A broad-spectrum antibacterial peptide cAMP-919nc derived from bacteria and its applications

Abstract

The present invention relates to the technical field of the application of antimicrobial peptides, and particularly relates to a broad-spectrum bacterium-derived antimicrobial peptide cAMP-919nc and its application. The antimicrobial peptide cAMP-919nc is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1. This antimicrobial peptide exhibits excellent antibacterial effects against a variety of bacteria, especially the minimum inhibitory concentration against Staphylococcus aureus is as low as 4 μg / mL. Under different pH conditions, this antimicrobial peptide also shows significant bactericidal activity against the Gram-negative pathogenic bacterium Acinetobacter baumannii. In addition, this peptide has extremely low cytotoxicity, showing good safety. The present invention provides an antimicrobial peptide with excellent performance and has broad application potential, and can be used to develop various antimicrobial products such as antimicrobial drugs, disinfectants, and biological preservatives. The present invention opens up a new direction for the research and application of antimicrobial peptides and has important scientific and practical value.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial peptide applications, and particularly relates to a broad-spectrum bacterial-derived antibacterial peptide cAMP-919nc and its applications. Background Art

[0002] As a naturally occurring antibacterial molecule, antibacterial peptides have become an important research direction for solving the problem of antibiotic resistance in recent years due to their broad-spectrum antibacterial activity and low tendency to induce drug resistance. Different from traditional antibiotics, antibacterial peptides exhibit significant antibacterial and bactericidal effects on a variety of pathogenic microorganisms through unique mechanisms of action such as disrupting cell membrane integrity or interfering with intracellular functions.

[0003] Despite the excellent antibacterial properties of antibacterial peptides, they still face many challenges in practical applications, such as insufficient environmental stability, relatively high potential toxicity to host cells, and relatively limited selectivity for specific pathogenic bacteria. To overcome these limitations, various modification methods have been developed in recent years, including acetylation, amidation, cyclization, and non-natural amino acid substitution. These strategies have significantly improved the stability, biological activity, and targeting of antibacterial peptides. However, most of the currently known antibacterial peptides have a relatively narrow antibacterial spectrum. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a broad-spectrum bacterial-derived antibacterial peptide cAMP-919nc and its applications.

[0005] The present invention adopts the following technical solutions:

[0006] First, the present invention provides a broad-spectrum bacterial-derived antibacterial peptide cAMP-919nc, which is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1. The molecular formula of the antibacterial peptide cAMP-919nc is C 111 H 175 O 20 N 31 , and its molecular weight is 2262.36.

[0007] The present invention provides a broad-spectrum antibacterial peptide cAMP-919nc derived from bacteria, which not only exhibits broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, but also maintains good activity under the conditions of pH = 2, pH = 7.4, and pH = 8.4. In addition, toxicity evaluation shows that the toxicity of cAMP-919nc to human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B is much lower than that of conventional antibacterial peptides, providing a good safety basis for its clinical application. Antibacterial peptides have broad application prospects in the fields of anti-infective drugs, disinfectants, and biological preservatives.

[0008] The minimum inhibitory concentration of the antibacterial peptide cAMP-919nc of the present invention against Gram-positive Staphylococcus aureus and Gram-negative Acinetobacter baumannii is as low as 4 μg / mL and 16 μg / mL respectively, showing significant antibacterial potential. As a functional polypeptide derived from bacteria, cAMP-919nc of the present invention has excellent properties and wide application value, providing new ideas and technical support for the key issues in the clinical transformation of antibacterial peptides.

[0009] Secondly, the present invention provides the application of the antibacterial peptide cAMP-919nc in the preparation of antibacterial products for inhibiting Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii.

[0010] Furthermore, the antibacterial product is a drug or a bacteriostatic agent.

[0011] Specifically, the antibacterial peptide cAMP-919nc can be used as a broad-spectrum antibacterial drug for treating various diseases caused by drug-resistant bacterial infections, especially suitable for combating multidrug-resistant strains, including Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa. The antibacterial peptide cAMP-919nc of the present invention can still maintain high antibacterial performance in extreme environments, significantly superior to the clinical manifestations of existing antibacterial peptides or antibiotics.

[0012] Specifically, the bacteriostatic agent can be used in the fields of medical and health, food preservation, personal care products and environmental disinfection, etc.

[0013] Furthermore, the antibacterial product takes the antibacterial peptide cAMP-919nc as the only active ingredient.

[0014] Furthermore, the minimum inhibitory concentrations of the antibacterial peptide cAMP-919nc against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii are 4 μg / mL, 32 μg / mL, 16 μg / mL and 16 μg / mL in sequence.

[0015] Furthermore, the bacteriostatic agent is obtained by dissolving the antibacterial peptide cAMP-919nc in sterile water.

[0016] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0017] Furthermore, the excipient is an excipient required for preparing one of aqueous extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] Broad-spectrum and high efficiency: The antimicrobial peptide cAMP-919nc has significant antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, especially showing remarkable effects on the clinically multi-drug resistant strains Staphylococcus aureus and Acinetobacter baumannii.

