A class of de novo designed antimicrobial peptides and their applications

Abstract

The present invention provides a class of antimicrobial peptides designed from scratch and applications thereof, wherein the antimicrobial peptides have an α-helical structure, are composed of 5 Ks or Rs, 2 Gs and 6 Ls, and hydrophobic residues and hydrophilic residues are interspersed every 2-3. The antimicrobial peptides designed from scratch in the present invention have a broad antimicrobial spectrum, strong antimicrobial activity, good stability, and low cytotoxicity, providing a possibility for discovering new antimicrobial drugs and having good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and relates to a class of antimicrobial peptides designed from scratch and applications thereof. Background Art

[0002] Shigella is a Gram-negative bacterium, and the disease caused by it is called shigellosis, or bacillary dysentery. Once infected, Shigella will enter the small intestine and colonize there, then enter the large intestine, directly invade the colon mucosa to produce enterotoxins, cause cell damage and then lead to abdominal cramps, diarrhea, fever, etc. At present, especially in developing countries, shigellosis seriously threatens people's life and health. According to biochemical reactions and serological reactions, Shigella can be divided into 4 types, including: Shigella dysenteriae (Shigella dysenteriae, S.dysenteriae), Shigella boydii (Shigella boydii, S.boydii), Shigella flexneri (Shigella flexneri, S.flexneri), and Shigella Sonnei (Shigella Sonnei, S.Sonnei). Studies have shown that infection with S.flexneri accounts for about 70% of Shigella cases and is the most widely distributed in the world. At present, the most commonly used drugs for the treatment of Shigella infection are fluoroquinolones (norfloxacin, ciprofloxacin, etc.), cephalosporins (ceftazidime, ceftriaxone, etc.), macrolides (azithromycin, etc.), etc. However, the resistance of Shigella to these drugs is becoming more and more common, and these drugs can also cause a variety of side effects, such as damage to human liver and kidney function, irritation of the gastrointestinal tract, neurotoxicity, and allergic reactions.

[0003] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides that exist in various life forms (animals, plants, microorganisms, etc.), usually composed of 10 to 50 amino acids, with the advantages of broad antimicrobial spectrum, wide sources, and low resistance. Studies have shown that antimicrobial peptides have good antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can also mediate inflammatory responses, promote cytokine release, cell proliferation, wound healing, etc. Therefore, antimicrobial peptides have the potential to replace antibiotics and become a new generation of antimicrobial drugs. Summary of the invention

[0004] In view of the above technical problems, the purpose of the present invention is to provide a class of antimicrobial peptides designed from scratch and their applications, which have strong antimicrobial activity, good stability, low cytotoxicity and good application prospects.

[0005] The technical solution adopted by the present invention to achieve the technical purpose is:

[0006] The invention provides a class of antimicrobial peptides designed from scratch, which have an alpha-helical structure and consist of 5 Ks or Rs, 2 Gs and 6 Ls, wherein hydrophobic residues and hydrophilic residues are interspersed every 2-3.

[0007] Preferably, the antimicrobial peptide and its corresponding amino acid sequence are as follows:

[0008] GLK-1: GLLKLLKKLLGKK;

[0009] GLK-2: GLLLKKLKKLLGK;

[0010] GLK-4: LGKLGKKLLKKLL;

[0011] GLR-1: RGGLLRLLLRLLRR;

[0012] GLR-4: LLRGLGRLRRLLR;

[0013] GLR-5: LGRGLLRLRRLLR.

[0014] More preferably, it also includes: an amino acid sequence having the same function as the above antimicrobial peptide after one or more amino acids are replaced and / or deleted and / or added.

[0015] Preferably, the C-terminus and N-terminus of the antimicrobial peptide are modified; the modified part comprises at least one of a chemical modification, a targeting part, a fluorescent dye and a protein tag; the chemical modification comprises at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation and ubiquitination; the targeting part comprises at least one of a ligand, a receptor and an antibody; the fluorescent dye comprises FITC; the protein tag comprises at least one of His, Flag, GST, Myc, GFP, MBP and HA.

