Antibacterial combined peptide and application thereof

Abstract

The invention relates to the technical field of biology, and provides an antibacterial combined peptide and application thereof, the amino acid sequence of the antibacterial combined peptide is one of the following: Meta180: X1LRRX1LX2ILLX3RI, Meta204: IWRX4X4IRRLLRX4I, Meta203: IRVX4X4RR X5LRWLR, X1, X2, X3, X4 and X5 are random sites, X1 is L or I, X2 is Q or T, X3 is R or K, X4 is I or V, and X5 is V or L; the Meta180, the Meta204 and the Meta203 are respectively a mixture of all selected sites in the random sites. The antibacterial combined peptide can effectively act on various bacteria such as escherichia coli, pseudomonas aeruginosa and staphylococcus aureus, and has the characteristics of broad-spectrum antibacterial activity and low toxicity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a class of antimicrobial combination peptides and their applications. Background Technology

[0002] With the increasing overuse of antibiotics, drug-resistant bacteria are spreading rapidly, seriously threatening global public health security. Antimicrobial peptides, due to their natural bactericidal mechanisms and extremely low risk of resistance, are considered ideal candidates to replace antibiotics.

[0003] CN110305194A discloses an antimicrobial polypeptide with the following amino acid sequence: Gly–Lys–Ile–Lys–Ile–Gly–Ile–Asn–Gly–Phe–Gly–Arg–Ile–Gly–Arg–Leu–Val–Ala–Arg–Val. CN115433258A discloses an antimicrobial polypeptide and its application. It utilizes a compound probiotic fermentation method to enhance the antimicrobial activity against dominant bacteria causing prickly pear spoilage in walnut meal. The changes in antimicrobial active polypeptides during fermentation were investigated, and antimicrobial active polypeptides FGGDSTHP, ALGGGY, YVVPW, and PLLRW were screened. The interaction mechanism between the antimicrobial polypeptide and phenolic compounds was further analyzed, and the optimal antimicrobial combination YVVPWSA was obtained to enhance the synergistic antimicrobial activity against *P. victoriae*. However, its antimicrobial activity is usually only applicable to one type of pathogen or a limited range of pathogens, and its broad-spectrum activity remains insufficient.

[0004] CN119285699A discloses an antimicrobial polypeptide derived from walnut meal and its application. Using walnut meal as raw material, the antimicrobial polypeptide is extracted and separated, which can effectively improve the utilization rate of walnut meal, reduce resource waste, provide new polypeptide types for the field of antimicrobial polypeptides, and provide new resources for the development of novel antimicrobial agents, bacterial lysates, food preservatives, and anti-infective drugs.

[0005] However, due to their complex synthesis process and high cost, they are difficult to widely use in clinical and public health systems. To overcome this bottleneck, simplifying the synthesis process of antimicrobial peptides, reducing synthesis costs, and improving the practicality and accessibility of antimicrobial peptides will provide a sustainable, cost-effective solution for the upgrading of antimicrobial drugs, which has significant social implications. Summary of the Invention

[0006] This invention constructs an antimicrobial combination peptide with synergistic antibacterial effects, effectively targeting a variety of drug-resistant bacterial strains. Through formulation optimization and process simplification, the final antimicrobial combination peptide is obtained in a one-pot process using mixed amino acids during solid-phase synthesis, eliminating the need for pure product formulation and complex purification steps such as HPLC. This significantly reduces synthesis costs to 1 / 30th that of traditional antimicrobial peptides. This technological breakthrough not only improves the practicality and accessibility of antimicrobial peptide formulations but also provides a sustainable, cost-effective solution for the upgrading of antimicrobial drugs, possessing significant social implications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A class of antimicrobial combination peptides, wherein the amino acid sequence of the antimicrobial combination peptide is one of the following: Meta180: X1LRRX1LX2ILLX3RI Meta204: IWRX4X4IRRLLRX4I Meta203: IRVX4X4RR X5LRWLR Among them, X1, X2, X3, X4 and X5 are random sites, X1 is L or I, X2 is Q or T, X3 is R or K, X4 is I or V, and X5 is V or L; Meta180, Meta204 and Meta203 are mixtures of all selected random sites.

