Antimicrobial peptide, antimicrobial product and application thereof for inhibiting multiple human pathogens

Abstract

The present invention belongs to the field of antimicrobial peptides, and specifically relates to an antimicrobial peptide, antimicrobial product and application that inhibits a variety of human pathogens. The antimicrobial peptide is named CAMP1229NC, and is obtained by capping the polypeptide shown in SEQ ID NO:1 with an N-terminal acetyl group and a C-terminal amino group. 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 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, an antimicrobial product and an application thereof, which can inhibit a variety of human pathogens. Background Art

[0002] The widespread use of antibiotics has greatly improved human health and effectively controlled bacterial infections. However, with the long-term overuse and inappropriate use of antibiotics, bacteria have gradually evolved resistance, making antibiotic resistance a major challenge to global public health. The emergence and spread of drug-resistant bacteria have made many infections that were once easily treatable difficult to control, and have even led to drug-resistant infections with high mortality rates. Therefore, there is an urgent need to develop new antimicrobial drugs to address the development of drug resistance.

[0003] Antimicrobial peptides are a class of natural immune molecules that are widely present in organisms and have broad-spectrum antimicrobial activity. They are composed of short chains of amino acids and are usually positively charged. They can bind to negatively charged bacterial cell membranes through electrostatic interactions, disrupting their integrity, leading to leakage of cell contents and bacterial death. Antimicrobial peptides not only have broad-spectrum antimicrobial activity against bacteria, but also exhibit inhibitory effects against fungi, viruses, and even certain cancer cells. Compared with traditional antibiotics, the mechanism of action of antimicrobial peptides is more complex, they are less likely to induce bacterial resistance, and they exhibit significant inhibitory effects against multiple resistant strains. Therefore, they are considered to be one of the new treatment strategies for dealing with drug-resistant bacterial infections. In addition, antimicrobial peptides also have immunomodulatory functions, can promote wound healing and regulate inflammatory responses, and therefore have attracted much attention in the biomedical field.

[0004] However, the widespread application of antimicrobial peptides still faces many challenges, mainly due to low antibacterial activity and high instability, which limit their widespread application in actual treatment. 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 for inhibiting multiple human pathogens, named CAMP1229NC, which is obtained by capping the N-terminus with an acetyl group (acetylation modification) and capping the C-terminus with an amino group (amidation modification) of the polypeptide shown in SEQ ID NO: 1. That is, the structure of CAMP1229NC is CH3CO-HKNLKKVKLLLKKLLYF-NH2.

[0007] The antimicrobial peptide exhibited significant antibacterial activity against Staphylococcus aureus standard strain ATCC12600, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae, Acinetobacter baumannii standard strain ATCC19606, as well as 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 were 2 μg / mL, 32 μg / mL, 8 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, and 2 μg / mL, respectively.

[0008] The present invention also provides the use of the antimicrobial peptide for inhibiting multiple human pathogens 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 CAMP1229NC.

[0019] Figure 2 This is the predicted structure of the antimicrobial peptide CAMP1229NC.

[0020] Figure 3 The MIC test results of the antimicrobial peptide CAMP1229NC 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 CAMP1229NC on the morphology of Staphylococcus aureus ATCC12600 using scanning electron microscopy, Figure 6 A is the control group, Figure 6 Middle B is the CAMP1229NC group.

[0024] Figure 7 These are the results of pH stability and pepsin stability experiments of the antimicrobial peptide CAMP1229NC.

[0025] Figure 8 Changes in mouse body weight during the experimental period of antimicrobial peptide CAMP1229NC's anti-infection activity in the mouse skin abrasion model.

[0026] Figure 9Bacterial load in mice 24 hours after treatment with the antimicrobial peptide CAMP1229NC. 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 CAMP1229NC

[0030] The antimicrobial peptide CAMP1229NC is obtained by solid-phase synthesis of the polypeptide shown in SEQ ID NO: 1 by additional acetyl blocking (acetylation modification) and amino blocking (amidation modification) at the N-terminus and C-terminus, respectively. The molecular formula of the antimicrobial peptide CAMP1229NC is C 107 H 183 N 27 O 20 , with a molecular weight of 2167.76 g / mol, a net charge of 7, 7 positive charges, an isoelectric point of 11, and a normalized hydrophobicity of -0.09. The chemical structure diagram is shown in Figure 1 shown.

[0031] The antimicrobial peptide CAMP1229NC 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: HKNLKKVKLLLKKLLYF (SEQ ID NO: 1).

[0034] Example 2: Determination of the Minimum Inhibitory Concentration (MIC) of the Antimicrobial Peptide CAMP1229NC 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 CAMP1229NC 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. The 96-well plate was incubated at 37°C for 24 hours, and bacterial growth was measured 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] MICs of the antimicrobial peptide CAMP1229NC against seven human pathogens 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 8 μg / mL and 32 μg / mL, respectively; the MICs against Acinetobacter baumannii ATCC19606 and resistant Acinetobacter baumannii bio-53272 were both 2 μg / mL; and the MICs against Staphylococcus aureus ATCC12600, resistant Staphylococcus aureus ATCC33591, and resistant Staphylococcus aureus ATCC33592 were 2 μg / mL, 4 μg / mL, and 8 μg / mL, respectively.

[0038] Example 3: Scanning electron microscopy observation of the bactericidal effect of the antimicrobial peptide CAMP1229NC 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 CAMP1229NC 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. The fixed 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 CAMP1229NC 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 CAMP1229NC

[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] The stability experiment of antimicrobial peptide CAMP1229NC is as follows 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 4 μg / mL.

[0045] Example 4: Anti-infection experiment of antimicrobial peptide CAMP1229NC 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 McFadden 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 CAMP1229NC. Two hours after infection, 20 μL of CAMP1229NC 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 CAMP1229NC experimental group slowed down; Figure 9This is the bacterial load of mice treated with the antimicrobial peptide CAMP1229NC 24 hours after treatment. The CFU count results showed that CAMP1229NC 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 that inhibits multiple human pathogens, 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.

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