Antibacterial peptide and application thereof

Abstract

The application belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide and application thereof. The antibacterial peptide is obtained by acetyl capping and amino capping modification of the amino acid sequence shown in SEQ ID NO. 1. The antibacterial peptide provided by the application has high inhibition activity on pathogenic bacteria Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and methicillin- and gentamicin-resistant Staphylococcus aureus, and has good pH stability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide and application thereof. BACKGROUND

[0002] Staphylococcus aureus is a gram-positive bacterium widely existing in nature and human body, commonly found in skin, nasal cavity and respiratory tract and the like. It is one of important pathogenic bacteria of human and animals, and can cause various infections from slight skin infection to severe systemic infection, such as pneumonia, endocarditis and toxic shock syndrome. At present, Staphylococcus aureus has become one of the main threats of clinical surgical infection. Especially, the emergence of drug-resistant Staphylococcus aureus makes many traditional antibiotics ineffective, such as methicillin and gentamicin, which brings severe challenges to clinical treatment. In addition, Staphylococcus aureus can form a biofilm, which further enhances its resistance to antibiotics and host immune system, leading to difficult eradication of infection.

[0003] Antibacterial peptides are a kind of natural immune molecules widely existing in organisms, which have broad-spectrum antibacterial activity. They are composed of short-chain amino acids, usually with positive charge, which can combine with the negatively charged bacterial cell membrane through electrostatic interaction, destroy its integrity, and lead to cell content leakage and bacterial death. Antibacterial peptides not only have broad-spectrum antibacterial activity against bacteria, but also show inhibitory effect on fungi, viruses and even some cancer cells. Compared with traditional antibiotics, the mechanism of action of antibacterial peptides is more complex, and it is difficult to induce bacterial drug resistance, and it shows significant inhibitory effect on many drug-resistant strains, so it is considered as one of the new treatment strategies for drug-resistant bacterial infection. In addition, antibacterial peptides also have immunomodulatory function, which can promote wound healing and regulate inflammatory response, so they are concerned in the field of biomedicine. In recent years, the research on antibacterial peptides against Staphylococcus aureus has attracted much attention, aiming to develop safer and more effective antibacterial drugs.

[0004] However, it still faces many challenges and difficulties in practical application, such as poor stability and the like. SUMMARY

[0005] To solve the above problems, the application provides an antibacterial peptide and application thereof.

[0006] An antibacterial peptide, which is obtained by acetyl capping and amino capping modification of the amino acid sequence shown in SEQ ID NO. 1.

[0007] Preferably, the modification refers to acetylation modification of the N-terminal of the amino acid sequence shown in SEQ ID NO. 1 and amide modification of the C-terminal.

[0008] The application of the antibacterial peptide in preparing an antibacterial product for inhibiting Staphylococcus aureus and drug-resistant Staphylococcus aureus.

[0009] Preferably, the antibacterial product is an antibacterial drug or an antibacterial agent.

[0010] Preferably, the antibacterial drug and the antibacterial agent both take the antibacterial peptide as the only effective component.

[0011] Preferably, the antibacterial drug further comprises a pharmaceutically acceptable excipient.

[0012] Preferably, the pharmaceutically acceptable excipient comprises a diluent.

[0013] Preferably, the diluent is sterile PBS, and the antibacterial drug is a solution obtained by mixing the sterile PBS and the antibacterial peptide at a ratio of 1 mL:0.5 mg-1.0 mg.

[0014] Preferably, the antibacterial drug is in the form of a water infusion, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.

[0015] Preferably, the antibacterial agent is mixed by mixing the sterile water and the antibacterial peptide at a ratio of 1 mL:1 μg-70 μg.

[0016] Compared with the prior art, the application has the following advantages:

[0017] The peptide shown in SEQ ID NO. 1 in the application is derived from a marine biofilm, which is a microbial community with strong resistance. After long-term evolution, the molecules including the peptide in the marine biofilm have the characteristics of strong stability, the chemical basis of which is that the arrangement of amino acids forms a stable structure, avoids the action site of protease and is not easy to be degraded by protease, thereby ensuring the stability. In addition, the peptide shown in SEQ ID NO. 1 is acetylated and amidated, thereby further improving the stability.

