An antibacterial peptide against drug-resistant Staphylococcus aureus and its application

Abstract

The present invention relates to the technical field of the application of antimicrobial peptides, and particularly relates to an antimicrobial peptide for inhibiting drug-resistant Staphylococcus aureus and its application. The antimicrobial peptide is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1. The antimicrobial peptide of the present invention is a novel cationic antimicrobial peptide, and the minimum inhibitory concentrations against the pathogenic bacteria Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 are 4 μg / mL and 32 μg / mL respectively, and its biosafety is extremely high. These characteristics make it show extremely high antibacterial value for medical drugs, providing alternative medicinal resources for clinical confrontation against these two human pathogenic bacteria in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of the application of antimicrobial peptides, and particularly relates to an antimicrobial peptide for inhibiting drug-resistant Staphylococcus aureus and its application. Background Art

[0002] Staphylococcus aureus is a common Gram-positive pathogenic bacterium and is widely regarded as an important pathogenic bacterium causing various serious infections. It widely exists in human skin, nasal cavity and throat, and usually shows an asymptomatic colonization state in individuals with normal immune function. However, when the host immunity declines or the skin barrier is damaged, Staphylococcus aureus can quickly invade the subcutaneous tissue, causing suppurative infections, skin inflammation, and even leading to systemic diseases such as sepsis, pneumonia and toxic shock syndrome. In recent years, due to the irrational use of antibiotics, the drug resistance of Staphylococcus aureus to a variety of conventional antibiotics has increased significantly. Especially methicillin-resistant Staphylococcus aureus, such as the clinical isolate Staphylococcus aureus ATCC33591, which has a drug-resistant phenotype to a variety of antibiotics, greatly reducing the treatment effect and seriously threatening the life and health of patients. The reference is: Lemaire S., Olivier A., Van Bambeke F., Tulkens P.M., Appelbaum P.C., Glupczynski Y. Restoration of susceptibility of intracellular methicillin-resistant Staphylococcus aureus to β-lactams: comparison of strains, cells, and antibiotics. Antimicrobial Agents and Chemotherapy, 2008, 52(3): 1246-1255. As a new candidate molecule for antimicrobial drugs, antimicrobial peptides have received extensive attention due to their unique bactericidal mechanism and the advantage of not easily inducing drug resistance. Antimicrobial peptides mainly achieve rapid bactericidal effects by destroying the integrity of the bacterial membrane, interfering with bacterial metabolism or interacting with specific bacterial targets. Compared with traditional antibiotics, antimicrobial peptides have the characteristics of small molecular weight, fast bactericidal speed, broad antibacterial spectrum, etc., and show great potential in dealing with drug-resistant strains. However, the clinical application of antimicrobial peptides still faces some challenges, including poor environmental stability, high toxicity to human cells, insufficient selectivity for specific pathogenic bacteria, etc. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides an antimicrobial peptide for inhibiting drug-resistant Staphylococcus aureus and its application.

[0004] The present invention adopts the following technical solutions:

[0005] First, the present invention provides an antibacterial peptide for inhibiting drug-resistant Staphylococcus aureus, which is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO. 1.

[0006] The present invention has developed a novel antibacterial peptide, which exhibits significant antibacterial activity against both Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591, and effectively overcomes the limitations of traditional antibacterial peptides. Through reasonable molecular design and modification, while ensuring high antibacterial ability, this antibacterial peptide shows good safety, especially with low cytotoxicity to human skin keratinocyte HaCaT cells and bronchial epithelial BEAS-2B cells.

[0007] Currently, there are limited effective treatment means clinically for Staphylococcus aureus, especially drug-resistant strains, and the antibacterial peptide provided by the present invention fills this gap. It can not only significantly inhibit the growth of Staphylococcus aureus, but also has advantages such as low production cost and strong environmental adaptability, providing important technical support for the development of clinical antibacterial drugs. Especially for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections, this antibacterial peptide shows excellent potential and can become a safe and highly effective new treatment method to solve the problem of antibiotic resistance faced clinically.

[0008] Secondly, the present invention provides the application of the antibacterial peptide in the preparation of antibacterial products for inhibiting Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0009] Furthermore, the antibacterial product is a drug or a bacteriostatic agent.

