Efficient and safe antibacterial peptide variant and application thereof

Abstract

The invention discloses an efficient and safe antibacterial peptide variant and application thereof, which is based on a natural antibacterial peptide Upin3.6 sequence (as shown in SEQ NO: 1), and a disordered N-terminal segment of the natural antibacterial peptide Upin3.6 sequence is replaced by basic amino acid arginine; repeating the two amyloid C-terminal sequences and adding three arginines to each of the two ends of the amyloid C-terminal sequences; and inserting a tetra-amino acid sequence DPDG (DPDG has the greatest tendency to form a beta-turn structure) between the two repeated amyloid C-terminal sequences, and adding three arginines to each of the two ends of the tetra-amino acid sequence DPDG. According to the antibacterial peptide, the activity and safety of the antibacterial peptide can be improved, the antibacterial peptide has good antibacterial performance on gram-negative bacteria and gram-positive bacteria, and the antibacterial peptide serving as a novel antibacterial agent has wide application prospects.

Description

[0001] This application is a divisional application. The filing date of the original application is February 14, 2023, the application number is 2023101110084, and the invention title is "An efficient and safe antimicrobial peptide variant and its application". Technical Field

[0002] The present invention belongs to the field of biotechnology, and particularly relates to an efficient and safe antimicrobial peptide variant and its application. Background Art

[0003] Antimicrobial peptides (AMPs), also known as host defense peptides, are widely present in the biological world and are important components of the body's innate immune system, forming the first line of defense against the invasion of pathogenic microorganisms by the host. Most of these active polypeptides have characteristics such as strong acid-base resistance, thermal stability, and broad-spectrum antibacterial properties. In 1980, Swedish scientist G. Boman et al. 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 significantly promoted 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 prohibited the addition of antibiotics to animal feed and continuously regulated the use of clinical antibiotics. In recent years, the research on new medical drugs and substances to replace antibiotics 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.

[0004] Today, antimicrobial peptides have been 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 immune molecules effectively retained in mammalian bodies 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 of biological organisms and can resist the invasion of pathogens. Antimicrobial peptides have various biological activities such as antibacterial, antifungal, antiviral, and cancer cell killing, and are not easily resistant.

[0005] At present, some satisfactory results have been achieved in the medical application of antimicrobial peptides, and many new drugs have gradually entered the pharmaceutical market. The most well-studied ones are tyrocidine, polymyxin, and lactacin. Tyrocidine is restricted for use in superficial wounds and upper respiratory tract infections; on the contrary, polymyxin can be used not only for the treatment of eye infections, but also for gastrointestinal infections and systemic infections caused by drug-resistant Gram-negative bacteria. Another effective one that plays a role in the treatment of complicated skin and skin structure infections caused by Staphylococcus aureus infections is daptomycin, which is often used in combination therapy to improve the treatment success rate. Nisin is used for dental care, the treatment of gastric ulcers, and the treatment of colon infections. The antimicrobial peptide drug MAI278 developed from 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 by the US Food and Drug Administration for marketing in September 2003. It can be used for the treatment of 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 has played an important role in the development of the livestock industry. However, their residues in animals and animal products, as well as the problem of drug resistance produced by pathogenic bacteria, have had a negative impact on human health and the environment. It can be seen that antimicrobial peptides, as the most promising substances to replace traditional antibiotics, have good application prospects in the pharmaceutical industry and food additives and other fields. Summary of the Invention

[0006] The main object of the present invention is to provide a highly efficient and safe antimicrobial peptide variant and its application. Research shows that the natural antimicrobial peptide Uperin3.6 derived from the skin gland secretion of Toadlet Uperoleiamjobergii (Australian toad) has good antibacterial activity against some bacteria, but has poor antibacterial effect against Escherichia coli (MIC value > 100 μg / mL). Therefore, based on the antimicrobial peptide Uperin3.6, the present invention obtained a variant with higher antibacterial performance than the natural antimicrobial peptide Uperin3.6 by modifying it, as a strong candidate for a new generation of antibacterial drugs.

