A Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 and its application

Abstract

The present invention discloses a Bacillus cereus aminomethyltransferase antimicrobial peptide BA14, whose amino acid sequence is HKKEMVLAWLQNQK, and the molecular weight of the antimicrobial peptide is 1753.092 Daltons. The antimicrobial peptide of the present invention has a significant inhibitory effect on Staphylococcus aureus, and can be used as a medicine or feed additive for preparing a drug for preventing or inhibiting Staphylococcus aureus infection. Studies have shown that the antimicrobial peptide BA14 of the present invention combined with vitamin D3 can kill or strongly inhibit the growth of Staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 and an application thereof. Background Art

[0002] Staphylococcus aureus ( Staphylococcus aureus ) belongs to the genus Staphylococcus, a Gram-positive bacterium, a common contaminating bacterium in food, and one of the five major foodborne pathogens. It can produce enterotoxins and cause food poisoning. Patients infected with Staphylococcus aureus will experience symptoms such as nausea, vomiting, cramping pain in the upper and middle abdomen, and diarrhea, among which vomiting is the most prominent. It is an important pathogen that causes animal infections.

[0003] At present, the treatment of bacterial diseases is still mainly based on antibiotics, but due to the abuse of antibiotics, bacterial resistance to antibiotics is becoming more and more serious, posing a major threat to human health. Antimicrobial peptides (AMPs) are a class of small molecule peptides that are widely present in organisms. They constitute part of the human body's first line of defense against pathogens and can effectively resist the invasion of pathogenic microorganisms. Unlike antibiotics that interfere with the metabolic process of pathogenic microorganisms, antimicrobial peptides mostly produce antibacterial effects by destroying the cell membrane on the surface of microorganisms and inducing leakage of cell contents, and are less likely to be affected by bacterial resistance. Antimicrobial peptides have broad-spectrum anti-biofilm activity, are conducive to regulating host immune responses, and are not prone to drug resistance. Therefore, they have unique advantages in the treatment of infectious diseases and are expected to become ideal anti-infective drugs. At present, people have discovered and isolated antimicrobial peptides from bacteria, fungi, higher plants, animals, etc.

[0004] Bacillus cereus is a Gram-positive, rod-shaped, facultative anaerobic bacterium that can form spores. It is often found in soil, water, and animal intestines. It can produce toxins that cause different types of gastrointestinal diseases or other diseases. Bacillus can also antagonize fungal pathogens through antibacterial, nutrient competition, site exclusion, parasitism or induction. At the same time, it can also produce a variety of active substances, such as low-molecular-weight peptides, lipopeptide antibiotics and antimicrobial proteins, which can effectively inhibit the growth of a variety of pathogenic microorganisms and have good research and application value. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art, provide a Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 and its application, and solve the problems in the above-mentioned background technology.

[0006] One of the technical solutions adopted by the present invention to solve the technical problem is: providing a Bacillus cereus aminomethyltransferase antimicrobial peptide BA14, whose amino acid sequence is shown in SEQ ID NO: 1:

[0007] SEQ ID NO: 1:

[0008] HKKEMVLAWLQNQK

[0009] The antimicrobial peptide BA14 has a molecular weight of 1753.092 Daltons, a positive charge of +2.25, and a total hydrophobicity ratio of 43%. APD3 predicts that the peptide may form an alpha helix.

[0010] Preferably, the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 is used in the preparation of an antibacterial drug, which is used to inhibit and / or kill Staphylococcus aureus.

[0011] The second technical solution adopted by the present invention to solve the technical problem is: providing an antibacterial drug, the active ingredient of which includes Bacillus cereus aminomethyltransferase antibacterial peptide BA14, and the amino acid sequence of the antibacterial peptide BA14 is SEQ ID NO: 1.

[0012] In a preferred embodiment of the present invention, the active ingredient of the antibacterial drug is Bacillus cereus aminomethyltransferase antibacterial peptide BA14, and the amino acid sequence of the antibacterial peptide BA14 is SEQ ID NO: 1.

[0013] In a preferred embodiment of the present invention, the antibacterial drug is used to inhibit and / or kill Staphylococcus aureus.

[0014] Preferably, the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 is used in the preparation of a feed additive, and the feed additive is used to inhibit and / or kill Staphylococcus aureus.

