Novel peptide derived from hirudo nipponia and uses thereof

Abstract

The present invention relates to a novel peptide derived from Hirudo nipponia, which has antimicrobial activity, antibiofilm activity, antibiotic sensitivity–enhancing activity, and anti-inflammatory activity. With antimicrobial activity, antibiofilm activity, antibiotic sensitivity–enhancing activity, and anti-inflammatory activity, the novel peptide derived from Hirudo nipponia according to the present invention can be advantageously applied as a material for various purposes in the pharmaceutical field, food field, cosmetic field, and agriculture and livestock industries. In addition, the novel antimicrobial peptide derived from Hirudo nipponia according to the present invention increases the sensitivity of antibiotics to multidrug-resistant bacteria, thereby markedly enhancing antimicrobial activity against multidrug-resistant bacteria when used in combination with antibiotics. Accordingly, a composition of the present invention comprising the peptide as an active ingredient can be advantageously used as a pharmaceutical composition for enhancing antibiotic sensitivity. In particular, the novel peptide of the present invention has very low hemolytic activity and cytotoxicity, and thus is safe for the human body. Moreover, the novel peptide of the present invention not only has excellent activity even under a high salt concentration, but also maintains activity even in a serum environment, thereby having high stability when applied in vivo.

Description

Novel peptide derived from Hirudoniphonia and uses thereof

[0001] The present invention relates to a novel peptide derived from Hirudo nipponia having antibacterial activity, antibiometabolic activity, antibiotic sensitivity-enhancing activity, and anti-inflammatory activity.

[0002] Hirudo nipponia, a native Korean leech, has been used for therapeutic purposes for over 100 years due to the numerous active components present in its body, particularly in its salivary glands. In Hirudo nipponia, saliva is secreted through small holes between calcified teeth, and the secretory effect of glandular cells is induced by neuropeptide and serotonin stimulation. Extensive studies on the transcriptome, genomics, and proteome of leeches have been conducted to identify and characterize the active components in the leech's saliva, particularly peptides and proteins. To date, more than 34 molecules have been identified in Hirudo nipponia, including peptides, phosphatidylcholine, pteridines, and other components.

[0003] Antimicrobial peptides (AMPs) are essential and universal components of innate immunity, acting as effective weapons against various pathogens, including bacteria, fungi, viruses, and protozoa. AMPs kill pathogens using non-specific mechanisms that disrupt cell membranes and reduce the likelihood of antibiotic resistance. Blood-sucking leeches (Phylum: Annelida) have garnered attention as a promising source of AMPs. AMPs derived from leeches are particularly attractive in the medical field due to their unique ability to store ingested blood without degrading for extended periods. The leech's immune system has evolved to inhibit the growth of exogenous pathogens ingested with food and to regulate the gut microbiome. Therefore, these AMPs are promising antimicrobial agents with low toxicity. Notable antimicrobial peptides (AMPs) from various leech species, such as theromyzin, theromacin, neuromacin, and hydramacin-1, exhibit potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. However, limitations such as a narrow spectrum of activity, potential toxicity to human cells, and sensitivity to proteolysis hinder therapeutic efficacy. This necessitates the discovery of new antimicrobial peptides (AMPs) with enhanced properties, including broader antimicrobial activity, higher stability, and reduced cytotoxicity.

[0004] Against this background, the inventors performed simultaneous screening of antimicrobial peptide (AMP) candidates derived from the salivary gland transcriptome genome database of Hirudoniphonia using in silico antimicrobial peptide (AMP) prediction, and confirmed that among 19 antimicrobial peptide candidates, a specific peptide (hirunipin 1-3) possesses excellent antimicrobial activity and antibiole activity against various pathogens, excellent synergistic activity when combined with existing antibiotics, and anti-inflammatory activity. In particular, the present invention was completed by confirming that the novel peptide (hirunipin 1-3) of the present invention can significantly enhance antimicrobial activity against multidrug-resistant bacteria when used in combination with antibiotics by increasing the sensitivity of multidrug-resistant bacteria to antibiotics.

[0005] Therefore, the objective of the present invention is to provide a novel peptide derived from Hirudo nipponia.

[0006] Another objective of the present invention is to provide a composition for enhancing antibiotic sensitivity comprising a novel peptide derived from Hirudo nipponia as an active ingredient.

[0007] Another objective of the present invention is to provide an antibiotic adjuvant composition comprising the novel peptide as an active ingredient.

[0008] Another objective of the present invention is to provide the above-mentioned antibiotic sensitivity-enhancing composition and an antibiotic composition comprising an antibiotic as an active ingredient.

[0009] In order to achieve the objectives of the present invention as described above,

[0010] The present invention provides a novel peptide composed of any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

[0011] In one embodiment of the present invention, the peptide may have antibacterial activity, antibiometabolic activity, antibiotic sensitivity enhancing activity or / and anti-inflammatory activity.

[0012] In one embodiment of the present invention, the peptide may have antibacterial activity against one or more bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and multidrug-resistant bacteria.

[0013] In one embodiment of the present invention, the multidrug-resistant bacteria may be one or more selected from the group consisting of multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Enterobacter cloacae, and multidrug-resistant Escherichia coli.

[0014] In one embodiment of the present invention, the peptide may have a minimum biofilm eradication concentration of 64 to 256 μg / mL against multidrug-resistant Acinetobacter baumannii.

[0015] In one embodiment of the present invention, the peptide can enhance the sensitivity to the antibiotic when used in combination with the antibiotics chloramphenicol, ciprofloxacin, tetracycline, or rifampicin.

[0016] In one embodiment of the present invention, the peptide has anti-inflammatory activity by inhibiting the production and mRNA expression of inflammatory cytokines TNF-α, IL-6, and MCP-1.

[0017] In addition, the present invention provides a composition for enhancing antibiotic sensitivity comprising, as an active ingredient, a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

[0018] In one embodiment of the present invention, the antibiotic is methicillin, oxacillin, norfloxacin, vancomycin, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, Cefadroxil, Cefazolin, Cephalotin, Cephalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin / clavulanate, Ampicillin / sulbactam, Piperacillin / tazobactam, Ticarcillin / clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Naldixic acid,Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), TMP-SMX), Sulfonamido chrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidi acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin / Dalfopristin, Thiamphenicol,It can be selected from the group consisting of tigecycline, tinidazole, and trimethoprim.

[0019] In one embodiment of the present invention, the peptide may have synergistic antibacterial activity against multidrug-resistant bacteria when used in combination with an antibiotic.

[0020] In one embodiment of the present invention, the multidrug-resistant bacteria may be one or more selected from the group consisting of multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Enterobacter cloacae, and multidrug-resistant Escherichia coli.

[0021] In addition, the present invention provides an antibiotic adjuvant composition comprising, as an active ingredient, a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

[0022] In addition, the present invention provides the above-mentioned antibiotic sensitivity-enhancing composition and an antibiotic composition comprising an antibiotic as an active ingredient.

[0023] In one embodiment of the present invention, the antibiotic is methicillin, oxacillin, norfloxacin, vancomycin, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, Cefadroxil, Cefazolin, Cephalotin, Cephalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin / clavulanate, Ampicillin / sulbactam, Piperacillin / tazobactam, Ticarcillin / clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Naldixic acid,Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), TMP-SMX), Sulfonamido chrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidi acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin / Dalfopristin, Thiamphenicol,It can be selected from the group consisting of tigecycline, tinidazole, and trimethoprim.

[0024] The novel peptide derived from Hirudo nipponia according to the present invention possesses antibacterial activity, antimicrobial biofilm activity, antibiotic sensitivity-enhancing activity, and anti-inflammatory activity; as such, it can be usefully utilized as a material for various applications in the pharmaceutical, food, cosmetic, and agricultural sectors. Furthermore, the novel antibacterial peptide derived from Hirudo nipponia according to the present invention can significantly enhance antibacterial activity against multidrug-resistant bacteria when used in combination with antibiotics by increasing antibiotic sensitivity to multidrug-resistant bacteria; thus, the composition of the present invention containing this as an active ingredient can be usefully utilized as a pharmaceutical composition for enhancing antibiotic sensitivity. In particular, the novel peptide of the present invention has the advantage of being safe for the human body due to its very low hemolytic activity and cytotoxicity. Additionally, the novel peptide of the present invention not only exhibits excellent activity even at high salt concentrations but also maintains activity in a serum environment, thereby possessing high stability when applied in vivo.

[0025] Figure 1a shows the experimental design and workflow for antimicrobial peptide prediction using in silico tools and ODT-HTS. Figure 1b shows the ratio of PI-positive bacterial cells to total bacterial cells for 19 antimicrobial peptides identified by the ODT-HTS method. Red bars represent the mean ± standard deviation of each sample from three independent experiments. Figure 1c shows the helical wheel diagram of the hirunipin peptide determined by the HeliQuest server (https: / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py). Positively charged residues are indicated by blue circles, negatively charged residues by red, hydrophilic residues by purple, amide residues by pink, hydrophobic residues by yellow, and small residues by gray. The N- and C-terminal regions of the peptide are indicated by red N and C, respectively. Z: Net charge, μH: Hydrophobic moment. 1d shows the change in cytoplasmic membrane potential of S. aureus KCTC 1621 treated with hirunifin peptide (16 μg / mL). 1e shows the concentration-dependent depolarization induced by hirunifin peptide. 1f shows the results of measuring the membrane uptake of N-phenyl-1-naphthylamine (NPN) in E. coli (KCTC 1682) in the presence of various concentrations of hirunifin peptide. 1g shows the results of measuring the hydrolysis of ortho-nitrophenyl-beta-D-galactosidase (ONPG) due to cytoplasmic β-galactosidase release in E. coli ML-35 cells treated with various concentrations of hirunifin peptide.

[0026] Figures 2a and 2b show the results of PI-inflow analysis using a flow cytometer after treating E. coli (KCTC 1682) and S. aureus (KCTC 1621), respectively, with hirunipin peptide (16 μg / mL) for 60 minutes. Figure 2c is an SEM image of E. coli (KCTC 1682) treated with hirunipin peptide (16 μg / mL). Scale bar: 200 nm.

[0027] Figure 3a shows representative 3D-ODT images of E. coli 1 hour after treatment with the control group and hirunipin 2. Figure 3b shows representative 2D-ODT and fluorescence images regarding PI internalization in E. coli. Figure 3c shows the results of quantitative analysis of PI fluorescence intensity 1 hour after treatment with the control group and hirunipin 2 in E. coli. Figure 3d shows time-lapse images using ODT-HTS over time after treatment with the control group and hirunipin 2 (1×MIC) in E. coli. Figure 3e shows segmented ODT images including PI signals following treatment with the control group and hirunipin 2 in E. coli. Figure 3f shows the results of quantitative analysis of the mean RI (mean refractive index) over time after treatment with the control group and hirunipin 2 (1×MIC) in E. coli. Figure 3g is E. These are the results of a quantitative analysis of cell numbers over time in E. coli after treatment with a control group and hirunipin 2 (1×MIC). 3h represents the results of an analysis of the PI-positive / total cell ratio over time after treatment with a control group and hirunipin 2 (1×MIC) in E. coli. Scale bar = 1 μm. All values ​​are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, and ***p < 0.001.

