Novel antibacterial peptide CR24, screening synthesis method and antibacterial application

Abstract

The invention discloses screening synthesis and antibacterial application of a novel antibacterial peptide CR24. According to the present invention, the screened novel antibacterial peptide CR24 is excavated from the whole genome of the sheep intestinal symbiotic bacterium Bacillus enteritidis B1 (Bacillus haynesii strain B1), and the novel antibacterial peptide CR24 is obtained through artificial synthesis; experiments show that the antibacterial peptide CR24 is a novel antibacterial peptide, has extremely low cytotoxicity and hemolytic activity, does not generate drug resistance, and is excellent in safety. The antibacterial peptide CR24 has broad-spectrum antibacterial activity on various pathogenic bacteria, can exert bactericidal activity by damaging a staphylococcus aureus cell membrane, and can promote skin wound healing caused by staphylococcus aureus infection when applied in vivo. In a word, the antibacterial peptide CR24 provided by the invention is a novel antibacterial peptide which is small in molecular weight, easy to artificially synthesize, high in antibacterial activity, safe and non-toxic, can be used for preparing feed additives, anti-infection drugs, food preservatives and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a novel antimicrobial peptide CR24, a screening and synthesis method and an antimicrobial application. Background Art

[0002] The misuse of antibiotics leads to high-level drug resistance in pathogenic microorganisms and the formation of multidrug resistance. In recent years, "super pathogens" with multidrug resistance have broken out one after another around the world, seriously threatening the life and health safety of humans and livestock. Although antibiotics are traditionally the main drug intervention for bacterial infections, the formation of multidrug-resistant bacteria has seriously hindered the effectiveness of antibiotic treatment. More and more infectious diseases have become difficult to treat, and sometimes even untreatable. However, there are very few chemically synthesized antibiotics under development at this stage, and most of them are derivatives of current antibiotics. Therefore, there is an urgent need to seek new antibacterial drugs from new sources.

[0003] Antimicrobial proteins or peptides have unique structural sequences, mechanisms of action, and highly effective bactericidal effects. They also have little cross-resistance with traditional antibiotics, and are therefore the best alternatives to antibiotics. Antimicrobial peptides are a class of low-molecular-weight polypeptides with antimicrobial activity, with a molecular weight usually around 2000-7000 and containing 20-60 amino acids. Antimicrobial peptides, as an important component of the natural immune defense system of organisms, are a class of active biological molecules that have endogenous immune responses in most organisms. To date, thousands of antimicrobial peptides with different structures have been identified. Because the antibacterial and bactericidal mechanisms of antimicrobial peptides are different from those of antibiotics and they have multiple antimicrobial mechanisms, they are safe and efficient when used as drug treatments, and the probability of pathogens developing resistance to them is very low. They have broad application prospects and have become a new generation of antibiotic alternatives.

[0004] At present, the vast majority of antimicrobial peptides come from mammals and insects. In addition, a variety of antimicrobial peptides have also been identified in amphibians. The intestines of livestock and poultry are colonized by hundreds of trillions of microorganisms. These intestinal symbiotic flora have opened up new avenues for the search for new antimicrobial peptides, and a variety of new antimicrobial substances have been discovered. Therefore, mining new antimicrobial peptides from intestinal symbiotic bacteria in livestock and poultry will provide a new solution to the problem of antibiotic resistance and the development of new antimicrobial drugs. Summary of the invention

[0005] One of the purposes of the present invention is to provide a novel antimicrobial peptide CR24, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0006] The second object of the present invention is to provide a method for screening and synthesizing an antimicrobial peptide CR24. The antimicrobial peptide CR24 is obtained by mining the whole genome of the sheep intestinal symbiotic bacteria - Bacillus haynesii strain B1, and then synthesized by solid phase synthesis and purified by HPLC.

[0007] Application of a novel antimicrobial peptide CR24 in antagonizing pathogenic bacteria, wherein the pathogenic bacteria include Staphylococcus aureus, Listeria monocytogenes, Enterococcus, Salmonella, Escherichia coli, and Pseudomonas aeruginosa.

[0008] Application of the novel antimicrobial peptide CR24 in promoting wound healing caused by Staphylococcus aureus infection.

