Antibacterial peptide with broad-spectrum antibacterial activity and application thereof

Abstract

The invention provides an antibacterial peptide with broad-spectrum antibacterial activity and application of the antibacterial peptide, and belongs to the field of biological medicines.The amino acid sequence of the antibacterial peptide AMP-27 is shown as SEQ ID NO.1; the antibacterial peptide AMP-27 has the characteristics of low concentration and strong bactericidal effect, and has broad-spectrum killing effect. The antibacterial peptide AMP-27 can also promote the healing speed of fungal infectious wounds, relieve inflammatory cell infiltration and enhance collagenous fiber deposition and tissue reconstruction; the invention provides a novel biotechnology and method for developing efficient and low-toxicity treatment of skin wound microbial infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an antimicrobial peptide with broad-spectrum antibacterial activity and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of widely sourced, natural or synthetic small-molecule polypeptides with broad-spectrum antimicrobial activity. They can rapidly inactivate invading microorganisms, particularly at mucosal surfaces and epithelial barriers, showing promising applications in the prevention and treatment of microbial infections such as Candida albicans infection at skin lesions. Current research on antimicrobial peptides mainly focuses on their potential as antibiotic alternatives, especially in the context of increasingly serious antibiotic resistance. Unlike traditional antibiotics, antimicrobial peptides have multiple mechanisms, including directly disrupting bacterial cell membranes, inhibiting biofilm formation, and immunomodulation. They exhibit good inhibitory effects against various drug-resistant bacteria and are less likely to induce bacterial resistance, offering significant advantages. Due to their low toxicity and good biocompatibility, antimicrobial peptides show broad prospects in clinical applications. AMPs with lengths between 3 and 50 have been reported to have potential therapeutic effects against fungi and drug-resistant bacteria, suggesting their potential as alternatives to antibiotics.

[0003] Currently, antimicrobial peptides are being researched and applied in various fields, including medicine, agriculture, food preservation, and aquaculture. In the pharmaceutical field, antimicrobial peptides are being developed for the treatment of systemic infections, skin infections, wound healing, and oral care. However, the practical application of antimicrobial peptides still faces challenges, such as poor in vivo stability, high cost, difficult production, and potential toxicity and immunogenicity. To overcome these obstacles, researchers are exploring strategies such as structural optimization, synthetic modification, and improved delivery systems to enhance the stability and safety of antimicrobial peptides and reduce production costs. In the future, with technological advancements and in-depth research, antimicrobial peptides are expected to play a more important role in anti-infective therapy.

[0004] Existing technologies have reported related antimicrobial peptides, such as SAAP-148, an artificially designed antimicrobial peptide derived from the natural antimicrobial peptide LL-37. Through modification of the amino acid sequence, its antimicrobial activity and stability have been enhanced. SAAP-148 is effective against a variety of drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), and vancomycin-resistant Enterococcus (VRE). Its mechanism of action primarily involves disrupting the integrity of the bacterial cell membrane, leading to leakage of cell contents and bacterial death. Studies have shown that SAAP-148 has strong penetrability through bacterial biofilms and can be used to treat difficult-to-treat chronic wound infections. Furthermore, it exhibits low toxicity and good biocompatibility in vitro and in animal models. Due to its potent antibacterial activity and relative safety, SAAP-148 is considered a promising new antibacterial therapy candidate that holds promise for combating antibiotic-resistant infections.

[0005] The research and development of antimicrobial peptides with strong bactericidal effects at low concentrations is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an antimicrobial peptide with broad-spectrum antibacterial activity and its applications.

[0007] The technical solution of the present invention is as follows: An antimicrobial peptide AMP-27, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] LKRVWKRVFKLLKRYWRQLPVRWWWRRSEQ ID NO.1.

[0009] The antimicrobial peptide AMP-27 can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0010] The aforementioned biomaterials related to the antimicrobial peptide AMP-27 are any one of the following: ① The nucleotide sequence encoding the above-mentioned antimicrobial peptide; ②A recombinant vector containing the nucleotide sequence described in ①; ③ Recombinant bacterial cells containing the nucleotide sequence described in ①.

[0011] The application of the above-mentioned antimicrobial peptide AMP-27 or the above-mentioned biomaterial in the preparation of products with the ability to kill drug-resistant bacteria.

[0012] According to a preferred embodiment of the present invention, the drug-resistant bacteria include one or more of the following: Candida albicans, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii.

