Antibacterial peptide CR24, screening and synthesis method and antibacterial application thereof

Abstract

The application discloses screening and synthesis of an antibacterial peptide CR24 and antibacterial application of the antibacterial peptide CR24. The antibacterial peptide CR24 is screened from a whole genome of a sheep intestinal symbiotic bacterium Bacillus haynesii strain B1, and is obtained through artificial synthesis. Experiments show that the antibacterial peptide CR24 is an antibacterial peptide, has extremely low cytotoxicity and hemolytic activity, does not produce 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 cell membrane of Staphylococcus aureus, and can promote skin wound healing caused by Staphylococcus aureus infection in in-vivo application. In a word, the antibacterial peptide CR24 provided by the application is an antibacterial peptide which is small in molecular weight, easy to artificially synthesize, strong in antibacterial activity, safe and non-toxic, and 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 application belongs to the technical field of biological medicine, and particularly relates to an antibacterial peptide CR24 and a screening and synthesis method and antibacterial application. BACKGROUND

[0002] The abuse of antibiotics leads to the generation of high levels of drug resistance of pathogenic microorganisms, and the formation of multi-drug resistance. In recent years, multi-drug resistant "super pathogenic bacteria" have broken out successively in various parts of the world, seriously threatening the life and health safety of human beings and livestock and poultry. Although antibiotics are the main drug intervention measures for bacterial infections in the past, the formation of multi-drug resistant bacteria seriously hinders the effectiveness of antibiotic treatment. More and more infectious diseases become difficult to treat, and sometimes even no medicine can be used, however, few chemical synthetic antibiotics are developed at the present stage, and most of them are derivatives of current antibiotics. Therefore, it is urgent to seek antibacterial drugs.

[0003] Antibacterial proteins or polypeptides have unique structural sequences, action mechanisms and efficient bactericidal effects, and basically do not produce cross drug resistance with traditional antibiotics, and thus are the best substitutes for antibiotics. Antibacterial peptides are a kind of low molecular weight polypeptides with antibacterial activity, the molecular weight is usually about 2000-7000, and contains 20-60 amino acids. Antibacterial peptides are an important part of the natural immune defense system of organisms, and are a kind of active biological molecules with endogenous immune response of most organisms. So far, several thousand antibacterial peptides with different structures have been identified. Since the antibacterial and bactericidal mechanisms of antibacterial peptides are different from those of antibiotics, antibacterial peptides have multiple antibacterial mechanisms, and thus are safe and efficient when used as drugs, and the probability of pathogenic bacteria developing drug resistance is very low, and have broad application prospects, and become a new generation of antibiotic substitutes.

[0004] At present, most of the antibacterial peptides are derived from mammals and insects, in addition, a variety of antibacterial peptides have also been identified in amphibians. The intestinal tract of livestock and poultry is colonized by hundreds of billions of microorganisms. These intestinal symbiotic flora open up a new way for finding antibacterial peptides, and a variety of antibacterial substances have been found. Therefore, mining antibacterial peptides from intestinal symbiotic bacteria of livestock and poultry will provide a new solution to the problem of antibiotic resistance and the development of antibacterial drugs. SUMMARY

[0005] One of the purposes of the application is to provide an antibacterial peptide CR24, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2.

[0006] The second object of the present application is to provide a screening and synthesis method of the antibacterial peptide CR24, which is obtained by mining the whole genome of the sheep intestinal symbiotic bacteria Bacillus haynesii strain B1, and then synthesized by using a solid-phase synthesis method, and obtained after HPLC purification.

[0007] The antibacterial peptide CR24 is applied in antagonizing pathogenic bacteria, including Staphylococcus aureus, Listeria monocytogenes, Enterococcus, Salmonella, Escherichia coli and Pseudomonas aeruginosa.

[0008] The antibacterial peptide CR24 is applied in promoting wound healing of Staphylococcus aureus infection.

[0009] The antibacterial peptide CR24 provided by the present application has broad-spectrum antibacterial activity, and various pathogenic bacteria involved include Staphylococcus aureus, Listeria monocytogenes, Enterococcus, Salmonella, Escherichia coli and Pseudomonas aeruginosa.

