Pleurocidin modified antimicrobial peptide, molecular design and construction method and its application

Abstract

The present invention belongs to the field of genetic engineering technology and specifically relates to a modified antimicrobial peptide, a molecular design and construction method, and its application. The modified antimicrobial peptide has an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2. The modified antimicrobial peptide can be used to prepare an antibacterial drug by using the natural antimicrobial peptide, Pleurocidin, as a template for molecular design. The present invention analyzes and designs the peptide based on the pathogenic mechanism of Vibrio anguillarum and the molecular structure and function of the antimicrobial peptide, thereby enhancing its antibacterial activity.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to a Pleurocidin modified antimicrobial peptide, a molecular design and construction method and an application thereof. Background Art

[0002] Vibrio anguillarum (Vibrio anguillarum), a Gram-negative bacterium widely distributed in marine and estuarine environments, primarily infects aquatic animals such as fish and crustaceans, causing hemorrhagic septicemia. Its high mortality and persistent infections in aquaculture have made it a focus of industry attention. V. anguillarum is a key pathogen causing vibriosis in fish, particularly posing a significant threat to commercial species such as eels, sea bass, and large yellow croaker. At a suitable water temperature of 25-30°C, V. anguillarum populations can double in 10-12 minutes. Research has demonstrated that V. anguillarum's pathogenicity involves multiple mechanisms. It utilizes pili, flagella, and outer membrane proteins to adhere to and colonize host tissues, employing biofilms to enhance persistence. A type III secretion system delivers effector proteins, releasing proteases and hemolysins to damage tissues and red blood cells. Furthermore, lipopolysaccharide and capsule circumvent immune clearance, while antioxidant enzymes mitigate oxidative stress. A two-component system and quorum sensing modulate virulence depending on the environment, ultimately leading to septicemia and multi-organ damage, forming a complete pathogenic network.

[0003] To control outbreaks caused by Vibrio anguillarum, many countries around the world have incorporated antibiotics into routine aquaculture prevention and control measures since the mid-20th century. However, the long-term, blind use of antibiotics has led to the rapid evolution of drug resistance in Vibrio anguillarum. In recent years, the resistance rate of clinically isolated Vibrio anguillarum strains to traditional antibiotics such as fluoroquinolones and sulfonamides has exceeded 60%, and some strains have even developed multidrug resistance. This drug resistance not only increases the difficulty of disease prevention and control, but also leads to longer aquaculture cycles and increased economic losses. In addition, the unique outer membrane structure and active efflux system of Vibrio anguillarum further limit the penetration efficiency of antibiotics, exacerbating the treatment dilemma. Faced with the failure of traditional antimicrobial strategies, the development of new antibacterial technologies, such as antimicrobial peptides, phage therapy, and immunomodulators, has become a core research direction for combating Vibrio anguillarum infections and promoting the sustainable development of the aquaculture industry.

[0004] The antimicrobial peptide pleurocidin is a cationic peptide isolated from the mucus secretions of the skin of winter flounder (Pseudopleuronectes americanus) and exhibits broad-spectrum antimicrobial activity. Pleurocidin is composed of 25 amino acids with a molecular weight of approximately 2.7 kDa. Its secondary structure exhibits a typical α-helical conformation in physiological environments. The helical wheel diagram shows a 180-degree polar distribution between the hydrophobic and hydrophilic regions, a key structural feature of its membrane function. Pleurocidin, enriched in arginine and lysine residues within the highly conserved cationic α-helical region of its structure, can generate strong electrostatic adsorption with negatively charged components of bacterial membranes, such as lipopolysaccharide (LPS) and phosphatidylethanolamine. This promotes peptide aggregation on the membrane surface and forms "ring-shaped pores," which then form ion channels in the bacterial membrane, leading to membrane permeabilization and exerting antimicrobial activity. Nuclear magnetic resonance imaging and molecular dynamics simulations reveal that pleurocidin recognizes bacterial membranes via two sites: the positively charged K7, K8, and K14 residues bind to the phosphate groups of LPS, while the hydrophobic F6 and L25 residues insert deep into the membrane's fatty acyl chains. Furthermore, pleurocidin can bind to bacterial plasmid DNA, potentially interfering with nucleic acid replication and transcription by intercalating into the DNA duplex or binding to base pairs. This dual mechanism of "membrane disruption plus intracellular targeting" is similar to that of the antimicrobial peptide NK-18, reducing the potential for bacterial resistance. Although in vitro studies have demonstrated its broad-spectrum killing activity against Gram-positive and Gram-negative bacteria and fungi, its selective killing mechanism against different pathogens requires further elucidation based on differences in membrane composition. Furthermore, the native peptide is susceptible to proteolysis and pH sensitivity in serum, limiting its translation into clinical applications.

