Antibacterial peptide and application thereof

Abstract

The invention belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide and application thereof. The antibacterial peptide provided by the invention is obtained by modifying an amino acid sequence as shown in SEQ ID NO.1 through acetyl end capping and amino end capping. The antibacterial peptide provided by the invention has efficient inhibitory activity on pathogenic bacteria staphylococcus aureus, methicillin-resistant staphylococcus aureus and methicillin-resistant and gentamicin-resistant staphylococcus aureus; the pH stability is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and in particular relates to an antimicrobial peptide and an application thereof. Background Art

[0002] Staphylococcus aureus is a Gram-positive bacterium that is widely found in nature and in the human body, commonly found on the skin, nasal cavity, and respiratory tract. It is a major pathogen in humans and animals, capable of causing a wide range of infections, from minor skin infections to severe systemic infections such as pneumonia, endocarditis, and toxic shock syndrome. Currently, S. aureus has become a major threat to surgical infections in the clinic. In particular, the emergence of drug-resistant S. aureus has rendered many traditional antibiotics, such as methicillin and gentamicin, ineffective against it, posing a significant challenge to clinical treatment. Furthermore, S. aureus can form biofilms, further enhancing its resistance to antibiotics and the host immune system, making the infection difficult to eradicate.

[0003] Antimicrobial peptides are a class of naturally occurring immune molecules widely found in organisms, exhibiting broad-spectrum antimicrobial activity. Composed of short chains of amino acids, they typically carry a positive charge and can bind to negatively charged bacterial cell membranes through electrostatic interactions, disrupting their integrity, leading to leakage of cellular contents and bacterial death. Antimicrobial peptides not only exhibit broad-spectrum antimicrobial activity against bacteria, but also exhibit inhibitory effects against fungi, viruses, and even certain cancer cells. Compared to traditional antibiotics, antimicrobial peptides have a more complex mechanism of action, are less likely to induce bacterial resistance, and exhibit significant inhibitory effects against multiple drug-resistant strains. Therefore, they are considered a novel therapeutic strategy for combating drug-resistant bacterial infections. Furthermore, antimicrobial peptides possess immunomodulatory properties, promoting wound healing and modulating inflammatory responses, and therefore have attracted considerable attention in the biomedical field. In recent years, research on antimicrobial peptides targeting Staphylococcus aureus has garnered significant attention, aiming to develop safer and more effective antimicrobial drugs.

[0004] However, it still faces many challenges and difficulties in practical applications, such as poor stability. Summary of the Invention

[0005] To solve the above problems, the present invention provides an antimicrobial peptide and its application.

[0006] An antimicrobial peptide is obtained by modifying the amino acid sequence shown in SEQ ID NO. 1 by performing acetyl-terminal and amino-terminal modifications.

[0007] Preferably, 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.

[0008] The antimicrobial peptide is used in the preparation of an antimicrobial product, and the antimicrobial product is used for inhibiting Staphylococcus aureus and drug-resistant Staphylococcus aureus.

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

[0010] Preferably, the antimicrobial drug and antimicrobial agent both contain the antimicrobial peptide as the sole active ingredient.

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

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

[0013] Preferably, 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.

[0014] Preferably, the dosage form of the antibacterial drug is an aqueous extract, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

[0015] Preferably, the antibacterial agent is mixed with the antimicrobial peptide by the sterile water at a material-liquid ratio of 1 mL: 1 μg~70 μg.

[0016] Compared with the prior art, the present invention is beneficial in that: The peptide represented by SEQ ID NO. 1 in this invention originates from marine biofilms, a highly stress-resistant microbial community. Through long-term evolution, molecules within marine biofilms, including peptides, possess a highly stable structure. This stability is due to the arrangement of amino acids, which creates a stable structure that avoids protease action sites, making it less susceptible to degradation by proteases and ensuring stability. Furthermore, the peptide represented by SEQ ID NO. 1 has been modified with acetylation and amidation to further enhance its stability.

[0017] The antimicrobial peptide provided by the present invention has highly effective inhibitory activity against the pathogenic bacteria Staphylococcus aureus ATCC12600, methicillin-resistant Staphylococcus aureus ATCC33591, and methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592. It has good pH stability and exhibits anti-infection activity in a mouse model. These properties make it exhibit antimicrobial value as a medical drug, providing an alternative medicinal resource for future clinical use against these human pathogens.

[0018] The antimicrobial peptide provided by the present invention also has the advantages of low production cost and high environmental stability, and provides important technical support for the development of clinical antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the chemical structure of the antimicrobial peptide CAMP77NC.

[0020] Figure 2 This is the predicted structure of the antimicrobial peptide CAMP77NC.

