Antibacterial peptide and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to an antibacterial peptide and application thereof, wherein the antibacterial peptide has broad-spectrum and high-efficiency antibacterial activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antimicrobial peptide and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), are considered essential components of the innate immune system. Antimicrobial peptides exhibit diverse sequence compositions and physical and chemical structures and properties, typically consisting of short amino acid sequences, generally fewer than 100 amino acid residues, a large proportion of which are positively charged amino acid residues.

[0003] Antimicrobial peptides are widely found in nature. Conocospira polypeptides, which possess antimicrobial activity, typically exhibit broad-spectrum activity against Gram-negative and Gram-positive bacteria, as well as fungi and molds. Furthermore, they can kill enveloped viruses, in vivo and in vitro parasites, and even cancer cells. Antimicrobial peptides can also regulate innate and adaptive immunity, potentially inhibiting inflammatory responses and reducing the proliferation of macrophages, neutrophils, eosinophils, and T lymphocytes.

[0004] The accelerating growth and global expansion of antibiotic resistance has intensified the need for the discovery of novel antibiotic drugs. Compared with traditional antibiotics, antimicrobial peptides have significant advantages, including slower development of resistance, broad-spectrum antimicrobial membrane activity, and the ability to modulate host immune responses.

[0005] Bacterial infections pose a serious threat to human health. In China, at least 3 million people are infected annually, directly resulting in approximately 33,000 deaths. Particularly in recent years, the overuse of traditional antibiotics and the accelerated evolution of multidrug-resistant bacteria have led to the development of drug resistance in many bacteria. Furthermore, driven by commercial interests, bacterial resistance is developing at an alarming rate compared to the speed of antibacterial drug development. Therefore, there is an urgent need for new antibacterial drugs to inhibit the growth of bacteria, especially multidrug-resistant bacteria. Summary of the Invention

[0006] This invention utilizes deep learning and molecular dynamics simulations to design a novel antimicrobial peptide and its variants based on artificial intelligence, exhibiting highly efficient and broad-spectrum antimicrobial activity. Furthermore, circular dichroism (CD) chromatography and Alphafold 2 three-dimensional structure prediction confirmed that the secondary structure of this antimicrobial peptide is primarily α-helical. Based on these findings, this invention was completed.

[0007] In a first aspect, the present invention provides an antimicrobial peptide, wherein the sequence of the antimicrobial peptide is selected from one or more of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the sequences are shown in Table 1.

[0008] Furthermore, certain sites in the amino acid sequence may be deleted or mutated, and the deleted or mutated amino acid sequence has 80%-100% homology with the original amino acid sequence, preferably 99%, 95%, 90%, 95%, or 80% homology.

[0009] Furthermore, one or more of the following amino acids in the amino acid sequence are mutated, and the amino acid mutation sites include: L17A, V14R, K11A, R16A, R13A, R10A, R9A, and R3A; the mutated sequence is shown in Table 3. Preferably, the amino acid sequence of the mutant is shown as 3344[A18R] to 3344[L1A] in Table 3.

[0010] Furthermore, the N-terminus of the amino acid sequence is truncated to DN1 or DN2; Furthermore, the C-terminus of the amino acid sequence is truncated to DC1 or DC2; Furthermore, the amino acid sequence is a D-type sequence in which the following mutations occur: d3344 or dV14R.

[0011] The antimicrobial peptide has highly efficient and broad-spectrum antimicrobial activity.

[0012] In a second aspect, the present invention provides a composition containing an antimicrobial peptide, wherein the antimicrobial peptide is the antimicrobial peptide described in the first aspect of the present invention.

[0013] Furthermore, the antimicrobial peptide composition can be prepared as a drug, antimicrobial agent, or bactericide.

[0014] Furthermore, the drug, antibacterial agent, or bactericide can be prepared as tablets, capsules, pills, powders, granules, sprays, fumigants, washes, injections, etc.

[0015] Thirdly, the present invention provides the use of an antimicrobial peptide in the preparation of a drug or preparation that inhibits bacterial growth, wherein the antimicrobial peptide is the antimicrobial peptide described in the first aspect of the present invention.

[0016] Furthermore, the bacteria are either Gram-negative or Gram-positive.

