Novel antibacterial polypeptide and variant thereof

Abstract

The present invention relates to a novel antibacterial polypeptide and, more specifically, to an antibacterial composition comprising the antibacterial polypeptide. The antibacterial polypeptide according to the present invention exhibits excellent antibacterial activity against not only Gram-positive bacteria, Gram-negative bacteria, and fungi, but also multidrug-resistant bacteria, and exhibits a dual antibacterial action mechanism through cell membrane disturbance and gDNA aggregation and thus is also expected to have a sensitivity recovery effect for existing antibiotics. Therefore, the antibacterial polypeptide can be effectively used in various fields such as pharmaceutical compositions, food compositions, and cosmetic compositions.

Description

Novel antimicrobial polypeptides and variants thereof

[0001] The present invention relates to a novel antimicrobial polypeptide, and more particularly, to an antimicrobial composition comprising the antimicrobial polypeptide.

[0002] Recent outbreaks of nosocomial infections and the spread of antibiotic-resistant bacteria have become serious global health threats. In particular, multidrug-resistant bacteria (MDRB), such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant Klebsiella pneumoniae, are significantly less responsive to existing antibiotics, making treatment extremely challenging. Consequently, there is a pressing need to develop next-generation antibacterial agents with low resistance to existing antibiotics and novel mechanisms of action.

[0003] Antimicrobial peptides are defense molecules based on the innate immune system of natural organisms. Their relatively short amino acid sequences and non-specific mechanisms of action, including electrostatic interactions and membrane disruption, allow them to rapidly kill pathogenic microorganisms. Unlike conventional antibiotics, antimicrobial peptides act on diverse targets, including cell walls, cell membranes, and genomes, reducing the risk of resistance and offering a broad antimicrobial spectrum. Consequently, they are attracting attention as next-generation antimicrobial candidates.

[0004] However, conventional antimicrobial peptides generally have low stability against proteases, in vivo toxicity, and limited production efficiency, limiting their commercialization as practical therapeutics. In particular, peptides with high antimicrobial activity tend to exhibit increased cytotoxicity and hemolytic activity, reducing their selectivity as therapeutics.

[0005] Accordingly, the development of recombinant antimicrobial polypeptides that maintain antimicrobial activity, ensure biosafety, and can be expressed and purified with high efficiency is becoming a critical technological challenge. Furthermore, there is a persistent need for novel antimicrobial agents that exhibit broad activity against a variety of pathogenic bacteria and exhibit synergistic effects when used in combination with existing antibiotics.

[0006] Accordingly, the inventors of the present invention have completed the present invention by developing a novel antibacterial polypeptide and its variants having excellent antibacterial activity and biosafety to overcome the limitations of the prior art.

[0007] Accordingly, the purpose of the present invention is to provide an antimicrobial polypeptide comprising a peptide represented by the amino acid sequence of SEQ ID NO: 2 or 7.

[0008] Another object of the present invention is to provide an antimicrobial polypeptide represented by the following structural formula:

[0009] [constitutional formula]

[0010] N'- WG X1GK X2KY X3PQ (X4) n GKQKYLKKARKYHRK -C' (SEQ ID NO: 15)

[0011] In the above formula,

[0012] The above N' is the N-terminus of the antimicrobial polypeptide,

[0013] The above C' is the C-terminus of the antimicrobial polypeptide,

[0014] wherein X1 is KYY, RVV or RII,

[0015] The above X2 is IKKYIQ, ILKYLY, VKQYVQ or IKQIIQ,

[0016] The above X3 is K or I,

[0017] The above n is 0 or 1,

[0018] If the above n is 1, X4 is S.

[0019] Another object of the present invention is to provide a composition comprising the above antimicrobial polypeptide.

[0020] Another object of the present invention is to provide a method for treating microbial infection, comprising the step of administering the antimicrobial polypeptide of the present invention to a subject in need thereof.

[0021] To achieve the above purpose, the present invention provides an antimicrobial polypeptide comprising a peptide represented by the amino acid sequence of SEQ ID NO: 2 or 7.

[0022] The present invention also provides an antimicrobial polypeptide represented by the following structural formula:

[0023] [constitutional formula]

[0024] N'- WG X1GK X2KY X3PQ (X4) n GKQKYLKKARKYHRK -C' (SEQ ID NO: 15)

[0025] In the above formula,

[0026] The above N' is the N-terminus of the antimicrobial polypeptide,

[0027] The above C' is the C-terminus of the antimicrobial polypeptide,

[0028] wherein X1 is KYY, RVV or RII,

[0029] The above X2 is IKKYIQ, ILKYLY, VKQYVQ or IKQIIQ,

[0030] The above X3 is K or I,

[0031] The above n is 0 or 1,

[0032] If the above n is 1, X4 is S.

