Database-Assisted Design of Antibacterial Peptides Targeting Escherichia coli, and Preparation Methods and Applications Thereof

Abstract

The present invention provides a database-assisted design of an antibacterial peptide targeting Escherichia coli, its preparation method and application. The antibacterial peptide (LGTK)7 has the sequence (Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys -NH2). By using the peptide library-assisted method to count the naturally occurring antibacterial peptides from Bacillus subtilis, including 46 antibacterial peptides from Bacillus subtilis, after analyzing the peptide library and removing lipopeptides, the average sequence length of the remaining antibacterial peptides is 27.58. The top 4 amino acids with the highest amino acid proportion are selected as G (9.50%), T (8.40%), K (7.60%), and L (7.50%). Taking (X1X2Y1Y2)n as the template, when X1 = L, X2 = G, Y1 = T, Y2 = K, and n = 7, the sequence length is 28, named antibacterial peptide (LGTK)7. This peptide is a non-natural peptide that can effectively inhibit Escherichia coli based on the Bacillus subtilis-derived antibacterial peptide database, has good biological activity and low hemolytic toxicity, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an α-helical antibacterial peptide that can effectively inhibit Escherichia coli, designed based on a Bacillus subtilis-derived antibacterial peptide database, and a preparation method and application thereof. Background Art

[0002] Since the advent of broad-spectrum antibiotics, they have always occupied a major position in the medical field. However, the long-term abuse of antibiotics has led to an increasing threat from drug-resistant bacteria. Many studies have shown that the emergence of antibiotic-resistant pathogens has made many dangerous communicable diseases more difficult to treat. Antibiotic resistance has become an urgent global problem. Through the research of scholars in various countries in recent years, it has been found that antibacterial peptides derived from bacteria are a kind of antibacterial drug with great potential to solve the global antibiotic resistance problem. Bacteriocins are small antibacterial peptides produced by bacteria. Most bacteriocins play a bactericidal role by irreversibly damaging the bacterial membrane, inducing the leakage of intracellular components of bacteria, and ultimately leading to cell death, so it is not easy to produce drug resistance. This characteristic makes bacterium-derived antibacterial peptides be considered as one of the potential antibiotic alternatives.

[0003] The conformations of bacterium-derived antibacterial peptides are diverse, which can be α-helix, β-sheet, linear extension or random coil, or can be cyclic peptides or a mixture of the above multiple conformations, but most of the conformations of antibacterial peptides are α-helix. Although natural bacterium-derived AMPs show superiority in overcoming multi-drug resistance, their development lacks design principles and is not systematic. Therefore, this process has become a "random" "black box trial and error" program. From this perspective, the development of non-natural peptides is to provide a wider AMP library. Taking Bacillus subtilis as an example, Bacillus subtilis (B. subtilis) can produce a variety of Bacillus subtilis-derived antibacterial peptides. Most of the bacteriocins produced by Bacillus subtilis can form pores in the bacterial cell membrane, resulting in cell death. However, the extraction cost of natural Bacillus subtilis antibacterial peptides is high and the activity is low. Therefore, it is very crucial to find or artificially design Bacillus subtilis antibacterial peptides with low toxicity and side effects, strong stability, low cost and high antibacterial characteristics. Database template-assisted design is a very effective development means. Establish a database using natural antibacterial peptides, select amino acid sequence templates with obvious statistical significance according to the type of amino acids (positively charged amino acids or hydrophobic amino acids), and conduct subsequent artificial screening. Compared with the traditional sequence modification method, this method can effectively reduce the number of peptides synthesized for research, and compared with the simple random combinatorial peptide library method, the antibacterial peptides designed by this method retain the immune characteristics possessed by natural antibacterial peptides. Summary of the Invention