[0008] The present invention also provides the use of the aforementioned antimicrobial combination peptide in the preparation of products for treating and / or preventing bacterial infections.

[0009] The products include one or more of the following: medical devices, implantable medical devices, drugs, excipients, food preservatives, and pet disinfectants.

[0010] The bacteria are Gram-positive and / or Gram-negative.

[0011] The bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis.

[0012] The present invention also provides nucleotide sequences encoding the aforementioned antimicrobial combination peptides, including DNA and RNA.

[0013] The present invention also provides an expression vector containing the nucleotide sequence described above.

[0014] The present invention also provides compositions containing the aforementioned antimicrobial combination peptides.

[0015] The composition contains one or more of a pharmaceutically or food-grade acceptable carrier, excipient, diluent, adjuvant, or medium.

[0016] The present invention also provides an antimicrobial product comprising the aforementioned antimicrobial combination peptide, the aforementioned nucleotide sequence, or the aforementioned composition. For example, using the antimicrobial combination peptide as a coating of an implantable medical device imparts antimicrobial efficacy to the implantable medical device.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an antimicrobial combination peptide with synergistic antimicrobial effects, which can effectively target a variety of drug-resistant strains, exhibiting broad-spectrum antimicrobial activity and low toxicity. This provides a sustainable, cost-effective solution for the upgrading of antimicrobial drugs and has significant social implications. Attached Figure Description

[0018] Figure 1 The ability of Meta180, Meta204 and Meta203 to target the outer membrane permeability of Acinetobacter baumannii pathogen at different concentrations.

[0019] Figure 2 The ability of Meta180, Meta204 and Meta203 to depolarize the plasma membrane of Acinetobacter baumannii pathogen at different concentrations. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0021] All raw materials used in the following specific implementation methods were purchased from the market.

[0022] Example 1 The synthesis of antimicrobial combination peptides specifically includes: 1. Swelling of dendrites: Weigh 2-chlorotriphenylmethyl chloride resin (2-CTC) and soak it in dichloromethane (DCM) to swell the resin for 1 hour. Then wash the resin three times with dimethyl thionamide (DMF) to remove residual solvent and impurities.

[0023] 2. Connecting the first amino acid: Take one or two protected amino acids (e.g., 1 eq of amino acid A and 1 eq of amino acid B in a 1:1 molar ratio) and react them with 1.5 eq of diisopropylethylamine (DIEA) in DMF for 2 hours to connect these amino acids to the resin.

[0024] 3. End-capping: After the resin is dried, it is washed three times with DMF, and then methanol and DIEA are added for end-capping treatment. The reaction is carried out for 1 hour to protect the amino groups on the resin.

[0025] 4. Removal of Fmoc protecting group: Use 20% piperidine in DMF to remove the Fmoc protecting group, react for 10 minutes, and repeat twice to ensure complete removal of the protecting group.

[0026] 5. Linking new amino acids: After washing the resin, add 3 eq of one or two amino acids (e.g., amino acid C and amino acid D in a 1:1 molar ratio) and 3 eq of HOBT and 3 eq of DIC, and react in DMF for 1.5 hours to link these amino acids to the peptide chain.

[0027] 6. Repeat steps 4 and 5 until all the required amino acids are added, and remove the N-terminal Fmoc protecting group. Wash the resin and dry it to prepare for the subsequent deprotection step.

[0028] 7. Deprotection and peptide chain cleavage: The reaction was carried out using 95% trifluoroacetic acid (TFA), 2% Tis (thiazolidinone), 2% EDT (ethylenediaminetetraacetic acid), and 1% water for 2 hours to cleave the protecting groups of the resin and peptide side chains.

[0029] 8. Filtration and precipitation: Filter the resin, wash the filtrate with ice-cold ether, centrifuge and retain the precipitate, which is the crude product. After freeze-drying, the final random peptide mixture is obtained.

