Antibacterial peptide cathpc3 derived from common cormorant and application thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method derived from the common cormorant ( Phalacrocorax carbo This study focuses on the cathelicidin family antimicrobial peptide CATHPc3 and its applications, particularly its selective inhibition of Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). The amino acid sequence of the antimicrobial peptide CATHPc3 is shown in SEQ ID NO:1. Experimental results show that CATHPc3 exhibits significantly better inhibitory and bactericidal activity against Gram-positive bacteria than against Gram-negative bacteria; it also demonstrates good antimicrobial activity and rapid bactericidal effect against MRSA. Further research indicates that CATHPc3 possesses low cytotoxicity, good salt tolerance and serum tolerance, and can inhibit bacterial biofilm formation and remove mature biofilms. This antimicrobial peptide can be used to prepare antimicrobial drugs, bactericides, antimicrobial agents, animal feed additives, cosmetics, and preservatives.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method derived from the common cormorant ( Phalacrocorax carbo The cathelicidin family antimicrobial peptide CATHPc3 and its applications, especially in the selective inhibition of Gram-positive bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA). Background Technology

[0002] Antibiotics, as a crucial means of anti-infective treatment, are widely used in clinical medicine and animal husbandry. However, with the long-term, excessive, and even irrational use of antibiotics, the problem of microbial resistance has become increasingly prominent, posing a major challenge to global public health. According to the 2025 Global Antimicrobial Resistance Surveillance Report, antimicrobial resistance continues to threaten the effectiveness of existing treatments. In particular, multidrug-resistant Gram-positive bacteria, represented by methicillin-resistant Staphylococcus aureus (MRSA), can cause various serious infections such as skin and soft tissue infections, pneumonia, and bacteremia, limiting clinical treatment options and seriously threatening human health and public health security. Related studies indicate that MRSA remains one of the most important drug-resistant pathogens globally.

[0003] Gram-positive and Gram-negative bacteria differ significantly in their cell surface structures. Gram-positive bacteria lack an outer membrane; their cell walls are primarily composed of thick peptidoglycan and abundant teichoic acid, exhibiting pronounced anionic characteristics on their cell surface. Related data and research progress indicate that this structure is closely related to bacterial virulence, drug resistance, and biofilm formation, and also provides a structural basis for the preferential electrostatic interaction between cationic antimicrobial peptides and these peptides. Therefore, developing antimicrobial molecules that preferentially act on Gram-positive bacteria, especially exhibiting high activity against MRSA, is of great significance.

[0004] Antimicrobial peptides are an important component of an organism's innate immune system, typically killing pathogens by disrupting cell membrane integrity, interfering with cell wall formation, or acting on intracellular targets. Cathelicidin family antimicrobial peptides are a class of host defense peptides widely distributed in vertebrates. Studies on avian cathelicidin have shown that members of this family have been identified in various avian species, but most reports indicate broad-spectrum antimicrobial activity against both Gram-negative and Gram-positive bacteria, or a preference for Gram-negative bacteria. There are no clear reports of avian-derived antimicrobial peptides with selective Gram-positive bacteria activity. Therefore, research on avian-derived antimicrobial peptides with a preference for Gram-positive bacteria has significant scientific importance and application prospects. Summary of the Invention

[0005] This invention addresses the problems of insufficient targeting of existing antimicrobial peptides against drug-resistant Gram-positive bacteria, lack of significant activity against methicillin-resistant Staphylococcus aureus (MRSA), or limited application stability. It provides a peptide derived from common cormorants (… Phalacrocorax carbo The cathelicidin family antimicrobial peptide CATHPc3 and its applications, especially in the selective inhibition of Gram-positive bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA).

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An antimicrobial peptide CATHPc3 derived from common cormorants, wherein CATHPc3 is a mature peptide derived from the C-terminus of the common cormorant cathelicidin precursor protein; wherein the amino acid sequence is shown in SEQ ID NO:1.

[0007] The antimicrobial peptide consists of 27 amino acid residues, has a molecular weight of 3069.79 Da, an isoelectric point of 12.50, and exhibits typical structural characteristics of a cationic antimicrobial peptide. Its amino acid sequence is shown in SEQ ID NO:1. The amino acid sequence of CATHPc3 is as follows: K 1 -R 2 -F 3 -W 4 -P 5 -L 6 -L 7 -R 8 -V 9 -A 10 -I 11 -K 12 -T 13 -V 14 -A 15 -T 16 -A 17 -V 18 -N 19 -I 20 -F 21 -K 22 -A 23 -I 24 -K 25 -G 26 -K 27 ; The nucleotide sequence encoding the antimicrobial peptide CATHPc3 is shown in SEQ ID NO:2.

