Antibacterial peptide for inhibiting drug-resistant staphylococcus aureus and application thereof

Abstract

The invention relates to the technical field of antibacterial peptide application, in particular to an antibacterial peptide for inhibiting drug-resistant staphylococcus aureus and application of the antibacterial peptide. The antibacterial peptide is obtained by performing N-terminal acetylation modification and C-terminal amidation modification on polypeptide as shown in SEQ ID NO.1. The invention further discloses a preparation method of the antibacterial peptide. The antibacterial peptide provided by the invention is a novel cationic antibacterial peptide, the minimum inhibitory concentrations of the antibacterial peptide to pathogenic bacteria staphylococcus aureus ATCC12600 and staphylococcus aureus ATCC33591 are respectively 4 [mu] g / mL and 32 [mu] g / mL, the biological safety is extremely high, and due to the characteristics, the antibacterial peptide shows extremely high medical drug antibacterial value, and can be used for preparing the antibacterial peptide. Alternative medicinal resources are provided for clinically resisting the two human pathogenic bacteria in the future.

Description

Technical Field

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

[0002] Staphylococcus aureus is a common Gram-positive pathogen that is widely considered to be an important pathogen that causes a variety of serious infections. It is widely present in human skin, nasal cavity and throat, and usually presents asymptomatic colonization in individuals with normal immune function. However, when the host's immunity is reduced or the skin barrier is damaged, Staphylococcus aureus can quickly invade the subcutaneous tissue, causing suppurative infection, skin inflammation, and even 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 phenotype of resistance to multiple antibiotics, which greatly reduces the therapeutic effect and seriously threatens the life and health of patients. References: Lemaire S., Olivier A., ​​Van Bambeke F., Tulkens PM, 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. Antimicrobial peptides, as a new type of antimicrobial drug candidate molecule, have attracted widespread attention due to their unique bactericidal mechanism and the advantage of not easily inducing drug resistance. Antimicrobial peptides kill bacteria rapidly by destroying the integrity of bacterial membranes, 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, and broad antibacterial spectrum, showing 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, and insufficient selectivity for specific pathogens. Summary of the invention

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

[0004] The present invention adopts the following technical solution: Firstly, the present invention provides an antimicrobial peptide for inhibiting drug-resistant Staphylococcus aureus. 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.

[0005] The present invention has developed a novel antimicrobial peptide, which exhibits significant antibacterial activity against Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591, and effectively overcomes the limitations of traditional antimicrobial peptides. Through reasonable molecular design and modification, the antimicrobial peptide exhibits good safety while ensuring efficient bactericidal ability, especially low cytotoxicity to human skin keratinocytes HaCaT cells and bronchial epithelial cells BEAS-2B cells.

[0006] At present, there are limited effective treatments for Staphylococcus aureus, especially drug-resistant strains, in clinical practice, and the antimicrobial peptide provided by the present invention fills this gap. It can not only significantly inhibit the growth of Staphylococcus aureus, but also has the advantages of low production cost and strong environmental adaptability, providing important technical support for the development of clinical antimicrobial drugs. In particular, for the treatment of methicillin-resistant Staphylococcus aureus MRSA infection, the antimicrobial peptide has shown excellent potential and can become a safe and efficient new treatment method to solve the current clinical antibiotic resistance problem.

[0007] Secondly, the present invention provides the use of the antimicrobial peptide in the preparation of an antimicrobial product, wherein the antimicrobial product is used to inhibit Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0008] Furthermore, the antibacterial product is a medicine or an antibacterial agent.

[0009] Specifically, the antimicrobial peptide can be used as a broad-spectrum antimicrobial drug for treating a variety of diseases caused by drug-resistant bacteria, especially for methicillin-resistant Staphylococcus aureus. The antibacterial agent can be used in the fields of medical care, food preservation, personal care products and environmental disinfection.

[0010] Furthermore, the antibacterial product contains the antibacterial peptide as the only active ingredient.

[0011] Furthermore, the minimum inhibitory concentrations of the antimicrobial peptide against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are 4 μg / mL and 32 μg / mL, respectively.

[0012] Furthermore, the antibacterial agent is obtained by dissolving the antimicrobial peptide in sterile water.

[0013] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0014] Furthermore, 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.

[0015] The antibacterial product is a medicine or an antibacterial agent.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a novel cationic antimicrobial peptide, named CAMP899NC, whose minimum inhibitory concentrations for pathogenic bacteria Staphylococcus aureus ATCC12600 and Staphylococcus aureus 33591 are 4 μg / mL and 32 μg / mL respectively, and it has extremely low cytotoxicity, and skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B still maintain 96.9% and 96.0% survival rates after being treated at a concentration of 128 μg / mL for 24 hours. These characteristics make it show extremely high antibacterial value of medical drugs, and provide alternative medicinal resources for future clinical treatment against these two human pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the amino acid composition of the antimicrobial peptide CAMP899NC.

