Antibacterial peptide for inhibiting various human pathogenic bacteria, antibacterial product and application

Abstract

The invention belongs to the field of antibacterial peptides, and particularly relates to an antibacterial peptide for inhibiting various human pathogenic bacteria, an antibacterial product and application. The antibacterial peptide is named as CAMP1229NC, and is obtained by performing N-terminal acetyl end capping and C-terminal amino end capping on polypeptide shown in SEQ ID NO: 1. The antibacterial peptide has remarkable antibacterial activity on a staphylococcus aureus standard strain ATCC12600, pseudomonas aeruginosa PAO1, klebsiella pneumoniae, an acinetobacter baumannii standard strain ATCC19606, methicillin-resistant staphylococcus aureus ATCC33591, methicillin-resistant and gentamicin-resistant staphylococcus aureus ATCC33592 and acinetobacter baumannii bio-53272 resistant to various antibiotics, and the antibacterial peptide can be used for preparing the antibacterial peptide. The limitation of the traditional antibacterial peptide is effectively overcome.

Description

Technical Field

[0001] The present invention belongs to the field of antimicrobial peptides, and in particular relates to an antimicrobial peptide capable of inhibiting a variety of human pathogens, an antimicrobial product and an application thereof. Background Art

[0002] The widespread use of antibiotics has greatly improved human health and effectively controlled bacterial infectious diseases. However, with the long-term abuse and improper use of antibiotics, bacteria have gradually evolved resistance, making antibiotic resistance a major challenge facing global public health. The emergence and spread of drug-resistant bacteria have made many infections that were once easy to treat difficult to control, and even led to drug-resistant infections with high mortality rates. Therefore, there is an urgent need to develop new antimicrobial drugs to cope with 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-chain amino acids, usually positively charged, and can bind to negatively charged bacterial cell membranes through electrostatic interaction, destroying their integrity, leading to leakage of cell contents and bacterial death. Antimicrobial peptides not only have broad-spectrum antimicrobial activity against bacteria, but also show inhibitory effects on fungi, viruses and even certain cancer cells. Compared with traditional antibiotics, the mechanism of action of antimicrobial peptides is more complex, less likely to induce bacterial resistance, and exhibits significant inhibitory effects on a variety of 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 new antimicrobial peptide that can effectively inhibit a variety of human pathogens and has a broad-spectrum antibacterial effect. The technical solution is as follows: The present invention provides an antimicrobial peptide for inhibiting a variety of human pathogens, named CAMP1229NC, which is obtained by capping the polypeptide shown in SEQ ID NO: 1 with an acetyl group at the N-terminus (acetylation modification) and an amino group at the C-terminus (amidation modification), that is, the structure of CAMP1229NC is CH 3 CO-HKNLKKVKLLLKKLLYF-NH 2 .

[0006] The antimicrobial peptide exhibited significant antibacterial activity against the standard strain of Staphylococcus aureus ATCC12600, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae, standard strain of Acinetobacter baumannii ATCC19606, methicillin-resistant Staphylococcus aureus ATCC33591, methicillin-resistant and gentamicin-resistant Staphylococcus aureus ATCC33592, and Acinetobacter baumannii bio-53272 resistant to multiple antibiotics, 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-resistant 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.

[0007] The present invention also provides the use of the antimicrobial peptide for inhibiting multiple human pathogens in the preparation of antimicrobial products, wherein the antimicrobial products are used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii or Klebsiella pneumoniae.

[0008] Furthermore, the antibacterial product is an antibacterial drug or a bacteriostatic agent.

[0009] Furthermore, the antibacterial product contains the antibacterial peptide as the only active ingredient.

[0010] 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.

[0011] Furthermore, the antibacterial drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the dosage form of the antibacterial drug is an aqueous extract, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

[0013] The present invention also provides an antibacterial product, comprising the antibacterial peptide. Further, the antibacterial product has the antibacterial peptide as the only active ingredient. Further, the antibacterial product is an antibacterial drug or an antibacterial agent.

