Antibacterial peptide with double structural domains

Abstract

The invention relates to the technical field of antibacterial peptides, in particular to an antibacterial peptide with double structural domains. According to the invention, a new AMP (adenosine monophosphate) sequence is obtained from a protein sequence of Mesorhizobium sp., and the AMP sequence has double structural domains. According to the present invention, the independent research on the beta folding structure domain and the alpha spiral structure domain separately reveals that the two structure domains of the double-structure-domain antibacterial peptide have the potential of independently performing the antibacterial activity for the first time, and the beta folding region has the potential of inhibiting the formation of the microbial film and removing the mature microbial film, and particularly has significant antibacterial activity on fungi; the antibacterial activity on bacteria is weak, so that the damage to intestinal flora can be reduced. The invention provides a new thought and an experimental basis for designing the ultrashort antibacterial peptide, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and in particular to an antimicrobial peptide with double domains. Background Art

[0002] In recent years, antibiotics have achieved remarkable results in the treatment of microbial infectious diseases, bringing great breakthroughs to the development of medicine. However, with the widespread use of antibiotics, the problem of antimicrobial resistance (AMR) has gradually intensified and become a major challenge to global public health. AMR can lead to a series of public health problems such as decreased treatment effects, increased treatment costs, higher mortality rates, and increased pressure on medical resources.

[0003] Antimicrobial peptides (AMPs) have gradually gained attention due to their broad-spectrum and highly effective antimicrobial activity, unique mechanism of action, and low likelihood of inducing significant drug resistance in microorganisms. They are expected to become the next generation of drugs for treating microbial infections. A large amount of data shows that AMPs are significantly superior to traditional antibiotics in terms of the probability of inducing microbial production and the degree of inducing microbial resistance, which is of great significance in alleviating the problem of microbial resistance.

[0004] AMPs are usually cationic polypeptides, including abundant α-helical or β-folded structures. Unlike traditional antibiotics, AMPs mainly achieve their antibacterial and bactericidal effects by destroying the integrity of microbial membranes and causing the loss of cell contents. In addition, there is some evidence that AMPs can interact with targets within microbial cells and achieve their antibacterial and bactericidal effects by interfering with microbial energy metabolism, protein synthesis, DNA replication, and translation. Although some AMPs have shown two bactericidal mechanisms, there is currently no evidence that there are differences in the sequences or structures that exert different bactericidal mechanisms. Summary of the invention

[0005] First, the present invention discovered a new AMP sequence, whose amino acid sequence is RRYCRTYWRYGRLRRRCYRRRVWIWFRL (shown in SEQ ID No. 1, named mesoricin1). The AMP sequence is derived from Rhizobium ( Mesorhizobium sp.) The AMP sequence has 28 amino acids. Through structural analysis, the present invention found that the 3rd to 21st amino acids form a β-fold conformation, and the 22nd to 28th amino acids form an α-helical conformation, so the AMP sequence has a double domain.

[0006] Furthermore, based on the β-folded domain of the sequence shown in SEQ ID No.1, the present invention reduces the sequence length from 28 amino acids to 19 amino acids while retaining the antibacterial activity through domain identification and modification, and obtains an antibacterial peptide with an amino acid sequence of YCRIYWRHGRLKRRCFRRG (shown in SEQ ID No.2).

[0007] Compared with the original sequence, this antimicrobial peptide has significantly improved biofilm inhibition and mature biofilm clearance activity, and significantly reduced cytotoxicity, showing better biosafety, significantly reducing the difficulty of peptide chain synthesis and production costs, and providing greater structural regulation space for subsequent targeted delivery.

[0008] Furthermore, based on the α-helical domain of the sequence shown in SEQ ID No.1, the present invention obtains a series of ultrashort antimicrobial peptides through reasonable design, and the amino acid sequence thereof is one of the following: VWIWFRL (shown in SEQ ID No.3), RWIWFRL (shown in SEQ ID No.4), VFIWFRL (shown in SEQ ID No.5), VKIWFRL (shown in SEQ ID No.6), VKRWFRL (shown in SEQ ID No.7), VWIWFRF (shown in SEQ ID No.8), VWIWKRR (shown in SEQ ID No.9), VWIWCRR (shown in SEQ ID No.10) or VKIWFRL (shown in SEQ ID No.11).

[0009] Furthermore, the present invention provides a product containing any of the above antimicrobial peptides.

[0010] Preferably, the product is a medicine or a fungicide.

[0011] In some embodiments, the drug further includes a pharmaceutically acceptable excipient.

[0012] Preferably, the drug is a targeted drug.

[0013] Furthermore, the present invention provides the use of the antimicrobial peptide and the product in antibacterial activities; preferably, the antimicrobial activity is antifungal activity; more preferably, the fungus is an enveloped yeast.

[0014] Furthermore, the present invention provides the use of the antimicrobial peptide in the preparation of antimicrobial products.

[0015] Preferably, the product is a medicine or a fungicide.

