An antibacterial peptide of marine origin, mutants thereof, bacteriostatic compositions and uses thereof

Abstract

The application belongs to the field of polypeptides, and particularly relates to a marine-derived antibacterial peptide, a mutant thereof, an antibacterial composition and application. The application screens a marine-derived antibacterial peptide, and optimizes net charge, hydrophobicity and amphiphilicity of the antibacterial peptide through mutation. Through experimental testing, the four antibacterial peptides have differential antibacterial effects on different gram-negative bacteria, wherein the antibacterial peptide mutant shown in SEQ ID NO. 2 has the best antibacterial effect and low hemolysis, and has a positive application prospect.

Description

Technical Field

[0001] This invention belongs to the field of polypeptides, specifically relating to a marine-derived antimicrobial peptide, its mutants, antimicrobial compositions, and applications. Background Technology

[0002] The widespread use of antibiotics has led to bacterial resistance, posing a serious threat to public health and highlighting the urgent need for novel antimicrobial strategies. Among these, antimicrobial peptides have shown great potential and are considered the most promising next-generation antimicrobial drugs in the post-antibiotic era.

[0003] Antimicrobial peptides typically consist of fewer than 50 amino acid residues. They are rich in positively charged amino acids such as lysine and arginine, while having a lower proportion of negatively charged amino acids such as aspartic acid and glutamic acid, thus often carrying a net positive charge. This cationic property promotes their interaction with the negatively charged bacterial cell membrane, enabling them to exert a direct bactericidal effect through membrane disruption mechanisms, thereby exhibiting broad-spectrum biological activity. Antimicrobial peptides can target a variety of microorganisms, including bacteria, fungi, and viruses, and their mode of action makes them less likely to induce bacterial resistance than traditional antibiotics. These characteristics make antimicrobial peptides an important candidate for addressing the increasingly serious threat of drug-resistant bacterial infections.

[0004] However, natural antimicrobial peptides still suffer from drawbacks such as low in vivo activity, high toxicity, unclear toxicology, and immature stability, which are the biggest obstacles to their application. Therefore, it is possible to optimize the overall performance of antimicrobial peptides based on net charge, hydrophobicity, and amphiphilicity, thereby improving their activity and reducing their toxicity. This also helps to gain a deeper understanding of the relationship between various parameters of antimicrobial peptides and their antimicrobial activity. Summary of the Invention

[0005] In view of this, the purpose of this invention is to screen antimicrobial peptides with high activity against specific pathogens or with broad-spectrum antimicrobial activity. This invention screened an antimicrobial peptide of marine origin and optimized its net charge, hydrophobicity and amphiphilicity through mutation, and verified its antibacterial effect.

[0006] This invention provides an antimicrobial peptide of marine origin, the amino acid sequence of which is shown in any one of SEQ ID NO. 1-4. The antimicrobial peptides shown in SEQ ID NO. 2-4 are mutants of the antimicrobial peptide shown in SEQ ID NO. 1, with mutation sites A8K, L20K, and A8K / L20K, respectively.

[0007] The present invention also provides the use of the marine-derived antimicrobial peptide in the preparation of an antimicrobial composition for inhibiting Gram-negative bacteria.

[0008] Furthermore, the antibacterial composition is a combination of Gram-negative antimicrobial drugs, disinfectants, preservatives, feed additives, or daily chemical detergents.

[0009] Furthermore, the Gram-negative bacteria are selected from one or more of Gardnerella vaginalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Salmonella.

[0010] The present invention also provides an antibacterial composition comprising the aforementioned marine-derived antimicrobial peptide.

[0011] Furthermore, the antibacterial composition is a combination of Gram-negative antimicrobial drugs, disinfectants, preservatives, feed additives, or daily chemical detergents.

[0012] Furthermore, the Gram-negative bacteria are selected from one or more of Gardnerella vaginalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Salmonella.

[0013] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0014] The marine-derived antimicrobial peptides screened in this invention are newly discovered antimicrobial peptides with good antibacterial effects. This invention also optimizes their net charge, hydrophobicity, and amphiphilicity through mutation. The antimicrobial peptide shown in SEQ ID NO. 2 has a significant antibacterial effect against Gram-negative bacteria, including Gardnerella vaginalis, and at the same time, the antimicrobial peptide has low hemolytic toxicity. Attached Figure Description

[0015] Figure 1 Tertiary structure and amphiphilicity analysis of antimicrobial peptides.

[0016] Figure 2 This is a characterization diagram of the hemolytic toxicity of antimicrobial peptides. Detailed Implementation

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0018] Example 1: Sources and properties of antimicrobial peptides

[0019] An antimicrobial peptide with the sequence LGSGAKGALRKPYNSIVKYLNKLKCYKVAIDIPTGLD (SEQ ID NO. 1) was screened from a marine microbiome database using a random forest machine learning approach. This antimicrobial peptide consists of 37 amino acids, with an average molecular weight of 40008.71, an isoelectric point (PI) of 11.52, and an average GRAVY coefficient of -0.08. Subsequent analysis of the α-helix structure of this antimicrobial peptide revealed that its α-helix exhibits significant amphiphilicity (e.g., ...). Figure 1 (As shown).

[0020] Example 2: Mutation Optimization of Antimicrobial Peptides

[0021] By using point mutation, a specific amino acid of the antimicrobial peptide shown in SEQ ID NO. 1 was mutated to lysine (K) to optimize the net charge, hydrophobicity, and amphiphilicity of the antimicrobial peptide. The sequences and properties of the antimicrobial peptide and its mutants are shown in Table 1.