[0014] Compared with the prior art, the beneficial effects of the present invention include at least: 1. The antimicrobial peptide provided by the present invention has highly effective inhibitory activity against pathogenic bacteria Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. It also has a high antibacterial effect on drug-resistant Staphylococcus aureus and drug-resistant Acinetobacter baumannii. It has good pH stability and exhibits anti-infection activity in a mouse model. These characteristics make it show the antibacterial value of medical drugs, and provide alternative medicinal resources for future clinical use against these human pathogens.

[0015] 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

[0016] Figure 1 Schematic diagram of the chemical structure of the antimicrobial peptide CAMP1229NC.

[0017] Figure 2 This is the predicted structure of the antimicrobial peptide CAMP1229NC.

[0018] Figure 3 The MIC test results of the antimicrobial peptide CAMP1229NC against 7 pathogens.

[0019] Figure 4 The growth of Klebsiella pneumoniae, Pseudomonas aeruginosa PAO1, Acinetobacter baumannii ATCC19606 and resistant Acinetobacter baumannii bio-53272 was measured when MIC was determined.

[0020] Figure 5 To determine the growth of Staphylococcus aureus ATCC12600, resistant Staphylococcus aureus ATCC33591 and resistant Staphylococcus aureus ATCC33592 when MIC was determined.

[0021] 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.

[0022] Figure 7 These are the results of the pH stability and pepsin stability experiments of the antimicrobial peptide CAMP1229NC.

[0023] 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.

[0024] Fig. 9Bacterial load in mice treated with the antimicrobial peptide CAMP1229NC 24 hours later. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but 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.

[0026] Staphylococcus aureus ATCC12600, Acinetobacter baumannii ATCC19606, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae, 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.

[0027] Example 1: Physicochemical properties and preparation of antimicrobial peptide CAMP1229NC The antimicrobial peptide CAMP1229NC is obtained by performing additional acetyl blocking (acetylation modification) and amino blocking (amidation modification) at the N-terminus and C-terminus of the polypeptide shown in SEQ ID NO: 1 during solid phase synthesis. The molecular formula of the antimicrobial peptide CAMP1229NC is C 107 H 183 N 27 O 20 , molecular weight is 2167.76 g / mol, net charge number is 7, with 7 positive charges, isoelectric point is 11, normalized hydrophobicity is -0.09, chemical structure diagram is as follows Figure 1 shown.

[0028] The antimicrobial peptide CAMP1229NC was synthesized by Shanghai Bioengineering Co., Ltd. through solid phase chemical synthesis with a purity of more than 95%. The lower molecular weight makes CAMP1229NC have a lower production cost.

[0029] 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 indicates that the peptide is a typical α-helical peptide with amphipathic characteristics, which is conducive to its interaction with bacterial membranes.

[0030] The amino acid sequence involved is: HKNLKKVKLLLKKLLYF (SEQ ID NO: 1).

[0031] Example 2: Determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide CAMP1229NC against seven human pathogens The MIC of antimicrobial peptides was determined by the broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991.

[0032] Seven pathogens were inoculated into sterile CAMHB liquid culture medium and cultured overnight at 37°C with shaking. The pathogens were inoculated into fresh CAMHB liquid culture medium at a 1% inoculation rate and cultured until the logarithmic growth phase. The bacterial concentration was adjusted to 1×10 5 CFU / mL was used to obtain the bacterial solution. Then 180 μL of the bacterial solution was transferred to a 96-well plate. The antimicrobial peptide CAMP1229NC powder was dissolved in sterile water and diluted to a 2-fold antimicrobial peptide solution with concentrations of 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL and 640 μg / mL. 20 μL of antimicrobial peptide solutions of different concentrations were added to the bacterial solution in the 96-well plate, respectively, so that the antimicrobial peptide concentrations in the bacterial solution were 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL and 64 μg / mL. After the 96-well plate was incubated at 37°C for 24 h, the bacterial growth was detected by an ELISA instrument. MIC was defined as the minimum antimicrobial peptide concentration at which no bacterial growth was detected. Sterile water was used as the control group CK, and 3 replicates were set for each group of experiments.