[0007] The highly efficient and safe antimicrobial peptide variant of the present invention is based on the amyloid region C-terminal sequence (the 8th to 17th amino acids of Uperin3.6) of the natural antimicrobial peptide Uperin3.6 (as shown in sequence SEQ NO:1), and replaces the original disordered N-terminal (the 1st to 7th amino acids) sequence of Uperin3.6 with 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 as shown in sequence SEQ NO:2.

[0008] The highly efficient and safe antimicrobial peptide variant of the present invention repeats the amyloid C-terminal sequence of Uperin3.6 (shown in SEQ NO:1, amino acids 8 to 17 of Uperin3.6) and adds 3 arginines at each end to obtain the antimicrobial peptide variant Uperin3.6C-C-6R, and its amino acid sequence is shown in SEQ NO:3.

[0009] The highly efficient and safe antimicrobial peptide variant of the present invention repeats the amyloid C-terminal sequence of Uperin3.6 (shown in SEQ NO:1, amino acids 8 to 17 of Uperin3.6) and adds 3 arginines at each end, and inserts the four-amino acid sequence DPDG (which has a strong tendency to form a β-turn and inhibits amyloid aggregation) between the two repeated amyloid C-terminal sequences of Uperin3.6 to obtain the antimicrobial peptide variant Uperin3.6C-6R-DPDG, and its amino acid sequence is shown in SEQ NO:4.

[0010] Furthermore, the C-terminus of the antimicrobial peptide is modified by amidation, and the N-terminus is modified by acetylation.

[0011] Furthermore, the modified antimicrobial peptide Uperin3.6-N 7R has a molecular weight of 2307.79 Da; Uperin3.6C-C-6R has a molecular weight of 3307.04 Da; Uperin3.6C-6R-DPDG has a molecular weight of 3691.38 Da.

[0012] The polypeptide sequence encoding provided by the present invention is also within the protection scope of the present invention.

[0013] The application of the antimicrobial peptide variant of the present invention is to prepare antimicrobial preparations and related preparations such as anticancer preparations with the antimicrobial peptide variant.

[0014] The antimicrobial preparation has inhibitory and killing effects on both Gram-negative bacteria and / or Gram-positive bacteria. Specifically, it includes Escherichia coli, Bacillus subtilis, Micrococcus luteus, Escherichia fergusonii, Staphylococcus epidermidis, Pseudomonas aeruginosa, Listeria innocua, etc.

[0015] The physicochemical properties of wild-type and modified antimicrobial peptides were evaluated and analyzed. The physicochemical property evaluation and analysis included the minimum inhibitory concentration (MIC) value, aggregation kinetics, secondary structure analysis, membrane interaction analysis, and hemolytic activity analysis. The MIC value was the minimum concentration value for inhibiting Gram-positive bacteria such as Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, and Listeria innocua, as well as Gram-negative bacteria such as Escherichia coli, Escherichia fergusonii, and Pseudomonas aeruginosa. Aggregation kinetics involved incubating the antimicrobial peptide with thioflavin T (ThT). The β-sheets formed by the aggregation of the antimicrobial peptide could specifically bind to thioflavin T (ThT), resulting in a change in fluorescence intensity. The change in the fluorescence intensity of the bound thioflavin T was used to indicate the change in the number of aggregates formed. Secondary structure analysis was to detect the far-ultraviolet circular dichroism spectrum of the antimicrobial peptide in PBS buffer and 2,2,2-trifluoroethanol (TFEA) in a simulated membrane environment. Membrane interaction analysis was to detect the changes in the bacterial cell membrane after incubation of the antimicrobial peptide with bacteria by scanning electron microscopy. Hemolytic activity analysis was to evaluate the killing effect of the antimicrobial peptide on normal mammalian cells by the activity of the antimicrobial peptide to disrupt the erythrocyte membrane and release hemoglobin.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] Based on the existing natural antimicrobial peptide sequence, the present invention inserts a sequence with a specific structure and effect (DPDG with a strong tendency to form β-turns and inhibit amyloid aggregation) and adds a sequence with a specific effect (three consecutive Arg that can inhibit amyloid aggregation) at the end for modification. Short peptides are obtained by artificial synthesis, and the relevant physicochemical properties such as the antibacterial activity of the obtained series of short peptides are measured to evaluate whether they are AMPs and their activity intensity. Based on the existing AMP sequence and antibacterial activity information, the present invention rationally designs and modifies them and verifies them through experiments, which can efficiently and rapidly screen out antimicrobial peptides and solve problems such as low activity of some natural AMPs and low success rate of modification.