[0005] Currently, the main methods for antimicrobial peptide molecular design include: 1) amino acid modification; 2) peptide chain cyclization modification; 3) active fragment splicing; 4) biological model simulation design; 5) structural parameter optimization; 6) computer-aided design; 7) targeted sequence design; and 8) combinatorial library screening. Structural parameter prediction plays a key role in guiding the design of antimicrobial peptide molecules. Structural optimization of antimicrobial peptides to meet diverse production and daily needs will become a new trend in antimicrobial peptide research and development.

[0006] Based on the above theory, the pathogenic mechanism of Vibrio anguillarum and the molecular structure and function relationship of the antimicrobial peptide Pleurocidin were analyzed and designed as the basic template, which is of great significance for improving the antibacterial activity. Summary of the Invention

[0007] The present invention provides a Pleurocidin modified antimicrobial peptide, a molecular design and construction method and an application thereof, which improves its antibacterial activity by combining the pathogenic mechanism of Vibrio anguillarum and the molecular structure and functional relationship of the antimicrobial peptide Pleurocidin as a basic template for analysis and design.

[0008] The specific technical solutions are as follows:

[0009] The first object of the present invention is to provide a Pleurocidin modified antimicrobial peptide, wherein the amino acid sequence of the Pleurocidin modified antimicrobial peptide is selected from SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] A second object of the present invention is to provide a method for molecular design and construction of the Pleurocidin modified antimicrobial peptide, comprising the following steps: the amino acid sequence of the Pleurocidin modified antimicrobial peptide is obtained from the National Center for Biotechnology Information (NCBI) of the United States, and molecular design is performed using the natural antimicrobial peptide Pleurocidin as a template.

[0011] Furthermore, the molecular design and construction method of the Pleurocidin modified antimicrobial peptide comprises the following steps: amino acid substitution of the natural antimicrobial peptide Pleurocidin from the perspective of amphiphilicity, and evaluating the activity of the modified peptide (Pleurocidin modified antimicrobial peptide) by combining functional prediction with detection of actual antibacterial effects.

[0012] The third object of the present invention is to provide a use of the Pleurocidin modified antimicrobial peptide in the preparation of antibacterial infection drugs.

[0013] Furthermore, the bacteria include Gram-negative bacteria and Gram-positive bacteria.

[0014] Furthermore, the Gram-negative bacteria include Vibrio anguillarum.

[0015] Taking the outer membrane structural characteristics of Vibrio anguillarum as the main target site, the natural antimicrobial peptide Pleurocidin was rationally designed using the amino acid replacement molecular design scheme. The physicochemical properties, antimicrobial activity, transmembrane region and secondary structure of the derived peptide (Pleurocidin modified antimicrobial peptide) were preliminarily judged in combination with antimicrobial peptide computational analysis software and antimicrobial peptide prediction tools.

[0016] The fourth object of the present invention is to provide a nucleic acid molecule encoding the Pleurocidin modified antimicrobial peptide.

[0017] A fifth object of the present invention is to provide a recombinant microorganism that expresses the Pleurocidin modified antimicrobial peptide.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention studies the molecular interaction mechanism between the natural antimicrobial peptide Pleurocidin and key components of the cell membrane and cell wall of Vibrio, and further optimizes the natural antimicrobial peptide Pleurocidin using molecular design and computer-aided methods, thereby further improving the activity of the antimicrobial peptide and reducing cytotoxicity and hemolysis.