[0028] Figure 4a shows the experimental design of real-time ODT imaging of biofilm formation and the color map of RI. Figures 4b through 4k show merged or segmented 3D ODT images of multidrug-resistant Acinetobacter baumannii (MDRAB) biofilms treated with hirunipin 1, 2, 3, LL-37, and a control. Figures 4l through 4n show the results of quantitative analysis for the dry mass, surface area, and volume of multidrug-resistant Acinetobacter baumannii (MDRAB) biofilms treated with hirunipin 1, 2, 3, LL-37, and a control. Figure 4o shows the percentage of biofilm eradication of multidrug-resistant Acinetobacter baumannii (MDRAB) treated with hirunipin 1, 2, 3, and LL-37. Scale bar = 20 μm. All values ​​are expressed as mean ± standard deviation.

[0029] Figures 5a to 5d show time-death curves for the combined treatment of hirunipin 2 and antibiotics at single or synergistic concentrations on multidrug-resistant Acinetobacter baumannii (MDRAB). CHL represents chloramphenicol, CIP represents ciprofloxacin, TET represents tetracycline, and RIF represents rifampicin. The Y-axis represents CFU on a log scale. Values ​​are expressed as the mean ± standard error of three independent experiments and are statistically significant at *p < 0.05. Figure 5e shows the biofilm formation inhibition rate (%) for the combined treatment of hirunipin 2 and antibiotics at single or synergistic concentrations on multidrug-resistant Acinetobacter baumannii (MDRAB). 1: Control group, 2: 0.1% Triton-X100, 3: Hirunipin 2 (4 μg / mL), 4: Chloramphenicol (128 μg / mL), 5: Hirunipin 2 (4 μg / mL) + Chloramphenicol (128 μg / mL), 6: Hirunipin 2 (4 μg / mL), 7: Ciprofloxacin (32 μg / mL), 8: Hirunipin 2 (4 μg / mL) + Ciprofloxacin (32 μg / mL), 9: Hirunipin 2 (8 μg / mL), 10: Tetracycline (2 μg / mL), 11: Hirunipin 2 (8 μg / mL) + Tetracycline (2 μg / mL), 12: Hirunipin 2 (8 μg / mL), 13: Rifampicin (1 μg / mL), 14: Hirunipin 2 (8 μg / mL) + rifampicin (1 μg / mL). Values ​​are expressed as the mean ± standard error of three independent experiments and are statistically significant at *p < 0.05.Figures 5f to 5i represent the results of measuring the fluorescence intensity of PI using a flow cytometer following the combined treatment of hirunipin 2 and antibiotics at single or synergistic concentrations with multidrug-resistant Acinetobacter baumannii (MDRAB) (5f: hirunipin 2 (4 μg / mL), CHL (128 μg / mL), and hirunipin 2 (4 μg / mL) + CHL (128 μg / mL); 5g: hirunipin 2 (4 μg / mL), CIP (32 μg / mL), and hirunipin 2 (4 μg / mL) + CIP (32 μg / mL); 5h: hirunipin 2 (8 μg / mL), TET (2 μg / mL), and hirunipin 2 (4 μg / mL) + TET (2 μg / mL); 5i: hirunipin 2 (16 μg / mL), RIF (1 μg / mL), and hirunipin 2 (16 μg / mL) + RIF (1 μg / mL)). CHL means chloramphenicol, CIP means ciprofloxacin, TET means tetracycline, and RIF means rifampicin.

[0030] Figure 6 shows the results of measuring the hemolysis rate (%) of sheep red blood cells according to treatment with different concentrations of hirunipin and melittin.

[0031] Figure 7 shows the results of measuring cell viability (%) according to treatment with different concentrations of hirunipin and melittin (7a: mouse macrophage RAW 264.7, 7b: mouse fibroblast NIH-3T3, 7c: human keratinocyte HaCat).

[0032] Figure 8 shows the results of evaluating the drug resistance of hirunipin 2, tetracycline, and ciprofloxacin against E. coli (KCTC1682) through multiple passage resistance experiments.

[0033] Figures 9a to 9c show the quantification of inflammatory cytokine release in LPS-stimulated mouse macrophages treated with different concentrations of hirunipin 2 using sandwich ELISA (9a: TNF-α, 9b: IL-6, 9c: MCP-1), and 9d shows the results of measuring the mRNA expression levels of inflammatory cytokines via RT-PCR.

[0034] In one embodiment, the present invention relates to a novel peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

[0035] In the present invention, "peptide" means a polymer composed of two or more amino acids connected by amide bonds (or peptide bonds).

[0036] The peptide of the present invention may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3, and may include an amino acid sequence having sequence homology of at least 75%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3, and may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

[0037] Additionally, functional variants of the peptide of the present invention may also be included. The functional variants include biological equivalents of the peptide sequence described herein (any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3). For example, additional changes may be made to the amino acid or polynucleotide sequence of the peptide to further improve the binding affinity and / or other biological properties of the peptide. Such modifications include deletion, insertion, and / or substitution of amino acid sequence residues of the peptide and may be based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge size, etc.

[0038] In addition, the peptide of the present invention can be obtained by various methods widely known in the field. For example, it can be prepared by using a polynucleotide recombination and protein expression system, by synthesizing in vitro through chemical synthesis such as peptide synthesis, and by cell-free protein synthesis methods.

[0039] In the present invention, the peptide having the amino acid sequence of SEQ ID NO. 1 was named 'Hirunipin 1'; the peptide having the amino acid sequence of SEQ ID NO. 2 was named 'Hirunipin 2'; and the peptide having the amino acid sequence of SEQ ID NO. 3 was named 'Hirunipin 3'.

[0040] A peptide composed of any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3 of the present invention may have antibacterial activity, antibiometabolic activity, antibiotic sensitivity enhancing activity or / and anti-inflammatory activity.

[0041] The novel peptide of the present invention may have antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria.

[0042] The above Gram-positive bacteria may be any Gram-positive bacteria known in the art, including Staphylococcus, Listeria, Streptococcus, Corynebacterium, Lactobacillus, Clostridium, Enterococcus, Erysipelothrix, and Bacillus, and preferably may be Staphylococcus aureus and Staphylococcus epidermidis, but are not limited thereto.

[0043] The above Gram-negative bacteria may be any Gram-negative bacteria known in the art, including Escherichia coli, Pseudomonas, Salmonella, Leptospira, and Rickettsia, and preferably Escherichia coli and Pseudomonas aeruginosa, but are not limited thereto.

[0044] The above antibiotic-resistant bacteria refers to bacteria (strains) that are resistant to antibiotics, and the above antibiotics may include, for example, β-lactam antibiotics, aminoglycoside antibiotics, quinolone antibiotics, peptide antibiotics, glycopeptide antibiotics, tetracycline antibiotics, rifamycin antibiotics, lincomycin antibiotics, and macrolide antibiotics, but are not specifically limited thereto.

[0045] In one embodiment of the present invention, the antibiotic-resistant bacteria may be multidrug-resistant bacteria that exhibit resistance to various antibiotics, and preferably may be multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Enterobacter cloacae, and multidrug-resistant Escherichia coli, but are not limited thereto.

[0046] The novel peptide of the present invention has excellent antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Enterobacter cloaca and multidrug-resistant Escherichia coli.

[0047] The novel peptide of the present invention may have antimicrobial biofilm activity.

[0048] In one embodiment of the present invention, the novel peptide may have a Minimal Biofilm Eradication Concentration against multidrug-resistant Acinetobacter baumannii of 64 to 256 μg / mL.

[0049] The novel peptide of the present invention may have antibiotic sensitivity-enhancing activity.

[0050] In one embodiment of the present invention, the peptide exhibits synergistic activity in which the antibacterial activity against multidrug-resistant bacteria is significantly increased compared to the use of the antibacterial agent alone when used in combination with existing antibiotics such as chloramphenicol, ciprofloxacin, tetracycline, or rifampicin, and exhibits a strong synergistic effect in inhibiting biofilm formation, thereby having activity that enhances the sensitivity of antibiotics.

[0051] The novel peptide of the present invention may have anti-inflammatory activity.

[0052] In one embodiment of the present invention, the peptide can effectively inhibit the production and mRNA expression of inflammatory cytokines TNF-α, IL-6, and MCP-1.

[0053] The peptide of the present invention is safe for the human body due to its very low hemolytic activity and cytotoxicity. In addition, the peptide of the present invention not only exhibits excellent activity even at high salt concentrations but also maintains activity in a serum environment, thus possessing high stability when applied in vivo.

[0054] In another aspect, the present invention relates to a pharmaceutical composition for enhancing antibiotic sensitivity, comprising as an active ingredient a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

[0055] The peptide of the present invention can have synergistic antimicrobial activity against multidrug-resistant bacteria when used in combination with antibiotics.

[0056] In the present invention, 'antibiotic' is methicillin, oxacillin, norfloxacin, vancomycin, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, Cefadroxil, Cefazolin, Cephalotin, Cephalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin / clavulanate, Ampicillin / sulbactam, Piperacillin / tazobactam, Ticarcillin / clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Naldixic acid,Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), TMP-SMX), Sulfonamido chrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidi acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin / Dalfopristin, Thiamphenicol,It may be one or more selected from the group consisting of tigecycline, tinidazole, trimethoprim, combinations thereof, and derivatives thereof, and preferably one or more selected from the group consisting of chloramphenicol, tetracycline, ciprofloxacin, and rifampicin, but the types are not particularly limited.

[0057] In another aspect, the present invention relates to an antibiotic adjuvant composition comprising, as an active ingredient, a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

[0058] The peptide of the present invention has synergistic activity against multidrug-resistant bacteria when used in combination with antibiotics, so it can be used to assist antibiotics.

[0059] The antibiotic adjuvant composition of the present invention may be administered at the time of antibiotic administration, together with the antibiotic, before antibiotic treatment, or after antibiotic treatment, and may be used as an adjuvant to enhance the antimicrobial activity of the antibiotic. It is preferable that the antibiotic adjuvant composition has a synergistic effect with respect to the antimicrobial activity of the antibiotic, and it is preferable that it increases the antimicrobial activity of the antibiotic against multidrug-resistant bacteria.

[0060] In another aspect, the present invention relates to the above-mentioned antibiotic sensitivity-enhancing composition and an antibiotic composition comprising an antibiotic as an active ingredient.