[0015] The third technical solution adopted by the present invention to solve the technical problem is: providing a feed additive, the effective ingredient of which includes Bacillus cereus aminomethyltransferase antimicrobial peptide BA14, and the amino acid sequence of the antimicrobial peptide BA14 is SEQ ID NO: 1.

[0016] In a preferred embodiment of the present invention, the active ingredient of the feed additive is the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14, and the amino acid sequence of the antimicrobial peptide BA14 is SEQ ID NO: 1.

[0017] In a preferred embodiment of the present invention, the feed additive is used to inhibit and / or kill Staphylococcus aureus.

[0018] Preferably, the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 is used in the preparation of a food preservative, and the food preservative is used to inhibit and / or kill Staphylococcus aureus.

[0019] The fourth technical solution adopted by the present invention to solve the technical problem is: providing a food preservative, the active ingredient of which includes Bacillus cereus aminomethyltransferase antimicrobial peptide BA14, and the amino acid sequence of the antimicrobial peptide BA14 is SEQ ID NO: 1.

[0020] In a preferred embodiment of the present invention, the active ingredient of the food preservative is the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14, and the amino acid sequence of the antimicrobial peptide BA14 is SEQ ID NO: 1.

[0021] In a preferred embodiment of the present invention, the food preservative is used to inhibit and / or kill Staphylococcus aureus.

[0022] The present invention uses APD3, Pymol2.0, SWISS-MODEL and other software and websites to perform bioinformatics prediction on the protein sequence of aminomethyltransferase with antibacterial activity of Bacillus cereus, and obtains a new amino acid sequence polypeptide BA14. 3 The antibacterial activity against Staphylococcus aureus was tested. Flow cytometry and TEM were used to observe the degree of damage to the bacterial cell membrane by BA14. The genomic DNA of Staphylococcus aureus was extracted to verify its effect on bacterial DNA. The experimental results showed that the peptide had a significant inhibitory effect on Staphylococcus aureus.

[0023] The antimicrobial peptide BA14 will damage bacteria through the following effects: the antimicrobial peptide BA14 can penetrate the cell membrane, bind to the bacterial genomic DNA, inhibit the synthesis of bacterial DNA, and thus cause bacterial death.

[0024] The antimicrobial peptides of the present invention can be synthesized by methods known to those skilled in the art, such as solid phase synthesis, and purified by methods known to those skilled in the art, such as high performance liquid chromatography.

[0025] The implementation of the present invention has the following beneficial effects:

[0026] The antimicrobial peptide BA14 of the present invention has a significant inhibitory effect on Staphylococcus aureus and is related to vitamin D 3 The combined effect is better. Its antibacterial mechanism is to first penetrate the bacterial cell membrane, then combine with the bacterial genomic DNA, inhibit the synthesis of bacterial DNA, and thus cause bacterial death. 3 It can be made into antibacterial drugs, feed additives, food preservatives, etc. for preventing or treating diseases caused by Staphylococcus aureus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of the structure of antimicrobial peptide BA14.

[0028] Figure 2 This is a comparison chart for the determination of the minimum bactericidal concentration (MBC) of the antimicrobial peptide BA14 against Staphylococcus aureus.

[0029] Among them, the concentrations of AF antimicrobial peptide BA14 were 0μg / mL, 500μg / mL, 250μg / mL, 125μg / mL, 62.5μg / mL, and 31.25μg / mL, respectively.

[0030] Figure 3 Combination of antimicrobial peptide BA14 with vitamin D 3 Comparison chart of minimum bactericidal concentration (MBC) determination for Staphylococcus aureus.

[0031] Among them, the concentrations of AF antimicrobial peptide BA14 were 0μg / mL, 500μg / mL, 250μg / mL, 125μg / mL, 62.5μg / mL, and 31.25μg / mL, respectively.

[0032] Figure 4 Antimicrobial peptide BA14 and antimicrobial peptide BA14 combined with vitamin D 3 Time-kill kinetic curves against Staphylococcus aureus.

[0033] Figure 5 To observe the effect of antimicrobial peptide BA14 on the cell membrane permeability of Staphylococcus aureus using transmission electron microscopy.

[0034] Figure 6 Flow cytometry observation of antimicrobial peptide BA14 combined with vitamin D 3 Effects on the cell membrane permeability of Staphylococcus aureus.