[0008] The nucleotide sequence encoding the CATHPc3 precursor is shown in SEQ ID NO:2, wherein nucleotides 760-840 constitute the coding region of the mature CATHPc3 peptide. The nucleotide sequence of the CATHPc3 precursor is as follows: atg tcc ctg caa cac tcc tgg tgt cat cct gcg ggt ccc tgc gcc gcc ctg tcc ctc cct 60 gcg ccc cgc cgg gga gcg ggg aca gtc ccc ggg ggc tca cgg tcc cac aga cac ccc caa 120 ccc cag acg agg ggc tcc gag ggc tgg gga cac ccc acg agc ggg gcc gtg tcc ccg agg 180 ggg acc aga cgt gct acc gcc tgc ctg tgg gac acg ctc ggg acc ccc caa ccc cgt gtc 240 tcc ccc cca aaa aac tgc cac tct gtg ggc tgg ggc cag gca ttt ccg ggg ggg ggc agc 300 cga gcc ccg ggg cgg gtt ccc caa cct cct ggg ggg gat aaa agg ggc cgc ggg ggg tgg 360 cgg tgg ggg aca agg atg ccg agc tcc tgg gtg ctg gtg ctg gcg gtg ctg ggg ggg gcc 420 tgt gcc ctc ccc gcc ccg gcc ccc ctc gcc tac acc cag gcg ctg gcc cag gcc gtc gac 480 tcc ttc aac cag cgc ccc gag gtg cag aac gtc ttc agg ctg ctc agc gcc gac ccc gag 540 ccc aac ccg gac gtc cag ctc agc tcc ctg cag cgc ctc aac ttc acc atc atg gag aca 600 cga tgc tcg gtg cgc tcc ggc gcc cgc ccc gac agc tgc gag ttc aag gat gac ggg gtc 660 atc aag gac tgc tcg gcg ccc gtg ccc gtg ccc cag cgc ggc ggc cac ccc gcg ctc gac 720 atc acc tgc gtg gat tcc acc gtg gat ccc gtc cgt gtc aag cgc ttc tgg ccg ctg ctg 780 agg gtg gcc atc aag acg gtg gcc acc gcc gtc aac atc ttt aag gcc atc aag ggg aag 840 tga gac tcc ccc cct cct cga ggt gtt gcc atc tca ggg atc ccc cct atg aca ccc acc 900 tcc cca aat aa 911 The application of the common cormorant-derived antimicrobial peptide CATHPc3, specifically its use in the preparation of a medicament for inhibiting and / or killing Gram-positive bacteria.

[0009] The Gram-positive bacteria are positive bacteria or positive-tolerant bacteria; among them, the positive bacteria are one or more of Enterococcus faecalis, Bacillus cereus, and Staphylococcus aureus.

[0010] The positive resistant bacteria were methicillin-resistant Staphylococcus aureus.

[0011] The application of the common cormorant-derived antimicrobial peptide CATHPc3, specifically its use in the preparation of a medicament for inhibiting and / or disrupting bacterial biofilms.

[0012] The bacterial biofilm is a Staphylococcus aureus biofilm and / or methicillin-resistant Staphylococcus aureus biofilm.

[0013] The application of the antimicrobial peptide CATHPc3 derived from common cormorants in the preparation of antimicrobial agents, animal feed additives, cosmetics, or preservative products.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides for the first time the cathelicidin family antimicrobial peptide CATHPc3 derived from common cormorants, enriching the resources of poultry-derived natural antimicrobial peptides; (2) The antimicrobial peptide CATHPc3 of the present invention has a clear structure, a clear amino acid sequence, a small molecular weight, and a high isoelectric point. It has typical characteristics of cationic antimicrobial peptides, which is conducive to interaction with negatively charged bacterial surface components.

[0015] (3) Experimental results show that the antimicrobial peptide CATHPc3 of the present invention has a significantly better inhibitory and killing effect on Gram-positive bacteria than on Gram-negative bacteria, showing a selective preference for Gram-positive bacteria, among which it has better antimicrobial activity and killing effect on MRSA. (4) The antimicrobial peptide CATHPc3 of the present invention has antibacterial biofilm activity, which can inhibit biofilm formation and / or destroy existing biofilms, and is especially suitable for the prevention and treatment of MRSA-related biofilm infections; (5) The antimicrobial peptide CATHPc3 of the present invention has low cytotoxicity and good salt tolerance and serum tolerance, suggesting that it has good application potential in complex physiological environments. (6) Based on its antibacterial activity and physicochemical and biological properties, CATHPc3 can be applied to pharmaceuticals, bactericides, antimicrobial agents, animal feed, cosmetics and preservation. Attached Figure Description

[0016] Figure 1 The graph shows the detection results of the geometric mean (GM value) of the minimum inhibitory concentration (MIC) of antimicrobial peptides CATHPc2 and CATHPc3 against Gram-positive and Gram-negative bacteria.

