Antimicrobial peptides consisting of transmembrane peptide domains and alkaline or polar amino acids at both ends thereof and variants thereof

Abstract

The present invention relates to a fusion polypeptide which is obtained by binding a polypeptide comprising a transmembrane domain (TMD) and a polypeptide of general formula I at the C-terminus and N-terminus of the polypeptide, and which can provide antibacterial activity against Gram-positive, Gram-negative, and multi-drug resistant bacteria.

Description

Technical Field

[0001] The present invention relates to an antimicrobial peptide, which is composed of a transmembrane domain in a membrane protein of a cell membrane and an intracellular organelle membrane located in a cell and basic or polar (uncharged) amino acids at both ends of the domain. Specifically, the antibacterial activities of various peptides artificially synthesized with peptides existing in nature having such characteristics against Gram-positive, Gram-negative, and multi-drug resistant bacteria are confirmed, and each of the peptides and an antibacterial composition containing each of the peptides are provided, etc. Background Art

[0002] The biggest problem with antibiotics used as a treatment means for bacterial infections is the emergence of antibiotic resistance in bacteria. According to a report by the Wellcome Trust of the UK government, by 2050, the number of deaths globally due to superbugs that cannot be treated with existing antibiotics will exceed 10 million people. Therefore, among the causes of death, superbug infections will become a threat that surpasses cancer.

[0003] In addition, the World Health Organization (WHO) has selected carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Enterobacteriaceae sp. strains as bacteria at the emergency level among many multi-drug resistant bacteria, and announced that the Gram-negative superbugs that are resistant to carbapenem antibiotics, known as the last line of antibiotics, should be given the highest priority for solution.

[0004] As described above, the biggest problem with antibiotics used by humans so far is the emergence of bacterial resistance. Therefore, many studies are underway to solve this problem. In particular, to overcome bacterial antibiotic resistance, antimicrobial peptides (AMPs) are being actively developed. Although various antimicrobial peptides derived from host defense proteins of animals or humans have been developed, they have not been put into practical use due to problems such as the possibility that bacteria may develop resistance to the human immune system, a short half-life in the blood, toxicity shown to humans, and effective drug efficacy compared to existing antibiotics. As an example, magainin, an antimicrobial peptide developed in the United States in 1999, requested approval from the FDA after a Phase 3 clinical trial but was rejected because its drug efficacy was not superior to that of existing antibiotics. Also, daptomycin was approved by the US Food and Drug Administration in 2003, and oritavancin was approved in 2014, but both peptides only show antibacterial activity against Gram-positive bacteria and can only be applied to skin infection diseases.

[0005] To solve this problem, the present inventors confirmed in the prior invention that peptides (KU-5878, AMPR-11) containing the second transmembrane domain of the Romo1 protein located in the inner mitochondrial membrane show antibacterial activity against Gram-positive, Gram-negative, and multi-drug resistant bacteria, and thus can be applied to the treatment of a wide range of bacterial infectious diseases, and applied for patents and published papers. However, although KU-5878 contains a transmembrane peptide domain, one basic amino acid is contained at each of the two ends of the domain, so its antibacterial activity is not excellent. As a further study, the present inventors found that a peptide composed of a transmembrane peptide domain in a membrane protein, a total of three or more basic amino acids at both ends of the domain, and some polar (uncharged) amino acids has more excellent antibacterial activity than the prior invention, and confirmed that it shows antibacterial activity against Gram-positive, Gram-negative, and multi-drug resistant bacteria and can be applied to the treatment of a wide range of bacterial infectious diseases, thereby completing the present invention. Summary of the Invention

[0006] Technical Problem

[0007] The technical problem to be solved by the present invention is to provide a fusion polypeptide, which is an antimicrobial peptide composed of a transmembrane peptide domain in a cell, a total of three or more basic amino acids at both ends of the domain, and some polar amino acids Figure 1) It is formed by binding a polypeptide containing a transmembrane domain (TMD) composed of 15 to 30 amino acids and terminal amino acids with more than 1 at each of the C-terminus and N-terminus of the polypeptide and a total of more than 3 amino acids at both termini.

[0008] Moreover, an object of the present invention is to provide an antibiotic containing the antimicrobial peptide as an active ingredient, and food and feed additives, cosmetic compositions, biopesticides, and quasi-drug compositions for antibacterial use containing the antimicrobial peptide.

[0009] However, the technical problems to be solved by the present invention are not limited to the above problems, and those skilled in the art will clearly understand other problems not mentioned through the following description.

[0010] Technical Solution

[0011] An embodiment of the present invention provides a fusion polypeptide formed by binding a polypeptide containing a transmembrane domain (TMD) composed of 15 to 30 amino acids and terminal amino acids with more than 1 at each of the C-terminus and N-terminus of the polypeptide and a total of more than 3 amino acids at both termini.