[0061] The 'antibiotic composition' of the present invention preferably exhibits antibacterial activity against the following pathogens, but is not limited thereto: Acinetobacter baumannii, Actinomyces (e.g., Actinomyces israelii and Actinomyces naeslundii), Aeromonas (e.g., Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria) and Aeromonas caviae), Anaplasma phagocytophilum, and Alkaline genus Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus sp. (e.g., Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (e.g., Bacteroides fragilis), Bartonella sp. (e.g., Bartonella bacilliformis and Bartonella henselae), Bifidobacterium sp., Bordetella Genus (Bordetellasp.) (e.g., Bordetellella pertussis,Bordetella parapertussis and Bordetella bronchiseptica), Borrelia genus (e.g., Borrelia recurrentis and Borrelia burgdorferi), Brucella genus (e.g., Brucellus abortus, Brucellus canis, Brucellus melintensis and Brucellus suis), Burkholderia genus (e.g., Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter genus (e.g., Campylobacter jejuni), Campylobacter coli, Campylobacter lari, and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (e.g., Corynebacterium diphtheria, Corynebacterium jeikeum, and Corynebacterium), Clostridium sp. (e.g.,Clostridium perfringens, Clostridium difficile, Clostridium botulinum, and Clostridium tetani), Eikenella corrodens, Enterobacter species (e.g., Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae), enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and urinary tract-causing bacteria Escherichia coli, including opportunistic E. coli such as uropathogenic E. coli), Enterococcus sp. (e.g., Enterococcus faecalis and Enterococcus faecium), Ehrlichia sp. (e.g., Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus Genus (Haemophilus sp.) (e.g.,Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus), Helicobacter genus (e.g., Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae), Kingella kingii, Klebsiella genus (e.g., Klebsiella pneumoniae, Klebsiella Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (e.g., Mycobacterium leprae, Mycobacterium Mycobacterium intracellulare, Mycobacterium avium,Mycobacterium bovis and Mycobacterium marinum), Mycoplasma sp. (e.g., Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (e.g., Nocardia asteroides, Nocardia cyriacigeorgica, and Nocardia brasiliensis), Neisseria sp. (e.g., Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas sigeloid shigelloides), Prevotellasp., Porphyromonassp., Prevotella melaninogenica, Proteussp. (e.g., Proteus vulgaris and Proteus mirabilis), Providenciasp. (e.g., Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Salmonella Genus (Salmonella sp.) (e.g., Salmonella enterica,Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium, Serratia sp. (e.g., Serratia marcesans and Serratia liquifaciens), Shigella sp. (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis) epidermidis), Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus genus (e.g., Streptococcus pneumoniae, Spectinomycin-resistant serotype 6B Streptococcus pneumoniae, Streptomycin-resistant serotype 9V Streptococcus pneumoniae, Erythromycin-resistant serotype 14 Streptococcus pneumoniae), Optochin-resistant serotype 14 Streptococcus pneumoniae serotype 14Streptococcus pneumoniae),Rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, and streptomycin-resistant serotype 9V pneumoniae Streptococcus (Streptomycin-resistant serotype 9V Streptococcus pneumoniae), Optochin-resistant serotype 14 Streptococcus pneumoniae, Rifampicin-resistant serotype 18C Streptococcus pneumoniae, Penicillin-resistant serotype 19F Streptococcus pneumoniae or Trimethoprim-resistant serotype 23F Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus Mutans (Streptococcus mutans), pyogenic streptococci (Streptococcus pyogenes),Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci and Streptococcus anginosus, Group G streptococci, Spirillum minus minus), Streptobacillus moniliformi, Treponema genus (e.g., Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum), Veillonellasp., Vibrio genus (e.g., Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus), Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus,Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela, and Vibrio furnisii), Yersinia genus (e.g., Yersinia enterocolitica and Yersinia pestis), and Xanthomonas maltophilia.

[0062] The antibiotic composition of the present invention is intended to kill bacteria or inhibit bacterial growth, and preferably is intended to prevent or treat infectious diseases caused by pathogens.

[0063] The above infectious diseases are any one or more selected from the group consisting of food poisoning, impetigo, cellulitis, scalded skin syndrome, mastitis, bacteremia, sepsis, staphylococcal pneumonia, endocarditis, osteomyelitis, staphylococcal sepsis, toxic shock syndrome, hospital-acquired pneumonia, urinary tract infection, systemic infection (bacterial dermatitis and sepsis), skin and soft tissue infection, surgical infection, intra-abdominal infection, pulmonary infection (including that in patients with cystic fibrosis), Helicobacter pylori (and alleviation of the above complications involving peptic ulcer disease, gastric cancer, etc.), diabetic foot infection, osteomyelitis, and central nervous system infection. It may be an infectious disease, but is not specifically limited to this.

[0064] The term "prevention" as used in the present invention may refer to any act of administering the antimicrobial peptide of the present invention to an individual to suppress or delay the onset of bacterial disease caused by Gram-positive bacteria, Gram-negative bacteria, or antibiotic-resistant bacteria.

[0065] The term "treatment" as used in the present invention may refer to any act of administering the antimicrobial peptide of the present invention to an individual suspected of developing a bacterial disease caused by Gram-positive bacteria, Gram-negative bacteria, or antibiotic-resistant bacteria to improve or benefit the symptoms of said disease.

[0066] As used in the present invention, the term "individual" may refer to any animal, including humans, that has developed or is likely to develop a bacterial disease caused by Gram-positive bacteria, Gram-negative bacteria, or antibiotic-resistant bacteria. Such animals may include not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, but are not limited thereto.

[0067] The antibiotic composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. Carriers usable in the present invention may include slowly metabolized macromolecules such as liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Examples may include salts of inorganic acids such as hydrochloride, hydrobromide, phosphate, and sulfate; pharmaceutically acceptable salts such as salts of organic acids such as acetate, propionate, malonate, and benzoate; liquids such as water, brine, glycerol, and ethanol; and auxiliary materials such as hydrates, emulsifiers, or pH buffers. Pharmaceutically acceptable carriers are described in the literature [Remingtion's Pharmaceutical Sciences, Mack Publishing Company, 1991].

[0068] The antibiotic composition of the present invention may be formulated into a unit-dose formulation suitable for administration into a patient's body according to conventional methods in the pharmaceutical field, preferably in a formulation useful for administration of peptide drugs, and may be administered orally or by a parenteral administration route including, but not limited to, the skin, intravenously, intramuscularly, intra-arterially, intramedullaryly, intrathecally, intraventricularly, pulmonaryly, transdermally, subcutaneously, intraperitoneally, intranasally, gastrointestinally, topically, sublingually, vaginally, or rectal route using administration methods conventionally used in the industry.

[0069] The antibiotic composition of the present invention can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation.

[0070] The peptide, which is the active ingredient in the antibiotic composition of the present invention, can actually be administered in various parenteral formulations. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as bases for suppositories.

[0071] The total effective amount of the antimicrobial peptide, which is the active ingredient in the antibiotic composition of the present invention, may be administered to a patient as a single dose in the form of a bolus or by infusion over a relatively short period, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period. Since the effective dose for the patient is determined by considering various factors such as the route of administration and the number of treatments, as well as the patient's age and health condition, a person with ordinary knowledge in the art would be able to determine an appropriate effective dose for a specific use of the peptide as a pharmaceutical composition, taking these points into account.

[0072] Accordingly, the novel peptide having the above-described characteristics of the present invention can be used for various purposes and applications requiring antibacterial activity, antimicrobial biofilm activity, antibiotic sensitivity enhancement activity, and anti-inflammatory activity. Specifically, it can be used as a material for antibiotics, pharmaceuticals, quasi-pharmaceuticals, cosmetics, food, feed, and biopesticides, as well as for coating agents, packaging agents, preservatives, preservatives (cosmetic preservatives, food preservatives, pharmaceutical preservatives, etc.), and additives (pharmaceutical additives, food additives, cosmetic additives, feed additives, etc.). For example, in pharmaceuticals, it can be used for purposes such as antibiotics or anti-contamination agents; in food, for preservation or antibacterial purposes; in agriculture, for antibacterial, sterilization, and disinfection purposes; and in cosmetics or household goods, for use as a coating agent for items requiring antibacterial properties, but it is not limited to the aforementioned purposes.

[0073] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0074]

[0075] <Example>

[0076]

[0077] 1. Materials and Methods

[0078]

[0079] Illumina library preparation and sequencing

[0080] Total RNA was isolated from frozen samples using the Qiagen RNA Isolation Kit, and the mRNA from the total RNA was converted into a library template using the TruSeq RNA Sample Prep Kit v2. This procedure included purification, strand synthesis, end repair, adapter ligation, and PCR enrichment. The enriched libraries were quantified and sequenced using the Illumina Hi-Seq 4000 and Illumina Novo-Seq 6000, respectively. Finally, the sequenced reads were processed using sequencing control software, and the output was a paired-end FASTQ format file. The entire process was performed by Macrogen Korea (http: / www.macrogen.com).

[0081]

[0082] Assembly and differential gene expression

[0083] Complete fragment read sequences were preprocessed using BBduk v38.26 to remove low-quality chimeric sequences and adapter contaminants. The processed reads underwent de novo assembly using Trinity v2.11.0 and were remapped to reference using Bowtie2 v.2.3.3 to filter out expressed contigs. Additionally, sequence clustering CD-HIT-EST v4.8.13 was performed to reduce fragmented contigs that were very similar to iso-forms, and the sequences were converted into protein sequences using TransDecoder v5.5.0 (https: / / github.com / TransDecoder / TransDecoder / releases). The protein sequences were annotated with Trinotate v3.2.2 (http: / / trinotate.github.io) to obtain functional terms such as gene ontology (GO), KEGG pathway, and protein domain. Finally, the completeness of the assembled transcript was evaluated using the BUSCO metazoa_odb10.2021-02-24 dataset.

[0084]

[0085] Antimicrobial Peptide (AMP) Prediction and Classification

[0086] The complete amino acid sequences prepared from the transdecoder underwent antimicrobial peptide (AMP) prediction analysis using the proposed bioinformatics strategy. Peptide characteristics regarding molecular propensity (based on physicochemical properties) and aggregation propensity (in vitro and in vivo) were determined, and antimicrobial peptide (AMP) predictions were established using a predefined bioinformatics strategy with previously defined parameters. In addition to this prior strategy, the allergenicity of the peptides was also determined using Allerdictor software. Finally, antimicrobial peptides (AMPs) were mapped using the CAMP database and classified as either novel antimicrobial peptides (AMPs) or known antimicrobial peptides (AMPs). To classify the predicted antimicrobial peptides (AMPs) as novel, the sequences were mapped to the CAMP database using two programs: PatMatch (no mismatch) for sequences 20 bp or less in length and BLASTP (1E-05) for sequences 20 bp or longer. BLAST results were filtered for similarity scores ≥ 90. Sequences with similarity observed at a given cutoff value were considered known antimicrobial peptides (AMPs), and other sequences were considered novel antimicrobial peptides (AMPs). Finally, novel and known antimicrobial peptides (AMPs) were manually validated for successive extensions of amino acids to account for low-complexity regions and assembly artifacts.

[0087]

[0088] Synthesis of 19 Estimated Antimicrobial Peptides (AMPs)

[0089] The 19 presumed antimicrobial peptides (AMPs) used in the experiment were synthesized by Dandicure Ltd (Ochang, Korea) via Fmoc-based solid-phase peptide synthesis. The mass of the purified peptides was determined using MALDI-TOF mass spectrometry. The calculated and measured peptide weights were consistent, confirming that all 19 peptides were accurately synthesized.

[0090]

[0091] Microbial strains and cells

[0092] Several bacterial strains were used in this experiment. Standard staining included Escherichia coli (KCTC 1682), Pseudomonas aeruginosa (KCTC 1637), Staphylococcus aureus (KCTC 1621), and Staphylococcus epidermidis (KCTC 1917), supplied by the Korea Culture Collection Center (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Clinically isolated multidrug-resistant bacteria included multidrug-resistant Klebsiella pneumoniae (MDRKP 328-83), multidrug-resistant Acinetobacter baumannii (MDRAB 329-53), multidrug-resistant Pseudomonas eruginosa (MDRPA 314-69), multidrug-resistant Enterobacter cloaca (MDRE 328-84), and multidrug-resistant Escherichia coli (MDREC 329-66). All bacterial strains were stored in 20% glycerol at -80°C. For subsequent experiments, bacteria were recovered and cultured in the corresponding concentrated medium at 37°C. Mouse macrophages RAW264.7, mouse fibroblasts NIH-3T3, and human keratinocytes HaCat cells were cultured in DMEM supplemented with fetal bovine serum (Sigma-Aldrich, MO, US), 100 U / mL penicillin, and 0.1 mg / mL streptomycin at 37°C in the presence of 5% CO2.

