Antibacterial peptide RH22 and application thereof

Abstract

The invention discloses an antibacterial peptide RH22 and application thereof, the amino acid sequence of the antibacterial peptide RH22 is KLPFQR, and the molecular weight of the antibacterial peptide is 787.4704 Daltons. The antibacterial peptide disclosed by the invention has an obvious inhibition effect on bacillus cereus and can be used for preparing medicines, food preservatives or feed additives for preventing or inhibiting bacillus cereus infection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to an antimicrobial peptide RH22 and its applications. Background Technology

[0002] Bacillus cereus is a Gram-positive bacillus widely found in nature. It grows aerobically or facultatively anaerobicly, forms heat-resistant spores, and possesses unique biological characteristics and potential pathogenicity. Bacillus cereus is a common microorganism in soil, water, plant surfaces, and animal intestines, and is also present in food. Due to its heat resistance and spore-forming ability, it easily contaminates starchy or protein-rich foods such as rice, dairy products, and meat. It multiplies at room temperature and produces toxins, making it one of the important pathogens causing food poisoning.

[0003] Currently, antibiotics remain the primary treatment for bacterial diseases, but antibiotic overuse has led to a serious problem—drug resistance. Antimicrobial peptides (AMPs) are a class of multifunctional small-molecule proteins renowned for their broad-spectrum antibacterial, antiviral, and antifungal activities. These peptides are ubiquitous in various life forms, including animals, plants, and microorganisms, and have attracted significant scientific interest due to their unique mechanisms of action and potential applications. Unlike traditional antibiotics, AMPs primarily target bacterial cell membranes, disrupting them through multiple pathways and eliminating bacteria at multiple targets, significantly reducing the likelihood of bacterial resistance development. As an alternative to traditional antibiotics, AMPs have been widely used in medicine, animal husbandry, and the food industry.

[0004] Therefore, finding a novel antimicrobial peptide that can inhibit and kill Bacillus cereus without producing drug resistance is a technical challenge that urgently needs to be solved in this industry. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antimicrobial peptide RH22 and its application, thereby solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] One of the technical solutions adopted by the present invention to solve its technical problem is to provide an antimicrobial peptide RH22, whose amino acid sequence is KLPFQR, as shown in SEQ ID NO: 1.

[0008] The antimicrobial peptide RH22 has a molecular weight of 787.4704 Daltons, a positive charge of +2, and a total hydrophobicity of 33%. APD3 analysis suggests that the peptide may form an α-helix.

[0009] Preferably, the above-mentioned antimicrobial peptide RH22 is used in the preparation of an antimicrobial drug for inhibiting and / or killing Bacillus cereus.

[0010] The second technical solution adopted by the present invention to solve its technical problem is: to provide an antibacterial drug, the effective ingredient of which includes antimicrobial peptide RH22, the amino acid sequence of which is SEQ ID NO: 1.

[0011] Preferably, the above-mentioned antimicrobial peptide RH22 is used in the preparation of aquatic feed additives, which are used to inhibit and / or kill Bacillus cereus.

[0012] The third technical solution adopted by the present invention to solve its technical problem is: to provide an aquatic feed additive, the effective ingredient of which includes antimicrobial peptide RH22, the amino acid sequence of which is SEQ ID NO: 1.

[0013] Preferably, the above-mentioned antimicrobial peptide RH22 is used in the preparation of food preservatives, which are used to inhibit and / or kill Bacillus cereus.

[0014] The fourth technical solution adopted by the present invention to solve its technical problem is: to provide a food preservative, the effective ingredient of which includes antimicrobial peptide RH22, the amino acid sequence of which is SEQ ID NO: 1.

[0015] Antimicrobial peptide RH22 primarily acts on bacterial cell membranes, increasing cell membrane permeability and leading to leakage of intracellular ions, metabolites, and other cellular contents. This results in loss of cell membrane integrity and disruption of membrane structure and function. Simultaneously, it also acts on bacterial DNA, inhibiting DNA replication and synthesis. These changes ultimately lead to bacterial cell death.

