Antibacterial peptide for inhibiting staphylococcus aureus and pseudomonas aeruginosa and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an antibacterial peptide for inhibiting staphylococcus aureus and pseudomonas aeruginosa and application of the antibacterial peptide. The antibacterial peptide CAMP648NC is a novel cationic antibacterial peptide, the minimum inhibitory concentrations of the antibacterial peptide CAMP648NC to pathogenic bacteria staphylococcus aureus and pseudomonas aeruginosa are 16 [mu] g / mL and 32 [mu] g / mL respectively, the antibacterial peptide CAMP648NC has extremely low cytotoxicity, and after human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B are treated by the antibacterial peptide CAMP648NC with the concentration of 128 [mu] g / mL for 24 hours, the survival rates of 110.4% and 87.3% are still kept. Due to the characteristics, the compound shows extremely high medical drug antibacterial value, and alternative medicinal resources are provided for clinically resisting the two human pathogenic bacteria in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to antibacterial peptides that inhibit Staphylococcus aureus and Pseudomonas aeruginosa and their applications. Background Art

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. It is a common foodborne pathogenic microorganism. Staphylococcus aureus often colonizes the skin, nasal cavity, throat, gastrointestinal tract, carbuncles, and suppurating sores of humans and animals, and is also ubiquitous in environments such as air and sewage. Reports have shown that after Staphylococcus aureus colonizes the human skin, it can invade the subcutaneous tissue and directly act on nerve cells, causing skin itching. The frequency of drug resistance generation of Staphylococcus aureus is frequent. Its production of exotoxins can cause human toxin diseases, and can also cause scalded skin syndrome and toxic shock syndrome. Pseudomonas aeruginosa PAO1, also known as Pseudomonas aeruginosa, belongs to the genus Pseudomonas and is an aerobic Gram-negative bacillus. Under normal circumstances, it generally does not cause disease, but can cause disease when the body's resistance is low, and can also cause the death of patients. Systemic Pseudomonas aeruginosa infection can lead to septicemia, and patients have fever, jaundice, splenomegaly, and can develop pneumonia, urinary tract infection, and meningitis. Pseudomonas aeruginosa has greatly increased its antibiotic resistance due to the formation of biofilms, seriously hindering clinical treatment.

[0003] Antibacterial peptides, also known as antimicrobial peptides, are widely distributed in organisms and are part of the natural immune defense system. Antibacterial peptides are different from conventional antibiotics. They are protein products encoded by a specific gene and thus have a unique antibacterial mechanism. Nevertheless, the application of antibacterial peptides in clinical practice still faces challenges, such as strong cytotoxicity and lack of selectivity for specific strains. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide antibacterial peptides that inhibit Staphylococcus aureus and Pseudomonas aeruginosa and their applications.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides an antibacterial peptide that inhibits Staphylococcus aureus and Pseudomonas aeruginosa. The antibacterial peptide is CAMP648NC, and the antibacterial peptide is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide sequence shown in SEQ ID NO.1.

[0007] The antimicrobial peptide CAMP648NC is a novel cationic antimicrobial peptide. The minimum inhibitory concentrations against the pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa are 16 μg / mL and 32 μg / mL respectively. It has extremely low cytotoxicity. The skin keratinocyte HaCat and bronchial epithelial cell BEAS-2B still maintain survival rates of 110.4% and 87.3% after being treated with 128 μg / mL of the antimicrobial peptide CAMP648NC for 24 hours. 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.

[0008] The present invention also provides an application of the antimicrobial peptide CAMP648NC in the preparation of an antibacterial product for inhibiting Staphylococcus aureus and Pseudomonas aeruginosa.

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

[0010] Furthermore, the antibacterial product takes the antimicrobial peptide CAMP648NC as the only active ingredient.

[0011] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide CAMP648NC against Staphylococcus aureus is 16 μg / mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 32 μg / mL.

[0012] Furthermore, the bacteriostatic agent is obtained by dissolving the antimicrobial peptide CAMP648NC in sterile water.

[0013] Furthermore, the concentration of the antimicrobial peptide CAMP648NC in the bacteriostatic agent is 16 μg / mL to 32 μg / mL.