[0093]

[0094] Minimal Inhibitory Concentration (MIC)

[0095] The MIC of the peptide was measured using the CLSI (Clinical and Laboratory Standards Institute) liquid medium microdilution method. Briefly, bacteria were grown to the intermediate logarithmic scale and diluted in Mueller-Hinton liquid medium (MHB) (Difco, USA). The bacterial suspension (1×10⁻⁶) 6CFU / well) was added to 96-well microtitration plates with serial dilutions of the sample. The plates were incubated at 37°C for 24 hours, and bacterial growth inhibition was evaluated by measuring optical density using an ELISA plate reader. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the sample that prevents visible bacterial growth.

[0096]

[0097] Salt and serum sensitivity analysis

[0098] The salt sensitivity of the peptides was analyzed. E. coli (KCTC 1682) and S. aureus (KCTC 1621) were cultured in the presence of various final concentrations of physiological salts (150 mM NaCl, 4.5 mM KCl, 6 μM NH4Cl, 8 μM ZnCl2, 1 mM MgCl2, 2 mM CaCl2, 4 μM FeCl3) or human serum (20%). MIC values ​​in the physiological environment were determined as described above, and data were obtained from three independent assays performed in triplicate.

[0099]

[0100] Circular dichroism (CD) spectroscopy

[0101] The secondary structure of the hirunipin peptide was evaluated in a membrane-mimicking environment with a final peptide concentration of 150 μM by performing circular dichroism (CD) analysis using a Jasco-715 spectropolarimeter (Jasco). CD spectra were measured at room temperature in the wavelength range of 190–250 nm with a path length of 1 mm. The percentage of α-helical content was calculated using the equation proposed by McLean et al., %α-helical content = -100 (θ 222 It was calculated using + 3000) / 33000.

[0102]

[0103] Reverse-phase high-performance liquid chromatography (RP-HPLC)

[0104] For the experiment, an HPLC system (SHIMAZU model SPD-M20 230V) equipped with a UV detector (215 nm) and a manual injector (20 μL) was used, and 5 μg of antimicrobial peptide dissolved in distilled water was injected into a Vydac C18 column (5 mm; 4.6 mm × 250 mm). The column was equilibrated with 0.05% (v / v) TFA / water and eluted with a linear gradient of acetonitrile at a flow rate of 1.0 mL / min.

[0105]

[0106] Hemolysis and cytotoxicity analysis

[0107] The hemolytic activity of the antimicrobial peptide of the present invention was measured using sheep red blood cells (sRBCs). Sheep red blood cells were diluted in PBS to a final concentration of 4% (v / v). Then, 100 μL of the peptide solution (1–128 μM) was added to a 96-well plate containing 100 μL of the red blood cell suspension. After incubation at 37°C for 1 hour and centrifugation at 1000g for 10 minutes, 100 μL of the supernatant was transferred to another 96-well plate. Released hemoglobin was measured at 450 nm using a microplate reader. The hemolytic rate was calculated using the following formula: Hemolytic rate = 100 × [(A–A0) / (A t -A0)] Here, A is the absorbance of the peptide sample at 540 nm, and A0 and A tThe results were 0% and 100% hemolysis in PBS and 0.1% Triton X-100, respectively. The cytotoxicity of the antimicrobial peptide of the present invention against mouse macrophages RAW264.7, mouse fibroblasts NIH-3T3, and human keratinocytes HaCat was measured by the MTT method. RAW264.7, NIH-3T3, and HaCat cells were plated in 96-well plates in 100 mL of medium at a density of 10,000 cells per well and cultured overnight. The cells were then exposed to peptides at various concentrations (1–64 μg / ml) for 48 hours. Subsequently, 100 μL of MTT solution (0.5 mg / ml) was added to each well. After incubation for an additional 4 hours, the formazan formed from the MTT was incubated in 150 μL of DMSO for 15 minutes. Then, the absorbance at 550 nm was measured using a microplate reader. The percentage of cell viability was calculated using the following formula: Percentage of cell viability = [(A-A0) / (A t -A0)]×100%, where A, A0 and A t represents the absorbance of the peptide, DMSO, and control, respectively.

[0108]

[0109] Drug resistance research

[0110] Drug resistance experiments were performed as described in the previous report. First, the minimum inhibitory concentrations (MICs) of hirunipin 2 and the antibiotic were measured, and then the concentrations of each sub-MIC (0.5×MIC) of Escherichia coli (KCTC 1682) were 1×10⁻¹⁰ as MHB. 6 The MIC of the next passage was measured after dilution to CFU / mL. The above procedure was repeated for 15 passages. Ciprofloxacin and tetracycline were used as control antibiotics.

[0111]

[0112] Membrane depolarization analysis

[0113] The depolarization effect of the antimicrobial peptide of the present invention on the cytoplasmic membrane of S. aureus (KCTC 1621) was confirmed using the fluorescent dye diSC3-5 (3,3′-dipropylthiadicarbocyanine iodide). Specifically, S. aureus (KCTC 1621) was harvested during the middle logarithmic phase and washed three times with 5 mM HEPES buffer (pH 7.4, containing 20 mM glucose), and then OD in the same buffer 600 This was resuspended to 0.05. The cell suspension was incubated with 0.4 μM diSC3-5 for 90 minutes to allow most dye molecules to accumulate on the cytoplasmic membrane. Cytoplasmic and extracellular K + To equilibrate the concentration, KCl was added to the cell suspension containing disSC3-5 to obtain a final concentration of 100 mM, and then the peptide aliquot was added to 2 mL of the suspension and the cells were incubated at room temperature for 15-30 minutes. Fluorescence was recorded at an excitation wavelength of 622 nm and an emission wavelength of 670 nm using a Model RF-5301 PC fluorescence spectrophotometer (Shimadzu, Japan).

[0114]

[0115] Analysis of outer and inner membrane permeability

[0116] Changes in the integrity of the outer membrane (OM) of E. coli (KCTC 1682) due to the presence of the antimicrobial peptide of the present invention were determined by the absorption of N-phenylnaphthalen-1-amine (NPN), a fluorescent dye sensitive to the outer membrane. Specifically, to prepare a bacterial suspension, E. coli KCTC 1682 cells were grown to the mid-logarithmic phase and washed three times in 5 mM HEPES buffer (pH 7.4) containing 20 mM glucose and 5 mM KCN. The cells were then diluted in the same buffer to an optical density of 0.05 at 600 nm. NPN was dissolved in acetone to prepare a 1 mM stock solution, and 30 μL of this solution was added to the bacterial suspension to achieve a final concentration of 10 μM. The fluorescence of NPN (excitation λ = 350 nm, emission λ = 420 nm) was measured until a stable level was reached. Then, peptides of various concentrations were added to the suspension, and fluorescence was monitored over time until no further increase was observed. The inner membrane permeability of E. coli ML-35 in the presence of antimicrobial peptides was evaluated by measuring β-galactosidase activity using ONPG (o-nitrophenyl-β-D-galactoside), a substrate of cytoplasmic β-galactosidase, as previously described. E. coli ML-35 cells were grown to the mid-logarithmic phase and resuspended in sample buffer (10 mM sodium phosphate, 100 mM NaCl, pH 7.4) containing 1.5 mM ONPG to achieve an optical density of 0.5 at 600 nm. The effect of the peptides on inner membrane permeability was evaluated by measuring absorbance at 405 nm. An increase in absorbance reflects the hydrolysis of ONPG into o-nitrophenol, a yellow compound. ONPG can enter the cell through the permeated inner membrane and is cleaved by the cytoplasmic enzyme β-galactosidase.

[0117]

[0118] Flow cytometry analysis

[0119] Damage to membrane integrity induced by the antimicrobial peptide of the present invention was evaluated using flow cytometry of E. coli (KCTC 1682) and S. aureus (KCTC 1621) stained with the membrane impermeability dye PI. E. coli (KCTC 1682) and S. aureus (KCTC 1621) were cultured in LB broth until reaching the intermediate logarithmic phase, then washed and 2×10⁶ in PBS. 5 The bacterial suspension was resuspended to a density of CFU / mL. The bacterial suspension was incubated with the sample and 10 μg / ml of the fluorescent dye propidium iodide (PI) in a dark room at 37°C for 1 hour. The sample was then centrifuged and washed twice with PBS to remove any remaining dye. The fluorescence of PI, which indicates membrane damage, was measured using a FACS flow cytometer (Agilent, ACEA Bioscience Inc., San Diego, CA) with a laser excitation wavelength of 488 nm.

[0120]

[0121] Scanning Electron Microscope (SEM) Imaging

[0122] The membrane morphological changes of E. coli (KCTC 1682) treated with the antimicrobial peptide of the present invention were visualized using SEM as previously described. E. coli (KCTC 1682) cells at the intermediate logarithmic phase (OD) 600The cells were grown to a value of 0.1 and exposed to peptides (1×MIC or 2×MIC) for 4 hours. Then, the cells were collected and fixed overnight in 2.5% (w / v) glutaraldehyde at 4°C. After fixation, the cells were dehydrated in a series of ethanol solutions (50, 70, 90, 100%) for 10 minutes each, followed by dehydration in a mixture of ethanol and tert-butanol (1:1, v / v) for 15 minutes, and then in pure tert-butanol for 15 minutes. The samples were dried in a critical point dryer using liquid CO2, coated with gold-palladium, and observed using a scanning electron microscope (Zeiss Gemini 500 Field).

[0123]

[0124] 3D-ODT Imaging and Quantitative Analysis for E. coli

[0125] ODT-HTS was performed using a low-coherence HT system (Tomocube) capable of simultaneously monitoring multiple unlabeled bacterial cells or various antimicrobial peptide (AMP) treatment conditions over time. The device is equipped with a motorized ×40 NA 0.95 objective lens (UPLXAPO40X, Olympus), a custom condenser lens (NA = 0.72, working distance = 30 mm), and a 450 nm LED illumination module combined with a digital micromirror device in the pupil plane. The device is equipped with a stage-top incubator (STXG-WSKMXA22B-E, TOKAI HIT). The optimal imaging point is set with a laser wavelength of 780 nm and a range of +100 to -300 μm. The optimal imaging point can be focused using an autofocus system, which is digitally authenticated and controlled by operating software (TomoStudioX, Tomocube). Escherichia coli at 1.0 × 10⁻⁶ in PBS in custom 96-well or 6-well plates containing PI (2 μg / mL) 7Prepared at cells / mL. Peptide (32 μg / mL) was added to each well.

[0126] For peptide screening, quantitative imaging of bacterial cells was performed using ODT-HTS 2 hours after peptide treatment. PI-positive cells were also detected using fluorescence imaging, and time-lapse monitoring of bacterial cells using ODT-HTS was performed for 2 hours at 10-minute intervals under the same conditions as peptide treatment. The number of bacterial cells and PI-positive cells was counted based on RI values ​​and the ratio of PI-positive cells calculated per total cell using TomoAnalysis version 1.7.13 (Tomocube).

[0127]

[0128] ODT Imaging and Quantitative Analysis of MDRAB Biofilms

[0129] For ODT imaging of biofilms, multidrug-resistant Acinetobacter baumannii (MDRAB) (329-53) cells were inoculated and cultured in MHB medium. Bacterial cells were harvested by centrifugation, washed with PBS, and then placed in 1.0 × 10⁻⁶ 6-well glass-bottom plates with high-performance #1.5 cover glass. 6 The cells were resuspended in fresh MHB medium at a concentration of cells / mL. Antimicrobial peptide (AMP) was prepared at 64 μg / mL in MHB medium and added to each well. ODT imaging was performed every 2 hours for 12 hours to monitor the morphological dynamics of the biofilm. 3D RI tomography of the biofilm was visualized using TomoAnalysis version 1.7.13 (Tomocube), and biophysical properties such as volume, dry mass, and surface area were quantified at an FOV set to 80×80 μm.