[0021] Figure 3 These are the MIC test results of the antimicrobial peptide CAMP77NC against Staphylococcus aureus ATCC12600, drug-resistant Staphylococcus aureus ATCC33591 and drug-resistant Staphylococcus aureus ATCC33592.

[0022] Figure 4 To determine the growth of Staphylococcus aureus ATCC12600, resistant Staphylococcus aureus ATCC33591 and resistant Staphylococcus aureus ATCC33592 when MIC was determined.

[0023] Figure 5 To observe the effect of antimicrobial peptide CAMP77NC on the morphology of Staphylococcus aureus ATCC12600 using scanning electron microscopy, Figure 5 A is the control group, Figure 5 Middle B is the CAMP77NC group.

[0024] Figure 6 These are the results of pH stability and pepsin stability experiments of the antimicrobial peptide CAMP77NC.

[0025] Figure 7 Changes in mouse body weight during the experimental period of antimicrobial peptide CAMP77NC's anti-infection activity in the mouse skin abrasion model.

[0026] Figure 8 Figure 2 shows the bacterial load in mice treated with the antimicrobial peptide CAMP77NC 24 hours after skin abrasion model.

[0027] Figure 9 Figure 2 shows the bacterial load in the peritoneal lavage fluid of mice treated with the antimicrobial peptide CAMP77NC in the peritonitis model 24 hours later.

[0028] Figure 10 Figure 2 shows the bacterial load in the liver of mice treated with the antimicrobial peptide CAMP77NC 24 hours after peritonitis model.

[0029] Figure 11 Figure 2 shows the bacterial load in the kidneys of mice treated with the antimicrobial peptide CAMP77NC in a peritonitis model 24 hours later. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0031] In the present invention, Staphylococcus aureus ATCC12600, methicillin-resistant Staphylococcus aureus ATCC33591 and methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592 were all purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0032] The CAMHB liquid culture medium and the sterile LB liquid culture medium in the present invention were both purchased from Haibo Biotechnology Co., Ltd.

[0033] The present invention provides an antimicrobial peptide for inhibiting drug-resistant Staphylococcus aureus, named CAMP77NC, which is obtained by capping the polypeptide shown in SEQ ID NO.1 with an acetyl group at the N-terminus and an amino group at the C-terminus. The N-terminal acetyl capping is acetylation modification, and the C-terminal amino capping is amidation modification. The structure of CAMP77NC is CH3CO-LFKKWKTPWWKFVLLQRA-NH2.

[0034] The antimicrobial peptide exhibits significant antibacterial activity against the standard strain of Staphylococcus aureus ATCC12600, methicillin-resistant Staphylococcus aureus ATCC33591, and methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592, effectively overcoming the limitations of traditional antimicrobial peptides.

[0035] The minimum inhibitory concentrations of the antimicrobial 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.

[0036] The antibacterial drug is used to treat diseases caused by drug-resistant bacterial infection.

[0037] The antibacterial agent can be used in the fields of medical health, food preservation, personal care products or environmental disinfection.

[0038] Example 1 Physicochemical properties and preparation of antimicrobial peptide CAMP77NC The antimicrobial peptide CAMP77NC is obtained by solid-phase synthesis of the polypeptide shown in SEQ ID NO.1 with additional acetyl and amino blocking at the N-terminus and C-terminus, respectively. The molecular formula of the antimicrobial peptide CAMP77NC is C 123 H 182 N30 O 21 , with a molecular weight of 2416.94 g / mol, a net charge of 5, 5 positive charges, an isoelectric point of 12, and a normalized hydrophobicity of -0.31. The chemical structure diagram is shown in Figure 1 shown.

[0039] The antimicrobial peptide CAMP77NC is synthesized by Sangon Biotech (Shanghai) Co., Ltd. via solid-phase chemical synthesis with a purity exceeding 95%. Its low molecular weight allows for a low production cost.

[0040] The online tool AlphaFold2 cannot predict the structure of modified peptides. The protein structure prediction of the polypeptide shown in SEQ ID NO.1 is performed to reflect its structure. The prediction results are as follows Figure 2 As shown, it is shown that part of the polypeptide region shown in SEQ ID NO. 1 presents an α-helical structure and has amphipathic characteristics, which facilitates its interaction with bacterial membranes.

[0041] The amino acid sequence involved is: LFKKWKTPWWKFVLLQRA, recorded as SEQ ID NO.1.

[0042] Example 2 Determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide CAMP77NC against three strains of Staphylococcus aureus The MICs of antimicrobial peptides were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines: Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991.

[0043] The three pathogens were Staphylococcus aureus ATCC12600, drug-resistant Staphylococcus aureus ATCC33591 and drug-resistant Staphylococcus aureus ATCC33592.