[0018] Figure 2 These are the MIC test results of the antimicrobial peptide CAMP899NC against Staphylococcus aureus and Staphylococcus aureus 33591.

[0019] Figure 3 The bacterial growth was measured when the MIC of the antimicrobial peptide CAMP899NC was measured against Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591. Rows a to c were the three replicates of the MIC of the antimicrobial peptide CAMP899NC against Staphylococcus aureus ATCC1260, rows d to f were the three replicates of the MIC of the antimicrobial peptide CAMP899NC against Staphylococcus aureus ATCC33591, and columns 1 to 6 were the The concentrations of the antimicrobial peptide CAMP899NC should be 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, and 2μg / mL; columns 1 to 3 of row g are Staphylococcus aureus ATCC12600+sterile water; columns 1 to 3 of row h are Staphylococcus aureus ATCC33591+sterile water; columns 4 to 6 of row g are antimicrobial peptide CAMP899NC+LB liquid culture medium; columns 4 to 6 of row h are sterile water+LB liquid culture medium.

[0020] Figure 4These are the results of scanning electron microscopy observation of the effect of the antimicrobial peptide CAMP899NC on the morphology of Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591. A is the scanning electron microscopy image of Staphylococcus aureus ATCC12600, B is the scanning electron microscopy image of Staphylococcus aureus ATCC12600 after treatment with the antimicrobial peptide CAMP899NC, and C is the scanning electron microscopy image of Staphylococcus aureus ATCC33591 after treatment with the antimicrobial peptide CAMP899NC.

[0021] Figure 5 These are the results of the cytotoxicity experiment of the antimicrobial peptide CAMP899NC.

[0022] Figure 6 These are photos of the MTT staining plate of the cytotoxicity experiment of the antimicrobial peptide CAMP899NC. Columns 1 to 4 are HACAT cells treated with the antimicrobial peptide CAMP899NC at concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL, respectively. Columns 5 to 8 are BEAS-2B cells treated with the antimicrobial peptide CAMP899NC at concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL, respectively. Rows a to c are repeated three times. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0024] The pathogenic bacteria used in the following examples are from: Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 were purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0025] Example 1: Physicochemical properties and preparation of antimicrobial peptide CAMP899NC.

[0026] The antimicrobial peptide CAMP899NC is obtained by modifying the natural polypeptide shown in SEQ ID NO.1 by acetylation at the N-terminus and amidation at the C-terminus. The molecular formula of the antimicrobial peptide CAMP899NC is C 81 H 135 O 15 N 21, molecular weight is 1642.04g / mol, net charge number is 4, with 4 positive charges, its isoelectric point is 14, and normalized hydrophobicity is 1.04. Containing only 13 amino acids and a molecular weight of only 1642.04g / mol, the production cost of CAMP899NC is very low. In short, the antimicrobial peptide CAMP899NC is a new type of cationic antimicrobial peptide with small molecular weight. The amino acid structure and related schematic diagrams are from the peptide website: https: / / www.allpeptide.com / jiegoutu.html. Since the online tool AlphaFold3 cannot predict the structure of modified peptides, the protein structure prediction of the unmodified peptide is performed to reflect the structure from the side. The prediction results are as follows Figure 1 As shown, the peptide is a typical α-helical peptide with amphipathic characteristics, which helps it interact with bacterial membranes. The website of AlphaFold3 is: https: / / golgi.sandbox.google.com / about.

[0027] SEQ ID NO. 1: IIKLWKTVFLRGK.

[0028] The antimicrobial peptide CAMP899NC in the present invention is synthesized by Sangon Biotech (Shanghai) Co., Ltd. through solid phase chemical synthesis, with a purity of more than 95%.

[0029] 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.

[0030] The MIC of antimicrobial peptides was determined by the broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991.

[0031] The two pathogens were inoculated into sterile LB liquid medium and cultured at 37°C overnight with shaking. The pathogens were inoculated into fresh LB liquid medium at a 1% inoculum and cultured until the exponential phase. The bacterial concentration was adjusted to 1×10 5cfu / mL to obtain bacterial solution. Then 180μL of bacterial solution was transferred to a 96-well plate. The antimicrobial peptide CAMP899NC powder was dissolved in sterile water and diluted to a 2-fold antimicrobial peptide solution. The concentrations of the 2-fold antimicrobial peptide solutions were 5μg / mL, 10μg / mL, 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL, 320μg / mL, 640μg / mL, 1280μg / mL and 2560μg / mL. 20μL of antimicrobial peptide solutions of different concentrations were added to the bacterial solution in the 96-well plate, respectively, so that the concentrations of antimicrobial peptides in the bacterial solution were 0.5μg / mL, 1μg / mL, 2μg / mL, 4μg / mL, 8μg / mL, 16μg / mL, 32μg / mL, 64μg / mL, 128μg / mL and 256μg / mL. After incubating the 96-well plate at 37°C for 20 hours, the bacterial growth was detected by an ELISA instrument. MIC was defined as the minimum antimicrobial peptide concentration for detecting bacterial growth. Sterile water was used as the control group CK, and 3 replicates were set for each group of experiments.