[0018] The antibacterial peptide provided by the application has high inhibitory activity on pathogenic bacteria Staphylococcus aureus ATCC12600, methicillin-resistant Staphylococcus aureus ATCC33591 and methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592, has good pH stability and shows anti-infection activity in a mouse model, and these characteristics make it show the value of a medical antibacterial drug, thereby providing an alternative drug resource for clinical resistance to these human pathogenic bacteria in the future.

[0019] The antibacterial peptide provided by the application also has the advantages of low production cost and high environmental stability, thereby providing important technical support for the development of clinical antibacterial drugs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a schematic diagram of the chemical structure of the antibacterial peptide CAMP77NC.

[0021] Figure 2 Figure 2 is a predicted structure of the antibacterial peptide CAMP77NC.

[0022] Figure 3 Figure 3 is the MIC determination results of the antibacterial peptide CAMP77NC against S. aureus ATCC12600, drug-resistant S. aureus ATCC33591 and drug-resistant S. aureus ATCC33592.

[0023] Figure 4 Figure 4 is the growth of S. aureus ATCC12600, drug-resistant S. aureus ATCC33591 and drug-resistant S. aureus ATCC33592 during the MIC determination.

[0024] Figure 5 Figure 5 is the scanning electron microscope observation of the effect of the antibacterial peptide CAMP77NC on the morphology of S. aureus ATCC12600, Figure 5 Figure 6A is the control group. Figure 5 Figure 6B is the CAMP77NC group.

[0025] Figure 6 Figure 7 is the results of the pH stability and pepsin stability experiments of the antibacterial peptide CAMP77NC.

[0026] Figure 7 Figure 8 is the body weight changes of mice in the antibacterial peptide CAMP77NC anti-infection activity experiment stage in the mouse skin abrasion model.

[0027] Figure 8 Figure 9 is the bacterial load of the antibacterial peptide CAMP77NC treated mice in the skin abrasion model after 24 hours.

[0028] Figure 9 Figure 10 is the bacterial load of the peritoneal lavage fluid of the antibacterial peptide CAMP77NC treated mice in the peritonitis model after 24 hours.

[0029] Figure 10 Figure 11 is the bacterial load of the liver of the antibacterial peptide CAMP77NC treated mice in the peritonitis model after 24 hours.

[0030] Figure 11 Figure 12 is the bacterial load of the kidney of the antibacterial peptide CAMP77NC treated mice in the peritonitis model after 24 hours. DETAILED DESCRIPTION

[0031] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0032] Staphylococcus aureus ATCC12600, methicillin-resistant Staphylococcus aureus ATCC33591 and methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592 in the present application are purchased from North China Biotech Co., Ltd.

[0033] The CAMHB liquid medium and the sterile LB liquid medium in the present application are purchased from Haibo Biotechnology Co., Ltd.

[0034] The present application provides an antibacterial peptide for inhibiting drug-resistant Staphylococcus aureus, named CAMP77NC, which is obtained by acetyl capping at the N-terminus and amino capping at the C-terminus of the polypeptide shown in SEQ ID NO. 1. The acetyl capping at the N-terminus is acetylation modification, and the amino capping at the C-terminus is amidation modification. The structure of CAMP77NC is CH3CO-LFKKWKTPWWKFVLLQRA-NH2.

[0035] The antibacterial peptide exhibits significant antibacterial activity against Staphylococcus aureus standard strain ATCC12600, methicillin-resistant Staphylococcus aureus ATCC33591 and methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592, effectively overcoming the limitations of traditional antibacterial peptides.

[0036] The minimum inhibitory concentrations of the antibacterial peptide against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and methicillin- and gentamicin-resistant Staphylococcus aureus are 4 μg / mL, 64 μg / mL and 32 μg / mL, respectively.

[0037] The antibacterial drug is used for treating diseases caused by drug-resistant bacteria infection.

[0038] The antibacterial agent can be used in the fields of medical health, food preservation, personal care products or environmental disinfection, etc.

