Marine-derived antibacterial peptide, mutant thereof, antibacterial composition and application

Abstract

The invention belongs to the field of polypeptides, and particularly relates to a marine-derived antibacterial peptide, a mutant thereof, an antibacterial composition and application. The marine-derived antibacterial peptide is obtained through screening, and the net charge, hydrophobicity and amphipathy of the marine-derived antibacterial peptide are optimized through mutation. Experimental tests show that the four antibacterial peptides have differentiated antibacterial effects on different gram-negative bacteria, and the antibacterial peptide mutant shown in SEQ ID NO. 2 has the best antibacterial effect, has low hemolytic activity and has a positive application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptides, and particularly relates to an antibacterial peptide of marine origin, mutants thereof, an antibacterial composition and application. BACKGROUND

[0002] The widespread use of antibiotics has led to the emergence of bacterial drug resistance, which poses a serious threat to public health, highlighting the urgent need for new antibacterial strategies. Among them, antibacterial peptides show great potential and are considered the most promising new generation of antibacterial drugs in the post-antibiotic era.

[0003] Antibacterial peptides are usually composed of less than 50 amino acid residues, and are often positively charged due to the high content of positively charged amino acids such as lysine and arginine, and the low content of negatively charged amino acids such as aspartic acid and glutamic acid. This cationic property promotes its interaction with the negatively charged bacterial cell membrane, enabling it to produce a direct bactericidal effect through a membrane disruption mechanism, thereby exerting broad-spectrum biological activity. Antibacterial peptides can target a variety of microorganisms such as bacteria, fungi, and viruses, and their mode of action makes them less likely to induce bacterial drug resistance than traditional antibiotics. These characteristics make antibacterial peptides an important candidate solution to the growing threat of drug-resistant bacterial infections.

[0004] However, natural antibacterial peptides still have the disadvantages of low in vivo activity, high toxicity, unclear toxicology, and immature stability, which are the biggest obstacles to their application. Therefore, based on net charge, hydrophobicity and amphiphilicity, it is possible to optimize the overall performance of antibacterial peptides, improve antibacterial peptide activity, and reduce antibacterial peptide toxicity, which will also help to better understand the relationship between various parameters of antibacterial peptides and antibacterial activity. SUMMARY

[0005] Therefore, the purpose of the present application is to screen antibacterial peptides with high activity against specific pathogenic bacteria or broad-spectrum antibacterial activity. The present application screens an antibacterial peptide of marine origin, and optimizes its net charge, hydrophobicity and amphiphilicity through mutation, and verifies its antibacterial effect.

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

[0007] The present application also provides the use of the antibacterial peptide of marine origin in the preparation of an antibacterial composition, and the antibacterial composition is used for inhibiting gram-negative bacteria.

[0008] Further, the antibacterial composition is a composition of gram-negative antibacterial drugs, disinfectants, preservatives, feed additives, or daily washing products, etc.

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

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

[0011] Further, the antibacterial composition is a gram-negative bacteria antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily chemical washing product, etc.

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

[0013] Compared with the prior art, the present application has at least the following beneficial effects: The marine-derived antibacterial peptide screened by the present application is the first discovered antibacterial peptide, and has good antibacterial effect; the present application also optimizes the net charge, hydrophobicity, and amphiphilicity through mutation, wherein the antibacterial peptide shown in SEQ ID NO. 2 has significant antibacterial effect on gram-negative bacteria including Gardnerella vaginalis, and the antibacterial peptide has low hemolytic toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 for the analysis of the tertiary structure and amphiphilicity of the antibacterial peptide.

[0015] Figure 2 for the characterization of the hemolytic toxicity of the antibacterial peptide. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application will be further described in detail below in combination with the drawings and specific embodiments. The examples of the embodiments are shown in the drawings. It should be understood that the specific examples described in the following embodiments of the present application are only exemplary illustrations of the specific embodiments of the present application, and are intended to explain the present application, but do not constitute a limitation on the present application.

