An antimicrobial peptide derived from deep-sea endosymbiotic methane-oxidizing bacteria and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to an antimicrobial peptide derived from deep-sea endosymbiotic methane-oxidizing bacteria and its application. The amino acid sequence of the antimicrobial peptide is shown by SEQ ID NO.1. The antimicrobial peptide of the present invention is a new type of antimicrobial peptide, which has a broad-spectrum antimicrobial activity against a variety of Gram-negative and Gram-positive pathogens, such as Pseudomonas aeruginosa, Listeria, Enterococcus faecalis and various types of Vibrio, with a minimum inhibitory concentration of 8.13-16.25 μM. In addition, the antimicrobial peptide has weak hemolytic activity and low cytotoxicity. The antimicrobial peptide provided by the present invention can replace antibiotics and is used in the prevention and treatment of various bacterial diseases, and as a safe and stable food additive or cosmetic preservative candidate.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the development and application of new deep-sea antimicrobial resources, specifically an antimicrobial peptide derived from deep-sea endosymbiotic methane-oxidizing bacteria and the application thereof. Background Art

[0002] The deep sea is characterized by extreme environmental conditions, including darkness, high pressure, oligotrophy, and low or extremely high temperatures (such as hydrothermal vents). These conditions have led to the development of unique life processes and forms in deep-sea organisms, resulting in the production of a wealth of novel genetic resources and metabolites, creating a treasure trove of new biological resources. Antimicrobial peptides, naturally occurring antimicrobial molecules, are being extensively studied as potential alternatives to antibiotics due to their broad-spectrum antimicrobial activity, low risk of drug resistance, and excellent biocompatibility.

[0003] As small-molecule polypeptides, antimicrobial peptides generally exert their bactericidal effects by producing membrane perforation effects or through non-membrane structural mechanisms. In the membrane perforation effect, positively charged antimicrobial peptides can interact with negatively charged components of bacterial membranes, inducing structural reorganization and forming channels through the cell membrane, leading to bacterial rupture and death. Furthermore, some antimicrobial peptides can bind to various target molecules within the cell after entry, inhibiting bacterial DNA replication, RNA transcription, protein translation, and other processes, thereby producing a bactericidal effect.

[0004] At the same time, antimicrobial peptides in nature generally have problems such as weak bactericidal activity and poor stability, which restrict their application. Expanding the sources of antimicrobial peptide isolation and targeted screening of new antimicrobial peptide resources with strong biological activity, high biocompatibility and stability have become important directions for the current development and application of antimicrobial peptides. At the same time, in deep-sea cold seeps and hydrothermal environments, large organisms represented by deep-sea mussels and tubeworms often form close chemo-energetic symbiotic relationships with specific microorganisms such as methanotrophs or sulfur-oxidizing bacteria to support each other in obtaining the required nutrients. Especially for symbiotic microorganisms, they are not only constantly facing the screening effect of the host, but also need to maintain a competitive advantage among complex and diverse environmental microorganisms and non-symbiotic bacteria, and develop defense mechanisms against non-symbiotic bacteria together with the host. Therefore, they have become an important source for exploring new antimicrobial peptide resources with strong biological activity, high biocompatibility and stability. Summary of the Invention

[0005] In order to obtain a new antimicrobial peptide with strong biological activity, high biocompatibility and stability, the present invention screened and obtained a new natural antimicrobial peptide Gp-Sym-AMP1 from the endosymbiotic methanotrophic bacteria of deep-sea mussels (Mytilidae: Bathymodiolinae), and clarified its efficacy and application.

[0006] The present invention adopts the following technical solutions:

[0007] First, the present invention provides an antimicrobial peptide Gp-Sym-AMP1 that inhibits a variety of Gram-negative and Gram-positive bacteria. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1, and the molecular formula of the antimicrobial peptide is C 177 H 254 N 46 O 43 S3, molecular weight is 3810.43Da.

[0008] The present invention also provides the use of the antimicrobial peptide Gp-Sym-AMP1 in the preparation of antibacterial drugs, wherein the antibacterial drugs have broad-spectrum antibacterial activity.

[0009] The present invention also provides the use of Gp-Sym-AMP1 in the preparation of functional foods, food preservatives, cosmetic preservatives, and feed additives.

