A highly effective and safe antimicrobial peptide variant and its application

Abstract

The present invention discloses an efficient and safe antimicrobial peptide variant and its application, which is based on the natural antimicrobial peptide Uperin3.6 sequence (as shown in sequence SEQNO:1), by replacing its disordered N-terminal segment with the basic amino acid arginine; repeating two amyloid C-terminal sequences and adding 3 arginines at both ends; and inserting a four-amino acid sequence DPDG (DPDG has the greatest tendency to form a β-turn structure) between the two repeated amyloid C-terminal sequences and adding 3 arginines at both ends. The antimicrobial peptide of the present invention can improve the activity and safety of the antimicrobial peptide, has good antimicrobial properties against both Gram-negative and Gram-positive bacteria, and has broad application prospects as a new antimicrobial agent.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an efficient and safe antimicrobial peptide variant and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are widely present in the biological world. They are important components of the body's innate immune system and constitute the first barrier for the host to defend against the invasion of pathogenic microorganisms. Most of these active peptides have the characteristics of strong acid and alkali resistance, thermal stability and broad-spectrum antibacterial. In 1980, Swedish scientist G. Boman and others discovered the world's first antimicrobial peptide, named Cecropins. Since the discovery of antibiotics, they have played an important role in the health of humans and animals, and have played an important role in promoting the production and growth of animals. However, due to the increasingly prominent problems of drug resistance and residues, more and more countries and regions have gradually implemented the prohibition of adding antibiotics to animal feed, and at the same time have continuously standardized the use of clinical antibiotics. In recent years, the research on new medical drugs and alternative antibiotic substances in animal feed has become a hot topic of concern and research, and the application research of antimicrobial peptides in medicine and animal production has also increased.

[0003] Today, antimicrobial peptides can be successfully isolated and classified from most organisms, from prokaryotes to humans. Antimicrobial peptides usually act on bacteria and play an important role in the natural immunity of eukaryotes. They are considered to be immune molecules effectively retained in mammals during ancient evolution. Therefore, as a class of polypeptide substances widely present in natural organisms, antimicrobial peptides can serve as the first line of defense for biological organisms and resist the invasion of pathogens. Antimicrobial peptides have multiple biological activities such as antibacterial, fungal, viral, anti-cancer and so on, and are not easy to develop drug resistance.

[0004] At present, the application of antimicrobial peptides in medicine has achieved some satisfactory results, and many new drugs have gradually entered the pharmaceutical market. The most fully studied are gramicidin, polymyxin and lactic acid. Gramicidin is limited to surface wounds and upper respiratory tract infections; on the contrary, polymyxin can be used not only to treat eye infections, but also gastrointestinal infections and systemic infections caused by drug-resistant Gram-negative bacteria. Another effective cyclic AMP that plays a role in the treatment of complex skin and skin structure infections caused by Staphylococcus aureus infection is daptomycin, which is often used in combination therapy to improve the success rate of treatment. Nisin is used for dental care, gastric ulcer treatment, and colon infection treatment. The antimicrobial peptide drug MAI278 developed by the amphibian antimicrobial peptide magainin is close to completing phase III clinical trials and shows good killing effects on viruses and tumor cells. Daptomycin is an anionic antimicrobial peptide developed by Cubit Pharmaceuticals and approved for marketing by the US Food and Drug Administration in September 2003. It can be used to treat skin infections and sepsis caused by Gram-positive bacteria such as Staphylococcus aureus. In addition, antibiotics are used as feed additives in animal production, which plays an important role in the development of animal husbandry. However, their residues in animals and animal products, as well as the drug resistance of pathogens, have a negative impact on human health and the environment. It can be seen that antimicrobial peptides, as the most promising substance to replace traditional antibiotics, have good application prospects in the fields of pharmaceutical industry and food additives. Summary of the invention

[0005] The main purpose of the present invention is to provide an efficient and safe antimicrobial peptide variant and its application. Studies have shown that the natural antimicrobial peptide Uperin3.6 derived from the skin gland secretions of Toadlet Uperoleiamjobergii (Australian toad) has good antimicrobial activity against some bacteria, but has a poor antibacterial effect on Escherichia coli (MIC value>100μg / mL). Therefore, the present invention is based on the antimicrobial peptide Uperin3.6, and through the modification of it, a variant with higher antimicrobial performance than the natural antimicrobial peptide Uperin3.6 is obtained, as a powerful alternative for the new generation of antimicrobial drugs.