[0039] Example 1

[0040] Physicochemical properties and preparation of antibacterial peptide CAMP77NC

[0041] The antibacterial peptide CAMP77NC is obtained by additional acetyl capping at the N-terminus and amino capping at the C-terminus of the polypeptide shown in SEQ ID NO. 1 during solid-phase synthesis. The molecular formula of the antibacterial peptide CAMP77NC is C123 H 182 N 30 O 21 , the molecular weight is 2416.94 g / mol, the net charge number is 5, it carries 5 positive charges, the isoelectric point is 12, the normalized hydrophobicity is -0.31, and the chemical structure schematic diagram is as shown in Figure 1

[0042] The antibacterial peptide CAMP77NC is synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. by solid-phase chemical synthesis method, and the purity is greater than 95%. The lower molecular weight makes CAMP77NC have lower production cost.

[0043] The online tool AlphaFold2 cannot predict the structure of the peptide with modification. The protein structure prediction of the polypeptide shown in SEQ ID NO. 1 is made to reflect its structure from the side, and the prediction result is shown in Figure 2 , which shows that the polypeptide shown in SEQ ID NO. 1 presents an alpha-helix structure in some regions, has the characteristics of amphiphilicity, and helps its interaction with the bacterial membrane.

[0044] The amino acid sequence involved is: LFKKWKTPWWKFVLLQRA, denoted as SEQ ID NO. 1.

[0045] Example 2

[0046] Determination of the minimum inhibitory concentration MIC of the antibacterial peptide CAMP77NC on three kinds of Staphylococcus aureus

[0047] The MIC determination of the antibacterial peptide adopts the broth microdilution method, and refers 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.

[0048] The three pathogenic bacteria are Staphylococcus aureus ATCC12600, drug-resistant Staphylococcus aureus ATCC33591 and drug-resistant Staphylococcus aureus ATCC33592.

[0049] The three pathogenic bacteria are inoculated in sterile CAMHB liquid medium, and after shaking culture at 37℃ overnight, the cultured pathogenic bacteria are inoculated into fresh CAMHB liquid medium at an inoculation amount of 1%, and the bacteria are cultured to the logarithmic growth phase, and the bacterial concentration is adjusted to 1×10 5 ​CFU / mL. Then 180 μL of the bacterial solution was transferred to a 96-well plate. The antibacterial peptide CAMP77NC powder was dissolved with sterile water and diluted into a serially diluted antibacterial peptide solution with concentrations of 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL and 640 μg / mL, respectively. 20 μL of antibacterial peptide solution with different concentrations was added to the bacterial solution in the 96-well plate, so that the concentration of antibacterial peptide in the bacterial solution was 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL and 64 μg / mL, respectively. After the 96-well plate was incubated at 37°C for 24 h, the bacterial growth was detected by an enzyme-labeled instrument, and the MIC was defined as the minimum antibacterial peptide concentration at which no bacterial growth was detected. Sterile water was used as a control group CK, and three replicates were set for each experiment.

[0050] The MIC of antibacterial peptide CAMP77NC against three human pathogenic bacteria was as shown in Table 1, and the bacterial growth during the determination of MIC was as shown in Figure 1. The results showed that the MIC of antibacterial peptide CAMP77NC against Staphylococcus aureus ATCC12600, drug-resistant Staphylococcus aureus ATCC33591 and drug-resistant Staphylococcus aureus ATCC33592 was 4 μg / mL, 64 μg / mL and 32 μg / mL, respectively. Figure 3 Figure 4 The MIC of antibacterial peptide CAMP77NC against three human pathogenic bacteria was as shown in Table 1, and the bacterial growth during the determination of MIC was as shown in Figure 1. The results showed that the MIC of antibacterial peptide CAMP77NC against Staphylococcus aureus ATCC12600, drug-resistant Staphylococcus aureus ATCC33591 and drug-resistant Staphylococcus aureus ATCC33592 was 4 μg / mL, 64 μg / mL and 32 μg / mL, respectively.

