An antibacterial peptide derived from a tailed virus class phage and application thereof

Abstract

The application discloses an antibacterial peptide derived from a Caudoviricetes bacteriophage and application thereof, and belongs to the technical field of bioinformatics and active peptide. The application screens an antibacterial peptide derived from a Caudoviricetes bacteriophage, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO:1, and specifically is MKTLRRLLRAIANRLGTIV. The application mines and identifies a novel antibacterial peptide with broad-spectrum antibacterial activity from macro-genome data, and verifies the antibacterial activity, and has important scientific significance and clinical application value.

Description

Technical Field

[0001] This invention belongs to the fields of bioinformatics and bioactive peptide technology, specifically relating to an antimicrobial peptide derived from tailed bacteriophages and its applications. Background Technology

[0002] The widespread use and even abuse of antibiotics has led to increasingly serious bacterial resistance, which has become a major challenge in the global public health field. In particular, the continuous emergence of common clinical pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii, and Klebsiella pneumoniae poses a serious threat to human health. These drug-resistant strains exhibit high resistance to multiple traditional antibiotics, leading to increased difficulty in clinical treatment, higher patient mortality rates, and significantly higher medical costs. Therefore, the development of novel, safe, and effective alternatives to antimicrobial drugs is urgently needed.

[0003] Antimicrobial peptides (AMPs) are a class of small polypeptides widely found in organisms in nature. They possess broad-spectrum antimicrobial activity, are less prone to inducing drug resistance, and have unique mechanisms of action, making them considered as alternatives to antibiotics. However, the number of known antimicrobial peptides is currently limited, especially novel antimicrobial peptides with potent antibacterial activity against clinically multidrug-resistant bacteria, which remain scarce. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide derived from tailed bacteriophages and to provide specific applications of this antimicrobial peptide, so as to make up for the shortcomings of the prior art.

[0005] Metagenomics technology has developed rapidly in recent years, providing an efficient technical means to discover novel antimicrobial peptides from the environmental or host microbiome. However, the number of antimicrobial peptides discovered based on metagenomics is still limited, especially those with experimentally verified antimicrobial activity. Therefore, discovering novel antimicrobial peptides from metagenomic data and validating their functions has significant scientific research value and promising clinical applications.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: A type of virus derived from the class Tailed Virus ( Caudoviricetes The antimicrobial peptide of bacteriophage, wherein the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1, specifically: MKTLRRLLRAIANRLGTIV.

[0007] The origin is from the class Caudata ( Caudoviricetes The screening method for antimicrobial peptides from bacteriophages includes the following steps: (1) Prediction: Antimicrobial peptides were predicted from metagenomic sequences using Macrel v1.5.0, amPEPpy v1.1.0 and existing deep learning pipeline methods; (2) Screening: Sequences predicted by at least two of the above methods are identified as candidate antimicrobial peptides; (3) Deduplication: The amino acid sequences were deduplicated to 100% identity using MMseqs2 v15.6f452, and representative sequences of peptide clusters were selected based on the maximum integrity of vOTUs; (4) Filtering: Candidate sequences are filtered and screened according to the following criteria: no matching with the public AMP database, fewer than 2 cysteine ​​residues, length not exceeding 50 amino acids, net charge ≥ +2, average hydrophobicity ≤ 1.5, to obtain the final target antimicrobial peptide sequence.

[0008] (5) Species annotation of metagenomically assembled phages was performed using VITAP v1.12. The antimicrobial peptide was derived from tailed viral phages.

[0009] The application of the antimicrobial peptide in the preparation of antimicrobial drugs.

[0010] The antimicrobial peptide is used in the preparation of drugs that inhibit Staphylococcus aureus, Acinetobacter baumannii, and / or Klebsiella pneumoniae.

[0011] The application of the antimicrobial peptide in the preparation of drugs that inhibit marine pathogens.

[0012] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention utilizes metagenomics and bioinformatics techniques to discover a novel antimicrobial peptide sequence, and its broad-spectrum antimicrobial activity against a variety of clinically relevant pathogens (including Gram-positive and Gram-negative bacteria) has been verified through in vitro antimicrobial experiments. Compared with traditional methods for extracting antimicrobial peptides from natural biological resources, this invention's metagenomics-based discovery strategy offers advantages such as high throughput, high efficiency, and the ability to discover novel peptides from unculturable microorganisms. This invention provides new candidate molecules for the development of novel antimicrobial drugs and has significant application value. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0014] Example 1: Screening of candidate antimicrobial peptides based on metagenomics This embodiment describes a specific method for screening antimicrobial peptide sequences from metagenomic data.

[0015] (1) Using Macrel v1.5.0, amPEPpy v1.1.0, and two deep learning pipeline methods, sequences predicted by at least two of the above four methods are identified as candidate antimicrobial peptides. The two deep learning pipeline methods are: (1) an antimicrobial peptide mining method based on a natural language processing fusion model, see the literature "Identification of antimicrobial peptides from the human gut microbiome using deep learning", published in Nature Biotechnology, March 3, 2022; (2) an interpretable deep learning and virtual evolution method based on the EvoGradient model, see the literature "Explainable deep learning and virtualevolution identifies antimicrobial peptides with activity against multidrug-resistant human pathogens", published in Nature Microbiology, January 17, 2025; (2) The amino acid sequences of candidate antimicrobial peptides were deduplicated to ensure 100% identity, and only one identical sequence was retained. Representative sequences of peptide clusters were selected based on the maximum integrity of vOTUs (viral operational taxonomic units).