[0016] In the present invention, the antimicrobial peptides include but are not limited to those obtained by solid phase peptide synthesis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention is from Rhizobium genus ( Mesorhizobium sp.) obtained a new AMP sequence, which has a dual domain. Through independent studies on the β-fold and α-helical domains, it was revealed for the first time that both domains of the dual-domain antimicrobial peptide have the potential to independently exert antimicrobial activity, among which the β-fold region has the potential to inhibit the formation of microbial membranes and remove mature microbial membranes, especially has significant antibacterial activity against fungi, and has weak antibacterial activity against bacteria, which may reduce the damage to intestinal flora. The present invention provides new ideas and experimental basis for the design of ultrashort antimicrobial peptides, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the amino acid sequence and corresponding spatial structure of mesoricin1.

[0019] Figure 2 is the circular dichroism spectrum of mesoricin1.

[0020] Figure 3 It is mesoricin1 C. neformans H99 bactericidal kinetic curve.

[0021] Figure 4 It is mesoricin1 C. neformans H99 biofilm inhibition and removal activity results, where A is the inhibition result and B is the removal result.

[0022] Figure 5 These are the cytotoxic results of mesoricin 1, where A is the toxicity to human red blood cells, B is the toxicity to HepG2, C is the toxicity to HT-29, and D is the toxicity to HEK-293.

[0023] Figure 6 It is mesoricin4 C. neformans H99 bactericidal kinetic curve.

[0024] Figure 7 It is mesoricin4 C. neformans H99 biofilm inhibition and removal activity results, where A is the inhibition result and B is the removal result.

[0025] Figure 8 These are the cytotoxic results of mesoricin4, where A is the toxicity to human red blood cells, B is the toxicity to HepG2, C is the toxicity to HT-29, and D is the toxicity to HEK-293. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0028] Example 1 Spatial structure analysis of mesoricin1 The amino acid sequence of mesoricin1 is shown in SEQ ID No. 1, and the spatial structure of meosoricin1 was predicted using AlphaFold2. Figure 1 As shown, the results showed that the 3rd to 21st amino acids formed a β-sheet conformation, and the 22nd to 28th amino acids formed an α-helical conformation.

[0029] Furthermore, mesoricin 1 with a purity of >95% was obtained by solid phase peptide synthesis, and the secondary structure of mesoricin 1 in water and TFE was detected using circular dichroism spectra. Figure 2 As shown, the results show that in water environment, it is mainly β-folding and random coil structure, and in TFE environment, it shows a certain 20% α-helix, which is close to the predicted result of the spatial structure.

[0030] Example 2 Antibacterial activity of mesoricin1 1. Detection of the antibacterial activity of mesoricin1 against 7 fungi and 5 bacteria The minimum inhibitory concentration (MIC) was detected by broth microdilution method. The microorganisms after MIC detection were inoculated on solid plate culture medium to detect the minimum bactericidal concentration (MBC). The results are shown in Table 1 below.

[0031] The results showed that mesoricin1 had good antibacterial and bactericidal activity against 7 representative fungi, but had no obvious antibacterial activity against bacteria, indicating that as an antibacterial drug it may have less impact on intestinal flora and is more beneficial for patients who need to maintain intestinal balance or need to take long-term medication.

[0032] Table 1 MIC and MBC of mesoricin against 7 representative fungi and 5 representative bacteria

[0033] Note: The 5 bacterial strains tested include Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Acinetobacter baumannii ATCC19606 and Pseudomonas aeruginosa CMCC-10104.

[0034] 2. Detection of the bactericidal kinetics of mesoricin1 1×10 5 CFU / mL C. neformans H99 were incubated with different concentrations of mesoricin1 at 35°C for 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 h, and the bactericidal effect was then detected using the plate colony counting method.

[0035] The results are as follows Figure 3 The results showed that even 1×MIC could kill all microorganisms within 6 hours, while 4×MIC mesoricin1 could kill all microorganisms within 2 hours. 4×MIC amphotericin B (AmB) could completely kill microorganisms within 60 minutes, while fluconazole (FLC) still failed to effectively reduce the bacterial load at the end of the experiment. These results show that mesoricin1 can effectively and quickly kill C. neformans H99.

[0036] Example 3 Inhibitory and Clearing Activity of Mesoricin1 on Fungal Biofilm Characterization of mesoricin1 inhibition using microbial film inhibition assay and microbial film removal assay C. neformans The ability of H99 to form biofilms and to remove mature microbial films.

[0037] The results are as follows Figure 4 As shown, mesoricin1 could inhibit biofilm formation by 80% at 4×MIC, but was unable to effectively eliminate microbial film at least at 16×MIC.

[0038] Example 4 Cytotoxicity of mesoricin1 The CCK8 assay was used to detect the cytotoxicity of mesoricin1 against four different cell types, including human red blood cells, HepG2, HT-29, and HEK-293.

[0039] The results are as follows Figure 5 As shown in the figure, the toxicity of mesoricin1 in each cell line showed different dose-dependencies. It also had a certain hemolytic activity against human red blood cells, with the half hemolytic concentration (HC 50 ) reached 134.2 μg / mL. Mesoricin1 also had a significant inhibitory effect on the growth of HT-29 and HEK-293 cells, with a half inhibitory concentration (HC 50 ) reached 16.82 and 16.13 μg / mL respectively, while for HepG2 cells, only about 60% of the cells survived at 128 μg / mL. The results showed that mesoricin1 has certain toxicity to mammalian cells.

