Antibacterial active peptide YC18 and antibacterial application

Abstract

The invention provides an antibacterial active peptide YC18 and antibacterial application, and belongs to the technical field of biological medicine. The invention provides an antibacterial peptide. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID No. 1. The active polypeptide YC18 has different degrees of antibacterial activity to various bacteria, especially has MIC of 6.25 mu g / ml to staphylococcus aureus and methicillin-resistant staphylococcus aureus, and is good in antibacterial effect and high in sterilization speed. The active polypeptide YC18 has the capacity of inhibiting and removing a biofilm, is low in hemolytic activity and free of cytotoxicity, does not easily generate drug resistance in the use process, has an obvious protection effect on an abdominal cavity infection model, and has the potential of being applied as an anti-infection drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an antibacterial active peptide YC18 and its antibacterial use. Background Art

[0002] Since the discovery of penicillin in the 1940s, the widespread use of antibiotics has achieved remarkable success in treating bacterial infections, but it has also led to a serious problem of antibiotic resistance. With the overuse of antibiotics, drug-resistant strains have continued to emerge, making many bacterial infections difficult to effectively treat with existing antibiotics. The emergence of multidrug-resistant bacteria, in particular, has led to a significant increase in serious infections and deaths in clinical practice, becoming a major challenge to global public health.

[0003] Against this backdrop, finding new antimicrobial drugs to address drug resistance has become a top priority. Antimicrobial peptides, as a non-traditional antibiotic, are considered an ideal choice for addressing antibiotic resistance due to their unique mechanism and broad biological activity. Antimicrobial peptides are a class of small-molecule polypeptides composed of a small number of amino acid residues, characterized by light weight, diverse charge distribution, and flexible three-dimensional structure. Unlike traditional antibiotics, antimicrobial peptides exert their antimicrobial effects by destroying bacterial cell membranes or interfering with intracellular functions. This mechanism makes it difficult for bacteria to develop drug resistance through a single mutation, thereby reducing the risk of resistance development.

[0004] Antimicrobial peptides are widely distributed in nature and have been found in everything from single-celled organisms to higher organisms. In organisms, antimicrobial peptides are not only an important component of the innate immune system, but also function as a primary antimicrobial defense mechanism in some organisms. For example, antimicrobial peptides in insects are highly effective in resisting pathogen invasion, while antimicrobial peptides (such as defensins) in mammals (including humans) also play a key role in the skin, mucous membranes, and immune cells. In addition, antimicrobial peptides have broad-spectrum antimicrobial activity and can effectively fight against Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, and even certain parasites, which provides broad prospects for their clinical application.

[0005] Antimicrobial peptides (AMPs) are ideal candidates for combating infections. However, since the discovery of AMPs is mainly experimentally driven, there is an urgent need to explore and discover new AMPs. Summary of the Invention

[0006] The present invention provides an antimicrobial active peptide YC18 and its antimicrobial use. The antimicrobial peptide exhibits significant antimicrobial activity against a variety of pathogenic bacteria and is not prone to inducing bacterial resistance.

[0007] The present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0008] In a preferred embodiment of the present invention, the antimicrobial spectrum of the antimicrobial peptide includes Gram-positive bacteria and Gram-negative bacteria.

[0009] In a preferred embodiment of the present invention, the antimicrobial spectrum of the antimicrobial peptide includes at least one of the following: Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

[0010] The present invention also provides an active polypeptide YC18 obtained by artificially synthesizing and then oxidatively folding the above antimicrobial peptide.

[0011] In a preferred embodiment of the present invention, the oxidative folding comprises placing the artificially synthesized antimicrobial peptide in a buffer solution and reacting at 25° C. for 24 hours;

[0012] The buffer comprises the following components at the following concentrations: Tris-HCl 0.1 M, NaCl 0.1 M, oxidized glutathione (GSSG) 0.5 mM, and reduced glutathione (GSH) 5 mM, pH = 9.4.

[0013] The present invention also provides the use of the active polypeptide YC18 in the preparation of antibacterial products.

[0014] In a preferred embodiment of the present invention, the antimicrobial spectrum of the antimicrobial product includes at least one of the following: Staphylococcus aureus, drug-resistant Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

[0015] In a preferred embodiment of the present invention, the drug-resistant Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

[0016] The present invention also provides an antibacterial product, the active ingredients of which include the above-mentioned antibacterial peptide or the above-mentioned active polypeptide YC18, and also include auxiliary materials.

[0017] The present invention also provides the use of the above-mentioned antimicrobial peptide, the above-mentioned active polypeptide YC18 or the above-mentioned antimicrobial product in the preparation of a drug for treating diseases caused by pathogenic bacteria.

