Antimicrobial peptide with broad-spectrum antibacterial effect, antimicrobial product and application

Abstract

The present invention belongs to the field of antimicrobial peptides, and specifically relates to an antimicrobial peptide, antimicrobial product and application with a broad-spectrum antimicrobial effect. The antimicrobial peptide is named CAMP502NC, and is obtained by capping the polypeptide shown in SEQ ID NO:1 with an N-terminal acetyl group and capping the C-terminal amino group. The antimicrobial peptide exhibits significant antimicrobial activity against Staphylococcus aureus standard strain ATCC12600, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae, Acinetobacter baumannii standard strain ATCC19606, methicillin-resistant Staphylococcus aureus ATCC33591, methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592 and Acinetobacter baumannii bio-53272 resistant to multiple antibiotics, effectively overcoming the limitations of traditional antimicrobial peptides.

Description

Technical Field

[0001] The present invention belongs to the field of antimicrobial peptides, and in particular relates to an antimicrobial peptide with broad-spectrum antibacterial effect, an antimicrobial product and applications. Background Art

[0002] The discovery and application of antibiotics are hailed as milestones in modern medicine, greatly reducing the mortality rate of diseases caused by bacterial infections. However, with the widespread use of antibiotics, the problem of bacterial resistance has become increasingly serious, becoming a major challenge to global public health. Antibiotic abuse, irrational use of drugs, and the lack of an effective resistance monitoring system have accelerated the generation and spread of drug-resistant bacteria. Currently, the rapid spread of drug-resistant bacteria has made the treatment of various infections increasingly difficult, especially drug-resistant strains in hospital infections. These drug-resistant bacteria do not respond well to conventional antibiotic treatment, resulting in prolonged infection, increased complications, and even life-threatening conditions. In recent years, antimicrobial peptides have become an important research direction for alternatives to antibiotics due to their unique antibacterial mechanism and lower risk of resistance development.

[0003] Antimicrobial peptides, a class of natural immune molecules widely present in organisms, are considered an important alternative to traditional antibiotics due to their broad-spectrum antimicrobial activity, low risk of developing drug resistance, and unique mechanism of action. Antimicrobial peptides are typically composed of 5-100 amino acids and have an amphipathic structure. They can exert their antibacterial effects through various mechanisms, such as disrupting bacterial cell membranes and interfering with protein synthesis. Unlike traditional antibiotics, the mechanism of action of antimicrobial peptides makes it difficult for bacteria to develop drug resistance through single gene mutations. For example, CN118546228B discloses an antimicrobial peptide that can be used to inhibit the growth of Streptococcus suis and exhibits a highly effective bactericidal effect against Streptococcus suis by increasing cell membrane permeability. CN112321698B discloses an antimicrobial peptide and its pharmaceutical composition, which are modified from the natural antimicrobial peptide PGLa-AM1 and have stronger antimicrobial effects and better performance, inhibiting the growth of Helicobacter pylori.

[0004] However, it still faces many challenges and difficulties in practical application, such as high toxicity and poor stability. Therefore, developing new antimicrobial drugs and optimizing the structure of antimicrobial peptides will be an important direction to deal with the increasingly serious problem of antibiotic resistance. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a novel antimicrobial peptide that can effectively inhibit a variety of human pathogens and has a broad-spectrum antibacterial effect. The technical solution is as follows:

[0006] The present invention provides an antimicrobial peptide with a broad-spectrum antibacterial effect, named CAMP502NC, which is obtained by capping the N-terminal acetyl group (acetylation modification) and the C-terminal amino group (amidation modification) of the polypeptide shown in SEQ ID NO:1, that is, the structure of CAMP502NC is CH3CO-ILKLSRFCKKLI-NH2.

[0007] The antimicrobial peptide exhibits significant antibacterial activity against Staphylococcus aureus standard strain ATCC12600, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae, Acinetobacter baumannii standard strain ATCC19606, methicillin-resistant Staphylococcus aureus ATCC33591, methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592, and multi-antibiotic-resistant Acinetobacter baumannii bio-53272, effectively overcoming the limitations of traditional antimicrobial peptides. Furthermore, the minimum inhibitory concentrations of the antimicrobial peptide against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, methicillin- and gentamicin-resistant Staphylococcus aureus, and drug-resistant Acinetobacter baumannii are 8 μg / mL, 64 μg / mL, 64 μg / mL, 16 μg / mL, 16 μg / mL, 32 μg / mL, and 32 μg / mL, respectively.

