Lipopolysaccharide response type nano antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses a lipopolysaccharide response type nano antibacterial peptide as well as a preparation method and application thereof, and belongs to the technical field of biology, and the amino acid sequence of the antibacterial peptide is as shown in SEQ ID No.1. Meanwhile, the invention further discloses application of the antibacterial peptide in preparation of drugs for treating gram-negative bacterium infectious diseases. The antibacterial peptide disclosed by the invention can be combined with lipopolysaccharide in a targeted manner to form nanofibers for capturing, and then the nanofibers are clustered and presented to phagocytes for phagocytosis, so that killing of beneficial bacteria in a host is fully avoided. In conclusion, the antibacterial peptide disclosed by the invention can target gram-negative bacteria to rupture membranes and sterilize, fully avoid mistakenly killing beneficial bacteria in a host body and effectively reduce the generation of drug resistance, so that the antibacterial peptide is a nano capture peptide with an immune regulation function, which has great development potential and relatively high application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a lipopolysaccharide-responsive nano antimicrobial peptide and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are an important part of the body's innate immune system. They are considered to be the most promising alternatives to antibiotics due to their membrane-breaking and bactericidal mechanisms that are not likely to induce bacterial resistance. Although antimicrobial peptides have shown incomparable superiority, they still face challenges in clinical application and animal husbandry production. On the one hand, traditional antimicrobial peptides often kill beneficial bacteria in the host due to their broad-spectrum antibacterial activity, and also have a killing effect on normal microbial flora in the intestine, thereby destroying the balance of the intestinal microecological environment and inducing severe secondary infections. On the other hand, after antibiotic-induced bacterial death, Gram-negative bacteria release membrane-bound lipopolysaccharide (LPS) into the blood. The uncontrolled systemic inflammatory response caused by endotoxemia can cause collateral damage to host tissues and interruption of physiological processes, which may lead to septic shock and high mortality to date. Therefore, mobilizing the body's immune function to synergistically resist and capture bacterial infection is an effective strategy that can further reduce the inflammatory response caused by lipopolysaccharide. Therefore, the new design of LPS-responsive self-assembling capture peptides is of great significance for solving the problem of drug resistance and finding alternatives to antibiotics. Summary of the invention

[0003] Based on the above problems, the purpose of the present invention is to provide a lipopolysaccharide-responsive nano antimicrobial peptide, which can be assembled into a nano-network structure in the presence of lipopolysaccharide, has a good killing effect on Gram-negative bacteria, and has good biocompatibility. At the same time, it can promote the phagocytosis of cell clusters by macrophages, and has the possibility of reducing bacterial resistance.

[0004] The technical solution adopted by the present invention is as follows: a lipopolysaccharide-responsive nano antimicrobial peptide FI2, whose amino acid sequence is shown in SEQ ID No.1.

[0005] Furthermore, the molecular formula of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 as described above is shown in formula (I):

[0006]

[0007] Furthermore, the self-assembly method of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 as described above has the following self-assembly conditions: placing the antimicrobial peptide FI2 with a concentration of 8-256 μM in deionized water containing lipopolysaccharide with a concentration of 20 μg / ml, and incubating at 37°C for 24 hours.

[0008] Another object of the present invention is to provide the use of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 as described above in the preparation of drugs for treating diseases caused by Gram-negative bacteria infections.

[0009] Furthermore, the Gram-negative bacteria are Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli or Salmonella typhimurium.

[0010] Another object of the present invention is to provide a drug as described above that is suitable for treating and / or preventing Gram-negative bacterial infection, wherein the drug contains a lipopolysaccharide-responsive nano antimicrobial peptide FI2 as described above.