[0130]

[0131] Quantitative analysis of fluorescence intensity

[0132] The fluorescence intensity of PI was analyzed using ImageJ software by applying a segmentation algorithm to mask cell boundaries and stained regions using Otsu's method, and then measuring the average integration density at the cell boundaries. The intensity values ​​quantified in the cells were averaged.

[0133]

[0134] Minimal biofilm eradication concentration (MBEC)

[0135] The biofilm clearance concentration (MBEC) of the peptide against the multidrug-resistant Pseudomonas eruginosa (MDRPA) strain 329-53 was determined using Innovotech's Calgary Biofilm Device (CBD). 1×10⁶ in 150 μL of Luria-Bertani (LB) medium 6A bacterial suspension of CFU / mL was added to each well of a 96-well microtiter plate (Innovotech, product code: 19111) with peg lids. The plates were incubated at 37°C for 24 hours with agitation at 110 rpm to form a biofilm on the pegs. The peg lids were rinsed with phosphate-buffered saline (PBS, 0.01 M) and transferred to new plates containing peptides of varying concentrations (200 μL per well). The plates were incubated at 37°C for 24 hours with shaking at 110 rpm to expose the biofilm to the peptides. The peg lids were rinsed again with PBS and transferred to recovery plates containing 200 μL of LB medium per well. The recovery plates were sonicated in a water bath for 10–15 minutes to detach the biofilm from the pegs. The recovery plates were incubated at 37°C for 24 hours with shaking at 110 rpm to allow viable bacteria to grow and produce turbidity. The Minimum Biofilm Evaporation Concentration (MBEC) was defined as the lowest peptide concentration that prevented turbidity in the recovery plates compared to a sterile control. The experiment was repeated three times, and the median value of each experiment was recorded.

[0136]

[0137] Confocal laser scanning microscopy (CLSM)

[0138] The biofilm removal activity of the antimicrobial peptide of the present invention against pre-formed biofilms of MDRAB (329-53) strains was confirmed by CLSM using LIVE / DEAD staining (SYTO9 / PI). Specifically, MDRAB (329-53) cells (1×10⁶ 6Plankton cells (CFU / ml) were inoculated into 24-well plates containing Mueller-Hinton liquid medium (MHB) and sterile discs. The plates were incubated at 37°C for 24 hours to allow biofilm formation on the discs. The discs were washed three times with phosphate-buffered saline (PBS) to remove plankton cells and transferred to new plates containing MHB and peptides (64 or 128 μg / ml). The plates were incubated at 37°C for 6 hours to expose the biofilms to the peptides. The discs were washed twice with PBS and stained with a mixture of SYTO 9 (6.7 μM) and propidium iodide (PI, 40 μM) in the dark at 37°C for 30 minutes. The biofilm structure and viability of the discs were visualized and analyzed using a confocal laser scanning microscope (CLSM) with a Zeiss LSM 710 Meta microscope and ZEISS Microscopy's ZEN 2009 Light Edition software.

[0139]

[0140] Synergy test by checkerboard assay

[0141] The synergistic effect of the antimicrobial peptide of the present invention in combination with existing antibiotics (chloramphenicol, tetracycline ciprofloxacin, and rifampicin) against MDRAB (329-53) was investigated by checkerboard analysis. Specifically, twofold serial dilutions of two antibiotics ranging from 1 / 8 MIC to 2 MIC were inoculated into each 96-well microtiter plate, with one antibiotic inoculated in the columns and the other in the rows. Subsequently, MDRAB (329-53) cells were 1 × 10⁶ 6Inoculated at a concentration of CFU / ml. Plates were incubated overnight under static conditions and visually observed. The fractional inhibitory concentration index (FICI) was calculated as follows: FICI = [(MIC of antibiotic combination) / (MIC of antibiotic alone)] + [(MIC of combined small molecule) / (MIC of small molecule alone)], where FICI ≤ 0.5 indicates a synergistic effect, 0.5 < FICI ≤ 1.0 indicates an additive effect, 1.0 < FICI ≤ 4.0 indicates no effect, and FICI > 4.0 indicates antagonism.

[0142]

[0143] Time Killing Assay

[0144] The bactericidal activity over time was performed using the antimicrobial peptide alone or in combination with an antibiotic against MDRAB (329-53). Specifically, MDRAB (329-53) suspension (1×10⁻⁶) 6 CFU / mL) was added in equal volumes of the antimicrobial peptide of the present invention and antibiotics, either alone or in combination at synergistic concentrations. Aliquots (5 μl) were periodically collected at different time points, diluted, and incubated on LB solid medium for 24 hours. The number of colonies on the LB solid medium was counted. Each test was performed at least three times, and the average value was recorded.

[0145]

[0146] Measurement of TNF-α, IL-6, and MCP-1 Release in LPS-Stimulated RAW264.7 Cells

[0147] The inhibition of TNF-α, IL-6, and MCP-1 production induced by the antimicrobial peptide of the present invention in RAW 264.7 cells stimulated with LPS (lipopolysaccharide) was confirmed using ELISA. RAW264.7 mouse macrophages (5×10⁶ 5Cells (100 μL / well) were plated and attached to a 96-well plate (100 μL / well). Then, the cells were stimulated for 24 hours with LPS containing Escherichia coli O111:B4 (100 ng / mL) with or without the peptide. The release of TNF-α, IL-6, and MCP-1 was quantified using a commercial ELISA kit (R&D Systems) according to the manufacturer's protocol.

[0148]

[0149] Reverse Transcription Polymerase Chain Reaction (RT-PCR)

[0150] As previously described, RT-PCR analysis was performed for the quantification of TNF-α, IL-6, and MCP-1. RAW264.7 cells were placed in 5×10⁴ well plates. 5Cells were plated at a concentration of cells / well and incubated overnight. The antimicrobial peptide of the present invention was added to the wells at concentrations of 8, 16, and 32 μg / mL. Cells for the quantification of TNF-α, IL-6, and MCP-1 were treated for 6 hours in DMEM supplemented with 10% bovine serum with or without 20 ng / mL LPS, with or without the peptide (negative control). Cells were detached from the wells and washed once with PBS. Total RNA was extracted using Trizol Reagent (Ambion). Then, 1 μg of total RNA was converted to cDNA using TakaRa Primescript™ Reverse Transcriptase (TakaRa). The primers used were purchased from Bioneer (see Table 1). The cDNA products were amplified using AccuPower® PCR PreMix (Bioneer). The amplification protocol consisted of an initial denaturation step of 5 minutes at 94°C and 30 subsequent denaturation cycles of 1 minute at 94°C. It was annealed at 55°C for 1.5 minutes, extended at 72°C for 1 minute, and finished with a final extension step of 5 minutes at 94°C, followed by denaturing at 94°C for 1 minute, annealing at 55°C for 1.5 minutes, and extending at 72°C for 1 minute, repeating this process 30 times, and then undergoing a final extension step of 5 minutes at 72°C.

[0151]

[0152] Primer sequences used for RT-PCR Genetic Primer Sequence (5'->3') TNF-α Forward CCTGTAGCCCACGTCGTAGC Reverse TTGACCTCAGCGCTGAGTTGIL-6 Forward ACAACCACGGCCTTCCCTACTT Reverse CACGATTTCCCAGAGAACATGTGMCP-1 Forward ATCCCAATGAGTAGGCTGGAGAGC Reverse CAGAAGTGCTTGAGGTGGTTGTGGAPDH Forward GACATCAAGAAGGTGGTGAA Reverse TGTCATACCAGGAAATGAGC

[0153]

[0154] Statistical analysis

[0155] Statistical analysis was performed using the Student t-test for comparisons between groups or one-way Analysis of Variance (ANOVA) with Tukey's multiple comparisons for comparisons between two or more groups. Statistical analysis was performed using SPSS ver. 22. A p-value < 0.05 was considered statistically significant. All values ​​were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8 or Origin 2018. Comparisons between groups were performed using unpaired and two-sided t-tests. All values ​​were expressed as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.

[0156]

[0157] 2. Results

[0158]

[0159] Sequencing and de novo transcriptome assembly

[0160] To identify putative antimicrobial peptides (AMPs) derived from Hirudo nipponia, sequential in vitro validations including silicopeptide prediction and peptide screening were performed using ODT-HTS (Fig. 1a). Initially, 44.96 GB of Illumina short reads from three tissues of Hirudo nipponia (head, salivary glands, and teeth) were assembled into 69,497 transcripts to generate 111 Mb (see Table 2). Of these, 29,203 (42.02%) transcripts were annotated with functional terms, and major transcripts were not mapped to the Uniport database. Most transcripts were found to be similar to transcripts of the human proteome. In addition, the transcript showed 95.18% completeness on the Metazoan BUSCO dataset (Manni, M., Berkeley, MR, Seppey, M. & Zdobnov, EM BUSCO: Assessing Genomic Data Quality and Beyond. Current Protocols 1, e323, doi:https: / doi.org / 10.1002 / cpz1.323 (2021).).

[0161]

[0162] De Novo again ) Transcriptome Assembly and Annotation Summary de novelo Transcriptome Assembly# Transcripts69497Total length (bp)111635342N502838Avg.length1606,33Max.length50191Min.length70GC%40.27Translated proteins206080SwissProt29,203 (42.02%)KEGG25,850 (37.20%)eggong179 (0.26%)Pfam24,635 (35.45%)GO28,693 (41.29%)GO BP25,576 (36.80%)GO CC26,982 (38.82%)GO MF25,233 (36.31%)No hit40,294 (57.98%)

[0163]

[0164] Prediction of antimicrobial peptides (AMPs) from transcripts

[0165] The entire assembled transcript was translated into 206,080 proteins, and the proteome was converted into 159,321 small peptides. As described in the section on the antimicrobial peptide (AMP) prediction method, small peptides with similarities observed at a given cutoff value were considered known antimicrobial peptides (AMPs), while other peptides were considered novel antimicrobial peptides (AMPs), resulting in 1,453 and 477 peptides, respectively (see Table 3). Of these, 259 peptides from the known antimicrobial peptides (AMPs) and 97 from the novel antimicrobial peptides (AMPs) were anti-inflammatory agents. Finally, 19 peptides were randomly selected for experimental screening. Thus, the features included in the antimicrobial peptide (AMP) prediction methodology encompassed the physicochemical and structural characteristics of the peptides, and a familiar web server was utilized for antimicrobial peptide (AMP) prediction. The antimicrobial peptide (AMP) properties of the peptide were systematically evaluated and used for experimental verification.

[0166]

[0167] 항균 펩타이드 특징에 대한 요약과 선택 컷오프 값. AIP(Anti-inflammatory peptides)#PropensityMethodsDescriptions / ParametersCutoff / Filters# of SequencesRaw SequenceTotal Given Proteins Sequences206,080Raw SequenceSmall Proteins / Peptides<= 100159,321MolecularEPESTFINDProteins contains potential protease clevage site!=013,578MolecularAMPAAntimicrobial Spots!=033,861MolecularPepstatsTotal Peptides<= 10033,861MolecularPepstatsPeptide Length<= 5021,252MolecularPepstatsSmall Proteins which have AMP peptides<= 5020,012MolecularPepstatsCharge>0.0030,051MolecularPepstats Isoelectric Point (pI)8.00 <= pI <= 12.0025,912Aggregation (In vivo)TangoAGG<= 500.0024,123Aggregation (In vivo)TangoHelix0.00<= Helix <= 25.0023,744Aggregation (In vivo)TangoBeta25.00<= Beta <= 100.0011,069Aggregation (In vitro)AggrescanNa4vSS-40.00<= Na4vSS <= 60.0031,177AllergenAllerdictorPredictionsNon-Allergen33,718HomologusBlastNovelNo Blast Hits33,384HomologusBlastKnownBlast Hits477CAMPAMPSupport Vector Machine (SVM) classifierAMP8,616CAMPAMPRandom Forest ClassifierAMP9,704CAMPAMPArtificial Neural Network (ANN) classifierAMP11,744CAMPAMPDiscriminant Analysis classifierAMP9,144CAMPAMPPredicted >=3 out of 4 classifier>=37,764Final ResultNovel: 1,453 (AIP: 259), Known: 477 (AIP: 97).

