Membrane-rupturing antibacterial peptide and application thereof

Abstract

The invention discloses a membrane-rupturing antibacterial peptide and application thereof. On the basis of an artificial intelligence screening method, a membrane-rupturing antibacterial peptide KLAKLLKLLLWAQNELDQKK is identified from a proteome of toads, the membrane-rupturing antibacterial peptide has three positive charges, and 50% of amino acid is hydrophobic amino acid. Through experimental tests, the antibacterial peptide can inhibit the growth of 10 different strains including gram negative strains and gram positive strains; meanwhile, the semi-maximum hemolysis concentration and the semi-maximum cytotoxicity concentration of the antibacterial peptide are both greater than 128 mu g / mL, and the antibacterial peptide has good biological safety. The antibacterial peptide plays an antibacterial role by destroying cell membranes, has the characteristic that the drug resistance of bacteria is not easily caused, and is an ideal development object of antibacterial drugs and antibacterial products such as disinfectants, detergents and feed additives.

Description

Technical Field

[0001] The invention relates to an antimicrobial peptide, in particular to a membrane-breaking antimicrobial peptide and application thereof in drug preparation, and belongs to the field of biomedicine. Background Art

[0002] Antimicrobial peptides are a class of short peptides that are widely present in the natural immune system of organisms. They have various mechanisms of action, the most important of which is the destruction of cell membranes, which is also the main feature that distinguishes antimicrobial peptides from traditional small molecule antibiotics. Due to the accelerated growth of antibiotic resistance and the global expansion of pathogenic bacteria, the need to find antimicrobial peptides with broad-spectrum bactericidal ability and not prone to drug resistance is becoming increasingly urgent. Membrane-breaking antimicrobial peptides have the ability to target and destroy cell membranes. This special antibacterial mechanism makes antimicrobial peptides have the potential to become a new generation of antimicrobial drugs that can treat diseases such as cancer, bacterial and viral infections, and have huge market potential. Although a certain number of antimicrobial peptides have been discovered by separating and extracting natural substances, filtering and screening genomes, the mechanisms of action of most antimicrobial peptides have not been thoroughly studied. Summary of the invention

[0003] The purpose of the present invention is to discover a new membrane-breaking antimicrobial peptide with broad-spectrum bactericidal ability and low cytotoxicity from the proteome. To this end, the present invention identifies a membrane-breaking antimicrobial peptide from the proteome of toads based on an artificial intelligence screening method.

[0004] The membrane-breaking antibacterial peptide obtained by the present invention has three positive charges and contains 20 amino acid residues. The specific sequence is: KLAKLLKLLLWAQNELDQKK (SEQ ID No: 1), wherein the GRAVY hydrophobicity is -0.39, and 50% of the amino acids are hydrophobic amino acids. The sequence wheel diagram is shown in FIG. Figure 1 shown.

[0005] The membrane-breaking antimicrobial peptide of the present invention can be synthesized by chemical methods such as polypeptide solid phase synthesis. According to experimental tests, the antimicrobial peptide can inhibit the growth of 10 different strains including Gram-negative strains and Gram-positive strains. At the same time, the half-maximum hemolytic concentration and half-maximum cytotoxic concentration of the antimicrobial peptide are both greater than 128 μg / mL, and has good biosafety.

[0006] In summary, the membrane-permeable antimicrobial peptide provided by the present invention has an inhibitory effect on a variety of bacteria, and has low hemolytic activity and cytotoxicity, good biosafety, and exerts its antibacterial effect by destroying the cell membrane, and is not likely to cause bacterial resistance. It is an ideal development target for antibacterial drugs and other antibacterial products (such as disinfectants, detergents, and feed additives). BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 It is a sequence wheel diagram of the antimicrobial peptide of the present invention.

[0008] Figure 2 This is the membrane-breaking activity experimental result of the antimicrobial peptide of the present invention. DETAILED DESCRIPTION

[0009] The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

[0010] Example 1 Determination of Minimum Inhibitory Concentration of Antimicrobial Peptides

[0011] In this example, the minimum inhibitory concentration (MIC) of antimicrobial peptides was determined using the broth microdilution method. The selected strains included four Gram-negative strains (Acinetobacterbaumannii ATCC 17978, Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, Escherichia coli DH5α) and six Gram-positive strains (Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Micrococcus luteus CMCC (B) 28001, Staphylococcus capitis ATCC 27842, Staphylococcus hominis ATCC 27844). The bacteria were cultured overnight at 37°C and the bacterial solution was diluted to 5×10 5 cfu / mL, and then 99μL of bacterial suspension was added to each well of the 96-well plate. The antimicrobial peptide was dissolved in cation-adjusted Mueller-Hinton medium with an initial concentration of 1mg / mL, and then diluted in a two-fold gradient concentration ranging from 0.125μg / mL to 128μg / mL, and 1μL was added to each well. The 96-well plate was placed in a 37°C incubator and incubated for 20 hours. The lowest peptide concentration at which no bacterial growth was observed was observed and recorded, which was the MIC of the peptide, as shown in Table 1. After testing, the antimicrobial peptide can inhibit the growth of 10 different strains and has a broad-spectrum antibacterial ability.