[0017] Figure 2 The figure shows the detection results of the bactericidal kinetics of the antimicrobial peptide CATHPc3 against methicillin-resistant Staphylococcus aureus (MRSA) USA300.

[0018] Figure 3 The image shows the results of detecting the cytotoxicity of the antimicrobial peptide CATHPc3 against HepG2 cells.

[0019] Figure 4 The image shows the detection results of the antimicrobial peptide CATHPc3 on HaCaT cell toxicity.

[0020] Figure 5The image shows the results of detecting the cytotoxicity of the antimicrobial peptide CATHPc3 on RAW264.7 cells.

[0021] Figure 6 The figure shows the detection results of the ability of the antimicrobial peptide CATHPc3 to remove mature biofilms of methicillin-resistant Staphylococcus aureus (MRSA) USA300.

[0022] Figure 7 The figure shows the results of detecting the inhibitory effect of the antimicrobial peptide CATHPc3 on biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) USA300.

[0023] Figure 8 The figure shows the results of detecting the effect of the antimicrobial peptide CATHPc3 on the minimum inhibitory concentration (MIC) of methicillin-resistant Staphylococcus aureus MRSAUSA300 under different salt ion conditions.

[0024] Figure 9 The figure shows the results of detecting the effect of the antimicrobial peptide CATHPc3 on the minimum inhibitory concentration (MIC) of methicillin-resistant Staphylococcus aureus (MRSA) USA300 under different mouse serum concentrations.

[0025] Figure 10 The figure shows the results of detecting the effect of the antimicrobial peptide CATHPc3 on the cell membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) USA300.

[0026] Figure 11 The figure shows the results of detecting the effect of the antimicrobial peptide CATHPc3 on the cell membrane permeability of methicillin-resistant Staphylococcus aureus (MRSA) USA300.

[0027] Figure 12 The figure shows the detection results of the effect of different membrane lipid components on the activity of the antimicrobial peptide CATHPc3 against MRSA USA300.

[0028] Figure 13 The figure shows the results of detecting the effect of different concentrations of lipopolysaccharide on the activity of the antimicrobial peptide CATHPc3 against MRSA USA300. Detailed Implementation

[0029] The following examples provide a more detailed description of specific embodiments of the present invention. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Example 1 Discovery of antimicrobial peptides CATHPc2 and CATHPc3 from common cormorants: cormorant( Phalacrocorax carboCormorants are a group of waterbirds that widely inhabit freshwater, estuarine, and near-shore environments, constantly exposed to a complex array of environmental microorganisms and potential pathogens. Compared to poultry and common model organisms, research reports on innate immune molecules in cormorants, especially cathelicidin family antimicrobial peptides, are relatively scarce, and systematic studies on cathelicidin antimicrobial peptides from common cormorants are lacking. Therefore, common cormorants can serve as an important source for screening novel antimicrobial peptides. Cathelicidin family precursor proteins typically consist of an N-terminal signal peptide, a cathelin-like conserved domain, and a C-terminal mature peptide region. The cathelin-like domain exhibits high conservation across different species, while the mature peptide region shows high variability and determines its main biological activity. Therefore, the conservation of the cathelin-like domain can be utilized to screen for new cathelicidin molecules through sequence similarity searches.

[0031] In this embodiment, the original pigeon ( Columba livia Using the cathelicidin precursor protein sequence (GenBank: PKK18081.1) as a query template, a search was conducted in the National Center for Biotechnology Information (NCBI) database for the common cormorant (GenBank: PKK18081.1). Phalacrocorax carbo BLAST homology search was performed. Combining the annotated cathelicidin-related sequences of the common cormorant in the NCBI database, a systematic search and bioinformatics analysis yielded two cathelicidin precursor protein sequences, named CATHPc2 precursor and CATHPc3 precursor, respectively. To determine the mature peptide region of these precursor proteins, the SignalP 6.0 online analysis tool was first used to predict their signal peptide cleavage sites. Then, MEGA 12.0 software was used to perform multiple sequence alignment analysis with cathelicidin precursor protein sequences of closely related representative bird species, and a comprehensive judgment was made based on the typical physicochemical characteristics of the mature peptide region. The analysis results showed that amino acid residues 254 to 280 at the C-terminus of the CATHPc3 precursor protein constitute a 27-amino acid candidate region of mature peptide. Amino acid residues 268 to 300 at the C-terminus of the CATHPc2 precursor protein constitute a 32-amino acid candidate region of mature peptide. The mature peptide regions of CATHPc2 and CATHPc3 are rich in basic amino acids such as lysine and arginine, and have a high positive charge and typical amphiphilic α-helix formation potential, which conforms to the general structural characteristics of mature antimicrobial peptides in the cathelicidin family. Therefore, they are named CATHPc2 and CATHPc3.