[0020] Environmental stability: In different pH environments, the antimicrobial peptide cAMP-919nc can maintain excellent activity.

[0021] Low cytotoxicity: Toxicity experiments show that the antimicrobial peptide of the present invention has low toxicity to human skin keratinocytes and bronchial epithelial cells and higher safety.

[0022] Broad application prospects: The antimicrobial peptide cAMP-919nc is not only suitable for the development of anti-infective drugs, but also can be used for the preparation of food preservatives, disinfectants and other antimicrobial products.

[0023] In summary, the present invention provides a functional polypeptide with novel origin and excellent performance in the field of antimicrobial peptides, providing important technical support and alternative resources for the development of antimicrobial drugs and the treatment of multi-drug resistant bacterial infections. Brief Description of the Drawings

[0024] Figure 1 It is the amino acid composition and structure of the antimicrobial peptide cAMP-919nc. A is the amino acid composition and B is the amino acid structure.

[0025] Figure 2 It is the MIC determination results of the antimicrobial peptide cAMP-919nc against various pathogenic bacteria. In the figure, S.aureus is Staphylococcus aureus, P.aeruginosa is Pseudomonas aeruginosa, A.baumannii 1 is Acinetobacter baumannii 1, and A.baumannii 2 is Acinetobacter baumannii 2.

[0026] Figure 3 It is the stability experiment results of the antimicrobial peptide cAMP-919nc.

[0027] Figure 4 It is the cytotoxicity experiment results of the antimicrobial peptide cAMP-919nc. Detailed Description of the Invention

[0028] The present invention will be described in detail below with reference to the 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.

[0029] The preservation numbers and sources of the pathogenic bacteria used in the following embodiments:

[0030] Staphylococcus aureus: ATCC12600.

[0031] Acinetobacter baumannii 1: ATCC19606.

[0032] Acinetobacter baumannii 2: ATCC53272, a multi-drug resistant strain.

[0033] Staphylococcus aureus and Acinetobacter baumannii were purchased from Beina Chuanglian Biotechnology Co., Ltd., Acinetobacter baumannii 2 was purchased from Beijing BioVector Science & Technology Co., Ltd., and Pseudomonas aeruginosa was from the laboratory.

[0034] The present invention provides a broad-spectrum antibacterial peptide cAMP-919nc against multi-drug resistant bacteria, which is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1. The antibacterial peptide cAMP-919nc exhibits high antibacterial activity under the conditions of pH = 2, pH = 7.4 and pH = 8.4, and can kill a variety of Gram-positive bacteria and Gram-negative bacteria including Staphylococcus aureus, such as Acinetobacter baumannii. The minimum inhibitory concentration value MIC of the antibacterial peptide against Staphylococcus aureus is 4 μg / mL. The antibacterial peptide cAMP-919nc has extremely high antibacterial value for medical drugs and can provide alternative medicinal resources for clinical confrontation against multi-drug resistant pathogenic bacteria.

[0035] Example 1: An antibacterial peptide cAMP-919nc and its preparation method.

[0036] The antibacterial peptide cAMP-919nc is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1. Its molecular formula is C 111 H 175 O 20 N 31 , with a molecular weight of 2262.36 Da, and the amino acid composition is as shown in A of Figure 1 .

[0037] SEQ ID NO.1: KFLSLRRKLKVFAGFHKF.

[0038] The amino acid structure and related schematic diagrams are from the specialized peptide website: https: / / www.allpeptide.com / jiegoutu.html. Since the online tool Acetylation AlphaFold3 cannot predict the structure of the modified peptide, the protein structure of the unmodified peptide of cAMP-919nc was predicted to reflect the structure of cAMP-919nc from the side. The prediction results show that the peptide is a typical α-helical peptide, as shown in Figure 1As shown in B, it has amphiphilic characteristics, which helps its interaction with the bacterial membrane. The AlphaFold3 website is: https: / / golgi.sandbox.google.com / about. Preparation method: The antimicrobial peptide cAMP-919nc was prepared by Shanghai Acetylation Chemical Engineering Biotechnology Co., Ltd. using solid-phase chemical synthesis method, and the purity reached more than 95% acetylation.

[0039] Example 2: Determination of the MIC of the antimicrobial peptide cAMP-919nc.

[0040] 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. The above-mentioned pathogenic bacteria were inoculated into sterile LB medium and cultured overnight at 37°C with shaking. Each pathogenic bacterium was inoculated into fresh LB medium at an inoculation amount of 1%, cultured until the exponential phase, and the cell concentration was adjusted to 1×10 5 cfu / mL to obtain the bacterial suspension. Then, 180 μL of the bacterial suspension was transferred to a 96-well plate. The antimicrobial peptide powder was dissolved in sterile water and diluted to a series of two-fold diluted antimicrobial peptide solutions. 20 μL was taken and added to the bacterial suspension in the 96-well plate, so that the antimicrobial peptide concentration range in the bacterial suspension was 0.5 - 256 μg / mL. After incubating the 96-well plate at 37°C for 24 hours, the growth of bacteria was detected by an enzyme-linked immunosorbent assay (ELISA) reader. The MIC was defined as the minimum antimicrobial peptide concentration for detecting bacterial growth. The experiment was set up with 3 replicates.

