Membrane-rupturing antibacterial peptide and application thereof

Abstract

The application discloses a membrane-damaging antibacterial peptide and application thereof. Based on an artificial intelligence screening method, a membrane-damaging antibacterial peptide KLAKLLKLLLWAQNELDQKK is identified from a protein group of a toad, the antibacterial peptide has 3 positive charges, and 50% of amino acids are hydrophobic amino acids. Experimental tests show that the antibacterial peptide can inhibit the growth of 10 different bacterial strains including gram-negative bacterial strains and gram-positive bacterial strains; meanwhile, the half maximum hemolytic concentration and the half maximum cytotoxicity concentration of the antibacterial peptide are greater than 128 mu g / mL, and the antibacterial peptide has good biological safety. The antibacterial peptide exerts antibacterial effect by damaging a cell membrane, has the characteristics of not easily leading to drug resistance of bacteria, and is an ideal antibacterial drug and a development object of antibacterial products such as disinfectants and detergents.

Description

TECHNICAL FIELD

[0001] The application relates to an antibacterial peptide, in particular to a membrane-destroying antibacterial peptide and application thereof in preparation of medicines, and belongs to the field of biological medicines. BACKGROUND

[0002] The antibacterial peptide is a kind of short peptide widely existing in the natural immune system of organisms, and has various mechanisms of action, and the most important one is to destroy the cell membrane, which is also the main feature distinguishing the antibacterial peptide from traditional small-molecule antibiotics. Due to the accelerated growth of antibiotic resistance and the global expansion of pathogenic bacteria, the demand for antibacterial peptides with broad-spectrum bactericidal ability and not prone to causing drug resistance is increasingly urgent. The membrane-destroying antibacterial peptide has the ability of targeted binding and destruction of the cell membrane, and this special bacteriostatic mechanism makes the antibacterial peptide have the potential to become a new generation of antibacterial drugs, which can treat diseases such as cancer, bacterial and viral infections, and has great market potential. Although a certain number of antibacterial peptides have been discovered through the separation and extraction of natural substances, the filtration and screening of genomes, etc., the mechanisms of action of most antibacterial peptides have not been thoroughly studied. SUMMARY

[0003] The purpose of the application is to mine a brand-new membrane-destroying antibacterial peptide with broad-spectrum bactericidal ability and low cytotoxicity from the proteome, and for this purpose, the application identifies a membrane-destroying antibacterial peptide from the proteome of a toad based on an artificial intelligence screening method.

[0004] The membrane-destroying antibacterial peptide obtained by the application has three positive charges and contains 20 amino acid residues, and 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 chart is as shown in the figure. Figure 1

[0005] The membrane-destroying antibacterial peptide of the application can be synthesized by a polypeptide solid-phase synthesis method or other chemical methods. Experimental tests show that the antibacterial peptide can inhibit the growth of a total of 10 different strains including gram-negative bacterial strains and gram-positive bacterial strains. At the same time, the half-maximum hemolytic concentration and the half-maximum cytotoxicity concentration of the antibacterial peptide are both greater than 128 mu g / mL, and the antibacterial peptide has good biological safety.

[0006] In summary, the membrane-destroying antibacterial peptide provided by the application has inhibitory effect on a variety of bacteria, has low hemolytic activity and cytotoxicity, has good biological safety, and exerts antibacterial effect by destroying the cell membrane, has the characteristics of not easily leading to drug resistance of bacteria, and is an ideal antibacterial drug and other antibacterial product (such as a disinfectant, a detergent) development object. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 is the sequence wheel chart of the antibacterial peptide of the application.​

