Antibacterial peptide for inhibiting propionibacterium acnes and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide for inhibiting propionibacterium acnes and application of the antibacterial peptide. The antibacterial peptide for inhibiting propionibacterium acnes is derived from marine organism hylophila, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1. The amino acid sequence is novel, has remarkable antibacterial activity on propionibacterium acnes, has the advantages of low hemolysis toxicity, low cytotoxicity and high safety, and can be used for preparing the antibacterial peptide for inhibiting propionibacterium acnes. Due to the characteristics, the compound has huge application potential in the aspect of treating acnes caused by propionibacterium acnes or removing acnes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antimicrobial peptide that inhibits Propionibacterium acnes and its application. Background Technology

[0002] Acne is a chronic inflammatory disease of the pilosebaceous unit characterized by comedones, papules, pustules, nodules, cysts, and scars. Propionibacterium acnes (…) Propionibacterium acnes Propionibacterium acnes is an opportunistic pathogen of acne, and its overgrowth is considered a major factor in the pathogenesis of acne. Clindamycin and doxycycline are commonly used clinically to treat acne. However, with the widespread use of antibiotics and the evolution of drug-resistant bacteria, drug resistance is becoming increasingly serious, urgently requiring the development of novel antibacterial drugs for the treatment of acne.

[0003] Antimicrobial peptides (AMPs), also known as host defense peptides, are important effectors of the innate immune system. They are usually composed of 2 to 50 amino acid sequences and have the advantages of rapid and efficient antibacterial activity, broad-spectrum antibacterial activity, and low risk of developing drug resistance. Due to their unique antibacterial mechanism and low risk of developing drug resistance, the development of novel antimicrobial peptides targeting Propionibacterium acnes for the prevention, treatment, or elimination of acne caused by the over-proliferation of Propionibacterium acnes is of great value.

[0004] Marine organisms possess unique living environments and metabolic mechanisms, making them a natural treasure trove of antimicrobial peptides. Threadworms (Trichoplax adhaerens Schulze), metazoans belonging to the phylum Platycodontia and class Trichoplax, are among the simplest known multicellular organisms. In their complex living environments and through a long process of biological evolution, the threadworm genome encodes a large number of novel antimicrobial peptides, representing a natural repository of antimicrobial peptides. With the development of artificial intelligence technology, the discovery of novel antimicrobial peptides derived from threadworms using AI for the prevention and treatment of acne or pimple removal shows significant development and application potential. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, this invention provides an antimicrobial peptide that inhibits Propionibacterium acnes and its applications. Based on the transcriptome of *Pseudomonas acnes*, this invention utilizes artificial intelligence technology to screen and obtain the antimicrobial peptide TAMPa-6, which inhibits Propionibacterium acnes. This antimicrobial peptide has a novel amino acid sequence, a secondary structure primarily composed of α-helices, and exhibits a significant inhibitory effect on Propionibacterium acnes. It is safe and non-toxic, and can be used to develop novel antimicrobial products or cosmetics that inhibit Propionibacterium acnes for the prevention and treatment of acne.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention first provides an antimicrobial peptide that inhibits Propionibacterium acnes; its amino acid sequence is shown in SEQ ID NO. 1.

[0007] Furthermore, the present invention provides a method for preparing the antimicrobial peptide that inhibits Propionibacterium acnes. The preparation method adopts the Fmoc solid-phase polypeptide synthesis method, in which Fmoc amino acids are subjected to repeated deprotection-coupling cycles to form resin peptides on resin. Then, the resin peptides are cleaved to obtain linear peptides, which are purified by high performance liquid chromatography to obtain antimicrobial peptides with a purity greater than 95%.

[0008] Furthermore, the present invention provides the application of the aforementioned antimicrobial peptide that inhibits Propionibacterium acnes in the preparation of antimicrobial products.

[0009] Furthermore, the present invention provides an antibacterial product comprising the aforementioned antimicrobial peptide that inhibits Propionibacterium acnes; the antibacterial product is a topical medication for treating acne or a product for preventing Propionibacterium acnes infection.

[0010] Preferably, the antibacterial product is a solution, spray, cream, or gel.

[0011] Furthermore, the present invention provides a skin care product comprising the aforementioned antimicrobial peptide or antimicrobial product.

[0012] Preferably, the skin care product is a face mask, serum, lotion, cream, spray, or gel.

[0013] Furthermore, the present invention also provides a composition comprising the aforementioned antimicrobial peptide, antimicrobial product, or skin care product.

[0014] The present invention has the following beneficial effects: (1) The antimicrobial peptide TAMPa-6 for inhibiting Propionibacterium acnes provided by the present invention has a novel amino acid sequence and structure. It has significant antibacterial activity against Propionibacterium acnes with a MIC of 2µg / mL. It also has the advantages of low hemolytic toxicity, low cytotoxicity and high safety. It has great value in the development of antimicrobial products or cosmetics for inhibiting Propionibacterium acnes.