[0012] Another embodiment of the present invention provides an antimicrobial polypeptide containing the fusion polypeptide.

[0013] Another embodiment of the present invention provides an antibiotic, food and feed additives, cosmetic compositions, biopesticides, and quasi-drug compositions for antibacterial use containing the polypeptide.

[0014] Effects of the Invention

[0015] Compared with existing antibiotics and peptides of existing inventions, the peptides and their variants of the present invention have high antibiotic resistance to more types of bacteria. In particular, the peptides of the present invention exhibit high antibacterial ability against bacteria with antibiotic resistance, thus overcoming the limitations in the application of existing antibiotics. Therefore, they can be provided as preventive or therapeutic agents for infectious diseases caused by multi-drug resistant bacteria. Accordingly, it is expected that this antimicrobial peptide can be used for various purposes, such as for drugs, for quasi-drugs, for food and feed additives, for pesticides, and for cosmetic additives, etc., for the purpose of preventing or treating a wide range of bacterial infectious diseases. Brief Description of the Drawings

[0016] Figure 1To provide a diagram of an antimicrobial peptide composed of a transmembrane domain (TMD) that penetrates a membrane and basic or polar (uncharged) amino acids at both ends of the domain. It is a diagram showing that the peptide kills bacteria by forming pores in the cell membranes of Gram-positive, Gram-negative, and multi-drug resistant bacteria.

[0017] Best Mode

[0018] One embodiment of the present invention provides a fusion polypeptide formed by binding a polypeptide containing a transmembrane domain (TMD) composed of 15 to 30 amino acids and terminal amino acids, each containing 1 amino acid at the C-terminus and N-terminus of the polypeptide and a total of more than 3 amino acids.

[0019] As a specific example of the present invention, the transmembrane domain (TMD) can be any one of SEQ ID NO: 39, 41, 54, 73 to 78.

[0020] As a specific example of the present invention, the terminal amino acids can be amino acids with 2 to 6 amino acids bound to each of the C-terminus and N-terminus.

[0021] As a specific example of the present invention, the terminal amino acids can be selected from the group consisting of basic amino acids and polar amino acids.

[0022] As a specific example of the present invention, the terminal amino acids can be any one of lysine, arginine, histidine, serine, threonine, cysteine, glutamine, asparagine, and tyrosine.

[0023] As a specific example of the present invention, the fusion polypeptide can be any one of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, 59, 61, 63, 64, 65, 66, 71, and 72.

[0024] Another embodiment of the present invention provides an antimicrobial polypeptide containing the fusion polypeptide.

[0025] As a specific example of the present invention, the modification of the N-terminus or C-terminus of the polypeptide can also include PEGylation, acetylation, carboxylation, lipidation, or amidation.

[0026] As a specific example of the present invention, the polypeptide may have antibacterial activity against one or more bacteria selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and multidrug-resistant bacteria.

[0027] Another embodiment of the present invention provides an antibiotic, a food additive, a feed additive, a cosmetic composition, a biopesticide preparation for antibacterial use, and a quasi-drug composition for antibacterial use, which contain the polypeptide as an active ingredient. Detailed implementation mode

[0028] One embodiment of the present invention provides a fusion polypeptide, which is formed by binding a polypeptide containing a transmembrane domain (TMD) composed of 15 to 30 amino acids and terminal amino acids each containing more than 1 amino acid at the C-terminus and N-terminus of the polypeptide, and a total of more than 3 amino acids at both ends.

[0029] The transmembrane domain is a peptide sequence region having transmembrane ability, and a peptide having transmembrane ability is a peptide that penetrates the lipid bilayer membrane of cell organelles existing inside the cell. The transmembrane domain (TMD) in the transmembrane peptide is composed of about 22 amino acids, and is composed of hydrophobic amino acids and some hydrophilic amino acids. The transmembrane domain may be one or more.

[0030] A large part of the genome is transmembrane proteins, and the transmembrane part can be predicted relatively accurately by computer algorithms. In the present invention, the transmembrane protein sequence information is derived from UniProt (www.uniprot.org).

[0031] The inventors of the present invention synthesized peptides of different lengths composed of a transmembrane domain and a total of more than 3 basic amino acids and some polar amino acids at both ends based on the information obtained by searching UniProt, confirmed their antibacterial activity, and completed the present invention by confirming the antibacterial activity of variants of the peptides (refer to Example 1 and Example 2).

[0032] In a specific embodiment of the present invention, as described above, the inventors synthesized peptides of different lengths existing in nature, composed of a transmembrane domain in human proteins and a total of more than 3 basic amino acids and polar amino acids at both ends, and used the minimum bactericidal concentration assay method to confirm the antibacterial activity of each peptide against Pseudomonas aeruginosa and Staphylococcus aureus. As a result, it was confirmed that all the synthesized peptides had antibacterial activity superior to that of the previously invented KU-5878 (refer to Example 2).