[0030] Example 2: Antimicrobial activity analysis of three AMPCs (antimicrobial combination peptides) Step 1: Inoculate the seven bacterial cultures [Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Acinetobacter baumannii ATCC 19606, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633] into sterile MHB medium and incubate overnight at 37˚C and 250 rpm for 18 hours.

[0031] Step 2: Prepare the three antimicrobial combination peptides to 128 µg / mL with PBS, and then serially dilute them with PBS at concentrations of 128, 64, 32, 16, 8, 4, 2, and 1 µg / mL. Add 50 µL of each concentration to the AH wells of a 96-well plate. Repeat each antimicrobial combination peptide preparation three times, adding to wells 1-9.

[0032] Step 3: Dilute the seven cultured bacteria to 5 × 10⁵ CFU / mL with PBS. (Due to the large number of bacterial species, only one bacterial culture is used per plate; Staphylococcus aureus will be used as an example later.) Add 50 µL of bacterial dilution to each of the AF wells containing the existing antimicrobial peptide dilution. The peptide concentrations (µg / mL) at this point are shown in Table 1 below.

[0033] Table 1. Final concentrations of the antimicrobial combination peptides Meta180, Meta204, and Meta203 in the MIC assay, in µg / mL. Step 4: Add 100 µL of PBS solution to wells A10-H10 as a negative control, and add 100 µL of bacterial culture (5 × 10⁵ CFU / mL) to wells A11-H11 as a positive control. Seal the 96-well plate with sealing film, place it in a self-sealing bag, and incubate overnight at 37˚C for 18 hours. Measure the OD600 values ​​of wells A1-G9 using a microplate reader. The minimum concentration corresponding to the lowest OD600 value in each antimicrobial combination peptide group is the minimum inhibitory concentration (MIC) of the corresponding bacteria.

[0034] Table 2. MICs (µg / mL) of the antimicrobial combination peptides Meta180, Meta204, and Meta203 against seven pathogens. The antimicrobial combination peptides Meta180, Meta204, and Meta203 are compositions of antimicrobial peptides with multiple sequences. These compositions are obtained directly through synthesis, rather than by synthesizing multiple antimicrobial peptides and then mixing them. In the table, the amino acid sequences of the peptides are represented by one random amino acid within parentheses, such as (LV) indicating L or V. For example, the specific sequence of the antimicrobial combination peptides might be as follows: Meta180 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 1~16, specifically: SEQ ID NO.1: LLRRLLQILLRRI SEQ ID NO.2: LLRRLLQILLKRI SEQ ID NO.3: LLRRLLTILLRRI SEQ ID NO.4: LLRRLLTILLKRI SEQ ID NO.5: LLRRILQILLRRI SEQ ID NO.6: LLRRILQILLKRI SEQ ID NO.7: LLRRILTILLRRI SEQ ID NO.8: LLRRILTILLKRI SEQ ID NO.9: ILRRLLQILLRRI SEQ ID NO.10: ILRRLLQILLKRI SEQ ID NO.11: ILRRLLTILLRRI SEQ ID NO.12: ILRRLLTILLKRI SEQ ID NO.13: ILRRILQILLRRI SEQ ID NO.14: ILRRILQILLKRI SEQ ID NO.15: ILRRILTILLRRI SEQ ID NO.16: ILRRILTILLKRI Meta204 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 17~24, specifically: SEQ ID NO.17: IWRIIIRRLLRVI SEQ ID NO.18: IWRIIIRRLLRII SEQ ID NO.19: IWRIVIRRLLRVI SEQ ID NO.20: IWRIVIRRLLRII SEQ ID NO.21: IWRVIIRRLLRVI SEQ ID NO.22: IWRVIIRRLLRII SEQ ID NO.23: IWRVVIRRLLRVI SEQ ID NO.24: IWRVVIRRLLRII Meta203 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 25~32, specifically: SEQ ID NO.25: IRVVIRRVLRWLR SEQ ID NO.26: IRVVIRRLLRWLR SEQ ID NO.27: IRVVVRRVLRWLR SEQ ID NO.28: IRVVVRRLLRWLR SEQ ID NO.29: IRRIVLRWLR SEQ ID NO.30: IRRVIIRRLLRWLR SEQ ID NO.31: IRVIVRRVLRWLR SEQ ID NO.32: IRVIVRRLLRWLR Table 2 shows that the three antimicrobial combination peptides of the present invention exhibit good antibacterial activity against seven different bacteria, demonstrating high efficacy and broad applicability.

