A novel antibacterial peptide CGV-AMP-4 from chicken enterobacteria phage and application thereof

Abstract

This invention discloses a novel antimicrobial peptide CGV-AMP-4 derived from chicken intestinal bacteriophage and its applications, belonging to the field of biotechnology. The antimicrobial peptide CGV-AMP-4 of this invention exhibits broad-spectrum antimicrobial activity, with particularly significant effects against Streptococcus suis type II. Simultaneously, this peptide demonstrates extremely high safety, exhibiting almost no hemolytic activity within several times the effective concentration range and very low cytotoxicity, showcasing great potential as a novel anti-infective drug.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel antimicrobial peptide CGV-AMP-4 derived from chicken intestinal bacteriophage and its applications. Background Technology

[0002] With the increasing intensification of global livestock farming, bacterial infectious diseases have become one of the main threats to animal health and livestock product safety. In poultry and livestock farming, pathogens such as Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus suis often cause serious infectious diseases. For example, Streptococcus suis type II not only causes meningitis, septicemia, and arthritis in pigs, resulting in huge economic losses, but also has significant public health implications, seriously threatening human health.

[0003] For a long time, livestock production has relied primarily on traditional chemical antibiotics to prevent and treat infections caused by these pathogens. However, the long-term unregulated and abusive use of antibiotics has led to serious bacterial resistance problems and the emergence of various "superbugs." At the same time, antibiotic residues in animal products and environmental pollution are becoming increasingly prominent issues. Therefore, developing novel antibacterial drugs that are highly effective, low in toxicity, and less likely to induce resistance has become an urgent problem that needs to be solved in the fields of biomedicine and animal husbandry and veterinary medicine.

[0004] Antimicrobial peptides, as an important component of the organism's innate immune system, are a class of polypeptides with broad-spectrum antimicrobial activity. Unlike traditional antibiotics that act on specific proteins or enzymes, antimicrobial peptides typically exert their effects by physically disrupting bacterial cell membranes or interfering with intracellular physiological processes, giving them significant advantages such as rapid antimicrobial action, good thermal stability, and extreme difficulty in bacterial resistance.

[0005] In recent years, with the development of metagenomics technology, the discovery of novel antimicrobial peptides from complex ecosystems (such as animal gut microbiomes and viromes) has become a research hotspot. The enterovirome, especially bacteriophages, contains an extremely rich array of bioactive sequences, making it a natural treasure trove for screening novel antimicrobial molecules. Compared to traditional eukaryotic antimicrobial peptides, bacteriophage-derived sequences exhibit significant heterogeneity. Because bacteriophages have long been engaged in a micro-evolutionary process of interaction with bacteria, the antimicrobial molecules they produce often possess novel amino acid arrangements and extremely low sequence homology. This structural uniqueness suggests that they may possess unique targets and superior physicochemical properties.

[0006] Therefore, screening and identifying novel antimicrobial peptides from chicken intestinal bacteriophages that have extremely low similarity to known peptide libraries and exhibit rapid bactericidal efficacy against specific pathogens such as Streptococcus suis type II is of great practical significance for developing a new generation of highly safe and non-drug-resistant biological antimicrobial agents. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a novel antimicrobial peptide CGV-AMP-4 derived from chicken intestinal bacteriophage and its application. The novel antimicrobial peptide CGV-AMP-4 of this invention has significant antimicrobial properties and high safety.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a novel antimicrobial peptide CGV-AMP-4 derived from chicken intestinal bacteriophage, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] This invention provides the application of the novel antimicrobial peptide CGV-AMP-4 in the preparation of antimicrobial agents.

[0010] This invention provides the application of the novel antimicrobial peptide CGV-AMP-4 in the preparation of drugs for the prevention and treatment of Streptococcus suis type II infection.

[0011] Preferably, the concentration of the novel antimicrobial peptide CGV-AMP-4 is 8-128 μg / mL.

[0012] It contains at least the following beneficial technical effects: In this invention, CGV-AMP-4 exhibited the most significant antibacterial activity against Streptococcus suis type II, with a minimum inhibitory concentration (MIC) of 8 μg / mL and a minimum bactericidal concentration (MBC) of 32 μg / mL. Even at the highest tested concentration of 128 μg / mL, CGV-AMP-4 only induced a very low level of hemolysis, with a hemolysis rate of less than 3%. CGV-AMP-4 showed low cytotoxicity at concentrations of 32 μg / mL and below, with a cytotoxicity rate remaining below 30%, indicating good cell tolerance within the effective antibacterial concentration range. Attached Figure Description

[0013] Figure 1 The time-dependent bactericidal curve of the antimicrobial peptide CGV-AMP-4 against Streptococcus suis type II is shown. Detailed Implementation

[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0017] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0019] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0020] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0021] Example 1 Screening of antimicrobial peptides First, phage genome sequences were obtained from the chicken gut phage database (doi.org / 10.5281 / zenodo.14684892), and protein-coding regions were predicted using Prodigal v2.6.3 software with the parameter set to -p meta. Short peptides with a length not exceeding 50 amino acids were then retained for subsequent analysis.