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

[0015] Furthermore, the excipients are the excipients required for preparing one of a water infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

[0016] Beneficial effects:

[0017] The present invention provides a novel antimicrobial peptide CAMP648NC against the human pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa, and this antimicrobial peptide CAMP648NC has the characteristics of low production cost, small molecular weight, good bacteriostatic effect and low cytotoxicity to human cells. Description of the drawings

[0018] Figure 1Growth curve for determining the minimum inhibitory concentration of antimicrobial peptide CAMP648NC against Staphylococcus aureus and Pseudomonas aeruginosa. Among them, rows A to C are three replicates for determining the minimum inhibitory concentration of antimicrobial peptide CAMP648NC against Staphylococcus aureus, and rows D to F are three replicates for determining the minimum inhibitory concentration of antimicrobial peptide CAMP648NC against Pseudomonas aeruginosa. The concentrations of antimicrobial peptide in the bacterial liquid from column 1 to column 6 are: 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL; in row G, columns 1 to 3 are 180 μL of Staphylococcus aureus bacterial liquid plus 20 μL of sterile water, and columns 4 to 6 in row G are 20 μL of antimicrobial peptide CAMP648NC with the corresponding column concentration plus 180 μL of LB medium; in row H, columns 1 to 3 are 180 μL of Pseudomonas aeruginosa bacterial liquid plus 20 μL of sterile water, and columns 4 to 6 in row H are 20 μL of sterile water plus 180 μL of LB medium.

[0019] Figure 2 Minimum inhibitory concentration graph of antimicrobial peptide CAMP648NC against Staphylococcus aureus and Pseudomonas aeruginosa.

[0020] Figure 3 Cytotoxicity result graph of antimicrobial peptide CAMP648NC. Detailed implementation mode

[0021] 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.

[0022] Example 1: Physicochemical properties and preparation of antimicrobial peptide CAMP648NC.

[0023] Antimicrobial peptide CAMP648NC is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide sequence shown in SEQ ID NO.1. The polypeptide sequence shown in SEQ ID NO.1 is all the small open reading frames encoded in the genome publicly available on China National Center for Bioinformation with the data number CRA006934 analyzed by the prodigal software; and a deep learning model for predicting polypeptide sequences is established based on CNN-BiLSTM-Attention; and this model is used to screen all the small open reading frames. The molecular formula of antimicrobial peptide CAMP648NC is C 58 H 104 N 14 O 10, with a molecular weight of 1157.56 g / mol, a net charge number of 3, carrying 3 positive charges, an isoelectric point of 11.28, and a normalized hydrophobicity of 1.078. In summary, the antimicrobial peptide CAMP648NC is a novel cationic antimicrobial peptide with a small molecular weight.

[0024] SEQ ID NO.1: KIKKLVKFL.

[0025] The antimicrobial peptide CAMP648NC was synthesized by Sangon Biotech Co., Ltd. through solid-phase chemical synthesis method, and the purity is greater than 95%.

[0026] Example 2: Determination of the minimum inhibitory concentration.

[0027] For the two pathogenic bacteria used in the experiment, the preservation number of Staphylococcus aureus is ATCC12600, and the preservation number of Pseudomonas aeruginosa is ATCC15692. Both strains were purchased from Beijing NaChuangLian Biotechnology Co., Ltd. The minimum inhibitory concentration of the antimicrobial peptide CAMP648NC was determined by the broth microdilution method.

[0028] Inoculate the two pathogenic bacteria into sterile LB liquid medium and culture them overnight at 37°C with shaking. Inoculate 1 ml of the pathogenic bacteria into fresh LB liquid medium at an inoculation volume ratio of 1:99, and culture until the exponential phase. Adjust the bacterial concentration to 1×10 5 cfu / mL to obtain the bacterial suspension. Then transfer 180 μL of the bacterial suspension to a 96-well plate. The antimicrobial peptide CAMP648NC powder was dissolved in sterile water and diluted to an antimicrobial peptide solution with serial dilutions of 2-fold. The concentrations of the antimicrobial peptide solution with serial dilutions of 2-fold are: 640 μg / mL, 320 μg / mL, 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL. Take 20 μL of the antimicrobial peptide solution with different concentrations as described above and add them to the bacterial suspension in the 96-well plate. The antimicrobial peptide concentrations in columns 1 to 6 are: 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL. After incubating the 96-well plate at 37°C for 20 hours, detect the bacterial growth by a microplate reader. The minimum inhibitory concentration is defined as the minimum antimicrobial peptide concentration at which no bacterial growth is detected. Each experiment was set with 3 replicates. The experimental pictures are as Figure 1 shown.