[0006] The highly effective and safe antimicrobial peptide variant of the present invention is based on the amyloid segment C-terminal sequence (the 8th to 17th amino acids of Uperin3.6) of the natural antimicrobial peptide Uperin3.6 (as shown in the sequence SEQ NO: 1), and the original disordered N segment (the 1st to 7th amino acids) sequence of Uperin3.6 is replaced by the basic amino acid arginine (which has a stronger inhibitory effect on aggregation than other amino acids) to obtain the antimicrobial peptide variant Uperin3.6-N 7R , and its amino acid sequence is shown in SEQ NO:2.

[0007] The highly effective and safe antimicrobial peptide variant of the present invention repeats the amyloid C-terminal sequence (the 8th to 17th amino acids of Uperin3.6) of Uperin3.6 (as shown in sequence SEQ NO: 1) and adds 3 arginines at both ends to obtain the antimicrobial peptide variant Uperin3.6C-C-6R, whose amino acid sequence is shown in sequence SEQ NO: 3.

[0008] The highly effective and safe antimicrobial peptide variant of the present invention repeats the amyloid C-terminal sequence of Uperin3.6 (as shown in sequence SEQ NO: 1) (the 8th to 17th amino acids of Uperin3.6) and adds 3 arginines at both ends thereof, and inserts a four-amino acid sequence DPDG (having a strong tendency to form β-turn and inhibit amyloid aggregation) between the two repeated Uperin3.6 amyloid C-terminal sequences to obtain the antimicrobial peptide variant Uperin3.6C-6R-DPDG, whose amino acid sequence is shown in sequence SEQ NO: 4.

[0009] Furthermore, the C-terminus of the antimicrobial peptide is modified by amidation, and the N-terminus is modified by acetylation.

[0010] Furthermore, the modified antimicrobial peptide Uperin3.6-N 7R The molecular weight of Uperin3.6C-C-6R is 3307.04Da; the molecular weight of Uperin3.6C-6R-DPDG is 3691.38Da.

[0011] The polypeptide sequence encoding provided by the present invention is also within the protection scope of the present invention.

[0012] The application of the antimicrobial peptide variant of the present invention is to prepare antimicrobial preparations and related preparations such as anticancer preparations using the antimicrobial peptide variant.

[0013] The antibacterial preparation has an inhibitory and killing effect on Gram-negative bacteria and / or Gram-positive bacteria, including Escherichia coli, Bacillus subtilis, Micrococcus luteus, Escherichia fergusonii, Staphylococcus epidermidis, Pseudomonas aeruginosa, Listeria monocytogenes, etc.

[0014] The physicochemical performance evaluation and analysis of the wild-type and modified antimicrobial peptides included minimum inhibitory concentration (MIC) value, aggregation kinetics, secondary structure analysis, membrane interaction analysis and hemolytic activity analysis. The MIC value is the minimum concentration value for inhibiting Gram-positive bacteria Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Listeria monocytogenes and Gram-negative bacteria Escherichia coli, Escherichia fergusonii and Pseudomonas aeruginosa; aggregation kinetics is to incubate the antimicrobial peptide with thioflavin T (ThT), and the β-pleated sheet formed by the aggregation of the antimicrobial peptide can specifically bind to thioflavin T (ThT), thereby changing the fluorescence intensity, and the change in the number of aggregates formed is indicated by the change in the fluorescence intensity of thioflavin T binding; secondary structure analysis is to detect the far-ultraviolet circular dichroism spectrum of the antimicrobial peptide in PBS buffer and 2,2,2-trifluoroethanol TFEA simulating a membrane environment; membrane interaction analysis is to detect the changes in bacterial cell membranes after the antimicrobial peptide is incubated with bacteria through a scanning electron microscope; hemolytic activity analysis is to evaluate the killing effect of the antimicrobial peptide on normal mammalian cells through the activity of the antimicrobial peptide in destroying the red blood cell membrane to release hemoglobin.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The present invention is based on the existing natural antimicrobial peptide sequence, inserts a sequence with a specific structure and effect (DPDG with a strong tendency to form β-turn and inhibit amyloid aggregation) and adds a sequence with a specific effect at the end (three consecutive Args that can inhibit amyloid aggregation) for modification, obtains short peptides through artificial synthesis, and measures the antimicrobial activity and other related physical and chemical properties of the obtained series of short peptides to evaluate whether they are AMPs and the intensity of their activity; the present invention rationally designs and modifies the existing AMP sequence and antimicrobial activity information, and verifies it through experiments, which can efficiently and quickly screen antimicrobial peptides and solve the current problems of low activity of some natural AMPs and low modification success rate.

