A cell-penetrating antimicrobial peptide against intracellular bacteria and its preparation method and application
By designing the cell-penetrating antibacterial peptide 2L2R2W, the problem that existing antibacterial agents are difficult to remove bacteria in cells is solved, and efficient bactericidal against Salmonella, Listeria monocytogenes and Staphylococcus aureus is achieved, and the antibacterial mechanism is low in macrophage toxicity and destruction of bacterial membranes is achieved.
Patent Information
- Application Number
- CN202410460261.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-17
- Publication Date
- 2025-04-22
- Estimated Expiration
- 2044-04-17
AI Technical Summary
Existing antibacterial agents are difficult to effectively remove intracellular bacteria because most antibiotics cannot penetrate mammalian cell membranes and antibacterial agents are prone to drug resistance.
A cell-penetrating antimicrobial peptide 2L2R2W is designed, which can penetrate mammalian cells and exert antibacterial effects by destroying bacterial membranes by selecting specific amino acid sequences and hydrophobic amino acid terminal labeling techniques.
Antibacterial peptide 2L2R2W has strong antibacterial activity against common intracellular bacteria such as Salmonella, Listeria monocytogenes and Staphylococcus aureus. It can efficiently penetrate macrophages and perform excellent bactericidal on intracellular bacteria, and has low toxicity to macrophages.
Smart Images
Figure CN118388589B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to a cell-penetrating antimicrobial peptide 2L2R2W for resisting intracellular bacteria, and a preparation method and application thereof. Background Art
[0002] Many bacterial pathogens can already invade mammalian cells. These bacteria are difficult to eliminate and cause repeated infections. These bacteria are called intracellular bacteria, such as Salmonella, Listeria monocytogenes, Mycobacterium tuberculosis, Staphylococcus aureus, etc. After intracellular bacteria invade mammalian cells, they escape the attack of the host immune system on the one hand, and are protected from the bactericidal effect of antibiotics on the other hand. In addition, most antibiotics have poor permeability to mammalian cells, limited intracellular accumulation concentrations, and antibiotic resistance. Therefore, there is an urgent need for new antibacterial agents that can replace antibiotics to solve the problem of intracellular bacteria.
[0003] Antimicrobial peptides have multiple biological activities against bacteria, fungi, etc. and their mechanism of action is different from that of antibiotics, so they are regarded as potential substitutes for antibiotics. However, most antimicrobial peptides do not have the ability to penetrate mammalian cell membranes and cannot solve the problem of intracellular infection caused by some bacteria. Summary of the invention
[0004] Based on the above shortcomings, the purpose of the present invention is to provide a cell-penetrating antimicrobial peptide 2L2R2W against intracellular bacteria, which is a polypeptide that enters mammalian cells through membrane translocation or endocytosis, solving the problem of poor anti-intracellular bacterial effect.
[0005] The technical solution adopted by the present invention is as follows: a cell-penetrating antimicrobial peptide 2L2R2W for resisting intracellular bacteria, whose amino acid sequence is shown in SEQ ID No. 1, and whose C-terminus is amidated with -NH2.
[0006] Furthermore, the molecular formula of the antimicrobial peptide 2L2R2W is shown in formula (I),
[0007]
[0008] Another object of the present invention is to provide a method for preparing the cell-penetrating antimicrobial peptide 2L2R2W against intracellular bacteria as described above, as follows: by symmetrical sequence design, two consecutive Trp are selected and placed in the center of the sequence to provide the cell-penetrating ability required for intracellular sterilization; then, two consecutive Leu are selected and placed at both ends of the sequence by hydrophobic amino acid terminal labeling technology to provide the hydrophobicity and stability required for antibacterial activity; finally, two consecutive Arg are selected and placed between Trp and Leu to provide the positive charge required for antibacterial activity; thus, a polypeptide is designed, the amino acid sequence of which is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2; then, the polypeptide is synthesized by solid phase chemical synthesis, the polypeptide is identified by mass spectrometry and purified by reversed-phase high performance liquid chromatography, and then the antibacterial activity, cytotoxicity and cell penetration ability are determined, and finally named as cell-penetrating antimicrobial peptide 2L2R2W.
