Application of Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16 in the preparation of antibacterial drugs

Abstract

The invention discloses a Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16, whose amino acid sequence is HSHMNHMFALLRLAAK, and the molecular weight of the antimicrobial peptide is 1877.27 Daltons. The antimicrobial peptide of the invention has a significant inhibitory effect on hemolytic Staphylococcus aureus, and can be used as a drug for preparing a drug for preventing or inhibiting hemolytic Staphylococcus aureus infection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16 in the preparation of antibacterial drugs. Background Art

[0002] Staphylococcus haemolyticus is a gram-positive bacterium belonging to the genus Staphylococcus and is the second most commonly isolated pathogen in blood culture. It accounts for 20% of clinical coagulase-negative staphylococcal infections and is the second most frequent and severe bacterial isolate in clinical infections. It is associated with hospital-acquired infections with implanted medical devices and infections in premature infants and has the highest levels of multidrug resistance, including heteroresistance to glycopeptides, limiting treatment options.

[0003] At present, the treatment of bacterial diseases is still mainly based on antibiotics, but the abuse of antibiotics has led to a serious problem - drug resistance. Antimicrobial peptides (AMPs) are a class of small molecule peptides that are widely present in organisms. They are an important component of the innate immunity of organisms and can effectively resist the invasion of pathogenic microorganisms. Unlike antibiotics that interfere with the metabolic process of pathogenic microorganisms, antimicrobial peptides are short-chain polypeptides that exist naturally in the body. They exert their antibacterial effects by directly destroying bacterial cell membranes or interfering with the physiological functions of bacteria, and are less likely to be affected by bacterial resistance. Compared with the current main antibacterial drugs, antimicrobial peptides have broad-spectrum antibacterial activity and are less likely to develop drug resistance than other antibacterial drugs. Antimicrobial peptides also have a certain inhibitory effect on some viruses, parasites, tumor cells, etc. Therefore, antimicrobial peptides have the potential to become a new type of antibacterial agent.

[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 the use of Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16 in the preparation of antibacterial drugs, 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 the use of Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16 in the preparation of antimicrobial drugs, the antimicrobial drugs are used to inhibit and / or kill hemolytic Staphylococcus, the amino acid sequence of the antimicrobial peptide BM16 is shown in SEQ ID NO: 1:

[0007] SEQ ID NO: 1: HSHMNHMFALLRLAAK

[0008] The antimicrobial peptide BM16 has a molecular weight of 1877.27 Daltons, a positive charge of +2.75, and a total hydrophobicity ratio of 56%. APD3 predicts that the peptide may form an alpha helix.

[0009] 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 2,3-diphosphoglycerate mutase antimicrobial peptide BM16, and the amino acid sequence of the antimicrobial peptide BM16 is SEQ ID NO: 1.

[0010] In a preferred embodiment of the present invention, the active ingredient of the antibacterial drug is the Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16, and the amino acid sequence of the antimicrobial peptide BM16 is SEQ ID NO: 1.

[0011] In a preferred embodiment of the present invention, the antibacterial drug is used to inhibit and / or kill hemolytic Staphylococcus.

[0012] The present invention uses software and websites such as APD3, Pymol2.0, SWISS-MODEL to perform bioinformatics prediction on the protein sequence of 2,3-diphosphoglycerate mutase with antibacterial activity of Bacillus cereus, and obtains a new amino acid sequence of antimicrobial peptide BM16. The antibacterial activity of BM16 combined with β-carotene on hemolytic Staphylococcus aureus was studied; and the degree of damage to the cell membrane of hemolytic Staphylococcus aureus by BM16 was observed by flow cytometry and TEM; at the same time, the genomic DNA of hemolytic Staphylococcus aureus was extracted to verify the effect of antimicrobial peptide BM16 on bacterial DNA. The experimental results show that the antimicrobial peptide BM16 combined with β-carotene has a significant inhibitory effect on hemolytic Staphylococcus aureus.

[0013] The antimicrobial peptide BM16 can damage bacteria through the following actions: the antimicrobial peptide BM16 can penetrate the cell membrane, bind to the bacterial genomic DNA, inhibit the synthesis of bacterial DNA, and thus cause bacterial death.

[0014] 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.

[0015] The implementation of the present invention has the following beneficial effects:

[0016] The antimicrobial peptide BM16 of the present invention has a significant inhibitory effect on hemolytic Staphylococcus aureus, and the effect is better when used in combination with β-carotene. 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. The antimicrobial peptide BM16 of the present invention can be made into an antimicrobial drug for preventing or treating diseases caused by hemolytic Staphylococcus aureus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the structure of the antimicrobial peptide BM16.

[0018] Figure 2 This is the minimum bactericidal concentration (MBC) of the antimicrobial peptide BM16 combined with β-carotene against hemolytic Staphylococcus aureus.