[0044] The three pathogens were inoculated into sterile CAMHB liquid medium and cultured overnight at 37°C with shaking. The cultured pathogens were then inoculated into fresh CAMHB liquid medium at a 1% inoculum volume and cultured until the logarithmic growth phase. The bacterial concentration was adjusted to 1×10 5The bacterial suspension was obtained by measuring the CFU / mL. 180 μL of the bacterial suspension was then transferred to a 96-well plate. The antimicrobial peptide CAMP77NC powder was dissolved in sterile water and diluted serially two-fold to produce solutions with concentrations of 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL, and 640 μg / mL. Twenty μL of each solution of different concentrations was added to the bacterial suspension in the 96-well plate, resulting in concentrations of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 64 μg / mL, respectively. After incubation at 37°C for 24 hours, bacterial growth was assessed using a microplate reader. The MIC was defined as the minimum antimicrobial peptide concentration at which no bacterial growth was detected. Sterile water served as the control group (CK). Each experiment was repeated three times.

[0045] MICs of the antimicrobial peptide CAMP77NC against three human pathogens Figure 3 As shown in Figure 2, the bacterial growth during MIC determination was as follows: Figure 4 The results showed that the MICs of the drug against Staphylococcus aureus ATCC12600, drug-resistant Staphylococcus aureus ATCC33591, and drug-resistant Staphylococcus aureus ATCC33592 were 4 μg / mL, 64 μg / mL, and 32 μg / mL, respectively.

[0046] Example 3 Scanning electron microscopy observation of the bactericidal effect of antimicrobial peptide CAMP77NC on Staphylococcus aureus Staphylococcus aureus ATCC12600 was cultured in sterile LB liquid medium at 37°C overnight and diluted to 1×10 8CFU / mL. Subsequently, the bacterial suspension was divided into two groups: one group was treated with the antimicrobial peptide CAMP77NC at a concentration of 5× the MIC, and an untreated group served as a negative control. All samples were incubated at 37°C for 1 hour. After incubation, the samples were centrifuged at 5000 rpm for 3 minutes at room temperature, the supernatant discarded, and the bacteria washed twice with sterile PBS buffer (pH 7.0). After washing, the pellet was resuspended in 200 μL of 2.5% glutaraldehyde solution and fixed overnight at 4°C. After fixation, the samples were centrifuged at 4000 rpm for 10 minutes, the supernatant discarded, and the bacteria were evenly plated on microscope slides. Subsequently, the samples were washed three times with sterile PBS buffer, each for 10 minutes. After washing, the samples were dehydrated in a graded ethanol series consisting of 30%, 50%, 70%, 80%, and 100% ethanol, with two 10-minute treatments in 100% ethanol. After dehydration, the slides were treated sequentially with 50% and 100% ethyl isovalerate, respectively. The 50% ethyl isovalerate solution consisted of ethyl isovalerate and ethanol in a 1:1 volume ratio. Following the dehydration and displacement steps, 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 morphological changes in S. aureus under the different treatment conditions.

[0047] Imaging results such as Figure 5 As shown, the experimental results showed that Staphylococcus aureus treated with the antimicrobial peptide CAMP77NC showed obvious cell membrane damage or rupture, while the bacterial cell structure in the control group was intact and there was no obvious change.

[0048] Example 4 Stability experiment of antimicrobial peptide CAMP77NC The PBS buffer was adjusted to pH 2, 7.4, and 8 with HCl and NaOH, respectively. The antimicrobial peptide powder was dissolved in the adjusted sterile PBS buffer to a peptide solution with a concentration of 640 μg / mL. After incubation at 37°C for 1 h, the MIC value of the treated antimicrobial peptide against Staphylococcus aureus ATCC12600 was detected according to the method described in Example 2 to evaluate the stability of the antimicrobial peptide under different pH conditions.

[0049] For the pepsin stability assay, pepsin was dissolved in sterile PBS solution at pH 2 to a final concentration of 100 μg / mL. The peptide was then dissolved in the pepsin solution to a concentration of 640 μg / mL. The mixture was incubated at 37°C for 1 h and then boiled at 100°C for 15 min to inactivate pepsin. The MIC value of the treated antimicrobial peptide against Staphylococcus aureus ATCC12600 was then determined according to the method described in Example 2 to evaluate the pepsin hydrolysis resistance of the antimicrobial peptide.

[0050] The stability experiment of antimicrobial peptide CAMP77NC is as follows Figure 6 As shown in the results, different pH and pepsin had no significant effect on the activity of antimicrobial peptides, and the MIC was stable at 8 μg / mL.

