A broad-spectrum antibacterial and low-toxicity antimicrobial peptide WP-4, WP-6, and their combinations and applications

Abstract

This invention discloses broad-spectrum antibacterial peptides WP-4 and WP-6, their compositions, and applications, belonging to the fields of antimicrobial peptide technology and biomedicine. This invention obtains a series of antimicrobial peptides by truncating and rationally modifying template peptides to balance the relationship between charge number, hydrophobicity, and amphiphilicity. By measuring antibacterial activity, stability, hemolytic activity, and cytotoxicity, two novel antimicrobial peptides, WP-4 and WP-6, with high activity and low toxicity were obtained, and their amino acid sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 7. The novel antimicrobial peptides obtained by this invention possess broad-spectrum antibacterial activity, good stability under temperature, pH, salt ion, pepsin, and serum conditions, and exhibit no significant hemolytic activity and low cytotoxicity. They are characterized by rapid bactericidal action and low likelihood of inducing drug resistance. Furthermore, the antimicrobial peptides of this invention can enter cells and have a strong inhibitory effect on intracellular bacteria, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of antimicrobial peptide technology and biomedicine, specifically relating to broad-spectrum antimicrobial and low-toxicity antimicrobial peptides WP-4 and WP-6, their combinations and applications. Background Technology

[0002] The long-term, irregular use of antibiotics has led to an increasing number of antibiotic resistance cases, seriously threatening global public health. Therefore, there is an urgent need to develop promising antibiotic alternatives to address the challenges posed by unavoidable antibiotic resistance. Antimicrobial peptides primarily act on the bacterial cell membrane; this unique antibacterial mechanism makes them highly promising in preventing and controlling infections caused by drug-resistant bacteria, and they are considered one of the most promising antibiotic alternatives. However, the poor stability and low in vivo efficacy of many primary or natural antimicrobial peptides severely hinder their clinical application.

[0003] Based on the properties of antimicrobial peptides, template antimicrobial peptides were truncated and rationally modified to balance the relationship between charge number, hydrophobicity and amphiphilicity, resulting in two novel antimicrobial peptides with broad application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide two antimicrobial peptides, WP-4 and WP-6, which have broad-spectrum antibacterial activity, low toxicity, and are not prone to inducing drug resistance, as well as their combinations and applications.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides novel antimicrobial peptides WP-4 and WP-6, which possess broad-spectrum antibacterial activity, low toxicity, and are less prone to inducing drug resistance. These peptides are obtained by optimizing the charge number, hydrophobicity, and amphiphilicity of the non-natural antimicrobial peptide P8 (amino acid sequence at positions 29-40) through point mutation. The amino acid sequence of P8 is shown in SEQ ID NO.1, and the amino acid sequence of WP-1 is shown in SEQ ID NO.2. The preparation method employs solid-phase chemical synthesis to synthesize WP-4 and WP-6, achieving a purity of ≥95%. The antimicrobial peptides are either WP-4 or WP-6, wherein the amino acid sequence of WP-4 is shown in SEQ ID NO.5, and the amino acid sequence of WP-6 is shown in SEQ ID NO.7.

[0006] The present invention also proposes the application of the aforementioned antimicrobial peptide in the preparation of antibacterial drugs.

[0007] Furthermore, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria are selected from one or more of the following: Escherichia coli ATCC25922, Escherichia coli ETEC7, Escherichia coli EPEC28, Escherichia coli PCN033, Salmonella ATCC14028, Salmonella C79-13, Actinobacillus pleuropneumoniae 4074, and Pasteurella 9261. The Gram-positive bacteria are selected from one or more of Staphylococcus aureus ATCC29213, Staphylococcus aureus 1213M4A, Streptococcus suis SC19, Streptococcus suis 0810, Streptococcus suis LXJ, and Streptococcus suis HN105.

[0008] Furthermore, the drug is an anti-intracellular bacterial drug, and the intracellular bacterial bacteria is Salmonella Typhimurium ATCC14028.

[0009] The present invention also proposes an antimicrobial composition comprising the aforementioned antimicrobial peptide, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0010] Furthermore, the composition is an oral formulation, an injectable formulation, or a topical formulation.

[0011] The present invention also proposes the application of the aforementioned antimicrobial peptide in the preparation of feed additives, which are used to inhibit pathogenic bacteria in and outside farmed animals.

[0012] Furthermore, the pathogenic bacteria include Gram-negative and / or Gram-positive bacteria of farmed animal origin, as well as intracellular parasites.

