Melittin reduced cell viability. Compared with the parent peptide, truncated LR-24 showed reduced cytotoxicity.

[0044] Table 4. Cytotoxicity (cell survival rate) of antimicrobial peptides 3. Antibacterial activity The minimum inhibitory concentrations (MICs) of GG-28 and its truncated peptide against eight different bacterial strains are shown in Tables 5 and 6, which summarize the results for Gram-negative and Gram-positive bacteria, respectively. The results indicate that LR-24 has inhibitory effects on both Gram-negative and Gram-positive bacteria at low concentrations (2–16 μM).

[0045] To more intuitively compare the antibacterial activities of different short peptides, this invention calculated the geometric mean (GM) of the MIC values ​​of GG-28 and the truncated peptide against the tested microbial strains, reflecting the average killing effect on the microbial strains. A smaller GM value indicates that the peptide can maintain an average killing effect against clinically typical microorganisms at a lower concentration (see Table 7). The results showed that the truncated peptide LR-24 had a better average killing effect on the tested clinically typical microorganisms than the parent peptide and Melittin.

[0046] This invention further introduces the therapeutic index (TI) of antimicrobial peptides by calculating the ratio of GM to MHC, thus evaluating the targeting selectivity of antimicrobial peptides against pathogenic microorganisms and host cells. A higher TI value indicates greater cell selectivity. Although LR-24 retains hemolytic activity and cytotoxicity, its TI value is higher than that of GG-28. This result indicates that LR-24 possesses good cell selectivity and application potential.

[0047] Table 5. MIC values ​​(µM) of antimicrobial peptides against Gram-negative bacteria. Table 6. MIC values ​​(µM) of antimicrobial peptides against Gram-positive bacteria. Table 7. Therapeutic potential of peptides Note: a) The peptide concentration that causes 10% hemolysis is the minimum hemolytic concentration (MHC). When the hemolytic concentration is >128 µM, use 256 µM for calculation. When the hemolytic concentration is still greater than 10% at 2 µM, use 2 µM to calculate the therapeutic index. b) The geometric mean of the MIC values ​​of the peptide. c) The therapeutic index = MHC / GM. The larger this value, the better the selectivity of the antimicrobial peptide.

[0048] 4. Stability assessment 4.1 Salt Ion Stability The results are shown in Tables 8 and 9. In the presence of salt ions, the antibacterial activity of LR-24 was not affected, demonstrating good tolerability. The MIC value of the parent peptide increased by 1-2 times.

[0049] Table 8. MIC values ​​(µM) of antimicrobial peptides against Cronobacter sakazakii ATCC 12868 under different salt ion environments. Table 9. MIC values ​​(µM) of antimicrobial peptides against Staphylococcus albus CGMCC 1.490 under different salt ion environments. 4.2 Serum stability Serum contains a large number of free radicals, including serum enzymes and various anions that can affect the efficacy of antimicrobial peptides. Therefore, it is necessary to detect the changes in the antimicrobial activity of antimicrobial peptides at different serum concentrations. The results are shown in Tables 10 and 11. The MIC of LR-24 did not increase significantly with increasing serum concentration, while the parent peptide showed unstable antimicrobial properties in the serum environment. In a 50% serum environment, the MIC of the parent peptide against Staphylococcus aureus CGMCC 1.490 increased threefold.

[0050] Table 10. MIC values ​​(µM) of antimicrobial peptides against Cronobacter sakazakii ATCC 12868 under different serum conditions. Table 11. MIC values ​​(µM) of antimicrobial peptides against Staphylococcus albus CGMCC 1.490 under different serum concentrations. 4.3 Enzyme stability While basic and hydrophobic amino acids endow antimicrobial peptides with high antibacterial activity, they also lead to the enzymatic degradation of these peptides, hindering their ability to replace antibiotics. Therefore, it is necessary to detect changes in the antimicrobial activity of antimicrobial peptides against representative strains under different enzymatic conditions. The results are shown in Tables 12 and 13. The MIC value of LR-24 did not change in the presence of enzymes such as pepsin.

[0051] Table 12. MIC values ​​(µM) of antimicrobial peptides against Cronobacter sakazakii ATCC 12868 under different enzyme treatments. Table 13. MIC values ​​(µM) of antimicrobial peptides against Staphylococcus albus CGMCC 1.490 under different enzyme treatments. 4.4 Thermal stability The results are shown in Tables 14 and 15. The MICs of the parent peptide and LR-24 did not change under various temperature treatments.

