Antimicrobial peptides for inhibiting Staphylococcus aureus and Pseudomonas aeruginosa and applications thereof

Abstract

The present invention belongs to the field of biomedicine, and more specifically, to antimicrobial peptides that inhibit Staphylococcus aureus and Pseudomonas aeruginosa and their applications. The antimicrobial peptide CAMP648NC is a new type of cationic antimicrobial peptide with minimum inhibitory concentrations of 16 μg / mL and 32 μg / mL for pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa, respectively. It has extremely low cytotoxicity, and human skin keratinocytes HaCat and bronchial epithelial cells BEAS‑2B still maintain survival rates of 110.4% and 87.3% after being treated with the antimicrobial peptide CAMP648NC at a concentration of 128 μg / mL for 24 hours. These characteristics make it exhibit extremely high antimicrobial value as a medical drug, providing alternative medicinal resources for future clinical use against these two human pathogens.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to an antimicrobial peptide for inhibiting Staphylococcus aureus and Pseudomonas aeruginosa and an application thereof. Background Art

[0002] Staphylococcus aureus, a member of the genus Staphylococcus, is a representative Gram-positive bacterium and a common foodborne pathogen. It commonly colonizes the skin, nasal cavity, throat, stomach, carbuncle, and purulent sores of humans and animals, and is also ubiquitous in the air, sewage, and other environmental environments. Reports indicate that after colonizing human skin, S. aureus can invade the subcutaneous tissue, directly targeting nerve cells and causing itching. S. aureus frequently develops drug resistance, and its exotoxins can cause toxic diseases in humans, including 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. While generally nonpathogenic under normal circumstances, it can become pathogenic and potentially fatal when the body's immune system is weakened. Systemic Pseudomonas aeruginosa infection can lead to sepsis, fever, jaundice, splenomegaly, pneumonia, urinary tract infection, and meningitis. The formation of biofilms in Pseudomonas aeruginosa significantly increases its antibiotic resistance, severely hindering clinical treatment.

[0003] Antimicrobial peptides, also known as antimicrobial peptides, are widely distributed throughout the body and form part of the innate immune defense system. Unlike conventional antibiotics, antimicrobial peptides are proteins encoded by specific genes and therefore possess unique antimicrobial mechanisms. Despite this, clinical application of antimicrobial peptides still faces challenges, such as strong cytotoxicity and a lack of selectivity for specific bacterial strains. Summary of the Invention

[0004] In order to solve the above technical problems, the object of the present invention is to provide antimicrobial peptides for inhibiting Staphylococcus aureus and Pseudomonas aeruginosa and applications thereof.

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

[0006] The present invention provides an antimicrobial peptide for inhibiting Staphylococcus aureus and Pseudomonas aeruginosa. 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.

[0007] The antimicrobial peptide CAMP648NC is a novel cationic antimicrobial peptide with minimum inhibitory concentrations of 16 μg / mL and 32 μg / mL against the pathogens Staphylococcus aureus and Pseudomonas aeruginosa, respectively. It exhibits extremely low cytotoxicity, with survival rates of 110.4% and 87.3% in skin keratinocytes (HaCat) and bronchial epithelial cells (BEAS-2B) respectively, maintained after 24 hours of treatment with 128 μg / mL of CAMP648NC. These properties make it highly valuable as a medical antimicrobial drug, providing a potential alternative for future clinical use against these two human pathogens.

[0008] The present invention also provides a use of the antimicrobial peptide CAMP648NC in the preparation of an antimicrobial product, wherein the antimicrobial product is used to inhibit Staphylococcus aureus and Pseudomonas aeruginosa.

[0009] Furthermore, the antibacterial product is a medicine or an antibacterial agent.

[0010] Furthermore, the antibacterial product contains the antibacterial 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 antibacterial agent is obtained by dissolving the antimicrobial peptide CAMP648NC in sterile water.

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

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

[0015] Furthermore, the auxiliary material is an auxiliary material required for preparing one of water extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

[0016] Beneficial effects:

[0017] The present invention provides a novel antimicrobial peptide CAMP648NC targeting human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. The antimicrobial peptide CAMP648NC has the characteristics of low production cost, small molecular weight, good antibacterial effect and low toxicity to human cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 The minimum inhibitory concentration of the antimicrobial peptide CAMP648NC against Staphylococcus aureus and Pseudomonas aeruginosa.

[0020] Figure 3 This is the cytotoxicity result diagram of the antimicrobial peptide CAMP648NC. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they 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.