[0033] MICs of the antimicrobial peptide CAMP1229NC against seven human pathogens Figure 3 As shown in Figure 2, bacterial growth during MIC determination is as follows: Figure 4-Figure 5 The results showed that the MICs for Klebsiella pneumoniae and Pseudomonas aeruginosa PAO1 were 8 μg / mL and 32 μg / mL, respectively, the MICs for Acinetobacter baumannii ATCC19606 and resistant Acinetobacter baumannii bio-53272 were both 2 μg / mL, and the MICs for Staphylococcus aureus ATCC12600, resistant Staphylococcus aureus ATCC33591, and resistant Staphylococcus aureus ATCC33592 were 2 μg / mL, 4 μg / mL, and 8 μg / mL, respectively.

[0034] Example 3: Scanning electron microscopy observation of the bactericidal effect of the antimicrobial peptide CAMP1229NC on Staphylococcus aureus 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 solution was divided into two groups: one group was added with the antimicrobial peptide CAMP1229NC at a concentration of 5 × MIC, 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, the supernatant was discarded, and the bacteria were 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 overnight at 4°C. The fixed samples were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacteria were evenly spread on a microscope slide. Subsequently, the samples were washed three times with sterile PBS buffer, each washing for 10 min. After washing, the samples were dehydrated in a gradient ethanol series (30%, 50%, 70%, 80% and 100%), and were treated twice in 100% ethanol for 10 min each time. After dehydration, the slide samples were treated with 50% ethyl isovalerate (ethyl isovalerate:ethanol, 1:1) and 100% ethyl isovalerate, respectively. 2 The samples were processed by 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.

[0035] The imaging results are as follows 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.

[0036] Example 4: Stability test of antimicrobial peptide CAMP1229NC 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.

[0037] For the pepsin stability assay, pepsin was dissolved in a 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, it was 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.

[0038] The stability experiment of antimicrobial peptide CAMP1229NC is as follows Figure 7 As shown in the figure, different pH and pepsin had no significant effect on the activity of antimicrobial peptides, and the MIC was stable at 4 μg / mL.

[0039] Example 4: Anti-infection experiment of antimicrobial peptide CAMP1229NC in mouse skin abrasion model Staphylococcus aureus ATCC12600, ATCC33591 and ATCC33592 were inoculated into LB liquid medium and cultured at 37°C under shaking until the logarithmic growth phase. Subsequently, they were washed twice with sterile PBS and resuspended to OD 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 back area that the mice could not touch with their mouths or limbs was shaved and disinfected, and a 12×12 mm superficial abrasion was prepared. After the wound was rinsed with sterile saline, 20 μL of 1×10 8 CFU / mL of mixed bacterial suspension was used for infection. 2 h after infection, 20 μL of CAMP1229NC at a concentration of 32 μg / mL was dripped onto the wound for treatment. Three mice were set up in each group, and all mice were housed individually to avoid cross contamination, and were adaptively housed for 5 days before the experiment. Mice were killed 24 h after the experiment, and 5×5 mm skin tissue was taken from the infected site and homogenized using a grinder (25 Hz). 10-fold continuous gradient dilutions were spread on agar plates, and 3 parallels were set up for each plate, cultured at 37°C for 24 h, and colony counts were performed according to GB / T 5750.12-2023. At the same time, changes in mouse weight were recorded at each experimental stage.

[0040] Figure 8 The weight changes of mice during the experimental period. The weight loss trend of mice in the CAMP1229NC experimental group slowed down; Fig. 9This is the bacterial load of mice treated with the antimicrobial peptide CAMP1229NC 24 hours later. The CFU count results showed that CAMP1229NC effectively inhibited the growth of Staphylococcus aureus and reduced the bacterial load by one order of magnitude.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope 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.

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

4. The use according to claim 3, characterized in that The antibacterial drug is used to treat diseases caused by infection with drug-resistant bacteria; the antibacterial agent is used in medical care, food preservation, personal care products or environmental disinfection.

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

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