[0010] Specifically, the antibacterial peptide can be used as a broad-spectrum antibacterial drug for treating various diseases caused by drug-resistant bacteria infections, especially suitable for methicillin-resistant Staphylococcus aureus. The bacteriostatic agent can be used in fields such as medical and health, food preservation, personal care products, and environmental disinfection.

[0011] Furthermore, the antibacterial product takes the antibacterial peptide as the only active ingredient.

[0012] Furthermore, the minimum inhibitory concentrations of the antibacterial peptide against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are 4 μg / mL and 32 μg / mL, respectively.

[0013] Furthermore, the bacteriostatic agent is obtained by dissolving the antibacterial peptide in sterile water.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] Further, the adjuvant is an adjuvant required for preparing one of a water infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

[0016] The antibacterial product is a drug or a bacteriostatic agent.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention provides a novel cationic antibacterial peptide named CAMP899NC. Its minimum inhibitory concentrations against the pathogenic bacteria Staphylococcus aureus ATCC12600 and Staphylococcus aureus 33591 are 4 μg / mL and 32 μg / mL respectively. It has extremely low cytotoxicity. After being treated at a concentration of 128 μg / mL for 24 hours, the skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B still maintain survival rates of 96.9% and 96.0%. These characteristics make it show extremely high antibacterial value for medical drugs, providing alternative medicinal resources for clinical treatment of these two human pathogenic bacteria in the future. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the amino acid composition of the antibacterial peptide CAMP899NC.

[0020] Figure 2 It is the result of MIC determination of the antibacterial peptide CAMP899NC against Staphylococcus aureus and Staphylococcus aureus 33591.

[0021] Figure 3 It is the bacterial growth situation when determining the MIC of the antibacterial peptide CAMP899NC against Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591. Among them, rows a to c are three replicates for determining the MIC of the antibacterial peptide CAMP899NC against Staphylococcus aureus ATCC1260, and rows d to f are three replicates for determining the MIC of the antibacterial peptide CAMP899NC against Staphylococcus aureus ATCC33591. Columns 1 to 6 correspond to the concentrations of the antibacterial peptide CAMP899NC of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL in sequence; columns 1 to 3 in row g are Staphylococcus aureus ATCC12600 + sterilized water; columns 1 to 3 in row h are Staphylococcus aureus ATCC33591 + sterilized water; columns 4 to 6 in row g are the antibacterial peptide CAMP899NC + LB liquid medium; columns 4 to 6 in row h are sterilized water + LB liquid medium.

[0022] Figure 4Results of the effect of antimicrobial peptide CAMP899NC on the morphology of Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 observed by scanning electron microscopy. A is the scanning electron micrograph of Staphylococcus aureus ATCC12600, B is the scanning electron micrograph of Staphylococcus aureus ATCC12600 treated with antimicrobial peptide CAMP899NC, and C is the scanning electron micrograph of Staphylococcus aureus ATCC33591 treated with antimicrobial peptide CAMP899NC.

[0023] Figure 5 Results of the cytotoxicity experiment of antimicrobial peptide CAMP899NC.

[0024] Figure 6 Photograph of the MTT staining plate for the cytotoxicity experiment of antimicrobial peptide CAMP899NC. Columns 1 to 4 are HACAT cells treated with antimicrobial peptide CAMP899NC at concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL in sequence. Columns 5 to 8 are BEAS-2B cells treated with antimicrobial peptide CAMP899NC at concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL in sequence. Rows a to c are three replicates. Detailed implementation mode

[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] Sources of pathogenic bacteria used in the following examples:

[0027] Both Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 were purchased from Beijing NaChuangLian Biotechnology Co., Ltd.

[0028] Example 1: Physicochemical properties and preparation of antimicrobial peptide CAMP899NC.