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

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

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

[0011] In this example, the broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of antimicrobial peptides. The selected strains included four Gram-negative strains (Acinetobacter baumannii Acinetobacter baumannii Acinetobacter baumannii ATCC 17978, Escherichia coli ATCC 19606, Escherichia coli Escherichia coli Staphylococcus aureus ATCC 25922, Staphylococcus aureus DH5α) and six Gram-positive strains (Staphylococcus aureus Bacillus subtilis ATCC 25923, Micrococcus luteus ATCC 6538, Bacillus subtilis Staphylococcus capitis ATCC 6633, Micrococcus luteus Staphylococcus hominis CMCC(B) 28001, Staphylococcus capitis Figure 2 ATCC 27842, Staphylococcus hominis ​ ATCC27844). The bacteria were cultured at 37°C overnight and diluted to 5×10 5 cfu / mL, and then 99 μL of bacterial suspension was added to each well of a 96-well plate. The antimicrobial peptide was dissolved in cation-adjusted Mueller-Hinton medium with an initial concentration of 1 mg / mL, and then diluted in a two-fold gradient ranging from 0.125 μg / mL to 128 μg / mL, with 1 μL 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 the antimicrobial peptide on rat red blood cells, fresh rat blood was centrifuged at 500 g for 5 minutes, and the red blood cell pellet was retained and resuspended in PBS buffer (pH 7.4). The red blood cell solution was diluted and added to a 96-well plate at a density of 1 x 10 8 cells per well. The antimicrobial peptide was dissolved in PBS and added to the well plate after being diluted in a two-fold gradient, with a final concentration ranging from 0.125 μg / mL to 128 μg / mL. Triton X-100 at 10 mg / mL was used as a positive control to induce 100% hemolysis, and 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 was: (OD 450, peptide - OD 450, PBS ) / (OD 450, positive - OD 450, PBS ), where OD 450, peptide represents the absorbance at 450 nm of the current concentration of the antimicrobial peptide treated well, OD 450, PBS represents the absorbance at 450 nm of the PBS solution treated well, and OD 450, positive represents the absorbance at 450 nm of the positive control well. Each concentration was measured in triplicate to obtain the Half Maximal Hemolysis Concentration (HC 50 ) and the Minimum Hemolytic Concentration (MHC) value that caused 10% hemolysis. The tested antimicrobial peptide had an HC 50 > 128 μg / mL, and an MHC > 128 μg / mL, indicating good biological safety.

[0016] Example 3 Cytotoxicity assay of the antimicrobial peptide

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

[0018] Example 4 Membrane disruption activity of antibacterial peptides

[0019] DPhPE was resuspended with DPhPG lipid in chloroform and mixed at a 3:1 molar ratio. Chloroform was evaporated under a stream of nitrogen and the lipid membrane was hydrated with 600 μL Tris (20 mM, pH 7.4) containing 70 mM calcein. After 10 freeze-thaw cycles, the liposomes were squeezed through a filter 20 times. 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 calcein-encapsulated liposomes were collected and added to 90 μL Tris (20 mM, pH 8) containing different concentrations of polypeptides (1, 5, 10, 15 μM) at 10 μL each, and detected using an Infinite M Nano fluorescence spectrophotometer for 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 disruption ability of the antibacterial peptides. The results of the liposome disruption by the antibacterial peptides at four different concentrations are shown in ​ Figure 5. The antibacterial peptide at 5 μM can quickly release calcein from the liposomes, and the leakage ratio can reach 80% at 10 μM and above, indicating that the antibacterial peptide provided by the present application has strong membrane disruption ability.

Claims

1. A membrane disrupting antibacterial peptide, characterized in that, The amino acid sequence of the film-disrupting antibacterial peptide is as follows: KLAKLLKLLLWAQNELDQKK.

2. Use of the film-disrupting antibacterial peptide of claim 1 in the preparation of an antibacterial drug, wherein the bacteria against which the antibacterial drug is effective are one or more of the following: Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, Escherichia coli.

3. Use according to claim 2, wherein the compound is ###0002### The effective concentration of the film-disrupting antibacterial peptide in the antibacterial drug is greater than or equal to 8 μg / mL.

4. An antibacterial agent, characterized by, An antibacterial product comprising the film-disrupting antibacterial peptide of claim 1.

5. Use of the film-disrupting antibacterial peptide of claim 1 in the preparation of an antibacterial product, wherein the bacteria against which the antibacterial product is effective are one or more of the following: Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, Escherichia coli.

6. Use according to claim 5, wherein The antibacterial product is a disinfectant or a detergent.

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