[0011] Beneficial effects and advantages of the present invention: The antimicrobial peptide FI2 of the present invention has a significant inhibitory effect on Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium, but has no significant effect on Gram-positive bacteria such as Staphylococcus aureus, but has a significant capture effect on Escherichia coli under physiological conditions, and promotes the phagocytic ability of macrophages to bacterial clusters, which can not only capture bacteria and trigger the innate immune system to achieve antibacterial purposes, but also target Gram-negative bacteria to break the membrane and kill bacteria, fully avoiding the accidental killing of beneficial bacteria in the host, and effectively reducing the generation of drug resistance. In addition, the antimicrobial peptide FI2 of the present invention does not show hemolysis at the maximum test concentration in the presence or absence of lipopolysaccharide. In summary, the antimicrobial peptide FI2 of the present invention is a lipopolysaccharide-responsive self-assembling antimicrobial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the mass spectrum of antimicrobial peptide FI2;

[0013] Figure 2 is the chromatogram of antimicrobial peptide FI2;

[0014] Figure 3 It is the critical aggregation concentration of antimicrobial peptide FI2 and the linear fitting value diagram of the critical aggregation concentration;

[0015] Figure 4 Negative staining images of antimicrobial peptide FI2, including (a) control group LPS, (b) control group FI2, (c) control group FI2 and LPS co-incubated for 12 h;

[0016] Figure 5 Negative staining images of antimicrobial peptide FI2 co-incubated with E. coli; (a) control group, (b, c, d) control group FI2 and LPS co-incubated for 2 hours;

[0017] Figure 6 This is a graph showing the determination of the hemolytic activity of the antimicrobial peptide FI2;

[0018] Figure 7This is a graph showing the determination of the cytotoxicity of the antimicrobial peptide FI2;

[0019] Figure 8 The antimicrobial peptide FI2 promotes macrophage phagocytosis of E. coli 25922;

[0020] Fig. 9 To promote the phagocytosis of Escherichia coli by macrophages, (a) DAPI staining of the control group, (b) EGFP staining of the control group, (c) merge of the control group, (d) DAPI staining of the FI2-treated group; (e) EGFP staining of the FI2-treated group, (f) merge of the FI2-treated group. 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 peptide FI2: In coiled-coil peptides, the seven-residue pattern usually described by the heptapeptide abcdefg is repeated many times along the central axis of the helix. The residues at positions a and d or any i and i+3 positions are arranged along the same ridge of the helix. Here, the FI zipper is filled in the a and d positions of the heptapeptide with the α-helix rule as the hydrophobic group of the overall set chain. At the same time, lipopolysaccharide on the outer membrane surface of Gram-negative bacteria and teichoic acid and uronic acid teichoic acid on the cell wall surface of Gram-positive bacteria are both anionic substances, which can attract each other with antimicrobial peptides through electrostatic interaction. The net positive charge is an important factor affecting the capture of bacteria by antimicrobial peptides. In order to avoid the mutual repulsion between positive charges and affect the formation of nanofibers and further increase their tendency, positively charged arginine is filled in other positions of heptapeptide b and e. The two parts are separated by alanine to form an alternating hydrophobic structure to form a FRAIRAX sequence template, which is used to further utilize anion and cation complementarity between peptides to form a nanostructure, where X represents arginine and aspartic acid. The peptide FRAIRAX sequence template is repeated twice, forming a discrete ridge of repeated residues along the helical axis and presenting a one-to-one central symmetry in the overall structure. Finally, the peptide sequence (FRAIRAD)2(RARFARI)2 was designed; the amino acid sequence of the peptide is shown in Table 1.

[0024] Table 1 Amino acid sequence of lipopolysaccharide-responsive nanoantimicrobial peptide FI2

[0025]

[0026] Its molecular structure is shown in formula (I),

[0027]

[0028] Example 2

[0029] Synthesis of lipopolysaccharide-responsive nanoantimicrobial peptide FI2 by solid-phase chemical synthesis

[0030] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. Weigh 3g of RINK resin (substitution degree 0.3mmol / g) in a 150ml reactor and soak it with 50ml of dichloromethane (DCM). After 2 hours, wash the resin with 3 times the volume of the resin in nitrogen-dimethylformamide (DMF), and then drain it. Repeat this four times, and drain the resin for standby use. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor, and shake it on a decolorization shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with 3 times the volume of the resin in DMF, and then drain it;

[0031] 2. Take a small amount of resin and test it with the ninhydrin (Nine Wells Hydrated Ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1min). If the resin has color, it means that the deprotection is successful. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) in a 50ml centrifuge tube, add 20ml of DMF to dissolve it, then add 3ml of N, N-diisopropylcarbodiimide (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;