[0016] (3) Representative sequences were filtered and screened according to the following criteria: (1) No matching with the public AMP database; (2) Fewer than 2 cysteine ​​residues; (3) Length not exceeding 50 amino acids; (4) Net charge ≥ +2; (5) Average hydrophobicity ≤ 1.5.

[0017] (4) After the above screening, a target antimicrobial peptide sequence was obtained, as shown in SEQ ID NO:1, specifically: MKTLRRLLRAIANRLGTIV.

[0018] (5) Species annotation of metagenomically assembled phages was performed using VITAP v1.12. The antimicrobial peptide was derived from tailed viral phages.

[0019] Example 2: A species derived from the class Tailed Viruses ( Caudoviricetes Determination of the minimum inhibitory concentration (MIC) of antimicrobial peptides from bacteriophages This embodiment determined the minimum inhibitory concentration (MIC) of an antimicrobial peptide against six common pathogens. Staphylococcus aureus ATCC 12600, ATCC 33591, ATCC 33592, Klebsiella pneumoniae ATCC 13883, and Acinetobacter baumannii ATCC 19606 were all purchased from the American Type Culture Collection (ATCC); Acinetobacter baumannii bio-53272 was purchased from BioVector.

[0020] The antimicrobial peptides against Staphylococcus aureus were determined using the 96-well plate microbroth dilution method, following the CLSI M07-A10 standard operating procedure. Staphylococcus aureus ATCC 12600, ATCC 33591, ATCC33592), Acinetobacter baumannii ( Acinetobacter baumannii ATCC 19606, bio-53272) and Klebsiella pneumoniae ( Klebsiella pneumoniae ) MIC concentration of ATCC 13883.

[0021] The experimental method is as follows: The frozen bacterial strain was inoculated into cationic-regulated Mueller Hinton II broth (CAMHB) liquid medium and cultured overnight for 18 h in a constant temperature shaker at 37°C and 180 rpm to obtain the logarithmic growth phase bacterial suspension. The logarithmic growth phase bacterial suspension was then used to adjust the OD of the bacterial suspension. 600 Value, diluted and calibrated to an inoculation concentration of 5 × 10⁻⁶. 5 CFU / mL. The antimicrobial peptide was dissolved in sterile ultrapure water and serially diluted twofold to prepare antimicrobial peptide test solutions with final concentrations of 2, 4, 8, 16, 32, and 64 μg / mL.

[0022] The antibacterial experiment was conducted in 96-well plates, with four groups: (1) Blank control group: 200 μL sterile CAMHB medium; (2) Positive control group: 180 μL bacterial suspension and 20 μL sterile ultrapure water; (3) Negative control group: 180 μL sterile CAMHB medium and 20 μL antimicrobial peptide solution with a concentration of 64 μg / mL; (4) Experimental group: 180 μL bacterial suspension and 20 μL serially diluted antimicrobial peptide solution. The 96-well plates were placed in a constant temperature incubator at 37℃ and statically incubated for 24 h. The OD of each well was detected using a microplate reader. 600 value.

[0023] The minimum inhibitory concentration (MIC) is defined as the lowest concentration that can completely inhibit the visible growth of bacteria after incubation at 37°C for 24 hours, i.e., the OD of the experimental group. 600 Values ​​compared to the negative control group OD 600The values ​​showed no significant difference. All experiments were independently repeated three times.

[0024] The MIC determination results of the antimicrobial peptides against the six bacterial strains are shown in the table below: Table 1. Determination of the minimum inhibitory concentration (MIC) of an antimicrobial peptide derived from tailed bacteriophages against six bacteria.

[0025] Experimental results show that the virus provided by this invention is derived from the class Tailed Viruses (Virgospora spp.) Caudoviricetes The bacteriophage antimicrobial peptide exhibits antibacterial activity against Staphylococcus aureus (including drug-resistant strains), Klebsiella pneumoniae, and Acinetobacter baumannii. As shown in Table 1, the MIC value of this antimicrobial peptide against Staphylococcus aureus ATCC 12600 is 4 μg / mL, the MIC value against drug-resistant strains ATCC 33591 and ATCC 33592 is 32 μg / mL, the MIC value against Klebsiella pneumoniae is 8 μg / mL, and the MIC values ​​against Acinetobacter baumannii ATCC 19606 and bio-53272 are 16 μg / mL and 16 μg / mL, respectively. These results indicate that the antimicrobial peptide has broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, and shows significant antimicrobial effects, particularly against commonly seen drug-resistant strains in clinical settings.

[0026] Furthermore, this antimicrobial peptide exhibits significant antibacterial activity against both Gram-positive bacteria (such as Staphylococcus aureus) and Gram-negative bacteria (such as Acinetobacter baumannii and Klebsiella pneumoniae), and its mechanism of action mainly relies on cationic charge-mediated membrane adsorption and hydrophobicity-mediated membrane insertion. The antimicrobial peptide sequence has a high net positive charge (+5) and moderate average hydrophobicity (≤1.5), consistent with the typical characteristics of a broad-spectrum antimicrobial peptide. Since the cell membrane structure of marine pathogens (such as Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus) is highly similar to that of terrestrial Gram-negative bacteria and is also rich in negatively charged phospholipids, this antimicrobial peptide also possesses the potential to inhibit these marine pathogens.

[0027] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this invention. It should be understood that the above descriptions are merely specific embodiments of this invention and are not intended to limit the scope of this invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the protection scope of this invention.

Claims

1. An antimicrobial peptide derived from tailed bacteriophages, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. The use of the antimicrobial peptide according to claim 1 in the preparation of drugs that inhibit Staphylococcus aureus, Acinetobacter baumannii and / or Klebsiella pneumoniae.

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