[0013] More preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), the Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae, the Escherichia coli is carbapenem-resistant Escherichia coli (CRE), and the Acinetobacter baumannii is pan-drug-resistant Acinetobacter baumannii (MDR-AB).

[0014] An antimicrobial agent comprising the aforementioned antimicrobial peptide AMP-27.

[0015] According to a preferred embodiment of the present invention, the antibacterial agent further contains excipients.

[0016] More preferably, the excipients include one or more of hydrogels, liposomes, and nanomaterials.

[0017] A drug for treating pneumonia, sepsis, or skin wound infections caused by drug-resistant or non-drug-resistant bacteria, comprising the aforementioned antimicrobial peptide AMP-27.

[0018] According to a preferred embodiment of the present invention, the drug further contains excipients.

[0019] More preferably, the excipients include one or more of hydrogels, liposomes, and nanomaterials.

[0020] According to a preferred embodiment of the present invention, the pathogenic bacteria include one or more of the following: Candida albicans, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii.

[0021] More preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), the Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae (CRKP), the Escherichia coli is carbapenem-resistant Escherichia coli (CRE), and the Acinetobacter baumannii is pan-drug-resistant Acinetobacter baumannii (MDR-Ab).

[0022] According to a preferred embodiment of the present invention, the drug is administered by intravenous injection, nebulized inhalation, or topical application.

[0023] The beneficial effects of the present invention include at least the following: This invention provides an antimicrobial peptide AMP-27, the amino acid sequence of which is shown in SEQ ID NO.1. AMP-27 possesses the characteristic of strong bactericidal effect at low concentrations, exhibiting a broad-spectrum killing effect against clinical fungi (such as Candida albicans) and drug-resistant bacteria (such as MRSA, CRKP, CRE, MDR-Ab). AMP-27 can also promote the healing rate of fungal-infected wounds, reduce inflammatory cell infiltration, and enhance collagen fiber deposition and tissue reconstruction. This invention provides a new biotechnology and method for developing highly effective and low-toxicity treatments for microbial infections of skin wounds. It opens up new possibilities for the development of new anti-infective drugs, lays the foundation for the development of corresponding therapeutic products, and provides feasible methods for clinical medical applications. Attached Figure Description

[0024] Figure 1 The destructive effects of the novel antimicrobial peptide AMP-27 on C. albicans were shown under scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0025] Figure 2 Erythrocytotoxicity of the novel antimicrobial peptide AMP-27.

[0026] Figure 3 The therapeutic effect of the novel antimicrobial peptide AMP-27 on skin lesions.

[0027] Figure 4 HE and Masson staining of skin lesions treated with the novel antimicrobial peptide AMP-27. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] bacterial source Candida albicans, methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRE), and pan-drug-resistant Acinetobacter baumannii (MDR-AB) were all provided by the Department of Laboratory Medicine, Second Hospital of Shandong University. Other personnel skilled in the art can obtain the above-mentioned strains commercially to replicate the relevant experiments of this invention.

[0031] Bacterial cryopreservation The glycerol cryopreservation method was used. 500 μL of logarithmic phase bacterial culture was mixed with 500 μL of 50% sterile glycerol solution at a 1:1 ratio, and the mixture was pipetted to form a bacterial suspension, which was then frozen at -80℃.

[0032] Bacterial resuscitation and culture Take the glycerol-frozen bacterial strain, thaw it rapidly at room temperature, streak or spread it onto nutrient agar plates, and incubate it in a 30℃ incubator for 72 hours. Use a loop to pick 3-4 colonies and add them to liquid culture medium, and incubate overnight in a shaker at 220 rpm and 30℃ until the logarithmic phase.

[0033] Example 1 Design and Synthesis of Novel Antimicrobial Peptide AMP-27 1. Design of a novel antimicrobial peptide AMP-27 The synthetic antimicrobial peptide SAAP-148, derived from the human cathelicidin family LL-37, was selected as the main design subject for the novel antimicrobial peptide. Based on the SAAP-148 sequence, tryptophan (W) and arginine (R) were added, while two lysine residues KK were deleted from the original antimicrobial peptide sequence, generating the novel antimicrobial peptide AMP-27 with the amino acid sequence LKRVWKRVFKLLKRYWRQLPVRWWWRR.

[0034] 2. Synthesis and storage of antimicrobial peptides All antimicrobial peptides were synthesized by Jier Biochemical Company using a polypeptide solid-phase synthesis method. The lyophilized peptides were stored at -20℃ and dissolved as needed.