[0017] The modified antimicrobial peptides provided by the present invention have the following advantages: the series of antibacterial peptides have inhibitory and killing effects on Gram-negative bacteria and Gram-positive bacteria, specifically including Escherichia coli, Bacillus subtilis, Micrococcus luteus, Escherichia fergusonii, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Listeria monocytogenes, and have good application prospects. The modified antimicrobial peptides of the present invention can be used to prepare antibacterial drugs (antibacterial inhibitors, such as inhibitors against Gram-negative bacteria or inhibitors against Gram-positive bacteria). Because bacterial cells and cancer cells have many similarities, many antimicrobial peptides also show anticancer activity, so the anticancer application of the modified antimicrobial peptides of the present invention is also within the scope of protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Aggregation kinetics of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0019] Figure 2 Far-UV circular dichroism spectra of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0020] Figure 3 This is a scanning electron micrograph of the interaction between the natural antimicrobial peptide Uperin3.6 mutant and bacteria.

[0021] Figure 4 This is a comparison of the hemolytic activity of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0022] Figure 5 is the hemolytic rate of the natural antimicrobial peptide Uperin3.6 and its mutants. DETAILED DESCRIPTION

[0023] The present invention is not limited to the following specific implementations, and any design structure and concept of the present invention, with simple changes or modifications, falls within the protection scope of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0024] The present invention is further described in detail below with reference to specific examples.

[0025] The experimental drugs used in the present invention can all be purchased through commercial channels. The models and specifications of some experimental instruments used in the present invention are described below.

[0026] Antimicrobial peptide: The antimicrobial peptide used in the design of the present invention is prepared and synthesized by Shanghai Jier Biochemical Company using Fmoc solid phase synthesis, with a purity of more than 98%.

[0027] The bacterial species and source models used: Escherichia coli (E.coli) and Bacillus subtilis (B.subtilis) were provided by the laboratory; Micrococcus luteus (M.luteus), Staphylococcus epidermidis (S.epidermidis), Pseudomonas aeruginosa (P.aeruginosa), Listeria innocua (L.innocua), Salmonella typhimurium (S.typhimurium), and Bacillus cereus (B.cereus) were purchased from Beijing Biobo Biotechnology Co., Ltd.

[0028] Reagents used: PBS buffer, pure water, DMSO, ampicillin (100 mg / mL), and thioflavin.

[0029] Culture medium: LB liquid culture medium (sterilized at 121°C for 20 min).

[0030] Experimental instruments: SpectraMax M5 fully automatic microplate reader (MeiGu Molecular Instruments (Shanghai) Co., Ltd.), shaking incubator (Shanghai Minquan Instrument Co., Ltd.), circular dichroism spectrometer.

[0031] The original amino acid sequence of Uperin3.6 is shown in SEQ NO:1; the antimicrobial peptide variant Uperin3.6-N 7R The amino acid sequence of the antimicrobial peptide variant Uperin3.6C-C-6R is shown in SEQ NO: 2; the amino acid sequence of the antimicrobial peptide variant Uperin3.6C-6R is shown in SEQ NO: 3; the amino acid sequence of the antimicrobial peptide variant Uperin3.6C-6R-DPDG is shown in SEQ NO: 4.

[0032] Example 1: Design of antibacterial peptides

[0033] Table 1 below shows the sequences of the designed antimicrobial peptides.

[0034] Table 1. Peptide sequences designed and modified based on the natural antimicrobial peptide Uperin3.6 (the text is the amyloid C-terminal sequence of Uprin3.6, and the italics are the insertion and addition sequences)

[0035] name sequence C-terminal modification N-terminal modification UP3.6 GVIDAAKKVVNVLKNLF Amidation Acetylation <![CDATA[UP3.6-N 7R ]]> RRRRRRRKVVNVLKNLF Amidation Acetylation UP3.6C-C-6R RRRKVVNVLKNLFKVVNVLKNLFRRR Amidation Acetylation UP3.6C-6R-DPDG RRRKVVNVLKNLFPDDGKVVNVLKNLFRRR Amidation Acetylation

[0036] When the natural antimicrobial peptide Uperin3.6 and its variants were synthesized, their C-terminus was amidated and their N-terminus was acetylated. N-terminal acetylation and C-terminal amidation can reduce the total charge of the peptide, so its total solubility may be reduced, and it is closer to the parent protein, enhancing its ability to enter cells. And because terminal acetylation and amidation produce a mimic that is closer to the natural protein, the stability of the peptide can also be improved, thereby improving its resistance to exopeptidases such as proteases, endoplasmic reticulase and synthetase. Therefore, these modifications can improve the biological activity of the peptide.