[0009] The novel antimicrobial peptide CR24 provided by the present invention has a broad-spectrum antimicrobial activity, and the multiple pathogens involved include Staphylococcus aureus, Listeria monocytogenes, Enterococcus, Salmonella, Escherichia coli, Pseudomonas aeruginosa, etc.

[0010] The antimicrobial peptide CR24 provided by the invention has extremely low cytotoxicity and hemolytic activity, does not produce drug resistance and has good safety.

[0011] The antimicrobial peptide CR24 provided by the present invention can exert a bactericidal effect by destroying bacterial cell membranes.

[0012] The antimicrobial peptide CR24 provided by the present invention has the ability to promote the healing of wounds infected by Staphylococcus aureus.

[0013] Beneficial effects:

[0014] The present invention provides a novel antimicrobial peptide CR24. The antimicrobial peptide has the characteristics of small molecular weight, simple structure, non-cytotoxicity, non-hemolytic, etc. It has high-efficiency and broad-spectrum antimicrobial activity, can kill a variety of common clinical Gram-positive and Gram-negative pathogens, is not easy to develop drug resistance, and has the ability to promote the healing of wounds infected with Staphylococcus aureus when used in vivo. Therefore, the antimicrobial peptide CR24 has excellent characteristics, is cheap to prepare, and is easy to produce. It can be used to prepare feed additives, anti-infective drugs, food preservatives, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The purity and molecular weight identification results of the antimicrobial peptide CR24 after synthesis of the present invention are as follows: (A) HPLC purity identification of the antimicrobial peptide CR24 after synthesis; (B) ESI-MS identification of the antimicrobial peptide CR24 after synthesis;

[0016] Figure 2The antibacterial effect and bactericidal curve of the antimicrobial peptide CR24 of the present invention on Staphylococcus aureus (A) Antibacterial effect of the antimicrobial peptide CR24 on Staphylococcus aureus; (B) Visual changes of the antimicrobial peptide CR24 against Staphylococcus aureus after 24 hours; (C) Bactericidal curve of the antimicrobial peptide CR24 against Staphylococcus aureus;

[0017] Figure 3 The hemolytic activity of the antimicrobial peptide CR24 of the present invention on mouse and sheep erythrocytes (A) the hemolytic activity of the antimicrobial peptide CR24 on mouse erythrocytes; (B) the hemolytic activity of the antimicrobial peptide CR24 on sheep erythrocytes;

[0018] Figure 4 The cytotoxic effect of the antimicrobial peptide CR24 of the present invention on RAW264.7 and L929 cells (A) The cytotoxic effect of the antimicrobial peptide CR24 on RAW264.7 cells; (B) The cytotoxic effect of the antimicrobial peptide CR24 on L929 cells;

[0019] Figure 5 The induced resistance of the antimicrobial peptide CR24 of the present invention;

[0020] Figure 6 The bactericidal mechanism of the antimicrobial peptide CR24 against Staphylococcus aureus of the present invention (A) The effect of the antimicrobial peptide CR24 on the leakage of intracellular nucleic acid of Staphylococcus aureus; (B) The effect of the antimicrobial peptide CR24 on the leakage of intracellular protein of Staphylococcus aureus; (C) The effect of the antimicrobial peptide CR24 on the permeability of the cell membrane of Staphylococcus aureus; (D) The effect of the antimicrobial peptide CR24 on the integrity of the cell membrane of Staphylococcus aureus observed by scanning electron microscopy;

[0021] Figure 7 The therapeutic effect of the antimicrobial peptide CR24 of the present invention on Staphylococcus aureus skin lesions (A) Mouse processing and treatment process; (B) Changes in mouse wounds after antimicrobial peptide CR24 treatment; (C) Changes in skin wound area at different time points (day 0, day 4 and day 7) after antimicrobial peptide CR24 treatment; (D) Staphylococcus aureus load in mouse skin wounds after antimicrobial peptide CR24 treatment; (E) H&E staining and Masson staining of skin wound tissue 7 days after treatment. DETAILED DESCRIPTION

[0022] The present invention is further illustrated by some specific embodiments below. It should be clear that the embodiments described below are only part of the embodiments of the present invention, rather than all the embodiments, but the present invention is not limited to the scope of the embodiments.