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

[0011] The antibacterial peptide CR24 provided by the present application can play a bactericidal role by destroying the bacterial cell membrane.

[0012] The antibacterial peptide CR24 provided by the present application has the ability to promote wound healing of Staphylococcus aureus infection.

[0013] Beneficial effects:

[0014] The present application provides an antibacterial peptide CR24. The antibacterial peptide has the characteristics of small molecular weight, simple structure, no cytotoxicity, no hemolytic activity, etc. It has high-efficiency broad-spectrum antibacterial activity, can kill various clinically common gram-positive and negative pathogenic bacteria, is not easy to produce drug resistance, and has the ability to promote wound healing of Staphylococcus aureus infection in vivo. Therefore, the antibacterial peptide CR24 has excellent properties, is cheap to prepare, is convenient to produce, can be applied to prepare feed additives, anti-infection drugs and food preservatives, etc., and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For the purity and molecular weight identification results of the antibacterial peptide CR24 synthesized in the present application, (A) HPLC purity identification of the antibacterial peptide CR24 after synthesis; (B) ESI-MS identification of the antibacterial peptide CR24 after synthesis;

[0016] Figure 2Antibacterial effect of antibacterial peptide CR24 on S. aureus and bactericidal curve (A) Antibacterial effect of antibacterial peptide CR24 on S. aureus; (B) Change in eye observation after 24h of antibacterial peptide CR24 against S. aureus; (C) Bactericidal curve of antibacterial peptide CR24 on S. aureus;

[0017] Figure 3 Hemolytic activity of antibacterial peptide CR24 on mouse and sheep red blood cells (A) Hemolytic activity of antibacterial peptide CR24 on mouse red blood cells; (B) Hemolytic activity of antibacterial peptide CR24 on sheep red blood cells;

[0018] Figure 4 Cytotoxic effect of antibacterial peptide CR24 on RAW264.7 and L929 cells (A) Cytotoxic effect of antibacterial peptide CR24 on RAW264.7 cells; (B) Cytotoxic effect of antibacterial peptide CR24 on L929 cells;

[0019] Figure 5 Induction of resistance of antibacterial peptide CR24;

[0020] Figure 6 Bactericidal mechanism of antibacterial peptide CR24 on S. aureus (A) Effect of antibacterial peptide CR24 on intracellular nucleic acid leakage of S. aureus; (B) Effect of antibacterial peptide CR24 on intracellular protein leakage of S. aureus; (C) Effect of antibacterial peptide CR24 on cell membrane permeability of S. aureus; (D) Effect of antibacterial peptide CR24 on cell membrane integrity of S. aureus observed by scanning electron microscope;

[0021] Figure 7 Therapeutic effect of antibacterial peptide CR24 on skin injury of S. aureus (A) Mouse treatment and treatment process; (B) Change in mouse wound after treatment with antibacterial peptide CR24; (C) Change in skin wound area size at different time points (0 day, 4 days and 7 days) after treatment with antibacterial peptide CR24; (D) Bacterial load of S. aureus in mouse skin wound after treatment with antibacterial peptide CR24; (E) H&E staining and Masson staining of skin wound tissue 7 days after treatment. DETAILED DESCRIPTION

[0022] The present application will be further described below by some specific embodiments. It should be clear that the embodiments described below are only a part of the embodiments of the present application, but not all the embodiments of the present application, and the present application is not limited in the scope of the described embodiments.

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

[0024] Screening of antibacterial peptide CR24

[0025] The -80℃ frozen sheep intestinal-derived Bacillus haynesii B1 was streaked on TSB plates, cultured overnight at 37℃ to form single colonies, then a single colony was picked into 3ml TSB liquid medium, cultured at 37℃, 200rpm for 12h, the bacterial cells were collected, washed twice with PBS, then the genomic DNA of Bacillus haynesii B1 was extracted according to the bacterial genome extraction kit instructions, and then sent to the company for determination of the whole genome sequence of Bacillus haynesii B1. Subsequently, the open reading frames (ORF) less than 100bp in the whole genome of Bacillus haynesii B1 were selected, and the online software APD3, CAMPR3 and Ex PASy were used to predict the antibacterial peptide sequences, and their basic characteristics, 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 by I-TASSER. Through comprehensive screening, CR24 was finally selected for synthesis.