[0029] The minimum inhibitory concentrations of the antimicrobial peptide CAMP648NC against Staphylococcus aureus and Pseudomonas aeruginosa are as Figure 2 shown, which are 16 μg / mL and 32 μg / mL respectively.

[0030] Example 3: Cytotoxicity determination of the antimicrobial peptide CAMP648NC.

[0031] Cytotoxicity experiments of antimicrobial peptide CAMP648NC were carried out using human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B. The above cells were respectively inoculated into high-glucose DMEM complete medium containing 10% fetal bovine serum by volume. Placed in a cell culture incubator at 37 °C and 5% CO 2 cultivation, observe the cell state daily and change the medium in time. When changing the medium, first discard the old medium, wash three times with PBS at 37 °C, and then add fresh complete culture medium. Cultivate until the logarithmic phase. Take the cells growing in the logarithmic phase, discard the supernatant of the medium, wash with PBS, add trypsin with a mass-volume percentage of 0.25% and digest for 3 min, terminate the digestion with complete medium, and centrifuge at a low speed of 800 rpm at room temperature for 5 min. Discard the supernatant, pipette the cells with 1 mL of complete medium to prepare a cell suspension, and count. Dilute the cell suspension to 1.58×10 5 cell / mL according to the counting results, inoculate into a 96-well plate, 100 μL per well, and culture overnight in a 37 °C incubator.

[0032] Dissolve and dilute antimicrobial peptide CAMP648NC with complete medium to form 350 μL of working solutions with concentrations of 128 μg / mL, 96 μg / mL, and 64 μg / mL respectively. After aspirating the culture medium from the cells after overnight culture, add 100 μL of 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 inoculating cells. Set 3 replicates for each concentration and culture in a 37 °C incubator for 24 h. After 24 h of drug administration and culture, add MTT reagent, incubate in a 37 °C incubator 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.

[0033] After detection, the cytotoxicity results of antimicrobial peptide CAMP648NC are as Figure 3 shown. When the treatment concentration of antimicrobial peptide CAMP648NC reaches 128 μg / mL, the survival rates of skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B are 110.4% and 87.3% respectively. Its extremely low cytotoxicity gives it excellent medical treatment potential.

[0034] 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 know the basic creative concepts. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

Claims

1. An antimicrobial peptide for inhibiting Staphylococcus aureus and Pseudomonas aeruginosa, characterized in that: The antimicrobial peptide is CAMP648NC, which is obtained by modifying the N-terminus of the polypeptide sequence shown in SEQ ID NO.1 by acetylation and amidation of the C-terminus.

2. Use of the antimicrobial peptide CAMP648NC according to claim 1 in the preparation of antimicrobial products, characterized in that: The antibacterial product is used to inhibit Staphylococcus aureus and Pseudomonas aeruginosa.

3. The use according to claim 2, characterized in that: The antibacterial product is a medicine or an antibacterial agent.

4. The use according to claim 3, characterized in that: The antibacterial product contains the antibacterial peptide CAMP648NC as the only active ingredient.

5. The use according to claim 2, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide CAMP648NC against Staphylococcus aureus is 16 μg / mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 32 μg / mL.

6. The use according to claim 3, characterized in that: The antibacterial agent is obtained by dissolving the antimicrobial peptide CAMP648NC in sterile water.

7. The use according to claim 6, characterized in that: The concentration of the antimicrobial peptide CAMP648NC in the antibacterial agent is 16 μg / mL to 32 μg / mL.

8. The use according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. The use according to claim 3, 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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