[0010] An antimicrobial peptide Gp-Sym-AMP1, characterized in that the amino acid sequence of the antimicrobial peptide includes the amino acid sequence shown in SEQ ID NO.1.

[0011] The antimicrobial peptide Gp-Sym-AMP1 is used for inhibiting Gram-negative bacteria or Gram-positive bacteria.

[0012] The antimicrobial peptide Gp-AMP1 is derived from symbiotic methane-oxidizing bacteria in deep-sea mussels.

[0013] The antimicrobial peptide Gp-Sym-AMP1 is used in medicine or antibacterial preparations with the antimicrobial peptide as an active ingredient.

[0014] The drug also includes pharmaceutically acceptable excipients.

[0015] The auxiliary material is an auxiliary material required for preparing one of water extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

[0016] The antimicrobial peptide Gp-Sym-AMP1 is used in the preparation of feed additives.

[0017] The antimicrobial peptide Gp-Sym-AMP1 is used in the preparation of functional foods and food preservatives.

[0018] Application of the antimicrobial peptide Gp-Sym-AMP1 in the preparation of cosmetic preservatives.

[0019] The antimicrobial peptide Gp-Sym-AMP1 is a medicine, an antibacterial preparation, a feed additive, a functional food, a food preservative or a cosmetic preservative.

[0020] The antimicrobial peptide Gp-Sym-AMP1 of the present invention has the following advantages and beneficial effects:

[0021] The antimicrobial peptide Gp-Sym-AMP1 of the present invention exhibits broad-spectrum antimicrobial activity against pathogens including Pseudomonas aeruginosa, Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis, and various Vibrio species, with minimum inhibitory concentrations ranging from 8.13 to 16.25 μM and a minimum inhibitory concentration of 8.13 μM. The antimicrobial peptide Gp-Sym-AMP1 of the present invention can disrupt bacterial membrane structures, leading to bacterial death. The antimicrobial peptide Gp-Sym-AMP1 of the present invention also exhibits weak hemolytic activity and low cytotoxicity, demonstrating good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The amino acid composition and spatial structure diagram of the antimicrobial peptide Gp-Sym-AMP1, A is the amino acid composition, and B is the amino acid structure.

[0023] Figure 2 This is the mass spectrometry identification diagram of the antimicrobial peptide Gp-Sym-AMP1.

[0024] Figure 3 The graph shows the membrane disruption effect of Gp-Sym-AMP1 on Bacillus cereus, Pseudomonas aeruginosa, and Vibrio anguillarum at the minimum inhibitory concentration (8.13 µM) (using phosphate buffered saline as a control).

[0025] Figure 4 Figure 3 is a graph showing the in vitro cytotoxicity and hemolytic activity of Gp-Sym-AMP1. A is a graph showing the cytotoxicity of different concentrations of antimicrobial peptides on mouse mononuclear macrophages (RAW 264.7). B is a graph showing the cytotoxicity of different concentrations of antimicrobial peptides on human immortalized keratinocytes (HACAT). C is the quantitative results of the hemolytic activity of different concentrations of antimicrobial peptides on rabbit blood cells. D is a graph showing the hemolytic activity results of antimicrobial peptides. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples can all be obtained from commercial sources.

[0027] The following examples are used to further illustrate the present invention:

[0028] Example 1: Physicochemical Properties and Synthesis of Antimicrobial Peptide Gp-Sym-AMP1

[0029] The antimicrobial peptide Gp-Sym-AMP1 was isolated from the symbiotic methanotrophic bacteria in the gill cells of deep-sea mussels. Its amino acid sequence is represented by SEQ ID NO.1 and Figure 1 As shown in A, the molecular formula is and the molecular weight is 3810.43 Da. According to ExPASy (https: / / www.expasy.org / ), its isoelectric point is 9.8, the net charge is +4, and the overall average hydrophilicity is 0.192.

[0030] SEQ ID NO.1: GFRLCFSLSCLRRFGFFSCTLFSRNFVNFYSS

[0031] The structure of the antimicrobial peptide Gp-Sym-AMP1 was predicted from NetWheels (http: / / lbqp.unb.br / NetWheels / ) and I-TASSER (https: / / zhanggroup.org / I-TASSER / ) and visualized using PyMol software (https: / / pymol.org / ). It was predicted that the antimicrobial peptide Gp-AMP1 has a typical α-helical structure, as shown in the following figure. Figure 1 As shown in B.