[0035] The results are shown in Table 1. Using peptide prediction analysis software, the amino acid sequences of antimicrobial peptides SAAP148 and AMP-27 were compared. It was found that the two have the same positive charge, but AMP-27 has a higher total hydrophobicity, stronger hydrophobic association, enhanced ability to bind to the membrane, better antimicrobial ability, and lower normalized hydrophobic moment, resulting in lower biotoxicity.

[0036] Table 1 Physicochemical properties of the antimicrobial peptide prediction calculation website

[0037] Example 2 The antibacterial effect of AMP-27 was determined using the minimum inhibitory concentration (MIC). Take the bacterial culture in the logarithmic growth phase, wash the cells twice with PBS buffer, and adjust the concentration to 1×10⁻⁶ using a McFarland turbidimeter. 6 CFU / mL, diluted to 1×10⁻⁶ with SDB medium. 3CFU / mL. Weigh the required amount of lyophilized antimicrobial peptide powder, dissolve it thoroughly in PBS buffer, and prepare a stock solution. Dilute the stock solution to the highest possible drug concentration using SDB liquid medium. Take a sterile 96-well plate and add 100 μL to the first column for each subsequent drug dilution. Add 100 μL of the diluted bacterial suspension to each well for each drug concentration. Incubate the 96-well plate at 30 °C for 72 h. The lowest drug concentration at which no bacterial colony growth occurs is the minimum inhibitory concentration (MIC) of the drug for that bacterium (see Table 2).

[0038] The inventors also designed an antimicrobial peptide AMP-29 based on the SAAP-148 sequence, by adding tryptophan (W) and arginine (R) without deleting the two lysine KK residues in the original antimicrobial peptide sequence. The amino acid sequence is: LKRVWKRVFKLLKRYWRQLKKPVRWWWRR (SEQ ID NO.3). The inventors also tested the antimicrobial effect of the antimicrobial peptide AMP-29 using the above method, as shown in Table 2.

[0039] Table 2. Minimum inhibitory concentrations against strains (unit: μg / ml, n=3)

[0040] The results are shown in Table 2. All three antimicrobial peptides have broad-spectrum antimicrobial activity. Compared with the antimicrobial activity of the original antimicrobial peptide SAAP-148, AMP-27 has the best antimicrobial effect and has the characteristic of strong bactericidal activity at low concentrations.

[0041] Example 3 Electron microscopy verifies the disruptive effect of the novel antimicrobial peptide AMP-27 on bacterial membranes. Prepare 1×10 3 The bacterial suspension at CFU / mL was washed twice with PBS buffer and resuspended in PBS buffer. Antimicrobial peptide solutions were used to treat the bacterial suspensions (untreated group and AMP-27 group, treated with MIC concentration). The suspensions were incubated at 30°C for 4 hours, centrifuged, and the supernatant was discarded. The suspensions were washed twice with PBS buffer and resuspended in fixative. After subsequent fixation, dehydration, and drying, images were obtained under scanning electron microscopy and transmission electron microscopy.

[0042] like Figure 1As shown in the scanning electron microscopy images, the control group of *C. albicans* fungi had a regular morphology and a smooth surface. The AMP-27 treatment group caused damage to the fungal membrane, resulting in membrane depressions, breaks, and cell deformation. Transmission electron microscopy results showed that the control group of *C. albicans* fungi had an intact cell structure, a complete and clearly defined cell membrane, and a relatively homogeneous internal structure. The AMP-27-treated group of *C. albicans* fungi showed irregular changes in the cell membrane, with abnormal structures such as vacuoles appearing inside the fungus, indicating damage to the internal ultrastructure of the cells.

[0043] Example 4 The drug toxicity of AMP-27 was determined using human erythrocyte hemolytic activity assay. Red blood cells from healthy individuals were collected, washed twice, and resuspended in PBS buffer to prepare a 4% red blood cell suspension. 500 μL of each antimicrobial peptide solution at different concentration gradients (1, 2, 4, 8, 16, 36, 64, 128 μg / ml) were mixed with 500 μL of the red blood cell suspension and incubated at 37°C for 1 h. After centrifugation and photographing, 100 μL of the supernatant was aspirated into a 96-well plate, and the absorbance (OD 540 nm) of the supernatant was measured using a microplate reader. A complete hemolysis group (treated with Triton-100) was set up as a positive control, and an untreated group (treated with PBS buffer) was set up as a negative control. The hemolysis rate was calculated to assess the hemolytic activity of the antimicrobial peptides.