[0051] Example 3

[0052] Scanning electron microscope observation of the bactericidal effect of antibacterial peptide CAMP77NC on Staphylococcus aureus

[0053] Staphylococcus aureus ATCC12600 was cultured in sterile LB liquid medium at 37°C overnight, and then diluted to 1×10 8 ​CFU / mL. Subsequently, the bacterial solution was divided into two groups: one group was treated with the antimicrobial peptide CAMP77NC at a concentration of 5xMIC, and the untreated group was used as a negative control. All samples were incubated at 37°C for 1 h. After incubation, the samples were centrifuged at 5000 rpm for 3 min at room temperature, and the supernatant was discarded. The bacterial pellet was washed twice with sterile PBS buffer at pH 7.0. After washing, the pellet was resuspended in 200 μL of 2.5% glutaraldehyde solution and fixed at 4°C overnight. The fixed samples were centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The bacteria were then evenly spread on a microscope slide. The samples were then washed three times with sterile PBS buffer for 10 min each time. After washing, the samples were dehydrated in a gradient series of ethanol at 30%, 50%, 70%, 80%, and 100%, with two 10-min treatments in 100% ethanol. After dehydration and replacement, the samples were treated using CO2 critical point drying. Finally, the dried samples were gold-coated and imaged using a scanning electron microscope (Tescan Vega3) to observe the morphological changes of S. aureus under different treatment conditions.

[0054] The imaging results are shown in FIG. 4. Figure 5 As shown in FIG. 4, the experimental results show that S. aureus treated with the antimicrobial peptide CAMP77NC exhibits obvious cell membrane damage or rupture, while the control group of bacterial cells has an intact structure and no obvious changes.

[0055] Example 4

[0056] Stability experiment of antimicrobial peptide CAMP77NC

[0057] The PBS buffer was adjusted to pH values of 2, 7.4, and 8 using HC1 and NaOH, respectively. The antimicrobial peptide powder was dissolved in the adjusted sterile PBS buffer to a concentration of 640 μg / mL. After incubation at 37°C for 1 h, the MIC value of the treated antimicrobial peptide against S. aureus ATCC12600 was detected according to the method described in Example 2 to evaluate the stability of the antimicrobial peptide under different pH conditions.

[0058] For pepsin stability assay, pepsin was dissolved in sterile PBS solution at pH 2 to a final concentration of 100 μg / mL, and then the peptide was dissolved in the above pepsin solution to a concentration of 640 μg / mL. After incubation at 37 °C for 1 h, the pepsin was inactivated by boiling at 100 °C for 15 min. Then the MIC value of the treated antibacterial peptide against S. aureus ATCC12600 was determined according to the method described in Example 2 to evaluate the pepsin hydrolysis resistance of the antibacterial peptide.

[0059] The stability experiment of antibacterial peptide CAMP77NC was performed as shown in Table 1, and different pH and pepsin had little effect on the activity of the antibacterial peptide, and the MIC was stable at 8 μg / mL. Figure 6

[0060] Example 5

[0061] Mouse skin abrasion model experiment of antibacterial peptide CAMP77NC

[0062] S. aureus ATCC12600, ATCC33591 and ATCC33592 were inoculated into LB liquid medium respectively and cultured at 37 °C with shaking until the logarithmic growth phase. Then, they were washed twice with sterile PBS and resuspended to OD 600 600. The three strains with OD 600 600 were mixed at a volume ratio of 1:1:1 to obtain a mixed bacterial suspension. The concentration of the mixed bacterial suspension was determined using a McFarland turbidimeter and adjusted to 1×10 8 CFU / mL. After isoflurane anesthesia, the dorsal area of 6-week-old Kunming female mice that could not be contacted by mouth or limbs was depilated and disinfected to prepare a 12×12 mm superficial abrasion. After washing the wound with sterile saline, 20 μL of mixed bacterial suspension with a concentration of 1×10 8 CFU / mL was added for infection. After 2 h of infection, 20 μL of CAMP77NC with a concentration of 32 μg / mL was added to the wound for treatment, and 20 μL of sterile PBS was added to the control group. Each group had 3 mice, and all mice were housed separately to avoid cross contamination and acclimated for 5 days before the experiment. The mice were sacrificed after 24 h of experiment, and 5 mm×5 mm skin tissue was taken from the infection site and homogenized using a grinder at 25 Hz. The 10-fold serial gradient dilution was plated on agar plates, with 3 parallels for each gradient, incubated at 37 °C for 24 h, and the colony count was performed according to GB / T 5750.12-2023. At the same time, the body weight change of the mice was recorded at each experimental stage.

