A natural, broad-spectrum and high-efficiency antimicrobial peptide derived from fungi and application thereof

Abstract

The application discloses a natural, broad-spectrum and high-efficiency antibacterial peptide derived from a fungus and application thereof, and belongs to the technical field of biotechnology. The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 1, contains 13 amino acids, the molecular weight is 1808.25 Da, the isoelectric point is 12.02, the instability index is 31.03, the average hydrophilicity is -0.377, the hydrophobicity is 49.12, and the transmembrane capacity is -7.5 kcal / mol. The antibacterial peptide has broad-spectrum bacteriostatic activity, and has high bacteriostatic activity on G + and G ‑ Clinical drug-resistant strains have high bacteriostatic activity, and have great clinical drug development value; the antibacterial peptide is screened from the human intestinal symbiotic fungus Mucor circinatus, has the advantages of no hemolytic activity, low cytotoxicity, high in-vivo bacteriostatic activity and high stability, and has application safety; the antibacterial peptide also has strong bacteriostatic activity on common infection bacteria of human skin, such as Staphylococcus aureus and Pseudomonas aeruginosa, and intestinal pathogenic bacteria such as Salmonella and Shigella, and has wide application prospects of developing the antibacterial peptide into an antibacterial drug for human body.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a natural, broad-spectrum, and highly effective antimicrobial peptide derived from fungi and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of small-molecule polypeptides widely found in organisms in nature. They typically contain 10-80 amino acids and exhibit good inhibitory or killing effects against various microorganisms, including bacteria, fungi, and viruses. With the overuse and abuse of antibiotics in recent years, bacterial resistance and even multidrug resistance have become frequent occurrences, posing a bottleneck challenge in the treatment of bacterial infections. Due to their high antibacterial activity, broad antibacterial spectrum, and low tendency to induce resistance, antimicrobial peptides offer a promising alternative to antibiotics.

[0003] Antimicrobial peptides are widely derived, including from animals, plants, and microorganisms. Fungi, as an emerging source of peptides with therapeutic potential, offer a significant advantage in novel antimicrobial peptide discovery due to their species diversity. The human gut is a crucial site for bacterial-fungal symbiosis, where complex synergistic and antagonistic interactions exist. Based on these symbiotic relationships, rapid characterization using a combination of computational and experimental methods, and screening for broad-spectrum, highly effective novel antimicrobial peptides from the fungal genome, are of great significance for applications such as the defense against clinical bacterial infections. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a natural, broad-spectrum, and highly effective antimicrobial peptide derived from fungi and its applications.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a broad-spectrum, highly effective antimicrobial peptide derived from fungi, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0006] Based on the above technical solution, the antimicrobial peptide has a molecular weight of 1808.25 Da, an isoelectric point of 12.02, an instability index of 31.03, an average hydrophilicity of -0.377, a hydrophobicity of 49.12, and a transmembrane activity of -7.5 kcal / mol.

[0007] In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the above-mentioned antimicrobial peptide.

[0008] Based on the above technical solution, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient, wherein the pharmaceutically acceptable carrier and / or excipient includes fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, and flavoring agents.

[0009] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0010] Based on the above technical solution, the pharmaceutical preparations further include tablets, capsules, granules, drop pills, oral liquid preparations, injections, and aerosols.

[0011] The present invention also provides the use of the above-mentioned antimicrobial peptides, pharmaceutical compositions, and pharmaceutical preparations in the preparation of medicaments for the prevention or treatment of bacterial infections.

[0012] Based on the above technical solution, the application further includes the prevention or treatment of skin bacterial infections and intestinal bacterial infections.

[0013] Based on the above technical solution, the bacteria further include Gram-positive bacteria and Gram-negative bacteria.

[0014] Based on the above technical solution, further, Gram-positive bacteria include Staphylococcus aureus; Gram-negative bacteria include Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella, and Shigella.