[0037] Example 2: Determination of minimum inhibitory concentration (MIC) of antibacterial peptides

[0038] The determination of the minimum inhibitory concentration (MIC) was carried out according to the standard method of CLSI. The single colony of the strain was picked into the LB liquid medium with an inoculation stick, placed in a constant temperature shaking incubator at 37°C and 220rpm for overnight culture, and then prepared into a 104CFU / mL bacterial solution. 100μL of bacterial solution was added to a 96-well plate, and the antimicrobial peptide was diluted by a 2-fold dilution method. 100μL of antimicrobial peptide was added to each well to make the final concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625μg / mL, respectively. The negative control group replaced 100μL of the antimicrobial peptide solution with an equal amount of PBS buffer, and the positive control group replaced 100μL of the antimicrobial peptide solution with an equal amount of ampicillin. There were three parallel samples for each treatment. The 96-well plate was placed in a constant temperature shaking incubator at 37°C and 220rpm for 24h until a turbid liquid visible to the naked eye appeared in the negative control well. The concentration of antimicrobial peptide that can completely inhibit bacterial growth (wells are clear) is the MIC value of the antimicrobial peptide against the bacteria.

[0039] Table 2 shows the MIC values ​​(μg / mL) of the natural antimicrobial peptide Uperin3.6 and its mutants against various bacteria.

[0040] Table 2. MIC values ​​of Uperin3.6 and its designed mutants against various bacteria (μg / mL)

[0041]

[0042] The antimicrobial activity test results showed that compared with the natural antimicrobial peptide Uperin3.6, the antimicrobial peptide mutant UP3.6-N designed by us 7R , Uperin3.6C-C-6R and Uperin3.6C-6R-DPDG had significantly enhanced antibacterial activities.

[0043] Example 3: Aggregation kinetics of antimicrobial peptides

[0044] The β-pleated sheet aggregate structure formed by proteins or polypeptides can specifically bind to thioflavin T (ThT), thereby changing the fluorescence intensity. The change in fluorescence intensity of thioflavin T binding can indicate the change in the number of aggregates formed. Detection of Uperin3.6, Uperin3.6-C, Uperin3.6-N, Uperin3.6-N 7R, Uperin3.6C-6R-DPDG aggregation at different concentrations (20μM, 50μM, 100μM), ThT fluorescence kinetic samples were placed in a 96-well black ELISA plate, and three sets of parallel experiments and three sets of control experiments were set up for each sample. The ThT fluorescence intensity was measured at 25°C on a SpectraMax M5 multi-detection reader (Molecular Devices Limited), with an excitation wavelength of 440nm, an emission wavelength of 480nm, and a slit wavelength of 2nm. Before each measurement, the sample was shaken for 3 seconds to mix, and the fluorescence was read every two minutes.

[0045] Figure 1 The results of aggregation kinetics monitoring of the natural antimicrobial peptide Uperin3.6 and its mutants show that the C-terminal peptide segment of the truncated natural antimicrobial peptide Uperin3.6 aggregated; the disordered N-terminal peptide segment itself did not aggregate, but it inhibited the aggregation of the C-terminus; and the designed antimicrobial peptide mutants did not aggregate.

[0046] Example 4: Determination of the secondary structure of antimicrobial peptides

[0047] Far-ultraviolet circular dichroism spectroscopy was used to record the spectra of peptides in PBS aqueous solution, 10% trifluoroethanol TFEA, and 20% trifluoroethanol TFEA, respectively, with a wavelength range of 200-250 nm, a step length of 1 nm, and an optical path of 1 cm.

[0048] Figure 2 Far-ultraviolet circular dichroism spectra of natural antimicrobial peptide Uperin3.6 and its mutants. The results show that natural antimicrobial peptide Uperin3.6 forms a small amount of helical structure in the simulated membrane environment, indicating that it can interact with the membrane, but the intensity is not strong; while the designed antimicrobial peptides are induced to form more helical structures in the simulated membrane environment, indicating that they interact more strongly with the membrane. Therefore, it is inferred that the designed antimicrobial peptides can interact more strongly with the bacterial cell membrane, fold into a more regular secondary structure and insert into the bacterial membrane, thereby causing more serious membrane damage and cell death.

