Tick antibacterial peptide and application thereof in preparation of anti-infective drugs

Abstract

The invention provides tick antibacterial peptide and application thereof in preparation of anti-infection drugs, and belongs to the technical field of antibacterial peptide. The tick antibacterial peptide KN-17 is screened from a hard tick shoulder protruding genome on the basis of bioinformatics and an AI technology, and the amino acid sequence is shown as SEQ ID NO: 1. The tick antibacterial peptide KN-17 has antibacterial activity on bacteria (including clinical drug-resistant strains) and fungi, is relatively weak in cytotoxicity and has no obvious hemolytic activity, and a new means is provided for preparation of anti-infective drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of antimicrobial peptides, and specifically relates to a tick antimicrobial peptide and application thereof in the preparation of anti-infection drugs. Background Art

[0002] Antibiotic resistance refers to the increased resistance of bacteria and other microorganisms to antibiotics, which makes them ineffective in inhibiting or killing them. According to the World Health Organization (WHO) and other public health agencies, if the problem of antibiotic resistance is not controlled, antibiotic-resistant bacteria may become a major threat to global health.

[0003] Antimicrobial peptides are a class of small molecule polypeptides that exist in nature. They are usually composed of 10 to 50 amino acids and are widely found in animals, plants and microorganisms. Antimicrobial peptides usually kill bacteria by binding to bacterial cell membranes, causing membrane rupture. They kill bacteria quickly, so they are not prone to drug resistance and are an ideal alternative to antibiotics.

[0004] Ticks are carriers and transmitters of many pathogens, but they themselves do not cause disease. Patent publication number CN104031133A discloses an antimicrobial peptide protein derived from Lepidium sylvaticum that has antibacterial properties against several specific bacteria, such as Staphylococcus aureus, Bacillus brevis and Salmonella typhimurium, and has antibacterial effects on Micrococcus luteus and Pseudomonas aeruginosa, but the antibacterial activity is not obvious, and there is no antibacterial activity against Escherichia coli. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a tick antimicrobial peptide, which has antimicrobial activity not only against a variety of bacteria but also against fungi.

[0006] The present invention provides a tick antimicrobial peptide KN-17, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] Preferably, the C-terminus of the tick antimicrobial peptide KN-17 is amidated.

[0008] The invention provides an antibacterial preparation, comprising the tick antibacterial peptide KN-17 and auxiliary materials.

[0009] Preferably, the concentration of the tick antimicrobial peptide KN-17 is not less than 3.2 μg / ml or 3.2 μg / g.

[0010] The invention provides a broad-spectrum antibacterial drug, comprising the tick antibacterial peptide KN-17 and pharmaceutically acceptable excipients.

[0011] The present invention provides the use of the tick antimicrobial peptide KN-17 or the antimicrobial preparation in preparing a medicine for preventing and / or treating bacterial and / or fungal infections.

[0012] Preferably, the bacteria include at least one of the following: Staphylococcus aureus (S. aureus), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli).

[0013] Preferably, the bacteria include clinical drug-resistant strains and clinical non-drug-resistant strains.

[0014] Preferably, the fungus comprises Candida albicans (C. albicans).

[0015] Preferably, the dosage form of the drug includes at least one of the following: oral dosage form, injection dosage form, external application dosage form and spray dosage form.

[0016] The present invention provides a tick antimicrobial peptide KN-17, the amino acid sequence of which is shown in SEQ ID NO: 1. The present invention predicts the full ORFs of the genome of Ixodes scapularis by TBtools, and the obtained candidate polypeptide sequences are interactively verified by the deep learning antimicrobial peptide prediction model (iAMPCN-master) and the AI ​​learning antimicrobial peptide prediction model (Macrel) method, and the predicted antimicrobial peptides are selected therefrom. The present invention verifies the antimicrobial activity of the antimicrobial peptides predicted by chemical synthesis under in vitro conditions, and the results show that the tick antimicrobial peptide KN-17 has good antimicrobial activity not only against clinically resistant strains and clinically non-resistant strains of bacteria (Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli), but also against fungi (Candida albicans), and the antibacterial MIC value is 3.125-25 μg / ml. In addition, the tick antimicrobial peptide KN-17 has low cytotoxicity and only exhibits significant antimicrobial activity at 200 μg / ml; the tick antimicrobial peptide KN-17 has no significant hemolytic activity. It can be seen that the tick antimicrobial peptide KN-17 has a broad-spectrum antimicrobial activity and good drug safety, and can be used to treat diseases caused by a variety of bacterial and fungal infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The amphipathic helical structure diagram of the tick antimicrobial peptide KN-17 predicted by the present invention;

[0018] Figure 2 The amphipathic helical structure diagram of the tick antimicrobial peptide RK-17 predicted by the present invention;

[0019] Figure 3 The cytotoxicity test results of tick antimicrobial peptide KN-17 at different concentrations;

[0020] Figure 4 This is the result of the hemolytic activity test of tick antimicrobial peptide KN-17. DETAILED DESCRIPTION

[0021] The invention provides a tick antimicrobial peptide KN-17, whose amino acid sequence is shown in SEQ ID NO: 1 (KNVLKPCWKFLRRFLRK).