[0015] Compared with the prior art, the present invention has the following advantages: The fungal antimicrobial peptide MC of this invention has broad-spectrum and highly efficient antibacterial activity, and also exhibits strong antibacterial activity against clinically resistant strains. The antimicrobial peptide MC has the advantages of no hemolytic activity, low cytotoxicity, and high stability. The antimicrobial peptide MC has strong antibacterial activity against common skin infection bacteria, and has potential application value in the development of agents for the prevention and treatment of skin bacterial infections. The antimicrobial peptide MC has strong antibacterial activity against common human intestinal infection bacteria, and also has broad application prospects in the development of agents for the prevention and treatment of intestinal pathogenic bacterial infections. Attached Figure Description

[0016] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0017] Figure 1 This diagram illustrates the leakage of contents from *Escherichia coli* (A), *Salmonella typhi* (B), *Shigella* (C), *Staphylococcus aureus* Newman (D), *Acinetobacter baumannii* (E), and *Pseudomonas aeruginosa* (F) caused by the fungal antimicrobial peptide MC of this invention. Figure 2 The results of the hemolysis rate test of the fungal antimicrobial peptide MC of this invention on sheep erythrocytes; Figure 3 This is a diagram showing the cytotoxicity of the fungal antimicrobial peptide MC of this invention against human lung cancer cells A549. Figure 4 The diagram shows the effect of the fungal antimicrobial peptide MC of the present invention on the survival of the animal model *Staphylococcus aureus* MU50 (A), *Pseudomonas aeruginosa* (B), *Acinetobacter baumannii* (C), and *Salmonella typhi* (D) after infection. Detailed Implementation

[0018] To better understand the present invention, the following detailed description, in conjunction with specific embodiments and accompanying drawings, provides a further detailed description of a natural, broad-spectrum, and highly effective antimicrobial peptide MC derived from fungi and its applications.

[0019] Example 1: Screening of fungal antimicrobial peptides MC: Through the study of human symbiotic fungi, Mucor genus Mucor circinatus Whole genome sequence analysis was performed using the optimized antimicrobial peptide prediction program Deep-AmPEP30 (… https: / / cbbio.online / AxPEP / ) Antimicrobial peptides with high antimicrobial activity (>99.8%) and fewer than 15 amino acids were screened; the DBAASP database was used for further analysis. https: / / www.dbaasp.org / tools?page=linear-amp-prediction Antimicrobial peptides showing positive antimicrobial activity were screened based on their predictive indices for antimicrobial activity against different bacteria. Furthermore, CellPM was used to predict the transmembrane activity of antimicrobial peptides, screening for short peptide sequences with strong transmembrane activity (transfer energy < -5.0). Through a series of screening methods, antimicrobial peptides from the genus *Mucor* were selected. Mucor circinatus The short peptide sequence Ser-Lys-Arg-Phe-Lys-Trp-Ile-Arg-Lys-Ile-Leu-Phe-Trp (SEQ ID NO.1) was obtained, and the short peptide was chemically synthesized with a purity higher than 95%.

[0020] The fungal antimicrobial peptide MC of the present invention has 13 amino acid residues, and the specific amino acid sequence is: Ser-Lys-Arg-Phe-Lys-Trp-Ile-Arg-Lys-Ile-Leu-Phe-Trp. The single-letter abbreviation of the amino acid sequence is SKRFKWIRKILFW. The molecular weight of the fungal antimicrobial peptide MC is 1808.25 Da, and the isoelectric point is 12.02.

[0021] Example 2: Assay of the antibacterial activity of fungal antimicrobial peptides MC The strains involved in this embodiment include Gram-positive Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureusNewman) and Gram-negative bacteria Pseudomonas aeruginosa ( Pseudomonas aeruginosa PAO1), Escherichia coli ( Escherichia coli JM109), Acinetobacter baumannii ( Acinetobacter baumannii ATCC19606), Klebsiella pneumoniae ( Klebsiella pneumoniae 700603), Salmonella typhi ( Salmonella typhi Ty2) and Shigella ( Shigella m90T).