[0019] in, Figure 2 The concentrations of AF antimicrobial peptide BM16 in the samples were 0 μg / mL, 15.6 μg / mL, 7.8 μg / mL, 3.9 μg / mL, 1.95 μg / mL, and 0.98 μg / mL, respectively.

[0020] Figure 3 This is the time-killing kinetic curve of the antimicrobial peptide BM16 combined with β-carotene against hemolytic Staphylococcus.

[0021] Figure 4 The effect of antimicrobial peptide BM16 on the cell membrane permeability of hemolytic Staphylococcus aureus.

[0022] Figure 5 Flow cytometry was used to observe the effect of antimicrobial peptide BM16 combined with β-carotene 3 on the cell membrane permeability of hemolytic Staphylococcus aureus.

[0023] Figure 6 This is the circular dichroism spectrum change diagram of the antimicrobial peptide BM16 under different conditions.

[0024] Figure 7 This is the gel electrophoresis diagram of the antimicrobial peptide BM16 and the genomic DNA of hemolytic Staphylococcus aureus.

[0025] Among the bands 1-9, the mass ratios of antimicrobial peptide BM16 / DNA were 25 / 4, 25 / 8, 25 / 10, 25 / 12, 25 / 14, 25 / 16, 25 / 18, 25 / 20, and 25 / 25, respectively.

[0026] Band 10 is the control group DNA.

[0027] Figure 8This is the circular dichroism spectrum change diagram after the antimicrobial peptide BM16 binds to the genomic DNA of hemolytic Staphylococcus aureus. DETAILED DESCRIPTION

[0028] 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.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] Example 1 Screening of antimicrobial peptide BM16

[0032] Bioinformatics prediction was performed on the protein sequence of 2,3-diphosphoglycerate mutase with antibacterial activity from 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 HSHMNHMFALLRLAAK (as shown in SEQ ID NO: 1) with the best antibacterial properties was finally screened out and chemically synthesized (synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd.), and the antibacterial activity was verified.

[0033] Table 1: Prediction analysis of antimicrobial peptides in Bacillus cereus

[0034]

[0035] Example 2 Determination of the minimum bactericidal concentration (MBC) of antimicrobial peptide BM16 combined with β-carotene

[0036] Hemolytic 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 BM16 was dissolved in phosphate buffer, mixed with β-carotene at a molar ratio of 10:1, and incubated with the bacteria at 37°C for 2 h. The minimum inhibitory concentration (MBC) refers to the lowest concentration of antimicrobial peptide that can kill bacteria after incubation at 37°C. Figure 2As shown in the figure, the minimum bactericidal concentration (MBC) of the antimicrobial peptide BM16 combined with β-carotene against hemolytic Staphylococcus aureus was 1.95 µg / mL.

[0037] Example 3 Time kill curve determination of antimicrobial peptide BM16 combined with β-carotene

[0038] Hemolytic Staphylococci were 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 peptide and mix with bacteria in equal volume at 37℃ and incubate them separately. Take samples every 30 minutes and apply them on the plate. After incubation at 37℃ overnight, record the total number of colonies. The results show that the antimicrobial peptide BM16 combined with β-carotene can kill hemolytic Staphylococcus aureus within 1 hour (such as Figure 3 as shown).

[0039] Example 4 Effect of antimicrobial peptide BM16 on the cell membrane permeability of hemolytic Staphylococcus aureus

[0040] Transmission electron microscopy (TEM): dilute 5 mL to 10 5-6 CFU / mL of hemolytic Staphylococcus aureus was mixed with 1×MBC antimicrobial peptide BM16 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 4 shown).

[0041] like Figure 4 A in Figure 4 As shown in Figure B, the cell membrane of hemolytic Staphylococcus aureus that has not been treated with the antimicrobial peptide BM16 is intact, the intracellular tissue is evenly distributed, the surface is smooth and flat, and no content flows out. Figure 4 C in Figure 4 As shown in D, after treatment with the antimicrobial peptide BM16, a large number of hemolytic Staphylococci were observed to die, the cell membrane was destroyed, the cells shrank and deformed, and the intracellular substances were significantly reduced.

[0042] Flow cytometry: Hemolytic Staphylococcus 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, the antimicrobial peptide BM16 with a concentration of 1×MBC and 2×MBC was 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 PI dye with a concentration of 50 μg / mL 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 a data collection and analysis plan to analyze the sample by detecting the scattered light signal (SC) and the propidium iodide fluorescence signal (PI) (e.g. Figure 5 as shown).

[0043] like Figure 5 As shown in the figure, the mortality rate of hemolytic Staphylococcus in the control group was only 14.1%, indicating that the cell membrane structure was intact, PI could not pass through the cell membrane and enter the cell, and the bacterial mortality rate was low; the mortality rate of hemolytic Staphylococcus with the addition of 1×MBC concentration of antimicrobial peptide BM16 increased significantly to 73.3%; the mortality rate of hemolytic Staphylococcus in the experimental group with the addition of 2×MBC concentration of antimicrobial peptide increased slightly compared with 1×MBC, reaching 86.7%. It can be seen that the concentration of antimicrobial peptide BM16 is positively correlated with the mortality rate of hemolytic Staphylococcus, and increases in a dose-dependent manner.