[0029] Antimicrobial peptide CAMP899NC is obtained by N-terminal acetylation modification and C-terminal amidation modification of the natural polypeptide shown in SEQ ID NO.1. The molecular formula of the antimicrobial peptide CAMP899NC is C 81 H 135 O 15 N 21, with a molecular weight of 1642.04 g / mol, a net charge number of 4, carrying 4 positive charges, an isoelectric point of 14, and a normalized hydrophobicity of 1.04. It contains only 13 amino acids, and the molecular weight of only 1642.04 g / mol makes the production cost of CAMP899NC very low. In short, the antimicrobial peptide CAMP899NC is a novel cationic antimicrobial peptide with a small molecular weight. The amino acid structure and related schematic diagrams are from the website of Zhuanpeptide: https: / / www.allpeptide.com / jiegoutu.html. Since the online tool AlphaFold3 cannot predict the structure of peptides with modifications, the protein structure of the unmodified peptide was predicted to reflect the structure indirectly. The prediction results are as Figure 1 shown, indicating that the peptide is a typical α-helical peptide with amphiphilic characteristics, which helps its interaction with the bacterial membrane. The AlphaFold3 website is: https: / / golgi.sandbox.google.com / about.

[0030] SEQ ID NO.1: IIKLWKTVFLRGK.

[0031] The antimicrobial peptide CAMP899NC in the present invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd. through solid-phase chemical synthesis method, and the purity is greater than 95%.

[0032] Example 2: Determination of the minimum inhibitory concentration (MIC) and bactericidal effect of the antimicrobial peptide CAMP899NC against Staphylococcus aureus ATCC12600 and Staphylococcus aureus 33591.

[0033] The MIC of the antimicrobial peptide was determined by the broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, P.A. Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991.

[0034] The two pathogenic bacteria were inoculated into sterile LB liquid medium and cultured overnight with shaking at 37°C. The pathogenic bacteria were inoculated into fresh LB liquid medium at an inoculation amount of 1%, cultured to the exponential phase, and the bacterial concentration was adjusted to 1×10 5Obtain the bacterial suspension at cfu / mL. Then transfer 180 μL of the bacterial suspension into a 96-well plate. Dissolve the antimicrobial peptide CAMP899NC powder with sterile water and dilute it into an antimicrobial peptide solution with serial two-fold dilutions. The concentrations of the antimicrobial peptide solution with serial two-fold dilutions are: 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL, 640 μg / mL, 1280 μg / mL, and 2560 μg / mL. Take 20 μL of the antimicrobial peptide solution with different concentrations and add them to the bacterial suspension in the 96-well plate respectively, so that the concentrations of the antimicrobial peptide in the bacterial suspension are: 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, and 256 μg / mL. After incubating the 96-well plate at 37 °C for 20 hours, detect the bacterial growth by a microplate reader. The MIC is defined as the minimum concentration of the antimicrobial peptide for detecting bacterial growth. Use sterile water as the control group CK, and set 3 replicates for each experiment.

[0035] The MICs of the antimicrobial peptide CAMP899NC against Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 are as Figure 2 and Figure 3 shown, which are 4 μg / mL and 32 μg / mL respectively.

[0036] The bactericidal effect of the antimicrobial peptide CAMP899NC against Staphylococcus aureus was observed by scanning electron microscopy. The detection method is as follows:

[0037] After Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 were cultured overnight with shaking at 37 °C in sterile LB liquid medium, they were diluted to 1×10 8CFU / mL. Subsequently, the bacterial solution was divided into two groups: one group was added with the antibacterial peptide CAMP899NC at a concentration of 5×MIC, and the untreated group was used as a negative control. All samples were treated at 37°C for 1 hour. After treatment, they were centrifuged at 5000 rpm at room temperature for 3 minutes, the supernatant was discarded, and the bacteria were washed twice with sterile PBS buffer at pH = 7.0. After washing, the precipitate was resuspended in 200 μL of a 2.5% glutaraldehyde solution by volume and fixed overnight at 4°C. The fixed samples were centrifuged at 4000 rpm for 10 minutes, and after discarding the supernatant, the bacteria were evenly spread on a microscope slide. Subsequently, the samples were washed three times with sterile PBS buffer, 10 minutes each time. After washing, the samples were dehydrated successively in ethanol solutions with volume concentrations of 30%, 50%, 70%, 80%, and absolute ethanol, with two treatments in absolute ethanol, 10 minutes each time. After dehydration was completed, the slide samples were first treated with a 1:1 mixture of ethyl isovalerate / ethanol by volume, and then the slide samples were treated with ethyl isovalerate. After the dehydration and replacement steps were completed, the samples were treated using CO2 critical point drying. Finally, the dried samples were gold-plated and imaged using a Tescan Vega3 scanning electron microscope to observe the morphological changes of Staphylococcus aureus under different treatment conditions.