[0020] (2) The present invention adopts solid phase chemical synthesis method, standard Fmoc method, C 18 Purify by reverse phase high performance liquid chromatography, target purity ≥95%;

[0021] (3) This invention is not only expected to provide a new solution for the prevention and control of Vibrio anguillarum in aquaculture, but also to lay a solid theoretical foundation and technical support for the development and application of antimicrobial peptides, thereby promoting the sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 2 is a simulation diagram of the helical wheel of Pleurocidin and its derivative peptides in the examples of the present invention, where A, B, and C are the simulation results of Pleurocidin, P-1, and P-2, respectively. Yellow marks non-polar amino acids, red marks polar positively charged amino acids, and green marks polar uncharged amino acids.

[0023] Figure 2 Circular dichroism spectra of Pleurocidin and its derivative peptides P-1 and P-2 in the examples of the present invention;

[0024] Figure 3 This is a graph showing the cytotoxicity of Pleurocidin and its derivative peptides in the examples of the present invention. DETAILED DESCRIPTION

[0025] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0026] Example 1: Molecular Design and Construction of Pleurocidin-Modified Antimicrobial Peptide

[0027] Using the amino acid sequence of the mature peptide Pleurocidin as a template, Pleurocidin was molecularly designed from the perspective of amphipathicity. The physicochemical properties of the modified peptide were evaluated using the online prediction website Expasy (https: / / web.expasy.org / cgi-bin / ) and the Antimicrobial Peptide Database (https: / / aps.unmc.edu / ), and its secondary structure was predicted by Alphafold (https: / / alphafoldserver.com / ), so as to obtain derivative peptides (Pleurocidin-modified antimicrobial peptides) with good performance.

[0028] Amphiphilicity is a key property of antimicrobial peptides. Through the coordinated distribution of charge and hydrophobic regions, amphiphilicity drives electrostatic binding and hydrophobic insertion into bacterial membranes, forming transmembrane pores that induce cell death. Appropriate amphiphilic parameters (such as hydrophobic moment and charge-hydrophobic balance) can optimize antimicrobial activity while reducing toxicity to eukaryotic cells. Differences in conformation and membrane composition determine selective bacterial killing, making them key optimization targets in antimicrobial peptide molecular design.

[0029] Therefore, the derivative peptide P-1 was designed by replacing glycine G, phenylalanine F, valine V, and alanine A in the sequence with the basic amino acid lysine K to increase the hydrophilicity of the polypeptide; replacing glycine G in the sequence with alanine A, and replacing glycine A and lysine K with tryptophan W to increase the hydrophobicity of the polypeptide;

[0030] The derivative peptide P-2 was designed by replacing glycine G, alanine A, and leucine L in the sequence with basic amino acid lysine K to increase the hydrophilicity of the polypeptide; and replacing glycine A, glycine G, alanine A, and histidine in the sequence with tryptophan W to increase the hydrophobicity of the polypeptide.

[0031] By predicting the designed derivative peptides, two derivative peptides with better prediction results were comprehensively screened out, such as P-1 and P-2 in Table 1.

[0032] Table 1 Derivative peptide sequences and their physicochemical properties

[0033]

[0034] The synthesis of the above peptides was commissioned to Shanghai Jier Biochemical Co., Ltd. and confirmed by mass spectrometry and high-performance liquid chromatography.

[0035] The helical wheel simulation diagram of Pleurocidin and its derivative peptides is shown in the figure Figure 1As shown, A, B, and C are the simulation results of Pleurocidin, P-1, and P-2, respectively. Yellow marks are non-polar amino acids, red marks are polar positively charged amino acids, and green marks are polar uncharged amino acids.

[0036] Example 2: Circular dichroism (CD) determination

[0037] At room temperature, circular dichroism spectrometry was used to measure the characteristic spectra of the three antimicrobial peptides in sterile water, 60 mM SDS solution (simulating the negatively charged bacterial cell membrane), and 50% trifluoroethanol (TFE, simulating the hydrophobic environment of the bacterial cell membrane). The final concentration of the peptide solution was 0.2 mg / mL. A 5 mm thick quartz sample cell was used, and the scanning wavelength was 190-260 nm. Each peptide was measured in parallel three times and the average value was obtained. The results were subtracted from the solvent control. The results are shown in Figure 2. Figure 2 As shown, Figure 2 Circular dichroism spectra of Pleurocidin and its derivative peptides P-1 and P-2.