[0009] Another object of the present invention is to provide a use of the above-mentioned cell-penetrating antimicrobial peptide 2L2R2W against intracellular bacteria in the preparation of a drug for treating Gram-positive and / or Gram-negative bacterial infectious diseases.
[0010] Furthermore, in the application described above, the Gram-negative bacteria are Salmonella, Escherichia coli or Pseudomonas aeruginosa.
[0011] Furthermore, in the application described above, the Gram-positive bacteria are Listeria monocytogenes, Staphylococcus epidermidis or Staphylococcus aureus.
[0012] The present invention has the following advantages and beneficial effects: the cell-penetrating antimicrobial peptide 2L2R2W of the present invention has strong antibacterial activity against common intracellular bacteria such as Salmonella, Listeria monocytogenes, Staphylococcus aureus, etc.; it has low toxicity to macrophages, and can efficiently penetrate macrophages and has excellent bactericidal effects on Salmonella and Staphylococcus aureus in macrophages; in addition, it mainly exerts its antibacterial effect by destroying bacterial membranes, and has application potential against intracellular bacteria. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 Figure 2 shows the number of cell penetrating peptides with different charges, the number of cell penetrating peptides with different hydrophobic ratios, the amino acid composition of cell penetrating peptides, and the number of cell penetrating peptides with different lengths in the cell penetrating peptide library CPPsite 2.0, including (A) the number of cell penetrating peptides with different charges, (B) the number of cell penetrating peptides with different hydrophobic ratios, (C) the amino acid composition of cell penetrating peptides, and (D) the number of cell penetrating peptides with different lengths;
[0014] Figure 2 is the mass spectrum of the antimicrobial peptide 2L2R2W;
[0015] Figure 3 It is the high performance liquid chromatogram of the antimicrobial peptide 2L2R2W;
[0016] Figure 4 It is the cytotoxicity graph of the antimicrobial peptide 2L2R2W;
[0017] Figure 5 This is a graph showing the cell penetration ability of the antimicrobial peptide 2L2R2W;
[0018] Figure 6 The intracellular antibacterial activity of the antimicrobial peptide 2L2R2W, including (A) S. typhimurium 14028, (B) S. aureus 29213;
[0019] Figure 7 The antibacterial mechanism diagram of the antimicrobial peptide 2L2R2W against S.typhimurium 14028 under a scanning electron microscope;
[0020] Figure 8 This is the antibacterial mechanism diagram of the antimicrobial peptide 2L2R2W against S. aureus 29213 under a scanning electron microscope. DETAILED DESCRIPTION
[0021] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0022] Example 1
[0023] Design of antimicrobial peptides
[0024] The charge number, hydrophobic ratio, amino acid composition, and sequence length of 1166 cell-penetrating peptides in the cell-penetrating peptide database CPPsite 2.0 were statistically analyzed. The analysis results are shown in Figure 1 . The analysis results showed that: cell-penetrating peptides with a charge number of +5 appeared most frequently, appearing 148 times. Cell-penetrating peptides with a hydrophobic amino acid ratio of 41%-60% appeared most frequently, appearing 422 times. Among cationic amino acids, Arg appeared most frequently, accounting for 21.04%. Among hydrophobic amino acids, Leu appeared most frequently, accounting for 10.01%. In addition, although Trp accounted for only 3.18%, Trp played an important role in the penetration effect of cell-penetrating peptides, so Trp was also added to the design. Cell-penetrating peptides with a sequence length of 10-16 amino acid residues appeared most frequently, appearing 516 times.
[0025] Based on the above analysis results and the symmetrical sequence design of the antimicrobial peptide, two consecutive Trp were selected and placed in the center of the sequence to provide the cell penetration ability required for intracellular sterilization. Subsequently, two consecutive Leu were selected and placed at both ends of the sequence by hydrophobic amino acid terminal labeling technology to provide the hydrophobicity and stability required for antimicrobial activity. Finally, two consecutive Arg were selected and placed between Trp and Leu to provide the positive charge required for antimicrobial activity, and the C-terminus was amidated with -NH2. By selecting a symmetrical sequence design, better antimicrobial activity, stability and safety were obtained. The cell-penetrating antimicrobial peptide thus designed was named 2L2R2W, and the amino acid sequence is shown in Table 1.