[0022] In the present invention, the tick antimicrobial peptide KN-17 is predicted from the genome of the Ixodes persulcatus using TBtools software, and the candidate peptides are obtained by interactive verification using a deep learning antimicrobial peptide prediction model (iAMPCN-master) and an AI learning antimicrobial peptide prediction model (Macrel), from which the predicted antimicrobial peptide KN-17 is selected. The present invention proves through in vitro antibacterial experiments that the KN-17 peptide has broad-spectrum antibacterial and fungal activity.

[0023] In the present invention, the tick antimicrobial peptide KN-17 is preferably amidated at the C-terminus, which is beneficial to increase the positive charge of the antimicrobial peptide and enhance its antimicrobial effect. The present invention has no special restrictions on the preparation method of the tick antimicrobial peptide KN-17, and it can be obtained by chemical synthesis or genetic engineering recombination. In an embodiment of the present invention, the tick antimicrobial peptide KN-17 was synthesized by Hangzhou Gutuo Biotechnology Co., Ltd.

[0024] In the present invention, the tick antimicrobial peptide KN-17 forms a tertiary structure through spatial folding, and the positively charged amino acids on the cell membrane phospholipid molecules form electrostatic adsorption and bind to the membrane of bacteria or fungi, while the hydrophobic amino acids in the tick antimicrobial peptide KN-17 are inserted into the membrane of bacteria or fungi, thereby destroying the cell membrane and completing the bactericidal effect.

[0025] The invention provides an antibacterial preparation, comprising the tick antibacterial peptide KN-17 and auxiliary materials.

[0026] In the present invention, the concentration of the tick antimicrobial peptide KN-17 is preferably not less than 3.2 μg / ml or 3.2 μg / g, and can be 6.25 μg / g, 12.5 μg / g, 25 μg / g or 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml. The present invention has no special restrictions on the types of the excipients, and conventional excipients of antibacterial preparations known in the art can be used, such as hydrogels, polyvinyl alcohol / chitosan electrospun fiber membranes, liposomes, silver nanoparticles, peptide-chitosan copolymers, etc.

[0027] The invention provides a broad-spectrum antibacterial drug, comprising the tick antibacterial peptide KN-17 and pharmaceutically acceptable excipients.

[0028] In the present invention, the effective concentration of the tick antimicrobial peptide KN-17 is the same as that of the antimicrobial preparation, which is not described in detail. The present invention does not impose any special restrictions on the types of the pharmaceutically acceptable excipients, and the excipients of antimicrobial peptide drugs known in the art can be used.

[0029] The present invention provides the use of the tick antimicrobial peptide KN-17 or the antimicrobial preparation in preparing a medicine for preventing and / or treating bacterial and / or fungal infections.

[0030] In the present invention, the bacteria preferably include at least one of: Staphylococcus aureus (S.aureus), Acinetobacter baumannii (A.baumannii), Pseudomonas aeruginosa (P.aeruginosa) and Escherichia coli (E.coli). The bacteria preferably include clinical resistant strains and clinical non-resistant strains. In an embodiment of the present invention, Staphylococcus aureus (S.aureus) ATCC 6538, Acinetobacter baumannii (A.baumannii) ATCC 19606, Pseudomonas aeruginosa (P.aeruginosa) ATCC9027, Escherichia coli (E.coli) ATCC25922, Candida albicans (C.albicans) ATCC10231 were used as clinical non-resistant strains to carry out antibacterial activity tests, and the MIC value was 3.125-25 μg / mL. At the same time, antibacterial experiments were carried out with clinically resistant strains of Staphylococcus aureus (S. aureus) MRSA-Z, MRSA 11, MRSA22, Acinetobacter baumannii (A. baumannii) CRAB and clinically resistant strains of Pseudomonas aeruginosa (P. aeruginosa) CRPA. The MIC values ​​were 3.125-6.25 μg / mL, which are close to the antibacterial ability of vancomycin.