[0168]

[0169] 19개의 추정 항균 펩타이드(AMP)의 항균 활성

[0170] In this experiment, the antimicrobial activity of 19 putative antimicrobial peptides was tested against standard bacterial strains of two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis) (see Table 4). Melittin, a known bee venom antimicrobial peptide, was used as a positive control. The minimum inhibitory concentration (MIC) of the 19 putative antimicrobial peptides (AMPs) was measured using the liquid medium microdilution method. Among these 19 peptides, only three types—LP8, LP9, and LP12—were confirmed to have promising antimicrobial activity with minimum inhibitory concentration (MIC) values ​​ranging from 32 to 128 μg / mL (Table 4). In this invention, LP8, LP9, and LP12 were named Hirunipin 1, 2, and 3, respectively. In particular, hirunipin 2 exhibited the highest antibacterial activity, showing a minimum inhibitory concentration (MIC) value similar to melittin (see Table 5).

[0171] In addition, the antimicrobial effect of hirunipin was verified using a novel peptide screening method based on ODT technology. In this experiment, multiple samples of 19 antimicrobial peptides (AMPs) were monitored simultaneously using ODT-HTS. Hirunipin 1, 2, and 3 resulted in a significant reduction in bacterial cells and a high proportion of PI-positive apoptotic cells (see Fig. 1b). These results suggest that hirunipin 1, 2, and 3 are highly effective antimicrobial peptides (AMPs) and exhibit effective antimicrobial effects compared to other peptides. Consequently, three antimicrobial peptides (AMPs), hirunipin 1, 2, and 3, were identified in Hirudoniphonia using a predictive framework and verified using the novel approach, ODT-HTS.

[0172]

[0173] List of candidate antimicrobial peptides identified in the Hiruniponia genome assembly via computational algorithms Peptide Amino Acid Sequence Length (aa) Molecular Weight (Da) LP1KLTPKPAKKVKKVVKF161839.47LP2FGQKVNCILNFICYKYLRI LV212677.4LP3RRRTCKKEDGTCLGRKVQYKKYLQKK263214.98LP4KDVTGYWVRERQCKLNSKPCVGRNTQRRKYLNK334025.84LP5LLAHVSKGFKLSMQKSKKPSLPIKT252767.56LP6LSKGGFKGKPERAMAHHLKNIRKKLSKSIMKS323607.57LP7IIIIKMHKTRKEKTQSILSAPVGLLTTL283134.06LP8 (Hirunipin 1)LLKKLLRSLIKPAIMKIINTPPQEKLQK283256.32LP9 (Hirunipin 2)MKSINLKKVVKSIIKKILNSSF222519.31LP10KKLLKKLEKISKKKKKDKKRGRGSPMTSYAILYDGA364137.24LP11VLNVQSIMSWNKRVGGMILNLKT232602.31LP12 (Hirunipin 3)YFKIIKFLPKALKCLLPQKHKEHGNL263108.01LP13NITDYNKKKVRLLPRFIFPLG212533.18LP14LNTPRPELSISQRLLIKNFILII232692.44LP15ARSIKKVIFSSSSRVCPPLKMLKLRPCFKKVPKLDGQ374187.37LP16F RSQFPICKNLVNHTKFVCQKKPLSKLMKCKKALDR364279.45LP17PKKVRKSFKKLRNFSNKNSNIYGLPA263035.72LP18MANPVNSIKRIIKKLKKERKCKPNL252950.83LP19QKFNGKSYALAKKMKKRQVKFLNRILDQL293495.41

[0174]

[0175] Antimicrobial activity of candidate antimicrobial peptides Peptide Minimum Inhibitory Concentration (MICa): mg / mL Gram-negative bacteria Gram-positive bacteria Escherichia coli [KCTC 1682] Pseudomonas aeruginosa [KCTC 1637] Staphylococcus aureus [KCTC 1621] Staphylococcus epidermidis [KCTC 1917] LP1>128>128>128>128 LP2>128>128>128>128 LP3>128>128>128>128 LP4>128>128>128>128 LP5>128>128>128 LP6>128>128>128 LP7>128>128>128>128 LP8 (Hirunipin 1)64326432LP9 (Hirunipin 2)32323232LP10>128>128>128>128LP11>128>128>128>128LP12 Hirunipin 3)12812812864LP13>128>128>128>128LP14>128>128>128>128LP15>128>128>128>128LP16>128>128>128>128LP17>128>128>128>128LP18>128>128>128>128LP19>128>128>128>128 Melittin 32321632

[0176]

[0177] molecular structure

[0178] In this experiment, the tertiary structure of hirunipin (hirunipin 1, 2, and 3) was predicted using the I-TASSER server. Hirunipin 1 and 3 exhibited long α-helices in the N-terminal region, whereas hirunipin 2 had a long α-helice in the middle region. Additionally, the α-helix wheel diagram of the peptides was constructed using the HeliQuest server. As a result, as shown in Figure 1c, the hydrophobic residues (L, I, Y) of the peptides are concentrated on one side of the helix, while the hydrophilic residues (K and R) are located on the opposite side, forming an amphiphilic structure characteristic of natural antimicrobial peptides (AMPs). As shown in the α-helix wheel diagram (Figure 1c), the hydrophilic and hydrophobic regions are less prominent in hirunipin 1 compared to the other two peptides. This suggests that the hydrophilicity of hirunipin 1 may be lower than that of hirunipin 2 and 3. This is consistent with the hydrophobic moment (μH) value, a quantitative indicator of hydrophilicity. To verify the predicted structure, the secondary structure of the hirunipin peptide was measured using CD spectroscopy in aqueous buffer, 50% TFE, and 30 mM SDS. The CD spectrum indicated that the hirunipin peptide took on a random coiled form in aqueous buffer and exhibited a negative peak at 200 nm. In contrast, in 50% TFE and 30 mM SDS, which mimics a membrane environment, the hirunipin peptide took on a typical α-helical form with two negative peaks around 208 nm and 222 nm. Hirunipin 2 and 3 exhibited significantly higher α-helical content than hirunipin 1.

[0179]

[0180] hydrophobicity

[0181] Hydrophobicity is an important structural parameter that can affect the antimicrobial activity of natural antimicrobial peptides (AMPs). Reverse-phase high-performance liquid chromatography (RP-HPLC) is an accurate method for determining the hydrophobicity of peptides, and retention time directly reflects the hydrophobic structural state of the peptides. In this experiment, the antimicrobial activity of three types of hirunipins was investigated by performing RP-HPLC. According to the results of this experiment, the retention times (hydrophobicity) of the three types of hirunipins were in the order of hirunipin 1 (20.89 min) < hirunipin 3 (21.56 min) < hirunipin 2 (27.25 min), which suggests that hirunipin 2 exhibited the highest level of hydrophobicity.

[0182]

[0183] Hemolytic activity

[0184] The hemolytic activity of hirunipin was investigated in sheep red blood cells at concentrations ranging from 1 to 128 μg / mL. The hirunipin peptide exhibited significantly low hemolytic activity. At the highest test concentration of 128 μg / mL, the hirunipin peptide was found to cause lysis in less than 10% of red blood cells (see Fig. 6). Meanwhile, melittin was found to lyse approximately 100% of red blood cells at a concentration of 16 μg / mL. Through these results, it was confirmed that the hirunipin peptide of the present invention exhibits very low cytotoxicity and high serum stability.

[0185]

[0186] In vitro cytotoxicity

[0187] A major limitation of antimicrobial peptides (AMPs) in clinical applications is their potential cytotoxicity. Therefore, in this study, the cytotoxicity of hirunipin peptide was evaluated in RAW264.7 (mouse macrophages), NIH-3T3 (mouse fibroblasts), and HaCat (human dermal keratinocytes) cells using the MTT assay. As shown in Figure 7, the cell viability of RAW264.7, NIH-3T3, and HaCat cells was >70% upon exposure to 32 μg / mL of hirunipin peptide. The IC50 of hirunipin peptide for RAW264.7, NIH-3T3, and HaCat cells 50 The values ​​(concentrations that induce 50% cell death) were 45–60, 50.6–57.6, and 42–64 μg / mL, respectively (see Table 6). These results suggest that hirunipin peptide exhibited relatively low cytotoxicity in mammalian cells, indicating its potential as a therapeutic material.

[0188]

[0189] IC50 of hirunipin peptide against RAW264.7, NIH-3T3, and HaCat cells 50 Value Cell IC 50 (μg / mL) Hirunipin 1 Hirunipin 2 Hirunipin 3 Melittin RAW264.74546603.7 NIH-3T350.657.6522.1 HaCat42>64.0>64.06.2

[0190]

[0191] Salt and serum resistance

[0192] The antimicrobial activity of antimicrobial peptides (AMPs) can be inhibited by high salt concentrations. Therefore, the salt resistance of hirunipin peptides was evaluated by measuring the minimum inhibitory concentration (MIC) against Escherichia coli (KCTC 1682) and Staphylococcus aureus (KCTC 1621) in the presence of physiological levels of monovalent, divalent, and trivalent salts. The results showed that hirunipin 1 and 3 were less effective in killing bacteria under high salt conditions, whereas hirunipin 2 maintained or enhanced its antimicrobial activity in the presence of salt ions (see Table 7). Serum contains many proteases and peptide-binding proteins, such as albumin, which can reduce the in vivo efficacy of antimicrobial peptides (AMPs). To evaluate the serum stability of hirunipin peptides, activity against E. coli and S. aureus was measured in 25% human serum. As a result, the activity of hirunipin peptide was found to be reduced by 2 to 4 times (see Table 7). This suggests that the antimicrobial peptide is resistant to serum protease degradation.

[0193]

[0194] In the presence of physiological salts and human serum E. coli and S. aureus Minimum Inhibitory Concentration (MIC) Values ​​of Hirunipin Peptide Against E. coli (KCTC 1682) Peptide Control 150 mM NaCl 4.5 mM KCl 6 μM NH4Cl 1 mM MgCl2 2.5 mM CaCl2 4 μM FeCl3 25% Serum MIC (μg / mL) against E. coli (KCTC 1682) Hirunipin 164 256 256 28256 256 128128 Hirunipin 2321 688 32886 4 Hirunipin 3128 128 1286 4256 3232256 MIC (μg / mL) against S. aureus(KCTC1621) Hirunipin 16425625625625625664256 Hirunipin 23232321632323264 Hirunipin 3128256256256256256>256>256

[0195]

[0196] Antimicrobial activity against clinically isolated multidrug-resistant bacteria

[0197] Multidrug-resistant Gram-negative bacteria pose a serious threat to global health as they cause infections that are very difficult to treat with conventional antibiotics. In this study, the antimicrobial activity of hirunipin peptides was evaluated against clinically isolated multidrug-resistant Gram-negative pathogens such as P. aeruginosa, A. baumannii, K. pneumoniae, E. cloacae, and E. coli. As a result, hirunipin 2 exhibited significantly higher antimicrobial activity compared to hirunipin 1 and 3, and demonstrated antimicrobial activity similar to or superior to that of melittin, the positive control. In particular, hirunipin 2 showed twice the antimicrobial activity compared to melittin against multidrug-resistant Klebsiella pneumoniae (MDRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), and multidrug-resistant Pseudomonas eruzinosa (MDRPA) (see Table 8).