[0032] The MICs of the antimicrobial peptide CAMP899NC against Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 are shown in Figure 2 Figure 2 and Figure 3 As shown, they are 4 μg / mL and 32 μg / mL, respectively.

[0033] Scanning electron microscopy was used to observe the bactericidal effect of the antimicrobial peptide CAMP899NC on Staphylococcus aureus. The detection method was as follows: Staphylococcus aureus ATCC12600 and Staphylococcus aureus ATCC33591 were cultured in sterile LB liquid medium at 37°C overnight and diluted to 1×10 8CFU / mL. Subsequently, the bacterial solution was divided into two groups: one group was added with the antimicrobial 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, the samples were centrifuged at 5000rpm for 3 minutes at room temperature, 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 200μL of 2.5% glutaraldehyde solution and fixed overnight at 4°C. The fixed samples were centrifuged at 4000rpm for 10 minutes, the supernatant was discarded, and the bacteria were evenly spread on a microscope slide. Subsequently, the samples were washed three times with sterile PBS buffer, each time for 10 minutes. After washing, the samples were dehydrated in 30% ethanol solution, 50% ethanol solution, 70% ethanol solution, 80% ethanol solution and pure ethanol in turn, and treated twice in pure ethanol for 10 minutes each time. After dehydration, the slides were treated with a 1:1 volume ratio of ethyl isovalerate / ethanol solution and then with ethyl isovalerate. 2 The samples were processed by 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 S. aureus under different treatment conditions.

[0034] The experimental results showed that the Staphylococcus aureus treated with the antimicrobial peptide CAMP899NC showed obvious cell membrane damage or rupture, while the bacterial cell structure in the control group was intact and there was no obvious change. Figure 4 .

[0035] Example 3: Cytotoxicity assay of the antimicrobial peptide CAMP899NC.

[0036] The cytotoxicity experiment of the antimicrobial peptide CAMP899NC was carried out using human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B.

[0037] The above cells were inoculated in high-glucose DMEM complete medium containing 10% fetal bovine serum, which also contained 10000U / mL penicillin and 10000μg / mL streptomycin, referred to as complete medium. The cells were placed in a cell culture incubator at 37°C and 5% CO. 2 Culture, observe the cell status daily and change the medium in time. When changing the medium, discard the old medium first, wash three times with 37℃ PBS, and then add fresh complete culture medium. Culture until the logarithmic phase.

[0038] Take cells in logarithmic growth, discard the culture supernatant, wash with PBS, add 0.25% trypsin to digest for 3 minutes, terminate digestion with complete culture medium, and centrifuge at room temperature for 5 minutes at 800rpm. Discard the supernatant, blow the cells with 1mL complete culture medium to prepare cell suspension, and count. Dilute the cell suspension to 158,000 cells / mL according to the counting results, inoculate into 96-well plates, 100μL per well, i.e. 15,800 cells / well, and culture overnight in a 37℃ incubator.

[0039] Complete medium was used to dissolve and dilute the antimicrobial peptide CAMP899NC to form 350 μL of antimicrobial peptide working solutions with concentrations of 128 μg / mL, 96 μg / mL, and 64 μg / mL, respectively. The culture medium was removed from the cells after overnight culture, and 100 μL of antimicrobial peptide working solutions of different concentrations were added to the experimental group, and 100 μL of complete medium was added to the control group. The blank was complete medium without inoculation of cells. Three replicates were set for each concentration and cultured in a 37°C incubator for 24 hours.

[0040] After 24 hours of drug administration and culture, MTT reagent was added and incubated in a 37°C incubator for 4 hours. The supernatant was carefully aspirated, 100 μL of DMSO was added to the wells and shaken, and the plate was read at a wavelength of 570 nm on a microplate reader.

[0041] The cytotoxicity of the antimicrobial peptide CAMP899NC was tested. Figure 5 and Figure 6 As shown, when the treatment concentration of the antimicrobial peptide CAMP899NC reached 128 μg / mL, the survival rates of skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B were 110.4% and 87.3%, respectively. Its extremely low cytotoxicity gives it excellent medical therapeutic potential.

[0042] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall 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 for inhibiting Staphylococcus aureus and methicillin-resistant 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 or an antibacterial agent.

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 2 in the preparation of antimicrobial products, characterized in that: The minimum inhibitory concentrations of the antimicrobial peptide to Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are 4 μg / mL and 32 μg / mL, respectively.

6. The use of the antimicrobial peptide according to claim 3 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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