[0033] Furthermore, by synthesizing peptides with both terminal portions of the peptides shown in SEQ ID NO: 1 and 2 of the present invention deleted to confirm the antibacterial activity results, it was confirmed that the antibacterial activity decreased, and it was confirmed that peptides without basic or polar amino acids at both ends of the cell transmembrane domains shown in SEQ ID NO: 39 and 41 lost antibacterial activity. Therefore, it was confirmed that the basic or polar amino acid sequences at both ends of the cell transmembrane domains are important for antibacterial activity (refer to Example 3).

[0034] In another specific embodiment of the present invention, the inventors confirmed the antibacterial activity of synthetic peptides by artificially repeatedly binding basic amino acids to both ends of the cell transmembrane domain. As a result, it was found that peptide groups synthesized by binding a total of 3 or more lysines (K) or arginines (R) to both sides of the cell transmembrane domain exhibited excellent antibacterial activity (refer to Example 4).

[0035] In a specific embodiment of the present invention, the inventors confirmed the antibacterial activity of the fusion polypeptide of the present invention against Enterococcus faecalis, Escherichia coli, Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Staphylococcus aureus, multidrug-resistant Enterococcus faecalis, and multidrug-resistant Acinetobacter baumannii. As a result, it was found that the representative peptides exhibited broad antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and multidrug-resistant bacteria (refer to Example 5 and Example 6).

[0036] Moreover, in yet another specific embodiment of the present invention, the inventors confirmed the antibacterial activity by synthesizing peptides composed of a total of 3 or more basic amino acids and polar amino acids at the transmembrane domain and both ends of the domain of other species except humans. As a result, it was found that the peptides with common characteristics exhibited antibacterial activity regardless of the species (refer to Example 7).

[0037] Furthermore, in another specific embodiment of the present invention, the inventors confirmed that even when the amino acid sequences bound to both ends were bound to other transmembrane peptides, excellent antibacterial activity was exhibited (refer to Example 8).

[0038] In this specification, "peptide" refers to a linear molecule in which amino acid residues are bound to each other by peptide bonds. It can be prepared by chemical synthesis methods well known in the art, especially solid-phase synthesis technology or liquid-phase synthesis technology.

[0039] As a specific example of the present invention, the transmembrane domain may be composed of 17 to 25 amino acids, and more specifically, it may be any one of SEQ ID NO: 39, 41, 54, 73 to 78.

[0040] The terminal amino acids bind to the C-terminus and N-terminus of the transmembrane peptide domain, each terminus containing more than 1 amino acid and a total of more than 3 amino acids. Specifically, the C-terminus and N-terminus may each contain 3 to 6 amino acids, so the two termini can bind at least 1 to at most 6 amino acids.

[0041] The terminal amino acids are selected from basic amino acids and polar amino acids, and the terminal amino acids can be any one of lysine, arginine, histidine, serine, threonine, cysteine, glutamine, asparagine, and tyrosine.

[0042] In this case, the terminal amino acids binding to the C-terminus and N-terminus only need to contain more than 1 amino acid at each of the two termini and the total number of amino acids at the two termini is more than 3, and each amino acid is independent of each other. Even if the peptides binding to the C-terminus and N-terminus have different amino acid compositions and different lengths, they are included in the present invention. As an example of the terminal amino acids, it can be a structure in which one terminus of the transmembrane domain binds 1 amino acid and the other terminus binds more than 2 amino acids.

[0043] As a specific example of the present invention, the fusion polypeptide can be any one of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, 59, 61, 63, 64, 65, 66, 71, and 72.

[0044] As another example of the present invention, the modification of the polypeptide can be PEGylation, acetylation, carboxylation, lipidation, or amidation. More specifically, the amino terminus can be bound to protecting groups such as acetyl, fluorenylmethyloxycarbonyl, formyl, palmitoyl, myristoyl, stearoyl, and polyethylene glycol (PEG). The carboxyl terminus of the peptide can be modified with hydroxyl (-OH), amino (-NH2), azide (-NHNH2), etc. Moreover, the terminus of the peptide of the present invention or the R-residue (R-group) of the amino acid can be bound to fatty acids, oligosaccharide chains, all nanoparticles (gold particles, liposomes, heparin, hydrogels, etc.), amino acids, carrier proteins, etc.

[0045] As another example of the present invention, the amino acids constituting the peptide can each independently be L-type or D-type amino acids, and can be radioactive or fluorescently labeled amino acid analogs.

[0046] As another example of the present invention, the peptide may have antibacterial activity against one or more bacteria selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and multi-drug resistant bacteria.

[0047] Another embodiment of the present invention provides an antibacterial polypeptide comprising the fusion polypeptide.

[0048] As described above, the fusion polypeptide further comprises a transmembrane peptide domain and amino acid residues at both ends, and can have an antibacterial effect by penetrating the cell membrane and lysing bacteria. Therefore, the present invention can provide a polypeptide having antibacterial ability including the fusion polypeptide.