[0004] Based on the above deficiencies, the present invention provides a non-natural peptide based on a Bacillus subtilis-derived antimicrobial peptide database to solve the problems of complex synthesis, high cost, poor stability, and high toxicity of α-helical antimicrobial peptides. Contrary to the traditional iterative design method, the combinatorial peptide library method applies bioinformatics of high-throughput combinatorial screening to help researchers identify and optimize novel leader sequences in the shortest time and at the lowest cost.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The preparation method of the non-natural peptide (LGTK)7 based on the Bacillus subtilis-derived antimicrobial peptide library of the present invention is as follows: By using the peptide library-assisted method to count the naturally occurring Bacillus subtilis-derived α-helical antimicrobial peptides, including 46 Bacillus subtilis-derived antimicrobial peptides, among which there are 16 lipopeptides and 30 amino acid peptides. As shown in Table 1, a new AMP library was synthesized. After bioinformatics analysis of the peptide library, the amino acid contents are I (5.50%), V (5.70%), L (7.50%), F (4.10%), C (7.40%), M (1.60%), A (7.40%), W (2.50%), G (9.50%), P (4.10%), T (8.40%), S (7.40%), Q (4.00%), N (2.90%), E (4.50%), D (2.70%), H (1.20%), K (7.60%), R (1.80%). Excluding lipopeptides, the average sequence length of natural Bacillus subtilis antimicrobial peptides is 27.58, among which G (9.50%), T (8.40%), K (7.60%), and L (7.50%) have the highest proportions. Therefore, the present invention is based on the α-helix template (XXYY) n and selects the top 4 amino acids with the highest amino acid proportions, leucine, glycine, threonine, and lysine, and repeats them seven times in the order of (LGTK) to form an α-helical structure with a sequence length of 28 and its C-terminus amidated. The amino acid sequence of this polypeptide is LGTKLGTKLGTKLGTKLGTKLGTKLGTK-NH2. Then, the polypeptide is synthesized by solid-phase chemical synthesis and named antimicrobial peptide (LGTK)7. (LGTK)7 is a non-natural peptide based on the Bacillus subtilis-derived antimicrobial peptide database that can effectively inhibit Escherichia coli and can be applied in the preparation of drugs for treating infectious diseases caused by Gram-negative bacteria.

[0007] Principle of the present invention: Design cationic peptide amphiphiles based on the α-helical protein folding principle, in which the amide proton between the peptide carbonyl O atom and the i-th and (i + 4)-th amino acid positions forms paired hydrogen bonds, forming a folded structure with a regular turn every 3.6 amino acids. This conformation is stabilized by paired hydrogen bonds within the peptide molecular backbone, and the hydrophobic interaction between amino acid side chains contributes greatly to the nucleation of this helical conformation. By obtaining the secondary structure of α-helical AMP stabilized by the hydrophobic interaction between adjacent i-th and (i + 4)-th amino acid side chains, and having good stability while having high antibacterial activity and low hemolytic activity.

[0008] Table 1 Antibacterial peptide library derived from Bacillus subtilis

[0009]

[0010]

[0011] Beneficial effects and advantages of the present invention: The antibacterial peptide (LGTK)7 of the invention has a stable structure. The antibacterial, hemolytic and stability activities of the synthesized antibacterial peptide were detected, and it was found that the antibacterial peptide (LGTK)7 showed good inhibitory effects on various strains such as Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentrations for Escherichia coli (25922) and Pseudomonas aeruginosa (PAO1) reached 8.0 μM and 4.3 μM respectively, and it had very low hemolytic activity. At the same time, (LGTK)7 still maintained a certain stability under serum and physiological salt ion conditions. In summary, (LGTK)7 is an antibacterial peptide with high application value. Description of the drawings

[0012] Figure 1 It is the high performance liquid chromatography diagram of antibacterial peptide (LGTK)7;

[0013] Figure 2 It is the mass spectrometry diagram of antibacterial peptide (LGTK)7. Detailed implementation manners

[0014] The present invention will be further described in detail below in conjunction with examples and drawings, but the implementation manners of the present invention are not limited thereto.

[0015] Example 1

[0016] Design of polypeptide

[0017] The amino acid sequence of polypeptide (LGTK)7 is:

[0018] Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys Leu Gly Thr Lys 1 5 10 15 20

[0020] Leu Gly Thr Lys Leu Gly Thr Lys-NH2 25 28

[0022] A series of cationic peptide amphiphiles were designed based on the ɑ-helix protein folding principle, in which the peptide carbonyl O atom and the amide proton between the i-th and (i+4)-th amino acid positions form paired hydrogen bonds, and a helical structure with regular turns every 3.6 amino acids was designed. The ɑ-helix template of X1X2Y1Y2 X1X2Y1Y2 X1X2Y1Y2 X1X2Y1Y2 X1X2Y1Y2 X1X2Y1Y2 X1X2Y1Y2-NH2 was designed. When X1=L, X2=G, Y1=T, and Y2=K, the polypeptide was named (LGTK)7, and its amino acid sequence is shown in Table 2.