[0016] The antimicrobial peptides of the present invention can be synthesized using methods known to those skilled in the art, such as solid-phase synthesis, and purified using methods known to those skilled in the art, such as high-performance liquid chromatography.

[0017] Implementing this invention has the following beneficial effects:

[0018] This invention uses trypsin to enzymatically hydrolyze bullfrog skin, and the resulting hydrolysate is used for peptidomics identification. Based on the identification results, online websites such as APD3 and I-TASSER (https: / / zhanggroup.org / I-TASSER / ) and software such as Pymol 2.0 are used to predict the peptides obtained from peptidomics identification using bioinformatics. A peptide with antibacterial activity, RH22, is obtained and synthesized using a solid-phase synthesis method. The antibacterial activity of RH22 against Bacillus cereus is studied. Nucleic acid-protein leakage experiments, propidium iodide staining analysis, and transmission electron microscopy are used to observe the degree of damage to the bacterial cell membrane by RH22 and the leakage of contents after cell membrane disruption. Molecular dynamics simulation experiments further confirm that RH22 can act on the bacterial cell membrane. Simultaneously, genomic DNA of Bacillus cereus is extracted to verify its effect on bacterial DNA. Experimental results show that the antimicrobial peptide RH22 has a significant inhibitory effect on Bacillus cereus.

[0019] The antibacterial mechanism of antimicrobial peptide RH22 is to act on the cell membrane, causing it to deform and shrink, increasing cell membrane permeability so that the peptide can pass through the bacterial cell membrane, bind to bacterial DNA, and inhibit DNA replication and synthesis, thereby inactivating the bacteria. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of the antimicrobial peptide RH22.

[0021] Figure 2 This is a control diagram for determining the minimum bactericidal concentration (MBC) of antimicrobial peptide RH22 against Bacillus cereus.

[0022] The concentrations of the AG antimicrobial peptide RH22 in the figure are 0 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL and 31.25 μg / mL, respectively.

[0023] Figure 3 The time-killing kinetic curve of antimicrobial peptide RH22 against Bacillus cereus.

[0024] Figure 4 The effect of antimicrobial peptide RH22 on the cell membrane permeability (nucleic acid leakage) of Bacillus cereus.

[0025] Figure 5 The effect of antimicrobial peptide RH22 on the cell membrane permeability of Bacillus cereus (protein leakage).

[0026] Figure 6 The effect of antimicrobial peptide RH22 on the cell membrane permeability of Bacillus cereus (propidium iodide staining analysis).

[0027] Figure 7 The effect of the antimicrobial peptide RH22 on the cell membrane permeability of Bacillus cereus (fluorescence microscopy image analyzed by propidium iodide staining).

[0028] A represents the control group (bright field);

[0029] B is the control group (fluorescence image);

[0030] C represents the control group (overlay plot);

[0031] D represents the treatment group (bright field);

[0032] E represents the processing group (fluorescence image);

[0033] F represents the processing group (overlay plot).

[0034] Figure 8 This is a transmission electron microscopy (TEM) image of the antimicrobial peptide RH22. Among them,

[0035] A represents untreated Bacillus cereus cells;

[0036] B represents Bacillus cereus cells treated with the antimicrobial peptide RH22.

[0037] Figure 9 This is a molecular dynamics simulation diagram of the antimicrobial peptide RH22.

[0038] Figure 10 This is a gel electrophoresis image of antimicrobial peptide RH22 and Bacillus cereus genomic DNA.

[0039] in,

[0040] Bands 1-6 represent antimicrobial peptide RH22 / DNA mass ratios of 100 / 1, 50 / 1, 25 / 1, 25 / 2, 25 / 4, and 25 / 8, respectively.

[0041] Band 7 represents the control group DNA.