[0018] The modified antimicrobial peptides provided by the present invention have the following advantages: This series of antibacterial polypeptides have inhibitory and killing effects on both Gram-negative and Gram-positive bacteria, specifically including Escherichia coli, Bacillus subtilis, Micrococcus luteus, Escherichia fergusonii, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Listeria innocua, and have good application prospects. The modified antimicrobial peptides of the present invention can be used to prepare antibacterial drugs (inhibitors against bacteria, such as inhibitors against Gram-negative bacteria or inhibitors against Gram-positive bacteria). Since there are many similarities between bacterial cells and cancer cells, and many antimicrobial peptides also exhibit anticancer activity, the anticancer application of the modified antimicrobial peptides of the present invention is also within the scope of protection. Description of the Drawings

[0019] Figure 1 It is the aggregation kinetics of natural antimicrobial peptide Uperin3.6 and its mutants.

[0020] Figure 2 It is the far-ultraviolet circular dichroism spectrum of natural antibacterial peptide Uperin3.6 and its mutants.

[0021] Figure 3 It is the scanning electron microscopy image of the interaction between the mutant of natural antibacterial peptide Uperin3.6 and bacteria.

[0022] Figure 4 It is the comparison chart of the hemolytic activities of natural antibacterial peptide Uperin3.6 and its mutants.

[0023] Figure 5 It is the hemolysis rate of natural antibacterial peptide Uperin3.6 and its mutants. Specific implementation manners

[0024] The present invention is not limited to the following specific implementation manners. Any simple changes or modifications made by adopting the design structure and concept of the present invention fall within the protection scope of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0025] The following further elaborates the present invention in detail with specific examples.

[0026] All the experimental drugs used in the present invention can be obtained through commercial channels. The models and specifications of some experimental instruments used in the present invention are described below.

[0027] Antibacterial peptides: The antibacterial peptides used in the design of the present invention were prepared and synthesized by Shanghai Gil Biochemical Co., Ltd. using Fmoc solid-phase synthesis, with a purity of over 98%.

[0028] The strains used and their source models: Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), provided by the laboratory; Micrococcus luteus (M. luteus), Staphylococcus epidermidis (S. epidermidis), Pseudomonas aeruginosa (P. aeruginosa), Listeria innocua (L. innocua), Salmonella typhimurium (S. typhimurium), Bacillus cereus (B. cereus), purchased from Beijing BioWin Biotechnology Co., Ltd.

[0029] The reagents used: PBS buffer, pure water, DMSO, ampicillin (100 mg / mL), thioflavin.

[0030] Culture medium: LB liquid medium (sterilized at 121 °C for 20 min).

[0031] Experimental instruments: SpectraMax M5 fully automatic microplate reader (Molecular Devices (Shanghai) Co., Ltd.), shaking incubator (Shanghai Minquan Instrument Co., Ltd.), circular dichroism spectrometer.

[0032] The original amino acid sequence of Uperin3.6 is shown in detail in Sequence SEQ NO: 1; the amino acid sequence of the antimicrobial peptide variant Uperin3.6-N 7R is shown in detail in Sequence SEQ NO: 2; the amino acid sequence of the antimicrobial peptide variant Uperin3.6C-C-6R is shown in detail in Sequence SEQ NO: 3; the amino acid sequence of the antimicrobial peptide variant Uperin3.6C-6R-DPDG is shown in detail in Sequence SEQ NO: 4.

[0033] Example 1: Design of antibacterial polypeptides

[0034] Table 1 below shows the sequences of the antibacterial peptides after design and modification.

[0035]

[0036] When the natural antimicrobial peptide Uperin3.6 and its variants are synthesized, their C-termini are amidated and their N-termini are acetylated. N-terminal acetylation and C-terminal amidation can reduce the total charge of the peptide, so its total solubility may decrease, and it makes the peptide closer to the parent protein, enhancing its ability to enter cells. And because the terminal acetylation and amidation generate mimetics closer to natural proteins, the stability of the peptide can also be improved, thereby increasing its resistance to exopeptidases such as proteases, peptidases and synthases. Therefore, these modifications can improve the biological activity of the peptide.

