Use of antimicrobial peptide mimetic amp-2f in the preparation of campylobacter inhibitors

Abstract

The application provides application of antibacterial peptide mimics AMP-2f in preparation of campylobacter inhibitors. The application firstly finds good antibacterial activity of the AMP-2f by determining minimum bacteriostatic concentration of the AMP-2f to campylobacter clinical strains. Then the antibacterial activity and mechanism of the AMP-2f are detected by in-vitro tests such as bactericidal kinetics, motility, chemotaxis, biofilm formation ability, adhesion and invasion ability and membrane targeting. Finally, it is found that the AMP-2f can effectively prevent the colonization of campylobacter in the intestinal tract of mice by preventive administration of the AMP-2f. The application clarifies the anti-campylobacter activity of the AMP-2f, establishes a systematic AMP-2f activity evaluation system, and confirms the potential of the AMP-2f as a campylobacter infection prevention and treatment compound, thereby providing important test basis and candidate molecules for development of new anti-campylobacter drugs.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial technology, and in particular relates to the application of the antimicrobial peptide mimic AMP-2f in the preparation of Campylobacter inhibitors. Background Technology

[0002] Campylobacter is an important foodborne and zoonotic pathogen. In recent years, Campylobacter has shown increasing resistance to various antimicrobial drugs, which not only harms the healthy development of the livestock industry but also poses a threat to human health and public safety.

[0003] According to a World Health Organization (WHO) survey, approximately 166 million people worldwide are infected with Campylobacter each year (PLOS Medicine, 2015, 12(12): e1001923). Of the nearly 600 million cases of foodborne zoonotic diseases reported globally in 2015, 52% were caused by the pathogen (Journal of the Hellenic Veterinary Medical Society, 2018, 67(2):65-82), with 30% of these cases related to poultry consumption. In Europe, Campylobacter infection is the most common cause of bacterial enteritis, accounting for more than 60% of all reported zoonotic diseases (EFSA journal European Food Safety Authority, 2023, 21(12): 8442). According to EU statistics, Campylobacter has surpassed Salmonella to become the leading foodborne pathogen, with 137,107 confirmed cases, equivalent to 43.1 cases per 100,000 people, and a case fatality rate of 0.04% (ESFA, 2022, 20(3): e07209). Statistics from the Chinese Center for Disease Control and Prevention show that as of 2019, the detection rate of Campylobacter jejuni among adult patients with diarrhea in my country was approximately 10%. As a symbiotic pathogen in the intestines of poultry, Campylobacter enters the food chain through contamination of poultry meat and processed products, leading to human infection and posing a serious threat to public health (Berliner und Munchener tierarztliche Wochenschrift, 2015, 128(3-4):132-140).

[0004] In poultry farming, antibiotics are mostly used to control bacterial diseases, but due to long-term improper use, the problem of bacterial resistance has become increasingly serious. Long-term selective pressure has led to a continuous increase in the resistance rate of Campylobacter to commonly used clinical antibiotics such as fluoroquinolones and macrolides (Journal of applied microbiology, 2016, 120(5): 1139-1173; Transboundary and emerging diseases, 2018, 65 Suppl 1: 30-48; Microbiology spectrum, 2022, 10(5): 1622-1667).

[0005] Between 2008 and 2014, Chinese scholars Wang (Antimicrob Chemother, 2016, 71(3): 666-669) et al. found that almost all of the 2976 Campylobacter isolates were resistant to fluoroquinolones. Italian scholar Giacomelli (Microb DrugResist, 2014, 20(2):181-188) isolated 60 Campylobacter strains from broilers. Drug sensitivity results showed that all broilers and 92.00% of turkeys carried Campylobacter strains with multidrug resistance, with resistance rates to quinolones and tetracyclines ranging from 65.00% to 100.00% in broilers. Currently, the increasingly serious phenomenon of antibiotic resistance has become a focal point of public health globally. Therefore, developing highly effective and safe novel antimicrobial agents is of great value in controlling Campylobacter infection in poultry. Summary of the Invention

[0006] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides the application of the antimicrobial peptide mimic AMP-2f in the preparation of Campylobacter inhibitors. This invention offers a solution for controlling bacterial resistance, thereby ensuring public health safety.

[0007] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of the antimicrobial peptide mimic AMP-2f, or a composition containing the antimicrobial peptide mimic AMP-2f, in the preparation of Campylobacter inhibitors, wherein the structure of the antimicrobial peptide mimic AMP-2f is shown in formula (I):

[0009]

[0010] (I).

[0011] As a specific implementation, the Campylobacter is Campylobacter coli or Campylobacter jejuni; preferably, the Campylobacter is selected from Campylobacter coli 1922C17.

[0012] Secondly, the present invention provides a method for inhibiting Campylobacter, comprising the step of contacting Campylobacter with an effective dose of an antimicrobial peptide mimic AMP-2f, wherein the structure of the antimicrobial peptide mimic AMP-2f is shown in formula (I):

[0013]

[0014] (I).

[0015] As a specific implementation, the contact is performed in vitro or under in vitro conditions for the purpose of inhibiting Campylobacter for non-therapeutic purposes.

[0016] As a specific implementation, the Campylobacter is present in food, on the surface of food processing equipment, in aquaculture environments, or in medical devices.

[0017] Thirdly, the present invention provides a composition for inhibiting Campylobacter, characterized in that the composition comprises an antimicrobial peptide mimic AMP-2f as an active ingredient, and a pharmaceutically, food-, or veterinary-acceptable carrier, the structure of which is shown in formula (I):

[0018]

[0019] (I).

[0020] As a specific embodiment, the composition is a powder, tablet, solution, emulsion, spray, or disinfectant.