[0013] The present invention also proposes a feed additive comprising the antimicrobial peptide as described in claim 1.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention uses the non-natural antimicrobial peptide P8 as a template and optimizes it through truncation and point mutation to obtain antimicrobial peptides WP-4 and WP-6. These antimicrobial peptides exhibit significant broad-spectrum antibacterial activity against Gram-negative bacteria such as Escherichia coli, Salmonella, Actinobacillus pleuropneumoniae, and Pasteurella multocida, as well as Gram-positive bacteria such as Staphylococcus aureus and Streptococcus suis. At the same time, they have excellent environmental stability, can tolerate different temperatures, acid and alkaline environments, and physiological salt ion systems, and have good tolerance to pepsin enzymatic hydrolysis and serum environment, thus solving the problems of easy degradation and poor stability of traditional antimicrobial peptides.

[0015] In addition, this antimicrobial peptide has extremely low hemolytic activity, low toxicity to mammalian cells, excellent biosafety, and time-dependent rapid bactericidal properties. It is also less likely to induce bacterial resistance, overcoming the shortcomings of traditional antibiotics that are prone to outbreaks of resistance. At the same time, it can effectively penetrate mammalian cell membranes and exert a significant inhibitory effect on intracellular parasites, filling the technological gap that conventional antimicrobial agents cannot act on intracellular bacteria.

[0016] The antimicrobial peptide of this invention combines the advantages of broad-spectrum antibacterial activity, high stability, low toxicity, resistance to drug resistance, and intracellular antibacterial activity, providing a new and high-quality candidate for the prevention and control of pathogenic bacteria in aquaculture and the development of antimicrobial drugs in the pharmaceutical field, with broad application prospects. Attached Figure Description

[0017] Figure 1 This is the secondary structure analysis of the template peptide P8, which has an α-helix structure at the C-terminus; Figure 2 It is the α-helix projection of antimicrobial peptides WP-1 to WP-7; Figure 3 This involves the secondary structure analysis of antimicrobial peptides WP-1 to WP-7; Figure 4 It is the hemolytic activity of the antimicrobial peptides WP-2~WP-7; Figure 5 This is an analysis of the toxicity of antimicrobial peptides WP-2~WP-7 to eukaryotic cells, where A is mouse macrophage RAW264.7 cells and B is African green monkey kidney cells Vero. Figure 6 The images show the time-kill curves of antimicrobial peptides WP-4 and WP-6 against two types of Escherichia coli, where A represents standard Escherichia coli ATCC25922 and B represents multidrug-resistant Escherichia coli PCN033. Figure 7 This is an evaluation of the induced resistance of antimicrobial peptides WP-4 and WP-6; Figure 8 It refers to the cell penetration ability of antimicrobial peptides WP-4 and WP-6; Figure 9 The antimicrobial activities of antimicrobial peptides WP-4 and WP-6 are shown, where A represents the antimicrobial activity of WP-4 and B represents the antimicrobial activity of WP-6. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0019] Example 1: Modification and Physicochemical Properties of Antimicrobial Peptides 1. Acquisition of template and initial peptide A random peptide library was screened using a bacterial surface display system, yielding the non-natural antimicrobial peptide P8, with the amino acid sequence HSYARVRPSCNHKAGTQVAFRMGLGLKSLAVLADERRFSA (SEQ ID NO.1). This antimicrobial peptide consists of 40 amino acids, and analysis revealed that its C-terminus is an α-helix structure (e.g., ...). Figure 1 (As shown).

[0020] Using the non-natural antimicrobial peptide P8 as a template, amino acids 29-40 at the C-terminus of the P8 antimicrobial peptide were extracted to obtain the antimicrobial peptide WP-1 (SEQ ID NO.2).

[0021] 2. Mutant Construction The amino acid sequence of the antimicrobial peptide WP-1 (SEQ ID NO.2) was modified by point mutation to optimize the charge number, hydrophobicity and amphiphilicity of the antimicrobial peptide. The sequence and properties of the antimicrobial peptide and its mutants are shown in Table 1, and the antimicrobial peptide mutants WP-2~WP-7 (SEQ ID NO.3~8) were obtained.

[0022] 3. Physicochemical properties and structural analysis The charge number, hydrophobicity, and amphiphilicity of the antimicrobial peptide WP-1 and its mutants WP-2 to WP-7 were determined, as shown in Table 1. Their α-helix projection and secondary structure analysis are as follows: Figure 2 and Figure 3 As shown.

[0023] Table 1. Sequences and physicochemical properties of antimicrobial peptide mutants As shown in Table 1, WP-1 has a charge of 0 and its hydrophobicity and amphiphilicity are significantly lower than those of the other mutants; WP-2 to WP-5 all have a charge of +6, and WP-6 to WP-7 have a charge of +5; WP-3 has the highest amphiphilicity among all mutants (0.948), and WP-6 has the highest hydrophobicity (0.726).