[0031] In the present invention, the fungus preferably includes Candida albicans (C.albicans). In the embodiment of the present invention, in the antibacterial activity experiment, the MIC value of the antimicrobial peptide against Candida albicans is 3.125-12.5 μg / mL.

[0032] In the present invention, the dosage form of the drug preferably includes at least one of the following: oral dosage form, injection dosage form, external application dosage form and spray dosage form. The oral dosage form preferably includes oral liquid, powder, tablet, capsule, etc. The injection dosage form preferably includes injection liquid. The external application dosage form preferably includes wet compress, hydrogel patch, external gel and ointment, etc.

[0033] The tick antimicrobial peptide provided by the present invention and its application in the preparation of anti-infection drugs are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Prediction of a tick antimicrobial peptide KN-17

[0036] High-quality genome data of Ixodes persulcatus (GCA_013358835.2) were downloaded from NCBI, and the full ORFs were predicted using TBtools to obtain protein, polypeptide or protein fragment sequences. The obtained sequences were initially filtered and screened to remove redundant and invalid sequences and output candidate polypeptide sequences.

[0037] The candidate peptides were interactively verified by the deep learning antimicrobial peptide prediction model (iAMPCN-master) and the AI ​​learning antimicrobial peptide prediction model (Macrel) method, and the predicted antimicrobial peptide KN-17 was selected, with the sequence of KNVLKPCWKFLRRFLRK (SEQ ID NO: 1).

[0038] At the same time, this example also predicts an antimicrobial peptide RK-15, the sequence of which is RKVRRCTRHVQRAAK (SEQ ID NO: 2).

[0039] Example 2

[0040] Structural prediction of tick antimicrobial peptides KN-17 and RK-15

[0041] The structure of tick antimicrobial peptide KN-17 was analyzed and the helical properties were calculated using the website (https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py).

[0042] The amphipathic helical structure of KN-17 peptide is shown in Figure 1 Among them, the blue amino acids are positively charged amino acids. These amino acids can form electrostatic adsorption with the negative charges on the phospholipid molecules on the cell membrane and bind to the membrane. The yellow amino acids are hydrophobic amino acids. Hydrophobic amino acids can be inserted into the membrane, thereby destroying the cell membrane and completing the bactericidal effect.

[0043] The amphipathic helical structure of RK-15 peptide is shown in Figure 2 The blue amino acids are positively charged amino acids, which can form electrostatic adsorption with the negative charges on the phospholipid molecules of the cell membrane and bind to the membrane. The yellow amino acids are hydrophobic amino acids, but the number of yellow amino acids is small, and it is difficult to insert into the membrane and cannot destroy the cell membrane. The expected bactericidal effect is poor.

[0044] Example 3

[0045] Synthesis of tick antimicrobial peptides KN-17 and RK-15

[0046] According to the amino acid sequence of tick antimicrobial peptides KN-17 and RK-15, Hangzhou Gutuo Biotechnology Co., Ltd. was commissioned to chemically synthesize them. At the same time, the C-terminal amino acid was amidated (-NH2) and modified for in vitro experiments to verify their specific antimicrobial activity.

[0047] Example 4

[0048] Determination of the antibacterial activity of tick antimicrobial peptides KN-17 and RK-15

[0049] 1. Activate different standard strains purchased from Guangdong Microbial Culture Collection Center, inoculate them onto solid culture media (LB culture media for bacterial culture, YM culture media for fungal culture), and invert and culture them in a 37°C constant temperature incubator for 18 to 24 hours. Then pick a single colony on the solid culture medium and add it to a newly prepared and high-temperature sterilized liquid culture medium, and culture it in a constant temperature shaker at 180rpm and 37°C until the logarithmic growth phase. Take 2ml of the logarithmic growth phase bacterial solution and centrifuge it at 3500rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the bacterial solution with 0.9% saline, wash the fresh bacterial solution repeatedly 3 times, and finally resuspend the bacterial solution with an appropriate amount of saline. Use a pipette to take 200μl of the bacterial solution and add it to a sterile 96-well plate. Use an enzyme reader to detect its light absorbance at 600nm, and calculate it according to 1OD 600 =1×10 9 CFU / ml was used to calculate the bacterial concentration. The bacterial concentration was diluted to 2×10 CFU / ml using RPMI1640 medium (containing 10% fetal bovine serum for bacterial culture) or 1 / 4YM+3 / 4RPMI1640 medium (containing 10% fetal bovine serum for fungal culture). 5 CFU / ml. The drug was then diluted using a two-fold gradient dilution method. 100 μl of saline was added to a sterile 96-well plate in advance. 200 μl of the sample to be tested (200 μg / ml) was added to the first well. 100 μl was pipetted and added to the second well, mixed and diluted in sequence. 100 μl was discarded from the last well. Finally, 100 μl of a 2×10 5 CFU / ml bacterial solution was pipetted and mixed, and then placed in a 37°C constant temperature incubator for 16 to 24 hours. The light absorption value of the bacterial solution at 600nm was detected by an ELISA instrument, and the wells where no bacterial growth was detected were taken as the minimum inhibitory concentration, i.e., the MIC value.