[0035] Figure 7 This is the circular dichroism spectrum change diagram of antimicrobial peptide BA14 under different conditions.

[0036] Figure 8 This is the gel electrophoresis diagram of the antimicrobial peptide BA14 and Staphylococcus aureus genomic DNA.

[0037] Among them, the mass ratios of antimicrobial peptide BA14 / DNA in bands 1-9 were 25 / 4, 25 / 8, 25 / 10, 25 / 12, 25 / 14, 25 / 16, 25 / 18, 25 / 20, and 25 / 25, respectively, and band 10 was the control group DNA.

[0038] Fig. 9 This is the circular dichroism spectrum change diagram after the antimicrobial peptide BA14 binds to the genomic DNA of Staphylococcus aureus. DETAILED DESCRIPTION

[0039] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1 Screening of antimicrobial peptide BA14

[0043] Bioinformatics prediction was performed on the protein sequence of the aminomethyltransferase with antibacterial activity of Bacillus cereus using software and websites such as APD3, Pymol2.0, and SWISS-MODEL. The charge and hydrophobicity of the amino acid sequence were analyzed using APD3 (Table 1), and the three-dimensional structure was predicted using Pymol2.0 and Swiss-Model (e.g. Figure 1 As shown in SEQ ID NO: 1), the amino acid sequence HKKEMVLAWLQNQK (as shown in SEQ ID NO: 1) with the best antibacterial properties was finally screened out and chemically synthesized (by Beijing Zhongke Yaguang Biotechnology Co., Ltd.), and the antibacterial activity was verified.

[0044] Table 1: Prediction analysis of antimicrobial peptides in Bacillus cereus

[0045]

[0046] Example 2 Antimicrobial peptide BA14 combined with vitamin D 3 Minimum bactericidal concentration (MBC) determination

[0047] Staphylococcus aureus (provided by the Key Laboratory of Marine Biotechnology, Shantou University, Guangdong Province) was cultured at 37°C for 12 h until the logarithmic growth phase and diluted to 10 in 0.01 M pH 7.2 phosphate buffer. 5-6 CFU / mL. The antimicrobial peptide BA14 was dissolved in phosphate buffer and mixed with vitamin D 3 The solutions were mixed at a molar ratio of 20:1, mixed with bacteria in equal volumes at 37°C and incubated for 2 h. The minimum inhibitory concentration (MBC) refers to the lowest concentration of antimicrobial peptides that can kill bacteria after incubation at 37°C. Figure 2 , Figure 3 As shown, antimicrobial peptide BA14 and antimicrobial peptide BA14 combined with vitamin D 3 The minimum bactericidal concentrations (MBC) against Staphylococcus aureus were 62.50 µg / mL and 31.25 µg / mL, respectively.

[0048] Example 3 Antimicrobial peptide BA14 combined with vitamin D 3 Time kill determination

[0049] Staphylococcus aureus was cultured at 37°C for 12 h until the logarithmic growth phase and diluted to 10 in 0.01 M pH 7.2 phosphate buffer. 5-6 CFU / mL. Take 1×MBC concentration of antimicrobial peptide BA14 and vitamin D 3 The mixed solution was mixed with bacteria in equal volumes at 37°C and then incubated separately. Samples were taken every 30 minutes and plated. The total number of colonies was recorded after incubation at 37°C overnight. 3 Can kill Staphylococcus aureus within 1.5 hours (such as Figure 4 as shown).

[0050] Example 4 Effect of antimicrobial peptide BA14 on the cell membrane permeability of Staphylococcus aureus

[0051] Transmission electron microscopy (TEM): dilute 5 mL to 10 5-6 CFU / mL of Staphylococcus aureus liquid was mixed with 1×MBC antimicrobial peptide BA14 in equal proportions, incubated at 37°C for 2 hours, centrifuged at 12000 r / min for 10 minutes, removed the supernatant and washed three times with PBS, added 2 mL of 2.5% glutaraldehyde solution, fixed at 4°C for 12 hours, and dehydrated with ethanol. After embedding, the sample was baked at 70°C for 24 hours, and a 70 nm thin glass slide was prepared on a copper grid, stained with lead citrate and uranyl acetate, and the ultrastructure of the bacteria (such as Figure 5 shown).