[0018] Beneficial Effects: The present invention provides an antimicrobial peptide comprising 18 amino acid residues with an average molecular weight of 2153.57 Da. Its full sequence is SEQ ID No. 1: YCRSVCGRKRCFTYCKGK-NH2. The four cysteines in the antimicrobial peptide fold to form two disulfide bonds, and the peptide can undergo natural oxidative folding after artificial synthesis. The active peptide YC18 exhibits varying degrees of antimicrobial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. In particular, the MIC against both Staphylococcus aureus and methicillin-resistant Staphylococcus aureus was 6.25 μg / ml, demonstrating excellent antimicrobial efficacy and rapid bactericidal activity. The active peptide YC18 exhibits the ability to inhibit and eliminate biofilms, exhibits low hemolytic activity, is non-cytotoxic, and is not susceptible to developing drug resistance during use. Examples of the present invention also demonstrate that the active peptide YC18 exhibits significant protective effects in a peritoneal infection model, suggesting its potential as an anti-infective drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the bactericidal speed of YC18 and the positive drug vancomycin;

[0020] Figure 2 Schematic diagram of YC18's effect on inhibiting (A) and clearing (B) biofilm;

[0021] Figure 3 Effects of YC18 on the cell membrane morphology of Staphylococcus aureus. Figure A: The morphology of Staphylococcus aureus after being treated with YC18 under a transmission electron microscope; Figure B: The morphology of Staphylococcus aureus after being treated with YC18 under a scanning electron microscope.

[0022] Figure 4 The cytotoxicity and hemolytic activity of YC18 are shown in Figure 1. A: Cytotoxicity of YC18 against HEK293T cells, B: Cytotoxicity of YC18 against HaCaT cells, C: Cytotoxicity of YC18 against RAW264.7 cells, and D: Hemolytic activity of YC18 against mouse erythrocytes.

[0023] Figure 5 Figure 1 shows the stability of YC18 in plasma and the resistance of Staphylococcus aureus to YC18. (A) The stability of YC18 in plasma, (B) The resistance of Staphylococcus aureus to YC18 and vancomycin.

[0024] Figure 6 Figure 3. Changes in bacterial load in various tissues of YC18-treated mice after intraperitoneal infection. Figure A: Blood bacterial load, B: Lung bacterial load, C: Liver bacterial load, D: Spleen bacterial load. DETAILED DESCRIPTION

[0025] The present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID No. 1: YCRSVCGRKRCFTYCKGK-NH2.

[0026] The antimicrobial peptide comprises 18 amino acid residues with an average molecular weight of 2153.57 Da. Four cysteines fold to form two disulfide bonds. The antimicrobial peptide has a net charge of +6, with a 16 / 18 split of polar residues and a 2 / 18 split of nonpolar residues.

[0027] The antimicrobial peptide of the present invention has broad-spectrum antibacterial activity and has antibacterial activity against a variety of Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus and Bacillus subtilis among Gram-positive bacteria, and Escherichia coli and Pseudomonas aeruginosa among Gram-negative bacteria. It also has antibacterial activity against common drug-resistant strains of the aforementioned bacteria, such as methicillin-resistant Staphylococcus aureus, and has high antibacterial activity.

[0028] The present invention also provides an active polypeptide YC18 obtained by artificially synthesizing and then oxidatively folding the above antimicrobial peptide.

[0029] The present invention does not particularly limit the preparation method of the antimicrobial peptide, such as using common artificial synthesis methods in the art. For example, in one embodiment, Hangzhou Gutuo Biotechnology Co., Ltd. was commissioned to synthesize the antimicrobial peptide with a purity of more than 95%, synthesized by solid-phase Fmoc chemistry, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and confirmed by mass spectrometry MALDI-TOF-MASS.

[0030] The present invention performs oxidative folding on the artificially synthesized antimicrobial peptide, and the oxidative folding comprises placing the artificially synthesized antimicrobial peptide in a buffer solution and reacting at 25° C. for 24 hours; the buffer solution comprises components with the following concentrations: Tris-HCl 0.1M, NaCl 0.1M, GSSG 0.5mM and GSH 5mM, with a pH of 9.4.

[0031] The present invention also provides the use of the active polypeptide YC18 in the preparation of antibacterial products.

[0032] In one embodiment of the present invention, it was confirmed that the MICs of the active polypeptide YC18 against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were 6.25 μg / ml, 6.25 μg / ml, 25 μg / ml, 25 μg / ml and 25 μg / ml, respectively.