[0044] Example 5 Determination of the secondary structure of antimicrobial peptide BM16 by circular dichroism

[0045] The average residue molar ellipticity of the antimicrobial peptide BM16 was measured using a Jasco810 spectropolarimeter (Jasco, Tokyo) CD at a scan speed of 100 nm / min at 25°C. The antimicrobial peptide BM16 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.

[0046] like Figure 6As shown in the figure, in the PBS environment, the antimicrobial peptide BM16 has a positive peak at 189 nm and negative peaks at 197 nm and 226 nm, which indicates that the secondary conformation of the antimicrobial peptide BM16 in the PBS environment is mainly α-helix. In the SDS environment, there is a positive peak at 191 nm and two negative peaks at 208 nm and 222 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.

[0047] Example 6 Interaction between antimicrobial peptide BM16 and bacterial DNA

[0048] The interaction between the antimicrobial peptide BM16 and the genomic DNA of hemolytic Staphylococcus aureus was studied by DNA gel retardation method. Hemolytic Staphylococcus aureus was cultured in 50 mL nutrient broth at 37°C for 12 h, and the optical density ratio of bacteria at 260 and 280 nm (OD 260 / OD 280 ≥ 1.90) to evaluate the purity of the extracted genomic DNA. Next, 10 µL of DNA (20 ng / µL) was mixed with the antimicrobial peptide BM16 at 25°C to make the antimicrobial peptide BM16 / DNA mass ratios 25 / 4, 25 / 8, 25 / 10, 25 / 12, 25 / 14, 25 / 16, 25 / 18, 25 / 20, 25 / 25, and 0, respectively, as shown in Figures 1-10. After mixing, the mixture was incubated at 37°C for 2 h. Then, 8 µL of each sample was electrophoresed on a 1% agarose gel, and the gel retardation was observed under ultraviolet light using the GelDoc XR gel imaging system (Bio-Rad, USA).

[0049] like Figure 7 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 hemolytic Staphylococcus aureus in the control group showed the brightest and clearest bands. This shows that the antimicrobial peptide BM16 can interact with the genomic DNA of hemolytic Staphylococcus aureus.

[0050] Example 7 Effect of the interaction of antimicrobial peptide BM16 with bacterial DNA on its secondary structure

[0051] The average residue molar ellipticity of the antimicrobial peptide BM16 was measured using a Jasco810 spectropolarimeter (Jasco, Tokyo) CD at a scanning speed of 100 nm / min at 25°C. The antimicrobial peptide BM16 was dissolved in 25 mM sodium dodecyl sulfate (SDS) to a final concentration of 0.20 mg / mL. Hemolytic Staphylococcus DNA (20 ng / μL) and the antimicrobial peptide BM16 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. Figure 8 as shown).

[0052] After DNA was incubated with the antimicrobial peptide BM16, the positive peak at 191 nm and the two negative peaks at 208 nm and 222 nm still existed, but the spectrum was overall expanded compared with that in the SDS environment, indicating that the antimicrobial peptide BM16 interacted with the genomic DNA of hemolytic Staphylococcus aureus and could cause a certain change in the proportion of the secondary structure conformation of the antimicrobial peptide.

[0053] In summary, the present invention provides a new antimicrobial peptide BM16. The minimum bactericidal concentration (MBC) of the antimicrobial peptide BM16 combined with β-carotene against hemolytic Staphylococcus is 1.95 µg / mL, which can kill hemolytic Staphylococcus within 1 hour, indicating that it has a strong inhibitory effect on hemolytic Staphylococcus. Studies have found that the antimicrobial peptide BM16 of the present invention can penetrate the cell membrane of bacteria, bind to bacterial genomic DNA, inhibit the synthesis of bacterial DNA, and thus cause bacterial death.

[0054] 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. Application of Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16 in the preparation of antibacterial drugs, characterized in that: The amino acid sequence of the antimicrobial peptide BM16 is shown in SEQ ID NO:

1. The antimicrobial drug is used to inhibit and / or kill hemolytic Staphylococcus.

2. An antibacterial drug, characterized in that: The effective component comprises Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16, and the amino acid sequence of the antimicrobial peptide BM16 is SEQ ID NO:

1.

3. The antibacterial drug according to claim 2, characterized in that: The effective component is Bacillus cereus 2,3-diphosphoglycerate mutase antimicrobial peptide BM16, and the amino acid sequence of the antimicrobial peptide BM16 is SEQ ID NO:

1.

4. The antibacterial drug according to claim 2 or claim 3, characterized in that: The antibacterial drug is used for inhibiting and / or killing hemolytic Staphylococcus.

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