[0050] The formula of the culture medium involved in the experiment is as follows:

[0051] LB liquid medium: Tryptone: 10 g / L; Yeast extract: 5 g / L; Sodium chloride (NaCl): 10 g / L;

[0052] LB solid medium: Tryptone: 10 g / L; Yeast extract: 5 g / L; Sodium chloride (NaCl): 10 g / L; Agar powder: 15 g / L;

[0053] YM liquid culture medium: YM powder 21g / L. YM powder was purchased from item number MB0072, company: Meilun Biotechnology

[0054] YM solid medium: YM 21g / L; agar powder: 15g / L;

[0055] RPMI1640 culture medium: purchased from CORNING, 10-040-CV.

[0056] Table 1 MIC values ​​of KN-17 against several bacterial and fungal strains

[0057] Strain name MIC (μg / ml) Staphylococcus aureus (S. aureus) ATCC6538 3.125 Acinetobacter baumannii (A.baumannii) ATCC19606 3.125 Pseudomonas aeruginosa ATCC9027 25 Escherichia coli (E.coli) ATCC25922 6.25 Candida albicans ATCC10231 12.5

[0058] The results showed that KN-17 had a broad-spectrum antibacterial activity, especially against Staphylococcus aureus (S. aureus) ATCC6538 and Acinetobacter baumannii (A. baumannii) ATCC19606, with a MIC of 3.125 μg / ml.

[0059] 2. Activity of antimicrobial peptides: Determine the minimum inhibitory concentration of KN-17 against different clinical drug-resistant strains. The MIC determination method is the same as above.

[0060] Table 2 MIC values ​​of KN-17 against different clinical drug-resistant strains

[0061] Bacteria strain MIC (μg / ml) Clinical drug-resistant strain MRSA-Z of Staphylococcus aureus (S. aureus) 6.25 Clinical drug-resistant strain MRSA11 of Staphylococcus aureus (S. aureus) 3.125 Clinical drug-resistant strain MRSA22 of Staphylococcus aureus (S. aureus) 3.125 Clinical drug-resistant strains of Acinetobacter baumannii (A.baumannii) 3.125 Clinical drug-resistant strains of Pseudomonas aeruginosa (CRPA) 12.5

[0062] Note: Clinical drug-resistant bacteria MRSA-Z, MRSA 11 and MRSA22 of Staphylococcus aureus (S. aureus) are disclosed in the prior art 1 (Identification and Characterization of RK22, a Novel Antimicrobial Peptide from Hirudinaria manillensis against Methicillin Resistant Staphylococcus aureus, DOI: 10.3390 / ijms241713453);

[0063] The clinical drug-resistant bacteria CRAB of A. baumannii and the clinical drug-resistant bacteria CRPA of P. aeruginosa are disclosed in prior art 2 (James, Mwangi, Yizhu, et al. The antimicrobial peptide ZY4 combats multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii infection. [J]. Proceedings of the National Academy of Sciences of the United States of America, 2019. DOI: 10.1073 / pnas.1909585117.).

[0064] The results showed that KN-17 also had strong antibacterial activity against clinical drug-resistant bacteria of Staphylococcus aureus and Acinetobacter baumannii. The MICs of clinical drug vancomycin against MRSAA2, MRSAA3, and MRSA A4 were 1.56μg / ml, 3.125μg / ml, and 0.39μg / ml, respectively; the antibacterial activity of KN-17 against some clinical drug-resistant bacteria was close to or equivalent to that of vancomycin.

[0065] The MIC of the antibacterial activity of RK-15 peptide against the above standard strains was simultaneously tested according to the above scheme. The results showed that it did not show any antibacterial activity even at the highest tested concentration of 50 μg / ml.

[0066] Example 5

[0067] Cytotoxicity assay of tick antimicrobial peptide KN-17

[0068] 1) Take out the human immortalized keratinocytes (HaCaT cells) frozen in the liquid nitrogen tank in the laboratory and put them into a preheated 37℃ water bath for rapid thawing. Centrifuge at 1500rpm for 5min at room temperature, then discard the supernatant on a sterile clean bench and resuspend them in DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Then transfer them to a cell culture bottle and culture them in a 5% carbon dioxide, 37℃ constant temperature cell culture incubator.