[0032] Subsequent BLAST alignment analysis of the mature peptide candidate sequences showed that the CATHPc2 and CATHPc3 mature peptide regions had high similarity to several avian cathelicidin-like sequences. However, most of the highly similar sequences obtained by BLAST search mainly originated from predicted proteins or uncharacterized proteins in genome or transcriptome sequencing projects, rather than cathelicidin antimicrobial peptides that have been isolated, purified, structurally identified, and functionally verified. Given the high sequence diversity of the mature peptide regions of the cathelicidin family and the lack of experimental functional characterization in most existing similar sequences, this invention identifies the mature peptides CATHPc2 and CATHPc3 from common cormorants as novel cathelicidin antimicrobial peptide candidates.

[0033] Example 2 Preparation and identification of antimicrobial peptides CATHPc2 and CATHPc3: (1) Preparation of antimicrobial peptides CATHPc2 and CATHPc3: The mature peptide amino acid sequences of CATHPc2 and CATHPc3 were deduced from the coding sequence of the cathelicidin precursor protein of common cormorant based on the screening of the above-mentioned antimicrobial peptides. The full sequences of CATHPc2 and CATHPc3 were chemically synthesized using an automated peptide synthesizer (433A, Applied Biosystems). The synthesized crude peptides were desalted and purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the target peaks were collected and lyophilized for later use. The above process was outsourced to the company.

[0034] (2) Identification of antimicrobial peptides CATHPc2 and CATHPc3: The molecular weight of the peptides was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF); their purity was determined by high-performance liquid chromatography (HPLC); their isoelectric point was determined by isoelectric focusing electrophoresis; and their amino acid sequences were determined by an automated amino acid sequencer. Antimicrobial peptide CATHPc3 was identified as a small molecule peptide composed of 27 amino acid residues, with its amino acid sequence shown in SEQ ID NO:1, a molecular weight of 3069.79 Da, and an isoelectric point of 12.50. Antimicrobial peptide CATHPc2 was identified as a small molecule peptide composed of 32 amino acid residues, with a molecular weight of 3767.45 Da and an isoelectric point of 12.85. The amino acid sequences of CATHPc2 and CATHPc3 are as follows: CATHPc2: Q 1 -R 2 -G 3 -R 4 -F5 -G 6 -R 7 -F 8 -L 9 -S 10 -R 11 -I 12 -R 13 -R 14 -F 15 -R 16 -P 17 -K 18 -I 19 -S 20 -I 21 -H 22 -I 23 -Q 24 -A 25 -S 26 -G 27 -S 28 -L 29 -R 30 -L 31 -G 32 ; CATHPc3: K 1 -R 2 -F 3 -W 4 -P 5 -L 6 -L 7 -R 8 -V 9 -A 10 -I 11 -K 12 -T 13 -V 14 -A 15 -T 16 -A 17 -V 18 -N 19 -I 20 -F 21 -K 22 -A 23 -I 24 -K 25 -G 26 -K 27 ; CATPC2 and CATPC3 were prepared according to the above description and identified.

[0035] Example 3 Antimicrobial activity assay of antimicrobial peptides CATHPc2 and CATHPc3: The minimum inhibitory concentrations (MICs) of antimicrobial peptides CATHPc2 and CATHPc3 against different test strains were determined using the broth microdilution method. First, each test strain listed in Table 1 was inoculated into MH liquid medium and cultured at 37°C with shaking until the logarithmic growth phase. Subsequently, the bacterial culture was diluted to 2 × 10⁻⁶ using fresh MH liquid medium. 5 CFU / mL was prepared for use. In a sterile 96-well plate, 90 μL of MH liquid medium was added to well 1, and 50 μL of MH liquid medium was added to each of the remaining wells. 10 μL of a polypeptide sample solution (2 mg / mL) prepared with sterile pure water and filtered through a 0.22 μm membrane was added to well 1. After mixing, 50 μL was added to well 2, and this was repeated serially, diluting 50 μL at well 8. Well 9 was used as a negative control. Subsequently, 50 μL of the diluted bacterial solution was added to each well, and the plates were incubated at 37°C with gentle shaking for 18 h. The absorbance was measured at 600 nm. The lowest sample concentration at which no visible bacterial growth was observed was taken as the MIC value.