[0042] Figure 11 The fluorescence spectrum shows the competitive binding of the antimicrobial peptide RH22 to EB. Detailed Implementation

[0043] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] Example 1: Identification and Screening of Antimicrobial Peptide RH22

[0047] After drying and crushing, bullfrog skin was enzymatically hydrolyzed with trypsin at a material-to-liquid ratio of 1:15 (w / v) for 2 hours at a temperature of 55°C. The enzyme dosage was 5000 U / g. After hydrolysis, the enzyme was inactivated at 100°C for 15 minutes, cooled, and centrifuged at 10000 r / min for 10 minutes. The supernatant was collected and ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 3000 Da. The supernatant with a molecular weight <3000 Da was collected, freeze-dried, and then subjected to peptide omics analysis.

[0048] Chromatographic column: 75μm id×25cm, NanoViper C18 1.9μm, 100A

[0049] Mobile phase A: 0.1% formic acid (FA);

[0050] Mobile phase B: 0.1% FA, 80% ACN acetonitrile;

[0051] Flow rate: 2.50 μL / min;

[0052] Analysis time for each component: 7 min;

[0053] The original file was searched using software in the Lithobates catesbeianus protein database. The search results were then used to screen identified peptide sequences using the APD3 online server. The peptide structures were predicted using the Swiss-model server, revealing an antimicrobial peptide with strong antibacterial activity against Bacillus cereus. The sequence was KLPFQR, named RH22, with a molecular weight of 787.4704 Daltons. A schematic diagram of the structure of antimicrobial peptide RH22 is shown below. Figure 1 As shown.

[0054] Example 2: Determination of Minimum Bactericidal Concentration (MBC)

[0055] Bacillus cereus was cultured at 37°C for 12 hours to reach the logarithmic growth phase, and then diluted to a final concentration in 0.01M pH 7.2 phosphate buffer. 5-6 CFU / mL. The antimicrobial peptide was dissolved in phosphate buffer, mixed with an equal volume of bacteria at 37°C, and incubated for 3 hours. The minimum bactericidal concentration (MBC) refers to the lowest concentration of antimicrobial peptide that can kill bacteria after incubation at 37°C. For example... Figure 2 As shown, the minimum bactericidal concentration (MBC) of the antimicrobial peptide RH22 against Bacillus cereus is 62.5 μg / mL.

[0056] Example 3: Time-kill curve determination

[0057] Bacillus cereus was cultured at 37°C for 12 hours to reach the logarithmic growth phase, and then diluted to a final concentration of 10 μL in 0.01 M pH 7.2 phosphate buffer. 5-6 CFU / mL. Take an equal volume of antimicrobial peptide (1×MBC concentration) and mix it with bacteria at 37°C. Incubate at 37°C, taking samples every 30 minutes and plating. Incubate overnight at 37°C and record the total colony count. For example... Figure 3 As shown, the antimicrobial peptide RH22 showed a significant effect starting at 1 hour, followed by a continuous decline, and completely killed Bacillus cereus within 3 hours, indicating that the antimicrobial peptide RH22 has a significant inhibitory effect on Bacillus cereus as the treatment time increases.

[0058] Example 4: Effect of antimicrobial peptide RH22 on the cell membrane permeability of Bacillus cereus (nucleic acid and protein leakage analysis)

[0059] Nucleic acid leakage: 200 μL of Bacillus cereus strain stored at -20℃ was inoculated into LB medium and cultured at 37℃ and 200 rpm in a shaker until the logarithmic growth phase. The culture was then diluted to 10⁻¹⁰ in sterile PBS. 5-6 CFU / mL was mixed with peptides of 1×MBC and 2×MBC concentrations in equal proportions, and the OD was measured every 30 minutes using a multi-functional microplate reader. 260 The absorbance at 260 nm was used to assess the amount of nucleic acid leakage from the bacteria. An equal volume of sterile PBS mixed with bacteria in a uniform ratio served as a blank control. The absorbance at 260 nm can be used to estimate the amount of nucleic acid leaked from the cytoplasm. Figure 4 As shown, the absorbance value of the culture medium treated with antimicrobial peptide RH22 increased with the increase of antimicrobial peptide RH22 concentration, indicating that the amount of nucleic acid increased in a dose-dependent manner.