[0035] Example 3: Investigation into the antibacterial mechanism of AMPCs Step 1: NPN (1-naphthylaminobenzene) uptake assay to determine the effect of the antimicrobial combination peptide on the extracellular membrane permeability of Acinetobacter baumannii.

[0036] Logarithmically growing microbial cells were harvested (5,000 rpm, 5 min) and diluted to OD 600 = 0.2 in 5 mM HEPES buffer (pH = 7.4, containing 5 mM glucose). The bacterial suspension was further incubated with 10 μM NPN in the dark for 30 min. Subsequently, different concentrations of peptides were added (final peptide concentrations of 128–1 µg / mL, serially diluted 2-fold), and NPN fluorescence was detected (excitation λ = 350 nm, emission λ = 420 nm).

[0037] The results are as follows Figure 1 As shown, the fluorescence signal detected after treatment with AMPCs increased significantly, indicating that the permeability of the bacterial outer membrane was significantly enhanced, and that AMPCs exerted their bactericidal effect through membrane interaction mechanisms.

[0038] Step 2: DiSC3-5 staining was used to determine the effect of the antimicrobial combination peptides on the depolarization of the inner membrane of Acinetobacter baumannii.

[0039] Bacteria in mid-log phase were harvested and washed three times with 5 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer, then resuspended in buffer containing 0.1% glucose to an OD600 of 0.07. DiSC3-5 was added to a final concentration of 0.4 μM, and the mixture was incubated at 37°C in the dark for 90 min. Subsequently, 300 mM KCl was added and incubated for 20 min, followed by treatment with different concentrations of peptide. Changes in membrane potential were assessed by monitoring fluorescence (excitation wavelength 622 nm, emission wavelength 670 nm) until the fluorescence signal stabilized. The results were recorded, background fluorescence was subtracted, and the data were normalized.

[0040] The results are as follows Figure 2 As can be seen, the fluorescence signal was significantly increased after treatment with AMPCs, indicating that the inner membrane was significantly depolarized and lost its potential. AMPCs exerted their bactericidal effect through membrane interaction mechanism.

Claims

1. A class of antimicrobial combination peptides, characterized in that, The amino acid sequence of the antimicrobial combination peptide is one of the following: Meta180: X1LRRX1LX2ILLX3RI Meta204: IWRX4X4IRRLLRX4I Meta203: IRVX4X4RR X5LRWLR Among them, X1, X2, X3, X4 and X5 are random sites, X1 is L or I, X2 is Q or T, X3 is R or K, X4 is I or V, and X5 is V or L; Meta180, Meta204 and Meta203 are mixtures of all selected random sites.

2. The use of the antimicrobial combination peptide according to claim 1 in the preparation of products for treating and / or preventing bacterial infections.

3. The application according to claim 2, characterized in that, The products include one or more of the following: medical devices, implantable medical devices, drugs, excipients, food preservatives, and pet disinfectants.

4. The application according to claim 2, characterized in that, The bacteria are Gram-positive and / or Gram-negative.

5. The application according to claim 2, characterized in that, The bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis.

6. The nucleotide sequence encoding the antimicrobial combinatorial peptide of claim 1, characterized in that, Including DNA and RNA.

7. An expression vector containing the nucleotide sequence of claim 6.

8. A composition containing the antimicrobial combination peptide of claim 1.

9. The composition according to claim 8, characterized in that, The composition contains one or more of a pharmaceutically or food-acceptable carrier, excipient, diluent, adjuvant, or medium.

10. An antibacterial product, characterized in that, It includes the antimicrobial combination peptide of claim 1, the nucleotide sequence of claim 7, or the composition of claim 8.

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