Double-target antifungal peptide as well as preparation method and application thereof

Abstract

The invention provides two double-target antibacterial peptides as well as a preparation method and application thereof, and belongs to the field of bioengineering, the sequences of the double-target antibacterial peptides are shown as NLR-2 and NIR-3, and the N end of the double-target antibacterial peptides is connected with carboxyl of 3-(1-naphthyl) acrylic acid. The two antifungal peptides disclosed by the invention have good bactericidal activity (1 mu M) on cryptococcus neoformans under physiological conditions, and meanwhile, have a relatively good mildew-proof effect on corn feed, and can effectively reduce the toxin yield of the corn feed caused by fusarium graminearum. Therefore, the antifungal peptide provided by the invention has a good bactericidal function, can target a fungal membrane structure, achieves the effects of efficient membrane rupture and sterilization, and can be used as a novel mildew preventive.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to two dual-target nano antifungal peptides and preparation methods and applications thereof. Background Art

[0002] As the primary component of the host's own defense system, antimicrobial peptides (AMPs) are widely distributed in animals, plants, and microorganisms, and are considered to be one of the most promising alternatives to antibiotics. Membrane-active antimicrobial peptides (AMPs) selectively destroy the intact structure of microbial membranes and are considered to be a new generation of antibiotics for multidrug-resistant pathogenic fungi. Accurate understanding of the interaction between membrane-active drugs and biofilm components is crucial to reveal their molecular mechanisms and optimize their targeting specificity, and is also a prerequisite for reducing potential side effects. Ergosterol is a key sterol component in the cell membrane of fungi (including molds and yeasts). The reduction of ergosterol levels in fungal cell membranes can easily lead to damage to their cell membranes or even death, so most antifungal drugs target key enzymes involved in the ergosterol biosynthesis pathway. Dual-target drug design is considered to be a more effective strategy that can exert synergistic inhibition advantages similar to combination therapy in disease systems, thereby achieving a "1+1>" therapeutic effect. The construction of new antifungal peptides and compounds developed in this way is expected to improve the efficacy of antifungal peptides, reduce toxicity, and reduce drug resistance. Summary of the invention

[0003] Based on the above background technology problems, the purpose of the present invention is to provide two dual-target antifungal peptides that can self-assemble into nanoparticles in ultrapure water and have good killing effects on Cryptococcus neoformans and Fusarium graminearum in a physiological environment. In addition, the antifungal peptides of the present invention can effectively destroy the fungal cell membrane structure and kill fungi.

[0004] The technical scheme adopted by the present invention is as follows: two dual-target nano antifungal peptides NLR-2 and NLR-3, whose amino acid sequences are shown in SEQ ID No. 1, and whose N-termini are connected to the carboxyl group of 3-(1-naphthyl) acrylic acid.

[0005] Another object of the present invention is to provide a method for preparing two dual-target nano antifungal peptides NLR-2 and NLR-3, as follows:

[0006] Step S1: Using the heptad repeat sequence template (LKAIRRR) n (n=2, 3) as a hydrophobic scaffold to provide hydrophobic force;

[0007] Step S2: selecting 3-(1-naphthyl) acrylic acid to connect with it to construct a polypeptide;

[0008] Step S3: using solid phase chemical synthesis and mass spectrometry identification to complete the preparation of the peptides, and then measuring the nanomorphology, in vitro cytotoxicity, hemolytic activity, and bactericidal activity of the peptides, and finally naming them as antimicrobial peptides NLR-2 and NLR-3;

[0009] Furthermore, the self-assembly method of the two dual-target nano antifungal peptides NLR-2 and NLR-3 as described above is as follows: the concentration is 64 μM, and they can self-assemble into nanostructures by incubation at 37° C. for 24 hours.

[0010] Another object of the present invention is to provide the use of the above-mentioned dual-target nano antifungal peptides NLR-2 and NLR-3 in preventing corn mildew caused by Fusarium graminearum.