[0033] In one embodiment of the present invention, it was also confirmed that the active polypeptide YC18 has a fast bactericidal speed, the ability to inhibit and remove biofilms, low hemolytic activity and no cytotoxicity, and is not prone to drug resistance during use.

[0034] The present invention also provides an antibacterial product, the active ingredients of which include the above-mentioned antibacterial peptide or the above-mentioned active polypeptide YC18, and also include auxiliary materials.

[0035] The present invention also provides the use of the above-mentioned antimicrobial peptide, the above-mentioned active polypeptide YC18 or the above-mentioned antimicrobial product in the preparation of a drug for treating diseases caused by pathogenic bacteria.

[0036] In one embodiment of the present invention, it was confirmed that the active polypeptide YC18 has a significant protective effect on the abdominal infection model and has the potential to be used as an anti-infective drug.

[0037] To further illustrate the present invention, an antimicrobial active peptide YC18 provided by the present invention and its antimicrobial uses are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0038] The YC18 polypeptide used in the examples of the present invention was synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. with a purity of over 95%. It was synthesized by solid-phase Fmoc chemistry, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and confirmed by MALDI-TOF-MASS mass spectrometry.

[0039] Conditions for oxidative folding: Tris-HCl 0.1 M, NaCl 0.1 M, GSSG:GSH = 1:10, pH = 9.4.

[0040] The reaction was continued at 25°C for 24 h, and then the product was purified by RP-HPLC and confirmed by MALDI-TOF-MASS.

[0041] Example 1: Antibacterial activity of polypeptide YC18 against different strains

[0042] The experiment used the micro-dilution method to determine the minimum inhibitory concentration (MIC). The specific experimental operation was as follows: the fresh culture solution was measured for OD 600 The value of (OD 600 ≈1×10 9 CFU / mL), and then the bacterial concentration was adjusted to 2×10 5 CFU / mL. 100 μL of physiological saline was added to a sterile 96-well plate in advance. The sample to be tested was added to the first column and then serially diluted by two times. Three parallel wells were set up, and the last column was a control group without drug. 100 μL of the above bacterial suspension (final concentration 1×105 CFU / mL), mixed thoroughly, and incubated in a 37°C incubator for 18 hours. The absorbance at 600 nm was measured using a microplate reader, and the lowest drug concentration that completely inhibited bacterial growth was defined as the minimum inhibitory concentration (MIC).

[0043] As shown in Table 1, YC18 has different degrees of antibacterial activity against Staphylococcus aureus (S. aureus ATCC 6538), methicillin-resistant Staphylococcus aureus (MRSA-Z, the First Affiliated Hospital of Kunming Medical University, which has been disclosed in the article: A designed antimicrobial peptide with potential ability against methicillin-resistant Staphylococcus aureus.), Bacillus subtilis (B. subtilis ATCC 6633), Escherichia coli (E. coli ATCC 8739) and Pseudomonas aeruginosa (P. aeruginosa ATCC 27853). Its MIC against S. aureus is 6.25 μg / ml, the MIC against methicillin-resistant S. aureus is 6.25 μg / ml, the MIC against B. subtilis is 25 μg / ml, the MIC against E. coli is 25 μg / ml, and the MIC against Pseudomonas aeruginosa is 25 μg / ml. The MIC for Acinetobacter baumannii is greater than 50 μg / ml, and it can be considered that the MIC at this concentration is meaningless for antibacterial activity screening, that is, it is considered to have no antibacterial activity.

[0044] Table 1 MIC of YC18 against bacteria

[0045]

[0046] Example 2: Bactericidal kinetics of polypeptide YC18 against S. aureus ATCC 6538

[0047] Measure the OD of freshly cultured bacteria 600 The value of (OD 600 ≈1×10 9 CFU / mL) and then adjusted to 2×10 5CFU / mL. Equal volumes of YC18 (1, 4, 8× MIC) and the positive control drug vancomycin (VAN, 1, 4, 8× MIC) were added to the bacterial suspension. A sterile PBS group was set up as a negative control group, and the cells were cultured at 37°C with constant shaking (200 rpm). At 0, 15, 30, 60, 120, and 240 min, 10 μL of the reaction solution was aspirated and diluted 100-fold with 990 μL of normal saline. 50 μL of the dilution was evenly spread on an agar plate and incubated at 37°C for 24 hours before counting the colony forming units (CFU).

[0048] The results are as follows Figure 1 The results compared the killing rates of YC18 and the positive drug vancomycin at concentrations of 1, 4, and 8× MIC, indicating that YC18 had a fast killing rate.