[0023] Table 2 Amino acid sequence of polypeptide (LGTK) 7

[0024]

[0025] The length of the peptide (LGTK)7 sequence is 28 amino acids, Leu provides hydrophobicity, Lys increases the positive charge, and the charge number is +7. The antimicrobial peptide designed in this way has a stable α-helical structure, and has good stability while having efficient antibacterial activity and low hemolytic activity.

[0026] Example 2

[0027] Solid phase chemical synthesis of peptide (LGTK) 7

[0028] 1. Weigh 3 g of RINK resin (substitution degree 0.3 mol / g) into a 150 mL reactor and soak it with 50 mL of dichloromethane (DCM).

[0029] After 2.2 hours, the resin was washed with nitrogen-dimethylformamide (DMF) 3 times the volume of the resin, and then drained. This was repeated four times, and the resin was drained and set aside for use.

[0030] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF 3 times the volume of the resin and then drain it.

[0031] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0032] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30 ° C for reaction.

[0033] After 6.2 hours, a certain amount of acetic anhydride was used to cap the resin (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, and then washed four times with DMF 3 times the volume of the resin and dried for later use.

[0034] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it.

[0035] 8. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0036] 9. Weigh an appropriate amount of the second amino acid and an appropriate amount of HOBT into a 50mL centrifuge tube, add 25mL of DMF to dissolve them, then add 2.5mL of DIC and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.

[0037] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, the condensation is incomplete and the reaction should continue.

[0038] 11. After the reaction is complete, wash the resin four times with DMF, then drain, add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain to check whether the protection is removed.

[0039] 12. Follow steps 9-11 to connect the following amino acids.

[0040] 13. After the last amino acid is attached, remove the protection, wash four times with DMF, and then drain the resin with methanol. Then use a cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) to cut the peptide from the resin (add 10 mL of cutting solution per gram of resin), and centrifuge and precipitate four times with ice ether (cutting solution: ether = 1:9).

[0041] Finally, it was separated and purified by HPLC and then lyophilized to obtain a polypeptide with a certain purity.

[0042] 14. Purification conditions: stationary phase: C18; mobile phase configuration: PumpA: V(tfa) / V(water) = 1 / 1000, PumpB:

[0043] V(TFA) / v(acetonitrile) = 1 / 1000; flow rate: 10 mL / min, retention time: between 20 - 30 min, prepared twice.

[0044] 15. Example 3

[0045] Determination of antibacterial activity of antibacterial peptide (LGTK)7

[0046] 1. Determination of antibacterial activity: The method used to determine the minimum inhibitory concentration of the antibacterial peptide is the broth dilution method. Take out the test strain stored in the -20°C refrigerator, inoculate it in 10 mL of MHB medium and resuscitate for 8 h (constant temperature shaker at 37°C, 220 rpm), then transfer it to a new MHB and culture it in a constant temperature shaker at 37°C with a rotation speed of 220 rpm for 2 - 4 h until the logarithmic growth phase. Dilute the peptide solution by serial dilution with 0.2% BSA (containing 0.01% acetic acid), take 50 μL of the above solution and place it in a 96-well cell culture plate, and then add an equal volume of the test bacterial solution (10 6 CFU / mL) to each well. The well containing the bacterial solution without the peptide is used as the positive control, and the well without the bacterial solution and without the peptide is used as the negative control. Incubate at 37°C for 16 - 18 h. At the end of the incubation, observe and measure the optical density at 492 nm with an enzyme-linked immunosorbent assay reader. The antibacterial peptide concentration at which no microbial growth is observed visually and spectrophotometrically is the minimum inhibitory concentration of the antibacterial peptide. The detection results are shown in Table 3.

[0047] Table 3 Antibacterial activity of antibacterial peptide (LGTK)7 (μM)

[0048]

[0049]

[0050] It can be seen from Table 3 that antibacterial peptide (LGTK)7 shows high antibacterial activity against Gram-negative bacteria and almost no activity against Gram-positive bacteria and probiotics.