[0011] Beneficial effects and advantages of the present invention: The dual-target nano antifungal peptides NLR-2 and NLR-3 of the present invention form nanoparticles in a physiological environment, which are more conducive to destroying fungal cell membranes. They have direct bactericidal activity under physiological conditions and have a good killing effect on Cryptococcus neoformans and Fusarium graminearum. In summary, NLR-2 and NLR-3 are self-assembling antifungal peptides with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Mass spectra of NLR-2 (a) and NLR-3 (b);

[0013] Figure 2 are the chromatograms of NLR-2 (a) and NLR-3 (b);

[0014] Figure 3 Transmission electron microscopy nanostructured images of NLR-2 (a) and NLR-3 (b);

[0015] Figure 4 For the determination of NLR-2 and NLR-3 cytotoxicity;

[0016] Figure 5 It is the determination of hemolytic activity of NLR-2 and NLR-3;

[0017] Figure 6 The killing activity of NLR-2 and NLR-3 against Cryptococcus Neoformans 32609 and Fusarium graminearum 37687;

[0018] Figure 7 The effects of NLR-2 and NLR-3 on the outer membrane permeability of Cryptococcus Neoformans 32609 (a) and Fusarium graminearum 37687 (b) cells;

[0019] Figure 8 The effects of NLR-2 and NLR-3 on ergosterol on the cell membrane of Cryptococcus Neoformans 32609 (a) and Fusarium graminearum 37687 (b);

[0020] Fig. 9 Scanning electron microscopy observations of the morphological effects of NLR-2 and NLR-3 on Cryptococcus Neoformans 32609 (A) and Fusarium graminearum 37687 (B). (a) Physiological environment control group, (b) Physiological environment NLR-2 peptide treatment group, (c) Physiological environment NLR-3 peptide treatment group, (d) (e) (f) are partial enlarged images, respectively. 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 NLR-2 and NLR-3: Selection of heptad repeat sequence template (LKAIRRR) n (n=2, 3) as the hydrophobic core, and promote the self-assembly of peptide molecules by connecting 3-(1-naphthyl) acrylic acid; the naphthyl group can simultaneously dock with the dual-target active areas of SE and CYP 51 on the cell membrane, inhibit ergosterol synthesis, and thus enhance the membrane destruction ability; the amino acid sequence of the peptide is shown in Table 1.

[0024] Table 1 Amino acid sequences of dual-target nano antifungal peptides NLR-2 and NLR-3

[0025]

[0026] Example 2

[0027] Synthesis of dual-target nano antifungal peptides NLR-2 and NLR-3 by solid-phase chemical synthesis

[0028] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus using a peptide synthesizer. 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). After 2 hours, wash the resin with 3 times the volume of the resin in nitrogen-dimethylformamide (DMF), then drain it, and repeat this four times. Drain the resin and set it aside. 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, then drain it;

[0029] 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;

[0030] 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;

[0031] 4. Take a small amount of resin and test it with the ninhydrin (Nine Wells Hydrated Ninhydrin) method (two drops of each 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 shaker at 30℃ for reaction. After 1 hour, take a small amount of resin for detection and test it with the ninhydrin method (two drops of each 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;

[0032] 5. After the last amino acid is attached, remove the protection, wash with DMF four times, 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 polypeptide 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). Finally, use HPLC to separate and purify, and then freeze-dry to obtain a polypeptide with a certain purity;

[0033] 6. Add naphthyl acrylic acid, and the carboxyl group of 3-(1-naphthyl) acrylic acid condenses with the amino group of the last amino acid at the N-terminus;

[0034] 7. Identification of polypeptides: The polypeptides 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%.

[0035] Example 3

[0036] Transmission electron microscopy nano-characterization images of NLR-2 and NLR-3:

[0037] Nanomorphology analysis: To further analyze the nanomorphology of NLR-2 and NLR-3, the peptide (2.56 mM) was diluted to a concentration of 64 μM in deionized water and incubated in a 37°C incubator for 24 h. The samples were smeared on glass slides and coated with metal using a Polaron SC7640 sputter coater after natural drying. The samples were observed at 5 KV by a Hitachi S-4800SEM (Hitachi, Japan). The test results are shown in Figure 3 .