[0051] Example 5 Mouse skin abrasion model experiment with antimicrobial peptide CAMP77NC Staphylococcus aureus ATCC12600, ATCC33591 and ATCC33592 were inoculated into LB liquid medium and cultured at 37°C under shaking conditions until the logarithmic growth phase. Subsequently, they were washed twice with sterile PBS and resuspended to an OD of 600 The volume ratio of the three OD 600 The concentration of the mixed bacterial suspension was determined using a McFadden turbidimeter and adjusted to 1 × 10 8 CFU / mL. Six-week-old female Kunming mice were selected and anesthetized with isoflurane. The hair was removed and disinfected on the back area where the mice could not touch with their mouths or limbs. A 12×12 mm superficial abrasion was prepared. After rinsing the wound with sterile saline, 20 μL of 1×10 8 CFU / mL of mixed bacterial suspension was used for infection. 2 hours after infection, 20 μL of CAMP77NC at a concentration of 32 μg / mL was dripped onto the wound for treatment. The control group was dripped with 20 μL of sterile PBS. Each group had 3 mice, and all mice were housed individually to avoid cross-contamination. They were adaptively housed for 5 days before the experiment. The mice were killed 24 hours after the experiment, and 5 mm × 5 mm skin tissue was taken from the infected site and homogenized using a grinder at 25 Hz. The agar plate was spread on a 10-fold continuous gradient dilution, with 3 parallels for each gradient, cultured at 37 ° C for 24 hours, and the colony count was performed according to GB / T 5750.12-2023. At the same time, the weight changes of the mice were recorded at each experimental stage.

[0052] Figure 7 The weight changes of mice during the experimental period showed that the weight loss trend of mice in the CAMP77NC experimental group slowed down; Figure 8 This is the bacterial load of mice treated with the antimicrobial peptide CAMP77NC 24 hours after treatment. The CFU count results showed that CAMP77NC effectively inhibited the growth of Staphylococcus aureus and reduced the bacterial load by one order of magnitude.

[0053] Example 6 Mouse peritonitis model experiment with antimicrobial peptide CAMP77NC Staphylococcus aureus ATCC12600, ATCC33591 and ATCC33592 were inoculated into LB liquid medium and cultured at 37°C under shaking conditions until the logarithmic growth phase. Subsequently, they were washed twice with sterile PBS and resuspended to an OD of 600 The volume ratio of the three OD 600 The concentration of the mixed bacterial suspension was determined using a McFadden turbidimeter and adjusted to 1 × 10 9 CFU / mL. Female Kunming mice aged 6 to 8 weeks were selected and fasted for 12 hours after one week of adaptive culture. 200 μL of 1×10 9 CFU / mL of mixed bacterial suspension was used for infection. One hour after infection, CAMP77NC was injected into the abdomen of the mouse using a sterile syringe for treatment. When injecting CAMP77NC, CAMP77NC was prepared into a solution with a concentration of 0.7 mg / mL using sterile PBS solution, and 200 μL of 0.7 mg / mL solution was injected. The control group was injected with 200 μL of sterile PBS. There were 3 mice in each group, and all mice were housed individually to avoid cross contamination. 24 hours after the experiment, the mice were killed by cervical dislocation, and 5 mL of sterile PBS was used for peritoneal lavage. The peritoneal lavage fluid samples were collected, and the liver and kidney tissues of the mice were washed with sterile PBS. Sterile PBS was then added and the liver and kidneys were homogenized into a homogenate using a grinder at 25 Hz. After continuous 10-fold gradient dilution of peritoneal lavage fluid, liver and kidney tissue grinding fluid using sterile PBS, 100 μL of the dilution was evenly spread on LB agar plates. Three parallel plates were set for each gradient, and the plates were cultured at 37°C for 24 h. The colony count was performed according to GB / T 5750.12-2023.

[0054] Figure 9 The bacterial load of peritoneal lavage fluid of mice treated with antimicrobial peptide CAMP77NC for 24 hours. Figure 10 The bacterial load in the liver of mice treated with the antimicrobial peptide CAMP77NC 24 hours later. Figure 11 Figure 2 shows bacterial loads in the kidneys of mice treated with the antimicrobial peptide CAMP77NC 24 hours after treatment. CFU counts showed that CAMP77NC effectively inhibited the growth of Staphylococcus aureus, reducing bacterial loads in different areas of the mice by approximately one order of magnitude.

[0055] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0056] 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.

[0057] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

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 for inhibiting Staphylococcus aureus and drug-resistant Staphylococcus aureus.

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

5. The use according to claim 4, characterized in that The antibacterial medicine and antibacterial agent both use 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 agent is obtained by mixing sterile water and the antimicrobial peptide at a material-liquid ratio of 1 mL: 1 μg to 70 μg.

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