Pore-forming antibacterial peptide and application thereof

Abstract

The invention discloses a pore-forming antibacterial peptide and application thereof. According to the invention, screening is carried out from a proteome of toads by utilizing a self-developed machine learning screening process, and the membrane contact probability, the antibacterial ability, the physicochemical property and the like of a short peptide sequence are predicted, so that the pore-forming antibacterial peptide KKIASIIYAHVKALRARKILDNLKRLAANRP is obtained. Tests show that the antibacterial peptide can form holes in a membrane, destroy a cell membrane and play a broad-spectrum antibacterial role, has relatively low hemolytic activity and cytotoxicity, and is an ideal antibacterial drug development object.

Description

Technical Field

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

[0002] Pore-forming peptides are a class of polypeptides that can self-assemble into transmembrane pores and may function as ion channels, biosensors, etc. Due to the membrane-destructive characteristic of forming pores in the membrane, which leads to the leakage of cell contents, some pore-forming peptides can act as antimicrobial peptides to exert broad-spectrum antimicrobial effects. Compared with traditional small molecule antibiotics, this mechanism makes pore-forming antimicrobial peptides less likely to cause drug resistance, making them ideal targets for drug development. However, currently only some antimicrobial peptides are known to be antibacterial through pore-forming mechanisms, such as Melittin, Alamethicin, etc., and finding new pore-forming antimicrobial peptides remains a difficult task. Summary of the invention

[0003] The purpose of the present invention is to discover new pore-forming antimicrobial peptides with broad-spectrum bactericidal ability and low cytotoxicity from the proteome. To this end, the present invention uses a self-developed machine learning screening process to screen from the toad proteome, predict the membrane contact probability, antimicrobial ability, physicochemical properties, etc. of short peptide sequences, and obtain a pore-forming antimicrobial peptide.

[0004] The pore-forming antimicrobial peptide obtained by the present invention comprises 31 amino acid residues, is positively charged, and has the following sequence:

[0005] KKIASIIYAHVKALRARKILDNLKRLAANRP (SEQ ID No: 1).

[0006] The antimicrobial peptide can be synthesized by chemical methods such as polypeptide solid phase synthesis, and applied to antimicrobial drugs and other antimicrobial products (such as disinfectants, detergents, and feed additives).

[0007] According to experimental tests, the antimicrobial peptides of the present invention can form pores on the membrane, destroy the cell membrane, exert a broad-spectrum antibacterial effect, and show strong antibacterial ability against Gram-positive and Gram-negative bacteria. They have obvious inhibitory effects on Gram-positive and Gram-negative bacteria such as Bacillus subtilis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, and Escherichia coli. The minimum inhibitory concentration can reach 8 μg / mL, and they have low hemolytic activity and cytotoxicity. Moreover, antimicrobial peptides exert antibacterial effects by destroying cell membranes, and are not easy to cause bacterial resistance, so they are ideal objects for the development of antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the membrane-breaking activity experimental result of the antimicrobial peptide of the present invention.

[0009] Figure 2 It is the pore-forming signal of the antimicrobial peptide of the present invention. DETAILED DESCRIPTION

[0010] The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

[0011] Example 1 Determination of Minimum Inhibitory Concentration of Antimicrobial Peptides

[0012] In this example, the minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined by the microbroth dilution method. The selected strains included 4 Gram-negative strains (Acinetobacterbaumannii ATCC 17978, Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, Escherichia coli DH5α) and 4 Gram-positive strains (Bacillus subtilis ATCC 6633, Micrococcus luteus CMCC (B) 28001, Staphylococcus capitis ATCC 27842, Staphylococcus hominis ATCC27844). 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 11 different strains and has a broad-spectrum antibacterial ability.

[0013] Table 1. Minimum inhibitory concentrations of antimicrobial peptides against 8 strains

[0014]

[0015] Example 2 Determination of hemolytic activity of antimicrobial peptides

[0016] This example tests 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). The erythrocyte solution was diluted and 1×10 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.

[0017] Example 3 Cytotoxicity assay of antimicrobial peptides

[0018] 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 in the dark for 4 hours, and the absorbance (OD) was measured at a wavelength of 570nm. The formula for calculating the cell growth inhibition rate is: (OD 570,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 measured in triplicate for each concentration. The half-maximal cytotoxic concentration (CC) was fitted using Graphpad Prism software. 50 ) value. The CC 50 >128 μg / mL.

[0019] Example 4 Mechanism determination of antimicrobial peptides

[0020] 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 1 As shown, the antimicrobial peptides at a concentration of 5 μM and above can release 80% of calcein from the liposomes, indicating that the antimicrobial peptides provided by the present invention have a strong membrane-breaking ability.

[0021] DPhPE and DPhPG lipids (ratio 3:1) were dissolved in octane. Single-channel recording experiments were performed on an Orbit 16TC instrument (Nanion). The phospholipid membrane was formed on a MECA 16 recording chip, which contains 16 inert polymer circular microcavities with a diameter of 100 μm, each of which contains an independent Ag / AgCl microelectrode, which can simultaneously record 16 layers of artificial lipid bilayer membranes. Antimicrobial peptides (2mM) were dissolved in 20mM HEPES, 200mM KCl, pH 7.4 buffer, and the capacitance was recorded when they interacted with the phospholipid membrane. The data were recorded in Elements Data Reader (Elements) and collected at a sampling rate of 20kHz. The measurement results are shown in Figure 2 As shown, the single channel recording shows an obvious step-like curve, indicating that the antimicrobial peptide can be inserted into the artificial phospholipid membrane to form pores. In summary, the antimicrobial peptide provided by the present invention can exert antibacterial effects by destroying the cell membrane through pore formation, and has a relatively clear mechanism.

Claims

1. A pore-forming antimicrobial peptide, characterized in that: The pore-forming antimicrobial peptide has the following amino acid sequence: KKIASIIYAHVKALRARKILDNLKRLAANRP.

2. Use of the pore-forming 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 3, characterized in that The bacteria targeted by the antibacterial drug are one or more of the following bacteria: Bacillus subtilis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, and Escherichia coli.

5. The use according to claim 2, characterized in that The effective concentration of the pore-forming 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 pore-forming antimicrobial peptide according to claim 1.

7. Use of the pore-forming 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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