[0016] The present invention also provides a fusion protein comprising the antimicrobial peptide.

[0017] The present invention also provides a composition, wherein the active ingredient of the composition is the antimicrobial peptide.

[0018] The present invention also provides an antibacterial additive, the active ingredient of the antibacterial additive is the antibacterial peptide.

[0019] The present invention also provides the use of the antimicrobial peptide in the preparation of a broad-spectrum antimicrobial preparation.

[0020] Preferably, the broad-spectrum antibacterial preparation includes drugs that inhibit or kill Acinetobacter baumannii, Ralstonia solanacearum, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia, Shigella sonnei, Shigella flexneri, and Escherichia coli.

[0021] The beneficial effects of the present invention are:

[0022] The antimicrobial peptide designed from scratch in the present invention has a broad antimicrobial spectrum, strong antimicrobial activity, good stability, and low cytotoxicity, thus providing a possibility for discovering new antimicrobial drugs and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 .Amino acid analysis of antimicrobial peptides against Gram-negative bacteria.

[0024] Figure 2 .Analysis of antimicrobial peptide characteristic data.

[0025] Figure 3 .AF: 3D structure simulation of GLK-1, GLK-2, GLK-4, GLR-1, GLR-4, GLR-5, GL: helical wheel structure prediction of GLK-1, GLK-2, GLK-4, GLR-1, GLR-4, GLR-5.

[0026] Figure 4 .GLK-4 bactericidal kinetics.

[0027] Figure 5 The effect of GLK-4 on the morphology of S.flexneri was observed by scanning electron microscopy.

[0028] Figure 6 .The cytotoxicity of GLK-4 to HIEC and Hela cells.

[0029] Figure 7 .Effect of GLK-4 on the cytotoxicity of S.flexneri.

[0030] Figure 8 .Hemolytic toxicity of GLK-4. DETAILED DESCRIPTION

[0031] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0032] Example

[0033] 1. Antimicrobial peptide design

[0034] The APD3 database (https: / / aps.unmc.edu / database) was used to count the high-frequency amino acids of antimicrobial peptides with antimicrobial activity against Gram-negative bacteria. In order to design antimicrobial peptides with specific functions, 159 representative ones were selected from the APD3 database, and their characteristic data, such as length, charge number, hydrophobicity, structural characteristics, etc., were counted. The above data information was integrated to obtain different motifs, and then the different motifs were arranged and combined, and the different combinations were screened according to their frequency of appearance in the APD3 database, and finally the antimicrobial peptide sequences designed from scratch were obtained.

[0035] Depend on Figure 1 The statistical results show that the high-frequency amino acids in the antimicrobial peptides against Gram-negative bacteria include the hydrophilic amino acid - glycine (13.89%), the hydrophobic amino acid - leucine (7.64%) and the positively charged amino acid - lysine (9.86%).

[0036] 2. Analysis of the physicochemical properties of antimicrobial peptides

[0037] The APD3 database, helical wheel analysis software Heliquest (https: / / heliquest.ipmc.cnrs.fr / ), CAMP R3 The database (http: / / www.camp3.bicnirrh.res.in / prediction.php) and PEP-FOLD (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD4 / ) analyze the physicochemical properties of antimicrobial peptides, predict the possibility of exerting antimicrobial activity, and predict the structural characteristics of antimicrobial peptides.