[0060] Protein leakage: Dilute Bacillus cereus cultured to the logarithmic phase with sterile PBS solution to 10... 5-6 CFU / mL was mixed with peptides of 1×MBC and 2×MBC concentrations in equal proportions, and the concentrations were measured at OD500 every 30 minutes using a multi-functional microplate reader. 280 The absorbance at 280 nm was used to assess the amount of protein leakage from the bacterial cells. An equal volume of sterile PBS solution was mixed with bacteria in a specific ratio as a blank control. The absorbance at 280 nm can be used to estimate the amount of protein leaking from the cytoplasm. Figure 5 As shown, with the increase of antimicrobial peptide RH22 concentration, the absorbance value in the culture medium treated with antimicrobial peptide RH22 increases, and the protein content increases in a dose-dependent manner.

[0061] Example 5: Effect of antimicrobial peptide RH22 on bacterial PI uptake (propidium iodide staining analysis)

[0062] Take culture up to 10 6 -10 7 50 μL of a bacterial suspension with a concentration of CFU / mL was added to a 1.5 mL sterile centrifuge tube, followed by an equal volume of antimicrobial peptide solution at different concentrations, with sterile water as a blank control. After incubation at 37°C for 3 h, 100 μL of PI (final concentration 10 μM) was added and incubated in the dark for 15 min. The fluorescence intensity of the PI was detected at an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

[0063] Dilute to 10 6 -10 7 CFU / mL bacterial suspension was mixed with an equal volume of antimicrobial peptide to a final concentration of 1×MBC and incubated at 37°C. The incubation time was the same as in the fluorescence spectroscopy experiment. An equal volume of sterile PBS was used as a blank control. After incubation, 100 μL of the mixture was added to an equal volume of PI (final concentration 30 μM) dye, vortexed at 25°C, and incubated in the dark for 15 min. After staining, the mixture was washed and resuspended in sterile PBS to remove excess dye. 2 μL of bacterial suspension was added to a glass slide, covered with a coverslip and sealed. Images were captured using a fluorescence microscope, selecting the PI channel.

[0064] Propidium iodide (PI) is a DNA-binding dye that cannot penetrate the intact cell membrane of normal cells or early apoptotic cells. However, in late apoptotic cells and dead cells, PI can pass through the cell membrane and stain the cell nucleus red. Figure 6 As shown, the PI fluorescence signal of Bacillus cereus treated with antimicrobial peptide RH22 was significantly increased compared with the control group, indicating that antimicrobial peptide RH22 disrupted the cell membrane integrity of Bacillus cereus, allowing PI to bind to intracellular nucleic acids. Figure 7 As shown, the blank control group showed only a small amount of weak red fluorescence under the microscope, which was presumably due to the normal death of bacteria. However, after treatment with the antimicrobial peptide RH22 of 1×MBC, most of Bacillus cereus was marked with red fluorescence, indicating that the cell membrane was severely damaged and the bacteria died.

[0065] Example 6: Transmission electron microscopy analysis of antimicrobial peptide RH22

[0066] After activating the bacteria, take 1 mL of bacterial suspension and centrifuge at 10000 rpm for 1 min in a 1.5 mL centrifuge tube. Remove the supernatant, wash 3 times with sterile phosphate buffer, and resuspend to 10 mL. 8The bacterial cells were incubated at CFU / mL with an equal volume of antimicrobial peptide solution at 27°C for 3 hours. After incubation, the cells were centrifuged to obtain a precipitate, washed three times with sterile phosphate buffer, and then fixed with 2.5% glutaraldehyde for 12 hours. The fixative was then discarded, and the cells were washed three times with PBS buffer for 15 minutes each time. The samples were fixed with 1% osmium tetroxide solution for 1-2 hours. The osmium tetroxide waste solution was carefully removed, and the samples were rinsed three times with 0.1M phosphate-buffered saline (PBS, pH 7.4) for 15 minutes each time. Then, the samples were sequentially dehydrated in 30%, 50%, 70%, and 90% ethanol, with 15-minute intervals between each dehydration. Dehydration was then performed twice with 100% ethanol for 20 minutes each time. Finally, dehydration was performed twice with 100% acetone for 20 minutes each time. Pure embedding medium was poured into embedding plates, and the samples were inserted into the plates and baked in a 70°C oven for 12-48 hours for polymerization. The resin blocks were then removed, and ultrathin sections of 70-90 nm were cut using an ultramicrotome. These sections were then retrieved using a copper mesh, stained with uranium acetate for 8-15 minutes, stained with lead citrate for 5-10 minutes, and dried. The morphology was observed under a transmission electron microscope, and images were acquired for analysis. An equal volume of sterile phosphate buffer was used instead of the antimicrobial peptide solution as a blank control group.