[0049] As a specific example of the present invention, the modification of the N-terminus or C-terminus of the polypeptide may further include PEGylation, acetylation, carboxylation, lipidation, or amidation. More specifically, the amino terminus may be bound to a protecting group such as an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristoyl group, a stearoyl group, and polyethylene glycol (PEG). The carboxyl terminus of the peptide may be modified with a hydroxyl group (-OH), an amino group (-NH2), an azide group (-NHNH2), etc. In addition, the terminus of the peptide of the present invention or the R-residue (R-group) of the amino acid may be bound to fatty acids, oligosaccharide chains, all nanoparticles (gold particles, liposomes, heparin, hydrogels, etc.), amino acids, carrier proteins, etc.

[0050] As a specific example of the present invention, the polypeptide may have antibacterial activity against one or more bacteria selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and multi-drug resistant bacteria.

[0051] As yet another example of the present invention, the Gram-positive bacterium may be a bacterium belonging to a genus selected from the group consisting of Staphylococcus sp., Bacillus sp., Enterococcus sp., Streptomyces sp., and Streptococcus sp., and preferably, the Gram-positive bacterium may be one or more bacteria selected from the group consisting of Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, Streptomyces sindenensis, Enterococcus faecalis, and Streptococcus pneumoniae.

[0052] As another example of the present invention, the Gram-negative bacterium may be a bacterium belonging to a genus selected from the group consisting of Escherichia sp., Klebsiella sp., Acinetobacter sp., Pseudomonas sp., and Enterobacter sp., and preferably, the Gram-negative bacterium may be one or more bacteria selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter aerogenes.

[0053] As another example of the present invention, the multi-drug resistant bacteria are the Gram-positive or Gram-negative bacteria resistant to one or more antibiotics belonging to penicillins, carbapenems, cephalosporins, quinolones, macrolides, tetracyclins or glycopeptides. Preferably, the multi-drug resistant bacteria may be bacteria belonging to a genus selected from the group consisting of methicillin-resistant Staphylococcus sp., multidrug-resistant Pseudomonas sp., vancomycin-resistant Enterococcus sp., multidrug-resistant Klebsiella sp., multidrug-resistant Acinetobacter sp. and vancomycin-resistant Staphylococcus sp.

[0054] As yet another example of the present invention, the multi-drug resistant bacteria may be one or more bacteria selected from the group consisting of methicillin-resistant S. aureus, multidrug-resistant P. aeruginosa, multidrug-resistant A. baumannii, multidrug-resistant K. pneumoniae, vancomycin-resistant E. faecium and vancomycin-resistant S. aureus.

[0055] As another example of the present invention, the multi-drug resistant Pseudomonas aeruginosa may be resistant to one or more antibiotics selected from the group consisting of piperacilin, piperacilin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, and ciprofloxacin. The multi-drug resistant Acinetobacter baumannii may be resistant to one or more antibiotics selected from the group consisting of piperacilin, piperacilin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, and cefepime. The multi-drug resistant Klebsiella pneumoniae may be resistant to one or more antibiotics selected from the group consisting of piperacilin-tazobactam, ceftazidime, cefepime, imipenem, gentamicin, and ciprofloxacin.

[0056] As another example of the present invention, in addition to vancomycin, the vancomycin-resistant Enterococcus faecalis may also be resistant to one or more antibiotics selected from the group consisting of rifampin, tetracycline, gentamicin, erythromycin, streptomycin, and ampicillin. In addition to vancomycin, the vancomycin-resistant Staphylococcus aureus may also be resistant to one or more antibiotics selected from the group consisting of oxacillin, benzylpenicillin, ampicillin, and cefazolin.

[0057] Another embodiment of the present invention provides an antibiotic comprising the polypeptide as an active ingredient.

[0058] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating bacterial infectious diseases, comprising the antimicrobial peptide as an active ingredient.

[0059] In the present invention, "prevention" refers to any act of delaying the infection of the Gram-positive bacteria, Gram-negative bacteria, and / or multi-drug resistant bacteria, or delaying the onset of diseases caused by their infection, by administering the pharmaceutical composition of the present invention. "Treatment" refers to all acts of improving or beneficially changing the symptoms caused by the bacterial infection by administering the pharmaceutical composition of the present invention.

[0060] In the present invention, in addition to the fusion polypeptide or the antibacterial polypeptide, the pharmaceutical composition may further comprise one or more known antibiotics, and may also comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

[0061] In the present invention, a "carrier" is also referred to as a "vehicle", and refers to a compound that can easily introduce a protein or a peptide into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that can easily introduce many organic substances into cells or tissues of an organism.