[0026] Table 1 Amino acid sequences of antimicrobial peptides
[0027]
[0028] The molecular formula of the antimicrobial peptide 2L2R2W is shown in formula (I),
[0029]
[0030] Example 2
[0031] Synthesis of antimicrobial peptides by solid phase chemical synthesis
[0032] 1. Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 mL reactor and soak it with 50 mL of dichloromethane (DCM).
[0033] After 2.2 hours, the resin was washed with nitrogen-dimethylformamide (DMF) 3 times the volume of the resin, and then drained. This was repeated four times, and the resin was drained and set aside for use.
[0034] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF 3 times the volume of the resin and then drain it.
[0035] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.
[0036] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30 ° C for reaction.
[0037] After 6.2 hours, a certain amount of acetic anhydride was used to cap the resin (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, and then washed four times with DMF 3 times the volume of the resin and dried for later use.
[0038] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it.
[0039] 8. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.
[0040] 9. Weigh an appropriate amount of the second amino acid and an appropriate amount of HOBT into a 50mL centrifuge tube, add 25mL of DMF to dissolve them, then add 2.5mL of DIC and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.
[0041] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, the condensation is incomplete and the reaction should continue.
[0042] 11. After the reaction is complete, wash the resin four times with DMF, then drain, add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain to check whether the protection is removed.
[0043] 12. Follow steps 9-11 to connect the following amino acids.
[0044] 13. After the last amino acid is attached, remove the protection, wash four times with DMF, and then drain the resin with methanol. Then use 95 cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) to cut the peptide from the resin (add 10mL cutting solution per gram of resin), and centrifuge and precipitate four times with ice ether (cutting solution: ether = 1:9). Use LC-MS to determine the molecular weight (such as Figure 2 The molecular weight of the peptide was basically consistent with the theoretical molecular weight in Table 1, and then it was purified. Finally, it was separated and purified by HPLC, and then freeze-dried to obtain a polypeptide with a purity of >95% (as shown in Table 1). Figure 3 shown).
[0045] 14. Purification conditions:
[0046] Stationary phase: C18;
[0047] Mobile phase configuration:
[0048] Pump A: V(tfa) / V(water)=1 / 1000;
[0049] Pump B: V(TFA) / v(acetonitrile)=1 / 1000;
[0050] Flow rate: 10 mL / min;
[0051] Retention time: between 20-30 minutes;
[0052] Prepared twice.
[0053] Example 3
[0054] Determination of antibacterial activity of antimicrobial peptides
[0055] The minimum inhibitory concentration (MIC) of the peptide was determined by the standard microbroth dilution method. The bacteria were cultured in MHB medium at 37°C and 220 rpm overnight, and then the bacterial solution was transferred to new MHB medium and cultured until the logarithmic growth phase of the bacteria. Finally, the bacteria were diluted to ~2×10 5 CFU / mL. 50 μL of peptide and bacterial suspension with a final concentration of 1-64 μM was added to each well of a 96-well plate. In addition, only MHB medium was used as a negative control, and only bacteria and MHB medium were used as a positive control. The 96-well plate was placed in a 37°C constant temperature incubator for 18 to 20 hours, and the plate was read at 492 nm (OD 492 ) to determine the minimum inhibitory concentration. The test was repeated three times independently, with two replicates for each. The results are shown in Table 2.
[0056] Table 2 Minimum inhibitory concentration of antimicrobial peptide 2L2R2W (μM)
[0057]
[0058] It can be seen from Table 2 that the antimicrobial peptide 2L2R2W exhibits strong antibacterial activity against a variety of bacteria, including Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, and Staphylococcus epidermidis, with the minimum inhibitory concentration being 1-8 μM.
