An antimicrobial peptide with two domains

Abstract

The present invention relates to the field of antimicrobial peptide technology, and in particular to an antimicrobial peptide with a double domain. Mesorhizobium A new AMP sequence with two domains was identified from the protein sequence of the plant (Eubacterium sp.). Independent studies of the β-sheet and α-helical domains revealed for the first time that both domains of a dual-domain antimicrobial peptide have the potential to independently exert antimicrobial activity. The β-sheet region has the potential to inhibit microbial film formation and eliminate mature microbial films, exhibiting particularly significant antimicrobial activity against fungi, but less potent antimicrobial activity against bacteria, potentially reducing damage to the intestinal microbiota. This invention provides new insights and experimental basis for the design of ultrashort antimicrobial peptides and holds broad application prospects.

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 success in treating microbial infections, bringing significant breakthroughs to medical development. However, with the widespread use of antibiotics, antimicrobial resistance (AMR) has gradually intensified, becoming a major challenge to global public health. AMR can lead to a range of public health problems, including decreased treatment efficacy, increased treatment costs, higher mortality rates, and increased pressure on medical resources.

[0003] Antimicrobial peptides (AMPs) are gaining increasing attention due to their broad-spectrum, highly effective antimicrobial activity, unique mechanisms of action, and resistance to inducing significant microbial resistance. They are expected to become the next generation of drugs for treating microbial infections. Extensive data demonstrate that AMPs significantly outperform traditional antibiotics in both the probability of inducing microbial infection and the degree of inducing drug resistance, demonstrating their importance in alleviating the problem of microbial resistance.

[0004] AMPs are typically cationic polypeptides, including abundant α-helical or β-pleated structures. Unlike traditional antibiotics, AMPs primarily achieve their antibacterial and bactericidal effects by disrupting the integrity of microbial membranes and causing the loss of cellular contents. Furthermore, some evidence suggests that AMPs can interact with targets within microbial cells, achieving both antibacterial and bactericidal effects by interfering with processes such as microbial energy metabolism, protein synthesis, DNA replication, and translation. Although some AMPs have been shown to have two bactericidal mechanisms, there is currently no evidence that the sequences or structures underlying these mechanisms differ. 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). This AMP sequence is derived from Rhizobium genus ( Mesorhizobium sp.) in the protein sequence. This AMP sequence has 28 amino acids. Through structural analysis, the present invention found that amino acids 3-21 form a β-sheet conformation, and amino acids 22-28 form an α-helical conformation, thus this AMP sequence has a dual domain structure.

[0006] Furthermore, based on the β-pleated 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 antibacterial activity through domain identification and modification, and obtains an antibacterial peptide with the amino acid sequence 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 obtained a series of ultrashort antimicrobial peptides through reasonable design, whose amino acid sequences are as follows: 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 treatment; preferably, the antimicrobial agent is an antifungal agent; 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, 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:

[0018] The present invention is from Rhizobium genus ( Mesorhizobium A new AMP sequence with two domains was identified from the protein sequence of the plant (Eubacterium sp.). Independent studies of the β-sheet and α-helical domains revealed for the first time that both domains of a dual-domain antimicrobial peptide have the potential to independently exert antimicrobial activity. The β-sheet region has the potential to inhibit microbial film formation and eliminate mature microbial films, exhibiting particularly significant antimicrobial activity against fungi, but less potent antimicrobial activity against bacteria, potentially reducing damage to the intestinal microbiota. This invention provides new insights and experimental basis for the design of ultrashort antimicrobial peptides and holds broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the amino acid sequence and corresponding spatial structure of mesoricin1.

[0020] Figure 2 is the circular dichroism spectrum of mesoricin1.

[0021] Figure 3 It is mesoricin1 C. neformans H99 bactericidal kinetic curve.

[0022] Figure 4 It is mesoricin1 C. neformans H99 biofilm inhibition and clearance activity results, where A represents the inhibition result and B represents the clearance result.

[0023] Figure 5 These are the cytotoxicity results of mesoricin 1, where A represents the toxicity to human red blood cells, B represents the toxicity to HepG2, C represents the toxicity to HT-29, and D represents the toxicity to HEK-293.

[0024] Figure 6 It is mesoricin4 C. neformans H99 bactericidal kinetic curve.

[0025] Figure 7 It is mesoricin4 C. neformans H99 biofilm inhibition and clearance activity results, where A represents the inhibition result and B represents the clearance result.

[0026] Figure 8 These are the cytotoxicity results of mesoricin4, where A represents the toxicity to human red blood cells, B represents the toxicity to HepG2, C represents the toxicity to HT-29, and D represents the toxicity to HEK-293. DETAILED DESCRIPTION

[0027] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0028] In the examples provided herein, if specific techniques or conditions are not specified, the experiments were performed according to those described in literature in the field or according to the product instructions. Reagents or instruments used without manufacturer's indication are conventional products available through regular channels.

[0029] Example 1 Spatial structure analysis of mesoricin1

[0030] 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 amino acids 3-21 formed a β-sheet conformation and amino acids 22-28 formed an α-helical conformation.