[0036] The results are shown in Table 1. Figure 1 As shown in Table 2, CATHPc3 exhibited strong antibacterial activity against Gram-positive bacteria, with a MIC range of 4.68–37.5 μg / mL. It also showed some antibacterial activity against Gram-negative bacteria, but this activity was generally weak, with a MIC range of 18.75–100 μg / mL. Further calculations revealed that the geometric mean of the MIC for CATHPc3 against Gram-positive bacteria was 21.04 μg / mL, significantly lower than its geometric mean of 52.59 μg / mL against Gram-negative bacteria. CATHPc2 showed weaker antibacterial effects against both Gram-positive and Gram-negative bacteria compared to CATHPc3, with only significant bactericidal effects against Staphylococcus aureus CMCC26003 and Escherichia coli CMCC44102. Its geometric mean MIC for Gram-positive bacteria was 79.03 μg / mL, and its geometric mean MIC for Gram-negative bacteria was 80.01 μg / mL, showing no selective preference for Gram-positive bacteria. The above results indicate that CATHPc3 has strong antibacterial activity and exhibits significantly better inhibitory effects against Gram-positive bacteria than against Gram-negative bacteria, demonstrating a selective preference for Gram-positive bacteria.

[0037] Table 1. Antimicrobial activity of antimicrobial peptides CATHPc2 and CATHPc3

[0038] Table 2. Geometric mean MICs of antimicrobial peptides CATHPc2 and CATHPc3 against Gram-positive and Gram-negative bacteria.

[0039] Note: When the MIC value is greater than 100 μg / mL, it is calculated as 100 μg / mL in the geometric mean.

[0040] Example 4 Determination of the bactericidal rate of the antimicrobial peptide CATHPc3: Methicillin-resistant Staphylococcus aureus (MRSA) USA300, sensitive to CATHPc3, was selected as the test strain to determine the bactericidal kinetics of CATHPc3. MRSA USA300 was inoculated into MH liquid medium (Thermo Fisher Scientific, Inc., USA) and incubated at 37°C for 12 h, then diluted to 1×10⁻⁶ with fresh MH liquid medium. 6 A bacterial suspension was prepared at CFU / mL. CATP C3 dissolved in sterile deionized water was added to the bacterial suspension to a final concentration of 4×MIC (75 μg / mL). The treated bacterial suspension was incubated at 37°C with shaking. At 0, 15, 30, 60, 120, and 180 min, 50 μL of the suspension was taken, diluted 1000-fold, and then 50 μL of the diluted solution was spread onto the surface of LB agar medium. After incubation at 37°C overnight, the colony count was performed. Vancomycin was used as a positive control, and PBS buffer was used as a negative control.

[0041] The results are as follows Figure 2 As shown in Table 3, CATHPc3 exhibits rapid bactericidal activity against MRSA USA300, significantly reducing the number of viable bacteria within 15 minutes, with a bactericidal rate significantly faster than the positive control vancomycin. This indicates that CATHPc3 not only inhibits MRSA growth but also possesses strong rapid bactericidal ability.

[0042] Table 3. Bactericidal rate of antimicrobial peptide CATHPc3 against methicillin-resistant Staphylococcus aureus (MRSA) USA300

[0043] Example 5 Assay for the cytotoxicity of the antimicrobial peptide CATHPc3: The MTT assay was used to evaluate the in vitro cytotoxicity of CATPC3 against HepG2 cells, human immortalized keratinocytes (HaCaT) and mouse macrophages (RAW264.7). HepG2, HaCaT, and RAW264.7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum (Biological Industries, Israel) and 100 U / mL penicillin-streptomycin (Shanghai Yuanye Biotechnology Co., Ltd.), respectively. Before the formal experiments, the cells were seeded in 96-well plates at a density of 2 × 10⁶ cells / well. 4 Cells were cultured overnight at 37°C and 5% CO2. Then, different concentrations of CATHPc3 were added, and incubation continued for 24 h. After incubation, 20 μL of 5 mg / mL MTT solution was added to each well, and incubation continued for 4 h. The supernatant was discarded, and DMSO was added to dissolve formazan crystals. The absorbance was measured at 490 nm. The zero-adjustment group received only DMEM medium, and the negative control group received only cells without drugs. Cell viability was calculated using the following formula: C% = (A 样品 - A 调零 ) / (A 阴性对照 - A 调零 ) ×100%.