[0038] like Figure 2 As shown in Figure 1, 159 representative antimicrobial peptides were screened from the APD3 database and used as a model to calculate relevant parameters. It was found that the majority of antimicrobial peptides contained 13 amino acids ( Figure 2 E). According to previous statistics, the most common amino acids are leucine (L), glycine (G) and lysine (K). According to statistics, the most common antimicrobial peptides contain 5 positive charges ( Figure 2 B), so it was preliminarily determined that the designed antimicrobial peptide contained 5 K. In addition to positive charge, hydrophobicity is another basic element of natural antimicrobial peptides. The hydrophobic content is expressed as the ratio between the total hydrophobic residues and the total amino acids in the peptide. Figure 2C shows that the hydrophobic content of most peptides is between 41-50%, corresponding to 6 L in 13 amino acids. After determining 5 K and 6 L, 2 additional G need to be allocated. In summary, the antimicrobial peptide designed by the present invention consists of 5 K, 2 G and 6 L. Furthermore, the structures of these antimicrobial peptides are statistically analyzed, and most of them are α-helical structures ( Figure 2 D). Generally, there are two construction methods. One is to gather hydrophobic amino acids and hydrophilic residues on one side, and the other is to intersperse hydrophobic residues and hydrophilic residues every 2-3. According to database statistics, there is no motif consisting of 6 consecutive Ls, so the second construction method is used. Because studies have shown that the antimicrobial activity of antimicrobial peptides containing arginine (R) may be better than that of antimicrobial peptides containing K, antimicrobial peptides consisting of 5 Rs, 2 Gs and 6 Ls were also constructed.

[0039] After statistical analysis, a series of antimicrobial peptides were obtained, as shown in Table 1. After analyzing the probability scores predicted by the three models of random forest (RF), support vector machine (SVM), and artificial neural network (ANN), and the BI index predicted by the APD3 database, it can be seen that the parameters of the designed K series antimicrobial peptides are consistent. Studies have shown that the hydrophobic moment (μH) is related to antimicrobial activity. It is speculated that GLK-4 has the best antimicrobial activity among the K series antimicrobial peptides. The probability scores and μH predicted by the R series antimicrobial peptide combination model indicate that GLR-5 has the best antimicrobial activity among the R series antimicrobial peptides.

[0040] Table 1 Prediction of activity-related parameters of antimicrobial peptides

[0041]

[0042] a CAMP R3 The prediction was obtained from the database (http: / / www.camp3.bicnirrh.res.in / prediction.php); b Analyzed by Heliquest (https: / / heliquest.ipmc.cnrs.fr / ); c Predicted by APD3 database (https: / / aps.unmc.edu / home).

[0043] like Figure 3 As shown, the 3D models of GLK-1, GLK-2, GLK-4, GLR-1, GLR-4, and GLR-5 predicted by PEP-FOLD show that the antimicrobial peptides have a good α-helical structure. The size of the circle in the helical wheel structure predicted by Heliquest represents the volume of the amino acid, and the length and direction of the arrow represent the size and direction of the hydrophobic moment.

[0044] Next, we selected the antimicrobial peptides GLK-1, GLK-2, GLK-4, and GLR-5 with better predicted parameters, synthesized them, and measured their MICs and MBCs against different bacteria.

[0045] 3. Synthesis of antimicrobial peptides

[0046] The GLK-1, GLK-2, GLK-4, and GLR-5 used in this experiment were commissioned by Shanghai Jier Biochemical Co., Ltd. and synthesized using solid phase synthesis. After purification by high performance liquid chromatography and mass spectrometry, the purity was above 95%. Before use, they were dissolved in sterile water to prepare a stock solution.

[0047] 4. Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

[0048] The MIC was determined in a 96-well plate using the two-fold dilution method. 195 μL of the diluted bacterial solution was added to the first column, and 100 μL was added to each of the remaining wells. 5 μL of the antimicrobial peptide stock solution was added to the first column and mixed evenly. After two-fold dilution, 100 μL was removed from the last column. The final concentration of the sample was 0.5 μg / mL to 64 μg / mL, and the bacterial solution OD 600 =0.001. The samples were cultured at 37°C for 18 h, and the light absorption at 600 nm was detected by an enzyme-labeled instrument. Three parallel experiments were set up for each sample, and the independent experiments were repeated 3 times. MIC was defined as the lowest concentration that could inhibit bacterial growth.