[0052] like Figure 5 A in Figure 5 As shown in Figure B, the cell membrane of Staphylococcus aureus that has not been treated with the antimicrobial peptide BA14 is intact, the intracellular tissue is evenly distributed, the surface is smooth and flat, and no content flows out. Figure 5 C in Figure 5 As shown in D, after treatment with the antimicrobial peptide BA14, a large number of Staphylococcus aureus were observed to die, the cell membrane was destroyed, the cells shrank and deformed, and the intracellular substances were significantly reduced.

[0053] Flow cytometry: Staphylococcus aureus was cultured at 37°C for 12 h until the logarithmic growth phase. 1 mL of bacterial solution was taken and centrifuged at 12000 r / min for 1 min in a 1.5 mL centrifuge tube. The supernatant was removed and resuspended in 1 mL of sterile 0.01 M phosphate buffer. Repeat 3 times. After preparing the bacterial suspension in 0.01 M phosphate buffer, 1×MBC antimicrobial peptide BA14 and 2×MBC antimicrobial peptide BA14 were mixed with the bacterial solution in equal proportions (greater than 200 μL) and placed in a biochemical incubator at 37°C for 2 h. 0.01 M phosphate buffer was used as a blank control group. Iodinated pyridinium (PI) was used as a fluorescent dye. After the incubation was completed, an equal volume of 50 μg / mL PI dye was added to the mixture. After incubation at 4°C for 15 min, the mixture was loaded onto a flow cytometer for detection. The detection was set to obtain at least 10 4 Set up the data collection and analysis plan to analyze the samples by detecting the scattered light signal (SC) and the propidium iodide fluorescence signal (PI).

[0054] like Figure 6 As shown in the figure, the mortality rate of Staphylococcus aureus in the control group was only 15.3%, indicating that the cell membrane structure was intact and the bacterial mortality rate was low; the mortality rate of Staphylococcus aureus with the addition of 1×MBC concentration of antimicrobial peptide BA14 increased significantly to 63.7%; the mortality rate of Staphylococcus aureus in the experimental group with the addition of 2×MBC antimicrobial peptide BA14 increased compared with the 1×MBC concentration of antimicrobial peptide BA14, reaching 72.3%. It can be seen that the concentration of antimicrobial peptide BA14 is positively correlated with the mortality rate of Staphylococcus aureus.

[0055] Example 5 Determination of the secondary structure of antimicrobial peptide BA14 by circular dichroism

[0056] The average residue molar ellipticity of the antimicrobial peptide BA14 was measured using a Jasco810 spectropolarimeter (Jasco, Tokyo) CD at a scan speed of 100 nm / min at 25°C. The antimicrobial peptide BA14 was dissolved in 0.01 M PBS and 25 mM sodium dodecyl sulfate (SDS) to a final concentration of 0.20 mg / mL, and its spectrum was scanned from 180 to 280 nm using two scans.

[0057] like Figure 7 As shown in the figure, in the PBS environment, the antimicrobial peptide BA14 has a positive peak at 188 nm and negative peaks at 196 nm and 225 nm, which indicates that the secondary conformation of the antimicrobial peptide BA14 in the PBS environment is mainly α-helix. In the SDS environment, there is a positive peak at 190 nm and two negative peaks at 208 nm and 225 nm, which indicates that its secondary conformation is still α-helix in the SDS environment, and the antimicrobial peptide does not undergo structural conversion when it contacts the bacterial cell membrane.

[0058] Example 6 Interaction between antimicrobial peptide BA14 and bacterial DNA

[0059] The interaction between the antimicrobial peptide BA14 and the genomic DNA of Staphylococcus aureus was studied by DNA gel retardation method. Staphylococcus aureus was cultured in 50 mL nutrient broth at 37°C for 12 h, and the optical density ratio of the bacteria at 260 and 280 nm (OD 260 / OD 280 Next, 10 µL of DNA (20 ng / µL) was incubated with the antimicrobial peptide BA14 and vitamin D 3 The solutions were mixed at 25°C to make the mass ratios of antimicrobial peptide BA14 / DNA 25 / 4, 25 / 8, 25 / 10, 25 / 12, 25 / 14, 25 / 16, 25 / 18, 25 / 20, 25 / 25, and 0, respectively. Figure 8 1-10 in the figure, mix well and incubate at 37℃ for 2h. Then take 8 μL of each sample and perform electrophoresis on 1% agarose gel, and observe gel retardation under ultraviolet light using GelDoc XR gel imaging system (Bio-Rad, USA).

