A lactic acid bacteria-derived antimicrobial peptide and its database-assisted construction method and application

Abstract

The present invention discloses a lactic acid bacteria-derived antimicrobial peptide and a database-assisted construction method and application thereof, belonging to the field of bioengineering. The amino acid sequence of the antimicrobial peptide is shown in SEQ NO.1. The present invention utilizes bioinformatics resources and computational tools to assist in peptide design and a database prediction model to predict peptide activity, thereby screening the optimal candidate peptide sequence in the shortest time and at the lowest cost. The peptide is a non-natural peptide based on a lactic acid bacteria-derived antimicrobial peptide database that can effectively inhibit Gram-positive and Gram-negative bacteria, has good biological activity and low hemolytic toxicity, and has a therapeutic index of up to 168.98, thereby having good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a lactic acid bacteria-derived antimicrobial peptide and a database-assisted construction method and application thereof. Technical Background

[0002] Bacterial antimicrobial peptides have diverse conformations, ranging from α-helices to β-sheets, linear extensions to random coils, cyclic peptides, or a mixture of these. However, the majority of antimicrobial peptides adopt an α-helical conformation. Although natural bacterial AMPs have demonstrated superiority in overcoming multidrug resistance, their development lacks design principles and is unsystematic. Consequently, the process has become a random, black-box trial-and-error process. Under this perspective, the development of non-natural peptides aims to provide a broader AMP library. Currently, the most studied bacterial AMP producers are lactic acid bacteria (LABs), and AMPs produced by LABs are generally considered safe. Among them, nisin, produced by Streptococcus lactis, is currently approved for use as a food additive in many countries. However, its antimicrobial spectrum is narrow, with significant antibacterial activity against Gram-positive bacteria. Therefore, it is crucial to identify or artificially design bacterial antimicrobial peptides with broad antimicrobial spectrum, strong stability, and low cost. Traditional AMPs design methods are often costly and time-consuming, making it crucial to find new design strategies to reduce iteration costs. With the gradual maturity of bioinformatics and computer algorithms, AMPs databases and prediction models have begun to emerge. Using bioinformatics resources and computational tools to assist in peptide design, predict peptide activity, and screen for optimal candidate peptides has become a growing trend. Summary of the Invention

[0003] Based on the above shortcomings, the present invention provides a lactic acid bacteria-derived antimicrobial peptide (LAB 0.4) to solve the problems of high manual screening cost, low antimicrobial efficiency and high hemolysis of existing lactic acid bacteria-derived antimicrobial peptides. It has good antibacterial activity against both Gram-negative and Gram-positive bacteria, but low antibacterial activity against probiotics.

[0004] The technical solution adopted by the present invention is as follows: an antimicrobial peptide derived from lactic acid bacteria, the amino acid sequence of which is shown in SEQ NO.1.

[0005] Another object of the present invention is to provide a method for constructing a lactic acid bacteria-derived antimicrobial peptide as described above, which is based on database-assisted design and is as follows: by searching the two major lactic acid bacteria-derived antimicrobial peptide databases APD and DRAMP, a lactic acid bacteria-derived antimicrobial peptide library containing 129 sequences was established, as shown in Table 1 of this specification. After peptide library analysis, it was determined that the cationic amino acid: hydrophobic amino acid ratio was ≈1:4, the cationic amino acid + hydrophobic amino acid ratio was 68.20%, the average sequence length was 36 amino acids, and the cationic amino acid and hydrophobic amino acid with the highest amino acid ratio were K: 8.23%, G: 8.63%, and G: 8.63%, respectively. 14.00%, A: 11.09%, L: 6.25%, V: 6.05%. The sequence length was determined to be 25 according to the amino acid ratio. 120 sequences were generated using (ABCDE)5 as a template. The 120 sequences were screened using activity prediction tools in the three databases of DRAMP, CAMP, and DBAASP to obtain the most likely peptide sequence with antibacterial activity. The sequence was then mutated based on the genetic algorithm in the HeliQuest server to obtain ten sequences with a hydrophobic moment of 0.4. The database activity prediction tool was used again to screen the best candidate polypeptide. The amino acid sequence of the polypeptide is shown in SEQ NO.1. The polypeptide was then synthesized by solid-phase chemical synthesis, and then subjected to antibacterial activity assays, hemolytic activity assays, and stability assays. It was finally named antimicrobial peptide LAB 0.4.

[0006] Another object of the present invention is to provide a use of the lactic acid bacteria-derived antimicrobial peptide as described above in the preparation of a drug for treating and / or preventing Gram-negative bacteria and / or infectious diseases caused by Gram-negative bacteria.

[0007] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii or Pseudomonas aeruginosa.

[0008] Furthermore, the Gram-positive bacteria mentioned above are Staphylococcus aureus, Enterococcus faecalis or Bacillus subtilis.

[0009] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, wherein the drug contains the lactic acid bacteria-derived antimicrobial peptide as described above.