[0032] 3. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with 3 times the volume of the resin in DMF, and drain it for later use. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, 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, and then drain it;

[0033] 4. Take a small amount of resin and test it with the ninhydrin (Nine Wells Hydrated Ninhydrin) method (two drops of test A and test B, react at 100℃ for 1min). If the resin has color, it means that the deprotection is successful. Weigh the second amino acid and HOBT in a 50ml centrifuge tube, add 25ml of DMF to dissolve it, 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 30℃ shaker for reaction. After 1 hour, take a small amount of resin for detection and test it with the ninhydrin method (two drops of test A and test B, react at 100℃ for 1min). If the resin is colorless, it means that the reaction is complete; if the resin has color, it means that the condensation is incomplete, and the reaction is continued. After the reaction is complete, wash the resin with DMF four times, then drain it, add a certain amount of 20% piperidine (piperidine / DMF=1:4) to the reactor, and shake it on a decolorizing shaker for 20min to remove the Fmoc protecting group on the resin. After deprotection, wash with DMF four times and then drain to check whether the protection is removed;

[0034] 5. 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 liquid (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) to cut the polypeptide from the resin (add 10 ml of cutting liquid per gram of resin), and centrifuge and precipitate four times with ice ether (cutting liquid: ether = 1:9). Finally, use HPLC to separate and purify, and then freeze-dry to obtain a polypeptide with a certain purity;

[0035] 6. Peptide identification: The peptides obtained above were analyzed by electrospray mass spectrometry, such as Figure 1 The molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight in Table 1, and the purity of the polypeptide is greater than 95%.

[0036] Example 3

[0037] Nanoscale characterization of antimicrobial peptide FI2:

[0038] Critical aggregation concentration determination: In order to detect the ability of the antimicrobial peptide FI2 to form nanostructures at a lipopolysaccharide concentration of 20 μg / ml, the critical aggregation concentration (CAC) was determined using a 1-aniline-8-naphthalenesulfonic acid (ANS) fluorescent probe. 1 μL of ANS (final concentration of 1 mM, dissolved in 100% DMF) was added to different concentrations of polypeptides (dissolved in deionized water with an LPS concentration of 20 μg / ml) and incubated at 37°C for 24 hours. The mixed sample was transferred to a 96-well plate and the fluorescence spectrum was scanned using a fluorescence microplate reader with an excitation wavelength of 369 nm and an emission wavelength of 440 nm-550 nm. The CAC value of the polypeptide was then calculated using Origin software. The test results are shown in Figure 3 .

[0039] from Figure 3 (a) shows that when the LPS concentration is 20 μg / ml, the fluorescence intensity of the antimicrobial peptide FI2 rises sharply in the range of 440nm-550nm as the concentration increases, indicating the presence of nano-macromolecules in the solution. The formation of nanostructures was preliminarily determined, and then the Origin software was used for fitting analysis. Figure 3 As shown in (b), the CAC value of the antimicrobial peptide FI2 was 28.4 μM.

[0040] Example 4

[0041] Negative staining: After incubating the antimicrobial peptide FI2 with 20 μg / ml lipopolysaccharide for 12 h, 20 μL of the bacterial solution was placed on a copper mesh plate for 2 min and stained with 0.1% phosphotungstic acid for 10 s. Finally, the peptide morphology was detected after air drying under a Hitachi H-7650 transmission electron microscope (Hitachi H-7650, Japan). The test results are shown in Figure 4 .from Figure 4 (b, c) It can be seen that the peptide sample alone is in a dispersed state, and in the presence of lipopolysaccharide, a dense nanofiber structure is formed. Negatively stained bacterial TEM: Escherichia coli cells in the logarithmic growth phase were washed by centrifugation with PBS (10 mM) buffer and resuspended to OD 600 nm =0.4. E. coli cells were combined with peptide nanofibers (32 μM) and incubated at 37°C for 2 h. 20 μL of bacterial solution was placed on a copper mesh plate for 2 min. E. coli cells were stained with 0.1% phosphotungstic acid for 3 s. Finally, the air-dried bacterial morphology was detected under a Hitachi H-7650 transmission electron microscope (Hitachi H-7650, Japan). The test results are shown in Figure 5 .and Figure 5 (a) Compared with Figure 5 (b) It can be observed that the antimicrobial peptide FI2 is attached to the surface of Escherichia coli, which further verifies that the antimicrobial peptide FI2 targets the cell membrane surface of Gram-negative bacteria. Figure 5 (c, d) The antimicrobial peptide FI2 was incubated with Escherichia coli to assemble into nanofibers, which further captured the Escherichia coli.