[0062] In the present invention, a "diluent" is defined as a compound diluted in water, which not only stabilizes the bioactive form of the target protein or peptide, but also dissolves the protein or peptide. In the art, salts dissolved in buffer solutions are used as diluents. The commonly used buffer solution is phosphate buffered saline because it mimics the salt state of human body solutions. Since buffer salts control the pH of the solution at low concentrations, buffer diluents do not easily change the biological activity of compounds. The azelaic acid-containing compound used in the present invention can be administered to human patients as it is or as a pharmaceutical composition mixed with other components such as in combination therapy or mixed with a suitable carrier or excipient.

[0063] Moreover, the pharmaceutical composition of the present invention can be formulated into external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods. The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the required method. The dosage varies according to the patient's condition and body weight, the severity of the disease, the drug form, the administration route and time, but can be appropriately selected by those of ordinary skill in the art. For example, it can be administered in a form mixed with a pharmaceutically acceptable carrier at about 0.001 mg to 1000 mg. The pharmaceutical composition of the present invention can be administered once a day or in several divided doses as needed, and can be used alone or in combination with surgical procedures, hormone therapy, drug therapy and methods using biological response modifiers.

[0064] Moreover, the present invention provides a method for preventing or treating bacterial infectious diseases, which comprises the step of administering the antibacterial peptide to an individual.

[0065] Moreover, the present invention provides the use of the antibacterial peptide in the preparation of a drug for preventing or treating bacterial infectious diseases.

[0066] As an example of the present invention, the bacterial infectious disease may be one or more diseases selected from the group consisting of skin infection, food poisoning, otitis media, cystitis, peritonitis, urinary tract infection, mastitis, pneumonia, endocarditis, conjunctivitis, arthritis, endometritis, glanders, bacteremia, sepsis, osteomyelitis, and acne. Preferably, it may be pneumonia or sepsis.

[0067] Another embodiment of the present invention provides a food additive, a feed additive, a cosmetic composition, a biopesticide preparation for antibacterial use, and a quasi-drug composition for antibacterial use, each containing the polypeptide.

[0068] When the peptide of the present invention is used as an additive to food or feed, etc., the peptide may be added directly or used together with other foods, feeds, or their ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (preventing, health care, or treating infectious diseases by inhibiting the proliferation and growth of bacteria). Generally, when preparing feed, food, or beverage, the addition amount of the peptide raw material of the present invention is 15% by weight or less, preferably 10% by weight or less. However, when ingested for a long time for the purpose of health and hygiene or health management, the amount may be below the above range, and since there are no problems in terms of safety, the active ingredient may also be used in an amount above the above range. The types of the food and feed are not particularly limited.

[0069] The present invention can be variously modified and can have various embodiments. Therefore, the following specific embodiments will be shown in the drawings and described in detail. However, this is not intended to limit the present invention to a specific embodiment, and it should be understood to include all modifications, equivalents, and alternatives included in the spirit and technical scope of the present invention. When explaining the present invention, when it is judged that the specific description of related well-known technologies may obscure the gist of the present invention, the detailed description thereof will be omitted.

[0070] Examples

[0071] Example 1. Retrieving a peptide composed of a transmembrane domain and a total of three or more basic amino acids and some polar amino acids at both ends Figure 1

[0072] In the present invention, transmembrane protein sequence information was retrieved from UniProt (www.uniprot.org) containing protein sequence and functional information. The UniProt homepage was opened, the protein UniProt knowledgebase was accessed, and then the reviewed (Swiss-Prot) was accessed. Then, when accessing proteins with transmembrane function, 79,724 proteins were searched. In the case of human proteins, 20,422 proteins were searched, among which 5,217 transmembrane proteins were searched. If each transmembrane protein is accessed, the information of each protein can be confirmed, and at the same time, the peptide sequence of the transmembrane peptide domain can be confirmed. The present inventors first retrieved the transmembrane domain and its surrounding sequences among 5,217 transmembrane proteins and synthesized peptides composed of transmembrane domains having a structure similar to Example 2. Confirming the antibacterial activity of the peptide retrieved from UniProt and a total of more than 3 basic amino acids and some polar amino acids at both ends (Table 1).

[0073] Example 3. Confirming the antibacterial activity of a peptide with some amino acids deleted from both ends of the transmembrane domain in the peptide retrieved from UniProt

[0074] The present inventors synthesized 36 antimicrobial peptides composed of transmembrane peptide domains retrieved from UniProt and basic or polar amino acids at both ends thereof, and confirmed the antimicrobial activity of each peptide by the minimum bactericidal concentration (MBC) method (the information of each peptide is referred to Table 1 below). Confirming the antimicrobial activity of each of the peptides by the minimum bactericidal concentration method was carried out specifically as follows.

