Antibacterial peptide cAMP573 and application thereof

Abstract

The invention discloses an antibacterial peptide cAMP573 and application of the antibacterial peptide cAMP573. The amino acid sequence of the antibacterial peptide cAMP314 is as shown in SEQ ID NO: 1. The antibacterial peptide shows remarkable broad-spectrum antibacterial activity and is high in safety.

Description

Technical Field

[0001] The present invention relates to an antibacterial peptide cAMP573 and its application, belonging to the technical field of antibacterial peptides. Background Art

[0002] Antibiotics are one of the greatest discoveries in human history, and their emergence has saved the lives of countless patients. However, the abuse of antibiotics has led to the emergence of a large number of drug-resistant bacteria. Bacterial resistance to antibiotics has become a global public health problem, and a large number of people die every year due to the drug resistance of pathogenic bacteria. Among them, Escherichia coli causes the largest number of deaths, followed by Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter baumannii, Streptococcus pneumoniae, and Mycobacterium tuberculosis.

[0003] To alleviate the problem of antibiotic resistance, some alternative strategies for traditional antibiotic treatment have emerged. Currently, the main alternative treatment strategies include antibacterial peptides (AMPs), antibody-antibiotic conjugates (AACs), phage therapy, and microbiota-based therapies. Among them, antibacterial peptides have received extensive attention due to their strong antibacterial activity and less drug resistance.

[0004] Antibacterial peptides, also known as host defense peptides, usually consist of 2 - 50 amino acids and are a class of small polypeptides that can inhibit bacteria, fungi, and viruses. They are an important part of the innate immune system of organisms and are widely present in animals, plants, and microorganisms. The structures of AMPs are diverse, and the currently recognized main structures include (i) α-helix, (ii) β-sheet, (iii) αβ structure, or (iv) non-αβ structure. Antibacterial peptides can play a bactericidal role against many Gram-positive and Gram-negative bacteria. Their mechanisms of action mainly include directly killing bacteria by destroying the bacterial cell membrane and acting on important biological processes such as nucleic acid synthesis, cell wall synthesis, and enzyme synthesis inside the cell. Due to their diverse mechanisms of action, it is difficult for microorganisms to develop resistance to antibacterial peptides, and these characteristics make them one of the most promising candidate antibacterial drugs.

[0005] The early discovery of AMPs was through techniques such as material extraction, separation, purification, and mass spectrometry characterization to extract and identify from the body of a certain species or its metabolites. In addition, based on the principle of rational design, chemical modification means are used to find ideal polypeptides. In recent years, the application of integrated computational methods such as high-throughput sequencing technology, artificial intelligence technology, and molecular dynamics simulation has greatly improved the development efficiency of antibacterial peptides. The application of integrated computational methods depends on high-quality data, and the massive high-quality sequencing data in the metagenomics database is a solid foundation in the development process of antibacterial peptides.

[0006] The human gastrointestinal tract is a vast microbial ecosystem that harbors trillions of microorganisms, and its gut microbiome encodes highly diverse genes. Research shows that a large number of potential AMP families in the human gut microbiome remain to be deeply studied, and there is broad prospect in developing new antimicrobial peptides from them. However, in the existing technology, there are still relatively few AMP antimicrobial peptides screened from the gut microbiota. Summary of the Invention

[0007] The present invention provides an antimicrobial peptide cAMP573 and its applications, which can effectively solve the above problems.

[0008] An antimicrobial peptide cAMP573, wherein the amino acid sequence of the antimicrobial peptide cAMP573 is shown as SEQ ID NO:1.

[0009] An antimicrobial agent, which comprises an effective dose of the antimicrobial peptide cAMP573 described above.

[0010] An antimicrobial drug, which comprises an effective dose of the antimicrobial peptide cAMP573 described above.

[0011] A food disinfectant, which comprises an effective dose of the antimicrobial peptide cAMP573 described above.

[0012] An antimicrobial composition, which comprises an effective dose of the antimicrobial peptide cAMP573 described above.

[0013] An application of the antimicrobial peptide cAMP573 described above in the preparation of an antimicrobial agent.