[0024] like Figure 2 (Projection diagram of α-helices of WP-1~WP-7) As a whole, WP-1 has no basis for antibacterial activity due to its dispersed charge and lack of clear amphiphilic partitions; after point mutation, WP-2~WP-7 all form amphiphilic α-helices, with positively charged residues (R / K) concentrated on one hydrophilic side and hydrophobic residues (L / V / W / F / I) concentrated on the other hydrophobic side.

[0025] Example 2: Determination of the antimicrobial activity of antimicrobial peptides 1. Preparation of antimicrobial peptides The antimicrobial peptides WP-1 to WP-7 were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. using solid-phase chemical synthesis method, with a purity of ≥95%.

[0026] 2. Measurement Method The minimum inhibitory concentrations (MICs, μg / mL) of antimicrobial peptides against a variety of Gram-negative and Gram-positive bacteria were determined using the micro-broth 2-fold dilution method. The specific steps are as follows: (1) The pathogen to be tested was revived on agar medium and cultured overnight in a constant temperature incubator at 37℃. After three consecutive subcultures, it was ready for use.

[0027] (2) Use an inoculation loop to scrape fresh colonies from the agar medium into a McFarland turbidity tube containing 4.5 mL of physiological saline, and calibrate to a McFarland turbidity standard of 0.5. At this point, the bacterial count is approximately 1 × 10⁻⁶. 8 CFU / mL. Then dilute 100-fold with MH liquid medium, at which point the bacterial count is approximately 1×10⁻⁶. 6 CFU / mL, for later use.

[0028] (3) Add 200 μL of pre-prepared antibiotic or antimicrobial peptide solution to column 1 of a sterile 96-well plate, and 100 μL of LMH liquid culture medium to columns 2 to 10. Use a pipette to transfer 100 μL of antibiotic or antimicrobial peptide solution from column 1 to column 2, mix well, and then transfer 100 μL of antibiotic or antimicrobial peptide solution from column 2 to column 3, and so on. Discard 100 μL of antibiotic or antimicrobial peptide solution from column 10. Add 200 μL of pathogenic bacterial suspension (positive control) to column 11, and add 200 μL of LMH liquid culture medium (blank control) to column 12.

[0029] (4) Add 100 μL of the prepared pathogenic bacterial solution to each of columns 1 to 10, and shake to mix. At this point, the bacterial solution concentration in each well is approximately 5 × 10⁻⁶. 5 CFU / mL. After inoculating the 96-well plate, the bacterial growth was observed after culturing it in a 37℃ incubator for 18 hours.

[0030] (5) Result interpretation criteria: The concentration of the first clearing well in ascending order of antibiotic or antimicrobial peptide solution concentration is the minimum inhibitory concentration (MICs).

[0031] 3. Measurement Results The MICs of antimicrobial peptides WP-1 to WP-7 are shown in Table 2. WP-1 showed no significant antimicrobial activity against any of the tested strains (MICs were all >100 μg / mL). Except for antimicrobial peptide WP-1, antimicrobial peptides WP-2 to WP-7 showed strong antimicrobial activity against both Gram-negative and Gram-positive bacteria, and the inhibitory effects on different strains varied.

[0032] Among Gram-negative bacteria, Pasteurella multocida 9261 was highly sensitive to all WP-2 to WP-7 strains (MICs = 0.78 μg / mL); Escherichia coli ETEC and EPEC were even more sensitive to WP-6 and WP-7 strains (MICs as low as 1.56 μg / mL). Among Gram-positive bacteria, Streptococcus suis strains exhibit relatively high resistance (some MICs reach 50~100 μg / mL), while Staphylococcus aureus MICs against WP-2~WP-7 are mostly around 12.5 μg / mL.

[0033] Table 2. Antimicrobial activity of antimicrobial peptides (MICs, μg / mL) Example 3: Stability determination of antimicrobial peptides 1. Processing Condition Design To simulate various environments in the actual application of antimicrobial peptides, the following conditions were set for temperature, pH, enzymatic hydrolysis, serum, and salt ion treatment: (1) Dispense the prepared antimicrobial peptide solution into EP tubes and treat them at different temperatures (20℃, 40℃, 60℃, 80℃ and 100℃) for 30 min; (2) Dispense the prepared antimicrobial peptide solution into EP tubes and incubate for 2 hours under different pH conditions (2, 4, 6, 8, 10 and 12). After incubation, adjust to the initial pH. (3) In order to evaluate the pepsin stability of the antimicrobial peptide, the prepared antimicrobial peptide solution was dispensed into EP tubes, and the antimicrobial peptide was mixed with 100 μg / mL pepsin (dissolved in sterile water, pH=2.0), incubated at 37°C for 2 h, and the pH of the solution was adjusted to 8.0 to terminate the reaction. (4) Dispense the prepared antimicrobial peptide solution into EP tubes, and incubate the antimicrobial peptide with 50% serum at 37°C for 2h, 4h, and 8h. (5) In order to evaluate the effect of salt ions on the stability of antimicrobial peptides, the prepared antimicrobial peptide solutions were dispensed into EP tubes, and physiological concentrations of salt ions were added to MHB medium. The final concentrations of salt ions were: 150 mM NaCl, 4.5 mM KCl, 6 μM NH4Cl, 8 μM ZnCl2, 1 mM MgCl2, 2.5 mM CaCl2 and 4 μM FeCl3.