[0022] The experiment used a two-fold serial dilution method to determine the minimum inhibitory concentration, which is the lowest drug concentration that can inhibit bacterial growth and reproduction.

[0023] The MIC value determination process is as follows: 1. Prepare freshly streaked bacterial colonies. Scratch a small amount of the bacterial clone and place it in 1.5 mL of sterile water. Adjust the bacterial concentration to OD using a UV spectrophotometer. 600 =0.4, dilute the bacterial culture 200 times with an appropriate volume of MH medium, and adjust to OD. 600 =0.002; Mix well and dispense 75 μL of bacterial culture per well into a 96-well plate; 2. Dissolve the fungal antimicrobial peptide MC in DMSO at a concentration of 5 mg / mL; prepare 300 μL of the antimicrobial peptide in MH medium with a concentration gradient of 0 to 200 μg / mL; mix well by pipetting and add 75 μL / well to each well of a 96-well plate containing the bacterial culture to be tested, with three replicates for each concentration. 3. Incubate the 96-well plate in a constant temperature shaker at 37 °C and 180 rpm for 24 h; 4. The absorbance of the bacterial solution at 600 nm is measured by an ELISA reader. The minimum inhibitory concentration (MIC) is the average of the concentrations of the wells from which no bacterial growth is detected and the concentrations of the samples in adjacent wells.

[0024] The antibacterial activity of the fungal antimicrobial peptide MC was verified by three repeated experiments, as shown in Table 1. Its MIC value was 12.5-25 μg / mL.

[0025] Table 1. Results of antimicrobial activity of fungal antimicrobial peptide MC

[0026] Example 3: The MIC value determination method in Example 2 was used to perform antibacterial experiments on clinically resistant isolates. The strains involved in this embodiment include Gram-positive Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus MU50) and Gram-negative bacteria Pseudomonas aeruginosa ( Pseudomonas aeruginosa57006), Escherichia coli ( Escherichia coli MDR was used as a control experiment with meropenem, imipenem, ciprofloxacin, piperacillin, kanamycin, ampicillin, and polymyxin.

[0027] The antibacterial activity of the fungal antimicrobial peptide MC was verified by three repeated experiments, as shown in Table 2. Its MIC value was 10-25 μg / mL.

[0028] Table 2. Antimicrobial activity of fungal antimicrobial peptide MC against clinically multidrug-resistant strains

[0029] Example 4: Determination of the thermal and pH stability of fungal antimicrobial peptides (MC) The strain involved in this embodiment is the Gram-positive Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus MU50).

[0030] The experimental procedure is as follows: 1. Prepare 1×PBS phosphate buffer solution, filter through a 0.22 μm filter membrane and sterilize; use this as the stock solution, and adjust the pH of the solutions to 2, 4, 6, 8, 11, and 13 sequentially with HCl or NaOH; prepare 0.1M sodium acetate solution, filter through a 0.22 μm filter membrane and sterilize; use this as the stock solution, and adjust the pH of the solutions to 2, 4, 6, 8, 11, and 13 sequentially with HCl or NaOH. 2. pH stability: Prepare 500 μL of antimicrobial peptide MC containing 400 μg / mL using the above solution, mix well, and let stand at room temperature (25℃) for 1 h; Thermal stability: Prepare 500 μL of antimicrobial peptide MC containing 400 μg / mL using 1×PBS, mix well, and place in a 4℃ refrigerator for 1 h; place at room temperature of 25℃ for 1 h; and heat in a water bath at 50℃, 70℃ and 90℃ for 1 h respectively. 3. The MIC values ​​of the treated antimicrobial peptide MC and the untreated antimicrobial peptide MC were determined using the method in Example 2.

[0031] The results are shown in Table 3-4. Three repeated experiments showed that the MIC values ​​of the heat-treated antimicrobial peptide remained stable. The MIC values ​​of the antimicrobial peptide remained stable under neutral, weakly acidic, and weakly alkaline pH treatments. Strongly acidic and strongly alkaline pH treatments had a slight impact on the stability of the antimicrobial peptide MC.