[0075] First, Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (NCCP 15872) were cultured overnight at 37 °C and 200 rpm in an LB (1% tryptone, 0.5% yeast extract, 1% sodium chloride) liquid medium or a 3% (w / v) Tryptic Soy Broth (TSB) liquid medium, and then cultured again for 2 hours under the same conditions for the second time. A strain solution was prepared by diluting with a 10 mM sodium phosphate solution so that the final concentration of each strain after the second culture reached 5×10 5 CFU / ml.

[0076] Next, 100 μl of each peptide with different concentrations (final concentration: 0 μg / ml - 200 μg / ml of the peptide) was inoculated into each well of a 96-well microplate, 100 μl of the prepared strain solution was added and mixed, and the reaction was carried out in an incubator at 37 °C for 1 hour.

[0077] Then, 25 g / L of LB or 30 g / L of Tryptic Soy (TS) and 15 g / L of agar were dissolved in distilled water and sterilized. Then, 20 ml was taken and poured into a 100 mm round plate, and it was left to solidify at room temperature for more than 1 day to prepare LB or TS agar plates. 10 μl of the reaction solution of the peptide and the strain was smeared in a specified size, and it was cultured in an incubator at 37 °C for 18 hours, and then colony formation was confirmed on the plate. The minimum bactericidal concentration was defined as the lowest concentration of the peptide at which no colonies were formed. The experimental results are shown in Table 1 below.

[0078] Table 1

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] (Underline: Amino acid sequence of transmembrane domain)

[0087] As shown in Table 1, it was confirmed that 36 antimicrobial peptides composed of transmembrane domains retrieved from UniProt and basic or polar amino acids at both ends thereof had excellent antimicrobial activity. The inventors found in previous studies that peptides containing the second transmembrane domain of Romo1 protein had antimicrobial activity, and in subsequent studies, it was found that the KU-5878 peptide composed of 21 amino acid residues had the most excellent antimicrobial activity (MBC value for Pseudomonas aeruginosa: 100 μg / ml; MBC value for Staphylococcus aureus: 100 μg / ml). It was confirmed that compared with KU-5878, the peptides found in the present invention had 1.25 - 100 times higher antimicrobial activity against Pseudomonas aeruginosa and 1.25 - 25 times higher antimicrobial activity against Staphylococcus aureus than KU-5878.

[0088] Example 5. Confirming the antibacterial activity of representative peptides among the antibacterial peptides identified in Tables 1 and 3 against various bacteriaExample 6. Confirming the antibacterial activity of representative peptides among the antibacterial peptides identified in Tables 1 and 3 against multi-drug resistant bacteria

[0089] In order to confirm the antibacterial activity of peptides lacking the two terminal portions of the transmembrane domain among the peptides retrieved from UniProt, the COX20-1 peptide was selected as a representative from the synthetic peptides shown in Table 1 as described above, and its antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus was determined using the minimum bactericidal concentration assay. The minimum bactericidal concentration assay was the same as the method in Example 2. Three peptides (SEQ ID NO: 37, 38, 39) with amino acid sequences lacking the two terminal portions of amino acids of the COX20-1 peptide were synthesized, and the antibacterial activity of each peptide was compared and confirmed by the minimum bactericidal concentration assay (information on each peptide is referred to Table 2 below). As the same experiment, two peptides (SEQ ID NO: 40, 41) with amino acid sequences lacking the two terminal portions of amino acids of the HIG1A-1 peptide were synthesized, and the antibacterial activity of each peptide was compared and confirmed by the minimum bactericidal concentration assay (information on each peptide is referred to Table 2 below).

[0090] Table 2

[0091]

[0092]

[0093]

[0094] As shown in Table 2 as described above, it was confirmed that when partial amino acids at both ends of the transmembrane domain were deleted, the antibacterial activity of the peptide decreased compared to the peptide (SEQ ID NO: 1 or 2) that initially exhibited antibacterial activity. In particular, peptides without basic or polar amino acids at both ends of the cell transmembrane peptide domain had low antibacterial activity, and thus could not be determined by the minimum bactericidal concentration assay (SEQ ID NO: 39, 41). Therefore, it can be seen that the basic amino acid and polar amino acid sequences at both ends of the transmembrane domain affect antibacterial activity.

[0095] Example 4. Confirming the antibacterial activity of peptides synthesized by artificially binding basic amino acids at both ends of the transmembrane domain

[0096] In the present invention, it was confirmed that compared with KU-5878 found in existing research, the antibacterial peptide group composed of the transmembrane peptide domain and basic or polar amino acids at both ends thereof exhibited more excellent antibacterial activity (Example 2 above). These antibacterial peptide groups are peptides containing amino acid sequences existing in nature. In this example, peptides having similar characteristics to these antibacterial peptide groups and composed of amino acid sequences not existing in nature were synthesized, and the antibacterial activity of each peptide was compared and confirmed by the minimum bactericidal concentration assay (information on each peptide is referred to Table 3 below).