[0008] The present invention also provides the use of the antimicrobial peptide with broad-spectrum antibacterial effect in the preparation of an antimicrobial product, wherein the antimicrobial product is used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii or Klebsiella pneumoniae-like bacteria.

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

[0010] Furthermore, the antibacterial product contains the antibacterial peptide as the only active ingredient.

[0011] Furthermore, the antibacterial drug is used to treat diseases caused by infection with drug-resistant bacteria; the antibacterial agent can be used in the fields of medical care, food preservation, personal care products or environmental disinfection.

[0012] Furthermore, the antibacterial drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage form of the antibacterial drug is an aqueous extract, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

[0014] The present invention also provides an antimicrobial product comprising the antimicrobial peptide. Furthermore, the antimicrobial product comprises the antimicrobial peptide as the sole active ingredient. Furthermore, the antimicrobial product is an antimicrobial drug or bacteriostatic agent.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least:

[0016] 1. The antimicrobial peptides provided by the present invention have highly effective inhibitory activity against pathogenic bacteria Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. They also have a high antibacterial effect against drug-resistant Staphylococcus aureus and drug-resistant Acinetobacter baumannii. They have good pH stability and exhibit anti-infection activity in mouse models. These properties make them exhibit antibacterial value as medical drugs and provide alternative medicinal resources for future clinical use against these human pathogens.

[0017] 2. The antimicrobial peptides provided by the present invention also have the advantages of low production cost and high environmental stability, providing important technical support for the development of clinical antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the chemical structure of the antimicrobial peptide CAMP502NC.

[0019] Figure 2 This is the predicted structure of the antimicrobial peptide CAMP502NC.

[0020] Figure 3 The MIC test results of the antimicrobial peptide CAMP502NC against 7 pathogens.

[0021] Figure 4 The growth of Klebsiella pneumoniae, Pseudomonas aeruginosa PAO1, Acinetobacter baumannii ATCC19606 and resistant Acinetobacter baumannii bio-53272 was determined when MIC was determined.

[0022] Figure 5 To determine the growth of Staphylococcus aureus ATCC12600, resistant Staphylococcus aureus ATCC33591 and resistant Staphylococcus aureus ATCC33592 when MIC was determined.

[0023] Figure 6 To observe the effect of antimicrobial peptide CAMP502NC on the morphology of Staphylococcus aureus ATCC12600 using scanning electron microscopy, Figure 6 A is the control group, Figure 6 Middle B is the CAMP502NC group.

[0024] Figure 7 These are the results of pH stability and pepsin stability experiments of the antimicrobial peptide CAMP502NC.

[0025] Figure 8 The weight changes of mice during the experimental period of the anti-infection activity of the antimicrobial peptide CAMP502NC in the mouse skin abrasion model.

[0026] Figure 9 Bacterial load in mice 24 hours after treatment with the antimicrobial peptide CAMP502NC. DETAILED DESCRIPTION

[0027] 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.

[0028] Staphylococcus aureus ATCC12600, Acinetobacter baumannii ATCC19606, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae-like bacteria, methicillin-resistant Staphylococcus aureus ATCC33591, methicillin- and gentamicin-resistant Staphylococcus aureus ATCC33592, and drug-resistant Acinetobacter baumannii bio-53272 were purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0029] Example 1: Physicochemical properties and preparation of antimicrobial peptide CAMP502NC

[0030] The antimicrobial peptide CAMP502NC is obtained by solid-phase synthesis of the polypeptide shown in SEQ ID NO: 1 by additional acetyl end-capping (acetylation modification) and amino end-capping (amidation modification) at the N-terminus and C-terminus, respectively. The molecular formula of the antimicrobial peptide CAMP502NC is C 71 H 127 N 19 O 14 S has a molecular weight of 1502.95 g / mol, a net charge of 4, carries 4 positive charges, an isoelectric point of 11, and a normalized hydrophobicity of 0.73. The chemical structure diagram is shown in FIG. Figure 1 shown.

[0031] The antimicrobial peptide CAMP502NC 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.

[0032] The online tool AlphaFold2 cannot predict the structure of modified peptides. It predicts the protein structure of unmodified peptides based on the structure reflected by the side. The prediction results are as follows Figure 2 As shown, this peptide is a typical α-helical peptide with amphipathic characteristics, which is conducive to its interaction with bacterial membranes.

[0033] The amino acid sequence involved is: ILKLSRFCKKLI (SEQ ID NO: 1).

[0034] Example 2: Determination of the Minimum Inhibitory Concentration (MIC) of the Antimicrobial Peptide CAMP502NC against 7 Human Pathogens

[0035] 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.