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

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

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

[0040]

[0041] The results of the antibacterial activity detection showed that compared with the natural antimicrobial peptide Uperin3.6, the antibacterial activities of the antimicrobial peptide mutants UP3.6-N 7R 、Uperin3.6C-C-6R and Uperin3.6C-6R-DPDG designed and modified by us were significantly enhanced.

[0042] Example 3: Determination of the aggregation kinetics of antimicrobial peptides

[0043] The β-sheet aggregate structure formed by proteins or polypeptides can specifically bind to thioflavin T (ThT), thus causing a change in fluorescence intensity. The change in the fluorescence intensity of the ThT binding can be used to indicate the change in the number of aggregates formed. Detect Uperin3.6, Uperin3.6-C, Uperin3.6-N, Uperin3.6-N 7R, The aggregation of Uperin3.6C-6R-DPDG at different concentrations (20 μM, 50 μM, 100 μM) was measured. The ThT fluorescence kinetics samples were placed in a 96-well black microplate. For each sample, three sets of parallel experiments and three sets of control experiments were set up. The ThT fluorescence intensity was measured at 25 °C on a SpectraMax M5 multi-detection reader (Molecular Devices Limited), with an excitation wavelength of 440 nm, an emission wavelength of 480 nm, and a slit wavelength of 2 nm. Before each measurement, the sample needed to be vibrated for 3 seconds to mix evenly, and the fluorescence was read every two minutes.

[0044] Figure 1 This is the monitoring result of the aggregation kinetics of the natural antimicrobial peptide Uperin3.6 and its mutants. The aggregation kinetics results showed that the C-terminal peptide segment of the truncated natural antimicrobial peptide Uperin3.6 aggregated; the disordered N-terminal peptide segment did not aggregate by itself, but it inhibited the aggregation of the C-terminal; while none of the designed and modified antimicrobial peptide mutants aggregated.

[0045] Example 4: Determination of the secondary structure of antimicrobial peptides

[0046] Far-UV circular dichroism spectra were recorded for the peptides in PBS aqueous solution, 10% trifluoroethanol (TFEA), and 20% trifluoroethanol (TFEA) respectively. The wavelength range was 200 - 250 nm, the step size was 1 nm, and the optical path was 1 cm.

[0047] Figure 2 These are the far-UV circular dichroism spectra of the natural antimicrobial peptide Uperin3.6 and its mutants. The results showed that the natural antimicrobial peptide Uperin3.6 formed a small amount of helical structure in the simulated membrane environment, indicating that it could interact with the membrane, but the strength was not strong; while the designed and modified antimicrobial peptides were induced to form more helical structures in the simulated membrane environment, indicating that their interaction with the membrane was stronger. Therefore, it was inferred that the designed and modified antimicrobial peptides could interact more strongly with the bacterial cell membrane, fold into a more regular secondary structure and insert into the bacterial membrane, resulting in more serious membrane damage and cell death.

[0048] Example 5: Analysis of the interaction between antimicrobial peptides and bacterial cell membranes - SEM characterization

[0049] The bacteria were cultured in LB at 37 °C until the exponential phase, centrifuged at 1000 rpm for 10 min, washed twice with 10 mM PBS, and resuspended to an OD600 of 0.2. The cell suspension was incubated with different polypeptides at 1×MICs at 37 °C for 30 min. The control group was run without peptides. After incubation, the cells were collected by centrifugation at 5000 rpm for 5 min at 4 °C, and then washed 3 times with PBS. Then, the bacterial cells were fixed with 2.5% (v / v) glutaraldehyde at 4 °C overnight. The fixed samples were washed twice with PBS and then dehydrated in a graded ethanol solution (50, 70, 90, and 100%) for 15 min (twice at 100% concentration and once for the rest of the concentrations). Then they were transferred to a mixture of absolute ethanol and tert-butanol (1:1, v / v) for 20 min, and then transferred to pure tert-butanol for 30 min. After freeze-drying and gold plating, they were observed with a scanning electron microscope.