[0010] The present invention has the following advantages and beneficial effects: the present invention utilizes bioinformatics resources and computational tools to assist in peptide design and a database prediction model to predict peptide activity, enabling the screening of optimal candidate peptide sequences in the shortest time and at the lowest cost. The antimicrobial peptide LAB 0.4 of the present invention has a stable structure. Antimicrobial, hemolytic, and stability activity tests of the synthesized antimicrobial peptide revealed that the antimicrobial peptide LAB 0.4 has a high inhibitory effect against Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, and the like, but has low antimicrobial activity against probiotics and very low hemolytic activity. The therapeutic index is as high as 168.98. Furthermore, the antimicrobial peptide LAB 0.4 maintains a certain degree of stability under serum and physiological salt ion conditions. In summary, the antimicrobial peptide LAB 0.4 is an antimicrobial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the HPLC chromatogram of antimicrobial peptide (LAB 0.4);

[0012] Figure 2 This is the mass spectrum of the antimicrobial peptide (LAB 0.4). DETAILED DESCRIPTION

[0013] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1

[0015] Design of antimicrobial peptide (LAB 0.4)

[0016] A library of 129 sequences of lactic acid bacteria-derived antimicrobial peptides was established by searching the APD and DRAMP databases for lactic acid bacteria-derived antimicrobial peptides, as shown in Table 1. Peptide library analysis determined a cationic amino acid:hydrophobic amino acid ratio of approximately 1:4, with a cationic + hydrophobic amino acid ratio of 68.20%. The average sequence length was 36 amino acids. The highest amino acid ratios for cationic and hydrophobic amino acids were K (8.23%), G (14.00%), A (11.09%), L (6.25%), and V (6.05%), respectively. Based on the amino acid ratios, a sequence length of 25 was determined. Using (ABCDE)5 as a template, 120 sequences were generated. These 120 sequences were screened using activity prediction tools from the DRAMP, CAMP, and DBAASP databases to identify the most likely peptide sequences with antimicrobial activity. Sequences were then mutated using a genetic algorithm in the HeliQuest server to generate ten sequences with a hydrophobic moment of 0.4. The optimal candidate peptides were again identified using database activity prediction tools. The final antimicrobial peptide amino acid sequences are shown in Table 2.

[0017] Table 1 Antimicrobial peptide library derived from lactic acid bacteria

[0018]

[0019]

[0020]

[0021]

[0022] Table 2 Antimicrobial peptide (LAB 0.4) amino acid sequence

[0023]

[0024]

[0025] The antimicrobial peptide (LAB 0.4) has a sequence length of 25, a cationic amino acid:hydrophobic amino acid ratio of 1:4, and Lys provides a positive charge of +5. The antimicrobial peptide designed using this method maximizes antimicrobial activity while also having low hemolytic activity.

[0026] Example 2

[0027] 1. The peptides are synthesized one by one from the C-terminus to the N-terminus using a synthesis instrument. The first step is to connect Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) to Wang resin, and then remove the Fmoc group to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide) is added, and this process is followed from the C-terminus to the N-terminus until the synthesis is completed, obtaining a resin with the side chain protection of the Fmoc group removed;

[0028] 2. Add a cutting agent to the polypeptide resin obtained above, react in the dark at 20°C for 2 hours, filter, wash the precipitate with FA (trifluoroacetic acid), mix the filtrate and washing solution evenly, concentrate using a rotary evaporator, then add 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, then collect the precipitate, rinse the precipitate with anhydrous ether, and dry under vacuum to finally obtain the polypeptide. The cutting agent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0029] 3. Use 0.2 mol / L sodium sulfate (adjusted to pH = 7.5 with phosphoric acid) to equilibrate the column for 30 minutes, dissolve the polypeptide in 90% acetonitrile aqueous solution, filter, and perform gradient elution (eluent: methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30) on a C18 reversed-phase atmospheric pressure column at a flow rate of 1 mL / min and a detection wave of 220 nm. Then, collect the main peak and lyophilize.

[0030] 4. Peptide identification: The obtained peptide was analyzed by electrospray mass spectrometry. The mass spectrum (see attached Figure 1 ) is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide is greater than 95% (see Appendix Figure 2 ).

[0031] Example 3

[0032] Bioactivity assay of antimicrobial peptides

[0033] 1. Antibacterial Activity Determination

[0034] The bacteria were cultured in cation-adjusted MHB broth at 37°C and 220 g in a shaking incubator until the logarithmic growth phase and diluted to an OD of 600nm =0.4(3×10 8 -9×10 8 CFU mL -1 The bacterial solution was diluted 1000 times before use. Equal volumes (50 μL) of bacterial suspension and solutions containing different concentrations of amphiphiles (0.25×10 -6 -128×10 -6 A bovine serum albumin solution (BSA, 0.2%; acetic acid, 0.01%) was added to a round-bottomed, clear polypropylene 96-well plate. MHB medium containing bacteria served as a positive control, while MHB medium without bacteria served as a negative control. The 96-well plate was incubated in a 37°C incubator for 18-24 hours. The minimum inhibitory concentration (MIC) was determined by visual inspection and microplate reader OD. 492 nm The minimum concentration at which no bacterial growth was observed was 1.5 OD. Each assay was performed in three independent replicates, with two replicates per replicate. The results are shown in Table 3.