[0017] Furthermore, the Gram-negative bacteria include: Pseudomonas aeruginosa ATCC27853, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 25922, Stenotrophomonas maltophilia CGMCC 1.1788, Bacillus lysinophilus YC36, and Porphyromonas gingivalis ATCC33277.

[0018] Furthermore, the Gram-positive bacteria include: Staphylococcus aureus CMCC(B) 26003, Listeria bio-59992, and Streptococcus mutans ATCC25175.

[0019] Preferably, the antimicrobial peptide inhibits Pseudomonas aeruginosa ATCC27853, Porphyromonas gingivalis ATCC33277, and Streptococcus mutans ATCC25175. Attached Figure Description

[0020] Figure 1 The secondary structure of the antimicrobial peptide was predicted by Alphafold 2.

[0021] Figure 2 These are the results of circular dichroism characterization of the antimicrobial peptide. Detailed Implementation

[0022] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0024] Example 1: Chemical Synthesis of Antimicrobial Peptides All antimicrobial peptides used in the experiment were synthesized using solid-phase Fmoc chemical synthesis. Besides various amino acids with protecting groups, other required raw materials included RinkAmide resin, piperidine, DMF, DCM, HCTU, DIPEA, trifluoroacetic acid, triisopropylsilane, and anhydrous diethyl ether. The direction of peptide synthesis proceeded from the C-terminus to the N-terminus, with C-terminal amidation modification.

[0025] Specific implementation steps: First, RinkAmide resin was swollen overnight in a DMF:DCM = 1:1 solution. Then, 20% piperidine solution was added and reacted for 30 minutes to remove the Fmoc protecting group from the resin. Next, the correct amino acids were added sequentially according to the desired amino acid sequence. DMF was used as the solvent for amino acid coupling, and HTCU and DIPEA acted as condensing agents and activators of the amino acids, respectively. The reaction time was 1 hour. Then, the Fmoc protecting group on the amino acids was removed again, and the next amino acid was coupled, repeating the cycle until the last amino acid. After the peptide sequence was synthesized, the peptide was cleaved from the resin using a lysis buffer of trifluoroacetic acid:triisopropylsilane:water = 9:0.5:0.5 for 3 hours. The synthesized antimicrobial peptide was then precipitated using diethyl ether. The peptide was dissolved in an iodine solution with a concentration of 1 mg / mL to oxidize the Acm-protected cysteine, reacting for 30 minutes. Ascorbic acid was then added to terminate the oxidation reaction, followed by C... 18 Separation and purification were performed using a reverse-phase column chromatography. Electrospray ionization mass spectrometry verified the correct molecular weight of the peptide, and high-performance liquid chromatography identified a purity >95%.

[0026] The obtained antimicrobial peptide sequences are shown in Table 1.

[0027] Table 1. Formation of antimicrobial peptides

[0028] The secondary structures of five antimicrobial peptides were predicted using alphafold 2, and the results showed that α-helices were present in all of them. The secondary structure model that generated 3344 was as follows: Figure 1 As shown.

[0029] Example 2: Determination of the bioactivity of antimicrobial peptides The bioactivity of antimicrobial peptides is expressed by testing the minimum inhibitory concentration (MIC) of the antimicrobial peptide. The MIC refers to the minimum concentration at which the drug can inhibit the growth of bacteria in the culture medium. The bacteria tested in this invention include Gram-positive bacteria such as Staphylococcus aureus CMCC(B) 26003, Listeria bio-59992, and Streptococcus mutans ATCC25175, as well as Gram-negative bacteria such as Pseudomonas aeruginosa ATCC27853, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 25922, Stenotrophomonas maltophilia CGMCC 1.1788, Lysozyme YC36, and Porphyromonas gingivalis ATCC33277.

[0030] After reviving the bacterial strain, it was subcultured once, and a single colony was picked and cultured in MH liquid medium until it reached the logarithmic growth phase. The antimicrobial peptide to be tested was prepared into a solution of a specific concentration. In a 96-well plate, 100 µL of medium was added to each well. In rows A, B, and C, the first column, 100 µL of the first antimicrobial peptide to be tested was added. The solution was diluted using a micro-two-fold dilution method until the last well. Then, 100 µL of the diluted bacterial suspension was added (the final CFU / mL is approximately 1 x 10⁻⁶). 6 The final volume of each well was 200 µL. Negative and positive controls were set up to ensure bacterial growth. The 96-well plates were then incubated at 37°C for 18 hours, and colony growth was observed. The results showed that 3344 exhibited significant broad-spectrum antibacterial activity, and its MIC values ​​are shown in Table 2.