[0198]

[0199] Antimicrobial activity of hirunipin peptide against clinically isolated multidrug-resistant bacteria Multidrug-resistant bacteria Minimum Inhibitory Concentration (MIC: g / mL) Hirunipin 1 Hirunipin 2 Hirunipin 3 Melittin MDRKP (328-83) > 128 128 > 128 128 MDRAB (329-53) > 128 64 > 128 32 MDRPA (314-69) 64 16 > 128 32 MDRE (328-84) > 128 32 > 128 64 MDREC (329-66) 64 16 128 32 Clinically isolated multidrug-resistant bacteria were obtained from Chosun University Hospital, Korea MDRKP: Multidrug-resistant Klebsiella pneumoniae MDRAB: Multidrug-resistant Acinetobacter baumannii MDRPA: Multidrug-resistant Pseudomonas aeruginosa MDRE: Multidrug-resistant Enterobacter Cloaca MDREC: Multidrug-resistant E. coli

[0200]

[0201] Antimicrobial mechanism

[0202] In order to gain insight into the antimicrobial mechanism of hirunipin peptide, membrane depolarization, outer and inner membrane permeability analysis, ortho-nitrophenyl-β-D-galactoside (ONPG) analysis, PI-inflow analysis using a flow cytometer, scanning electron microscopy (SEM), and optical diffraction tomography (ODT) were performed in this experiment.

[0203] First, the effect of the hirunipin peptide on membrane integrity was evaluated, and then the membrane potential of S. aureus (KCTC 1621) was measured using 3,3'-dipropylthiocarbamate anthocyanin iodide (diSC3-5), a membrane potential-sensitive dye. This dye accumulates on the cell membrane and extinguishes its own fluorescence when mixed with S. aureus cells. However, when the membrane is depolarized by the peptide or 0.1% Triton, the dye is released and fluorescence increases. Buforin-2 and melittin were used as intracellular and membrane-targeting antimicrobial peptides (AMPs), respectively. Fluorescence changes were monitored for 500 seconds after the addition of 16 μg / mL of the hirunipin peptide (see Fig. 1d). As expected, buforin-2 did not affect the membrane potential. On the other hand, similar to melittin, the hirunipin peptide induced significant membrane depolarization at 16 μg / mL. Hirunipin 2 was the most potent peptide, inducing nearly 100% depolarization at 4–8 μg / mL, while hirunipin 1 and 3 induced approximately 60% depolarization at 16 μg / mL (see Fig. 1e). Depolarization was concentration-dependent for all peptides. These results indicate that hirunipin 2 possesses the most potent membrane-disrupting activity among the three peptides, which is consistent with antimicrobial activity.

[0204] N-phenyl-1-naphthylamine (NPN) fluorescence was utilized to investigate the effect of antimicrobial peptides (AMPs) on the permeability of the bacterial outer membrane. When the fluorescent dye NPN is disrupted by antimicrobial peptides (AMPs), it gains access to the membrane environment, which is generally hydrophobic, resulting in a significant increase in fluorescence intensity. In this experiment, the hirunipin peptide was found to increase the outer membrane permeability of E. coli in a dose-dependent manner, similar to melittin (see Fig. 1f). Additionally, the permeability of the bacterial inner membrane was evaluated using E. coli ML-35. When the inner membrane is disrupted by antimicrobial peptides (AMPs), β-galactosidase is released, hydrolyzing ONPG to produce yellow o-nitrophenol. Consequently, the hirunipin peptide, similar to melittin, was found to increase the inner membrane permeability in a dose-dependent manner (see Fig. 1g).

[0205] To evaluate the potential of hirunipin peptides to penetrate bacterial cell membranes, a PI-inflow assay was performed using a flow cytometer. PI is a dye that cannot pass through intact membranes but can enter cells with damaged membranes and bind to nucleic acids. In the absence of peptide treatment, the percentages of exposed E. coli (KCTC1682) and S. aureus (KCTC 1621) cells exhibiting PI fluorescence were 6.9% and 9.5%, respectively (see Figures 2a and 2b). This indicates that the cell membranes were not damaged. After treatment with 16 μg / mL of hirunipin 1, 2, and 3, the percentages of PI-positive cells in E. coli increased to 47.4%, 92.1%, and 53.3%, respectively (see Figure 2a). Similarly, S. The percentage of PI-positive cells in S. aureus increased to 62.9%, 98.2%, and 73.7%, respectively (see Fig. 2b). These results suggest that the hirunipin peptide exerts antimicrobial effects against E. coli and S. aureus by disrupting cell membranes similarly to melittin. On the other hand, buforin-2 did not cause cell membrane damage, as indicated by the low proportion of PI-positive cells in E. coli (KCTC1682) and S. aureus (KCTC 1621) (7.6% and 9.6%, respectively). Furthermore, the PI fluorescence of hirunipin-2 was the highest among all peptides, a result consistent with its antimicrobial activity.

[0206] Furthermore, to investigate the effects of hirunipin peptides on the morphology and surface of bacterial cells, E. coli (KCTC1682) were exposed to 32 μg / mL of each peptide for 4 hours and analyzed using a scanning electron microscope (SEM). As a result, bacterial cells without peptide treatment exhibited a normal shape and a smooth membrane surface (see Fig. 2c). Buforin-2 did not damage the bacterial surface. In contrast, E. coli cells treated with hirunipin peptide (32 μg / mL) showed severe cell wall damage, including a rough membrane structure similar to melittin, cell wall pores, and irregularly shaped cells. Compared to hirunipin 1, hirunipin 2 and hirunipin 3 caused greater changes in the morphology and structure of bacterial cells.

[0207] Next, the bactericidal effect of hirunipin 2 was evaluated using 3D-ODT imaging. 3D-ODT images of single bacterial cells treated with or untreated with hirunipin 2 were obtained to visualize the intracellular region (1.355–1.390), inner membrane (1.347–1.348), and outer membrane (1.344–1.345) of E. coli as indicated on the RI map (see Fig. 3a). Representative ODT or fluorescence images showed that the average RI of the intracellular region increased in E. coli cells treated with hirunipin 2 compared to untreated cells (see Fig. 3b). Additionally, using fluorescence imaging and intensity quantification analysis, an increase in the PI signal in E. coli after treatment with hirunipin 2 was observed (see Figs. 3b and 3c). Previous studies have reported that the average RI of E. coli increases due to the aggregation of intracellular components after E. coli death. In this experiment, we analyzed segmented 3D ODT images of the internal membrane and intracellular components to confirm that the average RI of E. coli increased in real time (see Figs. 3d and 3e). Consequently, we observed that the PI intensity and average RI of the cells gradually increased and intracellular aggregation was promoted following treatment with hirunipin 2 (see Figs. 3d-3f). These results indicated that hirunipin 2 disrupts bacterial cell membranes and induces intracellular aggregation, ultimately leading to apoptosis. Furthermore, using ODT-HTS, we confirmed that the number of large bacterial cells gradually decreased and the proportion of PI-positive cells increased over time following treatment with hirunipin 2 (see Figs. 3g-3h).

[0208] In summary, the above results suggest that hirunipin 2 exerts an antibacterial effect by destroying bacterial cell membranes and inducing intracellular aggregation, which eventually leads to cell death.

[0209]

[0210] Antimicrobial biofilm activity

[0211] Biofilms are microbial communities that attach to surfaces and produce proteins, polysaccharides, and other substances. These biofilms can protect bacteria from antimicrobial agents and increase their resistance. It is estimated that approximately 65% ​​of all bacterial infections are associated with bacterial biofilms. To combat bacterial biofilms, antimicrobial agents must prevent biofilm formation and break down mature biofilms. Acinetobacter baumannii is an opportunistic pathogen that frequently causes outbreaks in medical facilities, particularly in intensive care units (ICUs). Acinetobacter baumannii can rapidly form biofilms on various surfaces, such as urinary catheters, bronchial epithelial cells, and non-biological materials. Furthermore, the mortality rate for patients with burn wound infections associated with multidrug-resistant Acinetobacter baumannii (MDRAB) is very high. Therefore, in this experiment, the effects of hirunipin peptide on the inhibition and eradication of biofilms of multidrug-resistant Acinetobacter baumannii (MDRAB) were investigated using the minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC).

[0212] MDRAB biofilm formation was visualized using segmented 3D ODT images. Additionally, the RI distribution of living bacterial cells (RI = 1.351–1.380) and the extracellular components of the biofilm (RI = 1.340–1.350) were used (see Fig. 4a). Gradually increasing biofilm formation in the control group over time was observed (see Figs. 4b and 4c). However, hirunipin 2 was shown to significantly inhibit living MDRAB and biofilms compared to the well-known anti-biometabolic peptide LL-37 (see Figs. 4j and 4k) (see Figs. 4f and 4g). Relatively speaking, hirunipin 1 and hirunipin 3 showed less anti-biometabolic activity and increased the intensity of MDRAB cells (see Figs. 4d–4e & 4h–4i). Quantitative analysis of biophysical properties such as dry mass, surface area, and biofilm volume showed that hirunipin 2 was significantly more effective than LL-37, hirunipin 1, or hirunipin 3 (see Figs. 4l-4n). These results indicate that hirunipin 2 inhibits biofilm formation and affects MDRAB growth. Consequently, the reduced dynamics of the biofilm after treatment with hirunipin 2 were visualized and analyzed using time-lapse unlabeled ODT monitoring to elucidate the anti-biotactic mechanism. Furthermore, the effect of hirunipin peptides on the eradication of MDRAB biofilms was investigated using multidrug-resistant Enterobacter cloacae (MBEC). Fig. 4o shows that hirunipin peptides remove pre-formed MDRAB biofilms in a concentration-dependent manner. Hirunipin 1, 2, and 3 exhibited activity in removing approximately 50% of mature MDRMA biofilms at concentrations of 64, 32, and 256 μg / mL, respectively (see Fig. 4o). Therefore, the antimicrobial biofilm activity of the hirunipin peptides was in the order of hirunipin 3 < hirunipin 1 < hirunipin 2. In particular, hirunipin 2 showed superior antimicrobial biofilm activity compared to LL-37, which is well known as a potent antimicrobial biofilm peptide (AMP).

[0213]

[0214] Tendency to develop antibiotic resistance

[0215] The development of antibiotic resistance has become an important evaluation factor for the development of new antibiotics. Therefore, in this experiment, the drug resistance of hirunipin 2 against E. coli (KCTC1682) was evaluated through a multiple passage resistance study. Hirunipin 2, ciprofloxacin, or tetracycline were continuously exposed to E. coli (KCTC1682) at concentrations below the minimum inhibitory concentration (sub-MIC). As a result, as shown in Figure 8, the fold change of the MIC of hirunipin 2 against E. coli (KCTC1682) remained stable and unchanged after 15 passages, confirming that hirunipin 2 can effectively prevent the development of bacterial resistance. On the other hand, the fold change of the MIC of tetracycline and ciprofloxacin increased 32-fold and 4096-fold, respectively, indicating that the development of bacterial resistance induced by tetracycline and ciprofloxacin was significant.