[0038] Depend on Figure 2 As can be seen, in aqueous solution, all peptides exhibit a distinct negative peak around 200 nm, a characteristic peak of random coil structure. In SDS and TFE environments, the secondary structures of the peptides undergo significant changes. Pleurocidin, P-1, and P-2 exhibit positive peaks around 193 nm and negative peaks around 209 nm and 222 nm, respectively. This indicates that the three peptides primarily exist as α-helical structures in the bacterial cell membrane simulation environment, consistent with the secondary structure predicted by Alphafold 2.

[0039] Example 3: Minimum inhibitory concentration MIC test determination

[0040] 1. Strain culture: Dip an inoculation loop into -80℃ frozen Vibrio anguillarum TS340431 and streak it on a Mueller-Hinton Broth (MHB) solid plate. Incubate the plate upside down in a 28℃ constant temperature incubator. After a single colony grows, pick a single colony and place it in 5 mL of fresh MHB liquid medium. Incubate the plate in a shaking incubator at 28℃ / 150 rpm until the logarithmic growth phase. Then dilute the bacterial solution to a concentration of 1×10 6 CFU / mL is reserved.

[0041] 2. Pretreatment of antimicrobial peptide solutions: Place the three antimicrobial peptide solutions (2 mg / mL) of Pleurocidin, P-1, and P-2 in a PCR instrument. After reacting at 20°C for 30 min, quickly remove the solution and place it on ice for 10 min, then allow it to equilibrate at room temperature.

[0042] 3. Determination of MIC by 2-fold dilution method: Select a sterile round-bottom 96-well plate, add 90 μL of sterile MH liquid medium to the first row, and add 50 μL of sterile liquid medium to each of the remaining rows. Then, add 10 μL of pre-treated peptide sample solution (2 mg / mL) to each well in the first row, pipette to mix, and then draw 50 μL from the first row of wells to the corresponding wells in the second row. Repeat the operation in sequence, and draw 50 μL from the last well and discard it. Set up 3 parallels for each group. Then, add 50 μL of pre-diluted bacterial solution to each well so that the bacterial solution concentration in each well is 5×10 5 CFU / mL, with a final volume of 100 µL per well. The peptide sample concentrations were ranked from high to low, including 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, and 0.78125 μg / mL. An equal volume of bacterial suspension served as a negative control, while the original peptide, pleurocidin, and the antibiotics neomycin sulfate and kanamycin served as positive controls. The 96-well plate was incubated at 28°C for 14 h. The UV absorbance of each well at 600 nm was measured using a microplate reader. Wells where no bacterial growth was detected were designated as the MIC. The results are shown in Table 2.

[0043] Table 2 Minimum inhibitory concentration of Pleurocidin and its derivative peptides against Vibrio anguillarum

[0044]

[0045] As can be seen from Table 2, the antibacterial activities of the derivative peptides P-1 and P-2 designed by the template against Vibrio anguillarum were significantly higher than those of the two antibiotics kanamycin and neomycin sulfate, and were 4 times and 2 times that of the parent peptide Pleurocidin, respectively. The results showed that the increase in amphiphilicity was beneficial to improving the antibacterial activity of antimicrobial peptides.

[0046] Example 4: Minimum bactericidal concentration MBC test determination

[0047] From each clear well (≥MIC) in the MIC test in Example 3, 20 μL of liquid was pipetted onto MHB solid medium. The plates were incubated upside down in a suitable incubator for 24 hours, and bacterial growth was observed. The minimum concentration corresponding to the plate without bacterial growth was considered the MBC of the peptide solution. The results are shown in Table 3.