[0023] Example 1 Screening and synthesis of antimicrobial peptide CR24

[0024] Screening of antimicrobial peptide CR24

[0025] Bacillus heinei B1 from sheep intestine frozen at -80℃ was streaked on TSB plates, cultured overnight at 37℃ until a single colony was formed, and then a single colony was picked into 3ml TSB liquid culture medium, cultured at 37℃, 200rpm shaking for 12h, and the bacteria were collected and washed twice with PBS. Then, according to the instructions of the bacterial genome extraction kit, the genomic DNA of Bacillus heinei B1 was extracted, and then sent to the company to determine the whole genome sequence of Bacillus heinei B1. Subsequently, the open reading frames (ORFs) less than 100bp in the whole genome of Bacillus heinei B1 were selected, and the antimicrobial peptide sequences were predicted using online software such as APD3, CAMPR3 and Ex PASy, and their basic properties, including helical structure or disulfide bond, positive net charge, protein binding potential>0kcal mol and peptide formation rate>0.9, were evaluated, and the secondary structure model of the polypeptide was predicted using I-TASSER. After comprehensive screening, CR24 was finally selected for synthesis.

[0026] Bacillus haynesii strain B1 is deposited in China Center for Type Culture Collection (CCTCC) on October 14, 2024, with a deposit number of CCTCC NO: M 20242179, and the deposit address is Wuhan University, Wuhan, China. The classification name is Bacillus haynesii strain B1, and the 16S rDNA sequence of Bacillus haynesii strain B1 is shown in SEQ ID NO.3.

[0027] Synthesis of antimicrobial peptide CR24

[0028] The screened antimicrobial peptide CR24, whose amino acid sequence is shown in SEQ ID NO.1, was sent to Nanjing Peptide Industry Biotechnology Co., Ltd. for solid phase synthesis based on Fmoc. After synthesis, the purity and retention time of the peptide were measured by reverse phase high performance liquid chromatography (RP-HPLC), and the molecular weight of the peptide was determined by high resolution mass spectrometry (ESI-MS). According to HPLC analysis, the purity of the peptide CR24 was greater than 95%, and ESI-MS analysis showed that its actual molecular weight was basically consistent with the measured molecular weight ( Figure 1 ).

[0029] Example 2 Antimicrobial spectrum of antimicrobial peptide CR24

[0030] The pathogenic strains to be tested, including Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Escherichia coli, Salmonella, Pseudomonas aeruginosa and Klebsiella pneumoniae, were inoculated in TSB and cultured overnight at 37°C in a shaker. The cells were collected and washed twice with PBS, and then the bacterial solution was diluted to 10 9CFU / mL, set aside. Dilute the antimicrobial peptide CR24 with distilled water to a concentration of 128μM, 64μM, 32μM, 16μM, 8μM, 4μM, and 2μM. In a sterile 96-well plate, take 50μL of each concentration of antimicrobial peptide CR24 and add it to the plate, and add 50μL of PBS to make the final concentration of the antimicrobial peptide 64μM, 32μM, 16μM, 8μM, 4μM, 2μM, and 1μM, and set up negative and positive controls. Finally, add the diluted bacterial solution to the wells containing different concentrations of antimicrobial peptides to make the final concentration of bacteria 5×10 5 CFU / mL, cover the plate, incubate in a 37°C incubator for 16 hours, and finally take 10 μL of solution from each well and drop it on the TSA plate, mark it, and incubate it in a 37°C incubator for 12 hours to observe whether there is colony growth. The lowest antibacterial substance concentration that completely inhibits bacterial growth is MIC. The experimental results are shown in Table 1. For all tested Gram-negative and Gram-positive pathogens, the antimicrobial peptide CR24 showed good antibacterial activity, with a MIC range of 2-64 μM.