[0021] As a specific embodiment, the minimum inhibitory concentration of the antimicrobial peptide mimic AMP-2f in the composition is between 0.25 and 2 μg / mL.

[0022] Fourthly, the present invention provides the use of the antimicrobial peptide mimic AMP-2f, or a composition comprising the antimicrobial peptide mimic AMP-2f, in the preparation of a medicament for the prevention or treatment of diseases caused by Campylobacter, the structure of the antimicrobial peptide mimic AMP-2f being shown in formula (I):

[0023]

[0024] (I).

[0025] As a specific implementation plan, the diseases mentioned include bacterial enteritis, etc.

[0026] The in vitro anti-Campylobacterial activity of the antimicrobial peptide mimic AMP-2f described in this invention, and the in vivo colonization of anti-Campylobacterial bacteria in the intestine, were studied. The experimental results showed that:

[0027] The AMP-2f described in this invention exhibits low inhibitory concentrations (MICs) of 0.25-2 μg / mL against multiple clinical strains of Campylobacter, demonstrating good antibacterial activity. Kinetic experiments showed that the colony count in the 0.5 μg / mL AMP-2f group was consistently lower than that in the control group. The 2 μg / mL AMP-2f group showed superior bactericidal efficacy over 64 μg / mL florfenicol (FFC) within 6 hours, and reduced the viable bacterial count below the detection limit within 24 hours, demonstrating excellent bactericidal potential. Live / dead staining results also showed that bacteria treated with AMP-2f exhibited abundant red fluorescent signals, indicating a significant killing effect on Campylobacter, effectively disrupting the integrity of the bacterial cell membrane and leading to bacterial death. The results of the post-antibiotic effect (PAE) test showed that AMP-2f exhibited a significant concentration-dependent PAE for Campylobacter: the 0.25 μg / mL treatment group recovered growth at 4.25 h, the 0.5 μg / mL group was prolonged to 5.08 h, while no colony growth recovery was observed in the 2 μg / mL group and the 64 μg / mL control group during the observation period, suggesting that high concentrations of AMP-2f can significantly prolong the PAE.

[0028] Biofilm formation assays showed that AMP-2f at 1 μg / mL significantly inhibited biofilm formation, indicating that AMP-2f can effectively intervene in the biofilm-related pathogenicity of Campylobacter. Motility assays showed that AMP-2f at ≥0.25 μg / mL significantly inhibited the motility of Campylobacter, with the inhibitory effect becoming more pronounced with increasing concentration, demonstrating a concentration-dependent relationship. Chemotaxis assays showed that Campylobacter exhibited a positive chemotactic response to α-ketoglutarate, confirming normal function of its chemotactic system; however, no chemotactic zone was formed after treatment with different concentrations of AMP-2f, indicating that this antimicrobial peptide is not a repellent substance recognized by the Campylobacter chemotactic system. Cytotoxicity assays showed that AMP-2f had high safety in IPEC-J2 cells (CC50 of 52 μg / mL), and its inhibitory effect on Campylobacter adhesion and invasion will be further evaluated. The results showed that 4 μg / mL AMP-2f significantly reduced bacterial adhesion in a concentration-dependent manner and lowered the invasion rate below the detection limit, effectively blocking the invasive infection of intestinal epithelial cells by Campylobacter.

[0029] Membrane action mechanism results showed that the surface of Campylobacter treated with AMP-2f ruptured, accompanied by leakage of contents. Furthermore, AMP-2f significantly increased the staining of fluorescent dyes PI and NPN, indicating that AMP-2f can disrupt the integrity of the bacterial cell membrane. Further investigation revealed that AMP-2f treatment significantly increased bacterial ΔpH and Δφ in a concentration-dependent manner, indicating that AMP-2f significantly dissipated bacterial PMF, providing further support for its membrane disruption characteristics. Therefore, we used the reactive oxygen species indicator DCFH-DA to assess ROS accumulation in AMP-2f-treated Campylobacter, and the results showed that AMP-2f induced significant ROS accumulation in Campylobacter. Simultaneously, electron transport chain (ETC) activity assays showed that low concentrations (0.25 μg / mL, 0.5 μg / mL) of AMP-2f significantly increased INT reduction in Campylobacter, while high concentrations did not show further enhancement, suggesting possible saturation or detection interference. These findings indicate that the antibacterial effect of AMP-2f is related to accelerating bacterial respiration. These results collectively demonstrate that AMP-2f induces the loss of key cellular components and oxidative stress in bacterial cells, ultimately leading to their death. To further investigate the membrane phospholipid components targeted by AMP-2f, bacterial membrane phospholipids (PG, LPS, PGN, PC, PE, PS) were added to the experiment for observation. The results showed that high concentrations of LPS (128 μg / mL) reduced the antibacterial activity of AMP-2f by 4-fold, while PG at low concentrations (4 μg / mL) also reduced the antibacterial activity of AMP-2f by 4-fold. Furthermore, with increasing PG concentrations, the antibacterial and bactericidal activities of AMP-2f significantly decreased. Next, we performed molecular dynamics (MD) simulations to evaluate the molecular interactions between AMP-2f and bacterial phospholipid PG. The system underwent rapid conformational rearrangement in the initial stage of the simulation, subsequently entering a stable fluctuation range and maintaining dynamic stability. The binding free energy ΔG_bind = -31.35 kcal / mol calculated by MM / PBSA indicates that AMP-2f and PG have a significant tendency to bind spontaneously. Energy decomposition further shows that the binding is mainly driven by van der Waals interactions and nonpolar solvation terms (ΔvdW = -32.58, ΔESA = -4.31 kcal / mol), suggesting that the stacking of hydrophobic segments and the increase of contact area are key factors for stabilizing the complex.