[0059] Example 4
[0060] Determination of cytotoxicity of antimicrobial peptides
[0061] Mouse macrophage RAW 264.7 cells were diluted to 4 × 10 5cells / mL, add 50 μL of cell suspension to columns 1-11 of the 96-well plate, and continue to culture overnight in a 37°C cell culture incubator containing 5% CO2 until the cells are completely attached and grow. Add 50 μL of a polypeptide with a concentration of 64-0.125 μM to columns 1-10 of a new 96-well plate, add 50 μL of cell culture medium to column 11 as a positive control, and add 100 μL of cell culture medium to column 12 as a negative control. Transfer the liquid from each well of the above 96-well plate to the corresponding well of the 96-well plate containing cells, and culture in a 37°C cell culture incubator containing 5% CO2 for 4 hours. Subsequently, add 25 μL of MTT with a concentration of 0.5 mg / mL to each well of the 96-well plate, and continue to culture in a 37°C cell culture incubator containing 5% CO2 for 3 hours. Finally, discard the supernatant, add 150 μL of DMSO to each well to dissolve the formazan crystals, and use a microplate reader at a wavelength of 570 nm (OD 570 ) to measure the absorbance value. The experiment was repeated three times independently. The cell viability calculation formula is as follows:
[0062] Cell viability (%) = [(sample OD 570 - Negative control OD 570 ) / (positive control OD 570 - Negative control OD 570 )]×100%
[0063] Figure 4 The cytotoxicity of antimicrobial peptide 2L2R2W on mouse macrophages RAW 264.7 was studied. In the concentration range of 0.125-32 μM, antimicrobial peptide 2L2R2W had little effect on the survival rate of mouse macrophages and still maintained above 70%, indicating that antimicrobial peptide 2L2R2W has good biocompatibility.
[0064] Example 5
[0065] Determination of cell penetration ability of antimicrobial peptides
[0066] Mouse macrophage RAW 264.7 cells were diluted to 2 × 10 5 cells / mL, 1 mL of cell suspension was added to the 24-well plate, and the cells were cultured overnight in a 37°C cell culture incubator containing 5% CO2 until the cells were completely attached to the wall. FITC-labeled antimicrobial peptide 2L2R2W was added and cultured for 2 h in a 37°C cell culture incubator containing 5% CO2, 0.4% trypan blue was added for 3 min to quench the extracellular fluorescence, and then the cells were collected after washing 2-3 times with PBS, and finally analyzed by flow cytometry.
[0067] Figure 5 As for the cell penetration ability of the antimicrobial peptide, at a concentration of 16 μM, the antimicrobial peptide 2L2R2W can penetrate into more than 80% of mouse macrophages, indicating that it has efficient cell penetration ability.
[0068] Example 6
[0069] Determination of intracellular bactericidal activity of antimicrobial peptides
[0070] Mouse macrophage RAW 264.7 cells were diluted to 2 × 10 5 cells / mL, add 1mL of cell suspension to the 24-well plate, and continue to culture overnight in a 37℃ cell culture incubator with 5% CO2 until the cells are completely attached to the wall. S.typhimurium14028 or S.aureus 29213 was cultured in MHB medium at 37℃ and 220rpm overnight, and then the bacterial solution was transferred to a new MHB medium and cultured until the logarithmic growth phase of the bacteria. S.typhimurium 14028 or S.aureus29213 was added to the 24-well plate containing cells and infected at 37℃ for 1h (cell: bacteria = 1:10). Then, gentamicin at a concentration of 100μg / mL was added and cultured at 37℃ for 1h to completely eliminate extracellular bacteria. After rinsing with PBS, different concentrations of antimicrobial peptides were added, and PBS was added to the control, and cultured at 37℃ for 4h. After washing with PBS, the cells were lysed with 0.25% Triton X-100 for 15 min, spread on MHA plates after dilution, and counted after incubation in a 37°C incubator overnight.