[0015] (3) The antimicrobial peptide TAMPa-6 provided by the present invention for inhibiting Propionibacterium acnes has a short sequence, which is convenient for synthesis and separation and purification, has low production cost, and high product purity, providing important technical support for the development of antimicrobial drugs or cosmetics. Attached Figure Description

[0016] Figure 1 This is a helical diagram of the antimicrobial peptide TAMPa-6 provided in the embodiments of the present invention; Figure 2The secondary structure of the antimicrobial peptide TAMPa-6 provided in the embodiments of the present invention was predicted by Alphafold 3; Figure 3 The circular dichroism chromatographic characterization results of the antimicrobial peptide TAMPa-6 provided in the embodiments of the present invention are shown; wherein, (a) H2O; (b) H2O:TFE=1:1; Figure 4 This refers to the diameter of the inhibition zone of the antimicrobial peptide TAMPa-6 provided in this embodiment of the invention against Propionibacterium acnes. Figure 5 The cytotoxicity of the antimicrobial peptide TAMPa-6 provided in the embodiments of the present invention; Figure 6 This refers to the hemolytic toxicity of the antimicrobial peptide TAMPa-6 provided in the embodiments of the present invention; Figure 7 This is the interaction between the antimicrobial peptide TAMPa-6 provided in this embodiment of the invention and the cell membrane; wherein, (a) are the states at 0 ns, 300 ns, 600 ns and 1000 ns respectively; (b) kinetic simulation time series diagram; Figure 8 The images are scanning electron microscope images of Propionibacterium acnes cell morphology treated with the antimicrobial peptide TAMPa-6; (a) shows normally cultured Propionibacterium acnes; and (b) shows Propionibacterium acnes treated with the antimicrobial peptide TAMPa-6 for 2 hours. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] Example 1: Discovery, synthesis, and physicochemical properties of the antimicrobial peptide TAMPa-6 1. Discovery of the antimicrobial peptide TAMPa-6 Starting from the protein sequences of *Nematocystis jirovecii*, proteins containing signal peptides were screened using SignalP-6.0, as signal peptides play an important role in processes such as protein secretion, and proteins containing signal peptides are more likely to produce active antimicrobial peptides. Next, these protein sequences were processed using Kmer (k=18) and then input into a self-constructed activity prediction model to form a peptide library. The library was then used for prediction by the Hydro-AMP website and screening for antimicrobial peptides with good activity by the DBAASP website. Finally, manual screening was performed based on three criteria: removal of sequences containing cysteine ​​residues, removal of sequences containing five or more identical amino acids, and stable predicted secondary structures. This yielded the *Nematocystis jirovecii*-derived antimicrobial peptide TAMPa-6, whose amino acid sequence is shown in SEQ ID NO. 1.

[0020] 2. Synthesis of the antimicrobial peptide TAMPa-6 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.

[0021] 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, dissolved in pure water, and then subjected to 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 of 98.3%.

[0022] 3. The physicochemical properties of the antimicrobial peptide TAMPa-6 are shown in Table 1.

[0023] Table 1. Sequence and physicochemical properties of the synthesized antimicrobial peptide TAMPa-6

[0024] The helical wheel diagram of the antimicrobial peptide TAMPa-6 is shown below. Figure 1 As shown.

[0025] This invention uses alphafold 3 to predict the secondary structure of the antimicrobial peptide TAMPa-6, and the results show the presence of an α-helix, wherein the generated secondary structure model is as follows. Figure 2 As shown in the figure. Circular dichroism spectroscopy was used to detect the antimicrobial peptide TAMPa-6, revealing negative absorption at 208 nm and 222 nm, and positive absorption at 190 nm, confirming the presence of an α-helix. The results are as follows. Figure 3 As shown.

[0026] Example 2: Antibacterial activity of antimicrobial peptide TAMPa-6 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 is Propionibacterium acnes ATCC6919.

[0027] After reviving the bacterial strain, it was subcultured once, and single colonies were picked and cultured in BHI 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. 200 µL of the antimicrobial peptide TAMPa-6 was added to the first column of rows A, B, and C. The solution was then diluted using a micro-two-fold dilution method until the last well. 100 µL of the diluted bacterial suspension was added (the final bacterial concentration was approximately 1 x 10⁻⁶). 6 (CFU / mL), ensuring a final volume of 200µL per well. Negative and positive controls were established to ensure bacterial growth. The 96-well plate was then incubated anaerobically at 37°C for 96 hours, and colony growth was observed. Then, 100µL of bacterial culture from wells with concentrations of 2MIC, 4MIC, 8MIC, and 16MIC were spread onto BHI agar plates and incubated anaerobically at 37°C for 96 hours, and the MBC value was recorded.