[0030] In vivo safety studies showed that 30 mg / kg of AMP-2f was a safe dose. Mice had normal body weight, normal blood routine and blood biochemical indicators, and no obvious pathological changes in the colon and cecum. Anticolonization studies showed that AMP-2f significantly reduced the number of Campylobacter in mouse feces. In the model group, the colonic tissue structure of mice was basically intact, but the cecum tissue showed mild pathological damage: disordered arrangement of mucosal epithelial cells with a small amount of epithelial cell shedding (yellow arrow); a significant reduction in the number of goblet cells in the mucosal layer (red arrow); and loose edema in the submucosa. After AMP-2f intervention, the colonic and cecum tissue structures of mice recovered well, and the pathological damage was significantly improved. In the positive drug control group, the colon showed moderate abnormalities, with a small amount of mucosal epithelial shedding and necrosis (yellow arrow); the cecum showed mild pathological changes, with loose edema in the submucosa (green arrow) and a small amount of inflammatory cell infiltration (black arrow). Compared with the model group and the positive drug control group, AMP-2f can effectively repair intestinal tissue structure damage, relieve mucosal edema and inflammatory infiltration, restore the number of goblet cells, and has a significant effect on improving and protecting against pathological damage to the colon and cecum.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0032] 1. Highly effective antibacterial activity: AMP-2f showed good minimum inhibitory concentration against 10 clinical strains of Campylobacter, demonstrating its highly effective antibacterial activity against clinical isolates of Campylobacter and overcoming the problem of decreased sensitivity of existing antibacterial drugs to some clinical strains.

[0033] 2. Systematic activity evaluation framework: This invention establishes a complete research chain from in vitro MIC screening and antibacterial mechanism analysis to in vivo anticolonization verification, providing a standardized technical solution for the preclinical evaluation of antimicrobial peptide candidate drugs.

[0034] 3. Significant in vivo preventive effect: Through a prophylactic dosing strategy, AMP-2f was shown to effectively block the colonization of Campylobacter in the mouse intestine, providing experimental evidence and candidate compounds for preventive intervention against Campylobacter infection.

[0035] 4. Clear clinical translation potential: Using clinical strains as research subjects ensures the clinical relevance of the experimental results and accelerates the translation process from laboratory to clinical application.

[0036] 5. Balancing safety and activity: The evaluation of antibacterial activity is combined with safety assessment, providing important data support for subsequent drug development studies. Attached Figure Description

[0037] Figure 1The results are from in vitro antibacterial activity tests: A represents the results of the bactericidal kinetic test; B represents the results of the live / dead staining test; C represents the results of the antibiotic post-effect test; and D represents the results of the biofilm formation inhibition test.

[0038] Figure 2 The results of in vitro motility tests are as follows: A is a visual representation of the motility test results; B is the chemotaxis test results; C is the motility diameter diagram; D is the cell safety test results; and E is the adhesion and invasion test results.

[0039] Figure 3 The results are as follows: A represents the results of scanning electron microscopy; B represents the results of bacterial cell membrane permeability assay; C represents the results of bacterial outer cell membrane permeability assay; D represents the results of membrane depolarization assay; E represents the results of proton kinetic potential assay; F represents the results of reactive oxygen species assay; and G represents the results of INT reduction assay.

[0040] Figure 4 The results are from in vivo safety tests; A represents the results of routine blood tests and blood biochemistry tests; B represents the changes in mouse body weight; and C represents the pathological analysis of liver, kidney, cecum, and colon tissues.

[0041] Figure 5 The results are as follows: A represents the changes in the bacterial load of Campylobacter in mouse feces; B represents the changes in colon length; C represents the results of the catalase test in intestinal tissue; D represents the results of the test of inflammatory factors in intestinal tissue; and E represents the results of the pathological analysis test of intestinal tissue. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection claimed by the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art.

[0043] Test strains and main reagents

[0044] The test strain, 1922C17, was isolated and identified in our laboratory.

[0045] Test reagents and main culture media: MUELLER-HINTON AGAR (MHA) and MUELLER-HINTON BROTH (MHB) were purchased from Oxoid; sterile defibrinated sheep blood was purchased from Zhengzhou Yikang Biotechnology; microaerophilic gas generator bags were purchased from Mitsubishi, Japan; CCDA culture medium was purchased from Qingdao Haibo Biotechnology.

[0046] The structure of the antimicrobial peptide mimic AMP-2f is shown in formula (I):

[0047]

[0048] (I).

[0049] The antimicrobial peptide mimic AMP-2f was donated by Professor Zhang En's research group at the Institute of Pharmaceutical Sciences, Zhengzhou University. Alternatively, it can be prepared using the method described in Chinese Patent ZL202210539862.6.

[0050] Example 1: Minimum Inhibitory Concentration Test

[0051] The resuscitation test strain was cultured on MHA agar plates under microaerophilic conditions (5% O2, 10% CO2, 85% N2, v / v) for 24 h, then subcultured onto fresh MHA agar plates and cultured for 18 h. A suitable amount of bacterial cells was scraped and added to 500 μL of MHB broth and mixed thoroughly. 150 μL of the bacterial suspension was added to a 90 mm plate containing 15 mL of MHB broth and diluted 100-fold. The bacterial suspension was then mixed using a multichannel pipette. 100 μL of the bacterial suspension was transferred from the plate to each well of a 96-well plate using a multichannel pipette, for a total of 11 wells. MHB blank broth was added to the 12th well as a negative control. 100 μL of the compound was added to the first well of the 96-well plate to achieve a final concentration of 1280 μg / mL. After mixing, 100 μL of the mixture was transferred to the next well for serial dilution. Dilutes were performed up to the 10th and 11th wells as positive controls. Place the 96-well plate in a 42°C incubator and incubate under microaerophilic conditions for 24 h.