[0052] Table 14. MIC values ​​(µM) of antimicrobial peptides against Cronobacter sakazakii ATCC 12868 under different temperature treatments. Table 15. MIC values ​​(µM) of antimicrobial peptides against Staphylococcus albus CGMCC 1.490 under different enzyme treatments. 5. Bacterial growth curve determination Growth curves of *Cronobacter sakazakii* ATCC 12868 and *Staphylococcus albus* CGMCC 1.490 were observed using GG-28 and LR-24 at 1×MIC concentrations. Results are as follows: Figure 1 and Figure 2 As shown, the absorbance of the control group increased continuously over time, indicating that the bacteria in the control group grew well and multiplied rapidly within the detection period. Compared with the control group, the bacteria in the treatment group reached their absorbance peak within 5-10 minutes, and then gradually decreased from 10-45 minutes. No obvious logarithmic growth phase was observed in the treatment group throughout the monitoring period. This indicates that after a brief period of growth of 0-10 minutes, the bacteria were inhibited by GG-28 and LR-24, subsequently exhibiting growth arrest.

[0053] 6. Inhibits biofilm formation Biofilms are three-dimensional microbial aggregates embedded in a self-generating extracellular macromolecular matrix. This matrix promotes bacterial adhesion and enhances surface adhesion, thus contributing to microbial persistence. However, due to their inherent resistance to environmental stressors, these biofilms significantly compromise operational safety in the food and medical industries. Figure 3 As shown, GG-28 and LR-24 can inhibit the formation of biofilms from *Sakazakii* ATCC 12868. Within the concentration range of 0.5–16 µM, LR-24 showed significantly stronger biofilm inhibition than GG-28 (P<0.05). At a concentration of 2 µM (1×MIC), LR-24 inhibited biofilm formation by 49.98 ± 0.89%. The strong biofilm inhibitory activity of LR-24 provides a theoretical basis for its application in preventing bacterial adhesion and surface colonization in medical device coatings and food processing equipment.

[0054] Example 3 Combined drug susceptibility test I. Experimental Methods 1. Antibacterial activity assay of antibiotics The procedure was performed according to Example 2, Section 1, "Antibacterial Activity Assay".

[0055] 2. Antimicrobial peptide combined with antibiotic antimicrobial assay Based on the modified Pankey method, the standard chessboard method is used for identification.

[0056] (1) Bacterial culture and preparation of bacterial inoculum: Same as in Example 2, "1. Antibacterial activity determination"; (2) Drug preparation: In MHB medium, the antibiotic was diluted to twice the maximum detectable concentration, and the antimicrobial peptide to be tested was diluted to four times the maximum detectable concentration. The maximum detectable concentration for both the antibiotic and the antimicrobial peptide was 2 × MIC of the corresponding test bacteria. (3) Drug dilution: Antibiotic dilution: Add 50 µL of MHB medium to each well of the 96-well plate. Add 50 µL of the antibiotic solution prepared in (2) to wells A12-G12 in columns 11 and 12. Add an additional 50 µL of antibiotic solution to well H11. Mix the solution in column 11 thoroughly and aspirate 50 µL to column 10 for a 2-fold serial dilution. Continue this process until column 2 is reached. Aspirate 50 µL and discard the solution. At this point, the concentration of the antibiotic decreases by a factor of 2 along the X-axis from column 12 to column 2. Dilution of antimicrobial peptides: Add 50 µL of the antimicrobial peptide solution prepared in (2) to wells H1-H11 in row H, mix well, and add 50 µL to row G. Repeat this process to make a 2-fold serial dilution to row B. Discard 50 µL. At this time, the concentration of antimicrobial peptides decreases by 2-fold along the Y-axis from row H to row B. The concentration of antibiotics in each column of the diluted 96-well plate is the same, and the concentration of antimicrobial peptides in each row is the same. (4) Bacterial inoculation: Add 50 µL of the bacterial inoculation solution prepared in (1) (except for H12) to the 96-well plate. Add 100 µL of culture medium to the H12 well as a negative control. At this time, the A1 well of the 96-well plate does not contain antibiotics and antimicrobial peptides and serves as a positive control. After incubating the 96-well plate at 37℃ for 18-20 h, the negative control wells serve as the contamination group of the 96-well plate. If no turbidity is observed, the absorbance is measured at 600 nm using an ELISA reader. The concentration at which the solution in the wells does not show obvious turbidity is taken as the minimum inhibitory concentration when used in combination. This experiment was independently repeated three times.

[0057] (5) Results Analysis: The interaction between the tested drugs was evaluated by calculating the partial inhibitory concentration index (FIC index). The formula for calculating the FIC index is as follows: ; Evaluation criteria: FIC index ≤0.5, 0.5~4 and >4 represent synergistic effect, no interaction and antagonistic effect, respectively.