[0048] Table 3 Minimum bactericidal concentrations of Pleurocidin and its derivative peptides against Vibrio anguillarum

[0049]

[0050] The minimum bactericidal concentration (MBC) is the lowest concentration of an antimicrobial substance capable of killing a certain percentage (usually 99.9%) of bacteria in a cultured test strain. Comparing the results in Tables 2 and 3, we can see that the derivative peptides P-1 and P-2 have a 99.9% killing effect against Vibrio anguillarum at 1xMIC and 2xMIC, respectively.

[0051] Example 5: Cytotoxicity test

[0052] 1. Caco-2 Cell Culture and Passaging: Caco-2 cells frozen at -80°C were rapidly thawed in a 37°C water bath and gently shaken until ice crystals completely melted. In a sterile fume hood, the thawed cell suspension was slowly added dropwise to a pre-prepared centrifuge tube containing an appropriate amount of complete culture medium and gently mixed. The suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in fresh culture medium and transferred to a cell culture flask. Sufficient culture medium was added to cover the cell surface and incubated in a 37°C, 5% CO2 incubator for 48 h. The old culture medium was then discarded, and the cells were washed three times with PBS to remove residual culture medium. 1 mL of trypsin solution was added for digestion and incubated in the incubator for 5 min. The digestion reaction was quickly terminated by adding DMEM complete culture medium (containing 20% ​​fetal bovine serum, 1% streptomycin-penicillin, and 79% DMEM basal medium). The supernatant was discarded by centrifugation at 1000 rpm for 5 min, and the pellet was resuspended in 2 mL of fresh culture medium. After pipetting evenly, the pellet was transferred to a new culture flask and incubated for 24 h. When the cells reached the third generation, they were washed with PBS and centrifuged to discard the supernatant. The cells were diluted to a concentration of 1 × 10 5 The cells were cultured in 96-well cell culture plates for 24 h, with 200 μL per well and 5 replicates per group.

[0053] 2. The peptide samples were diluted with DMEM complete medium to 6 concentration gradients of 100 μg / mL, 80 μg / mL, 60 μg / mL, 40 μg / mL, 20 μg / mL, and 1 μg / mL. The cell culture medium after 24 hours of culture was discarded, and peptide samples of different concentration gradients were added in sequence, 200 μL per well, 5 parallels per group, and an equal volume of DMEM complete medium was added to the blank control. The cells were cultured in the incubator for 24 hours. The peptide solution was then discarded, and 150 μL of MTT (0.5 mg / mL) solution was added to each well. The cells were stained in the incubator for 4 hours, and then DMSO was added to dissolve the crystals. The cell viability was detected by a microplate reader at a UV absorption wavelength of 570 nm. The results are as follows: Figure 3 shown.

[0054] Depend on Figure 3As shown in the results, Caco-2 cells maintained a good cell survival rate after treatment with antimicrobial peptides at concentrations of 1 to 100 μg / mL for 24 hours. At concentrations of 1 to 60 μg / mL, the cell survival rate was relatively high (greater than 95%), indicating that antimicrobial peptides have a certain promoting effect on cell growth. When the peptide concentration increased to 80 μg / mL and 100 μg / mL, the cell survival rate in the P-2-treated group dropped below 90%, indicating that P-2 has a certain toxic effect on cell growth under high concentration conditions. This may be due to the fact that tryptophan W is a highly hydrophobic amino acid, and the increased hydrophobicity leads to increased cytotoxicity of the antimicrobial peptide.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Pleurocidin modified antimicrobial peptide, characterized in that: The amino acid sequence of the Pleurocidin modified antimicrobial peptide is selected from SEQ ID NO: 1 or SEQ ID NO:

2.

2. Use of the Pleurocidin modified antimicrobial peptide according to claim 1 in the preparation of antibacterial infection drugs.

3. The use according to claim 2, characterized in that The bacteria are selected from Gram-negative bacteria and Gram-positive bacteria.

4. The use according to claim 3, characterized in that The Gram-negative bacteria is Vibrio anguillarum.

5. A nucleic acid molecule, characterized in that Encodes the Pleurocidin modified antimicrobial peptide as described in claim 1.

6. A recombinant microorganism, characterized in that The recombinant microorganism expresses the Pleurocidin modified antimicrobial peptide according to claim 1.

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