[0031] 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 spectroscopy. Figure 2 As shown, the results show that in water environment, it is mainly β-folded and random coil structures, while in TFE environment, it shows a certain 20% α-helix, which is close to the predicted results of the spatial structure.

[0032] Example 2 Antibacterial activity of mesoricin 1

[0033] 1. Detection of the antibacterial activity of mesoricin 1 against 7 fungi and 5 bacteria

[0034] The broth microdilution method was used to determine the minimum inhibitory concentration (MIC). Microorganisms tested after MIC were inoculated onto solid plate culture media to determine the minimum bactericidal concentration (MBC). The results are shown in Table 1 below.

[0035] The results showed that mesoricin 1 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.

[0036] Table 1 MIC and MBC of mesoricin against 7 representative fungi and 5 representative bacteria

[0037]

[0038] Note: The 5 bacterial strains tested include Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Acinetobacter baumannii ATCC19606 and Pseudomonas aeruginosa CMCC 10104.

[0039] 2. Detection of the bactericidal kinetics of mesoricin1

[0040] 1×10 5 CFU / mL C. neformans H99 cells 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 by plate colony counting.

[0041] The results are as follows Figure 3 As shown in the figure, 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.

[0042] Example 3 Inhibitory and Clearing Activity of Mesoricin 1 on Fungal Biofilms

[0043] Characterization of mesoricin-1 inhibition using microbial film inhibition and microbial film clearance assays C. neformans The ability of H99 to form biofilms and to remove mature microbial films.

[0044] The results are as follows Figure 4As shown in the results, mesoricin1 could inhibit biofilm formation by 80% at 4×MIC, but could not effectively eliminate microbial film at least at 16×MIC.

[0045] Example 4 Cytotoxicity of mesoricin1

[0046] The CCK8 assay was used to detect the cytotoxicity of mesoricin-1 against four different cell types, including human red blood cells, HepG2, HT-29, and HEK-293.

[0047] The results are as follows Figure 5 As shown in the figure, the toxicity of mesoricin1 in various cell lines 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 cells survived at 128 μg / mL. These results indicate that mesoricin-1 has a certain degree of toxicity to mammalian cells.

[0048] Example 5 Modification of β-sheet in mesoricin 1

[0049] In this example, mesoricin 1 was used as a template, and the β-sheet sequence (YCRTYWRYGRLRRRCYRRR) was selected and named mesoricin 2. Solid-phase peptide synthesis and microbial assays confirmed that the product exhibited no antibacterial activity. Single-point mutation optimization, targeting the antifungal index (AFI), was performed by mutating amino acid 13 of mesoricin 2 (R13K), resulting in mesoricin 3 (YCRTYWRYGRLKRRCYRRR). Solid-phase peptide synthesis and microbial assays confirmed that the product exhibited no antibacterial activity. Global optimization of mesoricin 3, targeting the AFI, resulted in mutations to amino acids T4I, Y8H, Y16F, and R19G, resulting in mesoricin 4 (YCRIYWRHGRLKRRCFRRG). Its antibacterial activity was tested by solid-phase peptide synthesis and microbial assays.

[0050] The results are shown in Table 2. Although the number of amino acid residues in meosoricin4 has been reduced from 28 to 19 compared to the original sequence, its antifungal activity has hardly decreased. This will significantly reduce the difficulty and cost of its synthesis, making it easier to modify and target its delivery.

[0051] Table 2 MICs and MBCs of mesoricin4 against 7 representative fungi and 5 representative bacteria

[0052]

[0053] The five bacterial strains tested are the same as those in Table 1.

[0054] Furthermore, mesoricin4, obtained by separation and mutation of the β domain of mesoricin1, was further analyzed. C. neformans The bactericidal kinetics test of H99 showed the following results: Figure 6 As shown. Mesoricin4 at concentrations above the MIC can completely kill the C. neformans H99, mesoricin 4 at 4× MIC can also kill pathogens within 3 hours. Its killing rate is lower than that of mesoricin 1.

[0055] Furthermore, the expression of mesoricin4 C. neformans H99 biofilm inhibition and mature biofilm removal activities 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, a significant improvement compared to mesoricin1 at the same concentration. At 2×MIC, mesoricin4 could reduce the metabolic activity of mature biofilms to 60%, while at 16×MIC, mesoricin4 could almost completely eliminate mature biofilms, also a significant improvement compared to mesoricin1.

[0056] 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, with IC values ​​of >256 μg / mL 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 mammalian cells than mesoricin1, demonstrating good biosafety.

[0057] Example 6 Modification of the α-helix in mesoricin 1

[0058] 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.

[0059] Table 3 MICs of mesoricin α-helix and mutant sequences (μg / ml)

[0060]

[0061] 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 various embodiments of the present invention.

Claims

1. Use of an antimicrobial peptide with a dual domain structure in the preparation of an antimicrobial product; the amino acid sequence of the antimicrobial peptide with a dual domain structure is RRYCRTYWRYGRLRRRCYRRRVWIWFRL.

2. The use according to claim 1, characterized in that The 3rd to 21st amino acids in the antimicrobial peptide with double domains form a beta sheet conformation, and the 22nd to 28th amino acids form an alpha helical conformation.

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