[0040] Example 5 Modification of β-fold in mesoricin1 In this example, mesoricin1 was used as a template, and the β-folded sequence (YCRTYWRYGRLRRRCYRRR) was taken and named mesoricin2. It was verified by solid-phase peptide synthesis and microbial experiments that no antibacterial activity was shown. Single-point mutations were optimized with the antifungal index (AFI) as the goal, and the 13th amino acid in mesoricin2 was mutated (R13K) to obtain mesoricin3 (YCRTYWRYGRLKRRCYRRR). It was verified by solid-phase peptide synthesis and microbial experiments that no antibacterial activity was shown. Mesoricin3 was globally optimized with AFI as the goal, and the mutated amino acids included T4I, Y8H, Y16F and R19G to obtain mesoricin4 (YCRIYWRHGRLKRRCFRRG), and the antibacterial activity was detected by solid-phase peptide synthesis and microbial experiments.

[0041] The results are shown in Table 2. Although the number of amino acid residues of meosoricin4 was reduced from 28 to 19 compared with the original sequence, its antifungal activity was hardly reduced. This will significantly reduce the difficulty and cost of its synthesis, making it easier to achieve modification and targeted delivery.

[0042] Table 2 MIC and MBC of mesoricin4 against 7 representative fungi and 5 representative bacteria

[0043] The five bacterial strains tested are the same as those in Table 1.

[0044] Furthermore, mesoricin4 was obtained by separation and mutation of the β domain of mesoricin1. C. neformansThe bactericidal kinetics test of H99 showed the following results: Figure 6 Mesoricin4 at concentrations above the MIC can completely kill the cells within at least 10 hours. C. neformans H99, mesoricin4 with 4×MIC can also kill pathogens within 3 hours. Its killing rate is lower than that of mesoricin1.

[0045] Furthermore, the expression of mesoricin4 C. neformans H99 biofilm inhibition and mature biofilm removal activity were tested, and the results were as follows Figure 7 The results showed that mesoricin4 could achieve nearly 50% biofilm inhibition activity at 2×MIC, which was significantly higher than mesoricin1 at the same concentration. Mesoricin4 at 2×MIC could reduce the metabolic activity of mature biofilm to 60%, while mesoricin4 at 16×MIC could almost completely eliminate mature biofilm, which was also significantly higher than mesoricin1.

[0046] Furthermore, the toxicity of mesoricin4 to human red blood cells, HepG2, HT-29 and HEK-293 was tested. Figure 8 As shown, mesoricin4 has an effect on HC of human red blood cells. 50 Reached >256 μg / mL, IC for HepG2 and HT-29 cells 50 were also >128 μg / mL, while the IC 50 The results showed that mesoricin4 had significantly lower cytotoxicity to mammals than mesoricin1, showing good biosafety.

[0047] Example 6 Modification of the α-helix in mesoricin1 In this example, mesoricin1 was used as a template, and the α-helix sequence (VWIWFRL) was taken to obtain an ultrashort antimicrobial peptide with only 7 amino acids. Single-point mutation and multi-point mutation were performed with the antifungal index AFI as the target, and a total of 8 amino acid sequences were obtained. C. albicans ATCC10231, E. coli ATCC25922 and S. aureus The MIC of ATCC25923 is shown in Table 3. These results indicate that the α-helix of mesoricin1 also has certain antibacterial activity, and its antibacterial activity can be enhanced after reasonable design to form ultrashort antimicrobial peptides.

[0048] Table 3 MIC of α-helix and mutant sequences of mesoricin / (μg / ml)

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antimicrobial peptide having two domains, characterized in that: Its amino acid sequence is RRYCRTYWRYGRLRRRCYRRRVWIWFRL.

2. The antimicrobial peptide according to claim 1, characterized in that The 3rd to 21st amino acids form a β-sheet conformation, and the 22nd to 28th amino acids form an α-helical conformation.

3. An antimicrobial peptide, characterized in that: Its amino acid sequence is YCRIYWRHGRLKRRCFRRG.

4. An antimicrobial peptide, characterized in that Its amino acid sequence is one of the following: VWIWFRL, RWIWFRL, VFIWFRL, VKIWFRL, VKRWFRL, VWIWFRF, VWIWKRR, VWIWCRR or VKIWFRL.

5. A product, characterized in that: The antimicrobial peptide according to any one of claims 1 to 4 is contained.

6. The product according to claim 5, characterized in that The product is a medicine or a fungicide.

7. The product according to claim 6, characterized in that The medicine also includes pharmaceutically acceptable excipients.

8. The product according to claim 6, characterized in that The medicine is a targeted drug.

9. Use of the antimicrobial peptide according to any one of claims 1 to 4 or the product according to any one of claims 5 to 8 in antibacterial treatment; preferably, the antibacterial agent is an antifungal agent.

10. Use of the antimicrobial peptide according to any one of claims 1 to 4 in the preparation of antimicrobial products.

Images