[0028] The experimental results are shown in Table 2. The results indicate that the antimicrobial peptide TAMPa-6 has significant antimicrobial activity against Propionibacterium acnes ATCC6919, with a MIC and MBC of 2 µg / mL and 16 µg / mL, respectively.

[0029] Table 2 MIC and MBC of TAMPa-6

[0030] Example 3: Determination of the inhibition zone of the antimicrobial peptide TAMPa-6 In a clean bench, take a solidified BHI agar plate and use a sterile pipette to draw 100 µL of Propionibacterium acnes suspension (bacterial concentration approximately 5 × 10⁻⁶). 7Add a drop (CFU / mL) to the center of the plate. Using a sterile cotton swab (after dipping it in the bacterial suspension, gently squeeze it against the tube wall to remove excess suspension), evenly spread the bacterial suspension across the entire plate surface. When spreading, start from one direction and gradually move to the opposite direction to ensure even distribution. Spread the suspension at least three times per plate, rotating it 60° after each spread to ensure uniform inoculation. After spreading, place the plate in a laminar flow hood with the lid open for 5-10 minutes to allow the bacterial suspension to be fully absorbed, preventing filter paper slippage during subsequent operations. Use sterile forceps to pick up sterile filter paper discs and immerse them in different concentrations of the antimicrobial peptide TAMPa-6 solution for 1-2 minutes to ensure full absorption. Remove the soaked filter paper discs with sterile forceps and gently blot away excess solution on sterile filter paper (to prevent solution dripping and contaminating the plate). The treated filter paper was placed on a BHI agar plate containing inoculated bacterial solution and anaerobically cultured at 37°C for 5 days. The diameter of the inhibition zone was measured by mutagenesis calipers.

[0031] The experimental results are shown in Table 2. The results indicate that the antimicrobial peptide TAMPa-6 has a good inhibitory effect on Propionibacterium acnes ATCC6919. The inhibition zone increased with increasing concentration of the antimicrobial peptide, reaching 16.42 mm at 0.5 mg / mL. (Inhibition zone diagram follows.) Figure 5 As shown.

[0032] Table 3. Antibacterial activity of different concentrations of TAMPa-6

[0033] Example 4: Cytotoxicity and hemolytic toxicity of the antimicrobial peptide TAMPa-6 The toxicity of the antimicrobial peptide TAMPa-6 to Hacat cells was determined using a CCK-8 assay kit (CCK-8, Dojindo, Kumamoto, Japan). Logarithmically growing cells were collected, counted, and the cell pellet was diluted and resuspended in complete culture medium. 10,000 Hacat cells were added to each well of a 96-well plate. Cells were incubated for 24 h in a 5% CO2 incubator at 37°C. The antimicrobial peptide was dissolved in sterile dd H2O and diluted to a high-concentration stock solution. The stock solution was serially diluted twofold with complete culture medium to a final test concentration of 10 times. Diluted antimicrobial peptides (final concentrations of 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg / ml) were added to each well, with three replicates for each concentration. A control group was added to complete culture medium. Cells were incubated for 24 h in a 5% CO2 incubator at 37°C. Add CCK-8 diluent (10-fold dilution of the stock solution with complete culture medium) to each well, add 5% CO2, and incubate at 37°C for 2-4 hours. Then measure the absorbance at 450 nm. Cell viability % = OD value of drug-treated group / OD value of control group * 100%.

[0034] Cytotoxicity assessment results such as Figure 5 As shown, at a concentration of 128 μg / mL, the cell viability of TAMPa-6 was still higher than 80%, which is much higher than the MIC value, indicating that TAMPa-6 has great application potential.

[0035] Human erythrocytes used in the hemolysis assay were purchased from HaemoScan, Netherlands. Blood samples were aliquoted and washed twice with 5 mL of washing buffer, then centrifuged at 2500 rpm for 10 minutes at 4°C. This step was repeated twice. The precipitate was resuspended in 5 mL of dilution buffer to a final concentration of 5%. The peptide was added to the diluted erythrocyte suspension, and nine concentrations were tested (maximum concentration 256 μg / mL, serially diluted twofold). PBS was used as a negative control (0% hemolysis), and 1% Triton X-100 was used as a positive control (100% hemolysis). The mixture was incubated at 37°C and 100 rpm for 1 hour. After incubation, the sample was centrifuged at 5000 rpm for 1 minute, and the absorbance of the supernatant at 540 nm was measured using a microplate reader to quantify hemolysis. This assay was performed in triplicate.

[0036] .

[0037] Hemolytic toxicity assessment results as follows Figure 6 As shown, TAMPa-6 exhibits hemolytic activity of less than 10% at 500 μg / mL, a concentration far exceeding the MIC value, indicating that TAMPa-6 has a high safety profile.