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

[0027] Synthesis of antibacterial peptide CR24

[0028] The screened antibacterial peptide CR24, with the amino acid sequence shown as SEQ ID NO. 1, was sent to Nanjing Peptide Biotechnology Co., Ltd. for Fmoc-based solid-phase synthesis. 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). HPLC analysis showed that the purity of the polypeptide CR24 was greater than 95%, and ESI-MS analysis showed that the actual molecular weight was basically consistent with the determined molecular weight. Figure 1

[0029] Example 2 Antimicrobial spectrum of antibacterial peptide CR24

[0030] The pathogenic bacterial strains to be tested, including Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Escherichia coli, Salmonella, Pseudomonas aeruginosa and Klebsiella pneumoniae, were inoculated in TSB, cultured overnight at 37℃ in a shaking incubator, the bacterial cells were collected, washed twice with PBS, then the bacterial solution was diluted to 10 9 ​CFU / mL, standby. The antibacterial peptide CR24 was diluted with distilled water to concentrations of 128 μΜ, 64 μΜ, 32 μΜ, 16 μΜ, 8 μΜ, 4 μΜ, 2 μΜ. In a sterile 96-well plate, 50 μΐ^of each concentration of antibacterial peptide CR24 was added to the wells, and 50 μΐ^of PBS was also added to the wells, so that the final concentration of the antibacterial peptide was 64 μΜ, 32 μΜ, 16 μΜ, 8 μΜ, 4 μΜ, 2 μΜ, 1 μΜ, and negative and positive controls were set up. Finally, the diluted bacterial solution was added to the wells containing different concentrations of antibacterial peptide, so that the final concentration of bacteria was 5 x 10 5 CFU / mL, the plate was covered, and incubated in a 37 °C incubator for 16 hours. Finally, 10 μΐ^of solution was taken from each well and dropped onto a TSA plate, which was labeled and incubated in a 37 °C incubator for 12 hours. The presence or absence of bacterial growth was observed, and the lowest concentration of antibacterial substance that completely inhibited bacterial growth was taken as the MIC. The experimental results are shown in Table 1. For all the tested Gram-negative and Gram-positive pathogenic bacteria, the antibacterial peptide CR24 showed good antibacterial activity, with a MIC range of 2-64 μΜ.

[0031] Table 1. Antibacterial spectrum of antibacterial peptide CR24

[0032]

[0033] Example 3. Antibacterial effect and bactericidal curve of antibacterial peptide CR24 on S. aureus

[0034] S. aureus USA300 was cultured in TSB medium at 37 °C and 200 r / min to the logarithmic growth phase, washed twice with PBS, and the concentration of the bacterial solution was adjusted to 1 x 10 5 CFU / mL. Then, antibacterial peptide CR24 was added to the culture medium, and the final concentration of the antibacterial peptide CR24 was 1 x MIC and 2 x MIC. The culture was incubated at 37 °C and 200 r / min, and during the incubation period, 100 μΐ^was taken at 0, 0.5, 1, 2, 4, 6, and 8 hours for 10-fold serial dilution, and then 100 μΐ^of the diluted solution was spread on a TSA plate and incubated at 37 °C overnight. The total number of bacteria per milliliter of culture medium was calculated. The results showed that the antibacterial peptide CR24 had very strong bactericidal activity against S. aureus USA300 Figure 2 A-B). The results of the time-killing kinetics experiment showed that compared with the control group, the number of S. aureus USA300 in the 2 x MIC concentration treatment group was significantly reduced, and no bacteria were detected after 6 h of incubation Figure 2 C). These results showed that the antibacterial peptide CR24 had bactericidal activity against S. aureus and could kill bacteria in a short time.