[0063] Figure 7 The body weight of the mice in the CAMP77NC experimental group decreased more slowly than that in the control group. Figure 8 ​The CFU counting results of the bacterial load of the mice treated with the antibacterial peptide CAMP77NC after 24 h showed that CAMP77NC effectively inhibited the growth of S. aureus, reducing the bacterial load by one order of magnitude.

[0064] Example 6

[0065] Mouse peritonitis model experiment of antibacterial peptide CAMP77NC

[0066] S. aureus ATCC12600, ATCC33591 and ATCC33592 were inoculated into LB liquid medium respectively and cultured at 37°C with shaking to the logarithmic growth phase. Then, they were washed twice with sterile PBS and resuspended to OD 600 of 0.6. The three strains with OD 600 of 0.6 were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension, the concentration of which was determined using a McFarland turbidimeter and adjusted to 1×10 9 CFU / mL. Six to eight-week-old female Kunming mice were selected, fasted for 12 h after one week of acclimation, and infected with 200 μL of the mixed bacterial suspension with a concentration of 1×10 9 CFU / mL by injecting it into the abdomen of the mice using a sterile syringe. After 1 h of infection, the mice were treated with CAMP77NC by injecting it into the abdomen using a sterile syringe. When injecting CAMP77NC, a sterile PBS solution was used to prepare a solution with a concentration of 0.7 mg / mL, and 200 μL of the solution with a concentration of 0.7 mg / mL was injected. The control group was injected with 200 μL of sterile PBS. Each group had 3 mice, and all mice were raised separately to avoid cross contamination. The mice were sacrificed by cervical dislocation after 24 h of the experiment, and 5 mL of sterile PBS was used to perform abdominal lavage. The abdominal lavage fluid sample was collected, and the liver and kidney tissues of the mice were washed with sterile PBS. Then, the liver and kidney tissues were homogenized into a homogenate using a grinder at 25 Hz. The abdominal lavage fluid, liver and kidney tissue homogenate were serially diluted by 10 times, and 100 μL of the diluted solution was evenly spread on LB agar plates. Each gradient had three parallel plates, which were incubated at 37°C for 24 h, and the colony count was performed according to GB / T 5750.12-2023.

[0067] Figure 9 The bacterial load of the abdominal lavage fluid of the mice treated with the antibacterial peptide CAMP77NC after 24 h, Figure 10 The bacterial load of the liver of the mice treated with the antibacterial peptide CAMP77NC after 24 h, Figure 11The bacterial load of the kidney of the mice treated with antibacterial peptide CAMP77NC after 24h. The CFU counting results showed that CAMP77NC effectively inhibited the growth of S. aureus, and the bacterial load of different parts of the mice was reduced by about 1 order of magnitude.

[0068] It should be noted that the present application claims involving numerical ranges should be understood as each numerical range of two endpoints and any one value between the two endpoints can be selected, in order to prevent repetition, the present application describes the preferred embodiments.

[0069] Although the preferred embodiments of the application have been described, those skilled in the art will, upon acquiring the basic creative concept, make further changes and modifications to these embodiments. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0070] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. An antimicrobial peptide, characterized in that The antimicrobial peptide is obtained by modifying the amino acid sequence shown in SEQ ID NO.1 by acetyl end-capping and amino end-capping.

2. The antimicrobial peptide according to claim 1, characterized in that The modification refers to acetylation modification of the N-terminus and amidation modification of the C-terminus of the amino acid sequence shown in SEQ ID NO.

1.

3. 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.

4. The use according to claim 3, characterized in that The antibacterial product is an antibacterial drug.

5. The use according to claim 4, characterized in that The antibacterial drug has the antibacterial peptide as the only active ingredient.

6. The use according to claim 5, characterized in that The antibacterial drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that The pharmaceutically acceptable excipients include diluents.

8. The use according to claim 7, characterized in that The diluent is sterile PBS, and the antimicrobial drug is a solution obtained by mixing the sterile PBS and the antimicrobial peptide in a ratio of 1 mL: 0.5 mg to 1.0 mg.

9. The use according to claim 5, characterized in that The dosage form of the antibacterial drug is water extract, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

10. The use according to claim 5, characterized in that The antibacterial drug is an antibacterial agent, which is obtained by mixing sterile water and the antibacterial peptide at a material-liquid ratio of 1 mL: 1 μg~70 μg.

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