[0042] Example 5

[0043] Determination of in vitro hemolytic activity, cytotoxicity and bactericidal activity of antimicrobial peptide FI2:

[0044] 1. Hemolytic activity assay: Collect 1 mL of fresh healthy human blood and centrifuge it (4°C, 1000×g, 5 min), then wash it three times with PBS (10 mM) buffer solution, discard the supernatant, collect the precipitated blood cells and resuspend them to 10 mL with PBS (10 mM) buffer solution. Subsequently, place an equal volume of diluted red blood cell suspension in a 96-well plate and mix it with polypeptide solutions of different concentrations. After incubation at 37°C for 1 hour, centrifuge (1000×g, 10 min), and aspirate the supernatant and transfer it to a new 96-well plate. The wells treated with 0.1% Triton X-100 serve as positive controls, and the wells without polypeptide treatment serve as negative controls. Use an enzyme-labeled instrument to set the wavelength to 570 nm to measure the absorbance value. The polypeptide concentration that causes 50% hemolytic activity is defined as cytotoxic. See attached for test results. Figure 6 .

[0045] pass Figure 6 It can be seen that the antimicrobial peptide FI2 (128 μM) still did not cause hemolytic toxicity at high concentrations, which indicates that the antimicrobial peptide FI2 has good biocompatibility.

[0046] 2. Cytotoxicity assay: The cells frozen in liquid nitrogen were revived and inoculated into a culture medium containing 10% fetal bovine serum and 1% double antibody, and subcultured at 37°C and 5% CO2. The cultured cells were digested with 0.25% trypsin and adjusted to 2-4×10 5 cells / mL. 50μL of cell suspension was mixed with 50μL of peptides of different concentrations in a 96-well plate and incubated at 37°C and 5% CO2 for 24h. Then 50μL of MTT (5mg / mL) was added to each well and incubated for another 4h. After the incubation, the supernatant was discarded, 100μL of DMSO was used to dissolve the crystals at the bottom of the well, and the absorbance of each well was measured at 570nm using an ELISA reader. The culture medium wells were used as blank controls. The test results are shown in Figure 7 .

[0047] from Figure 7 It can be seen that even after treatment with high concentrations of antimicrobial peptide FI2, the cell survival rates of HEK 293T and Caco-2 were greater than 50%, and the cell survival rate of RAW264.7 was greater than 50% at a concentration of 32 μM, indicating that the antimicrobial peptide FI2 has good biocompatibility and has the potential to become an antibiotic substitute.

[0048] 3. Determination of bactericidal activity: The minimum inhibitory concentration of antimicrobial peptides was determined by microdilution method. Different concentrations of peptides were added to BSA solution in a 96-well plate, and then an equal volume of a final concentration of 1×10 5 CFUmL -1The final peptide concentration in the 96-well plate ranged from 0.5 to 128 μM. After 24 hours of incubation, the absorbance value was measured using a microplate reader set at a wavelength of 600 nm. The test results are shown in Table 2.

[0049] Table 2 Antibacterial activity against 8 Gram-negative bacterial strains (μM)

[0050]

[0051] GM a : Geometric mean MIC value for Gram-negative bacteria

[0052] It can be seen from Table 2 that under physiological conditions, the antimicrobial peptide FI2 has bactericidal ability and exhibits strong antibacterial activity against Gram-negative bacteria.

