Pore-forming antibacterial peptide and use thereof

Abstract

The application discloses a pore-forming antibacterial peptide and application thereof. The pore-forming antibacterial peptide is screened from the proteome of a toad by using a self-developed machine learning screening process, the membrane contact probability, antibacterial ability, physicochemical properties and the like of a short peptide sequence are predicted, and a pore-forming antibacterial peptide KKIASIIYAHVKALRARKILDNLKRLAANRP is obtained. The antibacterial peptide can form a pore on a membrane, destroy a cell membrane, exert a broad-spectrum antibacterial effect, and has low hemolytic activity and cytotoxicity, and is an ideal antibacterial drug development object.

Description

TECHNICAL FIELD

[0001] The present application relates to an antibacterial peptide, in particular to a pore-forming antibacterial peptide and its application in the preparation of drugs, belonging to the field of biological medicine. BACKGROUND

[0002] Pore-forming peptides are a class of polypeptides that can self-assemble into transmembrane pores, which can function as ion channels, biosensors, etc. Due to the membrane-destroying feature of pore-forming leading to the leakage of cell contents, some pore-forming peptides can exert broad-spectrum antibacterial effects as antibacterial peptides. Compared with traditional small molecule antibiotics, this mechanism makes pore-forming antibacterial peptides less likely to lead to drug resistance, and is an ideal object for drug development. However, only a few antibacterial peptides are known to have antibacterial effects through pore-forming mechanism, such as Melittin and Alamethicin, and finding new pore-forming antibacterial peptides is still a challenge. SUMMARY

[0003] The purpose of the present application is to mine a completely new pore-forming antibacterial peptide with broad-spectrum bactericidal ability and low cytotoxicity from the proteome. To this end, the present application uses a machine learning screening process independently developed to screen the proteome of the toad, predicts the membrane contact probability, antibacterial ability, physicochemical properties, etc. of short peptide sequences, and obtains a pore-forming antibacterial peptide.

[0004] The pore-forming antibacterial peptide obtained by the present application contains 31 amino acid residues and is positively charged, and its sequence is:

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

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

[0007] Through experimental testing, the antibacterial peptide of the present application can form pores on the membrane, destroy the cell membrane, and exert broad-spectrum antibacterial effects, while showing strong bacteriostatic ability against Gram-positive and negative bacteria. It has obvious inhibitory effect on Bacillus subtilis, Micrococcus luteus, Staphylococcus capitis, human Staphylococcus, Acinetobacter baumannii, Escherichia coli, etc. Gram-positive and negative bacteria, and the minimum inhibitory concentration can reach 8 μg / mL, and has low hemolytic activity and cytotoxicity. Moreover, the antibacterial peptide exerts antibacterial effect by destroying the cell membrane, has the characteristic of not easily leading to drug resistance of bacteria, and is an ideal object for antibacterial drug development. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is the membrane-destroying activity experimental result of the antibacterial peptide of the present application.

[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 with reference to the accompanying drawings.

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

[0012] 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 (Acinetobacter baumannii ATCC 17978, Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, and Escherichia coli DH5α) and four Gram-positive strains (Bacillus subtilis ATCC 6633, Micrococcus luteus CMCC(B)28001, Staphylococcus capitis ATCC 27842, and 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 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 11 different strains and has broad-spectrum antimicrobial 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 the antibacterial peptide on rat red blood cells. Fresh rat blood was centrifuged at 500g for 5 minutes, 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 antibacterial peptide was dissolved in PBS and added to the plate in a two-fold dilution gradient, with a final concentration ranging from 0.125 pg / mL to 128 pg / mL. Triton X-100 at 10 mg / mL was used as a positive control to induce 100% hemolysis, while PBS was used as a blank control. After 1 hour of incubation, the plate was centrifuged at 500g for 5 minutes. Subsequently, the absorbance (OD) of the supernatant was measured at 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 450 nm of the current concentration of antibacterial peptide treated wells, OD 450,PBS represents the absorbance at 450 nm of the PBS solution treated wells, and OD 450,positive represents the absorbance at 450 nm of the positive control wells. 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 antibacterial peptide tested had a HC 50 > 128 pg / mL and a MHC > 128 pg / mL, indicating good biosafety.

[0017] Example 3 Cytotoxicity assay of antibacterial peptide

[0018] The Alamar Blue method was used to assess the cytotoxicity of the antibacterial peptide on HEK293T. 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 antibacterial peptide was dissolved in PBS buffer at pH 7.4, with a final concentration ranging from 1 pg / mL to 128 pg / mL in a two-fold dilution gradient. After 72 hours of co-treatment of cells with the antibacterial peptide, 10 pL 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 570 nm. The formula for calculating the cell growth inhibition rate is: (OD 570,peptide - OD 570,PBSOD 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, and each concentration was measured in triplicate. The Half Maximal Cytotoxic Concentration (CC 50 ) value was fitted using Graphpad Prism software. The CC 50 of the antibacterial peptide was >128 μg / mL.

[0019] Example 4 Mechanism assay of antibacterial peptide

[0020] 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 film was hydrated with 600 μL of Tris (20 mM, pH 7.4) containing 70 μM 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 of Tris (20 mM, pH 8) containing different concentrations of polypeptide (1, 5, 10, 15 μM) at 10 μL per sample. The fluorescence was measured using an Infinite M Nano fluorospectrophotometer for 45 min. 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 peptide. The results of the liposome disruption by the antibacterial peptide at four different concentrations are shown in Figure 1 Figure 6. The antibacterial peptide at a concentration of 5 μM and above can release 80% of the calcein from the liposomes, indicating that the antibacterial peptide provided by the present application has strong membrane disruption 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). Phospholipid membranes were formed on MECA 16 recording chips, which contain 16 inert polymer circular microcavities with a diameter of 100 μm, each containing an independent Ag / AgCl microelectrode, allowing the simultaneous recording of 16 artificial lipid bilayer membranes. The antibacterial peptide (2 mM) was dissolved in a 20 mM HEPES, 200 mM KCl, pH 7.4 buffer and the capacitance was recorded when it interacted with the phospholipid membrane. The data were recorded in Elements Data Reader (Elements) and collected at a sampling rate of 20 kHz. The results are shown in Figure 2 Figure 6, which shows that single-channel recording exhibits a clear step-like curve, indicating that the antibacterial peptide can insert into the artificial phospholipid membrane to form pores. The above illustrates that the antibacterial peptide provided by the present application can exert an antibacterial effect by pore-forming to destroy the cell membrane, and has a relatively clear mechanism.

Claims

1. A pore-forming antibacterial peptide, characterized in that, The amino acid sequence of the pore-forming antibacterial peptide is as follows: KKIASIIYAHVKALRARKILDNLKRLAANRP.

2. Use of the pore-forming 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: 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 pore-forming antibacterial peptide in the antibacterial drug is greater than or equal to 8 μg / mL.

4. An antibacterial agent, characterized by, The antibacterial drug comprises the pore-forming antibacterial peptide of claim 1.

5. Use of the pore-forming 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: 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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