[0044] The results are as follows Figure 2 As shown, AMP-27 exhibits lower hemolytic activity at effective bactericidal concentrations than the antimicrobial peptide SAAP-148, demonstrating better safety.

[0045] Example 5 In vivo experiments to detect the therapeutic effect of AMP-27 on fungal infected wounds Male BALB / c mice aged 6-8 weeks were selected and fed routinely for one week. Their backs were shaved and disinfected, and a full-thickness skin defect with a diameter of approximately 8 mm was created in the center of the skin. They were randomly divided into 5 groups: NC group (blank control group), CON group (model group), AMP-27 group (antimicrobial peptide group), HG group (hydrogel group), and HG@AMP-27 group (hydrogel-loaded antimicrobial peptide group). Except for the NC group, each group received an application of C. albicans suspension (1×10⁻⁶) to the wound. 6A fungal infection wound model was constructed using CFU / mL (50 μL). After successful modeling, each group received local drug treatment every other day: the NC and CON groups were treated with 100 μL of sterile normal saline (NS), the AMP-27 group was treated with 100 μL of 2.5 mg / kg peptide solution, the HG group was treated with 100 μL of HG, and the HG@AMP-27 group was treated with 100 μL of HG containing 2.5 mg / kg AMP-27. Treatment continued until one group's wound healed. Wound healing was recorded during the process. At the end of the experiment, mice were sacrificed, and wound tissue was collected for HE staining and Masson's trichrome staining. HE staining was used to observe inflammatory cell infiltration and epithelial regeneration, while Masson's trichrome staining was used to assess collagen fiber deposition and arrangement. Tissue sections were observed under a microscope and analyzed to evaluate the comprehensive effects of AMP-27 in anti-infection and promoting tissue repair.

[0046] The results are as follows Figure 3 and Figure 4 As shown, the experiment was terminated when the wound in the NC group was close to healing. Compared with the CON group, AMP-27 promoted the healing speed of fungal infected wounds, reduced inflammatory cell infiltration, and enhanced collagen fiber deposition and tissue reconstruction. The combined use of AMP-27 and hydrogel had better effects.

[0047] The antibacterial agent AMP-27 provided by this invention not only possesses strong bactericidal ability at low concentrations and better safety, and can cause cell membrane damage, but also can disrupt the ultrastructure of cells. AMP-27 can also promote the healing speed of fungal infected wounds, while reducing inflammatory cell infiltration and enhancing collagen fiber deposition and tissue reconstruction.

Claims

1. An antimicrobial peptide AMP-27, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The biomaterial related to the antimicrobial peptide AMP-27 according to claim 1 is any one of the following: ① The nucleotide sequence encoding the antimicrobial peptide of claim 1; ②A recombinant vector containing the nucleotide sequence described in ①; ③ Recombinant bacterial cells containing the nucleotide sequence described in ①.

3. The use of the antimicrobial peptide AMP-27 of claim 1 or the biomaterial of claim 2 in the preparation of products with the ability to kill drug-resistant bacteria.

4. The application as described in claim 3, characterized in that, The drug-resistant bacteria include one or more of the following: Candida albicans, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. Preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus, the Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae, the Escherichia coli is carbapenem-resistant Escherichia coli, and the Acinetobacter baumannii is pan-drug-resistant Acinetobacter baumannii.

5. An antibacterial agent, characterized in that, Includes the antimicrobial peptide AMP-27 as described in claim 1.

6. The antibacterial agent as described in claim 5, characterized in that, The antibacterial agent also contains excipients; Preferably, the excipients include one or more of hydrogels, liposomes, and nanomaterials.

7. A drug for treating pneumonia, sepsis, or skin wound infections caused by drug-resistant or non-drug-resistant bacteria, characterized in that, Includes the antimicrobial peptide AMP-27 as described in claim 1.

8. The drug as described in claim 7, characterized in that, The drug-resistant bacteria include one or more of the following: Candida albicans, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii; Preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus, the Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae, the Escherichia coli is carbapenem-resistant Escherichia coli, and the Acinetobacter baumannii is pan-drug-resistant Acinetobacter baumannii.

9. The drug as described in claim 7, characterized in that, The drug also contains excipients; Preferably, the excipients include one or more of hydrogels, liposomes, and nanomaterials.

10. The medicament as claimed in claim 7, characterized in that, The drug is administered via intravenous injection, nebulized inhalation, or topical application.

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