[0014] An application of the antimicrobial peptide cAMP573 described above in the preparation of an antimicrobial drug.

[0015] An application of the antimicrobial peptide cAMP573 described above in the preparation of a food disinfectant.

[0016] An application of the antimicrobial peptide cAMP573 described above in the preparation of an antimicrobial composition.

[0017] The beneficial effects of the present invention are:

[0018] The antimicrobial peptide cAMP573 involved in the present invention is originally derived from the fecal microbiome and obtained through screening and extraction. This antimicrobial peptide exhibits remarkable broad-spectrum antibacterial activity and can effectively inhibit the growth of a variety of pathogenic bacteria, specifically including but not limited to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Propionibacterium acnes, and Enterococcus faecalis, etc. These pathogenic bacteria are relatively common in clinical infections, and the broad-spectrum antibacterial characteristics of the antimicrobial peptide cAMP573 make it have broad application prospects in the field of antibacterial therapy.

[0019] The antibacterial peptide cAMP573 of the present invention is particularly outstanding in terms of safety. After being verified by multiple experiments, this antibacterial peptide has no hemolytic property, that is, it will not cause the rupture of red blood cells, thus avoiding the occurrence of hemolytic reactions. At the same time, cAMP573 also has no cytotoxicity and will not damage normal cells, ensuring its high safety during use. This characteristic gives cAMP573 significant advantages in the research and development and application of antibacterial drugs, providing a safer and more effective option for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is a flow chart for the screening of the antibacterial peptide cAMP573.

[0022] Figure 2 It is the molecular dynamics simulation and three-dimensional structure diagram of the antibacterial peptide cAMP573.

[0023] Figure 3 It is the HPLC detection result diagram of the antibacterial peptide cAMP573.

[0024] Figure 4 It is the LCMS detection result diagram of the antibacterial peptide cAMP573.

[0025] Figure 5 It is the determination diagram of the minimum inhibitory concentration of the antibacterial peptide cAMP573. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents the selected embodiments of the present invention.

[0027] Example 1

[0028] Based on the fecal microbiome metagenomic data of our company, this invention conducts the mining and screening of novel antimicrobial peptides. For the specific screening process, please refer to Figure 1 as shown. First, we adopted advanced deep learning models, including Attention, LSTM, and BERT, etc., to predict antimicrobial peptides (Reference: Ma Y, Guo Z, Xia B, Zhang Y, Liu X, Yu Y, Tang N, Tong X, Wang M, Ye X, etc., 2022. Identifying antimicrobial peptides from the human gut microbiome using deep learning. Nature Biotechnology 40: 921-931). In this step, we effectively removed those polypeptide sequences that were identical to the known antimicrobial peptide database to ensure the uniqueness and novelty of the screening results.

[0029] Next, through a series of rigorous analytical methods, including metaproteomic cross-validation, solubility analysis, cross-validation with Macrel and iAMPCN, correlation analysis, and molecular dynamics simulations, etc., we comprehensively evaluated and determined the novel candidate antimicrobial peptides for chemical synthesis. During this process, we conducted a comprehensive prediction of the properties and functions of each candidate antimicrobial peptide to ensure its potential antibacterial activity.

[0030] Finally, we screened out an antimicrobial peptide with significant potential from numerous candidate antimicrobial peptides and named it cAMP573. To further verify the actual antibacterial function of this antimicrobial peptide, we will conduct a detailed antibacterial function verification in subsequent experiments, with the expectation of providing strong experimental evidence and support for the development of novel antibacterial drugs.

[0031] Example 2 Physicochemical properties of the antimicrobial peptide

[0032] By using the Peptide package in R language, we conducted a detailed physicochemical property analysis of the candidate antimicrobial peptide cAMP573. The specific results are shown in detail in Table 1. The amino acid sequence of cAMP573 consists of RRWQPRPGWDNPVPAAKDTLTLPRWWWG (SEQ ID NO:1), and its length is 28 amino acid residues (28AA). The theoretical molecular weight of this antimicrobial peptide is 3443.92 Daltons, and the isoelectric point (pI) is 12.05, indicating that it is relatively stable in an alkaline environment. In addition, the hydrophobicity index of cAMP573 is -0.1821, showing that it has certain hydrophilic characteristics.