[0041] As Figure 2 shown, the MICs of the antimicrobial peptide cAMP-919nc against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii 1, and Acinetobacter baumannii 2 were detected to be 4 μg / mL, 32 μg / mL, 16 μg / mL, and 16 μg / mL, respectively.

[0042] Example 3: Determination of the stability of the antimicrobial peptide cAMP-919nc.

[0043] Sterile PBS solution was added to 1 mol / L HCl and NaOH solutions to prepare PBS solutions with pH = 2, pH = 7.4, and pH = 8.4. Then, the antimicrobial peptide was dissolved in PBS solutions with different pH values and incubated at 37°C for 1 h. After that, the change in the MIC of the antimicrobial peptide under different pH treatments was determined according to the method described in Example 2. As Figure 3The results shown indicate that pH has no effect on the action of the antimicrobial peptide. At pH = 2, pH = 7.4, and pH = 8.4, the MIC of the peptide remains unchanged, and the antimicrobial peptide can maintain stability and exert its function in different pH environments.

[0044] Example 4: Toxicity determination of antimicrobial peptide cAMP-919nc.

[0045] Cytotoxicity experiments of antimicrobial peptide cAMP-919nc were performed using human bronchial epithelial cells BEAS-2B and human keratinocyte HaCat. Cells BEAS-2B and HACAT were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured in a cell incubator at 37°C with 5% CO2. Cell passage culture: When the cells grew to 80% - 90%, the medium was removed, and the cells were washed 1 - 2 times with PBS. After removing the PBS, 1 ml of trypsin was added to digest for 1 - 3 min, and then 3 ml of complete medium was added to neutralize the trypsin to terminate digestion. The digested cells were transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. After pouring off the supernatant, 3 ml of medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 into culture dishes for culture. BEAS-2B and HACAT cells that grew to 80% - 90% in the culture dishes were digested with trypsin, centrifuged at 1000 rpm, the supernatant was removed, and the cells were resuspended with complete medium. The cells were seeded into 96-well plates, with 5000 cells seeded in each well. After the cells adhered, drugs with concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL were added, gently mixed, and then cultured in an incubator for 24 h. Then 10 μl of MTT with a concentration of 5 mg / ml was added, and the cells were continued to be cultured in the cell incubator for about 3 h. The medium was removed, 150 μl of DMSO was added to each well, shaken evenly, and the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The OD values of each well were read at a wavelength of 570 nm using an ELISA reader, and the cell survival rate was calculated using the following formula:

[0046] Cell survival rate (%) = (OD of sample group / OD of blank control group) × 100.

[0047] After detection, the cytotoxicity results of antimicrobial peptide cAMP-919nc are as Figure 4 shown. When the concentration of the antimicrobial peptide was as high as 96 μg / ml, the survival rate of human bronchial epithelial cells BEAS-2B was still greater than 90%. When the concentration of the antimicrobial peptide was increased from 0 to as high as 128 μg / ml, the antimicrobial peptide had no negative effect on the growth of human keratinocyte HaCat, indicating that the antimicrobial peptide cAMP-919nc has extremely low cytotoxicity and great potential for medical treatment.

[0048] Although the preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

Claims

1. A broad-spectrum antibacterial peptide cAMP-919nc derived from bacteria, characterized in that, The antimicrobial peptide cAMP-919nc is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide cAMP-919nc according to claim 1 in the preparation of antimicrobial products, characterized in that, The antimicrobial product is used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii.

3. Use of the antibacterial peptide cAMP-919nc according to claim 2 in the preparation of antibacterial products, characterized in that, The antimicrobial product is a drug.

4. Use of the antibacterial peptide cAMP-919nc according to claim 3 in the preparation of antibacterial products, characterized in that, The drug is a bacteriostatic agent.

5. Use of the antimicrobial peptide cAMP-919nc according to claim 2 in the preparation of an antimicrobial product, characterized in that, The antimicrobial product uses the antimicrobial peptide cAMP-919nc as the sole active ingredient.

6. Use of the antimicrobial peptide cAMP-919nc according to claim 4 in the preparation of an antimicrobial product, characterized in that, The bacteriostatic agent is obtained by dissolving the antimicrobial peptide cAMP-919nc in sterile water.

7. Use of the antibacterial peptide cAMP-919nc according to claim 3 in the preparation of antibacterial products, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. Use of the antibacterial peptide cAMP-919nc according to claim 7 in the preparation of antibacterial products, characterized in that, The excipients are the excipients required for preparing one of water infusions, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

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