[0071] Figure 6 The results show that the antimicrobial peptide 2L2R2W has an intracellular antibacterial activity. The killing rate of 16 μM 2L2R2W against Salmonella typhimurium and Staphylococcus aureus in mouse macrophages is over 90%, indicating that it has excellent anti-intracellular bacterial activity.
[0072] Example 7
[0073] Antimicrobial mechanism of antimicrobial peptides
[0074] S. typhimurium 14028 or S. aureus 29213 was cultured in MHB medium at 37°C and 220 rpm overnight, and then the bacterial solution was transferred to new MHB medium and cultured until the logarithmic growth phase of the bacteria. S. typhimurium 14028 or S. aureus 29213 was centrifuged and the MHB medium was discarded. The remaining bacteria were collected and washed three times with PBS (10 mM, pH = 7.4) and the bacteria were resuspended with PBS to an OD of 600=0.2. The antimicrobial peptide (control without antimicrobial peptide treatment) was incubated with bacteria at 37°C for 1 hour, then the bacteria were collected by centrifugation, 600 μL of glutaraldehyde (2.5%) was added to resuspend the bacteria, and fixed at 4°C overnight. The samples were dehydrated with different concentrations of ethanol (50%, 70%, 90% and 100%). The samples were replaced with a 1:1 mixture of ethanol and tert-butanol and tert-butanol for 15 minutes respectively. After the samples were dried with a freeze dryer, they were adhered to the sample plate with conductive tape, and a metal film was coated on the surface of the samples with a coating instrument. Finally, images were collected with a scanning electron microscope.
[0075] Figure 7 and Figure 8 The following are scanning electron micrographs of the antimicrobial peptide 2L2R2W on S.typhimurium 14028 and S.aureus 29213. Compared with the control, the bacterial membranes of S.typhimurium 14028 and S.aureus29213 treated with the antimicrobial peptide 2L2R2W showed wrinkles and ruptures, while the untreated bacterial membranes were smooth and intact, indicating that the antibacterial mechanism of the antimicrobial peptide 2L2R2W is to kill bacteria by destroying the bacterial membrane.
[0076] In summary, the antimicrobial peptide 2L2R2W has strong antibacterial activity against common intracellular bacteria such as Salmonella typhimurium, Listeria monocytogenes, and Staphylococcus aureus. It can efficiently penetrate macrophages and has excellent bactericidal effects on intracellular Salmonella and Staphylococcus aureus. It has low toxicity to macrophages and kills bacteria by destroying the bacterial membrane, and has extremely high application potential.
Claims
1. A cell-penetrating antimicrobial peptide 2L2R2W against intracellular bacteria, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2; its molecular formula is shown in formula (I), 2. The method for preparing a cell-penetrating antimicrobial peptide 2L2R2W against intracellular bacteria according to claim 1, characterized in that: The method is as follows: two consecutive Trp residues are selected and placed in the center of the sequence through symmetric sequence design to provide the cell penetration ability required for intracellular bactericidal killing; then two consecutive Leu residues are selected and placed at both ends of the sequence through hydrophobic amino acid terminal labeling technology to provide the hydrophobicity and stability required for antibacterial activity; finally, two consecutive Arg residues are selected and placed between Trp and Leu to provide the positive charge required for antibacterial activity; thus, a polypeptide is designed, and its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2; then, the polypeptide is synthesized by solid phase chemical synthesis, and the polypeptide is identified by mass spectrometry and purified by reversed-phase high performance liquid chromatography, and then subjected to antibacterial activity determination, cytotoxicity determination and cell penetration ability determination, and finally named as cell-penetrating antimicrobial peptide 2L2R2W.
3. Use of the anti-intracellular bacterial cell-penetrating antimicrobial peptide 2L2R2W according to claim 1 in the preparation of a medicament for treating Gram-positive and / or Gram-negative bacterial infectious diseases.
4. The use according to claim 3, wherein the Gram-negative bacteria are Salmonella, Escherichia coli or Pseudomonas aeruginosa.
5. The use according to claim 3, wherein the Gram-positive bacteria is Listeria monocytogenes, Staphylococcus epidermidis or Staphylococcus aureus.