[0033] To further explore the spatial structure of cAMP573, we used the advanced three-dimensional structure prediction tool AlphaFold3 to simulate it. As Figure 2 shown, the prediction results show that the three-dimensional structure of cAMP573 is mainly an α-helix structure, which is relatively common in antimicrobial peptides and helps to exert its function.

[0034] To evaluate the stability of cAMP573 in a dynamic environment, we performed molecular dynamics simulations using GROMACS software. During the simulation, we selected the AMBER99SB force field and the water molecule TIP3P model to ensure the accuracy and reliability of the simulation. The total simulation time was 100 nanoseconds (ns), which was long enough to observe the behavioral changes of the antimicrobial peptide in the simulated environment.

[0035] After the simulation, we analyzed the structural stability of cAMP573 by calculating the root mean square deviation (RMSD). RMSD is an important indicator to measure the structural changes of proteins, and the smaller its value, the more stable the structure. The simulation results show that the RMSD value of cAMP573 remained stable at about 0.5 nanometers (nm) throughout the simulation process. This result indicates that cAMP573 has good structural stability under the simulated conditions, providing strong support for its potential functions in practical applications.

[0036] Table 1 Polypeptide sequences and physicochemical properties

[0037]

[0038] Example 3 Synthesis and purification of antimicrobial peptides

[0039] Chemically synthesize antimicrobial peptides by solid-phase peptide synthesis (this service is commercially supported by Gil Biochemical (Shanghai) Co., Ltd.), determine the purity of the synthesized antimicrobial peptides by high-performance liquid chromatography (HPLC) technology, and use mass spectrometry detection to determine the accurate molecular weight of antimicrobial peptide cAMP573. Specifically, the chromatographic column model used for HPLC analysis is Sinochrom ODS-BP (specification: 4.6 mm inner diameter, 250 mm length, 5 μm particle size), mobile phase A is configured as an acetonitrile solution containing 0.1% trifluoroacetic acid, and mobile phase B is deionized water containing 0.1% trifluoroacetic acid. The flow rate during the entire analysis process is fixed at 1 ml / min, the detection wavelength is set at 220 nm, and the injection volume is 5 μl. When detecting samples by LCMS, the relevant parameter settings are as follows: Nebulizer Gas Flow is 1.5 L / min, CDL Temp (Collision Induced Dissociation Temperature) is 250 °C, CDL voltage is set at -20.0 V, Block Temp (Block Temperature) is 200 °C, Probe Bias (Probe Bias) is +4.5 kV, Detector (Detector Voltage) is 1.5 kV, T.Flow (Total Flow) is 0.2 ml / min, and the mobile phase composition ratio is 50% water and 50% acetonitrile. According to Figure 3 As can be seen from the HPLC detection results shown, the purity of the antimicrobial peptide cAMP573 obtained by chemical synthesis method exceeds 95%. In addition, the calculated theoretical molecular weight of antimicrobial peptide cAMP573 is 3443.92, while Figure 4 The mass spectrometry analysis results presented also show that the actual molecular weight of the synthesized antimicrobial peptide cAMP573 is also 3443.92, which is exactly the same as the theoretical molecular weight, further verifying the accuracy and reliability of the synthesized antimicrobial peptide.

[0040] Example 4 Activity Analysis of Antimicrobial Peptides

[0041] 4.1 Determination of Minimum Inhibitory Concentration (MIC)

[0042] In this experiment, we selected three typical bacteria for cultivation, namely Escherichia coli CICC 10667, Pseudomonas aeruginosa JCM 5962, and Staphylococcus aureus ATCC 6538. These bacteria were all cultivated using LB medium. In addition, we also selected four other bacteria for cultivation, including Staphylococcus epidermidis CMCC 26069, Streptococcus mutans ATCC 25175, Propionibacterium acnes ATCC 6919, and Enterococcus faecalis ATCC 19433. These bacteria were cultivated using BHI medium.