[0044] The results are as follows Figures 3 to 5 As shown in Table 4, CATHPc3 has different effects on the IC50 values ​​of HepG2, HaCaT, and RAW264.7 cells. 50 The concentrations were 85.4 μg / mL, 330.5 μg / mL, and 49.5 μg / mL, respectively. The results indicate that, within a dosage range comparable to its effective antibacterial concentration, CATHPc3 exhibits low overall toxicity to mammalian cells, particularly low toxicity to HaCaT cells, demonstrating good safety and application potential.

[0045] Table 4. Cytotoxicity of the antimicrobial peptide CATHPc3

[0046] Example 6 Antimicrobial peptide CATHPc3's anti-biofilm activity: (1) The ability of the antimicrobial peptide CATHPc3 to clear mature biofilms. The ability of CATHPc3 to clear mature biofilms of methicillin-resistant Staphylococcus aureus (MRSA) USA300 was evaluated by crystal violet staining. MRSA USA300 was inoculated into MH liquid medium and cultured at 37°C for 12 h, then diluted to 1×10⁻⁶ with fresh medium. 7CFU / mL. Add 100 μL of bacterial suspension to each well of a 96-well plate and incubate at 37℃ for 48 h to form a mature biofilm. After discarding the supernatant, wash three times with PBS, add 100 μL of CATHPc3 at different concentration gradients, and continue incubation for 24 h. After incubation, discard the supernatant, wash with PBS, add 50 μL of methanol for fixation, air dry, and then stain with 0.1% crystal violet. After discarding the staining solution, dissolve the crystal violet in anhydrous ethanol and measure the absorbance at 600 nm. The zero-adjustment group only added culture medium, and the negative control group only added bacterial suspension without drugs. The biofilm retention rate was calculated using the following formula: B% = (A 样品 - A 调零 ) / (A 阴性对照 - A 调零 ) × 100%.

[0047] The results are as follows Figure 6 As shown in Table 5, when the CATHPc3 concentration was 1×MIC, the retention rate of mature MRSA USA300 biofilm was approximately 57.22%; with increasing concentration, its scavenging effect further increased. This indicates that CATHPc3 has a good scavenging ability for mature biofilms.

[0048] Table 5. Scavenging ability of antimicrobial peptide CATHPc3 on mature biofilms

[0049] (2) Inhibitory effect of antimicrobial peptide CATHPc3 on biofilm formation. The inhibitory effect of CATHPc3 on biofilm formation of MRSA USA300 was evaluated by crystal violet staining. MRSA USA300 was inoculated into MH liquid medium and cultured at 37℃ for 12 h, then diluted with fresh medium to 2×10⁻⁶. 7 CFU / mL. Add 50 μL of bacterial suspension to each well of a 96-well plate, along with 50 μL of CATHPc3 at different concentration gradients, and co-incubate at 37℃ for 48 h. The remaining steps are the same as the mature biofilm removal experiment. The zero-conditioning group only added culture medium, and the negative control group only added bacterial suspension without any drugs. Biofilm formation rate was calculated using the following formula: B% = (A... 样品 - A 调零 ) / (A 阴性对照 - A 调零 ) × 100%.

[0050] The results are as follows Figure 7 As shown in Table 6, CATHPc3 significantly inhibited biofilm formation in MRSA USA300, with a biofilm formation rate of approximately 43.5% at a concentration of 8×MIC. This indicates that CATHPc3 has promising application prospects in the prevention and control of biofilm-related infections.

[0051] Table 6. Inhibitory effect of antimicrobial peptide CATHPc3 on biofilm formation

[0052] Example 7 Tolerance assay for antimicrobial peptide CATHPc3: (1) Determination of antibacterial activity under salt ion conditions. MRSA USA300 was inoculated into MH liquid medium and cultured at 37℃ for 12 h. Then, the bacterial culture was diluted to 2×10⁻⁶ with fresh MH liquid medium containing 150 mM NaCl, 4.5 mM KCl, 6 μM NH₄Cl, 1 mM MgCl₂, 2.5 mM CaCl₂ and 8 μM ZnCl₂, respectively. 5 CFU / mL. CATHPc3 sample solutions were then prepared using MH liquid medium containing the corresponding salt ions. The MIC values ​​of CATHPc3 under different salt ion conditions were determined using the two-fold dilution method described in Example 3. The antibacterial activity was observed after incubation at 37°C for 16–18 h.