[0034] Meanwhile, a control group was set up. The treatment conditions for the control group were: 37℃, physiological pH (usually pH 7.2~7.4), no enzymes, and no additional treatment.

[0035] 2. Measurement Method After the above-mentioned experimental and control groups were treated, the MICs of the antimicrobial peptides against Escherichia coli ATCC25922 were determined according to the micro-broth 2-fold dilution method in Example 2 to evaluate their stability.

[0036] 3. Measurement Results The stability results of the antimicrobial peptides are shown in Table 3. WP-1 showed no antimicrobial activity under all treatment conditions (MICs>100μg / mL). Except for antimicrobial peptide WP-1, antimicrobial peptides WP-2~WP-7 maintained good antimicrobial activity after treatment with different temperatures, different pH values, pepsin and 50% serum, as well as in different salt ion environments.

[0037] In the temperature, pH (2-8), and pepsin treatment groups, the MICs of WP-2~WP-7 antimicrobial peptides against Escherichia coli ATCC25922 were completely consistent with those of the blank control group (37℃, pH 7.4, no enzyme incubation) without any treatment, indicating that the antimicrobial peptides of this series are structurally stable and have no loss of antimicrobial activity under the above conditions, and have excellent stability.

[0038] In the high pH (10, 12) treatment groups, the MICs of some antimicrobial peptides increased slightly (e.g., WP-3 had MICs of 12.5 μg / mL at pH=10 and 12, and WP-4 had MICs of 12.5 μg / mL at pH=10).

[0039] In the 50% serum treatment group, the MICs of WP-5 increased to 50 μg / mL, and the MICs of WP-6 increased from 6.25 μg / mL to 12.5 μg / mL after 8 h of incubation. The MICs of other antimicrobial peptides remained below 25 μg / mL. In the salt ion environment treatment group, NaCl significantly inhibited WP-4 and WP-5 (MICs>100μg / mL), CaCl2 increased the MICs of most antimicrobial peptides to 25μg / mL, and the remaining salt ions had no significant effect on the antimicrobial activity of WP-2~WP-7.

[0040] Table 3. Stability results of antimicrobial peptides Example 4: Assay of hemolytic activity and cytotoxicity of antimicrobial peptides This section assesses the biosafety of antimicrobial peptides by measuring their hemolytic activity on mammalian erythrocytes and their toxicity to cultured mammalian cells in vitro, and calculates the selection index (SI) to screen for the best candidate peptides. The positive control is the antimicrobial peptide Melittin (an antimicrobial peptide with strong hemolytic and cytotoxic properties).

[0041] 1. Hemolytic activity assay This experiment used sterile defibrinated sheep erythrocytes to determine the hemolytic activity of antimicrobial peptides.

[0042] 1.1 Determination Method (1) Red blood cell preparation: Collect sheep red blood cells and wash them with PBS until the supernatant becomes colorless and transparent. Then dilute the sheep red blood cells with PBS to an 8% (v / v) red blood cell suspension for later use.

[0043] (2) 96-well plate loading Test peptide group: Add a series of concentration gradients of WP-2 to WP-7 antimicrobial peptide solutions to sterile 96-well plates; Positive drug control group: One column of wells was set up simultaneously, and Melittin (antimicrobial peptide) solution with the same concentration gradient was added as a toxicity reference standard; Positive control for the system: Set up one column of wells and add 0.1% Triton X-100 solution as a 100% hemolysis standard; Negative control: Set up one column of wells and add an equal volume of PBS solution as a background control for 0% hemolysis; An equal volume of 8% (v / v) red blood cell suspension was added to all wells to ensure that the final volume of each well was consistent.

[0044] (3) Incubation and detection After incubation at 37°C for 1 hour, the supernatant was centrifuged at 1000×g for 5 minutes at 4°C. The supernatant was then transferred to a new 96-well plate (flat bottom), and the absorbance (A) at 540 nm was measured using an ELISA reader. The hemolysis rate was then calculated.

[0045] Hemolysis rate (%) = [(A) peptide –A negative ) / (A positive –A negative ]×100%; Among them, A peptide A positive and A negative The values ​​represent the absorbance at 540 nm for the antimicrobial peptide sample or Melittin sample, the positive control, and the negative control, respectively.