[0038] from Figure 3 It can be seen that (a) NLR-2 and (b) NLR-3 form spherical nanoparticles under physiological conditions;

[0039] Example 4

[0040] In vitro cytotoxicity, hemolytic activity and bactericidal activity assays of NLR-2 and NLR-3:

[0041] 1. 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 5cells / mL. 50 μL of cell suspension was mixed with 50 μL of peptides of different concentrations in a 96-well plate and incubated at 37°C and 5% CO2 for 24 h. Then 25 μL MTT (5 mg / mL) was added to each well and incubated for another 4 h. After the incubation, the supernatant was discarded, 100 μL DMSO was used to dissolve the crystals at the bottom of the well, and the absorbance of each well was measured at 570 nm using an ELISA reader. The culture medium wells were used as blank controls. The test results are shown in Figure 4 .

[0042] from Figure 4 It can be seen that even after treatment with high concentrations of NLR-2 and NLR-3, the cell survival rate of HEK293T was greater than 50%, indicating that NLR-2 and NLR-3 have good biocompatibility and have the potential to become antibiotic substitutes.

[0043] 2. 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 HEPES inner membrane buffer (pH=7.4), discard the supernatant, collect the precipitated blood cells and resuspend them to 10 mL with HEPES inner membrane 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 with a wavelength set 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 5 .

[0044] pass Figure 5 It can be seen that NLR-2 and NLR-3 (128 μM) still did not cause hemolytic reaction at high concentrations, which indicates that NLR-2 and NLR-3 have good biocompatibility.

[0045] 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 CFU / mL of Cryptococcus Neoformans 32609 / Fusarium graminearum 37687, the final peptide concentration in 96-well plates ranged from 0.5 to 64 μM. After incubation at 30°C for 72 hours, 50 μL was aspirated and serially diluted in PBS. The minimum bactericidal concentration was determined using the plate count method to kill 99.99% of the bacteria. The test results are shown in Figure 6 .

[0046] Depend on Figure 6 It can be seen that under physiological conditions, antifungal peptides have bactericidal ability, 1μM NLR-2 and NLR-3 can kill Cryptococcus neoformans, 32μM NLR-2 and 16μM NLR-3 can kill Fusarium graminearum.

[0047] Example 5

[0048] Mechanism of action assay: Prepare a spore suspension of Fusarium graminearum / Cryptococcus neoformans and dilute the culture to 1 × 10 using HEPES buffer. 5 CFU / mL. Subsequently, it was mixed with N-phenyl-1-naphthylamine (NPN, Sigma-Aldrich) fluorescent probe and incubated at 30°C in the dark for 30 min. Then, different amounts of peptides (50 μL) were introduced into a 96-well plate. The fluorescence intensity was measured by using an F-4500 fluorescence spectrophotometer with an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The results are shown in Figure 7 .

[0049] from Figure 7 It can be seen that NLR-2 and NLR-3 can significantly increase the fluorescence intensity, which indicates that NLR-2 and NLR-3 can increase the permeability of the fungal outer membrane.

[0050] Example 6

[0051] The spore suspension of Fusarium graminearum was inoculated into 20 mL of PDB medium to make the spore concentration 1×10 6 CFU / mL, and placed in a constant temperature shaking incubator at 28°C, 150r / min, and cultured for 12h. Take a certain concentration of polypeptide and add it to the PDB medium of Fusarium graminearum that has been cultured for 12h, so that the polypeptide concentrations of the test group are 1×MIC and 2×MIC, respectively, and the control group is 1×MIC of sodium benzoate. Each concentration is repeated 3 times. Continue to culture in a constant temperature shaking incubator at 28°C, 150r / min, and natural light. When the entire culture period reaches 3d, stop the culture and wait for filtration.

[0052] Pick a single colony in PDB medium and culture it to the logarithmic growth phase. Then transfer the adjusted bacteria into PBS solution and wash the bacteria three times. The final dilution concentration is 1×10 6 CFU / mL. 1×MIC and 2×MIC peptides were added to the bacteria and incubated for 2 hours. The control group was amphotericin B with the MIC concentration, and the bacteria were collected by centrifugation.