[0058] II. Test Results 1. Antibacterial activity The results are shown in Table 16. Ciprofloxacin, gentamicin, and cefixime showed good antibacterial effects, but were lower than LR-24.

[0059] Table 16. MIC values ​​(µM) of antimicrobial peptides and antibiotics against Cronobacter sakazakii ATCC 12868 2. Synergistic effect The results are shown in Table 17. Although there was no significant synergistic effect between LR-24 and antibiotics, the combined use (except with polymyxin B sulfate) could reduce the MIC concentrations of both antibiotics and LR-24 by 1-2 times.

[0060] Table 17 Synergistic effect of LR-24 in combination with antibiotics against Cronobacter sakazakii ATCC 12868 Example 4: Study on the mechanism of action of antimicrobial peptides I. Experimental Methods 1. Localization of FITC-labeled peptides FITC-labeled peptides and propidium iodide (PI) were used to observe the localization of peptides in bacteria and the disruption of bacterial membranes, respectively. Logarithmic-phase *Cronobacter sakazakii* ATCC 12868 was incubated with 2×MIC FITC-labeled peptides at 37°C for 1 h. After treatment, bacteria were collected by centrifugation and washed with PBS. PI (final concentration 10 μg / mL) was added to the bacterial suspension and incubated at 4°C for 15 min. Centrifugation and washing were repeated. After staining, the samples were placed on clean glass slides, covered, and incubated overnight at 4°C. Images of the samples were acquired at 488 nm and 535 nm wavelengths using a Nanoinsights Multi-SIM super-resolution microscope.

[0061] 2. LPS binding test The binding ability of LR-24 to LPS derived from *E. coli* was investigated using the BODIPY-TR-cadaverine fluorescent probe (BC). First, LPS and BC were dissolved in 50 mM Tris buffer to final concentrations of 50 μg / mL and 5 μg / mL, respectively. The solutions were incubated in a shaker in the dark for 4 h to allow LPS to bind to the BC dye. Then, following the method described in Example 2, "1. Antimicrobial Activity Assay," LR-24 was serially diluted in Tris solution into 96-well plates. The light-incubated LPS-BC was mixed with equal volumes of different concentrations of antimicrobial peptides in the 96-well plates and incubated at 37°C for 1 h. Finally, fluorescence intensity was detected at an excitation wavelength of 580 nm and an emission wavelength of 620 nm. A bacterial suspension treated with melitrix venom served as a control group.

[0062] 3. Outer membrane permeability test The effect of LR-24 on bacterial outer membrane permeability was investigated using propionic acid nitrate (NPN). First, *Cronobacter sakazakii* ATCC 12868 in the logarithmic growth phase was centrifuged at 5000×g for 5 min to collect the bacterial cells. The cells were then washed 2-3 times with 5 mM HEPES buffer (containing 5 mM glucose, pH=7.4), and the bacterial resuspended and adjusted to OD200. 600 =0.4. Subsequently, the diluted bacterial suspension and NPN (final concentration 10 μM) were incubated in a horizontal shaker at 37°C for 30 min under light-protected conditions. After incubation, equal volumes of LR-24 at different concentration gradients were added, and fluorescence values ​​were recorded using a fluorescence spectrophotometer (excitation wavelength 350 nm, emission wavelength 420 nm). The bacterial suspension treated with melitoxin served as the control group.

[0063] 4. Inner membrane integrity test The effect of LR-24 on intima integrity was investigated using PI. First, *Cronobacter sakazakii* ATCC 12868 in the logarithmic growth phase was collected by centrifugation (5000×g, 5 min). The cells were washed 2-3 times with 10 mM PBS buffer (pH=7.4), then resuspended and adjusted to OD. 600 =0.4. Subsequently, a final concentration of 200 nM PI dye was added to the diluted bacterial suspension for later use. LR-24 was serially diluted in 96-well plates using 10 mM PBS buffer, and the bacterial suspension containing PI dye was added to each well and incubated for 30 min. Finally, the fluorescence intensity was detected at an excitation wavelength of 535 nm and an emission wavelength of 615 nm. The bacterial suspension treated with melitoxin served as the control group.