[0046] The definition of minimum hemolytic concentration (MHC) is the minimum concentration of antimicrobial peptide required to cause hemolysis in 10% of sheep erythrocytes.

[0047] 1.2 Measurement Results Results of hemolytic activity of antimicrobial peptides WP-2~WP-7 are as follows Figure 4As shown, the hemolysis rate in the 0.1% Triton X-100 positive control group was 100%, while the hemolysis rate in the PBS negative control group was close to 0%, proving the reliability of the experimental system. In the positive drug control, the antimicrobial peptide Melittin exhibited extremely strong hemolytic activity, with a hemolysis rate exceeding 10% at 6.25 μg / mL, and an MHC concentration of only 6.25 μg / mL. In contrast, the hemolytic activity of the antimicrobial peptides of this invention, from WP-2 to WP-7, was significantly lower than that of Melittin overall. Only WP-3 showed mild bleeding at high concentrations, while the hemolysis rates of the remaining peptides were all below 10% at high concentrations, and their MHC concentrations were much higher than those of Melittin, demonstrating excellent biocompatibility.

[0048] 2. Cytotoxicity assay The cytotoxicity of mammalian cells in this experiment was determined using the CCK-8 assay on mouse macrophage RAW264.7 cells and African green monkey kidney cells (Vero cells).

[0049] 2.1 Determination Method (1) Cell plating culture Mouse macrophages RAW264.7 and African green monkey kidney cells Vero were seeded in sterile 96-well plates and cultured in a 37°C, 5% CO2 incubator for 24 hours until the cells adhered and reached 80%–90% confluence.

[0050] (2) Sample addition treatment Test peptide group: Add a series of concentration gradients of WP-2 to WP-7 antimicrobial peptide solutions to the corresponding wells; Positive drug control group: A series of wells containing antimicrobial peptide (Melittin) solutions with concentration gradients (completely consistent with the concentration gradient of the test peptide) were set up simultaneously as a reference standard for cytotoxicity assessment; Positive control group: An equal volume of cell culture medium was added, without any peptides, as the baseline for normal cell growth (100% survival rate). Negative control group: Only an equal volume of cell culture medium was added, without cell seeding or the addition of any peptides, serving as a background control for absorbance.

[0051] All wells were replenished with cell culture medium until the final volume was consistent, and then incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0052] (3) Color development and detection Then, 10 μL of LCCK-8 solution was added to each well, gently shaken to mix, and incubated at 37°C for 2 hours. The absorbance (A) of each well at 450 nm was measured using a microplate reader, and the cell viability was calculated.

[0053] Cell viability (%) = [(A) peptide –A negative ) / (Apositive –A negative ]×100%; Among them, A peptide A positive and A negative The values ​​represent the absorbance at 450 nm for the antimicrobial peptide sample or Melittin sample, the positive control, and the negative control, respectively.

[0054] 2.3 Measurement Results Results of hemolytic activity of antimicrobial peptides WP-2~WP-7 are as follows Figure 5 As shown, the cell viability in the positive control group was close to 100%, and the absorbance in the negative control wells was close to 0, proving the reliability of the cell culture system and detection method. In the positive drug control group, the antimicrobial peptide Melittin exhibited extremely strong cytotoxicity, causing over 50% death in RAW264.7 and Vero cells at a concentration of 12.5 μg / mL, demonstrating significant damage to mammalian cells. The cytotoxicity of the antimicrobial peptides WP-2~WP-7 of this invention showed a clear concentration-dependent effect; with increasing antimicrobial peptide concentration, the cytotoxicity to RAW264.7 and Vero cells increased slightly, but at the same concentration, their cytotoxicity was far lower than that of Melittin. Within the effective antimicrobial concentration range, WP-2~WP-7 had no significant effect on the survival rate of either mammalian cell type, demonstrating excellent cell safety.

[0055] 3. Selection Index (SI) Calculation and Optimal Peptide Screening To screen for the antimicrobial peptides with the best therapeutic index, the selectivity index (SI) of antimicrobial peptides WP-2 to WP-7 on three cell types was calculated. The higher the selectivity index, the stronger the antimicrobial specificity and the better the biosafety of the antimicrobial peptide. The results are shown in Table 4. WP-4 and WP-6 have the highest selectivity index and are the best candidate antimicrobial peptides.

[0056] Table 4 Selection Index of Antimicrobial Peptides Note: a) MIC GM The geometric mean (GM) represents the minimum inhibitory concentration (MIC) of the antimicrobial peptide against different bacterial strains. When there is no antimicrobial activity at a concentration of 100 μg / mL, the geometric mean is calculated using 200 μg / mL. + Represents Gram-positive bacteria, G - MIC represents Gram-negative bacteria, and "All" represents all bacteria. It is used to measure the overall antimicrobial activity of antimicrobial peptides. GM The lower the value, the stronger the antibacterial activity.