[0035] Example 4. Hemolytic activity of antibacterial peptide CR24 on mouse and sheep red blood cells

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

[0037] Take the 2% red blood cell suspension obtained above, and then take 50 μL to add to the wells of a 96-well plate. Dilute the antimicrobial peptide CR24 twice with PBS, and then take 50 μL to add to the wells to make the final concentrations of 200, 100, 50, 25, 12.5, and 6.25 μM, respectively. Use 0.5% Triton X-100 as a positive control. After mixing well, incubate in a 37°C incubator for 1 hour, and then measure the OD using a spectrophotometer. 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] Frozen RAW264.7 cells and L929 cells were revived in DMEM medium containing 10% fetal bovine serum and incubated overnight at 37°C. After passage 2, cells in the exponential growth phase were obtained, digested and counted, and the concentration was adjusted to 1×10 5 cell / mL, and then 100 μL per well (i.e., each well contains 1×10 4The cells were inoculated into 96-well plates and incubated at 37°C for 24 h. The culture medium was replaced with fresh culture medium containing antibacterial peptide CR24 (to make the final concentration of antibacterial peptide CR24 12.5, 25, 50, 100 and 200 μM, respectively), and wells without antibacterial substances and without cells were set as controls. The plates were incubated at 37°C for 48 h, 10 μL of CCK8 detection reagent was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance at OD450 was measured, and the cell survival rate was calculated according to the formula: cell survival rate = [(absorbance OD450 of experimental wells - absorbance OD450 of blank wells) / (absorbance OD450 of control wells - absorbance OD450 of blank wells)] x 100%, to evaluate the cytotoxicity of antibacterial peptide CR24. 450

[0041] The results are shown in Table 1. Figure 4 As can be seen, antibacterial peptide CR24 had no significant effect on the survival rate of RAW264.7 cells and L929 cells at a concentration of 100 μM or less.

[0042] Example 6 Induction of resistance by antibacterial peptide CR24

[0043] Staphylococcus aureus USA300 was cultured in TSB medium at 37°C and 200 r / min to the logarithmic growth phase, and then the MIC of antibacterial peptide CR24, neomycin and vancomycin against Staphylococcus aureus USA300 was determined. In a 96-well plate, 100 μL of MH medium was added to each well, and then antibacterial peptide CR24, neomycin and vancomycin were added to each well at a final concentration of 0.25x, 0.5x, lx, 2x and 4x MIC, respectively. Then, 5x10 5 CFU / mL of Staphylococcus aureus USA300 was inoculated into each well, and the plates were incubated at 37°C for 16-20 h. The bacterial solution from the wells with sub-inhibitory concentration was transferred to a new culture medium and incubated at 37°C and 200 r / min overnight to determine the MIC of antibacterial peptide CR24, neomycin and vancomycin. This was repeated for 10 days. The results are shown in Table 2. Figure 5 As can be seen, sub-inhibitory concentrations of antibacterial peptide CR24 and vancomycin could not induce resistance in Staphylococcus aureus USA300 for 10 days of continuous induction, while the resistance of Staphylococcus aureus USA300 increased by 16 times after 4 days of continuous induction of neomycin, indicating that antibacterial peptide CR24 is less likely to develop resistance.

[0044] Example 7 Killing mechanism of antibacterial peptide CR24 against Staphylococcus aureus

[0045] Staphylococcus aureus USA300 was cultured in TSB medium at 37°C and 200 r / min to the logarithmic growth phase, and then the MIC of antibacterial peptide CR24, neomycin and vancomycin against Staphylococcus aureus USA300 was determined. In a 96-well plate, 100 μL of MH medium was added to each well, and then antibacterial peptide CR24, neomycin and vancomycin were added to each well at a final concentration of 0.25x, 0.5x, lx, 2x and 4x MIC, respectively. Then, 5x10 5 ​CFU / mL, and then the antimicrobial peptide CR24 was added to the culture medium to a concentration of 1×MIC. During the culture period, 500 μL of the bacterial suspension was collected at 0, 0.5, 1, 2, 4, 6, and 8 h, centrifuged at 10,000 rpm for 5 min, and the supernatant was measured for absorbance at UV wavelengths of 260 nm and 280 nm.