[0064] 5. β-galactosidase activity test The leakage of intracellular β-galactosidase in bacteria after LR-24 treatment was investigated using 2-nitrobenzene-β-D-galactopyranoside (ONPG) to evaluate the degree of intracellular leakage. First, *Cronobacter sakazakii* ATCC12868 was grown to the logarithmic phase using MHB medium containing 2% lactose. The cells were collected by centrifugation (5000×g, 5 min), and then washed 2-3 times with 5 mM HEPES buffer (pH=7.4) containing 20 mM glucose and 1.5 mM ONPG, and resuspended to allow OD to reach the target level. 600 =0.2 for later use. LR-24 was serially diluted in 5 mM HEPES buffer in a 96-well plate, and the prepared bacterial suspension was added to the 96-well plate. The absorbance at 420 nm was monitored every 180 s from 0 s to 7000 s. The bacterial suspension treated with melitoxin served as the control group.

[0065] 6. Respiratory chain dehydrogenase inhibition test The effect of LR-24 on the activity of bacterial respiratory chain dehydrogenases was investigated using red tetrazoline (RT). First, *Cronobacter sakazakii* ATCC 12868 bacteria grown to the logarithmic growth phase were centrifuged (5000×g, 5 min), washed 2-3 times with Tris buffer (pH=8.6, 50 mM), and resuspended to allow OD to reach the target concentration. 600 =0.4 for later use. Then, 1 mg / mL red tetrazoline, 0.1 M glucose, and 50 mM Tris buffer (pH 8.6) were mixed in a 1:1:1 ratio. LR-24 was then serially diluted in 96-well plates, and an equal volume of the above mixture was added to each well. Finally, the bacterial suspension was added to each well in the same volume. The plates were incubated at 37°C for 90 min, and the absorbance was measured at 492 nm. The bacterial suspension treated with melittin served as the control group.

[0066] 7. Measurement of intracellular ROS accumulation The effect of LR-24 on intracellular ROS accumulation in bacteria was investigated using 2',7'-dichlorofluorescein diacetate (DCFH-DA). First, *Cronobacter sakazakii* ATCC 12868 in the logarithmic growth phase was collected by centrifugation (5000×g, 5 min). The cells were then washed 2-3 times with 10 mM PBS buffer (pH=7.4), and the bacterial resuspended and adjusted to OD24. 600=0.4. Subsequently, a final concentration of 200 μM DCFH-DA was added to the resuspended bacterial solution and incubated at 37 ℃ for 40 min. LR-24 was serially diluted in a 96-well plate, and then an equal volume of the bacterial suspension incubated with the fluorescent dye was added to each well. After incubation at 37 ℃ for 30 min, the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm was measured using a fluorescence spectrophotometer. The bacterial suspension treated with melitoxin served as the control group.

[0067] 8. Scanning electron microscopy observation To visually observe the effects of truncated peptides on bacterial morphology, scanning electron microscopy (SEM) was used to observe the cell membrane morphology of bacteria treated with antimicrobial peptides. Bacterial cultures were cultured to the second generation, centrifuged, and resuspended to obtain OD values. 600 =0.25, incubated with LR-24 for 1 h, discarded the supernatant and washed and centrifuged. Then, 1 mL of 2.5% glutaraldehyde was added and incubated for 12 h, followed by washing the bacterial sludge with 50%, 70%, 90%, and 100% ethanol solutions for 10 min each time. Subsequently, the bacterial sludge was treated with a mixture of ethanol and tert-butanol for 30 min. Finally, it was treated with pure tert-butanol for 1 h, dried, coated, and observed and photographed using a scanning electron microscope.

[0068] II. Test Results 1. Observation using a super-high resolution microscope The results are as follows Figure 4 As shown, LR-24-treated bacteria maintained their rod-like structure. The diffuse distribution of green fluorescence within the bacterial cells indicates that AMPs not only disrupted the cell membrane but may also penetrate the cytoplasm, potentially altering the intracellular microenvironment. Similarly, the bacterial cells exhibited strong red fluorescence, indicating that PI successfully crossed the ruptured cell membrane and inserted into intracellular DNA. This observation suggests a severe loss of membrane integrity. Although LR-24 induces a bacterium-like morphology in bacterial cells, it still causes significant disruption to the cell membrane structure. This facilitates the entry of exogenous small molecules LR-24 (MW = 2846.55 Da) and PI (MW = 668 Da) into the bacteria.

[0069] 2. LPS binding test The results, shown in Table 18, indicate that fluorescence intensity increased with increasing AMP concentration, suggesting that BC competitively substituted for the LPS binding site. This indicates a concentration-dependent interaction between LR-24 and LPS. After truncation, LR-24 retained sufficient net positive charge, which may be the material basis for promoting the efficient binding of the antimicrobial peptide to the negatively charged LPS.