[0057] (b) MHC represents the minimum concentration of antimicrobial peptides that can cause hemolysis of more than 10% of sheep erythrocytes. It is used to measure erythrocyte toxicity; a higher MHC value indicates a lower risk of hemolysis and better safety.

[0058] c) The lowest concentration of antimicrobial peptide that induces death in more than 50% of eukaryotic cells (Vero / RAW264.7). Measuring mammalian cytotoxicity, IC50. 50 The higher the value, the lower the cytotoxicity and the better the safety.

[0059] d) Selection Index (SI) based on MHC / MIC GM or IC50 / MIC GM This indicates that the red blood cell SI = MHC / MIC GM Cell SI=IC 50 / MIC GM , of which MIC GM The SI (Inhibitory MIC) value is calculated using the antimicrobial peptide against all bacteria. A higher SI value indicates stronger antimicrobial specificity, meaning stronger bactericidal ability and lower toxicity to the host, and greater potential for drug development.

[0060] e)SI GM This represents the geometric mean of SI (Self-Sensitive Ingredient), specifically the geometric mean of SI in erythrocytes, Vero cells, and RAW264.7 cells. It comprehensively evaluates the overall safety-activity balance of antimicrobial peptides; a higher value indicates better overall performance.

[0061] The results show that the WP-6 has the best overall performance, and its MIC... GM (All) was 11.89 μg / mL, MHC > 200 μg / mL, and the IC50 of Vero / RAW264.7 cells was 11.89 μg / mL. 50 The SI values ​​were 162.80 μg / mL and 153.10 μg / mL, respectively. The SI values ​​for erythrocytes, Vero cells, and RAW264.7 cells were 33.64, 13.69, and 12.88, respectively. GM Up to 18.10; WP-4 is the second best candidate peptide, SI GM The value was 15.32; WP-3 had a high risk of hemolysis, and WP-2 had weak antibacterial activity and poor cell safety, resulting in poor overall performance.

[0062] Example 5: Determination of time-based sterilization curve The purpose of this embodiment is to determine the bactericidal rate and bactericidal time-dependent effect of the optimal candidate antimicrobial peptides WP-4 and WP-6, and to clarify the time-dependent characteristics of their bactericidal activity using the antimicrobial drug colistin as a control.

[0063] 1. Experimental Methods Overnight cultures of the target bacteria (Escherichia coli ATCC25922 and Escherichia coli PCN033) were transferred to blank MHB medium at a ratio of 1:100. After reaching the logarithmic growth phase, the bacterial concentration was adjusted to approximately 1×10⁻⁶. 6 CFU / mL, for later use.

[0064] The bacterial suspensions were divided into four groups: a blank control group (MHB medium + bacterial suspension only), a WP-4 group, a WP-6 group, and a colistin positive control group. An equal volume of antimicrobial peptide or colistin solution was mixed with the bacterial suspensions to achieve a final concentration of 4×MIC for both the antimicrobial peptide and colistin, and a final bacterial suspension concentration of 5×10⁻⁶. 5 CFU / mL.

[0065] All treatment groups were continuously incubated at 37℃ and 180 rpm in a constant temperature shaker for 12 h. During the incubation period, 500 μL of bacterial suspension was taken at eight time points: 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 12 h. The suspension was serially diluted 10-fold with sterile physiological saline, and 100 μL of the diluted solution was spread onto MHA agar medium and incubated overnight at 37℃. Colony counts were then performed. Each experimental group was set up in triplicate. The bactericidal activity of different drug treatment groups was represented by calculating and comparing the colony counts among the different treatment groups.

[0066] 2. Experimental Results The results are as follows Figure 6 As shown, both WP-4 and WP-6 exhibited time-dependent bactericidal activity, with significant bactericidal efficiency against Escherichia coli ATCC25922 and Escherichia coli PCN033. They could completely kill Escherichia coli ATCC25922 and PCN033 within 4 hours of incubation.

[0067] Example 6: Evaluation of Antimicrobial Peptide-Induced Drug Resistance The purpose of this embodiment is to evaluate the drug resistance potential of WP-4 and WP-6 through continuous subculture induction experiments, using the antibiotic ciprofloxacin as a positive control, and to clarify their advantage of not easily inducing bacterial resistance.

[0068] 1. Experimental Methods Escherichia coli ATCC25922 was cultured to the logarithmic growth phase and diluted with MHB medium to a concentration of 0.5-1×10⁻⁶. 6 CFU / mL, for later use.

[0069] The diluted bacterial culture was mixed with WP-4 or WP-6 at a concentration of 1 / 2×MIC and incubated for 12 h. The culture was then passaged 30 times consecutively under the same bacterial concentration, drug concentration, and incubation conditions. Ciprofloxacin at a concentration of 1 / 2×MIC was used as a positive control, and induction was performed simultaneously through continuous passages.

