Antibacterial peptide modified from signal peptide and application thereof

Abstract

The invention discloses an antibacterial peptide modified from a signal peptide and application of the antibacterial peptide, and belongs to the field of biological medicine. According to the present invention, an artificial intelligence model is adopted to perform mutation modification on a signal peptide, and the antibacterial peptide Perceptide17 modified from the signal peptide is provided, and the sequence of the antibacterial peptide Perceptide17 is VKKRLLFRKPLLKLFLLFGRRLKA. Tests show that the antibacterial peptide has good bacteriostatic activity on gram-positive strains and gram-negative strains, and the minimum inhibitory concentration can reach 1 mu M; the hemolytic activity is low, the half hemolytic concentration HC50 is greater than 128 mu M, the selectivity is shown in the interaction with a bacterial inner membrane and a human cell membrane, and the compound can be used for developing antibacterial drugs and antibacterial products.

Description

Technical Field

[0001] The present invention relates to an antimicrobial peptide and an application thereof, in particular to an antimicrobial peptide modified from a signal peptide and an application thereof in drug preparation, belonging to the field of biomedicine. Background Art

[0002] Drug resistance caused by antimicrobial drugs is threatening the therapeutic effects of drugs that humans rely on. Commonly used small molecule antibiotics have the problem of causing pathogen resistance during use, while antimicrobial peptides mainly exert their antimicrobial effects by destroying bacterial cell membranes, have broad-spectrum inhibitory effects on bacteria and other pathogens, and are not prone to drug resistance. Based on its characteristic of destroying cell membranes, one strategy for designing antimicrobial peptides is to modify existing signal peptide sequences. Signal peptides themselves have the characteristics of being localized in biological membranes, and play an important role in correctly guiding the processing and secretion of endogenous proteins in different organelles. For example, Porto et al. changed six residues of a signal peptide EcDBS1 in Escherichia coli by pattern replacement, and obtained an antimicrobial peptide EcDBS1R6 that destroyed bacterial cell membranes. Under an electron microscope, it can be observed that it caused the morphology of Pseudomonas aeruginosa to change and was accompanied by leakage of contents (BBA-General Subjects, 1864, 129633, 2020).

[0003] At the same time, since the mechanism of membrane destruction by antimicrobial peptides may be broad-spectrum, another problem currently faced by antimicrobial peptides is high hemolytic activity. Ideal antimicrobial peptide drug candidates should show species selectivity when taking effect, that is, only destroy the cell membranes of pathogens such as bacteria, but have no effect on human cells. Signal peptides themselves have species-related characteristics. Depending on the different secretion pathways, some signal peptides only work in Gram-positive or Gram-negative bacteria without affecting eukaryotic organisms. Therefore, antimicrobial peptides obtained from signal peptide modification are very likely to show selective characteristics. Summary of the invention

[0004] In order to obtain selective antimicrobial peptides, the present invention uses an independently developed artificial intelligence model to mutate the signal peptide, and provides an antimicrobial peptide Perceptide17 modified from the signal peptide, whose sequence is VKKRLLFRKPLLKLFLLFGRRLKA (SEQ ID No: 1).

[0005] Perceptide17 is derived from the known bacterial signal peptide MNKKRLLFRTPLDALFLLFGTALSA (SEQ ID No: 2). After the first M of the signal peptide is truncated, 7 mutations (N1V, T20R, T9K, D12L, S23K, A13K, A21R) are introduced using an artificial intelligence model to obtain Perceptide17, which has a length of 23 amino acids, a molecular weight of 2954.78, and carries 9 positive charges.

[0006] The antimicrobial peptide Perceptide17 can be synthesized by chemical methods such as peptide solid phase synthesis, and applied to antimicrobial drugs and other antimicrobial products (such as disinfectants, detergents, and feed additives).

[0007] After testing, the antimicrobial peptide Perceptide17 has good antibacterial activity against both Gram-positive and Gram-negative strains, including but not limited to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, with a minimum inhibitory concentration of 1 μM; and low hemolytic activity, with a half hemolytic concentration of HC 50 Greater than 128μM, it exhibits selectivity in its interaction with bacterial inner membranes and human cell membranes and can be used in the development of antibacterial drugs and antimicrobial products. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The three-dimensional structure of the antimicrobial peptide Perceptide17 predicted using AlphaFold2 in Example 1.

[0009] Figure 2 The heavy atom interaction ratio between the antimicrobial peptide Perceptide 17 and the bacterial inner membrane and the human cell membrane simulated by molecular dynamics in Example 2.

[0010] Figure 3 The results of the hemolytic activity test of the antimicrobial peptide Perceptide 17 in Example 4 are shown. DETAILED DESCRIPTION

[0011] The present invention will be further described below by way of embodiments in conjunction with the accompanying drawings. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0012] Example 1 Source and properties of antimicrobial peptides

[0013] The antimicrobial peptide Perceptide17 was evolved from the known bacterial signal peptide MNKKRLLFRTPLDALFLLFGTALSA based on an independently developed artificial intelligence model. First, since the first M of the signal peptide is translated from the start codon and has no direct connection with the function, M was removed. The sequence was then input into the artificial intelligence model, and 7 mutations were performed in sequence to obtain Perceptide17: VKKRLLFRKPLLKLFLLFGRRLKA, which is 23 amino acids in length, has a molecular weight of 2954.78, carries 9 positive charges, has an isoelectric point of 12.61, and contains 0.13 aromatic amino acids and 0.54 hydrophobic amino acids. The three-dimensional structure of Perceptide17 predicted using AlphaFold2 is shown below: Figure 1 As shown, a portion of its N-terminus is a random coil, and the remaining secondary structure is an α-helix.