[0053] Wash the cells with PBS solution for 3 times, collect the cells by centrifugation, take 0.1g wet cells, add 3mL 25% KOH-ethanol solution, vortex for 2min, and place in a water bath at 85℃ for 4h. After cooling to room temperature, add 1mL deionized water and 3mL n-heptane for extraction, vortex for 3min, and stand at room temperature for 1 hour to separate the layers. Take 1.5mL of the upper n-heptane layer, centrifuge at 12000rpm for 10min, and transfer 1mL of the supernatant to an EP tube for liquid chromatography analysis. The results are shown in Table 1. Figure 8 .

[0054] from Figure 8 a It can be seen that 2 μM NLR-2 and NLR-3 significantly reduced the ergosterol content of Cryptococcus Neoformans 32609. Figure 8 b It can be seen that 32 μM NLR-2 and 16 μM NLR-3 significantly reduced the ergosterol content of Fusarium graminearum 37687, which indicates that NLR-2 and NLR-3 effectively inhibited the synthesis of ergosterol in Crytococcus Neoformans 32609 and Fusarium graminearum 37687.

[0055] Example 7

[0056] The spore suspension of Fusarium graminearum was inoculated into PDB medium to make the spore concentration 1×10 6 CFU / mL, 28℃, 150r / min, culture for 72h, wash the mycelium with HEPES solution, take equal mass of mycelium and resuspend it in NLR-2 (32μM) and NLR-3 (16μM), incubate for 2h, and collect the mycelium. Pick a single colony of Cryptococcus neoformans and culture it in potato dextrose broth overnight, and transfer it to a new potato dextrose broth medium to grow to the mid-logarithmic phase. Then centrifuge the above fungal solution and resuspend it in phosphate buffer to a final concentration of OD 600nm =0.4, add the prepared antimicrobial peptide to a final concentration of 2 μM, and incubate at room temperature for 1 hour.

[0057] The mycelium / thalli were placed in 2.5% glutaraldehyde and fixed overnight at 4°C. After the mycelium was dehydrated in sequence (50%, 70%, 90% and 100%), 600 μL of tert-butyl alcohol-ethanol (1:1, v / v) and whole tert-butyl alcohol were added and mixed for 15 min, and then freeze-dried and coated. Finally, the samples were observed using a Hitachi S-4800 scanning electron microscope.

[0058] from Fig. 9It can be seen that in the control group, Cryptococcus neoformans and Fusarium graminearum maintained complete membrane structures with smooth and intact appearances. In the peptide-treated group, the antifungal peptides attached to the periphery of Cryptococcus neoformans and Fusarium graminearum, increasing the outer membrane permeability and penetrating the outer membrane to reach the plasma membrane, depolarizing the plasma membrane and thus destroying the integrity of the plasma membrane, leading to the leakage of fungal contents and the shrinkage of the fungus, thus causing the death of the fungus.

Claims

1. Two dual-target nano antifungal peptides NLR-2 and NLR-3, characterized by: Its amino acid sequence is shown in SEQ ID No. 1, and its N-terminus is connected to the carboxyl group of 3-(1-naphthyl) acrylic acid, so as to target two key enzymes (SE / CYP51) of ergosterol on the fungal membrane.

2. The method for preparing the two dual-target antifungal peptides NLR-2 and NLR-3 according to claim 1, characterized in that: Here are the steps: Step S1: Using the heptad repeat sequence template (LKAIRRR) n (n=2, 3) as a hydrophobic scaffold to provide hydrophobic force; Step S2: selecting 3-(1-naphthyl) acrylic acid to connect with it to construct a polypeptide; Step S3: The peptides were prepared by solid phase chemical synthesis and mass spectrometry, and then the nanomorphology, in vitro cytotoxicity, hemolytic activity, bactericidal activity, membrane fluidity and ergosterol content on the fungal membrane were measured. Finally, they were named antifungal peptides NLR-2 and NLR-3.

3. The self-assembly method of two dual-target antifungal peptides NLR-2 and NLR-3 according to claim 1, characterized in that: The concentration was 64 μM and incubated at 37°C for 24 hours to self-assemble into nanostructures.

4. The use according to claim 4, characterized in that: Under physiological conditions, it has a killing effect on Cryptococcus neoformans and Fusarium graminearum.

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