[0049] Based on the determination of MIC, take 1.5 μL of liquid with a concentration equal to or greater than the MIC well, drop it on the LB agar plate, dry it, invert it at 37°C, and observe the experimental results after incubation overnight. MBC is defined as the minimum drug concentration required to kill 99.9% of microorganisms.

[0050] Table 2 MIC test results statistics

[0051]

[0052] ND, not done.

[0053] Table 3 MBC measurement results statistics

[0054]

[0055] ND, not done.

[0056] The results are shown in Tables 2 and 3. The antibacterial activity of GLK-2 and GLR-5 is weak, and the MIC for most bacteria is > 64 μg / mL. The best activity is GLK-4, followed by GLK-1. Both have good antibacterial activity against S.flexneri, so GLK-4 was selected for subsequent research on S.flexneri.

[0057] 5. Bactericidal kinetics detection

[0058] The bacteria were cultured to the logarithmic growth phase and the liquid medium was diluted to OD 600 =0.002, add the diluted sample and bacterial solution in equal volumes to the centrifuge tube, so that the final concentration of the sample is 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, and the final concentration of the bacterial solution is OD 600 =0.001. Incubate at 37℃ for 30min, 60min, 90min, 120min, 180min, 240min, and 300min, dilute by appropriate multiples, take 100μL for coating, incubate the coated plates at 37℃, and calculate the number of bacteria. The number of bacteria contained in 1mL of bacterial solution = dilution multiple × 10 × number of bacteria on the plate.

[0059] The results showed that the bactericidal effect of GLK-4 on S.flexneri was concentration-dependent and time-dependent ( Figure 4 ). At a concentration of 4×MIC (8μg / mL), the bacteria can be completely killed in 90 minutes, and at a concentration of 2×MIC (4μg / mL), the bacteria can be killed in 120 minutes. At a concentration of 1 / 2×MIC (1μg / mL), it has a certain inhibitory effect on bacterial growth within 120 minutes.

[0060] 6. Stability analysis

[0061] (1) Determination of the stability of antimicrobial peptides under different temperature conditions

[0062] The 5.12 mg / mL sample prepared with sterile water was divided into centrifuge tubes and heated at 40°C, 70°C and 100°C for 30 min. After cooling to room temperature, the MIC of the sample against bacteria was measured, and the 5.12 mg / mL antimicrobial peptide solution placed at 25°C (normal temperature) was used as a control. The effect of different temperatures on the activity of antimicrobial peptides was determined by measuring the change in MIC.

[0063] Table 4 Effect of temperature on sample stability

[0064]

[0065] It can be seen from Table 4 that GLK-4 can maintain good antibacterial activity even in a high temperature environment of 100°C after pretreatment for 30 minutes.

[0066] (2) Determination of the stability of antimicrobial peptides under different pH conditions

[0067] Liquid culture media with pH values ​​of 5, 7, and 9 were prepared to dilute the bacterial suspensions. The final concentrations of the samples were 0.5 μg / mL to 64 μg / mL, and the MIC was determined in a 96-well plate.

[0068] Table 5 Effect of pH on sample stability

[0069]

[0070] As shown in Table 5, the activity was greatly affected when pH was 5. This may be because in an acidic environment, the positively charged amino acid side chains of GLK-4 were protonated, which affected the positive charge of GLK-4, so the antibacterial activity was affected. However, the MIC remained unchanged when pH was 9, indicating that GLK-4 has good alkali resistance.

[0071] (3) Determination of the stability of antimicrobial peptides under different ionic conditions (ion concentration under physiological conditions)

[0072] Prepared separately containing Ca 2+ The concentration was 2.5 mM, Mg 2+ The concentration is 0.9 mM, Na + The concentration is 140mM, K + The LB liquid culture medium with a concentration of 4.5 mM was sterilized and used for later use. The MIC of the antimicrobial peptide against bacteria was determined according to the above MIC determination method. The effect of different ion concentrations on the activity of the antimicrobial peptide was determined by measuring the MIC.

