Antibacterial peptide derived from symbiotic methane-oxidizing bacteria in deep sea and application of antibacterial peptide

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide sourced from symbiotic methane-oxidizing bacteria in deep sea and application of the antibacterial peptide. The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 1. The antibacterial peptide provided by the invention is a novel antibacterial peptide, has broad-spectrum antibacterial activity on a variety of gram-negative bacteria and gram-positive bacteria pathogens, such as pseudomonas aeruginosa, listeria monocytogenes, enterococcus faecalis and various vibrios, and has the minimum inhibitory concentration of 8.13-16.25 [mu] M. In addition, the antibacterial peptide has relatively weak hemolytic activity and relatively low cytotoxicity. The antibacterial peptide provided by the invention can replace antibiotics, is applied to prevention and treatment of various bacterial diseases, and is used as a safe and stable food additive or cosmetic preservative candidate substance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the development and application of novel antibacterial resources in the deep sea. Specifically, it is an antibacterial peptide derived from endosymbiotic methanotrophic bacteria in the deep sea and its application. Background Art

[0002] The deep sea has extreme environmental characteristics such as darkness, high pressure, oligotrophy, and low temperature or extreme high temperature (hydrothermal vents). Affected by this, deep-sea organisms often form unique life processes and life forms, producing a large number of novel gene resources and metabolites, and becoming a treasure trove for exploring novel biological resources. As a naturally occurring antibacterial molecule, antibacterial peptides have been widely studied as potential alternatives to antibiotics due to their broad-spectrum antibacterial activity, low risk of drug resistance, and good biocompatibility.

[0003] As small-molecule polypeptides, antibacterial peptides generally exert bactericidal effects by producing membrane perforation effects or by affecting mechanisms through non-membrane structures. In the membrane perforation effect, positively charged antibacterial peptides can interact with negatively charged components in the bacterial membrane structure, induce its structural reconstruction, form channels penetrating the cell membrane, and cause bacteria to rupture and die. In addition, some antibacterial peptides can also bind to various target molecules inside the cell after entering the cell, inhibiting processes such as bacterial DNA replication, RNA transcription, and protein translation, thereby producing bactericidal effects.

[0004] At the same time, antibacterial peptides in nature generally have problems such as weak bactericidal activity and poor stability, which restrict their application. Expanding the separation sources of antibacterial peptides and specifically screening for novel antibacterial peptide resources with strong biological activity, high biocompatibility, and stability has become an important direction for the development and application of antibacterial peptides. At the same time, in deep-sea cold seep and hydrothermal environments, large organisms represented by deep-sea mussels and tubeworms often form close chemo-symbiotic relationships with specific microorganisms such as methanotrophic bacteria or sulfur-oxidizing bacteria to mutually support and obtain the required nutrients. Especially for symbiotic microorganisms, they not only face the screening effect of the host at all times but also need to maintain a competitive advantage among complex and diverse environmental microorganisms and non-symbiotic bacteria, and produce a defense mechanism against non-symbiotic bacteria together with the host. Therefore, they have become an important source for exploring novel antibacterial peptide resources with strong biological activity, high biocompatibility, and stability. Summary of the Invention

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

[0006] The present invention adopts the following technical solutions: First, the present invention provides an antibacterial peptide Gp-Sym-AMP1 that inhibits a variety of Gram-negative and Gram-positive bacteria. The amino acid sequence of the antibacterial peptide is shown in SEQ ID NO.1, and the molecular formula of the antibacterial peptide is C 177 H 254 N 46 O 43 S3, with a molecular weight of 3810.43 Da.

[0007] The present invention also provides the use of the antibacterial peptide Gp-Sym-AMP1 in the preparation of antibacterial drugs, and the antibacterial drugs have broad-spectrum antibacterial activity.

[0008] The present invention also provides the use of Gp-Sym-AMP1 in the preparation of functional foods, food preservatives, cosmetic preservatives, and feed additives.

[0009] An antibacterial peptide Gp-Sym-AMP1, characterized in that the amino acid sequence of the antibacterial peptide includes the amino acid sequence shown in SEQ ID NO.1.

[0010] The antibacterial peptide Gp-Sym-AMP1 is used to inhibit Gram-negative or Gram-positive bacteria.

[0011] The antibacterial peptide Gp-AMP1 is derived from endosymbiotic methanotrophic bacteria in deep-sea mussels.

[0012] The application of the antibacterial peptide Gp-Sym-AMP1 is a drug or bacteriostatic preparation with the antibacterial peptide as the active ingredient.

[0013] The drug also includes pharmaceutically acceptable excipients.

[0014] The excipients are the excipients required for preparing one of aqueous infusions, powders, lotions, tinctures, oils, emulsions, ointments, plasters, or aerosols.

[0015] The application of the antibacterial peptide Gp-Sym-AMP1 in the preparation of feed additives.

[0016] The application of the antibacterial peptide Gp-Sym-AMP1 in the preparation of functional foods and food preservatives.