[0022] Table 1. Antimicrobial peptide sequences and physicochemical properties

[0023]

[0024] Example 3: Determination of the antimicrobial activity of antimicrobial peptides

[0025] The antimicrobial peptides shown in SEQ ID NO. 1-4 were all synthesized by solid-phase chemical synthesis by Sangon Biotech (Shanghai) Co., Ltd., and the resulting antimicrobial peptide samples had a purity of greater than 95%.

[0026] The antimicrobial effect of antimicrobial peptides was determined using the micro-broth dilution method. The specific steps are as follows:

[0027] (1) Resuscitate Escherichia coli, Pseudomonas aeruginosa, Salmonella and Klebsiella pneumoniae, streak onto LB agar plates and incubate at 37°C for 16 h. Pick single colonies and transfer them to shake tubes containing 3 mL of LB medium. Incubate at 37°C for 4 h, then dilute with LB medium to 10. 6 CFU / ml. Gardnerella vaginalis was revived overnight (24 h) using modified BHI agar plates, and then cultured on modified BHI medium for 18 h at 37°C and 5% CO2. The culture was then diluted to 10⁻¹⁰ CFU / ml using modified BHI medium. 6 CFU / ml.

[0028] (2) Antimicrobial tests against *Escherichia coli*, *Pseudomonas aeruginosa*, *Salmonella*, and *Klebsiella pneumoniae*. Sterilized 96-well plates were used for the tests, with three parallel controls. LB medium was used to dilute the antimicrobial peptide to the highest concentration to be tested. Different concentrations of antimicrobial peptide solutions were obtained using a two-fold microdilution method. 50 μL of each solution was added to the wells of the plate, followed by 50 μL of the prepared bacterial suspension. For the control without antimicrobial peptide, 50 μL of LB medium and 50 μL of bacterial suspension were added. For the blank control, 100 μL of LB medium was added. The plates were incubated at 37°C for 18 h. After incubation, the OD600 of each well was measured using a multifunctional microplate. The concentration that significantly inhibits microbial growth is the minimum inhibitory concentration (MIC) of the antimicrobial peptide.

[0029] (3) Gardnerella vaginalis antimicrobial test. A sterile 96-well culture plate was used for the test, with three parallel controls. Modified BHI medium was used to dilute the antimicrobial peptide to the maximum concentration to be tested. Different concentrations of antimicrobial peptide solutions were obtained using a two-fold microdilution method. 50 μL of each solution was added to the wells of the culture plate, followed by 50 μL of the prepared bacterial suspension. For the control without antimicrobial peptide, 50 μL of modified BHI medium and 50 μL of bacterial suspension were added. For the blank control, 100 μL of modified BHI medium was added. The plates were incubated at 37℃ and 5% CO2 for 18 h. After incubation, the OD600 of each well was measured using a multifunctional microplate. The concentration that significantly inhibits microbial growth is the minimum inhibitory concentration (MIC) of the antimicrobial peptide.

[0030] The antimicrobial peptide inhibition results are shown in Table 2. Among them, the antimicrobial peptides shown in SEQ ID NO. 1-4 have significant inhibitory effects on 5 Gram bacteria, and the antimicrobial peptide mutant shown in SEQ ID NO. 2 has the best antimicrobial effect.

[0031] Table 2. Minimum inhibitory concentrations (μg / mL) of antimicrobial peptides

[0032]

[0033] Example 4: Determination of hemolytic activity of antimicrobial peptides

[0034] Defibrinated sheep blood was used to assess hemolytic toxicity and evaluate the hemolytic activity of antimicrobial peptides. 2 mL of blood was collected and incubated at 800 × 10⁻⁶ mmol / L. gCentrifuge for 10 min, discard the supernatant, wash twice with PBS buffer, and dilute with PBS buffer to prepare a 2% red blood cell suspension for later use. Prepare antimicrobial peptide dispersions of 8, 16, 32, 64, and 128 μg / mL. Add 200 μL of red blood cell suspension and 200 μL of antimicrobial peptide dispersion to a 2 mL centrifuge tube. The negative control is 200 μL of red blood cell suspension and 200 μL of PBS buffer, and the positive control is 200 μL of red blood cell suspension and 200 μL of 2% Triton X-100 solution. Incubate at 37 °C for 1 h. After incubation, centrifuge at 800 × 10⁻⁶. g Centrifuge for 10 min, add 200 μL of the supernatant to a 96-well plate, and measure the absorbance at 540 nm using a microplate reader to calculate the hemolysis rate. Hemolysis rate (%) = (A... sample -A PBS ) / (A tritonX-100 -A PBS )×100%; A sample Absorbance of the mixture of antimicrobial peptides and erythrocytes, A PBS Negative control absorbance, A tritonX-100 : Positive control absorbance.

[0035] The results are shown in Table 3. Figure 2 As shown, the antimicrobial peptide represented by SEQ ID NO. 2 has relatively low hemolytic activity.

[0036] Table 3: Results of the antimicrobial peptide hemolysis test

[0037]

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. An antibacterial peptide of marine origin, characterized in that, The amino acid sequence of which is shown in any one of SEQ ID NO. 1-4.

2. Use of the marine-derived antibacterial peptide according to claim 1 for the preparation of an antibacterial composition, characterized in that, The bacteriostatic composition is used to inhibit gram-negative bacteria selected from one or more of Gardnerella vaginalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Salmonella.

3. Use according to claim 2, wherein the compound is ###0002### The bacteriostatic composition is a gram-negative bacteria antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily washing product.

4. A bacteriostatic composition characterized in that, The bacteriostatic composition is a gram-negative bacteria antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily washing product.

5. The bacteriostatic composition of claim 4, wherein The bacteriostatic composition is a gram-negative bacteria antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily washing product; the gram-negative bacteria are selected from one or more of Gardnerella vaginalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Salmonella.

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