[0022] The obtained short peptide sequences were input into four deep learning-based antimicrobial peptide prediction models: c_AMPs-prediction, AMPlify, amPEPpy v1.0, and AI4AMP for screening. Finally, the antimicrobial peptide CGV-AMP-4 derived from chicken intestinal phage was obtained, with the amino acid sequence: KGSILAGIASILTGFGLLGVSKKKRN.

[0023] The antimicrobial peptide was subsequently synthesized by Shanghai Sangon Biotech Co., Ltd. using a solid-phase synthesis method, achieving a purity of 90%, and was used for subsequent experimental verification.

[0024] Example 2 1. Experimental methods for determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC): To evaluate the in vitro antimicrobial activity of synthetic antimicrobial peptides against common poultry and livestock pathogens, the MIC and MBC of the antimicrobial peptide CGV-AMP-4 were determined using the microbroth dilution method. A total of 14 common pathogens were selected for the experiment, including *Rimeria anatipestifer*, *Escherichia coli* (ATCC25922), *Salmonella* (ATCC14028), *Proteus mirabilis*, *Staphylococcus aureus* (ATCC29213), *Manniella*, *Enterococcus faecalis* (ATCC29212), *Clostridium perfringens*, carbapenem-resistant *Klebsiella pneumoniae*, *Klebsiella pneumoniae* (ATCC46117), *Pseudomonas aeruginosa*, *Pasteurella multocida*, hemolytic *Staphylococcus*, and *Streptococcus suis* type II (ATCC43765).

[0025] Before the experiment, glycerol-preserved strains of each bacterium were streaked onto their respective solid media and incubated at 37 °C for 18–24 h. Single colonies were then picked and inoculated onto their corresponding liquid media for further culture. Specifically, *Riemerella anatipestifer* was cultured on TSA solid medium, *Clostridium perfringens* on TSC selective medium, *Klebsiella pneumoniae* resistant to carbapenems on BHI medium, *Streptococcus suis* type II on THB medium, and the remaining strains on MH medium.

[0026] During liquid culture, *Riemerella anatipestifer* was inoculated on TSB medium, *Clostridium perfringens* on FT anaerobic medium, *Klebsiella pneumoniae* resistant to carbapenems was inoculated on BHI medium, *Streptococcus suis* type II on THB medium, and the remaining strains were inoculated on MH liquid medium. *Clostridium perfringens* was cultured statically at 37 °C under anaerobic conditions, while the other strains were cultured with shaking at 37 °C and 180 rpm for 9–12 h. After the bacterial culture became turbid and entered the logarithmic growth phase, it was used for subsequent experiments.

[0027] The antimicrobial peptide CGV-AMP-4 was dissolved in sterile ultrapure water to prepare a stock solution of 256 μg / mL. A serial dilution system was set up in a 96-well microplate. 50 μL of the antimicrobial peptide stock solution was added to each well in columns 1 and 2, and 50 μL of the corresponding culture medium was pre-added to each of the remaining wells. Serial dilutions were performed twofold starting from column 2, up to column 12, with excess liquid discarded. After adding the bacterial culture, the final concentrations of the antimicrobial peptide in each well were 128, 64, 32, 16, 8, 4, 2, and 1 μg / mL, respectively.

[0028] Then, 50 μL of diluted bacterial suspension was added to each well to achieve a final concentration of 5 × 10⁻⁶. 5 The concentration of CFU / mL was used, and the total reaction volume was 100 μL. Meropenem was used as a positive control with the same concentration gradient, and sterile ultrapure water was used as a negative control. After incubating the 96-well plates at 37 °C for 24 h, bacterial growth was assessed by observing the clarity of the culture medium. The MIC was defined as the lowest concentration of antimicrobial peptide in the culture medium at which no visible bacterial growth was observed.

[0029] For the strain with the lowest MIC value, the MBC was further determined. 10 μL of culture medium was taken from wells from which no visible growth was observed, and evenly spread onto antibiotic-free solid culture plates. The plates were then incubated at 37 °C for 18–24 h. If no colony formation was detected on the plate, the corresponding antimicrobial peptide concentration was determined as the minimum bactericidal concentration.

[0030] The results of the MIC experiment are shown in Table 1: Table 1. MIC determination of CGV-AMP-4 against various bacteria CGV-AMP-4 exhibited the most significant antibacterial activity against Streptococcus suis type II, with a minimum inhibitory concentration (MIC) of 8 μg / mL. In contrast, the inhibitory effect of this antimicrobial peptide on other tested strains was relatively weak, with a MIC of 32 μg / mL against Enterococcus faecalis and Klebsiella pneumoniae, while the MIC against Salmonella (ATCC14028) and Staphylococcus aureus (ATCC29213) was 64 μg / mL. Further bactericidal experiments showed that the minimum bactericidal concentration (MIC) of CGV-AMP-4 against Streptococcus suis type II was 32 μg / mL.