[0032] Table 3. Results of thermostability test of fungal antimicrobial peptide MC

[0033] Table 4. pH stability test results of fungal antimicrobial peptide MC

[0034] Example 5: Determination of bacterial intracellular contents leakage induced by fungal antimicrobial peptide MC The strains involved in this embodiment include Gram-positive Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Newman) and Gram-negative bacteria Pseudomonas aeruginosa ( Pseudomonas aeruginosa PAO1), Escherichia coli ( Escherichia coli JM109), Acinetobacter baumannii ( Acinetobacter baumannii ATCC19606), Salmonella typhi ( Salmonella typhi Ty2) and Shigella ( Shigella m90T).

[0035] The experimental procedure is as follows: 1. Place the bacteria to be tested in BHI medium and incubate at 37 ℃ and 220 rpm for 12 h; then transfer them 1:100 to 2 mL of BHI medium and continue incubation at 37 ℃ and 220 rpm for 6 h. 2. Adjust the bacterial concentration to OD using PBS. 600 =0.1; 2.5 mL of bacterial culture was sequentially dispensed into 5 mL EP tubes, and antimicrobial peptide MC was added to the EP tubes at final concentrations of 10 μg / mL and 20 μg / mL, respectively. The tubes were then placed on a mixing rack and incubated at room temperature; a bacterial culture containing 20 μL of 0.01% Triton-X 100 was used as a positive control; 3. At 0 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, respectively, 200 μL of bacterial culture was pipetted into a 96-well plate (white plate), 2 μL of 1 mg / mL propidium iodide was added, and the mixture was mixed. The plate was then incubated at room temperature for 15 minutes. 4. Place the 96-well plate in a microplate reader and use an excitation wavelength of 540 nm and an emission wavelength of 621 nm to detect the staining of propidium iodide; The results are as follows Figure 1 As shown in the three repeated experiments, the staining intensity of propidium iodide increased with the extension of the incubation time of the antimicrobial peptide MC, indicating that the antimicrobial peptide MC can cause leakage of various bacterial intracellular contents.

[0036] Example 6: Determination of hemolysis rate of fungal antimicrobial peptide MC The experimental procedure is as follows: 1. Take an appropriate amount of sheep blood, dilute it 3 times with PBS buffer (pH 7.4), and aliquot 190 μL / well into a 96-well plate; 2. Prepare antimicrobial peptide MCs at different concentration gradients using PBS buffer (pH 7.4): 0 (negative control), 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μg / mL, with a buffer containing 0.2% Triton X-100 as a positive control. 3. Add 10 μL of antimicrobial peptide solution to the 96-well plate containing sheep blood; incubate at 37°C for 60 min. 4. Remove the 96-well plate, centrifuge (3000×g, 5 min, 4℃), transfer the supernatant to a new 96-well plate, and measure the absorbance at 567 nm using a microplate reader; 5. Antimicrobial peptide hemolysis rate is calculated as (OD of test wells). 567 - Negative pore OD 567 ) / (Positive well OD 567 - Negative pore OD 567 ) × 100% for calculation.

[0037] The results are as follows Figure 2 As shown, compared with the control group, the antimicrobial peptide MC did not cause significant hemolysis of erythrocytes at concentrations up to 800 μg / mL, with a hemolysis rate of less than 10%.

[0038] Example 7: Assay for the cytotoxicity of fungal antimicrobial peptides (MC) The Cell Counting Kit-8 (CCK-8) method was used to determine cytotoxicity. The experimental procedure is as follows: 1. Human lung cancer A549 cells were cultured in DMEM medium, cell counts were performed, and the cell concentration was adjusted to 7. 10 ^4 cells / mL; 2. Add 100 μL of PBS to the outermost edge of the 96-well plate to prevent cell sap evaporation and experimental errors; seed 100 μL / well of cell suspension into the remaining 96-well plates, and add DMEM complete medium to the remaining column as a blank group. Incubate the 96-well plates overnight at 37 ℃ and 5% CO2 (usually 12 h).