[0049] Example 3: YC18 has the ability to inhibit and clear biofilm

[0050] Biofilm inhibition: S. aureus ATCC 6538 bacterial suspension was diluted to 2×10 6 CFU / mL, take 100μL of diluted bacterial solution and add it to each well of a 96-well plate. YC18 and vancomycin were gradiently diluted with sterile PBS to prepare final concentrations of 1×MIC, 2×MIC, 4×MIC, 8×MIC and 16×MIC, respectively. 100μL was added to each well and mixed with an equal volume of bacterial solution. Three parallel wells were set up for each concentration, and sterile PBS was used as a control. The 96-well plate was cultured in a 37°C constant temperature incubator for 24 hours, the bacterial solution was removed and washed 3 times with sterile PBS. Methanol was added to fix for 15 minutes, the methanol was discarded and the plate was dried. 100μL of 0.1% crystal violet stain was added to each well for 5 minutes, the excess dye was gently rinsed with deionized water, 100μL of ethanol was added to each well after drying, and the plate was shaken at 37°C for 30 minutes. The absorbance at 600nm (OD 600 ).

[0051] Biofilm removal: dilute the bacterial suspension to 2 × 10 6CFU / mL, take 200μL of bacterial solution and add it to a 96-well plate and pre-culture at 37℃ for 24h. After removing the bacterial solution, wash 3 times with PBS, add polypeptide YC18 and positive control vancomycin to the final concentration of 1×MIC, 2×MIC, 4×MIC, 8×MIC and 16×MIC respectively. Set up 3 parallel wells for each concentration, and set up PBS as a negative control (NC) group. After continuing to culture at 37℃ for 24h, remove the bacterial solution and wash 3 times with sterile PBS. Add methanol to fix for 15min, discard the methanol and dry it. Add 100μL of 0.1% crystal violet stain to each well for 5min, gently rinse the excess dye with deionized water, add 100μL of ethanol to each well after drying, shake at 37℃ for 30min, and use an enzyme reader to measure the absorbance at 600nm (OD 600 ).

[0052] The results are as follows Figure 2 As shown, YC18 has the ability to inhibit and clear biofilms.

[0053] Example 4: YC18 disrupts bacterial membrane integrity

[0054] S. aureus ATCC 6538 was treated with YC18 at 1× MIC concentration and an equal volume of sterile PBS for 2 hours. The cells were centrifuged at 3500 rpm for 5 minutes, the supernatant discarded, and the pellet washed three times with sterile PBS. The pellet was then centrifuged and the supernatant discarded. 2.5% glutaraldehyde was slowly added to the pellet and fixed overnight at 4°C. After dehydration, the pellet was stained. Two identical samples were prepared and observed under transmission electron microscopy and scanning electron microscopy, respectively. The results are shown in Figure 2. Figure 3 As shown, YC18 disrupts the integrity of the S. aureus cell membrane.

[0055] Example 5: YC18 has cytotoxicity and low hemolytic activity

[0056] HEK293T, HaCaT and RAW264.7 cells from the cell bank of Kunming Institute of Zoology, Chinese Academy of Sciences were used to determine cytotoxicity. HaCaT cells were diluted to 10% in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. 5 Cells were then plated into 96-well plates and incubated in a 5% CO2 incubator for 24 hours. YC18 antimicrobial peptide was added at varying concentrations (3.125–100 μg / ml) in four replicate wells per group. 10% DMSO was used as a positive control. Cells were incubated under the same conditions for an additional 24 hours. 10 μL of CCK8 was added to the cells and incubated under the same conditions for an additional 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader. Cell viability was calculated.

[0057] Mature red blood cells were washed three times with normal saline and resuspended with normal saline. 100 μL of red blood cell suspension was incubated with an equal volume of YC18 at different concentrations at 37°C for 30 minutes, centrifuged at 3500 rpm for 5 minutes, and the supernatant was taken. The absorbance of the supernatant was measured at 450 nm. The value measured with sterile normal saline (negative control, NC) group represents 0 hemolysis, while the value measured with 1% (v / v) TritonX-100 (positive control, PC) group represents 100% hemolysis value. The hemolysis value of the test sample was calculated as the percentage of hemolysis induced by Tritonx-100.

[0058] The experimental results are as follows Figure 4 As shown, YC18 showed no significant cytotoxicity against HEK293T, HaCaT, and RAW264.7 cells at concentrations between 3.125 and 100 μg / ml. Many antimicrobial peptides with excellent antimicrobial activity also exhibit strong hemolytic activity, hindering their in vivo application. This study examined the hemolytic activity of YC18 against mature mouse erythrocytes and found no significant hemolytic activity at concentrations between 3.125 and 100 μg / ml.