[0036] Seven pathogens were inoculated into sterile CAMHB liquid medium and cultured overnight at 37°C with shaking. Pathogens were 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 5 The bacterial suspension was obtained using a 180 μL microplate reader. The antimicrobial peptide CAMP502NC powder was dissolved in sterile water and serially diluted 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.

[0037] The MICs of the antimicrobial peptide CAMP502NC against seven human pathogens are as follows Figure 3 As shown in Figure 2, the bacterial growth during MIC determination was as follows: Figure 4-Figure 5 The results showed that the MICs against Klebsiella pneumoniae and Pseudomonas aeruginosa PAO1 were both 64 μg / mL, the MICs against Acinetobacter baumannii ATCC19606 and resistant Acinetobacter baumannii bio-53272 were 16 μg / mL and 32 μg / mL, respectively, and the MICs against Staphylococcus aureus ATCC12600, resistant Staphylococcus aureus ATCC33591, and resistant Staphylococcus aureus ATCC33592 were 8 μg / mL, 16 μg / mL, and 32 μg / mL, respectively.

[0038] Example 3: Scanning electron microscopy observation of the bactericidal effect of the antimicrobial peptide CAMP502NC on Staphylococcus aureus

[0039] Staphylococcus aureus ATCC1260 was cultured in sterile LB liquid medium at 37°C overnight and diluted to 1×10 8 CFU / mL. Subsequently, the bacterial suspension was divided into two groups: one group was treated with the antimicrobial peptide CAMP502NC 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. Following 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 (30%, 50%, 70%, 80%, and 100%), including two 10-minute treatments in 100% ethanol. After dehydration, the slides were treated sequentially with 50% ethyl isovalerate (ethyl isovalerate:ethanol, 1:1) and then 100% ethyl isovalerate. 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 different treatment conditions.

[0040] Imaging results such as Figure 6 As shown, the experimental results showed that Staphylococcus aureus treated with the antimicrobial peptide CAMP502NC showed obvious cell membrane damage or rupture, while the bacterial cell structure in the control group was intact and there was no obvious change.

[0041] Example 4: Stability test of antimicrobial peptide CAMP502NC

[0042] 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.

[0043] 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 resistance of the antimicrobial peptide to pepsin hydrolysis.

[0044] Stability test of antimicrobial peptide CAMP502NC Figure 7 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.

[0045] Example 5: Anti-infection experiment of antimicrobial peptide CAMP502NC in mouse skin abrasion model

[0046] 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 three strains were mixed in a ratio of 1:1:1, and the concentration of the mixed bacterial suspension was measured using a McFarland turbidimeter and adjusted to 1×10 8 CFU / mL. Six-week-old female Kunming mice were selected and anesthetized by intraperitoneal injection of 4% chloral hydrate. 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 Infection was performed with a mixed bacterial suspension containing 100 CFU / mL of CAMP502NC. Two hours after infection, 20 μL of CAMP502NC at a concentration of 32 μg / mL was applied to the wound surface as a single droplet. Three mice were housed individually to prevent cross-contamination and acclimated for 5 days before the experiment. Mice were sacrificed 24 hours after the experiment, and 5 × 5 mm skin tissue was obtained from the infection site and homogenized using a grinder (25 Hz). A 10-fold serial dilution was plated onto agar plates, with triplicate replicates per plate. The plates were incubated at 37°C for 24 hours, and colony counts were performed according to GB / T 5750.12-2023. Mouse weight changes were recorded at each experimental stage.

[0047] Figure 8 The weight changes of mice during the experimental period showed that the weight loss trend of mice in the CAMP502NC experimental group slowed down; Figure 9This is the bacterial load of mice treated with the antimicrobial peptide CAMP502NC 24 hours after treatment. The CFU count results showed that CAMP502NC effectively inhibited the growth of Staphylococcus aureus and reduced the bacterial load by one order of magnitude.

[0048] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solutions of the present invention may be subjected to various simple modifications, including combining the various technical features in any other appropriate manner. These simple modifications and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide with broad-spectrum antibacterial effect, characterized in that: The polypeptide shown in SEQ ID NO: 1 is obtained by capping the N-terminal acetyl group and the C-terminal amino group.

2. 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, Pseudomonas aeruginosa, Acinetobacter baumannii or Klebsiella pneumoniae-like bacteria.

3. The use according to claim 2, characterized in that The antibacterial product is an antibacterial drug.

4. The use according to claim 3, characterized in that The antibacterial drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 2, characterized in that The antimicrobial product is a bacteriostatic agent.

6. The use according to claim 5, characterized in that The antibacterial agent is used in medical hygiene, food preservation or personal care products.

Images