[0012] Table 1. Minimum inhibitory concentrations of antimicrobial peptides against 10 strains

[0013]

[0014] Example 2 Determination of hemolytic activity of antimicrobial peptides

[0015] To test the hemolytic activity of antimicrobial peptides on rat erythrocytes, fresh rat blood was centrifuged at 500 g for 5 minutes, and the erythrocyte pellet was retained and resuspended in PBS buffer (pH 7.4). 8 The cells were added to a 96-well plate at a density of 10 cells. The antimicrobial peptide was dissolved in PBS and added to the plate in a two-fold gradient dilution, with the final concentration ranging from 0.125 μg / mL to 128 μg / mL. 10 mg / mL Triton X-100 was used as a positive control to induce 100% hemolysis, while PBS was used as a blank control. After incubation for 1 hour, centrifugation was performed at 500 g for 5 minutes. Subsequently, the absorbance (OD) of the supernatant was measured at a wavelength of 450 nm. The formula for calculating the percentage of hemolysis is: (OD 450,peptide -OD 450,PBS ) / (OD 450,positive -OD 450,PBS ), where OD 450,peptide Represents the absorbance at 450nm of the well treated with the current concentration of antimicrobial peptide, OD 450,PBS Represents the absorbance of the wells treated with PBS solution at 450nm, OD 450,positive Represents the absorbance of the positive control well at 450nm. Each concentration was measured three times to obtain the half maximum hemolysis concentration (HC 50 ) and the minimum hemolytic concentration (MHC) value that causes 10% hemolysis. 50 >128μg / mL, MHC>128μg / mL, with good biosafety.

[0016] Example 3 Cytotoxicity assay of antimicrobial peptides

[0017] The cytotoxicity of antimicrobial peptides to HEK293T cells was evaluated using the Alamar Blue method. HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C and 5% CO2. Staurosporine was used as a positive control for cell toxicity. The antimicrobial peptides were dissolved in PBS buffer at pH 7.4, and the final concentration range was a two-fold gradient concentration from 1μg / mL to 128μg / mL. After the cells were co-treated with the antimicrobial peptides for 72 hours, 10μL of Alamar Blue reagent was added to each well. Subsequently, the 96-well plate was incubated for 4 hours in the dark, and the absorbance (OD) was measured at a wavelength of 570nm. The formula for calculating the cell growth inhibition rate is: (OD570,peptide -OD 570,PBS ) / (OD 570,staurosporine -OD 570,PBS ), where OD 570,peptide Represents the absorbance of the antimicrobial peptide treated well at 570 nm, OD 570,PBS Represents the absorbance at 570 nm of the wells treated with PBS solution only, OD 570,staurosporine The absorbance of the wells treated with staurosporine at 570 nm was represented. Each concentration was measured in triplicate. The half-maximal cytotoxic concentration (CC) was fitted using Graphpad Prism software. 50 ) value. The CC 50 >128 μg / mL.

[0018] Example 4: Membrane disruption activity of antimicrobial peptides

[0019] DPhPE and DPhPG lipids were resuspended in chloroform and mixed at a molar ratio of 3:1. Chloroform was evaporated under a nitrogen stream, and the lipid film was hydrated with 600 μL Tris (20 mM, pH 7.4) containing 70 mM calcein. After 10 freeze-thaw cycles, the liposomes were squeezed 20 times with a filter. The liposomes were passed through two HiTrap desalting columns (AKTA Go system, Cytiva) at a flow rate of 0.2 mL / min to remove unencapsulated calcein. The liposomes encapsulated with calcein were collected, and 10 μL each was added to 90 μL Tris (20 mM, pH 8) solution containing different concentrations of peptides (1, 5, 10, 15 μM), and detected using an Infinite MNano fluorescence spectrophotometer for a total of 45 minutes. At the end of the experiment, 0.1% Triton X-100 was added to determine the maximum release of calcein, which was used to calculate the leakage ratio and characterize the membrane-breaking ability of the antimicrobial peptide. The results of lipid destruction by antimicrobial peptides at four different concentrations are shown in Figure 2. Figure 2 As shown, 5 μM antimicrobial peptide can cause liposomes to quickly release calcein, and the leakage rate of 10 μM and above can reach 80%, indicating that the antimicrobial peptide provided by the present invention has a strong membrane-breaking ability.

Claims

1. A membrane-permeable antimicrobial peptide, characterized in that: The membrane-permeable antimicrobial peptide has the following amino acid sequence: KLAKLLKLLLWAQNELDQKK.

2. Use of the membrane-permeable antimicrobial peptide according to claim 1 in the preparation of antimicrobial drugs.

3. The use according to claim 2, characterized in that The bacteria targeted by the antibacterial drug include Gram-positive bacteria and Gram-negative bacteria.

4. The use according to claim 2, characterized in that The bacteria targeted by the antibacterial drug are one or more of the following bacteria: Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, and Escherichia coli.

5. The use according to claim 3, characterized in that The effective concentration of the membrane-permeable antimicrobial peptide in the antimicrobial drug is greater than or equal to 8 μg / mL.

6. An antibacterial drug, characterized in that: The invention comprises the membrane-permeable antimicrobial peptide according to claim 1.

7. Use of the membrane-permeable antimicrobial peptide according to claim 1 in the preparation of antimicrobial products.

8. The use according to claim 7, characterized in that The antimicrobial product is a disinfectant, a detergent or a feed additive.

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