[0097] For KU-5878, the transmembrane domain corresponds to amino acids 58 to 77. There is an arginine (R) on the right side of the transmembrane domain, but no basic amino acid on the left side. However, there is a lysine (K) as a basic amino acid on the left side inside the transmembrane domain. In the present invention, the antibacterial activity of a peptide composed of a total of more than 3 basic amino acids and some polar amino acids on both sides of the transmembrane domain was confirmed. As shown in Table 3, the antibacterial activities of peptides synthesized by artificially binding 1 to 5 lysines (K) or arginines (R) to the transmembrane domains of HIG1A and COX20 proteins were measured.

[0098] Table 3

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] As shown in Tables 2 and 3, since peptides without basic or polar amino acids at both ends of the cell transmembrane domain have low antibacterial activity, they cannot be measured by the minimum bactericidal concentration assay. However, as shown in Table 3, it can be seen that the peptide groups synthesized by artificially binding 2 or more lysines or arginines respectively exhibit antibacterial activity superior to that of KU-5878.

[0105] Furthermore, as a result of further synthesizing the transmembrane domains of SEQ ID NO:54, 58, 60, 62, 64, 65, 66 and measuring the antibacterial activities of peptides synthesized by artificially binding 1 to 4 lysines (K) to both ends of the domains, it can be seen that when lysine is artificially bound, the antibacterial activity of each peptide increases.

[0106] Example 7. Confirming the antibacterial activity of a peptide composed of a transmembrane domain and a total of three or more basic amino acids and some polar amino acids at both ends corresponding to those in proteins of other species among partial peptides of the antibacterial peptides in Table 1

[0107] To confirm the types of bacteria against which the peptides with antibacterial activity confirmed in Examples 2 and 4 exert bactericidal power, 3 peptides, OPA1-1, HIG1C, and COX20-1, were selected from the synthetic peptides as shown in Table 1 above, and 2 peptides, HIG1A-0-K4 and HIG1A-0-R4, were selected from the synthetic peptides as shown in Table 3 as representatives, and the antibacterial activities against the bacteria shown in Table 4 below were measured using the minimum bactericidal concentration assay. The minimum bactericidal concentration assay was the same as the method in Example 2.

[0108] Specifically, each type of bacterium was cultured overnight in a 3% (w / v) Tryptic Soy Broth (TSB) liquid medium at 37 °C and 200 rpm, and then cultured again for 2 hours under the same conditions for the second time. The strain solution was prepared by diluting with a 10 mM sodium phosphate solution so that the final concentration of each strain after the second culture reached 5×10 5 CFU / ml.

[0109] Next, each peptide with different concentrations (final concentration: 0 μg / ml - 200 μg / ml of the peptide) was inoculated into each well of a 96-well microplate, 100 μl of the prepared strain solution was added and mixed, and the reaction was carried out in an incubator at 37 °C for 1 hour.

[0110] Then, 30 g / L of Tryptic Soy (TS) and 15 g / L of agar were dissolved in distilled water and sterilized, and then 20 ml was taken and poured into a 100 mm round plate, and it was allowed to solidify at room temperature for more than 1 day to prepare a TS agar plate. 10 μl of the reaction solution of the peptide and the strain was smeared in a specified size, and it was cultured in an incubator at 37 °C for 18 hours, and then colony formation was confirmed on the plate. The minimum bactericidal concentration was defined as the lowest concentration of the peptide at which no colonies were formed. The experimental results are shown in Table 4 below.

[0111] Table 4

[0112]

[0113] As shown in Table 4 above, it can be seen that the antimicrobial peptides composed of transmembrane peptide domains and basic or polar amino acids at both ends exhibit broad antimicrobial activities against both Gram-positive bacteria and Gram-negative bacteria.

[0114] Example 8. Confirming the antibacterial activity of a peptide synthesized by fusing the cell transmembrane domains and the sequences of the N-terminus and C-terminus at both ends of two or more different bacteriopeptides ​

[0115] To confirm the antimicrobial activities of the peptides whose antimicrobial activities were confirmed in Example 2 and Example 4 against multi-drug resistant bacteria, 3 peptides, namely OPA1-1, HIG1C, and COX20-1, were selected from the synthetic peptides as shown in Table 1 above, and 2 peptides, namely HIG1A-0-K4 and HIG1A-0-R4, were selected from the synthetic peptides as shown in Table 3 as representatives, and the antimicrobial activities against the bacteria shown in Table 5 below were determined using the minimum bactericidal concentration assay method. The minimum bactericidal concentration assay method was the same as that in Example 2.

[0116] On the other hand, the multi-drug resistant Pseudomonas aeruginosa and multi-drug resistant Acinetobacter baumannii in Table 5 below were bacteria isolated from patients at Anam Hospital, Korea University. It was confirmed that the multi-drug resistant Pseudomonas aeruginosa was resistant to antibiotics such as piperacilin, piperacilin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, and ciprofloxacin, and the multi-drug resistant Acinetobacter baumannii was resistant to antibiotics such as piperacilin, piperacilin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, and cefepime and were used in the experiment.