[0031] Table 2. Minimum inhibitory concentrations of antimicrobial peptides (unit: µg / mL)

[0032] Example 3: Selection and Synthesis of the 3344 Mutant The activity of antimicrobial peptides mainly depends on factors such as positive charge, hydrophobicity, amphiphilicity, and secondary structure. Based on this, each amino acid on 3344 was subjected to site-directed mutagenesis with alanine (Ala) and arginine (Arg) to obtain different single mutants for studying their structure-activity relationship.

[0033] Based on the bioactivity assay results of antimicrobial peptide 3344, it was found that the bioactivity was improved when amino acids at positions 3, 10, 13, 14, 16 and 17 were replaced. Therefore, we selected two sites for mutation to obtain different double mutants.

[0034] Antimicrobial peptide 3344 has 18 amino acids in its full length, which has certain drawbacks in terms of synthesis time and cost. Therefore, we partially removed its C-terminus and N-terminus.

[0035] Considering that linear peptides are easily degraded in vivo and readily broken down by various proteases, thus losing their activity, V14R, which has relatively good activity, was selected to replace all D-type amino acids in the 3344 matrix, in an attempt to improve stability while retaining activity. Its sequence is shown in Table 3.

[0036] Table 3 Selection of mutants

[0037]

[0038] Example 4: Bioactivity test of the 3344 mutant 3344 showed good antibacterial activity against both Gram-positive and Gram-negative bacteria. Although most of its single mutants showed decreased activity, some retained their activity. The increase in positive charge did not have a significant effect on activity. However, the replacement of hydrophobic residues such as phenylalanine with arginine resulted in a significant decrease in activity, and the replacement of lysine also led to varying degrees of activity reduction. Therefore, mutation modification should focus on hydrophobicity.

[0039] Alanine mutations can elucidate the structure-activity relationship of individual residues and reduce the number of polar residues to improve overall hydrophobicity. Based on these results, a large number of alanine mutants maintained activity, indicating that the positive charge at 3344 has relatively little impact on activity within the 7-9 range, while adjustments to hydrophobicity are more significant for maintaining and enhancing activity.

[0040] When designing the 3344 double mutant, single mutants with relatively good activity retention were selected and combined in pairs. The double mutant also retained activity comparable to 3344 in various bacteria.

[0041] To improve the stability of the d-type mutant, the activity of d3344 is significantly increased compared to 3344; and the activity can still be maintained after removing two residues from the carbon terminus. The minimum inhibitory concentration is shown in Table 4.

[0042] Table 4. Minimum inhibitory concentrations (MICs) of the 3344 mutant (unit: µg / mL)

[0043]

[0044]

[0045] Example 5: Analysis of the structure of antimicrobial peptide 3344 using circular dichroism spectroscopy Circular dichroism spectroscopy was used to detect antimicrobial peptide 3344. Negative absorptions were observed at 208 nm and 222 nm, and a positive absorption was found at 190 nm, confirming the presence of an α-helix. The results are as follows: Figure 1 and Figure 2 As shown.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is SEQ ID NO.

4.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antimicrobial peptide of claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that, The drug dosage form is tablet, capsule, pill, powder, granule, spray, fumigation, lotion, or injection.

4. An antibacterial agent, characterized in that, The antimicrobial agent comprises the antimicrobial peptide of claim 1.

5. The antibacterial agent as described in claim 4, characterized in that, The antibacterial agent is available in tablet, capsule, pill, powder, granule, spray, fumigant, lotion, or injection form.

6. The use of the antimicrobial peptide of claim 1, the pharmaceutical composition of claim 2, or the antimicrobial agent of claim 4 in the preparation of a drug or disinfectant that inhibits bacterial growth, characterized in that, The bacteria are Pseudomonas aeruginosa ATCC27853, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 25922, Stenotrophomonas maltophilia CGMCC 1.1788, Bacillus lysozyme YC36, or Porphyromonas gingivalis ATCC33277.

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