[0031] Table 1 Antimicrobial spectrum of antimicrobial peptide CR24

[0032]

[0033] Example 3 Antibacterial effect and bactericidal curve of antimicrobial peptide CR24 against Staphylococcus aureus

[0034] Staphylococcus aureus USA300 was cultured in TSB medium at 37°C and 200 r / min until the logarithmic growth phase, washed twice with PBS, and the bacterial solution concentration was adjusted to 1×10 5 CFU / mL. Then antimicrobial peptide CR24 was added to the culture medium, and the final concentration of antimicrobial peptide CR24 was 1×MIC and 2×MIC. Culture was carried out at 37°C and 200r / min. During the culture period, 100μL was taken for 10-fold serial dilution every 0, 0.5, 1, 2, 4, 6 and 8 hours, and then 100μL of the dilution was spread on the TSA plate and cultured overnight at 37°C. The total number of bacteria in each milliliter of culture medium was calculated. The results showed that antimicrobial peptide CR24 had an effective antimicrobial effect on Staphylococcus aureus USA300( Figure 2 AB) has strong bactericidal activity. The results of time-bactericidal kinetic experiments showed that compared with the control group, the number of Staphylococcus aureus USA300 in the 2×MIC concentration treatment group was significantly reduced, and no bacteria were detected after 6 hours of incubation ( Figure 2 C). These results indicate that the antimicrobial peptide CR24 has bactericidal activity against Staphylococcus aureus and is able to kill the bacteria in a short period of time.

[0035] Example 4 Hemolytic activity of antimicrobial peptide CR24 on mouse and sheep erythrocytes

[0036] Collect fresh blood from sheep and mice and inject it into the anticoagulation tube. Slowly inject the anticoagulated blood into the centrifuge tube and centrifuge at 1500r / min for 5min to separate the anticoagulant and plasma. After the centrifugation, aspirate the upper liquid and white blood cell layer, add an appropriate amount of PBS buffer, balance and centrifuge for the second time, centrifuge at 1500r / min for 5min to wash the red blood cells. After the second centrifugation, aspirate the upper liquid and white blood cell layer, add PBS buffer, centrifuge for the third time, centrifuge at 2000r / min for 10min to further wash the red blood cells. After the centrifugation, aspirate the upper clear liquid for the last time, add an appropriate amount of PBS buffer to the precipitated red blood cells, prepare a 2% red blood cell suspension, mix well, store in a 4℃ refrigerator for later use, and mix well when needed.

[0037] Take the 2% red blood cell suspension obtained by the above separation, then take 50 μL and add it to the wells of a 96-well plate, dilute the antimicrobial peptide CR24 twice with PBS, then take 50 μL and add it to the wells, so that the final concentrations are 200, 100, 50, 25, 12.5, and 6.25 μM, respectively, and use 0.5% Triton X-100 as a positive control. After mixing well, incubate in a 37°C incubator for 1 hour, and then use a spectrophotometer to measure OD 450 , according to H(%)=(OD 450 – OD of PBS group 450 ) / (1%Triton X-100 group OD 450 – OD of PBS group 450 )×100%, and calculate the hemolysis rate.

[0038] The results are as follows Figure 3 It can be seen that when the concentration of antimicrobial peptide CR24 was between 6.25 μM and 200 μM, no obvious hemolysis was observed on mouse and sheep red blood cells.

[0039] Example 5 Cytotoxicity of antimicrobial peptide CR24 on RAW264.7 and L929 cells

[0040] The frozen RAW264.7 cells and L929 cells were revived in DMEM medium containing 10% fetal bovine serum and incubated overnight at 37°C. After two generations, cells in the exponential growth phase were obtained, digested and counted, and the concentration was adjusted to 1×10 5 Then, 100 μL per well (i.e., each well contains 1×10 4Cells) were inoculated into 96-well plates and incubated at 37°C for 24 h. The culture medium was replaced with fresh culture medium containing antimicrobial peptide CR24 (the final concentration of antimicrobial peptide CR24 was 12.5, 25, 50, 100, and 200 μM, respectively). Wells without antimicrobial substances and cells were set as controls and incubated at 37°C for 48 h. After that, 10 μL of CCK8 detection reagent was added to each well. After incubation at 37°C for 1 hour, OD was measured. 450 The absorbance at the point was measured and the cell survival rate was calculated according to the formula: cell survival rate = [(absorbance OD450 of experimental well - absorbance OD450 of blank well) / (absorbance OD450 of control well - absorbance OD450 of blank well)] × 100% to evaluate the cytotoxic effect of the antimicrobial peptide CR24.