[0117] Moreover, the vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Staphylococcus aureus in Table 5 below were purchased from the National Culture Collection for Pathogens (NCCP). It was confirmed that the vancomycin-resistant Enterococcus faecalis was resistant to vancomycin, rifampin, tetracycline, gentamicin, erythromycin, streptomycin, and ampicillin, and the vancomycin-resistant Staphylococcus aureus was resistant to vancomycin, oxacillin, benzylpenicillin, ampicillin, and cefazolin and were used in the experiment.

[0118] Table 5

[0119]

[0120] As shown in Table 5, it can be seen that the antimicrobial peptide composed of a transmembrane peptide domain and basic or polar amino acids at both ends exhibits broad antibacterial activity against multi-drug resistant Gram-positive bacteria and multi-drug resistant Gram-negative bacteria.

[0121] ​ ​

[0122] In Example 1 above, the present inventors confirmed that a peptide composed of a transmembrane domain in a human protein and basic or polar amino acids at both ends thereof has excellent antibacterial activity. In order to confirm that peptides with the same motif in proteins of other species have antibacterial activity, peptides as shown in Table 6 below were synthesized, and the antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus were measured using the minimum bactericidal concentration assay method. The minimum bactericidal concentration assay method is the same as that of Example 2.

[0123] Table 6

[0124]

[0125]

[0126] As shown in Table 6 above, it can be seen that antibacterial peptides composed of a transmembrane peptide domain corresponding to that in proteins of other species and basic or polar amino acids at both ends thereof also have antibacterial activity.

[0127] ​ ​

[0128] The present inventors fused the transmembrane domains of SEQ ID NO: 5 and 60 in the antibacterial peptides of the above examples with the amino acid sequence (KAK---KLK) at both ends of the domain of SEQ ID NO: 40 to form a peptide, and then measured the antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus using the minimum bactericidal concentration assay method. The minimum bactericidal concentration assay method is the same as that of Example 2. As a result, it was confirmed that the transmembrane domains of SEQ ID NO: 5 and 60 themselves do not have antibacterial activity, but the new fusion peptides composed of these transmembrane domains and 4 basic amino acids at both ends showed excellent antibacterial activity (SEQ ID NO: 71, 72).

[0129] Table 7

[0130]

[0131]

[0132] As shown in Table 7 above, it was confirmed that the fusion peptides composed of a transmembrane peptide domain in the same pattern as that in the above examples and basic or polar amino acids at both ends thereof also all showed excellent antibacterial activity.

[0133] Thus far, the present invention has been described with reference to the preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the basic features thereof. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims

1. A fusion polypeptide, characterized in that, It is formed by binding a polypeptide containing a transmembrane domain composed of 15 to 30 amino acids and terminal amino acids each containing more than 1 and a total of more than 3 amino acids at the C-terminus and N-terminus of the polypeptide.

2. The fusion polypeptide according to claim 1, wherein The transmembrane domain is any one of SEQ ID NO: 39, 41, 54, 73 to 78.

3. The fusion polypeptide according to claim 1, wherein The terminal amino acids are amino acids each having 2 to 6 amino acids bound to the C-terminus and N-terminus.

4. The fusion polypeptide according to claim 1, wherein, The terminal amino acids are selected from the group consisting of basic amino acids and polar amino acids.

5. The fusion polypeptide according to claim 1, wherein The terminal amino acid is any one of lysine, arginine, histidine, serine, threonine, cysteine, glutamine, asparagine, and tyrosine.

6. The fusion polypeptide according to claim 1, wherein The fusion polypeptide is any one of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, 59, 61, 63, 64, 65, 66, 71, and 72.

7. An antibacterial polypeptide, characterized in that, It contains the fusion polypeptide according to claim 1.

8. The antibacterial polypeptide according to claim 7, characterized in that, The modification of the N-terminus or C-terminus of the polypeptide further includes polyethylene glycolylation, acetylation, carboxylation, lipidation, or amidation.

9. The antibacterial polypeptide according to claim 7, wherein The polypeptide has antibacterial activity against one or more bacteria selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and multidrug-resistant bacteria.

10. An antibiotic, characterized in that, It contains the polypeptide according to any one of claims 1 to 9 as an active ingredient.

11. A food additive, characterized in that, It contains the polypeptide according to any one of claims 1 to 9.

12. A feed additive, characterized in that, It contains the polypeptide according to any one of claims 1 to 9.

13. A cosmetic composition, characterized in that, It contains the polypeptide according to any one of claims 1 to 9.

14. A biological pesticide preparation for antibacterial, characterized in that, It contains the polypeptide according to any one of claims 1 to 9.

15. A quasi-drug composition for antibacterial use, characterized in that, It contains the polypeptide according to any one of claims 1 to 9.

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