[0059] Example 6: YC18 is stable in plasma

[0060] To evaluate the plasma stability of YC18, the peptide was incubated with 50% plasma for varying periods of time (0 to 12 hours). Antibacterial activity was then assessed. 100 μL of the peptide-50% plasma mixture was added to a 96-well plate, followed by 100 μL of bacterial suspension. The final peptide concentration ranged from 1.5625 to 100 μg / mL. Vancomycin, treated in the same manner, served as a control. After thorough mixing, the plates were incubated in a 37°C incubator for 18 hours. The absorbance at 600 nm was measured using a microplate reader. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that completely inhibited bacterial growth.

[0061] The results are as follows Figure 5 As shown in Figure A, YC18 still maintains antibacterial activity (MIC = 12.5 μg / mL) after incubation in 50% plasma for different time periods (0 to 12 h), indicating that it also has good stability in the plasma environment.

[0062] Example 7: YC18 is not prone to drug resistance

[0063] The experiment selected the standard strain of Staphylococcus aureus S.aureus ATCC 6538, and cultured continuously for 24 hours as one generation. First, monoclonal colonies were selected and placed in liquid culture medium containing polypeptide YC18 (1 / 4MIC). Then, they were passaged once every 24 hours, and 10 μL of bacterial liquid was added to fresh liquid culture medium each time. 2 ml of bacterial liquid was preserved every 5 generations, and the polypeptide concentration was doubled every 10 generations. After 35 consecutive generations, the MIC was determined, and the resistance to the antibiotic vancomycin was used as a control.

[0064] like Figure 5 As shown in B, Staphylococcus aureus is not easily resistant to YC18.

[0065] Example 8: YC18 can inhibit bacterial growth in a mouse intraperitoneal infection model

[0066] 200 μL of Staphylococcus aureus ATCC 6538 (1×10 9 CFU / ml) was intraperitoneally injected into male C57BL / 6 mice (n=6). 2 hours after inoculation, YC18 (2, 4 mg / kg) and vancomycin (4 mg / kg) were intravenously injected to observe their therapeutic effects. To evaluate the spread of bacteria to target organs, mice were killed 4 hours after inoculation of bacteria, and blood was collected from the eyeballs to evaluate the concentration of cytokines and bacteria. At the same time, organs such as the spleen, liver, and lungs were collected. After grinding, the bacterial concentration was smeared on the plate. The blood was centrifuged at 2000g for 20 minutes to collect plasma. The collected organs were serially diluted 1000 times in sterile PBS, and the homogenate was smeared on LB agar plates to determine the bacterial load. The results are shown in the figure. Figure 6 As shown, YC18 treatment significantly inhibited bacterial colonization in the blood and target organs compared with the control group (PBS) after treatment with YC18 or vancomycin (VAN). Taken together, these data confirm that YC18 inhibits the dissemination of S. aureus to target organs in vivo.

[0067] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide, characterized in that The amino acid sequence of the antimicrobial peptide is shown in SEQ ID No.

1.

2. The antimicrobial peptide according to claim 1, characterized in that The antibacterial spectrum of the antimicrobial peptide includes Gram-positive bacteria and Gram-negative bacteria.

3. The antimicrobial peptide according to claim 1 or 2, characterized in that The antimicrobial spectrum of the antimicrobial peptide includes at least one of the following: Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

4. The active polypeptide YC18 obtained by artificially synthesizing and oxidatively folding the antimicrobial peptide according to claims 1 to 3.

5. The active polypeptide YC18 according to claim 4, characterized in that The oxidative folding comprises placing the artificially synthesized antimicrobial peptide in a buffer solution and reacting at 25° C. for 24 hours; The buffer comprises the following components at the following concentrations: Tris-HCl 0.1 M, NaCl 0.1 M, oxidized glutathione 0.5 mM, and reduced glutathione 5 mM, with a pH of 9.

4.

6. Use of the active polypeptide YC18 according to claim 4 or 5 in the preparation of antibacterial products.

7. The use according to claim 6, characterized in that The antibacterial spectrum of the antibacterial product includes at least one of the following: Staphylococcus aureus, drug-resistant Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

8. The application according to claim 7, characterized in that: The drug-resistant Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

9. An antibacterial product, characterized in that: The active ingredient comprises the antimicrobial peptide according to claims 1 to 3 or the active polypeptide YC18 according to claim 4 or 5, and also comprises excipients.

10. Use of the antimicrobial peptide according to claims 1 to 3, the active polypeptide YC18 according to claim 4 or 5, or the antimicrobial product according to claim 9 in the preparation of a medicament for treating diseases caused by pathogenic bacteria.

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