[0067] like Figure 8 As shown, untreated Bacillus cereus cells are uniformly black, indicating a uniform distribution of electron cloud in the cytoplasm and an intact cell wall. However, after treatment with the antimicrobial peptide RH22, the cells appear lighter in color and unevenly transparent, indicating significant leakage of intracellular material, obvious gaps in the cell membrane, and cavities between the cell wall and cytoplasm, suggesting that the bacteria are about to lyse and die.

[0068] Example 7 Molecular Dynamics Simulation

[0069] The POPG phospholipid membrane structure file was obtained from the charmm-gui website. This file contains 324 POPG phospholipid molecules, and the POPG membrane size is 10nm × 10nm. The force field of the phospholipid molecules can be obtained from the aforementioned charmm-gui website; this force field is the charmm36 force field. The force field information of the peptide used in the simulation was obtained using the Gromacs pdb2gmx module. Gromacs commands were used to place the POPG-peptide complex at the center of the box, and all simulations were performed within a 10nm × 10nm × 14nm box packaged in Gromacs-2022.2. Periodic boundary conditions were used in all simulations to maintain a constant particle number. Subsequently, the system was subjected to 50,000 steps of energy minimization, and the energy-minimized system was subjected to 100ps of NVT equilibration. The cutoff distance for non-bonded interactions was set to... Long-range electrostatic forces were calculated using the particle mesh Ewald (PME) summation method. The temperature was maintained at 310 K using V-rescale, with a coupling constant of 0.1 ps. The pressure was maintained at 1 bar using Berendsen, and hydrogen bonds were constrained using the Lincs algorithm. The time step was set to 2 fs. Finally, a 200 ns simulation of the entire model system was performed, and data analysis was conducted using the simulation trajectory files. Figure 9 As shown, it can be observed that the antimicrobial peptide (blue part) penetrates into the phospholipid bilayer, which can play a role in penetrating or destroying the membrane structure.

[0070] Example 8 DNA Gel Electrophoresis

[0071] The interaction between the antimicrobial peptide RH22 and the genomic DNA of Bacillus cereus was investigated using the DNA gel retardation method. Bacillus cereus was cultured in 50 mL of nutrient broth at 37 °C for 12 h, and the interaction was studied using an optical density ratio (OD) of 260 and 280 nm. 260 / OD 280 The purity of the extracted bacterial genomic DNA was evaluated using a ≥1.90 ppm. Next, 10 μL of DNA (20 ng / μL) was mixed with the antimicrobial peptide RH22 at 25°C to achieve peptide / DNA mass ratios of 100 / 1, 50 / 1, 25 / 1, 25 / 2, 25 / 4, 25 / 8, and 0 / 1, respectively. After mixing, the mixture was incubated at 37°C for 3 h. Then, 8 μL of each mixture was electrophoresed on a 1% agarose gel, and gel retardation was observed under UV irradiation using a GelDoc XR gel imaging system (Bio-Rad, USA).

[0072] like Figure 10 As shown, when the ratios are 100 / 1, 50 / 1, 25 / 1, 25 / 2, 25 / 4, and 25 / 8, the clarity and brightness of the bands increase significantly with the increase of the ratio; the gel electrophoresis image of the genomic DNA of Bacillus cereus in the control group shows the brightest and clearest bands.