[0038] The experimental results showed that Staphylococcus aureus treated with the antibacterial peptide CAMP899NC exhibited obvious cell membrane damage or rupture, while the bacterial cell structure of the control group was intact and showed no obvious changes. The imaging results are shown in Figure 4 .

[0039] Example 3: Cytotoxicity assay of the antibacterial peptide CAMP899NC.

[0040] The cytotoxicity experiment of the antibacterial peptide CAMP899NC was carried out using human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B.

[0041] The above cells were respectively inoculated into high-glucose DMEM complete medium containing 10% fetal bovine serum, and the high-glucose DMEM complete medium containing 10% fetal bovine serum also contained 10000 U / mL penicillin and 10000 μg / mL streptomycin, which was simply called complete medium. They were placed in a cell incubator and cultured at 37°C and 5% CO2. The cell state was observed daily and the medium was changed in a timely manner. When changing the medium, the old medium was first discarded, and the cells were washed three times with PBS at 37°C, and then fresh complete culture medium was added. The cells were cultured until the logarithmic phase.

[0042] For cells in the logarithmic growth phase, discard the culture medium supernatant. After washing with PBS, add 0.25% trypsin and digest for 3 min. Terminate the digestion with complete medium and centrifuge at 800 rpm for 5 min at room temperature. Discard the supernatant, pipette the cells with 1 mL of complete medium to prepare a cell suspension, and count the cells. Dilute the cell suspension to 158,000 cells / mL according to the counting result, and inoculate it into a 96-well plate, 100 μL per well, that is, 15,800 cells per well, and culture overnight in an incubator at 37°C.

[0043] Dissolve and dilute the antimicrobial peptide CAMP899NC with complete medium to form 350 μL of antimicrobial peptide working solutions with concentrations of 128 μg / mL, 96 μg / mL, and 64 μg / mL respectively. Aspirate the culture medium from the cells after overnight culture. Add 100 μL of antimicrobial peptide working solutions with different concentrations to the experimental groups, add 100 μL of complete medium to the control group, and the blank is complete medium without inoculated cells. Set 3 replicates for each concentration and culture in an incubator at 37°C for 24 h.

[0044] Add MTT reagent 24 h after drug administration and incubate in an incubator at 37°C for 4 h. Carefully aspirate the supernatant, add 100 μL of DMSO to the wells and shake, and read the plate at a wavelength of 570 nm on an enzyme-linked immunosorbent assay (ELISA) reader.

[0045] After detection, the cytotoxicity results of the antimicrobial peptide CAMP899NC are as Figure 5 and Figure 6 shown. When the treatment concentration of the antimicrobial peptide CAMP899NC reaches 128 μg / mL, the survival rates of the skin keratinocyte HaCat and bronchial epithelial cell BEAS-2B are 110.4% and 87.3% respectively. Its extremely low cytotoxicity endows it with excellent medical treatment potential.

[0046] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments 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.

Claims

1. An antimicrobial peptide for inhibiting drug-resistant Staphylococcus aureus, characterized in that: The antimicrobial peptide is obtained by modifying the polypeptide shown in SEQ ID NO.1 by acetylation at the N-terminus and amidation at the C-terminus.

2. The use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial products, characterized in that: The antibacterial product is used to inhibit Staphylococcus aureus.

3. The use of the antimicrobial peptide according to claim 2 in the preparation of antimicrobial products, characterized in that: The antibacterial product is a medicine.

4. The use of the antimicrobial peptide according to claim 2 in the preparation of antimicrobial products, characterized in that: The antibacterial product uses the antibacterial peptide as the only active ingredient.

5. The use of the antimicrobial peptide according to claim 3 in the preparation of antimicrobial products, characterized in that: The drug is a bacteriostatic agent.

6. The use of the antimicrobial peptide according to claim 5 in the preparation of antimicrobial products, characterized in that: The antibacterial agent is obtained by dissolving the antimicrobial peptide in sterile water.

7. The use of the antimicrobial peptide according to claim 3 in the preparation of antimicrobial products, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The use of the antimicrobial peptide according to claim 7 in the preparation of antimicrobial products, characterized in that: The auxiliary material is an auxiliary material required for preparing an aqueous extract, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.

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