[0017] The application of the antibacterial peptide Gp-Sym-AMP1 in the preparation of cosmetic preservatives.

[0018] The drug or bacteriostatic preparation or feed additive or functional food and food preservative or cosmetic preservative of the antibacterial peptide Gp-Sym-AMP1.

[0019] The antibacterial peptide Gp-Sym-AMP1 of the present invention has the following advantages and beneficial effects: The antibacterial peptide Gp-Sym-AMP1 of the present invention has broad-spectrum antibacterial activity against pathogenic bacteria including Pseudomonas aeruginosa, Bacillus cereus, Listeria, Enterococcus faecalis and various Vibrio spp. The minimum inhibitory concentration is 8.13 - 16.25 μM, and the minimum inhibitory concentration is 8.13 μM. The antibacterial peptide Gp-Sym-AMP1 of the present invention can destroy the bacterial membrane structure, resulting in bacterial death. The antibacterial peptide Gp-Sym-AMP1 of the present invention also has weak hemolytic activity and small cytotoxicity, and has the characteristics of good biocompatibility. Brief Description of the Drawings

[0020] Figure 1 It is the amino acid composition and spatial structure diagram of the antibacterial peptide Gp-Sym-AMP1. A is the amino acid composition, and B is the amino acid structure.

[0021] Figure 2 It is the mass spectrometry identification diagram of the antibacterial peptide Gp-Sym-AMP1.

[0022] Figure 3 It is the diagram of the membrane-breaking 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).

[0023] Figure 4 It is the in vitro cytotoxicity and hemolytic activity diagram of Gp-Sym-AMP1. A is the cytotoxicity diagram of antibacterial peptides at different concentrations on mouse mononuclear macrophages (RAW 264.7), B is the cytotoxicity diagram of antibacterial peptides at different concentrations on human immortalized keratinocytes (HACAT), C is the quantitative result of the hemolytic activity of antibacterial peptides at different concentrations on rabbit blood cells, and D is the hemolytic activity result diagram of antibacterial peptides. Detailed Description of the Invention

[0024] The present invention will be described in detail below with reference to the drawings and specific implementation cases, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following implementation cases are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following implementation cases can all be obtained from commercial channels.

[0025] The following examples are used to further illustrate the present invention: Example 1: Physicochemical Properties and Synthesis of the Antibacterial Peptide Gp-Sym-AMP1 The antibacterial peptide Gp-Sym-AMP1 was isolated from intracellular symbiotic methanotrophic bacteria in the gill cells of deep-sea mussels, and its amino acid sequence is composed of SEQ ID NO.1 and Figure 1As shown in A, its molecular formula is [formula not provided in the original], with a molecular weight of 3810.43 Da. Predicted by ExPASy (https: / / www.expasy.org / ), its isoelectric point is 9.8, net charge is +4, and the total average hydrophilicity is 0.192.

[0026] SEQ ID NO.1: GFRLCFSLSCLRRFGFFSCTLFSRNFVNFYSS The structure of the antimicrobial peptide Gp-Sym-AMP1 was predicted by 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-helix structure, as specifically shown in Figure 1 B.

[0027] The antimicrobial peptide Gp-AMP1 can be artificially synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase peptide synthesis (SPPS) method.

[0028] After synthesis, it 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. After freeze-drying, the purified polypeptide was subjected to mass spectrometry identification ( Figure 2 ) and HPLC purity detection.

[0029] Example 2: Antibacterial Activity Detection The synthetic polypeptide Gp-Sym-AMP1 was first dissolved in 1×PBS (10 mM Na2HPO4, 2 mM NaH2PO4, 135 mM NaCl, 4.7 mM KCl), and diluted using the two-fold dilution method. The minimum inhibitory concentration (MIC) method was used to detect the antibacterial activity of the synthetic polypeptide Gp-Sym-AMP1 against Gram-positive bacteria (such as: 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 etc.). That is: the test bacteria were cultured in appropriate media and temperature conditions ( Bacillus subtilis : LB medium, 37 °C; Enterococcus faecalis : BHI medium, 37 °C; Listeria monocytogenes : TSA medium, 37 °C; Pseudomonas aeruginosa : LB medium, 37 °C;Vibrio parahaemolyticus : 2216E medium, 30 °C; Edwardsiella tarda : Cultured in TSA medium at 30 °C until the logarithmic growth phase, the cultured bacteria were respectively added to a 96-well culture plate, and the test bacterial solution was diluted to a final concentration of 1×10 6 CFU mL -1 (100 μL). Then, the same volume (100 μL) of the gradient-diluted polypeptide Gp-Sym-AMP1 solution was added to the 96-well culture plate to a final volume of 200 μL, and the final concentrations of polypeptide Gp-Sym-AMP1 were 130.00 μM, 65.04 μM, 32.53 μM, 16.25 μM, and 8.13 μM in sequence; 1×PBS was used as a negative control, and gentamicin (GM, final concentration of 7.19 μM, diluted with 1×PBS) was used as a positive control; the culture was continued for 16 h under the above medium and temperature conditions, and the OD 600 absorbance value of the bacteria in each well was measured.