[0073] Example 9: Fluorescence spectrum of DNA competitively bound to EB

[0074] The interaction between the antimicrobial peptide RH22 and the genomic DNA of Bacillus cereus was further analyzed using fluorescence spectroscopy to competitively bind to DNA. Bacillus cereus was cultured in 50 mL of nutrient broth at 37°C for 12 h, and the interaction was analyzed using an optical density ratio (OD) of 260 and 280 nm. 260 / OD 280The purity of bacterial genomic DNA extracted using the bacterial genomic DNA extraction kit was evaluated (≥1.90). The bacterial genomic DNA was then diluted to 50 μg / mL with 1×TE buffer, and the reaction was performed in a 96-well plate. First, 5 μL of DNA solution and 10 μL of 100 μg / mL EB solution were added to each well, mixed, and incubated at 37°C in the dark for 10 min to allow EB and DNA to bind and form an EB-DNA complex. Then, antimicrobial peptides were added to final concentrations of 1 / 2×MBC, 1×MBC, and 2×MBC, respectively. The blank control group was replaced with sterile deionized water. After mixing, the mixture was incubated at 37°C in the dark for 30 min. After incubation, the fluorescence spectrum of the EB-DNA complex in the samples was measured using a multi-mode microplate reader in the excitation wavelength range of 535 nm and the emission wavelength range of 570-750 nm.

[0075] Epstein-Barr (EB) is a cationic conjugated planar molecule that typically exhibits weak fluorescence in aqueous environments. However, when it intercalates with DNA base pairs with high affinity, its fluorescence intensity is significantly enhanced. Therefore, EB is used as a DNA fluorescent probe to study the interaction between DNA and small molecules or proteins using fluorescence spectroscopy. Furthermore, when it coexists with molecules that produce similar interactions, they compete for binding to DNA, causing changes in the fluorescence of the EB-DNA complex. Figure 11 As shown, the control group had the highest relative fluorescence intensity. As the concentration of antimicrobial peptide RH22 in the EB-DNA complex increased, the relative fluorescence intensity decreased significantly. This indicates that antimicrobial peptide RH22 can competitively bind to Bacillus cereus genomic DNA with EB, leading to the dissociation of EB molecules embedded in DNA, which in turn reduces the relative fluorescence intensity. Furthermore, the binding strength between the antimicrobial peptide and DNA gradually increases with the increase of the antimicrobial peptide concentration.

[0076] In summary, this invention provides a novel antimicrobial peptide, RH22, which exhibits a minimum bactericidal concentration of 62.5 μg / mL against Bacillus cereus and can completely kill the bacteria within 3 hours, demonstrating a strong inhibitory effect of RH22 on Bacillus cereus. The antimicrobial peptide RH22 of this invention acts on the cell membrane, causing it to deform and shrink, thus increasing cell membrane permeability and disrupting the bacterial cell membrane. This membrane rupture leads to leakage of contents, and simultaneously, it binds to bacterial DNA, thereby inactivating the bacteria.

[0077] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An antimicrobial peptide RH22, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The application of the antimicrobial peptide RH22 as described in claim 1 in the preparation of antimicrobial drugs, characterized in that: The antibacterial drug is used to inhibit and / or kill Bacillus cereus.

3. An antibacterial drug, characterized in that: Its active ingredient includes antimicrobial peptide RH22, the amino acid sequence of which is SEQ ID NO:

1.

4. The antibacterial drug as described in claim 3, characterized in that: The antibacterial drug is used to inhibit and / or kill Bacillus cereus.

5. The application of the antimicrobial peptide RH22 as described in claim 1 in the preparation of aquatic feed additives, characterized in that: The aquatic feed additive is used to inhibit and / or kill Bacillus cereus.

6. An aquatic feed additive, characterized in that: Its active ingredient includes antimicrobial peptide RH22, the amino acid sequence of which is SEQ ID NO:

1.

7. The application of the antimicrobial peptide RH22 as described in claim 1 in the preparation of food preservatives, characterized in that: The food preservative is used to inhibit and / or kill Bacillus cereus.

8. A food preservative, characterized in that: Its active ingredient includes antimicrobial peptide RH22, the amino acid sequence of which is SEQ ID NO: 1.

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