[0035] Table 3 Minimum inhibitory concentration of antimicrobial peptides (uM)

[0036]

[0037] Table 3 shows that the antimicrobial peptide (LAB 0.4) has good antibacterial activity against both Gram-negative and Gram-positive bacteria, but has low activity against probiotics.

[0038] 2. Hemolytic Activity Assay

[0039] (1) Collect 1 mL of blood from a healthy individual and store it in a sodium heparin anticoagulant tube;

[0040] (2) Centrifuge at 3000 g for 5-10 min, discard the supernatant, and collect the red blood cells;

[0041] (3) Rinse the collected red blood cells three times with PBS solution, and then resuspend the red blood cells with 10 times the volume of PBS solution;

[0042] (4) Add 80 μL of PBS solution to the first well of each row of a 96-well plate, and add 50 μL of PBS solution to the remaining wells. Add 20 μL of antimicrobial peptide stock solution (2.56 mM) to well 1 and mix thoroughly. Then, pipette 50 μL into well 2 and mix thoroughly. Repeat this process until well 10. After mixing, pipette 50 μL and discard.

[0043] (5) Add 50 μL of red blood cell suspension to wells 1 to 11 of a 96-well plate, and add 50 μL of 0.2% Triton X-100 to well 12, and mix thoroughly. Well 11 will serve as a negative control, while well 12 will serve as a positive control.

[0044] (6) Place in an incubator and incubate at 37°C for 1 hour, then centrifuge at 3000 g for 5-10 minutes;

[0045] (7) Aspirate the supernatant and transfer it to a new 96-well plate. 570 The absorbance value was measured using a microplate reader under the following conditions;

[0046] (8) Hemolytic activity was calculated according to the following formula:

[0047] Hemolysis rate (%) = [(sample OD 570 -Negative control OD 570 ) / (positive control OD 570 -Negative control OD 570 )]×100%

[0048] The minimum hemolytic concentration is the concentration at which the antimicrobial peptide causes a 10% hemolysis rate. The results are shown in Table 4.

[0049] Table 4 Determination of hemolytic activity of antimicrobial peptides

[0050]

[0051]

[0052] Table 4 shows that the antimicrobial peptide (LAB 0.4) had no hemolytic activity within the detection range. The therapeutic index was calculated using the ratio of the geometric mean of the minimum hemolytic concentration to the minimum inhibitory concentration, and the therapeutic index was 168.98.

[0053] 3. Stability determination: Each concentration (150×10 -3 M NaCl, 4.5 × 10 -3 M KCl, 2 × 10 -3 MCaCl2, 1×10 -3 M MgCl2, 6×10 -6 M NH4Cl, 8×10 -6 M ZnCl2, 4×10 -6 The peptides were dissolved in a BSA solution (M FeCl3) and the subsequent experimental procedures were consistent with the antimicrobial activity assay. To evaluate the effect of serum on antimicrobial activity, the peptides were incubated with different serum concentrations (100%, 50%) for 4 hours and then the antimicrobial activity was determined. The results are shown in Table 5.

[0054] Table 5 Antibacterial activity of antimicrobial peptides against Escherichia coli 25922 under physiological salt concentration and serum conditions (μM)

[0055]

[0056] According to the results in Table 5, it can be seen that the antimicrobial peptide (LAB 0.4) still maintains a certain antibacterial activity in the environment of physiological concentration of salt ions, 50% and 100% serum.

[0057] Combining all of these results, the antimicrobial peptide (LAB 0.4) obtained through bioinformatics analysis of the lactic acid bacteria-derived antimicrobial peptide library and optimized for parameters, combined with database activity prediction and screening tools, demonstrated excellent antimicrobial activity, low hemolysis rate, and stability. The therapeutic index of the antimicrobial peptide (LAB 0.4) was as high as 168.98, demonstrating its potential as an alternative to antibiotics and providing a viable approach for antimicrobial peptide design.

Claims

1. A lactic acid bacteria-derived antimicrobial peptide, characterized in that: Its amino acid sequence is shown in SEQ NO.

1.

2. Use of the lactic acid bacteria-derived antimicrobial peptide according to claim 1 in the preparation of a medicament for treating and / or preventing infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria; the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, or Acinetobacter baumannii and Pseudomonas aeruginosa; and the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, or Bacillus subtilis.

3. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The medicine contains the lactic acid bacteria-derived antimicrobial peptide according to claim 1.

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