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

[0023] The antimicrobial peptide CAMP648NC is obtained by modifying the N-terminus of the peptide sequence shown in SEQ ID NO.1 by acetylation and amidation. The peptide sequence shown in SEQ ID NO.1 was obtained by analyzing all small reading frames encoded in the genome with the data number CRA006934 published on the China National Center for Bioinformation using prodigal software; and establishing a deep learning model that can predict peptide sequences based on CNN-BiLSTM-Attention; and using this model to screen all small reading frames. The molecular formula of the antimicrobial peptide CAMP648NC is C 58 H 104 N 14 O 10The molecular weight is 1157.56 g / mol, the net charge number is 3, with 3 positive charges, its isoelectric point is 11.28, and the normalized hydrophobicity is 1.078. In conclusion, the antimicrobial peptide CAMP648NC is a new type of 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 with a purity greater than 95%.

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

[0027] The two pathogens used in the experiment, Staphylococcus aureus (accession number ATCC 12600) and Pseudomonas aeruginosa (accession number ATCC 15692), were purchased from Beina Chuanglian Biotechnology Co., Ltd. The minimum inhibitory concentration of the antimicrobial peptide CAMP648NC was determined using the broth microdilution method.

[0028] The two pathogens were inoculated into sterile LB liquid medium and cultured at 37°C overnight with shaking. 1 ml of pathogens was inoculated into fresh LB liquid medium at a volume ratio of 1:99 and cultured until the exponential phase. The bacterial concentration was adjusted to 1×10 5 cfu / mL to obtain the bacterial solution. Then 180μL of the bacterial solution was transferred to a 96-well plate. The antimicrobial peptide CAMP648NC powder was dissolved in sterile water and diluted to a 2-fold serial dilution of the antimicrobial peptide solution. The concentrations of the 2-fold serial dilution of the antimicrobial peptide solution were: 640μg / mL, 320μg / mL, 160μg / mL, 80μg / mL, 40μg / mL, and 20μg / mL. 20μL of the above antimicrobial peptide solutions of different concentrations were added to the bacterial solution in the 96-well plate. The antimicrobial peptide concentrations from the 1st column to the 6th column were: 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, and 2μg / mL. After incubating the 96-well plate at 37°C for 20 hours, the bacterial growth was detected by an enzyme-linked microplate reader. The minimum inhibitory concentration was defined as the minimum antimicrobial peptide concentration at which no bacterial growth was detected. Three replicates were set up for each group of experiments. The experimental pictures are shown in the figure. Figure 1 shown.

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

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

[0031] Cytotoxicity experiments were conducted on the antimicrobial peptide CAMP648NC using human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B. The above cells were inoculated in high-glucose DMEM complete culture medium containing 10% fetal bovine serum by volume. They were placed in a cell culture incubator at 37°C and 5% CO2, and the cell status was observed daily and the medium was changed in time. When changing the medium, the old medium was discarded first, and the cells were washed three times with 37°C PBS, and then fresh complete culture medium was added. Cultured to the logarithmic phase. Take the cells growing in the logarithmic phase and discard the culture supernatant. After washing with PBS, add 0.25% trypsin by mass volume for 3 minutes, terminate the digestion with complete culture medium, and centrifuge at room temperature at 800rpm for 5 minutes. Discard the supernatant, blow the cells with 1mL of complete culture medium to prepare a cell suspension, and count. Dilute the cell suspension to 1.58×10 according to the counting results. 5 cells / mL, inoculated into 96-well plates, 100 μL per well, and cultured in a 37°C incubator overnight.

[0032] The antimicrobial peptide CAMP648NC was diluted in complete culture medium to form 350 μL of working solutions at concentrations of 128 μg / mL, 96 μg / mL, and 64 μg / mL, respectively. After overnight incubation, the culture medium was aspirated and 100 μL of working solutions of varying concentrations were added to the experimental groups, while 100 μL of complete culture medium was added to the control group. A blank plate containing complete culture medium without cells was used. Three replicates were set for each concentration and incubated in a 37°C incubator for 24 hours. After 24 hours of dosing and incubation, MTT reagent was added and the cells were incubated in a 37°C incubator for 4 hours. The supernatant was carefully aspirated, and 100 μL of DMSO was added to the wells, shaken, and the plate was read at a wavelength of 570 nm on a microplate reader.

[0033] The cytotoxicity of the antimicrobial peptide CAMP648NC was tested. Figure 3 As shown, when the treatment concentration of the antimicrobial peptide CAMP648NC 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.

[0034] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional 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 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.

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 a bacteriostatic 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 one of water extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

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