[0052] Result Interpretation and Analysis: Before reading and reporting the MIC of the tested strain, check whether the bacteria in the positive control wells are growing well, whether the negative control wells are contaminated, and whether the MIC value of the quality control drug is within the quality control range. Visually inspect the lowest drug concentration in the clear well; this is the MIC of the tested bacteria.

[0053] The results of the minimum inhibitory concentration test showed that:

[0054] The results of the minimum inhibitory concentration (MIC) test are shown in Table 1. AMP-2f showed good antibacterial effects against 10 clinical strains, with MICs ranging from 0.25 to 2 μg / mL. One clinical strain, 1922C17, was randomly selected as the test strain for subsequent experiments.

[0055] Table 1. Results of Minimum Inhibitory Concentration

[0056]

[0057] Concentration: μg / mL

[0058] Example 2: Bactericidal Kinetics Test

[0059] The bacterial strain was picked and placed in a 5 ml EP tube containing liquid culture medium (OD600=0.1), diluted 1:100, mixed thoroughly, and aliquoted into glass tubes. Different concentrations of AMP-2f were added, and an equal volume of broth was added to the blank control group. 100 μL of the sample solution was placed in the first well of a 96-well plate, and the sample solution was subsequently diluted 10-fold with 1×PBS buffer. 10 μL of each diluted solution was added to blood agar medium; this was the colony count at 0 h. Colony counts were then performed at 2, 4, 6, 8, 10, 12, and 24 h, with 10 μL added each time. Three parallel controls were set up for each dilution gradient. The culture was incubated at 42°C for 24 h before colony counting.

[0060] Results of bactericidal kinetics test:

[0061] Clinical strain 1922C17 was selected for a bactericidal kinetic study to systematically evaluate the antibacterial activity of AMP-2f against Campylobacter. Results showed that 0.5 μg / mL AMP-2f significantly inhibited bacterial growth. Notably, 2 μg / mL AMP-2f exhibited superior bactericidal efficacy over 6 hours compared to 64 μg / mL florfenicol (FFC), and reduced viable bacterial counts below the detection limit within 24 hours, demonstrating excellent bactericidal potential. Figure 1 A).

[0062] Example 3: Live / Dead Staining Test

[0063] Take the bacterial culture in the logarithmic growth phase, centrifuge at 5000 rpm for 5 min, wash twice with PBS and resuspend, and finally resuspend in 200 μL of PBS to a final volume of 10. 9 CFU / mL. An equal volume of the compound was added, and an equal volume of PBS was added to the control group. After incubation for 4 h, the mixture was centrifuged at 5000 rpm for 5 min and washed twice with PBS. Then, different concentrations of the fluorescent dyes DAPI and PI were added, and the mixture was incubated in the dark for 5-10 min, centrifuged at 5000 rpm for 5 min, washed twice with PBS, and finally resuspended in 10 μL of 70% glycerol for observation under a fluorescence microscope.

[0064] The results of the live-dead staining test showed that:

[0065] The control group bacteria stained blue with DAPI, indicating that the bacterial cell membrane structure was intact and the cells were viable. The bacteria treated with AMP-2f showed a large amount of red fluorescence, indicating that AMP-2f has a significant killing effect on Campylobacter, effectively disrupting the integrity of the bacterial cell membrane and leading to bacterial death. Figure 1 B).

[0066] Example 4: Post-antibiotic effect (PAE)

[0067] Take 1.5 × 10⁻⁶ of the logarithmic growth phase bacterial culture. 7 CFU / mL, equal volumes of bacterial suspension and AMP-2f solutions of different concentrations were mixed. The mixed solutions were incubated in a 42°C shaker for 2 hours. Then, they were centrifuged at 1400 rpm for 5 minutes. The top layer of culture medium was removed with a pipette, and then an equal volume of fresh broth was added. After shaking and mixing, the mixture was centrifuged again. The mixture was washed three times with broth to thoroughly remove the drug. Finally, the culture system was restored to its original volume with broth and incubated in a 42°C shaker for another 5 minutes, which was the reconstruction time 0. At different time points, 0.1 mL of bacterial suspension was taken from each test tube, and an equal volume of solution was added to the test tube. The mixture was serially diluted 10-fold with sterile PBS, and the bacterial count was performed using a dropper. T and C were calculated from the bacterial growth kinetic curves according to PAE=TC. T is the time required for the bacterial value in the experimental group to be 10 times higher than the reconstruction time 0, and C is the time (in hours) required for the bacterial value in the control group to be 10 times higher than the reconstruction time 0.

[0068] The results of the antibiotic post-effects test showed that:

[0069] Post-antibiotic effect (PAE) refers to the phenomenon where bacterial growth remains inhibited even after brief exposure to antibiotics. For example... Figure 1 As shown in C, AMP-2f exhibited a significant concentration-dependent post-expansion (PAE) in Campylobacter: the 0.25 μg / mL treatment group recovered growth at 4.25 h, the 0.5 μg / mL group was prolonged to 5.08 h, while the 2 μg / mL group and the 64 μg / mL control group did not show any recovery of colony growth during the observation period, suggesting that the PAE was significantly prolonged at high concentrations.