[0060] like Figure 8 As shown in the figure, when the ratio is 25 / 4, the DNA band did not run out, but remained in the sample well. As the ratio dropped to 25 / 10, fuzzy bands appeared in the lanes, and the clarity and brightness of the bands increased significantly with the increase of the ratio. The gel electrophoresis of the genomic DNA of Staphylococcus aureus in the control group showed the brightest and clearest bands. This shows that the antimicrobial peptide BA14 can interact with the genomic DNA of Staphylococcus aureus.

[0061] Example 7 Effect of the interaction of antimicrobial peptide BA14 with bacterial DNA on its secondary structure

[0062] The antimicrobial peptide BA14 combined with vitamin D was measured again using a Jasco810 spectropolarimeter (Jasco, Tokyo) at a scanning speed of 100 nm / min at 25°C. 3 The average residue molar ellipticity. The antimicrobial peptide BA14 was dissolved in 25 mM sodium dodecyl sulfate (SDS) to a final concentration of 0.20 mg / mL. Staphylococcus aureus DNA (20 ng / μL) and the antimicrobial peptide BA14 were mixed and incubated at 25°C for 2 h, and its spectrum was scanned from 180 to 280 nm using two scans (e.g. Fig. 9 as shown).

[0063] After DNA was incubated with the antimicrobial peptide BA14, the positive peak at 190 nm and the two negative peaks at 208 nm and 225 nm still existed, but the spectrum was overall expanded compared with that in the SDS environment, indicating that the antimicrobial peptide BA14 interacted with the genomic DNA of Staphylococcus aureus and could cause a certain change in the proportion of the secondary structure conformation of the antimicrobial peptide.

[0064] In summary, the present invention provides a new antimicrobial peptide BA14, the minimum bactericidal concentration (MBC) of the antimicrobial peptide BA14 against Staphylococcus aureus is 62.50 µg / mL, and the antimicrobial peptide BA14 combined with vitamin D 3 The minimum bactericidal concentration (MBC) for Staphylococcus aureus is 31.25 µg / mL, which can kill Staphylococcus aureus within 1.5 hours, indicating that it has a strong inhibitory effect on Staphylococcus aureus. Studies have found that the antimicrobial peptide BA14 of the present invention can penetrate the bacterial cell membrane, bind to the bacterial genomic DNA, inhibit the synthesis of bacterial DNA, and thus cause bacterial death.

[0065] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A Bacillus cereus aminomethyltransferase antimicrobial peptide BA14, whose amino acid sequence is shown in SEQ ID NO:

1.

2. The use of the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 as claimed in claim 1 in the preparation of antibacterial drugs, characterized in that: The antibacterial drug is used for inhibiting and / or killing Staphylococcus aureus.

3. An antibacterial drug, characterized in that: The effective component comprises bacillus cereus aminomethyltransferase antimicrobial peptide BA14, and the amino acid sequence of the antimicrobial peptide BA14 is SEQ ID NO:

1.

4. The antibacterial drug according to claim 3, characterized in that: The antibacterial drug is used for inhibiting and / or killing Staphylococcus aureus.

5. The use of the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 in the preparation of a feed additive as claimed in claim 1, characterized in that: The feed additive is used for inhibiting and / or killing Staphylococcus aureus.

6. A feed additive, characterized in that: The effective component comprises bacillus cereus aminomethyltransferase antimicrobial peptide BA14, and the amino acid sequence of the antimicrobial peptide BA14 is SEQ ID NO:

1.

7. The feed additive according to claim 6, characterized in that: The feed additive is used for inhibiting and / or killing Staphylococcus aureus.

8. The use of the Bacillus cereus aminomethyltransferase antimicrobial peptide BA14 in the preparation of a food preservative as claimed in claim 1, characterized in that: The food preservative is used for inhibiting and / or killing Staphylococcus aureus.

9. A food preservative, characterized in that: The effective component comprises bacillus cereus aminomethyltransferase antimicrobial peptide BA14, and the amino acid sequence of the antimicrobial peptide BA14 is SEQ ID NO:

1.

10. The food preservative according to claim 9, characterized in that: The food preservative is used for inhibiting and / or killing Staphylococcus aureus.

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