[0014] Example 2 Molecular dynamics simulation to verify the selectivity of antimicrobial peptides

[0015] The complex interaction between antimicrobial peptides and cell membranes can be studied by molecular dynamics simulation. The antimicrobial peptide structure used in the simulation was derived from the AlphaFold2 prediction. The simulation system was built with CHARMM-GUI. In the initial system, the peptide was placed about 2 nm above the phospholipid bilayer membrane. Two systems, bacterial inner membrane and human cell membrane, were built for the antimicrobial peptide Perceptide17. The phospholipid composition was referenced by Pandi et al. (Nature Communications, 14: 7197, 2023). 150 mM NaCl was added to keep the system electrically neutral. The simulation process was run using GROMACS2018, with a force field of CHARMM36m and a step size of 2 fs. The Nose-Hoover temperature coupling algorithm was used to keep the temperature at 310 K, and the Parinello-Rahman pressure coupling algorithm was used to keep the system pressure stable at 1.0 bar. The cutoff value of the Lennard-Jones potential was 1.2 nm, and the Ewald method was used for long-range electrostatic interactions. The NVT and NPT equilibrium processes both used the CHARMM-GUI default steps. A 500 ns simulation was run for each system. The proportion of the heavy atoms of the antimicrobial peptide interacting with the heavy atoms of the phospholipids (i.e., within 3.5 angstroms) in the last 100 ns of each trajectory was calculated. The results are shown in Figure 2 As shown in the figure, three repeated simulations were performed for each system. The interaction ratio of the antimicrobial peptide Perceptide17 with the bacterial inner membrane was significantly higher than that with the human cell membrane, indicating that the antimicrobial peptide Perceptide17 may exert its antibacterial function by interacting with the bacterial inner membrane without affecting the human cell membrane, which is selective.

[0016] Example 3 Determination of Minimum Inhibitory Concentration of Antimicrobial Peptides

[0017] The bacterial species used in the minimum inhibitory concentration determination experiment include two Gram-negative strains and two Gram-positive strains, namely Escherichia coli ATCC25922, Pseudomonas aeruginosa PAO1, Staphylococcus aureus ATCC6538, and Bacillus subtilis ATCC6633. The minimum inhibitory concentration of antimicrobial peptides was determined using the Hancock microbroth dilution method. The four standard strains were activated by overnight culture and cultured at 37°C in a shaker at 220 rpm for about 3 hours, and then diluted to OD 625 The values ​​are in the range of 0.08-0.13.

[0018] The dry powder of the antimicrobial peptide Perceptide17 was synthesized by Wuhan Dangang Biotechnology Co., Ltd. through solid phase chemical synthesis. The relative molecular mass detected by mass spectrometry was correct and the purity was above 95%. The antimicrobial peptide solution stock was prepared with sterile PBS solution (pH 7.4) at a concentration of 512 μM. 100 μL of LB culture medium and 100 μL of antimicrobial peptide solution were added to each 96-well plate, and the antimicrobial peptide solution was prepared by double concentration gradient dilution to a final concentration of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μM. 100 μL of 100-fold diluted bacterial solution was added to each well, mixed well, and cultured in a 37°C incubator for 16 hours. The lowest antimicrobial peptide solution concentration value at which no obvious bacterial growth was observed by the naked eye is the minimum inhibitory concentration value of the antimicrobial peptide under this condition. The test results are shown in Table 1. The minimum inhibitory concentrations of Perceptide17 against the four strains are 8, 32, 2, and 1 μM, respectively, showing strong antibacterial activity.

[0019] Table 1. Minimum inhibitory concentration of antimicrobial peptides against four strains (unit: μM)

[0020]

[0021] Example 4 Determination of hemolytic activity of antimicrobial peptides

[0022] Fresh rat blood was collected, mixed in PBS buffer (pH 7.4), and centrifuged at 500g for 5 minutes to prepare a 10% red blood cell suspension. The antimicrobial peptide was prepared into a solution with a final concentration of 1-128μM using a two-fold gradient dilution method and added to 100μL of red blood cell suspension. After standing at 37°C for 1 minute, centrifuged at 2500g for 6 minutes, and 75μL of supernatant was added to each well of a 96-well plate. 1%, 0.1%, and 0.001% Triton X-100 were used as positive controls, and PBS was used as a blank control. The absorbance of the supernatant at a wavelength of 450nm was then measured using an enzyme reader. All tests were repeated three times. The hemolysis rate of each well was calculated according to (OD 450,多肽 -OD 450,PBS ) / (OD 450,阳性对照 -OD 450,PBS ) calculation, OD 450,多肽 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,阳性对照 Represents the absorbance of the positive control well at 450nm. The hemolytic activity test results of the antimicrobial peptide Perceptide17 are as follows Figure 3 As shown. Its half hemolytic concentration HC 50 Greater than 128μM, confirming that Perceptide17 has good biosafety.

Claims

1. An antimicrobial peptide, characterized in that: The antimicrobial peptide has the following amino acid sequence: VKKRLLFRKPLLKLFLLFGRRLKA.

2. Use of the 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: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis.

5. The use according to claim 2, characterized in that The effective concentration of the antimicrobial peptide in the antimicrobial drug is greater than or equal to 1 μM.

6. An antibacterial drug, characterized in that: The antimicrobial peptide according to claim 1.

7. Use of the 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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