[0070] During the passage process, the MIC values ​​of WP-4 and WP-6 and the MIC value of ciprofloxacin against Escherichia coli ATCC25922 were measured every two generations. Based on the obtained MIC values, resistance change curves were plotted to analyze the resistance induction trend.

[0071] 2. Experimental Results The results are as follows Figure 7 As shown, after 30 consecutive passages at a concentration of 1 / 2×MIC, the MIC value of ciprofloxacin against Escherichia coli ATCC25922 increased by 256-fold, indicating that the bacteria rapidly developed high resistance. In contrast, the MIC values ​​of WP-4 and WP-6 against Escherichia coli ATCC25922 only increased by 2-fold and 4-fold, respectively, far lower than the increase in resistance to ciprofloxacin, demonstrating that WP-4 and WP-6 are less likely to induce bacterial resistance and possess advantages for long-term application against drug resistance.

[0072] Example 7: Determination of the cell penetration ability of antimicrobial peptides The purpose of this embodiment is to verify whether WP-4 and WP-6 can penetrate mammalian cell membranes and enter the cell, providing a structural and functional basis for subsequent evaluation of their anti-intracellular bacterial activity.

[0073] 1. Experimental Methods Mouse macrophages RAW264.7 were seeded into 12-well plates pre-filled with cell spreaders and cultured in a 37°C, 5% CO2 cell culture incubator for about 24 hours until the cells adhered and grew.

[0074] FITC-labeled WP-4 and WP-6 at a final concentration of 25 μg / mL were added to 12-well plates and incubated in the dark (37℃, 5% CO2) for 4 h. RAW264.7 cells without antimicrobial peptide treatment were set as negative controls.

[0075] After incubation, perform cell staining according to the following steps, ensuring the entire process is conducted in the dark: (1) The cells were washed three times with sterile PBS to remove unbound FITC-labeled antimicrobial peptides WP-4 and WP-6.

[0076] (2) Prepare the dye according to the instructions of the red fluorescent staining kit (Dil) for cell membranes of Shanghai Beyotime Biotechnology Co., Ltd. Add 500 μL of dye to each well and incubate in the dark for 15 min to label the cell membrane with red fluorescence.

[0077] (3) The cells were washed twice with sterile PBS to remove excess Dil dye, and then fixed with 4% paraformaldehyde for 15 min.

[0078] (4) Remove paraformaldehyde, wash cells twice with sterile PBS, add DAPI (10 μg / mL) staining solution, incubate at 37℃ for 10 min, and label cell nuclei with blue fluorescence.

[0079] (5) Discard the DAPI staining solution, wash 3 times with PBS, take out the cell smear, fix it on a glass slide, observe and record the intracellular fluorescence distribution using a laser confocal scanning microscope, and analyze the localization of FITC-labeled WP-4 and WP-6.

[0080] 2. Experimental Results The results are as follows Figure 8 As shown, under laser confocal microscopy, DAPI fluoresces blue when it binds to the cell nucleus, Dil fluoresces red when it binds to the cell membrane, and FITC-labeled WP-4 and WP-6 fluoresce green. After co-incubation with RAW264.7 cells, significant green fluorescence was detected in both the cytoplasm and nucleus, while the negative control cells showed no green fluorescence, indicating that WP-4 and WP-6 can effectively penetrate mammalian cell membranes and enter the cell to exert their effects.

[0081] Example 8: Determination of the anti-intracellular bacterial activity of antimicrobial peptides The purpose of this embodiment is to evaluate the inhibitory activity of WP-4 and WP-6 against intracellular parasites (Salmonella typhimurium ATCC14028) based on their cell penetration ability.

[0082] 1. Experimental Methods (1) Preparation of bacterial culture: A single colony of Salmonella Typhimurium ATCC14028 was picked and cultured overnight at 37°C and 180 r / min in LB medium. The next day, it was transferred to fresh LB medium at a ratio of 1:100 and cultured until the logarithmic growth phase. After centrifugation at 6000 r / min for 5 min, the culture was washed three times with PBS and finally resuspended in DMEM medium. The bacterial concentration was adjusted to 1×10⁻⁶. 7 CFU / mL, bacterial suspension should be prepared and used immediately.

[0083] (2) Preparation of antimicrobial peptides: Using DMEM medium, antimicrobial peptides WP-4 and WP-6 were diluted to three concentration gradients of 6.25 μg / mL, 12.5 μg / mL and 25 μg / mL, respectively, for later use.