[0053] The results are as follows Figure 8 As shown in Table 7, in Na + K + NH 4+ and Zn 2+ Under the given conditions, the MIC of CATHPc3 remains essentially unchanged; Mg 2+ and Ca 2+ This can increase its MIC to 37.5 μg / mL. These results indicate that CATHPc3 maintains good antibacterial activity under most common salt ion environments and exhibits a certain degree of salt tolerance.

[0054] Table 7. MIC of antimicrobial peptide CATHPc3 against MRSA USA300 under different salt ion conditions

[0055] (2) Antibacterial activity assay under serum conditions. MRSA USA300 was inoculated into MH liquid medium and cultured at 37°C for 12 h. Then it was mixed with mouse serum (Shanghai Yuanye Biotechnology Co., Ltd.) to make the final concentrations of mouse serum 0%, 5%, 10% and 20%. The MIC values ​​of CATHPc3 under different serum concentrations were determined according to the method described in Example 3. The antibacterial effect was observed after incubation at 37°C for 16-18 h.

[0056] The results are as follows Figure 9As shown in Table 8, the MIC of CATHPc3 did not change significantly under conditions containing 5% and 10% mouse serum, but increased to 37.5 μg / mL under conditions containing 20% ​​mouse serum. This indicates that CATHPc3 can maintain good antibacterial activity under certain serum conditions and has good serum tolerance.

[0057] Table 8. MIC of antimicrobial peptide CATHPc3 against MRSA USA300 under different concentrations of mouse serum.

[0058] Example 8 Study on the antibacterial mechanism of antimicrobial peptide CATHPc3: (1) Effect of antimicrobial peptide CATHPc3 on bacterial cell membrane integrity. The effect of CATHPc3 on the cell membrane integrity of MRSA USA300 was detected by propidium iodide (PI) uptake assay. PI cannot penetrate intact cell membranes, but can only enter damaged cells and bind to nucleic acids to produce fluorescence. Therefore, its fluorescence intensity can reflect the degree of bacterial cell membrane damage. MRSA USA300 was inoculated into MH liquid medium and cultured at 37℃ for 12 h, then diluted with fresh medium to 1×10⁻⁶. 7 CFU / mL. PI probe (Sigma-Aldrich, USA) was added to the bacterial suspension to a final concentration of 10 μM, and incubated in the dark for 30 min. In a 96-well plate, 50 μL of CATPc3 solution at different concentration gradients and 50 μL of PI-treated bacterial suspension were added to each well. After incubation at 37°C for 60 min, fluorescence intensity was detected using a microplate reader at an excitation wavelength of 535 nm and an emission wavelength of 617 nm. The PBS group served as a negative control.

[0059] The results are as follows Figure 10 As shown in Table 9, the bacterial uptake of PI increased significantly with increasing CATHPc3 concentration, exhibiting a clear concentration-dependent effect. This indicates that CATHPc3 can disrupt the cell membrane integrity of MRSA USA300, thereby increasing cell membrane permeability.

[0060] Table 9. Effects of the antimicrobial peptide CATHPc3 on the cell membrane integrity of MRSA USA300 cells.

[0061] (2) Effect of antimicrobial peptide CATHPc3 on the uptake of hydrophobic probes on membrane surfaces. The changes in the interaction between bacteria and hydrophobic probes after CATHPc3 treatment were evaluated using an N-phenyl-1-naphthylamine (NPN) probe uptake assay. NPN is a hydrophobic fluorescent probe; its fluorescence enhancement can reflect changes in cell surface barrier properties or membrane environment. MRSA USA300 was inoculated into MH liquid medium and cultured at 37℃ for 12 h, then diluted to 1×10⁻⁶ with fresh medium. 8 CFU / mL. In a 96-well plate, add 80 μL of bacterial suspension to each well, along with 10 μL of CATPc3 solutions prepared with different concentration gradients in PBS and 10 μL of 100 μM NPN solution. Incubate at 37℃ for 60 min, then detect fluorescence intensity using a microplate reader at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The PBS group served as a negative control, while polymyxin B, LL-37, and Hc-CATH prepared with PBS served as positive controls.

[0062] The results are as follows Figure 11 As shown in Table 10, compared with LL-37, Hc-CATH, and polymyxin B, the NPN fluorescence enhancement in the CATHPc3 treatment group was smaller, suggesting that its impact on the membrane surface barrier structure sensitive to NPN probes is limited. Combined with the difference in antibacterial activity of CATHPc3 against Gram-positive and Gram-negative bacteria in Example 3, and the results of the lipid competition experiment in Example 9, it is suggested that CATHPc3 may be more inclined to interact with membrane lipid components related to the cell surface of Gram-positive bacteria, thus exhibiting a selective preference for Gram-positive bacteria.