[0032] The antimicrobial peptide Gp-AMP1 can be synthesized artificially by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method (SPPS).

[0033] After synthesis, the peptide was purified by reverse phase high performance liquid chromatography (RP-HPLC) to a purity of over 95%. The peptide chain structure was confirmed by MALDI-TOF mass spectrometry. The purified peptide was freeze-dried and then identified by mass spectrometry ( Figure 2 ) and HPLC purity test.

[0034] Example 2: Antibacterial activity detection

[0035] The synthetic peptide Gp-Sym-AMP1 was first dissolved in 1×PBS (10 mM Na2HPO4, 2 mM NaH2PO4, 135 mMNaCl, 4.7 mM KCl) and diluted using the two-fold dilution method. The minimum inhibitory concentration (MIC) method was used to test the activity of the synthetic peptide Gp-Sym-AMP1 against Gram-positive bacteria (e.g., Bacillus subtilis). Bacillus subtilis , Enterococcus faecalis Enterococcus faecalis and Listeria monocytogenes Listeria monocytogenes etc.), and Gram-negative bacteria (Pseudomonas aeruginosa Pseudomonas aeruginosa , Vibrio parahaemolyticus Vibrio Parahaemolyticus and Edwardsiella tarda Edwardsiella tarda That is, the bacteria to be tested are placed in suitable culture medium and temperature conditions ( Bacillus subtilis LB medium, 37°C; Enterococcus faecalis : BHI medium, 37 ℃; Listeria monocytogenes:TSA medium, 37 ℃; Pseudomonas aeruginosa LB medium, 37°C; Vibrio parahaemolyticus :2216E medium, 30 ℃; Edwardsiella tarda :TSA medium, 30 ℃) to the logarithmic growth phase, the cultured bacteria were added to 96-well culture plates, and the test bacteria were diluted with the corresponding culture medium to a final concentration of 1×10 6 CFU mL -1 (100 μL), then, the same volume (100 μL) of serially diluted peptide Gp-Sym-AMP1 solution was added to the 96-well culture plate to a final volume of 200 μl. The final concentrations of peptide Gp-Sym-AMP1 were 130.00 μM, 65.04 μM, 32.53 μM, 16.25 μM and 8.13 μM, respectively. 1× PBS was used as a negative control, and gentamicin (GM, final concentration of 7.19 μM, diluted in 1× PBS) was used as a positive control. The culture was continued for 16 h under the above culture medium and temperature conditions, and the OD value of the bacteria in each well was measured. 600 Absorbance value.

[0036] The results showed that Gp-Sym-AMP1 had good broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacterial strains (Table 1).

[0037] Example 3: Analysis of membrane rupture and sterilization mechanism

[0038] Bacillus cereus, Pseudomonas aeruginosa and Vibrio anguillarum cultured to the logarithmic growth phase under the temperature and culture medium conditions in Example 2 were resuspended in 1× PBS to a volume of approximately 1× 10 8 CFU mL -1 The cells were then incubated with 1× MIC of Gp-Sym-AMP1 for 1 hour at room temperature. The samples were then fixed overnight with pre-chilled electron microscopy fixative and washed three times with 1× PBS. After centrifugation, the cells were dehydrated in a gradient of 30%, 50%, 70%, 80%, and 100% ethanol, with each gradient dehydration lasting at least 10 minutes. After critical point drying using an automated dryer (Leica EM CPD300, Wetzlar, Germany), the cells were gold-sprayed and observed under a scanning electron microscope (Zeiss SUPRA 55, Oberkochen, Germany).

[0039] The results showed that after Gp-Sym-AMP1 treated the bacteria, the bacterial membrane structure was destroyed ( Figure 3 ), confirming that Gp-AMP1 exerts its bactericidal effect through membrane disruption.