[0070] Example 5 Biofilm formation experiment

[0071] Resuscitated and passaged Campylobacter were harvested from MH liquid medium, adjusted to OD600 to 1, and diluted 100-fold with MH. This solution was then mixed with different concentrations of AMP-2f and added to 96 wells at 200 μL / well, with four replicates per group. The plates were incubated at 42°C under microaerophilic conditions. After 72 h, the medium was discarded, and the plates were washed three times with PBS. Residual medium and air-drying bacteria were removed. 50 μL of methanol was added to the cleaned 96-well plates to fix the bacterial strain for 15 min. The fixative was removed and the plates were air-dried. 200 μL of 0.1% crystal violet solution was added to each well, and staining was performed at room temperature for 15 min. The crystal violet was discarded, and the plates were washed three times with PBS and air-dried. Elution buffer (anhydrous ethanol or 33% acetic acid) was added to dissolve the strain, and the plates were incubated at 37°C for 30 min. After complete elution and dissolution, the absorbance at 570 nm was measured using a microplate reader. Data were recorded to compare the biofilm formation ability of Campylobacter under different concentrations of AMP-2f.

[0072] The results of the biofilm formation experiment showed that:

[0073] The inhibitory effect of AMP-2f on biofilm formation of clinical strain 1922C17 was evaluated, and the results showed that ( Figure 1 (D) AMP-2f inhibited the formation of bacterial biofilms in a concentration-dependent manner, with 1 μg / mL showing a significant inhibitory effect, indicating that AMP-2f can effectively intervene in the biofilm-related pathogenicity of Campylobacter.

[0074] Example 6: Motion Performance Test

[0075] The revived bacterial strain was cultured overnight on MH plates at 42°C under microaerophilic conditions. A small number of colonies were picked and cultured on MH blood agar plates for 16-18 hours to induce vigorous logarithmic growth. A small amount of Campylobacter pure culture was added to MH broth and the concentration was adjusted to OD600 = 0.3. 5 μL of the bacterial culture was inoculated into the center of a 0.4% agar plate containing different concentrations of AMP-2f, with the pipette tip puncturing the agar plate. The plate was placed upright in an incubator and cultured at 42°C under microaerophilic conditions for 48 hours. Afterward, the agar plate was observed; the formation of a bacterial ring centered on the inoculation site indicated motility. The diameter of the bacterial ring represents its motility under in vitro conditions.

[0076] The results of the exercise test showed that:

[0077] Motility is an important function mediated by bacterial infection. By evaluating the effect of AMP-2f on the motility of Campylobacter, the results are as follows: Figure 2 As shown in A and C, AMP-2f at ≥0.25 μg / mL significantly inhibited the motility of Campylobacter, and the inhibitory effect became more pronounced with increasing concentration, showing a concentration-dependent relationship.

[0078] Example 7 Chemotaxis test

[0079] The revived and passaged Campylobacter was harvested using MHB and adjusted to OD600=1. The bacterial suspension was heated in a 37°C water bath for 10 min, then mixed with an equal volume of 0.8% agar (42°C) prepared with PBS, poured onto plates, and allowed to stand for 2 h. Filter paper discs of different concentrations of AMP-2f were prepared, and filter paper discs prepared with PBS and 1 mM ketoglutarate were used as controls. The prepared filter paper discs of different concentrations were placed on the surface of bacterial plates, and each plate was inverted under microaerophilic conditions at 42°C for 48 h before the results were observed.

[0080] Chemotaxis test results showed:

[0081] Chemotaxis is a key mechanism by which Campylobacter recognizes host signaling molecules and migrates in a directed manner. Chemotaxis assays show that ( Figure 2(B) The strain showed a positive chemotactic response to α-ketoglutarate, confirming that its chemotactic system functioned normally; however, no chemotactic zones were formed after treatment with different concentrations of AMP-2f, indicating that the antimicrobial peptide was not a repellent substance recognized by the Campylobacter chemotactic system.

[0082] Example 8 Adhesion and Invasion Test

[0083] Cells were resuscitated and passaged using DMEM containing 10% fetal bovine serum and 1% penicillin-dextrin. After washing twice with PBS and digesting with trypsin, cells were harvested using DMEM containing 10% fetal bovine serum and counted. The cell concentration was adjusted, and 100 μL was seeded into 96-well cell culture plates. 5 Cells / well were incubated overnight at 37°C. Different concentrations of AMP-2f were mixed with DMEM containing blood but no antibiotics. The original medium was carefully discarded, and medium containing AMP-2f was added. DMEM containing 1% DMSO was used as a control. Each group had 4 replicate wells. The cells were incubated in a CO2 incubator at 37°C for 2 hours. After that, MTT was added in the dark at 100 μL / well, and the cells were incubated in a CO2 incubator at 37°C for 3 hours. The medium was then carefully aspirated, and 100 μL of formazan dissolution solution was added. After the formazan dissolved, the OD570 was measured using a microplate reader. The data were recorded, and the effect of AMP-2f on the cells was analyzed to determine the safe concentration for use.

[0084] As described above, after cell resuscitation and passage, the cell concentration was adjusted and seeded into 24-well cell plates at a density of 4 × 10⁶ cells / well. 5 Cells / well, incubated overnight at 37°C. Campylobacter was harvested from DMEM medium containing blood but no antibiotics and diluted to 10⁻⁶ cells / well. 6 CFU / mL, mix AMP-2f with Campylobacter to make the bacterial count 5 × 10⁻⁶. 5 CFU / mL. Simultaneously, a mixture of 1% DMSO and Campylobacter was used as a control, and incubated at 37°C for 15 min. Cells in 24-well plates were washed twice with DPBS, and a mixture of AMP-2f and bacteria was added. The plates were then incubated at 37°C in a CO2 incubator. After 2 h, the cell culture medium in the wells was discarded, and the cells were washed three times with PBS. 1 ml of 0.1% Triton X-100 was added to lyse the cells. The lysate was serially diluted with PBS, and plate counts were performed to calculate the adhesion rate.