[0073] Table 6 Effect of ion concentration on sample stability

[0074]

[0075] As shown in Table 6, in the case of monovalent cations (Na + , K + ) in the presence of , the MIC increased twofold compared with the control group. 2+ ,Mg 2+ ) in the presence of , the MIC increased by 8 and 4 times, respectively, which may be related to the fact that metal ions hindered the electrostatic interaction between GLK-4 and bacterial cell membrane.

[0076] (4) Determination of the stability of antimicrobial peptides in the presence of serum

[0077] LB liquid culture medium containing 2% and 10% fetal bovine serum was prepared, and the MIC of the antimicrobial peptide against bacteria was determined according to the above MIC determination method. The effect of fetal bovine serum on the activity of the antimicrobial peptide was determined by measuring the MIC.

[0078] Table 7 Effect of serum on sample stability

[0079]

[0080] Table 7 shows that when GLK-4 was in the presence of 2% fetal bovine serum, the MIC increased to 2 times of that of the control group, and when GLK-4 was in the presence of 10% fetal bovine serum, the MIC increased to 4 times of that of the control group.

[0081] 7. Scanning Electron Microscope (SEM) Observation

[0082] Cultivate bacteria to the logarithmic growth phase, take a certain volume of bacterial solution into a centrifuge tube, centrifuge at 5000rpm, 4℃, centrifuge for 5min, remove the supernatant, and wash the bacteria three times with PBS. After resuspending the bacteria, dilute the bacterial solution to a concentration of OD 600 = 1, take 500 μL of bacterial solution and add equal volumes of samples with concentrations of 256 μg / mL, 128 μg / mL, and 64 μg / mL to make the final concentrations of the samples 128 μg / mL, 64 μg / mL, and 32 μg / mL (corresponding to bacterial solution concentrations of OD 600 =0.5 MBC, MIC, 1 / 2×MIC), 37℃ water bath for 1h, centrifuged, washed three times with PBS. 2.5% glutaraldehyde 500μL fixed for 4h, centrifuged, washed three times with PBS, prepared with 30%, 50%, 70%, 90%, 100% alcohol, gradient dehydration. Drop the bacterial liquid on tin foil, freeze-dry overnight, vacuum spray gold, and observe with scanning electron microscope.

[0083] The morphological changes of S.flexneri after 1 h of GLK-4 treatment are shown in Figure 2. Figure 5 As shown, the control group ( Figure 5 A) The bacterial surface is smooth and has a complete rod-like structure. After treatment with 32μg / mL GLK-4 ( Figure 5 B), obvious wrinkles began to appear on the bacterial surface. After treatment with 64μg / mL GLK-4 ( Figure 5 C), the bacteria were severely ruptured, showing a large gap, and the cell membrane of the bacteria with leaked contents appeared around them. After treatment with 128μg / mL GLK-4 ( Figure 5 D) The complete bacterial morphology is basically invisible, the bacteria are stuck to each other, and the contents are leaking.

[0084] 8. Cytotoxicity

[0085] To recover cells (Hela and HIEC), the cells were cultured in DMEM medium containing 10% fetal bovine serum. When the cells grew to more than 80% confluence, the cells were digested with trypsin, resuspended in medium, and diluted to 5×10 4cells·mL -1 , add 100 μL of cell suspension to each well of a 96-well plate and culture overnight (37°C, 5% CO2). Remove the supernatant and add 100 μL of samples of different concentrations (2 μg / mL to 128 μg / mL) and incubate in an incubator for 24 hours. After adding 10 μL of CCK-8 solution to each well, continue to incubate in the incubator for 2-4 hours, and measure the absorbance at 450 nm with an enzyme-labeled instrument.

[0086] Calculate cell survival rate: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0087] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution);

[0088] Ac: absorbance of control well (containing cells, culture medium, CCK-8 solution, but no drug);

[0089] Ab: absorbance of blank well (containing culture medium and CCK-8 solution, but not cells or drugs).