[0040] Example 4: Biosafety Testing

[0041] The cytotoxicity of Gp-Sym-AMP1 on mammalian cell lines mouse mononuclear macrophages (RAW264.7) and human immortalized keratinocytes (HaCaT) was evaluated using a CCK-8 kit. Gp-Sym-AMP1-treated cells were first adjusted to 2 × 10 cells per well. 5 Cells were cultured at a density of 100 cells / mL and incubated overnight at 37°C and 5% CO2. The culture medium was then replaced with fresh DMEM high-glucose medium containing various concentrations of antimicrobial peptides (final concentrations of 0, 8.13, 16.26, 32.52, and 65.04 μM) and incubated with the cells for 24 hours. Cell viability was determined according to the CCK-8 kit instructions and calculated according to the manufacturer's instructions. Furthermore, to further determine the hemolytic activity of Gp-Sym-AMP1, defibrinated rabbit blood was washed with 1× PBS until the supernatant was clear and resuspended in the same saline solution to a concentration of 4% (v / v). Gp-Sym-AMP1 was added to final concentrations of 0, 8.13, 16.25, 32.50, and 65.00 μM and incubated at 37°C for 1 hour. Negative and positive controls were treated with 1× PBS and 1% TritonX-100. Absorbance at 540 nm was recorded using a microplate reader. The formula for calculating the hemolysis rate is as follows: Hemolysis rate (100%) = (OD 540 Experimental group - OD 540 blank group) / (OD 540 Positive control group - OD 540 blank group) × 100.

[0042] The results showed that there was no significant cytotoxicity and hemolysis at the effective inhibitory concentration, indicating that Gp-Sym-AMP1 has good biocompatibility ( Figure 4 ).

[0043] In summary, the antimicrobial peptide Gp-Sym-AMP1 of the present invention exhibits broad-spectrum antimicrobial activity, with strong antimicrobial activity against both Gram-positive and Gram-negative bacteria. Furthermore, the antimicrobial peptide Gp-Sym-AMP1 exhibits weak hemolytic activity and minimal cytotoxicity. Therefore, the antimicrobial peptide Gp-Sym-AMP1 of the present invention has significant application advantages in the preparation of therapeutic drugs for bacterial infections and as a functional food additive, animal feed additive, food preservative, and cosmetic preservative.

[0044] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0045] Table 1 shows the antibacterial activity of the synthetic peptide Gp-Sym-AMP1

[0046] microorganism Latin name and Gram classification <![CDATA[MIC [μmol L −1 ]]]> Bacillus subtilis <![CDATA[ Bacillus subtilis G + ]]> 16.25 Enterococcus faecalis <![CDATA[ Enterococcus faecalis G + ]]> 8.13 Listeria monocytogenes <![CDATA[ Listeria monocytogenes G + ]]> 8.13 Bacillus cereus <![CDATA[ Bacillus cereus G + ]]> 16.25 Pseudomonas aeruginosa PAO1 <![CDATA[ Pseudomonas aeruginosa PAO1G - ]]> 8.13 Vibrio anguillarum <![CDATA[ Vibrio anguillarum G - ]]> 8.13 Vibrio alginolyticus <![CDATA[ Vibrio alginolyticus G - ]]> 8.13 Vibrio parahaemolyticus <![CDATA[ Vibrio Parahaemolyticus G - ]]> 8.13 Edwardsiella tarda <![CDATA[ Edwardsiella tarda G - ]]> 8.13

Claims

1. An antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria, characterized in that: The amino acid sequence of the antimicrobial peptide is the amino acid sequence shown in SEQ ID NO.

1.

2. A use of the antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria according to claim 1, characterized in that: The antimicrobial peptide Gp-Sym-AMP1 is used in medicine or antibacterial preparations with the antimicrobial peptide as an active ingredient.

3. The use according to claim 2, characterized in that The drug also includes pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that The auxiliary material is an auxiliary material required for preparing one of water extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

5. Use of the antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria according to claim 1 in the preparation of a feed additive.

6. Use of the antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria according to claim 1 in the preparation of a functional food additive.

7. Use of the antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria according to claim 1 in the preparation of a food preservative.

8. Use of the antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria according to claim 1 in the preparation of a cosmetic preservative.

9. A medicine, an antibacterial preparation, a feed additive, a functional food additive, or a cosmetic preservative comprising the antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria according to claim 1.

10. A food preservative comprising the antimicrobial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria according to claim 1.

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