[0216]

[0217] Synergistic antimicrobial activity and antibiole activity of existing antibiotics and hirunipin 2 against multidrug-resistant bacteria

[0218] Antibiotic resistance is a serious global threat that requires new strategies to combat infections. One promising strategy is to combine different drugs to achieve synergistic effects. Such combinations can also help overcome antibiotic resistance (AMR) and extend the lifespan of existing antibiotics. Furthermore, some antimicrobial peptides (AMPs) have been shown to enhance the activity of existing antibiotics in addition to their direct antimicrobial action. These AMP-antibiotic combinations can effectively treat infections caused by antibiotic-resistant bacteria and prevent the development of resistance.

[0219] In this experiment, the synergistic effects of Hirunipin 2 and some existing antibiotics against multidrug-resistant Acinetobacter baumannii (MDRAB) were investigated using a checkerboard assay. For this experiment, existing antibiotics acting on different targets, such as protein (chloramphenicol and tetracycline) and DNA synthesis (ciprofloxacin and rifampicin), were selected. The Partial Inhibition Index (FICI) indicates the bactericidal efficiency of the drug combination. A lower FICI value indicates a superior synergistic effect. The FICI values ​​for combinations of Hirunipin 2 and four existing antibiotics against MDRAB are detailed in Table 9. Hirunipin 2 demonstrated excellent synergy with all antibiotics tested against multidrug-resistant Acinetobacter baumannii (MDRAB). In particular, Hirunipin 2 showed the best synergy with chloramphenicol, with an FICI of 0.1875.

[0220] Furthermore, to investigate the effect of the combination of hirunipin 2 and antibiotics on the growth kinetics of multidrug-resistant Acinetobacter baumannii (MDRAB), a time-kill analysis of MDRAB growth kinetics was performed using hirunipin 2 alone or in combination with antibiotics at concentrations exhibiting a synergistic effect (FICI < 0.5). As shown in Figure 5, treatment with hirunipin 2 or antibiotics alone had no effect on killing bacteria for 4 hours. However, treatment with hirunipin 2 and antibiotics together showed a prominent synergistic and bactericidal effect within 30 to 60 minutes (see Figures 5a-5d). Through these results, it was confirmed that the combination of antibiotics and hirunipin 2 enhances cytotoxic activity in response to drug exposure.

[0221] In addition, the effect of hirunipin 2 on inhibiting biofilm formation of multidrug-resistant Acinetobacter baumannii (MDRAB) in combination with existing antibiotics was investigated. Biofilm biomass was measured using crystal violet staining. The results showed that treatment with hirunipin 2 or antibiotics alone did not significantly inhibit biofilm formation even at synergistic concentrations (see Fig. 5e). On the other hand, combinations of hirunipin 2 / chloramphenicol, hirunipin 2 / ciprofloxacin, hirunipin 2 / tetracycline, and hirunipin 2 / rifampicin demonstrated a strong synergistic effect in inhibiting biofilm formation of multidrug-resistant Acinetobacter baumannii (MDRAB) (see Fig. 5e).

[0222]

[0223] Synergistic antimicrobial activity against multidrug-resistant bacteria according to the combination of antibiotic and hirunipin 2 Antibiotic MICA[A]FICAMICB[B]FICBFICI Interpretation CHL 10241280.1256440.06250.1875 Synergy CIP 128320.256440.06250.3125 Synergy TET 1620.1256480.1250.25 Synergy RIF 1610.062564160.250.3125 Synergy CHL: Chloramphenicol, CIP: Ciprofloxacin, TET: Tetracycline, RIF: Rifampicin MIC: Minimum inhibitory concentration MICA: MIC of antibiotic alone (μg / mL); [A]: MIC of antibiotic combination (μg / mL).MICB: MIC of hirunipin 2 alone (μg / mL); [B]: MIC of hirunipin 2 combination (μg / mL).FICA: Partial inhibitory concentration of antibiotic ([A] / MICA)FICB: Partial inhibitory concentration of hirunipin 2 ([B] / MICB)FICI: Partial inhibitory concentration index, FICI = FICA + FICBFICI ≤ 0.5 indicates a synergistic effect, 0.5 < FICI ≤ 1.0 indicates an additive effect, 1.0 < FICI ≤ 4.0 indicates no effect, and FICI > 4.0 indicates an antagonistic effect.

[0224]

[0225] Mechanism of the synergistic effect of existing antibiotics and Hirunipin 2 against multidrug-resistant bacteria

[0226] To elucidate the underlying mechanism of the synergistic effect of the combination of hirunipin 2 and antibiotics on multidrug-resistant Acinetobacter baumannii (MDRAB), the cell membrane permeability of the multidrug-resistant Acinetobacter baumannii (MDRAB) was investigated using a flow cytometer. The results showed that cell membrane permeability was not affected even when cells were cultured at the minimum inhibitory concentration (MIC) of each antibiotic (Figure 5f-i). However, when cells were cultured at the minimum inhibitory concentration of hirunipin 2 (MIC, 64 μg / mL), fluorescence intensity increased significantly due to PI uptake and DNA binding, leading to a gradual decrease in cell membrane integrity (see Figure 5f-i). Even at synergistic concentrations (4, 8, and 16 μg / mL), complete membrane permeability did not occur even with treatment with hirunipin 2 alone. On the other hand, when multidrug-resistant Acinetobacter baumannii (MDRAB) was treated with 4 μg / mL hirunipin 2 and 128 μg / mL chloramphenicol at synergistic concentrations, complete membrane damage was observed (see Figs. 5f-i). Complete membrane permeability was also induced when treated with synergistic concentrations of hirunipin 2 / ciprofloxacin, hirunipin 2 / tetracycline, and hirunipin 2 / rifampicin (see Figs. 5f-i). These results suggest that hirunipin 2 enhances the bactericidal activity of antibiotics by increasing outer membrane permeability.

[0227]

[0228] Anti-inflammatory activity in LPS-stimulated mouse macrophages

[0229] The effect of hirunipin 2 on the production of inflammatory cytokines, including TNF-α, IL-6, and MCP-1, in LPS-stimulated RAW264.7 cells was investigated. As a result, 16 μg / mL of hirunipin 2 was found to significantly inhibit the production of TNF-α, IL-6, and MCP-1 in LPS-stimulated RAW 264.7 by 40%, 70%, and 90%, respectively (see Figs. 9a-9c). In addition, hirunipin 2 was found to significantly inhibit the mRNA expression of these cytokines in LPS-stimulated RAW264.7 cells (see Fig. 9d). Through the above results, it was confirmed that hirunipin 2 of the present invention possesses anti-inflammatory activity.

[0230]

[0231] [Abbreviations Used]

[0232] AMP: antimicrobial peptide

[0233] RP-HPLC: reversed-phase high performance liquid chromatography

[0234] CD: circular dichroism

[0235] MIC: minimum inhibitory concentration

[0236] sRBCs: sheep red blood cells

[0237] MTT: 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide

[0238] MDRKP: multidrug-resistant Klebsiella pneumonia

[0239] MDRAB: multidrug-resistant Acinetobacter baumannii

[0240] MDRPA: multidrug-resistant Pseudomonas aeruginosa

[0241] MDRE: multidrug-resistant Enterobacter cloacae

[0242] MDREC: multidrug-resistant Escherichia coli

[0243] NPN: N-phenylnaphthalen-1-amine

[0244] ONPG: ortho-nitrophenyl-β-D-galactopyranoside

[0245] diSC3-5: dipropylthiadicarbocyanine iodide

[0246]

[0247] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0248]

[0249] [National R&D projects that supported this invention]

[0250] [Project ID] 1345366488

[0251] [Assignment No.] 2020R1A6A1A06046235

[0252] [Ministry Name] Ministry of Education

[0253] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0254] [Research Project Name] University Key Research Institute Support Project

[0255] [Research Project Title] Ecological and Environmental Toxicology Research Institute

[0256] [Name of Project Performing Organization] Chungbuk National University

[0257] [Research Period] 2020.06.01 ~ 2029.05.31

[0258] [National R&D projects that supported this invention]

[0259] [Project ID] 1345370811

[0260] [Project No.] 2021RIS-001

[0261] [Ministry Name] Ministry of Education (P13)

[0262] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0263] [Research Project Name] Local Government-University Cooperation-Based Regional Innovation Project

[0264] [Research Project Title] (Chungbuk Regional Innovation Platform) Chungbuk National University

[0265] [Name of Project Implementing Organization] (Chungbuk Regional Innovation Platform) Chungbuk National University

[0266] [Research Period] May 1, 2022 ~ March 31, 2023

Claims

A novel peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3. In paragraph 1, The above peptide is a novel peptide characterized by having antibacterial activity, antibiometabolic activity, antibiotic sensitivity-enhancing activity, or anti-inflammatory activity. In paragraph 2, The above peptide is a novel peptide characterized by having antibacterial activity against one or more bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and multidrug-resistant bacteria. In paragraph 3, A novel peptide characterized in that the above-mentioned multidrug-resistant bacteria is one or more selected from the group consisting of multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Enterobacter cloaca, and multidrug-resistant Escherichia coli. In paragraph 2, The above peptide is a novel peptide characterized by a Minimal Biofilm Eradication Concentration against multidrug-resistant Acinetobacter baumannii of 64 to 256 μg / mL. In paragraph 2, The above peptide is a novel peptide characterized by enhancing antibiotic sensitivity when used in combination with antibiotics such as chloramphenicol, ciprofloxacin, tetracycline, or rifampicin. In paragraph 2, The above peptide is a novel peptide characterized by having anti-inflammatory activity through the inhibition of the production and mRNA expression of inflammatory cytokines TNF-α, IL-6, and MCP-1. A composition for enhancing antibiotic sensitivity, comprising as an active ingredient a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3. In paragraph 8, The above antibiotics are methicillin, oxacillin, norfloxacin, vancomycin, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, Cefadroxil, Cefazolin, Cephalotin, Cephalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin / clavulanate, Ampicillin / sulbactam, Piperacillin / tazobactam, Ticarcillin / clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Naldixic acid,Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), TMP-SMX), Sulfonamido chrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidi acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin / Dalfopristin, Thiamphenicol,A pharmaceutical composition for enhancing antibiotic sensitivity, characterized by being selected from the group consisting of tigecycline, tinidazole, and trimethoprim. In paragraph 8, A composition for enhancing antibiotic sensitivity, characterized in that the above peptide has synergistic antimicrobial activity against multidrug-resistant bacteria when used in combination with an antibiotic. In Paragraph 10, A composition for enhancing antibiotic sensitivity, characterized in that the above-mentioned multidrug-resistant bacteria are one or more selected from the group consisting of multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Enterobacter cloacae, and multidrug-resistant Escherichia coli. An antibiotic adjuvant composition comprising, as an active ingredient, a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3. A composition for enhancing antibiotic sensitivity according to claim 8 and an antibiotic composition comprising an antibiotic as an active ingredient. In Paragraph 13, The above antibiotics are methicillin, oxacillin, norfloxacin, vancomycin, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, Cefadroxil, Cefazolin, Cephalotin, Cephalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin / clavulanate, Ampicillin / sulbactam, Piperacillin / tazobactam, Ticarcillin / clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Naldixic acid,Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), TMP-SMX), Sulfonamido chrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidi acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin / Dalfopristin, Thiamphenicol,An antibiotic composition characterized by being selected from the group consisting of tigecycline, tinidazole, and trimethoprim.

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