[0085] After incubation for 2 hours, the cell culture medium in the wells was discarded, and the cells were washed three times with PBS. 1 mL of DMEM medium containing 100 mg / mL gentamicin was added to each well, and the cells were incubated at 37°C with 5% CO2 for 1 hour. Subsequently, the culture medium in the wells was discarded, and the cells were washed three times with sterile PBS. 1 mL of 0.1% Triton X-100 was added to lyse the cells. The lysates were serially diluted with PBS, and plate counts were performed to calculate the invasion rate.

[0086] Adhesion and invasion results show:

[0087] Given that adhesion and invasion are key steps in Campylobacter colonization and pathogenesis, directly determining its ability to infect host epithelial cells, AMP-2f exhibits high safety in IPEC-J2 cells (CC50 of 52 μg / mL). Figure 2 (D) to further evaluate its inhibitory effect on Campylobacter adhesion and invasion. Results showed that 4 μg / mL AMP-2f significantly reduced bacterial adhesion in a concentration-dependent manner, lowering the invasion rate below the detection limit, effectively blocking the invasive infection of intestinal epithelial cells by Campylobacter. Figure 2 E).

[0088] Example 9 Membrane Mechanism Experiment

[0089] Scanning electron microscopy: Overnight cultured bacteria were adjusted to OD600=0.50 using MH liquid medium and treated with different concentrations of AMP-2f for 5 h. Then, the bacteria were centrifuged at 4000 rpm for 10 min, washed twice with PBS, resuspended and fixed in electron microscopy fixative, and stored at 4°C.

[0090] Bacterial cell membrane permeability: After overnight culture, the selected bacterial strains were centrifuged at 4000 rpm for 8 min, washed twice with PBS, and resuspended until the OD600 reached 0.5. Then, they were treated with PI at a final concentration in the dark and incubated at 37°C with shaking for 30 min. Bacterial cells labeled with the fluorescent probe PI were added to black 96-well plates, followed by 10 μl of different concentrations of AMP-2f (with polymyxin as a control). The plates were incubated at 37°C for 1 h, with three replicates for each concentration. Finally, the fluorescence intensity was measured for 30 min at 37°C using an automated microplate reader with an excitation wavelength of 535 nm and an emission wavelength of 615 nm.

[0091] Bacterial cell membrane depolarization assay: Selected bacterial strains were cultured overnight, centrifuged at 4000 rpm for 8 min to collect the cells, washed three times with 5 mM HEPES buffer containing 5 mM glucose, and resuspended until OD600 reached 0.50. Then, the fluorescent probe 3,3'-dipropylthiocyanate and iodide DISC3-5 (0.5 μmol / L) were added, and the mixture was incubated at 37°C for 15 min on a shaker. 190 μl of the probe-loaded bacterial cells were added to a 96-well plate, and the fluorescence intensity was measured every 5 min using an automated microplate reader for a total of 15 min until the fluorescence intensity stabilized. Subsequently, 10 μl of different concentrations of AMP-2f and polymyxin were added as positive controls, with three replicates for each concentration. Finally, the fluorescence intensity was measured every 5 min using an automated microplate reader at an excitation wavelength of 622 nm and an emission wavelength of 670 nm for a total of 1 h.

[0092] Bacterial outer membrane permeability: After overnight culture, the selected bacterial strains were centrifuged at 3500 rpm for 3 min at 4 °C, washed twice with 5 mmol / L HEPES, diluted 100-fold with HEPES, and resuspended in 5 mmol / L HEPES (PH7). Under light-protected conditions, the fluorescent dye NPN and bacterial suspension were mixed, and 190 µL of fluorescently labeled bacterial cells were added to a 96-well plate. After incubation at 37 °C for 30 min, fluorescence was measured using a microplate reader with excitation wavelength of 350 nm and emission wavelength of 420 nm for 10 min.

[0093] Proton kinetic analysis: Collect the bacterial suspension from overnight culture by centrifugation at 4000 rpm for 8 min. Wash three times with 5 mM HEPES buffer containing 5 mM glucose and resuspend until OD600 reaches 0.50. Then add 10 μM of the pH fluorescent indicator BCECF-AM and incubate at 37°C for 15 min on a shaker. Add 190 μl of probe-loaded bacterial solution to a 96-well plate and measure the fluorescence intensity every 5 min using an automated microplate reader for a total of 15 min until the fluorescence intensity stabilizes. Then add 10 μl of AMP-2f at different concentrations to the reaction system, mix well, and measure every 5 min using an automated microplate reader. The excitation wavelength is 488 nm and the emission wavelength is 535 nm for a total of 1 h.

[0094] ROS: Take the overnight cultured bacterial suspension, centrifuge at 4000 rpm for 5 min, resuspend in PBS and adjust OD600 to 0.5, then add 10 μM of the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Incubate in a 37 °C water bath for 20-30 min, gently mixing every 3-5 min. Then centrifuge at 4000 rpm for 5 min at 4 °C, wash 3 times with PBS, and take 190 μL of the fluorescently labeled bacterial suspension and add it to 96 wells. Then add 10 μL of AMP-2f at different concentrations, with ROSUP as a positive control, and incubate at 37 °C for 1 h. Monitor the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using an automated microplate reader, and detect the fluorescence intensity after 1 h. Each treatment was performed in triplicate.

[0095] INT reduction assay: Overnight bacterial suspension was washed twice with PBS and the OD600 was adjusted to 0.50. The suspension was then stained with 1 mM INT. Different concentrations of AMP-2f and polymyxin were added as positive controls. 200 μl of the mixture was transferred to a 96-well plate, and the absorbance was measured at 490 nm at 37°C.