[0041] The results are as follows Figure 4 It can be seen that the antimicrobial peptide CR24 had no significant effect on the survival rate of RAW264.7 cells and L929 cells at concentrations of 100 μM and below.

[0042] Example 6 Induced resistance of antimicrobial peptide CR24

[0043] Staphylococcus aureus USA300 was cultured in TSB medium at 37°C and 200 r / min until the logarithmic growth phase. Subsequently, the MICs of antimicrobial peptide CR24, neomycin and vancomycin against Staphylococcus aureus USA300 were determined. In a 96-well plate, 100 μL of MH medium was added to each well, followed by the addition of antimicrobial peptide CR24, neomycin and vancomycin at final concentrations of 0.25×, 0.5×, 1×, 2× and 4× MIC, respectively. The concentration of bacterial solution in each well was about 5×10 5 CFU / mL Staphylococcus aureus USA300 was inoculated into each well, incubated at 37℃ for 16-20h, and then the bacterial solution of the sub-inhibitory concentration well was transferred to a new culture medium, incubated at 37℃ and 200r / min overnight, and the MIC values ​​of antimicrobial peptide CR24, neomycin and vancomycin were determined. This was repeated for 10 days. The results are as follows Figure 5 It can be seen that within 10 days of continuous induction, sub-inhibitory concentrations of antimicrobial peptide CR24 and vancomycin could not induce drug resistance in Staphylococcus aureus USA300, while the resistance of Staphylococcus aureus USA300 to neomycin increased 16 times after 4 days of continuous induction, indicating that antimicrobial peptide CR24 is not easy to induce drug resistance.

[0044] Example 7 Bactericidal mechanism of antimicrobial peptide CR24 against Staphylococcus aureus

[0045] Staphylococcus aureus USA300 was cultured in TSB medium at 37°C and 200 r / min until the logarithmic growth phase, and then washed twice with PBS. The bacterial solution concentration was adjusted to 1×10 5CFU / mL, and then antimicrobial peptide CR24 was added to the culture medium to a concentration of 1×MIC. During the culture period, 500 μL of bacterial suspension was taken at 0, 0.5, 1, 2, 4, 6, and 8 h, centrifuged at 10,000 r / min for 5 min, and the supernatant was taken to measure the absorbance value at ultraviolet absorption wavelengths of 260 nm and 280 nm.

[0046] The effect of antimicrobial peptide CR24 on the cell membrane permeability of Staphylococcus aureus USA300 was detected using a live and dead bacteria staining kit. Staphylococcus aureus before and after treatment with antimicrobial peptide CR24 was incubated with a dye solution containing DMAO and PI in the dark for 15 minutes, and then imaged using a confocal fluorescence microscope. Live bacteria were stained green with DMAO; dead bacteria were stained red with PI due to the destruction of the cell membrane and cell wall.

[0047] Scanning electron microscopy was used to observe the effect of antimicrobial peptide CR24 on the cell membrane integrity of Staphylococcus aureus USA300. 8 CFU / mL), added antimicrobial peptide CR24 with a final concentration of 10×MIC, cultured at 37°C for 2h and 4h, respectively, then centrifuged at 10000r / min for 5min, collected the bacteria, fixed with 0.25% glutaraldehyde, then dehydrated with 30%, 50%, 70%, 90%, and 100% ethanol, dried by vacuum freeze drying, and observed the bacterial morphology under a scanning electron microscope.

[0048] The leakage of nucleic acids and proteins in bacteria is used to judge the integrity of the cell membrane. The experiment showed that no nucleic acids and proteins were detected in the initial treatment, but after treatment with 1×MIC concentration of antimicrobial peptide CR24, OD 260 The value increased significantly ( Figure 6 A). Similarly, after treatment with antimicrobial peptide CR24, OD 280 The values ​​also increased significantly ( Figure 6 B) These results indicate that the antimicrobial peptide CR24 can disrupt the cell membrane and accelerate the leakage of nucleic acids and proteins from the bacterial cytoplasm.

[0049] The results of DMAO / PI double staining showed that after treatment with the antimicrobial peptide CR24 at 1×MIC concentration, the bacteria were stained with PI and showed red fluorescence. The red fluorescence was more obvious in the samples treated with 2×MIC, while the control bacteria were not stained ( Figure 6 C) These results indicate that CR24 treatment can increase the permeability of bacterial cell membranes.