[0039] 3. Remove the 96-well plate, remove the cell supernatant, and add 100 μL of DMEM complete medium containing DMSO control group and gradient concentrations of antimicrobial peptides respectively. Incubate at 37 ℃ and 5% CO2 for 24 h.

[0040] 4. Remove the supernatant cell solution, add 100 μL of fresh DMEM complete medium to each well, and then add 10% CCK-8 solution (be careful not to generate air bubbles, otherwise it will affect the accuracy of the spectrophotometer reading). Incubate the 96-well plate at 37 ℃ for 2 h.

[0041] 5. The absorbance at 450 nm was measured using an ELISA reader.

[0042] 6. Repeat the experiment three times.

[0043] Cell viability = (Antimicrobial peptide treatment group - Blank) / (DMSO control group - Blank) × 100% The results are as follows Figure 3 As shown, the antimicrobial peptide MC solution and A 549 After incubation, the cell suspension showed OD 450 The test results showed that no cytotoxicity was observed when the concentration of the antimicrobial peptide MC was less than or equal to 50 μg / mL.

[0044] Example 8: In vivo bactericidal activity assay of fungal antimicrobial peptide MC The strains involved in this embodiment include Gram-positive Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus MU50) and Gram-negative bacteria Pseudomonas aeruginosa ( Pseudomonas aeruginosa 57006), Acinetobacter baumannii ( Acinetobacter baumannii 102147), Salmonella typhi ( Salmonella typhi Ty2).

[0045] This experiment used an animal infection model, wax moth larvae, to determine the bactericidal activity of the fungal antimicrobial peptide MC in vivo. The experimental procedure is as follows: 1. Weigh 0.3-0.4 g of wax moth larvae and starve them in a 37 ℃ incubator for 12 h; randomly assign 15 wax moth larvae to a petri dish; 2. Collect fresh bacterial culture that has been cultured at 37 ℃ and 220 rpm for 12 h, and adjust the bacterial culture to OD using PBS. 600 =0.2; According to the half-lethal dose of different bacteria, the bacterial solution was diluted with different concentration gradients; 100 μL of bacterial solution was taken out and antimicrobial peptide MC was added to a final concentration of 200 μg / mL and mixed well; 5 μL of bacterial solution and bacterial solution + antimicrobial peptide were injected into the left hind leg of the wax moth larvae, 15 larvae in each group; the wax moths were placed in an incubator at 37 ℃ for incubation; 3. Using PBS as a blank control group, 5 μL was injected into the left hind leg of the wax moth larvae, with 15 larvae in each group; the wax moths were then incubated in a 37 ℃ incubator. 4. Record the mortality of wax moth larvae every 12 hours, up to 120 hours; 5. Repeat the experiment three times.

[0046] The results are as follows Figure 4 As shown, the antimicrobial peptide MC can significantly increase the survival rate of wax moth larvae after bacterial infection, that is, it has in vivo bactericidal activity.

[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; and these 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. A broad-spectrum, highly effective antimicrobial peptide derived from fungi, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

1.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide has a molecular weight of 1808.25 Da, an isoelectric point of 12.02, an instability index of 31.03, an average hydrophilicity of -0.377, an average hydrophobicity of 49.12, and a transmembrane activity of -7.5 kcal / mol.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an effective amount of the antimicrobial peptide of claim 1 or 2.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient, including fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, lubricants, and flavoring agents.

5. A pharmaceutical formulation comprising the pharmaceutical composition of claim 3 or 4.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparations include tablets, capsules, granules, pellets, oral liquid preparations, injections, and aerosols.

7. The use of the antimicrobial peptide of claim 1 or 2, the pharmaceutical composition of claim 3 or 4, or the pharmaceutical formulation of claim 5 or 6 in the preparation of a medicament for treating bacterial infections, characterized in that, The bacteria mentioned are Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, or Klebsiella pneumoniae.

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