[0096] Effects of different membrane phospholipid components on the antibacterial activity of AMP-2f: First, different membrane phospholipids were serially diluted in columns 1-10 of a 96-well plate using MH liquid medium. Then, different concentrations of AMP-2f prepared in MH liquid medium were added to rows A and H of the 96-well plate. Finally, 100 μL of diluted bacterial suspension was added to each well, mixed thoroughly, and incubated at 42°C for 18-20 h under microaerophilic conditions. The MIC values ​​were then observed.

[0097] Molecular dynamics simulations: Before molecular dynamics simulations, to ensure reasonable system energies, a 50,000-step energy minimization was performed using the steepest descent method. Subsequently, the positions of protein heavy atoms were constrained, and equilibrium phase simulations were performed sequentially in the NVT and NPT ensembles for 100 ps. Finally, a 100 ns unconstrained molecular dynamics production phase simulation was conducted. During the simulations, the Parrinello-Rahman method was used to maintain the pressure at 1 bar and the temperature at 310 K. Long-range electrostatic interactions were calculated using the Particle-Mesh-Ewald (PME) method. The boundary threshold for the long-range van der Waals (vdW) energy term was set to 1 nm. To maintain the stability of hydrogen-containing bonds, the SHAKE algorithm was used in the simulations. Trajectory coordinates were recorded every 10 ps.

[0098] The results of the membrane action mechanism experiment show that:

[0099] The mechanisms of action of antimicrobial peptides are complex, but most interact with bacterial membrane phospholipids, leading to membrane rupture, leakage of contents, and death. First, scanning electron microscopy results show that the surface of Campylobacter treated with AMP-2f ruptured, accompanied by leakage of contents. Figure 3 A). We further found that AMP-2f significantly increased the staining of fluorescent dyes PI and NPN, indicating that AMP-2f can disrupt the integrity of bacterial cell membranes (A). Figure 3 B, C).

[0100] Bacterial membrane disruption can be reflected by perturbations of the proton motive force (PMF). Therefore, we investigated AMP-2f-induced membrane dysfunction by measuring membrane potential (Δφ) and transmembrane proton gradient (ΔpH). First, we used the fluorescent probe DISC3-5 to investigate changes in Δφ in bacteria after AMP-2f treatment. The results showed that AMP-2f treatment significantly increased the fluorescence of DISC3-5 in bacteria. Figure 3D). Then, we used the fluorescent indicator BCECF-AM to investigate the effect of AMP-2f on bacterial ΔpH. The results showed that AMP-2f treatment significantly increased the bacterial ΔpH in a concentration-dependent manner. Figure 3 E). The above results indicate that the bacterial PMF induced by AMP-2f significantly dissipated, providing further support for its membrane disruption characteristics.

[0101] Considering the alterations in cell membrane integrity induced by AMP-2f, we hypothesized that it also affected the respiratory chain and metabolic activity within the cell membrane. Therefore, we used the reactive oxygen species indicator DCFH-DA to assess ROS accumulation in AMP-2f-treated Campylobacter. The results showed that AMP-2f induced significant ROS accumulation in Campylobacter (…). Figure 3 F). We then used iodonitrosyltetrazole chloride (INT) to detect the activity of the electron transport chain (ETC), a substrate that can be reduced by respiratory chain dehydrogenases to formazan, a red formaldehyde insoluble compound. The results showed that only low concentrations (0.25 μg / mL, 0.5 μg / mL) of AMP-2f significantly increased INT reduction in Campylobacter, while higher concentrations did not show a further enhancing effect. Figure 3 G) suggests potential saturation or detection interference. These findings indicate that the antibacterial effect of AMP-2f is related to accelerated bacterial respiration. These results collectively demonstrate that AMP-2f-induced loss of important cellular components and oxidative stress in bacterial cells ultimately lead to their death.

[0102] To further investigate the membrane phospholipid components targeted by AMP-2f, we added bacterial membrane phospholipid components (PG, LPS, PGN, PC, PE, and PS) to our experiments. The results showed that high concentrations of LPS (128 μg / mL) reduced the antibacterial activity of AMP-2f by four-fold, while even low concentrations of PG (4 μg / mL) reduced the antibacterial activity of AMP-2f by four-fold. Furthermore, with increasing PG concentration, both the antibacterial and bactericidal activities of AMP-2f significantly decreased. Next, we performed molecular dynamics (MD) simulations to evaluate the molecular interactions between AMP-2f and bacterial phospholipid PG. The system underwent rapid conformational rearrangement in the initial stage of the simulation, subsequently entering a stable fluctuation range and maintaining dynamic stability. The binding free energy ΔG_bind = -31.35 kcal / mol calculated by MM / PBSA (Table 2) indicates that AMP-2f and PG have a significant tendency to bind spontaneously. Energy decomposition further shows that the binding is mainly driven by van der Waals interactions and nonpolar solvation terms (ΔvdW = -32.58, ΔESA = -4.31 kcal / mol), suggesting that the stacking of hydrophobic segments and the increase of contact area are key factors for stabilizing the complex.

[0103] Table 2. Decomposition of molecular docking binding free energy (kcal / mol)

[0104]

[0105] Example 10 In vivo safety test

[0106] Four- to five-week-old C57 / BL mice were acclimatized for one week and then administered 100 μL of AMP-2f (30 mg / kg and 50 mg / kg) by gavage, while the control group was administered an equal volume of PBS by gavage. The weight of the mice was measured daily for seven consecutive days. On the eighth day, blood was collected from the orbital rim for complete blood count and blood biochemical tests. At the same time, liver, kidney, colon, and cecum tissues were collected for HE staining. Five mice were in each group.