[0084] (3) Infected cells: RAW264.7 cells were seeded in 24-well plates and cultured for 24 hours. The supernatant in the 24-well plates was slowly aspirated, and Salmonella Typhimurium culture was added at an MOI of 10 (bacteria:cells = 10:1). The 24-well plates were then placed in a carbon dioxide incubator (37°C, 5% CO2) and incubated for 1 hour to allow the bacteria to infect the cells.

[0085] Subsequently, the cells were washed twice with sterile PBS to remove any unattached extracellular bacteria. Gentamicin at a concentration of 100 μg / mL was added to each well of a 24-well plate, and the plates were incubated for 1 hour to kill any extracellular bacteria that had not yet entered the cells. The 24-well plates were then thoroughly washed three times with sterile PBS to remove any residual gentamicin and bacterial debris. The washing process was gentle, avoiding blowing up the cells at the bottom of the plate.

[0086] (4) Treatment of cells with antimicrobial peptides: Add different concentrations of WP-4 and WP-6 solutions to 24-well plates and incubate at 37°C and 5% CO2 for 4 hours. Set up infected cells with only DMEM medium as negative control.

[0087] (5) Cell lysis and intracellular bacterial count: After incubation, the 24-well plate was thoroughly washed twice with sterile PBS, and then 0.5% Triton X-100 was added to lyse the cells for 10 min to release intracellular bacteria. The lysate was serially diluted 10-fold with sterile physiological saline, and the diluted solution was spread on LB agar medium and incubated overnight at 37°C. After incubation, colony counts were performed to determine the number of viable intracellular bacteria.

[0088] 2. Experimental Results The results are as follows Figure 9 As shown, the inhibitory activity of WP-4 and WP-6 against intracellular Salmonella Typhimurium was concentration-dependent. As the concentration increased from 6.25 μg / mL to 25 μg / mL, the number of viable bacteria in the cells gradually decreased significantly. At a concentration of 25 μg / mL, the number of intracellular Salmonella Typhimurium decreased by more than 50%, indicating that WP-4 and WP-6 have significant anti-intracellular bacterial activity and can exert antibacterial effects within cells.

[0089] In summary, this invention, through truncation and rational modification of template peptides to optimize charge number, hydrophobicity, and amphiphilicity, yielded two antimicrobial peptides, WP-4 and WP-6, with broad-spectrum antimicrobial activity. These two antimicrobial peptides exhibit good stability under temperature, pH, pepsin, serum, and salt ion conditions, demonstrate good cell selectivity, are less prone to inducing drug resistance, and can inhibit intracellular bacterial proliferation, showing broad application prospects.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide is WP-4 or WP-6, wherein the amino acid sequence of WP-4 is shown in SEQ ID NO.5 and the amino acid sequence of WP-6 is shown in SEQ ID NO.

7.

2. The use of the antimicrobial peptide as described in claim 1 in the preparation of antibacterial drugs.

3. The application as described in claim 2, characterized in that, The bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria are selected from one or more of the following: Escherichia coli ATCC25922, Escherichia coli ETEC7, Escherichia coli EPEC28, Escherichia coli PCN033, Salmonella ATCC14028, Salmonella C79-13, Actinobacillus pleuropneumoniae 4074, and Pasteurella 9261. The Gram-positive bacteria are selected from one or more of Staphylococcus aureus ATCC29213, Staphylococcus aureus 1213M4A, Streptococcus suis SC19, Streptococcus suis 0810, Streptococcus suis LXJ, and Streptococcus suis HN105.

4. The application as described in claim 2, characterized in that, The drug is an anti-intracellular bacterial drug, and the intracellular bacterial bacteria is Salmonella Typhimurium ATCC14028.

5. An antibacterial composition, characterized in that, The composition comprises the antimicrobial peptide of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

6. The antibacterial composition according to claim 5, characterized in that, The composition is an oral preparation, an injectable preparation, or a topical preparation.

7. The application of the antimicrobial peptide as described in claim 1 in the preparation of feed additives, characterized in that, The feed additive is used to inhibit pathogenic bacteria inside and outside farmed animals.

8. The application as described in claim 7, characterized in that, The pathogens include Gram-negative and / or Gram-positive bacteria of farmed animal origin, as well as intracellular parasites.

9. A feed additive, characterized in that, The feed additive contains the antimicrobial peptide as described in claim 1.

10. The method for preparing the antimicrobial peptide according to claim 1, characterized in that, Using the C-terminal amino acid sequence WP-1 at positions 29-40 of the non-natural antimicrobial peptide P8 as a template, WP-4 and WP-6 were obtained by optimizing charge number, hydrophobicity, and amphiphilicity through point mutation. The amino acid sequence of P8 is shown in SEQ ID NO.1, and the amino acid sequence of WP-1 is shown in SEQ ID NO.

2. The preparation method uses solid-phase chemical synthesis to synthesize WP-4 and WP-6, and the purity of the synthesized antimicrobial peptides is ≥95%.

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