[0070] Table 18 Results of the binding capacity determination of antimicrobial peptides at different concentrations with LPS 3. Outer membrane permeability test The results are shown in Table 19. LR-24 exhibited a significant concentration-dependent effect on bacterial outer membrane permeability. At a concentration of 1×MIC, LR-24 showed stronger membrane permeability than Melittin. LR-24 could bind to LPS and induce local curvature changes in the LPS layer, altering the bacterial outer membrane structure and permeability, ultimately leading to bacterial cell death.

[0071] Table 19 Results of the assay of outer membrane permeability of different concentrations of antimicrobial peptides against Cronobacter sakazakii ATCC 12868 4. Inner membrane integrity test The results, as shown in Table 20, indicate that the endometrial permeability increased with increasing peptide concentration (2–128 µM), demonstrating that the effect of antimicrobial peptides on bacterial endometrial integrity is concentration-dependent. Cationic AMPs enhance membrane permeability through their effective membrane-disrupting activity against bacterial cells. This result is consistent with the strong red light observed in super-resolution microscopy, confirming the high fidelity of the super-resolution microscopy images.

[0072] Table 20 Results of the assay of intimal permeability of different concentrations of antimicrobial peptides against Cronobacter sakazakii ATCC 12868 5. β-galactosidase leakage test The effect of LR-24 on β-galactosidase release is as follows: Figure 5 As shown, the absorbance value increased continuously and in a time-dependent manner throughout the detection period. The sustained increase in absorbance over time indicates significant leakage of β-galactosidase. This phenomenon suggests that the function of intracellular β-galactosidase leaked by antimicrobial peptides is time-dependent. Ultra-high resolution microscopy revealed that antimicrobial peptide treatment disrupted the bacterial cell membrane structure. Increased cell membrane permeability allowed not only small-molecule fluorescent dyes and antimicrobial peptides to pass through, but also β-galactosidase with a molecular weight of 130 kDa. These results indicate that LR-24 induces cell membrane permeability, leading to the disruption of fundamental physiological functions of the cell membrane.

[0073] 6. Respiratory chain dehydrogenase test The activity of respiratory chain dehydrogenases was determined by the reduction of lipid-soluble TTC to a red analogue. Since the electron transfer required for TTC reduction is impaired, the decrease in absorbance is directly related to dehydrogenase inhibition. The inhibitory effect of LR-24 on respiratory chain dehydrogenases of Cronobacter sakazakii ATCC12868 is shown in Table 21. At the tested concentrations, the inhibitory effect of LR-24 was significantly stronger than that of melittin.

[0074] Table 21. Results of respiratory chain dehydrogenase activity assay of Cronobacter sakazakii ATCC 12868 under different concentrations of antimicrobial peptide treatment. 7. ROS accumulation test The results are shown in Table 22. The fluorescence intensity increased with the increase of antimicrobial peptide concentration, indicating that LR-24 promotes the accumulation of intracellular ROS in a concentration-dependent manner.

[0075] Table 22 Results of ROS accumulation assay for Cronobacter sakazakii ATCC 12868 under different concentrations of antimicrobial peptide treatment 8. Scanning electron microscopy observation While fluorescence dyeing and reduction detection alone can reveal the effects of LR-24 on bacterial membrane disruption and content leakage, they are insufficient to provide a direct understanding of the extent of bacterial damage after antimicrobial peptide treatment. To more directly observe the effects of antimicrobial peptides on bacteria, scanning electron microscopy was used to examine untreated and antimicrobial peptide-treated Cronobacter sakazakii ATCC 12868 samples. Figure 6 As shown in the scanning electron microscope (SEM) images, the control group had a rough surface, a clear outer membrane structure, and was generally full. The treated group's bacterial membrane showed a few pores. The LR-24-treated bacteria had wrinkled and sunken surfaces, were more severely adhered, and appeared shriveled, but still maintained an overall rod-like structure.

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antimicrobial peptide LR-24, characterized in that, The amino acid sequence of the antimicrobial peptide LR-24 is shown in SEQ ID NO.

2.

2. The application of the antimicrobial peptide LR-24 according to claim 1 in the preparation of antimicrobial drugs, characterized in that, The antibacterial drug has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria; The Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis; The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

3. An antibacterial drug, characterized in that, The antibacterial drug uses the antimicrobial peptide LR-24 as the active ingredient as described in claim 1.

4. The use of the antimicrobial peptide LR-24 according to claim 1 in the combined preparation of an antimicrobial composition with an antibiotic, characterized in that, The antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime; The antibacterial composition has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria; The Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis; The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

5. An antibacterial composition, characterized in that, The antibacterial composition uses the antimicrobial peptide LR-24 as described in claim 1 and an antibiotic as active ingredients.

6. The antibacterial composition according to claim 5, characterized in that, The antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

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