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

[0047] The effect of antimicrobial peptide CR24 on the cell membrane integrity of Staphylococcus aureus USA300 was observed using scanning electron microscopy. 8 CFU / mL), the antimicrobial peptide CR24 was added at a final concentration of 10×MIC, and the cells were cultured at 37°C for 2 h and 4 h, respectively. The cells were then centrifuged at 10,000 rpm for 5 min, and the cells were collected and fixed with 0.25% glutaraldehyde. Subsequently, the fixed cells were dehydrated with 30%, 50%, 70%, 90%, and 100% ethanol, and dried by vacuum freeze drying. The cell morphology was observed 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 during the initial treatment, but after treatment with 1×MIC concentration of antimicrobial peptide CR24, the OD value was significantly increased compared with the control group. 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 cell membranes 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 1×MIC concentration of antimicrobial peptide CR24, the bacteria were stained by PI and showed red fluorescence. The red fluorescence was more obvious in the 2×MIC treated samples, 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] To observe the effect of antimicrobial peptide CR24 on the cell membrane of S. aureus USA300 more intuitively, scanning electron microscopy was used to observe the effect of antimicrobial peptide CR24 on the cell membrane of S. aureus USA300. The SEM observation results showed that the untreated S. aureus USA300 maintained a smooth and regular membrane morphology, while the S. aureus USA300 treated with antimicrobial peptide CR24 for 2 h showed membrane damage, twisting, rupture and intracellular material leakage, and the damage was more serious after 4 h of treatment Figure 6 D). This result further proves that antimicrobial peptide CR24 causes bacterial death by destroying the bacterial cell membrane.

[0051] Example 8 Therapeutic effect of antimicrobial peptide CR24 on S. aureus skin injury

[0052] Twenty mice were randomly divided into four groups, five mice in each group. The mice were anesthetized using anesthetic, and the fur on the back of the mice was then 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 then 100 μL (1 x 10 8 CFU / mL) of S. aureus USA300 bacterial solution was injected into the wound. After 2 and 24 h, 100 μL of 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 using a digital vernier caliper, and photographs were taken on days 0, 4 and 7. On day 7, all mice were euthanized and killed, and the skin tissue was collected, fixed, prepared into paraffin sections, and subjected to H&E staining and Masson staining. At the same time, the infected skin on the back of the mouse was taken, homogenized and then spread on TSA containing oxacillin, and after incubation at 37°C overnight, the number of bacterial colonies was counted.

[0053] The results show that after treatment on day 0, day 4 and day 7, treatment with antimicrobial peptide CR24 significantly accelerated wound healing Figure 7 B-C). On the 7th day of treatment, the S. aureus USA300 load in the skin was detected, and the results showed that compared with the untreated group, the S. aureus USA300 load in the skin was significantly reduced after treatment with 10 mg / kg of antimicrobial peptide CR24 Figure 7 D). H&E staining showed that the 10 mg / kg antimicrobial peptide CR24 treatment group showed a better histological state compared with the untreated group, and in addition, according to Masson staining, the skin fibers of the 10 mg / kg antimicrobial peptide CR24 treatment group were arranged in an orderly manner, and the collagen density was significantly higher than that of the untreated group Figure 7 E). These results show that antimicrobial peptide CR24 exhibits good antibacterial performance in vivo, and the application of antimicrobial peptide CR24 to skin wounds can promote wound closure by accelerating reepithelialization.

[0054] As can be seen from the above examples, the present application provides a new antibacterial peptide CR24, and the screening process, synthesis method, antibacterial activity, safety, antibacterial mechanism and antibacterial application thereof are described. However, it can also be seen that the above examples are only preferred embodiments of the present application, and some modifications can be made based on the present application. Therefore, improvements and changes made without departing from the spirit and principles of the present application should also be considered within the scope of protection of the present application.

Claims

1. An 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 method for preparing the antimicrobial peptide CR24 according to claim 1, characterized in that: The antimicrobial peptide CR24 was synthesized by solid phase synthesis and purified by HPLC.

3. Use of the antimicrobial peptide CR24 according to claim 1 in the preparation of a drug for antagonizing pathogens, wherein the pathogens are Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Salmonella, Escherichia coli, and Pseudomonas aeruginosa.

4. Use of the antimicrobial peptide CR24 according to claim 1 in the preparation of a drug for promoting wound healing caused by Staphylococcus aureus infection.

Images