[0053] Example 6

[0054] Determination of the immunomodulatory ability of antimicrobial peptide FI2:

[0055] 1. Gentamicin protection test: In order to evaluate whether the antimicrobial peptide FI2 can promote the phagocytosis of bacterial clusters by macrophages under physiological conditions, the bacterial content in macrophages was detected by gentamicin protection test. Take the E. coli 25922 bacterial solution in the logarithmic growth phase, centrifuge at 3000 rpm for 5 minutes to collect the bacteria, wash them three times with PBS buffer, resuspend them in PBS buffer, and adjust the bacterial concentration to OD 600nm =0.4. 16μM and 32μM antimicrobial peptides FI2 and FF2 were added to the bacterial suspension and co-cultured at 37°C for 2 hours. The bacterial particles pretreated with peptides were added to the cell culture medium of RAW 264.7 and cultured at 37°C for 2 hours. The cell culture medium was aspirated and washed 3 times. A solution containing 50μg / mL of gentamicin was added to remove extracellular bacteria. 0.1% Triton X-100 was used for lysis for 10 minutes. 50μL of lysate was aspirated and 450μL of high-temperature sterilized PBS was added for gradient dilution. 100μL of the dilution was evenly inoculated on the MHA plate culture medium. Cultured overnight in a 37°C incubator, the colony count (CFU) of each sample was calculated, the experiment was repeated three times, and the test results are shown in Figure 8 .

[0056] from Figure 8 It can be seen that compared with the control group, pretreatment with 16μM and 32μM antimicrobial peptide FI2 significantly increased the internalization of E. coli particles in RAW 264.7 in a dose-dependent manner. This indicates that antimicrobial peptide FI2 can capture bacteria and further enhance the phagocytic ability of phagocytes.

[0057] 2. Macrophage phagocytosis of bacteria test: In order to visually observe the effect of antimicrobial peptide FI2 on promoting the phagocytosis of bacterial clusters by macrophages, the phagocytosis of Escherichia coli by RAW 264.7 was observed using a fluorescent inverted microscope. Prepare E. coli particles containing green fluorescent protein, collect the E. coli cells by centrifugation at 3000rpm for 5 minutes, and resuspend them in PBS buffer. Add a peptide solution with a final concentration of 16μM and co-culture with the bacterial suspension at 37°C for 2 hours. Add the bacterial particles pretreated with the peptide to the cell culture medium of RAW264.7, culture at 37°C for 2 hours, aspirate the cell culture medium and wash 3 times, add Triton X-100 to fix for 10 minutes, aspirate Triton X-100 and wash 3 times, and add an anti-fade agent containing DAPI. Observe with a fluorescent inverted microscope. The test results are shown in Fig. 9 .

[0058] The results are as follows Fig. 9 As shown in the figure, compared with the control, E. coli particles treated with antimicrobial peptide FI2 aggregated and the phagocytosis of aggregated E. coli particles by RAW 264.7 macrophages was enhanced. This indicates that the nano-capture peptide can capture bacteria and further promote the uptake of phagocytes.

[0059] Therefore, the antimicrobial peptide FI2 of the present invention can target and bind to lipopolysaccharide to form nanofibers for capture, and then present them in agglomerates to phagocytosis, thereby fully avoiding the killing of beneficial bacteria in the host.

Claims

1. A lipopolysaccharide-responsive nano antimicrobial peptide FI2, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The lipopolysaccharide-responsive nano antimicrobial peptide FI2 according to claim 1, characterized in that: Its molecular formula is shown in formula (I):

3. The self-assembly method of a lipopolysaccharide-responsive nano antimicrobial peptide FI2 according to claim 1, characterized in that: The self-assembly conditions were as follows: the antimicrobial peptide FI2 at a concentration of 8-256 μM was placed in deionized water containing lipopolysaccharide at a concentration of 20 μg / ml and incubated at 37° C. for 24 hours.

4. Use of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 according to claim 1 in the preparation of a drug for treating diseases caused by Gram-negative bacteria.

5. The use according to claim 4, characterized in that: The Gram-negative bacteria are Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli or Salmonella typhimurium.

6. A drug suitable for treating and / or preventing Gram-negative bacterial infection, characterized in that: The medicine contains the lipopolysaccharide-responsive nano antimicrobial peptide FI2 as described in claim 1.

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