[0051] 2. Determination of hemolytic activity: Draw 1 mL of blood from healthy individuals and store it in a 1 mL heparin sodium anticoagulant tube. Centrifuge at 3000 - 3500 rpm at 4°C for 5 - 10 min, discard the supernatant, collect the lower layer of red blood cells, wash them 3 times with sterile PBS solution (pH = 7.4), resuspend the red blood cells with 10 times the volume of PBS solution for standby. Then add 80 μL of PBS solution to the first well of each row in a 96-well plate (8 rows × 12 columns), and 50 μL of PBS solution to the remaining wells. Add 20 μL of the antimicrobial peptide stock solution (2.56 mM) to the first well, mix well, then add 50 μL of the solution taken from the first well to the second well, mix well, and perform serial two-fold dilutions up to the tenth well. Aspirate and discard 50 μL of the mixed solution, and place it in an incubator at 37°C for 1 h of constant temperature incubation. The red blood cell suspension treated with 0.1% Triton X-100 is used as the positive control, and the untreated red blood cell suspension is used as the negative control. Centrifuge (3000 - 3500 rpm, 4°C) for 10 min, aspirate the supernatant and transfer it to a new sterile 96-well plate.

[0052] The formula for calculating the hemolysis rate is as follows:

[0053] Hemolysis rate (%) = [(A - A0) / (A t - A0)] × 100%.

[0054] In the formula, A0: absorbance value of the PBS negative control group;

[0055] A t : absorbance value of the 0.1% Triton X-100 positive control group;

[0056] A: absorbance value of the compound treatment group.

[0057] The minimum hemolytic concentration is the concentration of the antimicrobial peptide when it causes a 10% hemolysis rate. The test results are shown in Table 4.

[0058] Table 4 Determination of the hemolytic activity of antimicrobial peptide (LGTK)7

[0059]

[0060] As shown in Table 4, peptide (LGTK)7 did not show hemolytic activity within the test range. Calculate its therapeutic index using the ratio of the geometric mean of its minimum hemolytic concentration and the minimum inhibitory concentration (MIC) against Escherichia coli. The therapeutic index is 36.3.

[0061] 3. Determination of stability: For each concentration (150 × 10 -3 M NaCl, 4.5 × 10 -3 M KCl, 6 × 10 -6 M NH4Cl, 1 × 10 -3M MgCl2, 8×10 -6 M ZnCl2, 4×10 -6 Salts of (M FeCl3) were dissolved in the BSA solution, and then the subsequent experimental steps were the same as those for the determination method of antibacterial activity. To evaluate the effect of serum on antibacterial activity, the peptide was co-incubated with different concentrations of serum (100%, 50%) for 4 hours, and then the antibacterial activity was measured. The detection results are shown in Table 5.

[0062] Table 5 Antibacterial activity (μM) of antibacterial peptide (LGTK)7 against Escherichia coli 25922 under physiological salt concentration and serum conditions

[0063]

[0064] According to the results in Table 5, it can be seen that antibacterial peptide (LGTK)7 still maintains a certain antibacterial activity in an environment of physiological concentration of salt ions, 50% and 100% serum.

[0065] Based on all the above results, using (XXYY)n as a template, a cationic peptide amphiphile was designed using the principle of α-helical protein folding, in which paired hydrogen bonds were formed between the carbonyl O atom of the peptide and the amide proton at the i-th and (i + 4)-th amino acid positions, forming a folded structure with a regular turn every 3.6 amino acids. The designed antibacterial peptide (LGTK)7 has a certain therapeutic index, indicating its potential to replace antibiotics.

Claims

1. Database-assisted design of antibacterial peptides targeting Escherichia coli, characterized in that: By using the peptide library-assisted method to count the naturally occurring Bacillus subtilis-derived α-helical antimicrobial peptides, a new peptide library was combined. Through bioinformatics analysis of the peptide library, the top 4 amino acids with the highest amino acid proportions were selected as G = 9.50%, T = 8.40%, K = 7.60%, and L = 7.50%, and (XXYY) n was used as a template. The four amino acids were repeated seven times in the order of LGTK. The C-terminus of the antimicrobial peptide was amidated with -NH2, and the amino acid sequence of the obtained antimicrobial peptide was: LGTKLGTKLGTKLGTKLGTKLGTKLGTK-NH2.

2. The preparation method of a database-assisted designed antibacterial peptide targeting Escherichia coli according to claim 1, wherein, The method is as follows: A series of cationic peptide amphiphiles were designed based on the ɑ-helical protein folding principle, in which paired hydrogen bonds are formed between the peptide carbonyl O atom and the amide proton at the i-th and (i + 4)-th amino acid positions, and a regular-turning helical structure is formed every 3.6 amino acids.

3. The application of a database-assisted designed antibacterial peptide targeting Escherichia coli in the preparation of a drug for treating infectious diseases caused by Gram-negative bacteria according to claim 1, wherein the Gram-negative bacteria are Pseudomonas aeruginosa, Escherichia coli or Salmonella typhimurium.

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