[0038] Example 5: Antimicrobial peptide TAMPa-6 exerts its bactericidal effect by disrupting bacterial cell membranes. Coarse-grained simulations were used to predict the interaction between the antimicrobial peptide TAMPa-6 and the bacterial cell membrane. The results are as follows: Figure 7 This indicates that the antimicrobial peptide TAMPa-6 comes into contact with and is attracted to the bacterial cell membrane. Then, scanning electron microscopy was used to observe the changes in the bacterial cell membrane after treatment with the antimicrobial peptide TAMPa-6, such as... Figure 8 As shown, the bacterial cell membrane exhibits significant shrinkage, indicating that the antimicrobial peptide TAMPa-6 exerts its bactericidal effect by disrupting the bacterial cell membrane.

[0039] Example 6: Antimicrobial products and compositions containing the antimicrobial peptide TAMPa-6 (1) Antibacterial gel: Dissolve 1g of antimicrobial peptide TAMPa-6 and 1.5g of sodium hyaluronate in an appropriate amount of pH 7.2 10mM PBS buffer. After complete dissolution, mix the two evenly, adjust the osmotic pressure to 300mOsm / L with sodium chloride, and bring the volume to 1L. Dispense into polyethylene tubes and sterilize by moist heat to obtain antibacterial gel. As sample 1 (2) Antimicrobial peptide cosmetics: antimicrobial peptide TAMPa-6 0.1%, sodium hyaluronate 0.1%, hydroxyethyl cellulose 0.05%, glycerin 5%, 1,3-propylene glycol 3%, 0.5% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, rose fragrance 0.5%, and the balance is water.

[0040] Dissolve the antimicrobial peptide in an appropriate amount of water according to the formula. This is component I.

[0041] According to the formula, dissolve glycerin, 1,3-propylene glycol, 1,2-hexanediol, and p-hydroxyacetophenone in an appropriate amount of purified water at 30-40°C. Then add hyaluronic acid and hydroxyethyl cellulose and stir until completely dissolved. This is component II.

[0042] Mix component I with component II and stir until homogeneous to obtain component III.

[0043] Rose fragrance was added to component III, and the volume was adjusted to the target volume with purified water to obtain the cosmetic product. This was used as sample 2.

[0044] (3) Antimicrobial peptide composition: 1-10 parts of antimicrobial peptide, 1-2 parts of sodium hyaluronate, and 880-985 parts of purified water.

[0045] Dissolve 0.5g of antimicrobial peptide TAMPa-6 and 0.2g of sodium hyaluronate separately in purified water. After complete dissolution, mix the two thoroughly and bring the volume to 100mL to obtain the antimicrobial peptide composition, which is sample 3.

[0046] Following the experimental method of Example 3, the antibacterial activity of Sample 1, Sample 2, and Sample 3 against Propionibacterium acnes ATCC6919 was determined. The inhibition zones were 18.25 mm, 22.63 mm, and 25.75 mm, respectively, showing good antibacterial activity against Propionibacterium acnes ATCC6919. This indicates good application prospects in treating acne caused by excessive proliferation of Propionibacterium acnes or in acne removal.

[0047] 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 antibacterial peptide that inhibits Propionibacterium acnes, characterized by: The amino acid sequence of the antibacterial peptide for inhibiting Propionibacterium acnes is shown as SEQ ID NO.

1.

2. The method of claim 1, wherein the antibacterial peptide for inhibiting P. acnes is prepared by the steps of: The Fmoc amino acid is subjected to repeated deprotection-coupling cycles to form a resin peptide on the resin by Fmoc solid-phase polypeptide synthesis method, and then the resin peptide is cleaved to obtain a linear peptide, which is purified by high performance liquid chromatography to obtain an antibacterial peptide with a purity of more than 95%. ​ 3. Use of the antibacterial peptide for inhibiting Propionibacterium acnes according to claim 1 in the preparation of an antibacterial product.

4. An antibacterial product comprising the antibacterial peptide of claim 1 that inhibits P. acnes, characterized by: The antibacterial product is an external medicine for treating acne, or a product for preventing Propionibacterium acnes infection.

5. The antimicrobial product of claim 4, wherein: The antibacterial product is a solution, a spray, a cream or a gel.

6. A skin care product, characterized by The skin care product contains the antibacterial peptide for inhibiting Propionibacterium acnes according to claim 1 or the antibacterial product according to any one of claims 4-5.

7. A skin care article according to claim 6 wherein: The skin care product is a mask, essence, emulsion, cream, spray or gel.

8. A composition characterized in that: The antibacterial peptide for inhibiting Propionibacterium acnes according to claim 1, the antibacterial product according to any one of claims 4-5, or the skin care product according to any one of claims 6-7.

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