[0049] Example 5: Analysis of the interaction between antimicrobial peptides and bacterial cell membranes - SEM characterization

[0050] Bacteria were grown in LB at 37°C to exponential phase, centrifuged at 1000 rpm for 10 min, washed twice with 10 mM PBS, and resuspended to OD600 of 0.2. The cell suspension was incubated with different peptides at 1×MICs for 30 min at 37°C. The control group was run without peptides. After incubation, cells were collected by centrifugation at 4°C and 5000 rpm for 5 min and then washed three times with PBS. Then, bacterial cells were fixed with 2.5% (v / v) glutaraldehyde at 4°C overnight, and the fixed samples were washed twice with PBS and then dehydrated in graded ethanol solutions (50, 70, 90 and 100%) for 15 min (twice for 100% concentration and once for each of the remaining concentrations). They were then transferred to a mixture of absolute ethanol and tert-butyl alcohol (1:1, v / v) for 20 min, and then transferred to pure tert-butyl alcohol for 30 min. After freeze-drying and gold plating, they were observed by scanning electron microscopy.

[0051] Figure 3 This is a scanning electron microscope image of the natural antimicrobial peptide Uperin3.6 mutant interacting with bacteria. The SEM results show that after the modified antimicrobial peptide came into contact with bacteria, the bacterial cell membranes were all ruptured and the contents flowed out. This further proves that the antimicrobial peptide designed and modified in the present invention exerts its antimicrobial effect by interacting with the bacterial cell membrane. The mechanism may be that the antimicrobial peptide acts on the bacterial membrane and inserts the hydrophobic part into the lipid bilayer core to significantly damage the bacterial membrane, resulting in membrane damage and cell death.

[0052] Example 6: Determination of hemolytic activity of antimicrobial peptides

[0053] The killing effect of antimicrobial agents on normal mammalian cells is generally evaluated by the hemolytic activity on red blood cells. Anticoagulants are added to fresh blood collected from mice. The blood is diluted with saline, transferred to a centrifuge tube, centrifuged at 4°C and 3000rpm for 5 minutes, the upper serum is discarded, and the red blood cell pellet is left. The red blood cells are centrifuged and washed three times with an equal amount of saline to remove the residual serum. The red blood cells after centrifugation and washing are diluted to 8% (v / v) with saline. Take 300μL of red blood cell solution into a centrifuge tube, and then add 300μL of a series of 2-fold dilution gradient antimicrobial peptide solutions. Use 100μL of saline as the negative control of the experiment, and use 100μL of 0.1% Triton X-100 solution as the positive control of the experiment. Place the centrifuge tube in a constant temperature shaker at 37°C and 220rpm for incubation for 1h. After the reaction is completed, centrifuge at 4°C and 3000rpm for 5min to precipitate the red blood cells. The upper clear liquid in the centrifuge tube was aspirated into a 96-well plate, and the absorbance at 540 nm was detected by an ELISA instrument. The hemolysis rate was calculated according to the following formula:

[0054] Hemolysis rate (%) = (Abs peptide - Abs negative) / (Abs positive - Abs negative) × 100%.

[0055] Figure 4 This is a comparison of the hemolytic activity of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0056] Figure 5 is the hemolytic rate of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0057] The results are as follows Figure 4 , Figure 5 As shown in the figure, the hemolytic activity of the antimicrobial peptide UP3.6C-C-6R was 5.54% at a concentration of 3.125 μg / mL (hemolysis was considered to be hemolysis when the hemolysis rate was greater than 5%); UP3.6C-C-6R could inhibit bacteria only at a concentration above 3.125 μg / mL, and hemolysis had already occurred at this concentration, indicating that the antimicrobial peptide did not have good safety; while the antimicrobial peptide UP3.6-N 7R The hemolytic activity of UP3.6C-6R-DPDG was less than 5% at a high concentration of 100 μg / mL. These results indicate that the antimicrobial peptide UP3.6-N 7R UP3.6C-6R-DPDG has high antibacterial activity while ensuring high safety. The insertion of the four-amino acid sequence DPDG can significantly reduce the hemolytic activity of UP3.6C-C-6R and improve the safety of antimicrobial peptides.

[0058] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An efficient and safe antimicrobial peptide variant, characterized in that: The antimicrobial peptide variant is based on the C-terminal sequence of the amyloid segment of the natural antimicrobial peptide Uperin3.6, and the original amino acid sequence of the disordered N-terminal segment of Uperin3.6 is replaced with the basic amino acid arginine to obtain the antimicrobial peptide variant Uperin3.6-N 7R , whose amino acid sequence is shown in SEQ NO:2; The C-terminus of the antimicrobial peptide is modified by amidation, and the N-terminus is modified by acetylation.

2. A use of the antimicrobial peptide variant according to claim 1, characterized in that: Using the antimicrobial peptide variant to prepare an antimicrobial preparation; The antibacterial preparation has an inhibitory and killing effect on one or more of Escherichia coli, Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella typhimurium.

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