[0063] Table 10. Effect of antimicrobial peptide CATHPc3 on NPN uptake

[0064] Example 9 Research on the binding ability of antimicrobial peptide CATHPc3 to membrane lipid components: The interaction between CATHPc3 and different membrane lipid components was analyzed using an exogenous lipid competition assay. The basic principle is that if a specific exogenously added lipid can bind to an antimicrobial peptide, it will competitively weaken the binding of the antimicrobial peptide to the bacterial surface, thereby leading to an increase in its MIC (micromicrobial MIC). MRSA USA300 was inoculated into MH liquid medium and cultured at 37°C for 12 h, then diluted with fresh medium to a concentration of 2.5 × 10⁻⁶. 5CFU / mL. In a sterile 96-well plate, 90 μL of MH liquid medium was added to well 1, and 50 μL of MH liquid medium was added to each of the remaining wells. 10 μL of CATHPc3 sample solution (2 mg / mL) prepared with sterile pure water and filtered through a 0.22 μm membrane was added to well 1, and 50 μL was added to well 2. This was repeated serially, with 50 μL diluted to well 8 and then discarded. Well 9 served as a control. Subsequently, 40 μL of diluted bacterial solution and 10 μL of lipid solutions of different concentrations were added to each well. The lipids included cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and lipopolysaccharide (LPS). The 96-well plate was placed in a 37°C incubator and slowly shaken for 18 h. The absorbance was measured at a wavelength of 600 nm. The lowest sample concentration at which no bacterial growth was observed to the naked eye was taken as the MIC value.

[0065] The results are as follows Figure 12 , Figure 13 As shown in Tables 11 and 12, the MIC of CATHPc3 against MRSA USA300 increased more significantly after the exogenous addition of cardiolipin and phosphatidylglycerol, suggesting a strong interaction between CATHPc3 and cardiolipin and phosphatidylglycerol. In contrast, phosphatidylethanolamine, phosphatidylcholine, and lipopolysaccharide had relatively weaker effects on the antibacterial activity of CATHPc3. These results indicate that CATHPc3 tends to interact more with anionic lipid components commonly found on the membrane surface of Gram-positive bacteria, while its interaction with lipopolysaccharide, a characteristic component of the outer membrane of Gram-negative bacteria, is weaker. This may be one of the important molecular bases for its selective preference for Gram-positive bacteria.

[0066] Table 11 MIC of antimicrobial peptide CATPc3 against MRSA USA300 under different phospholipid concentrations

[0067] Table 12 MIC of antimicrobial peptide CATPc3 against MRSA USA300 under different concentrations of lipopolysaccharide.

[0068] The embodiments described above are merely illustrative of preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Equivalent substitutions, modifications, or variations made by those skilled in the art without departing from the spirit and principles of the present invention should fall within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A common cormorant-derived antimicrobial peptide, CATHPc3, characterized in that, The antimicrobial peptide CATHPc3 is a mature peptide derived from the C-terminus of the common cormorant cathelicidin precursor protein; the amino acid sequence is shown in SEQ ID NO:

1.

2. The antimicrobial peptide CATHPc3 derived from common cormorants according to claim 1, characterized in that, The nucleotide sequence encoding the antimicrobial peptide CATHPc3 is shown in SEQ ID NO:

2.

3. The application of the antimicrobial peptide CATHPc3 derived from common cormorants as described in claim 1, characterized in that: The use of the common cormorant-derived antimicrobial peptide CATHPc3 in the preparation of drugs for inhibiting and / or killing Gram-positive bacteria.

4. The application according to claim 3, characterized in that, The Gram-positive bacteria are positive bacteria or positive-tolerant bacteria; among them, the positive bacteria are one or more of Enterococcus faecalis, Bacillus cereus, and Staphylococcus aureus.

5. The application according to claim 4, characterized in that, The positive resistant bacteria were methicillin-resistant Staphylococcus aureus.

6. The application of the antimicrobial peptide CATHPc3 derived from common cormorants as described in claim 1, characterized in that: The use of the common cormorant-derived antimicrobial peptide CATHPc3 in the preparation of drugs for inhibiting and / or disrupting bacterial biofilms.

7. The application according to claim 6, characterized in that, The bacterial biofilm is a Staphylococcus aureus biofilm and / or methicillin-resistant Staphylococcus aureus biofilm.

8. The application according to claim 1, characterized in that, The application of the antimicrobial peptide CATHPc3 derived from common cormorants in the preparation of antimicrobial agents, animal feed additives, cosmetics, or preservative products.

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