[0069] 2) CCK8 detection of sample cytotoxicity

[0070] After 1 to 3 days of cell culture, when cells are observed to cover the bottom of the culture flask under a microscope, discard the culture medium and wash once with PBS. Add 1 ml of trypsin to the cell culture flask and gently shake the flask. When cells are observed to fall off the flask, use fresh DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to terminate digestion and mix with a pipette. Count the cell suspension with a cell counter and adjust its concentration to about 1×10 5 / ml. Add the diluted cell suspension to a sterile 96-well plate, 100μl per well, and add 100μl of PBS buffer to the outermost well of the sterile 96-well plate to maintain the humidity in the plate. Culture in a 37℃ constant temperature cell culture incubator overnight to allow the cells to adhere to the wall. After the cells adhere to the wall, remove the culture medium with a pipette and add 200μl of the sample to be tested diluted with DMEM culture medium at different concentrations. The concentration gradient is set to 200μg / ml, 100μg / ml, 50μg / ml, 25μg / ml, 12.5μg / ml, 0μg / ml, and 4 replicates are set for each concentration. Place in a constant temperature incubator at 37℃ and 5% CO2 for 24 hours, and add 10μl CCK8 to each well. Continue to incubate in the incubator for about 2 to 4 hours, and use a multifunctional microplate reader to detect the absorbance at 450nm.

[0071] Results Figure 3 The results showed that the cytotoxicity of KN-17 was weak, and it only showed significant antibacterial activity at 200 μg / ml, while its minimum inhibitory concentration was 3.125-25 μg / ml.

[0072] Example 6

[0073] Determination of hemolytic activity of tick antimicrobial peptide KN-17

[0074] The collected fresh mouse blood was placed in an anticoagulant tube containing sodium citrate and mixed by inversion. The collected blood was mixed with sterilized Ash solution at a volume ratio of 1:1, centrifuged at 1500rpm for 5min, and the supernatant was discarded. Then the red blood cells were repeatedly washed with physiological saline for 3 to 5 times until the supernatant no longer appeared red after centrifugation. The red blood cells were resuspended in an appropriate amount of physiological saline, and the red blood cell concentration was adjusted so that the absorbance value of 100μl red blood cell suspension plus 100μl Triton X-100 (10%) at 540nm of the microplate reader was 0.7 to 0.8. Then the sample to be tested was prepared into 100μl of different gradient concentrations and added to a 96-well plate (each concentration was set to 3 replicates), and then 100μl of red blood cell suspension was added and incubated in a 37℃ constant temperature incubator for 30min, followed by centrifugation at 1500rpm for 5min, and the supernatant was aspirated and the light absorption value at 540nm was detected by a microplate reader. The concentrations of the samples to be tested were 0, 25 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml, and the positive drug was 10% TritonX-100 (PC).

[0075] Results Figure 4 Result analysis: The antimicrobial peptide KN-17 had no obvious hemolytic activity.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A tick antimicrobial peptide KN-17, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. The tick antimicrobial peptide KN-17 according to claim 1, characterized in that: The C-terminus of the tick antimicrobial peptide KN-17 is amidated.

3. An antibacterial preparation, characterized in that It comprises the tick antimicrobial peptide KN-17 as described in claim 1 or 2 and auxiliary materials.

4. The antibacterial preparation according to claim 3, characterized in that: The concentration of the tick antimicrobial peptide KN-17 is not less than 3.2 μg / ml or 3.2 μg / g.

5. A broad-spectrum antibacterial drug, characterized in that: It comprises the tick antimicrobial peptide KN-17 as claimed in claim 1 or 2 and pharmaceutically acceptable excipients.

6. Use of the tick antimicrobial peptide KN-17 according to claim 1 or 2 or the antimicrobial preparation according to claim 3 or 4 in the preparation of a medicament for preventing and / or treating bacterial and / or fungal infections.

7. The use according to claim 6, characterized in that: The bacteria include at least one of the following: Staphylococcus aureus (S. aureus), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli).

8. The use according to claim 7, characterized in that: The bacteria include clinical drug-resistant strains and clinical non-drug-resistant strains.

9. The use according to claim 6, characterized in that: The fungi include Candida albicans (C. albicans).

10. The use according to any one of claims 6 to 9, characterized in that: The dosage form of the drug includes at least one of the following: oral dosage form, injection dosage form, external application dosage form and spray dosage form.

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