[0030] The results showed that Gp-Sym-AMP1 had good broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacterial strains (Table 1).

[0031] Example 3: Analysis of the mechanism of membrane-breaking bactericidal action The Bacillus cereus, Pseudomonas aeruginosa, and Vibrio anguillarum cultured to the logarithmic growth phase under the temperature and medium conditions in Example 2 were resuspended with 1×PBS to about 1×10 8 CFU mL -1 , and then incubated with 1×MIC of Gp-Sym-AMP1 at room temperature for 1 hour. Subsequently, the samples were fixed with pre-cooled electron microscopy fixative overnight, washed 3 times with 1×PBS, and the centrifuged bacteria were dehydrated stepwise in ethanol at concentrations of 30%, 50%, 70%, 80%, and 100%, with at least 10 min of dehydration for each gradient. After critical point drying using an automatic dryer (Leica EM CPD300, Wetzlar, Germany), gold sputtering was performed, and observations were made under a scanning electron microscope (Zeiss SUPRA 55, Oberkochen, Germany).

[0032] The results showed that: It could be seen from the scanning electron microscope (SEM) that after treatment with Gp-Sym-AMP1, the bacterial membrane structure was damaged ( Figure 3 ), confirming that Gp-AMP1 exerted its bactericidal effect by membrane disruption.

[0033] Example 4: Biosafety detection The cytotoxicity of Gp-Sym-AMP1 against mammalian cell lines, murine mononuclear macrophages (RAW264.7) and human immortalized keratinocytes (HaCaT), was evaluated using a CCK-8 kit. Cells treated with Gp-Sym-AMP1 were first adjusted to a density of 2×10 5 cells per well and incubated overnight at 37 °C and 5% CO2. Then, the medium was replaced with fresh high-glucose DMEM medium containing different concentrations of the antimicrobial peptide (final concentrations of 0, 8.13, 16.26, 32.52, and 65.04 μM), and the cells were incubated for 24 hours. Cell viability was determined according to the instructions of the CCK-8 kit and calculated according to the guidelines provided by the manufacturer. In addition, to further determine the hemolytic activity of Gp-Sym-AMP1, defibrinated rabbit blood was selected, washed with 1×PBS until the supernatant was clear, and resuspended in the same physiological saline to a concentration of 4% (v / v). Gp-Sym-AMP1 was added at final concentrations of 0, 8.13, 16.25, 32.50, and 65.00 μM, and incubated at 37 °C for 1 h. Treatment with 1×PBS and 1% TritonX-100 served as negative and positive controls, respectively. Absorbance at 540 nm was recorded using a microplate reader. The hemolysis rate was calculated using the following formula: Hemolysis rate (100%) = (OD 540 experimental group - OD 540 blank group) / (OD 540 positive control group - OD 540 blank group) × 100.

[0034] The results showed that there was no significant cytotoxicity or hemolytic activity at effective antibacterial concentrations, indicating that Gp-Sym-AMP1 had good biocompatibility ( Figure 4 ).

[0035] In summary, the antimicrobial peptide Gp-Sym-AMP1 of the present invention has broad-spectrum antibacterial activity and strong antibacterial activity against both Gram-positive and Gram-negative bacteria. In addition, the antimicrobial peptide Gp-Sym-AMP1 has weak hemolytic activity and low 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 functional food additives, animal feed additives, food preservatives, cosmetic preservatives, etc.

[0036] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0037] Table 1 shows the antibacterial activity of synthetic polypeptide Gp-Sym-AMP1 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 antibacterial peptide Gp-Sym-AMP1 derived from deep-sea endosymbiotic methanotrophic bacteria, characterized in that, The amino acid sequence of the antimicrobial peptide includes the amino acid sequence shown in SEQ ID NO.

1.

2. The antibacterial peptide Gp-Sym-AMP1 according to claim 1, characterized in that, The antimicrobial peptide Gp-Sym-AMP1 is used to inhibit Gram-negative bacteria or Gram-positive bacteria.

3. The antibacterial peptide Gp-Sym-AMP1 according to claim 1, characterized in that, The antimicrobial peptide Gp-AMP1 is derived from endosymbiotic methanotrophic bacteria in deep-sea mussels.

4. Use of the antibacterial 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 applied to a drug or bacteriostatic preparation with the antimicrobial peptide as the active ingredient.

5. The application according to claim 4, wherein The drug also includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The excipient is an excipient required for preparing one of a water infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

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

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

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

10. A drug or bacteriostatic preparation or feed additive or functional food and food preservative or cosmetic preservative comprising the antimicrobial peptide Gp-Sym-AMP1 derived from endosymbiotic methanotrophic bacteria in deep sea according to claim 1.

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