[0017] Example 1: Source and properties of antibacterial peptide The antibacterial peptide with the sequence of LGSGAKGALRKPYNSIVKYLNKLKCYKVAIDIPTGLD (SEQ ID NO. 1) was screened from the marine microbiome database by the random forest machine learning method. The antibacterial peptide consists of 37 amino acids, with an average molecular weight of 40008.71, an isoelectric point (PI) of 11.52, and a hydrophilic average coefficient GRAVY of -0.08. Subsequently, the alpha-helix structure of the antibacterial peptide was analyzed, and it was found that the alpha-helix of the antibacterial peptide had obvious amphiphilicity (as shown in Figure 1 ).

[0018] Example 2: Mutation optimization of antibacterial peptide The specific amino acid of the antibacterial peptide shown in SEQ ID NO. 1 was mutated to lysine (K) by point mutation to optimize the net charge, hydrophobicity and amphiphilicity of the antibacterial peptide. The sequences and properties of the antibacterial peptide and its mutants are shown in Table 1.

[0019] Table 1: Sequences and physicochemical properties of antibacterial peptides Example 3: Determination of antibacterial activity of antibacterial peptides The antibacterial peptides shown in SEQ ID NO. 1-4 were synthesized by solid-phase chemical synthesis by Shengong Bioengineering (Shanghai) Co., Ltd., and antibacterial peptide samples with a purity of more than 95% were obtained.

[0020] The microbroth dilution method was used to determine the antibacterial effect of the antibacterial peptides, and the specific steps were as follows: (1) Resuscitate E. coli, P. aeruginosa, Salmonella and K. pneumoniae, streak to LB solid medium plate and incubate at 37°C for 16h, pick single colonies into a shaking tube containing 3mL LB medium, incubate at 37°C for 4h, then dilute with LB medium to 10 6 CFU / ml. Gardnerella vaginalis was resuscitated on modified BHI agar plates overnight (24h), and then cultured in modified BHI medium for 18h at 37°C and 5% CO2. The culture was diluted to 10 6 CFU / ml.

[0021] (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.

[0022] (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.

[0023] 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.

[0024] Table 2. Minimum inhibitory concentrations (μg / mL) of antimicrobial peptides Example 4: Determination of hemolytic activity of antimicrobial peptides 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. g Centrifuge 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. gCentrifugation for 10 min, 200 μL supernatant after centrifugation was added in 96-well plate, and the absorbance value was detected at 540 nm by using microplate reader, and the hemolysis rate was calculated. Hemolysis rate (%) = (A sample -A PBS ) / (A tritonX-100 -A PBS ) x 100%; A sample : absorbance of antibacterial peptide and red blood cell mixture, A PBS : absorbance of negative control, A tritonX-100 : absorbance of positive control.

[0025] The results are shown in Table 3, Figure 2 and the antibacterial peptide shown in SEQ ID NO. 2 has relatively low hemolysis.

[0026] Table 3: Results of antibacterial peptide hemolysis experiment Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and do not constitute a limitation on the content of the present application. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A marine-derived antimicrobial peptide, characterized in that, Its amino acid sequence is shown in any of SEQ ID NO. 1-4.

2. The use of the marine-derived antimicrobial peptide according to claim 1 in the preparation of an antibacterial composition, characterized in that, The antibacterial composition is used to inhibit Gram-negative bacteria.

3. The application as described in claim 2, characterized in that, The antibacterial composition is a Gram-negative antimicrobial agent, disinfectant, preservative, feed additive, or daily chemical detergent.

4. The application as described in claim 2, characterized in that, The Gram-negative bacteria are selected from one or more of Gardnerella vaginalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Salmonella.

5. An antibacterial composition, characterized in that, It contains the marine-derived antimicrobial peptide as described in claim 1.

6. The antibacterial composition according to claim 5, characterized in that, The antibacterial composition is a Gram-negative antimicrobial agent, disinfectant, preservative, feed additive, or daily chemical detergent.

7. The antibacterial composition according to claim 5, characterized in that, The antibacterial composition is used to inhibit Gram-negative bacteria.

8. The antibacterial composition according to claim 7, characterized in that, 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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