[0043] To ensure the accuracy and consistency of the experiment, we cultivated these bacteria in the corresponding LB or BHI medium until the logarithmic growth phase, which was specifically manifested as the OD 600 value being between 0.4 and 0.6. At this time, we diluted these bacteria to an OD 600 value of 0.1 using fresh medium, and then further diluted them 1000 times for subsequent inhibition experiments. The specific operation steps were as follows: Add 50 μL of the diluted bacterial solution to a 96-well plate for cultivation. At the same time, using the two-fold dilution method, add AMPs solutions with different concentration gradients to each well, with specific concentrations of 512, 256, 128, 64, 32, 16, 8, 4, and 2 μg / mL respectively, and add 50 μL for each concentration gradient. Subsequently, place these well plates in a constant temperature environment at 37 °C for 20 hours and measure their OD 600 value.

[0044] During the experiment, we set up negative controls and positive controls. The negative control was adding an antimicrobial peptide solution to fresh medium, while the positive control was adding a bacterial solution to fresh medium. Through these control experiments, we could more accurately judge the antibacterial effect of the antimicrobial peptide. The minimum inhibitory concentration (MIC) was defined as the lowest concentration at which the antimicrobial peptide could completely inhibit bacterial growth.

[0045] The experimental results showed that the minimum inhibitory concentrations of cAMP573 against seven indicator bacteria were as Figure 5As shown in Table 2. Specifically, the minimum inhibitory concentrations against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Propionibacterium acnes, and Enterococcus faecalis were 64, 256, 32, 32, 32, 64, and 256 μg / mL, respectively. Generally speaking, cAMP573 exhibited broad-spectrum antibacterial activity and had significant inhibitory effects on a variety of bacteria.

[0046] Table 2 Determination of minimum inhibitory concentration (MIC, μg / mL)

[0047]

[0048] 4.2 Prediction of hemolytic activity and cytotoxicity

[0049] To further comprehensively evaluate the safety of antibacterial peptide cAMP573 in applications, we used the advanced iAMPCN tool to conduct a detailed predictive analysis of its potential hemolytic and cytotoxic effects. iAMPCN is an antibacterial peptide prediction tool developed based on the convolutional neural network (CNN) architecture in deep learning technology. It can not only efficiently identify and predict the structure and function of antibacterial peptides but also has the ability to simultaneously evaluate various toxicities of antibacterial peptides. Through the comprehensive analysis of this tool, we obtained the specific prediction results shown in Table 3. The analysis data clearly showed that the antibacterial peptide cAMP573 did not exhibit any hemolytic or cytotoxic effects under experimental conditions, which provided strong data support for its safety in subsequent research and applications.

[0050] Table 3 Prediction of hemolytic activity

[0051]

[0052] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An antibacterial peptide cAMP573, characterized in that The amino acid sequence of the antimicrobial peptide cAMP573 is shown in SEQ ID NO:

1.

2. An antibacterial agent, characterized in that, The antimicrobial agent comprises an effective dose of the antimicrobial peptide cAMP573 as claimed in claim 1.

3. An antibacterial drug, characterized in that, The antimicrobial drug comprises an effective dose of the antimicrobial peptide cAMP573 as claimed in claim 1.

4. A food disinfectant, characterized in that, The food disinfectant comprises an effective dose of the antimicrobial peptide cAMP573 as claimed in claim 1.

5. An antibacterial composition, characterized in that, The antimicrobial composition comprises an effective dose of the antimicrobial peptide cAMP573 as claimed in claim 1.

6. Use of the antimicrobial peptide cAMP573 as claimed in claim 1 in the preparation of an antimicrobial agent.

7. Use of the antimicrobial peptide cAMP573 as claimed in claim 1 in the preparation of an antimicrobial drug.

8. Use of the antimicrobial peptide cAMP573 as claimed in claim 1 in the preparation of a food disinfectant.

9. Use of the antimicrobial peptide cAMP573 as claimed in claim 1 in the preparation of an antimicrobial composition.

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