[0031] 2. Hemolytic test Experimental methods: To detect the hemolytic activity of antimicrobial peptides, a red blood cell suspension was prepared using defibrinated sheep blood. The procedure was briefly as follows: defibrinated sheep blood was centrifuged at 1,000 r / min for 10 min, the supernatant was discarded, and the red blood cells were washed repeatedly with PBS buffer until the supernatant was clear. The red blood cells were then resuspended in PBS at a volume ratio of 1:10 to prepare the red blood cell suspension for later use.

[0032] In the experiment, 100 μL of erythrocyte suspension was mixed with an equal volume of antimicrobial peptide solutions of different concentrations in a 1.5 mL centrifuge tube and incubated at 37 ℃ for 1 h to assess the possibility of hemolysis induced by the antimicrobial peptides. After incubation, the mixture was centrifuged at 1,500 r / min for 10 min, and the supernatant was collected for detection.

[0033] 100 μL of supernatant was transferred to a 96-well plate, and the absorbance was measured at 540 nm using a microplate reader. PBS was used as a negative control, representing 0% hemolysis, and 1% Triton X-100 was used as a positive control, representing complete hemolysis. All experiments were performed in triplicate, and results are expressed as mean plus or minus standard deviation.

[0034] The results of the hemolytic test are shown in Table 2: Table 2 Results of hemolytic test Experimental results: Even at the highest test concentration of 128 μg / mL, CGV-AMP-4 only caused a very low level of hemolysis, with a hemolysis rate of less than 3%.

[0035] 3. Cytotoxicity assay Experimental methods: The potential cytotoxicity of peptides was detected using the CCK-8 assay. Cells were cultured in DMEM medium containing 10% fetal bovine serum. After reaching the logarithmic growth phase, cells were collected by trypsin digestion and resuspended in complete medium. The cell density was adjusted to 2 × 10⁶ cells / year. 5Cells / mL. 100 μL of the culture medium was then seeded into each well of a 96-well plate and cultured overnight at 37 °C to ensure adequate cell adhesion. The next day, the culture medium was discarded, and the cells were washed twice with PBS. Different concentrations of peptide solution (8-128 μg / mL) were added for treatment. A negative control group containing only cells and a blank control group without cells were also included, and incubation continued overnight. After treatment, the supernatant was discarded, and the cells were washed twice again with PBS. Fresh DMEM medium containing 10% CCK-8 reagent was added to each well, and the cells were incubated at 37 °C for 2 h. The absorbance of each well was then measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: Cell viability (%) = (Absorbance of sample wells) / (Absorbance of sample wells) (Absorbance of blank well) / (Absorbance of negative control well) (Absorbance of blank well) × 100%. All experiments were performed in triplicate, and cytotoxicity was expressed as 100% minus cell viability.

[0036] Table 3: Results of Cytotoxicity Experiments Table 3 Results of Cytotoxicity Tests Experimental results: Cytotoxicity assay results showed that CGV-AMP-4 exhibited low cytotoxicity at concentrations of 32 μg / mL and below, with the cytotoxicity rate remaining below 30%, indicating good cell tolerance within the effective antibacterial concentration range.

[0037] 4. Time-based sterilization kinetics experiment Experimental methods: Streptococcus suis type II was cultured under suitable conditions with shaking until the logarithmic growth phase. The turbidity of the bacterial solution was measured and converted to viable cell concentration. The bacterial solution was adjusted to 2 × 10⁻⁶ using serum-free THB liquid medium. 5 CFU / mL. Subsequently, 500 μL of bacterial culture was mixed with 500 μL of solution containing antimicrobial peptides to achieve a final peptide concentration of twice the minimum bactericidal concentration (MBC), while the concentration of the mixed bacterial culture was 1 × 10⁻⁶. 5 Mix CFU / mL thoroughly in a 1.5 mL centrifuge tube.

[0038] The mixture was incubated at a constant temperature of 37 °C, and samples were taken at multiple time points (0, 15, 30, 60, 120, 180, and 240 min). Each time point sample was serially diluted 10-fold, and 100 μL was spread onto the surface of THB solid medium plates. The plates were incubated at 37 °C for 18–24 h, and the colony-forming units (CFU) were recorded to calculate the relative bacterial viability.

[0039] The negative control group was treated with an equal volume of sterile water instead of the antimicrobial peptide. Three replicates were set up for each time point, and the entire experiment was independently repeated three times. The experimental results are shown in Figure 1 : Experimental results: Experimental results show that CGV-AMP-4 can completely kill the target strain within 30 minutes, achieving rapid and efficient antibacterial effects.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A novel antimicrobial peptide CGV-AMP-4 derived from chicken intestinal bacteriophage, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The application of the novel antimicrobial peptide CGV-AMP-4 according to claim 1 in the preparation of antimicrobial agents.

3. The use of the novel antimicrobial peptide CGV-AMP-4 according to claim 1 in the preparation of drugs for preventing and treating streptococcal type II infection in suis.

4. The application according to claim 2 or 3, characterized in that, The concentration of the novel antimicrobial peptide CGV-AMP-4 is 8-128 μg / mL.

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