[0090] like Figure 6 As shown in A and B, GLK-4 has little toxicity to HIEC cells. The IC 50 It is 59.13μg / mL. At a concentration of 32μg / mL (16×MIC), there is no significant effect on the growth of HIEC cells. At a concentration of 64μg / mL, the cell survival rate reaches about 43%. Figure 6 As shown in C and D, GLK-4 has low toxicity to Hela cells. The IC 50 It is 33.69μg / mL. At a concentration of 16μg / mL (8×MIC), there is no significant effect on the growth of Hela cells, and at a concentration of 32μg / mL, the cell survival rate reaches about 53%.

[0091] IX. Detection of the effect of antimicrobial peptides on the toxicity of S.flexneri cells

[0092] S.flexneri was inoculated in LB, cultured overnight, washed three times with PBS, collected by centrifugation, resuspended in DMEM cell culture medium, and diluted to an OD 600 = 0.01 for standby use. HIEC is set at 1.5×10 4Cells were inoculated into 96-well plates and incubated overnight. The cell culture medium was discarded and the cells were rinsed with PBS twice. 100 μL of pre-prepared bacterial suspension containing different concentrations of samples (1 μg / mL to 16 μg / mL) was added to each well, and the cells were infected with bacterial suspension without samples as a control. After continuing to culture in a 5% CO2 incubator for 4 hours, the LDH activity of the cells was detected according to the steps in the Promega LDH kit.

[0093] Lactate dehydrogenase (LDH) is abundant in the cytoplasm and cannot pass through the cell membrane under normal circumstances. After bacterial infection, cells are damaged or die, and LDH is released in the supernatant. By detecting extracellular LDH, the strength of bacterial virulence can be quantitatively analyzed. Figure 7 As shown in the figure, the toxicity of bacteria to cells is concentration-dependent. When the concentration of GLK-4 is 16μg / mL, it can significantly reduce the toxicity of S.flexneri to HIEC. It also has a certain effect on the toxicity of S.flexneri at concentrations of 4μg / mL and 8μg / mL.

[0094] 10. Hemolytic toxicity

[0095] Mouse erythrocytes were washed three times with PBS at 4°C (1000 x g, 10 min) until the supernatant was clear. 300 μL of the cell suspension was mixed with 300 μL of the serially diluted sample (4 μg / mL to 256 μg / mL) in a centrifuge tube and incubated at 37°C for 60 min. The mixture was centrifuged, and 150 μL of the supernatant was taken into a new 96-well plate and the absorbance was measured at 450 nm. In addition, the bacterial suspension (OD 600 = 0.01) were incubated with the peptides to assess the selectivity of the peptides.

[0096] like Figure 8 As shown, GLK-4 has little hemolytic toxicity, and its hemolytic toxicity to mouse erythrocytes does not exceed 10% at a concentration of 128 μg / mL, and its selective effect on bacteria is weak.

[0097] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A class of antimicrobial peptides designed from scratch, having an α-helical structure, consisting of 5 Ks or Rs, 2 Gs and 6 Ls, and hydrophobic residues and hydrophilic residues are interspersed every 2-3; the antimicrobial peptide and its corresponding amino acid sequence are as follows: GLK-1: GLLKLLKKLLGKK; GLK-4:LGKLGKKLLKKLL.

2. A composition, wherein the active ingredient of the composition is the antimicrobial peptide according to claim 1.

3. An antibacterial additive, wherein the active ingredient of the antibacterial additive is the antimicrobial peptide according to claim 1.

4. Use of the antimicrobial peptide according to claim 1 in the preparation of a broad-spectrum antimicrobial preparation, wherein the broad-spectrum antimicrobial preparation comprises drugs that inhibit or kill Acinetobacter baumannii, Ralstonia solanacearum, Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella sonnei, Shigella flexneri, and Escherichia coli.

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