[0107] Safety test results show:

[0108] After administering different concentrations of AMP-2f to mice via gavage, blood routine and blood biochemical tests showed that, compared with the control group, the serum urea (UREA) and creatinine (CREA) levels in the high-concentration AMP-2f group were significantly increased, suggesting that high-dose AMP-2f can induce kidney damage in mice and poses a certain risk of nephrotoxicity. No other blood routine and blood biochemical indicators showed significant abnormalities; and the low-dose AMP-2f group showed no significant differences in any of its indicators compared with the control group, indicating no adverse effects. Figure 4 A). Continuous monitoring of mouse body weight changes over one week showed that on day 7 of the experiment, the body weight of mice in the high-concentration AMP-2f group was significantly lower than that of the control group. Figure 4 B). Further observation of histopathological changes in mice: No abnormalities were observed in the morphology of the liver, kidney, cecum, and colon tissues of mice in the control group and the low-dose group; in the high-dose group, the liver showed that the hepatocytes were still arranged in a relatively regular manner, but the tissue structure was loose, accompanied by mild edema of multiple hepatocytes, and a small amount of edema to vacuolar degeneration; a small amount of inflammatory cell infiltration was observed in the kidney, colon, and cecum tissues, showing mild pathological damage overall. Figure 4 C). In summary, the above studies indicate that 30 mg / kg is a safe dosage of AMP-2f in mice and can be used as the preferred safe dosage for subsequent in vivo experiments.

[0109] Example 11 In vivo anti-colonization test

[0110] After 4-5 week old C57 / BL mice had been acclimatized for one week, they were given 100 μL of AMP-2f (30 mg / kg) by gavage, while the control group was given an equal volume of PBS by gavage. This was done for 3 consecutive days, followed by gavage of 10 μL of AMP-2f twice a day. 8CFU / mL Campylobacter was administered AMP-2f via gavage starting on day 2 after Campylobacter administration, for three consecutive days. The Campylobacter load in feces was measured daily. On day 4, mice were dissected, and colonic and cecal tissues were fixed in 4% paraformaldehyde for HE staining analysis. Simultaneously, the levels of inflammatory factors (IL-6, TNF-α, IL-1β, IFN-γ) in the colonic and cecal tissues were measured. Myeloperoxidase activity in the colon and cecum was detected using a catalase kit. Finally, the colon length of the mice was measured, with five mice in each group.

[0111] The results of the anti-colonization test showed that:

[0112] Prophylactic administration of AMP-2f significantly reduced the amount of Campylobacter in mouse feces, catalase activity, and inflammatory factor levels, and the colon length was significantly higher in the mouse control group than in the model control group. Figure 5 (A, B, C). Consistent with the trend of inflammatory factors, the colonic tissue structure of mice in the model control group was basically intact, but the cecal tissue showed mild pathological damage: disordered arrangement of mucosal epithelial cells with a small amount of epithelial cell shedding (yellow arrow); the number of goblet cells in the mucosal layer was significantly reduced (red arrow), and loose edema was visible in the submucosa. After AMP-2f intervention, the colonic and cecal tissue structures of mice recovered well, and the pathological damage was significantly improved. The colon of the positive drug control group showed moderate abnormalities, with a small amount of mucosal epithelial shedding and necrosis (yellow arrow); the cecum showed mild pathological changes, with loose edema in the submucosa (green arrow) and a small amount of inflammatory cell infiltration (black arrow). Figure 5 (D, E). Compared with the model group and the positive drug control group, AMP-2f can effectively repair intestinal tissue structure damage, alleviate mucosal edema and inflammatory infiltration, restore goblet cell count, and has a significant improving and protective effect on pathological damage to the colon and cecum.

[0113] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. The use of the antimicrobial peptide mimic AMP-2f, or a composition containing the antimicrobial peptide mimic AMP-2f, in the preparation of Campylobacter inhibitors, characterized in that, The structure of the antimicrobial peptide mimic AMP-2f is shown in formula (I): (I)。 2. The application according to claim 1, characterized in that, The Campylobacter is Campylobacter coli or Campylobacter jejuni; preferably, the Campylobacter is selected from Campylobacter coli 1922C17.

3. A method for inhibiting Campylobacter, characterized in that, The step includes contacting an effective dose of the antimicrobial peptide mimic AMP-2f with Campylobacter, the structure of which is shown in formula (I): (I)。 4. The method according to claim 3, characterized in that, The contact is performed in vitro or under in vitro conditions for the non-therapeutic purpose of Campylobacter inhibition.

5. The method according to claim 3, characterized in that, The Campylobacter is present in food, on the surface of food processing equipment, in aquaculture environments, or in medical devices.

6. A composition for inhibiting Campylobacter, characterized in that, The composition comprises an antimicrobial peptide mimic AMP-2f as the active ingredient, and a pharmaceutically, culinary, or veterinary-acceptable carrier, the structure of which is shown in formula (I): (I)。 7. The composition according to claim 6, characterized in that, The composition is a powder, tablet, solution, emulsion, spray, or disinfectant.

8. The composition according to claim 6 or 7, characterized in that, In the composition, the minimum inhibitory concentration of the antimicrobial peptide mimic AMP-2f is between 0.25 and 2 μg / mL.

9. The use of the antimicrobial peptide mimic AMP-2f, or a composition containing the antimicrobial peptide mimic AMP-2f, in the preparation of a medicament, characterized in that, The drug is used to prevent or treat diseases caused by Campylobacter, and the structure of the antimicrobial peptide mimic AMP-2f is shown in formula (I): (I)。 10. The application according to claim 9, characterized in that, The diseases mentioned include bacterial enteritis.

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