[0050] Figure 3 This is a scanning electron micrograph of the interaction between the natural antibacterial peptide Uperin3.6 mutant and bacteria. The SEM results showed that after the modified antibacterial peptide contacted the bacteria, the bacterial cell membranes were ruptured and the contents leaked out. This further proved that the designed and modified antibacterial peptide of the present invention exerted its antibacterial effect by interacting with the bacterial cell membrane. The mechanism might be that the antibacterial peptide acted on the bacterial membrane, inserted the hydrophobic part into the core of the lipid bilayer to significantly damage the bacteria's membrane, resulting in membrane damage and cell death.

[0051] Example 6: Determination of the hemolytic activity of antibacterial peptides

[0052] The killing effect of antibacterial agents on normal mammalian cells is generally evaluated by the hemolytic activity on red blood cells. Anticoagulant was added to the freshly collected mouse blood. The blood was diluted with physiological saline and transferred to a centrifuge tube. It was centrifuged at 3000 rpm for 5 min at 4 °C, and the upper serum was discarded, leaving the red blood cell pellet. The red blood cells were centrifuged and washed three times with an equal volume of physiological saline to remove the remaining serum. The centrifuged and washed red blood cells were diluted with physiological saline to 8% (v / v). Take 300 μL of the red blood cell solution into a centrifuge tube, and then add 300 μL of a series of antibacterial peptide solutions with a two-fold dilution gradient. 100 μL of physiological saline was used as the negative control for the experiment, and 100 μL of 0.1% Triton X-100 solution was used as the positive control for the experiment. The centrifuge tubes were placed in a constant temperature shaker at 37 °C and 220 rpm for incubation for 1 h. After the reaction ended, it was centrifuged at 3000 rpm for 5 min at 4 °C to precipitate the red blood cells. The supernatant in the centrifuge tube was aspirated into a 96-well plate, and the absorbance at 540 nm was detected with an enzyme-linked immunosorbent assay reader. The hemolysis rate was calculated according to the following formula:

[0053] Hemolysis rate (%) = (Abs peptide - Abs negative) / (Abs positive - Abs negative) × 100%.

[0054] Figure 4 It is a comparison chart of the hemolytic activities of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0055] Figure 5 They are the hemolysis rates of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0056] The results are as Figure 4 、 Figure 5 shown. The hemolytic activity of the antimicrobial peptide UP3.6C-C-6R is 5.54% at a concentration of 3.125 μg / mL (hemolysis is considered when the hemolysis rate is greater than 5%); UP3.6C-C-6R can only inhibit bacteria at a concentration above 3.125 μg / mL, and hemolysis has occurred at this concentration, indicating that this antimicrobial peptide does not have good safety; while the antimicrobial peptides UP3.6-N 7R and UP3.6C-6R-DPDG have hemolytic activities below 5% at a high concentration of 100 μg / mL. These results show that after design and modification, the antimicrobial peptides UP3.6-N 7R and UP3.6C-6R-DPDG ensure relatively high safety while having high antibacterial activities. 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 the antimicrobial peptide.

[0057] As mentioned above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, and all should be covered within 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 3 arginines are added to each end of its tandem repeat sequence to obtain the antimicrobial peptide variant Uperin3.6C-C-6R, and its amino acid sequence is as shown in SEQ NO: 3; Alternatively, the antimicrobial peptide variant is based on the C-terminal sequence of the amyloid segment of the natural antimicrobial peptide Uperin3.6, 3 arginines are added to each end of its tandem repeat sequence, and a four-amino acid sequence DPDG is inserted between the two repeated Uperin3.6 amyloid C-terminal sequences to obtain the antimicrobial peptide variant Uperin3.6C-6R-DPDG, and its amino acid sequence is as shown in SEQ NO:

4.

2. The antimicrobial peptide variant according to claim 1, characterized in that: The C-terminus of the antimicrobial peptide is modified by amidation, and the N-terminus is modified by acetylation.

3. An application of the antimicrobial peptide variant according to claim 1 or 2, characterized in that: An antimicrobial preparation is prepared with the antimicrobial peptide variant.

4. The application according to claim 3, characterized in that: The antimicrobial preparation has inhibitory and killing effects on Gram-negative bacteria and / or Gram-positive bacteria.

5. The application according to claim 4, characterized in that: The Gram-negative bacteria and / or Gram-positive bacteria include one or more of Escherichia coli, Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Listeria innocua, Salmonella typhimurium.

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