[0050] In order to more intuitively observe the effect of antimicrobial peptide CR24 on the cell membrane of Staphylococcus aureus USA300, scanning electron microscopy was used to observe the effect of antimicrobial peptide CR24 on the cell membrane of Staphylococcus aureus USA300. The SEM observation results showed that the untreated Staphylococcus aureus USA300 maintained a smooth and regular membrane morphology, while the Staphylococcus aureus USA300 treated with antimicrobial peptide CR24 for 2 hours showed membrane damage, distortion, fracture and leakage of intracellular substances, and the damage was more serious after 4 hours of treatment ( Figure 6 D) This result further proves that the antimicrobial peptide CR24 causes bacterial death by destroying the bacterial cell membrane.

[0051] Example 8 Therapeutic effect of antimicrobial peptide CR24 on Staphylococcus aureus skin lesions

[0052] 20 mice were randomly divided into 4 groups, 5 mice in each group. The mice were anesthetized with anesthetics, and the hair on the back of the mice was shaved to fully expose the skin. A small circular wound with a diameter of about 8 mm was made on the back with surgical scissors, and 100 μL (1×10 8 CFU / mL) of Staphylococcus aureus USA300 solution was injected into the wound. After 2 and 24 hours, 100 μL CR24 (5 mg / Kg), CR24 (10 mg / Kg) and vancomycin (5 mg / Kg) were injected into the modeling area on the back of the mice, and the blank group was injected with normal saline (100 μL). The size of the skin wound was measured every day with a digital vernier caliper, and photographed on days 0, 4 and 7. On the 7th day, all mice were euthanized, skin tissues were collected, fixed, paraffin sections were prepared, and H&E staining and Masson staining were performed. At the same time, the infected skin on the back of the mouse was taken, homogenized and coated on TSA containing benzylpenicillin, and the number of colonies was counted after culturing at 37°C overnight.

[0053] The results showed that treatment with antimicrobial peptide CR24 significantly accelerated wound healing on days 0, 4, and 7 after treatment ( Figure 7 BC). On the 7th day of treatment, the bacterial load of Staphylococcus aureus USA300 in the skin was detected. The results showed that compared with the untreated group, the bacterial load of Staphylococcus aureus USA300 in the skin was significantly reduced after treatment with 10 mg / kg antimicrobial peptide CR24 ( Figure 7 D). H&E staining showed that the 10 mg / kg antimicrobial peptide CR24-treated group showed better histological status compared with the untreated group. In addition, according to Masson staining, the skin fibers of the 10 mg / kg antimicrobial peptide CR24-treated group were neatly arranged, and the collagen density was significantly higher than that of the untreated group ( Figure 7 E). These results suggest that the antimicrobial peptide CR24 exhibits good antimicrobial properties in vivo and that application of the antimicrobial peptide CR24 to skin wounds can promote wound closure by accelerating re-epithelialization.

[0054] It can be seen from the above examples that the present invention provides a new antimicrobial peptide CR24, and describes its screening process, synthesis method, antimicrobial activity, safety, antimicrobial mechanism and antimicrobial application. However, it can also be seen that the above examples are only preferred embodiments of the present invention, and some modifications can be made thereto on the basis of the present invention. Therefore, improvements and changes made without departing from the spirit and principles of the present invention should also be regarded as the scope of protection of the present invention.

Claims

1. A novel antimicrobial peptide CR24, characterized in that: The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

2. The novel antimicrobial peptide CR24 according to claim 1, characterized in that: It was obtained by mining the whole genome of Bacillus haynesii strain B1, a sheep intestinal symbiotic bacterium, and then synthesized by solid phase synthesis and purified by HPLC to obtain the antimicrobial peptide CR24.

3. Use of the novel antimicrobial peptide CR24 according to claim 1 in antagonizing pathogenic bacteria.

4. The use according to claim 3, characterized in that The pathogenic bacteria include Staphylococcus aureus, Listeria monocytogenes, Enterococcus, Salmonella, Escherichia coli, and Pseudomonas aeruginosa.

5. Use of the novel antimicrobial peptide CR24 according to claim 1 in promoting the healing of wounds infected by Staphylococcus aureus.

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