[0033] The present invention also provides an antibacterial composition comprising the above antibacterial polypeptide.

[0034] The present invention also provides an antibacterial pharmaceutical composition comprising the above antibacterial polypeptide.

[0035] The present invention also provides an antibacterial food composition comprising the antibacterial polypeptide.

[0036] The present invention also provides an antibacterial cosmetic composition comprising the antibacterial polypeptide.

[0037] The present invention also provides an antibacterial feed composition comprising the above antibacterial polypeptide.

[0038] The present invention also provides a method for treating a microbial infection, comprising the step of administering to a subject in need thereof an antimicrobial polypeptide comprising a peptide represented by the amino acid sequence of SEQ ID NO: 2 or 7.

[0039] The present invention also provides a method for treating microbial infection, comprising the step of administering an antimicrobial polypeptide represented by the following structural formula to a subject in need thereof:

[0040] [constitutional formula]

[0041] N'- WG X1GK X2KY X3PQ (X4) n GKQKYLKKARKYHRK -C' (SEQ ID NO: 15)

[0042] In the above formula,

[0043] The above N' is the N-terminus of the antimicrobial polypeptide,

[0044] The above C' is the C-terminus of the antimicrobial polypeptide,

[0045] wherein X1 is KYY, RVV or RII,

[0046] The above X2 is IKKYIQ, ILKYLY, VKQYVQ or IKQIIQ,

[0047] The above X3 is K or I,

[0048] The above n is 0 or 1,

[0049] If the above n is 1, X4 is S.

[0050] The antimicrobial polypeptide according to the present invention exhibits excellent antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, fungi, and even multidrug-resistant bacteria. Furthermore, it possesses a dual antimicrobial mechanism of action through cell membrane disruption and gDNA aggregation, and thus is expected to restore sensitivity to existing antibiotics. Therefore, the antimicrobial polypeptide can be usefully utilized in various fields, including pharmaceutical compositions, food compositions, and cosmetic compositions.

[0051] Figure 1a is a diagram showing a vector map of vector pET-NGT-A001 according to the present invention.

[0052] Figure 1b is a diagram showing a vector map of vectors pET-NGT-A002 to pET-NGT-A007 according to the present invention.

[0053] Figure 2a is a diagram showing the results of confirming the expression pattern and purification of the antimicrobial polypeptide NGT-A001 according to the present invention.

[0054] Figure 2b is a diagram showing the results of confirming the temperature-dependent expression pattern of the antimicrobial polypeptide NGT-A001 according to the present invention.

[0055] Figure 2c is a diagram showing the results of confirming the expression pattern of antimicrobial polypeptides NGT-A002 to NGT-A007 according to the present invention.

[0056] Figure 3 is a diagram showing the results of evaluating the antibacterial activity of the antibacterial polypeptide NGT-A001 according to the present invention against Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0057] Figure 4 is a diagram showing the results of confirming the antibiotic combination effect of the antimicrobial polypeptide NGT-A001 according to the present invention against multidrug-resistant bacteria.

[0058] Figure 5a is a diagram showing the results of an analysis using a scanning electron microscope of the effect of the antimicrobial polypeptide NGT-A001 according to the present invention on the cell membrane of a microorganism.

[0059] Figure 5b is a diagram showing the results of a gDNA aggregation test of the antimicrobial polypeptide NGT-A001 according to the present invention.

[0060] Figure 6a is a diagram showing the results of measuring the hemolytic activity of the antimicrobial polypeptide NGT-A001 according to the present invention.

[0061] Figure 6b is a diagram showing the results of confirming the cytotoxicity of the antimicrobial polypeptide NGT-A001 according to the present invention against a skin keratinocyte cell line.

[0062] Figure 7 is a diagram showing the results of confirming the cytotoxicity of an ointment containing the antimicrobial polypeptide NGT-A001 according to the present invention.

[0063] Hereinafter, the present invention will be described in detail.

[0064] According to an aspect of the present invention, the present invention provides an antimicrobial polypeptide comprising a peptide represented by the amino acid sequence of SEQ ID NO: 2 or 7.

[0065] In the present invention, "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds. The peptide may be prepared using chemical synthesis methods known in the art, preferably using solid-phase synthesis techniques, but is not limited thereto.

[0066] In a specific embodiment of the present invention, the antimicrobial polypeptide may be represented by the amino acid sequence of SEQ ID NO: 1. The antimicrobial polypeptide represented by the amino acid sequence of SEQ ID NO: 1 is named 'NGT-A001'. The NGT-A001 may be encoded by the nucleic acid sequence of SEQ ID NO: 8, but the scope of the present invention is not limited thereto.

[0067] Preferably, the antimicrobial polypeptide may be composed of an amino acid sequence of SEQ ID NO: 2 or 7.

[0068] The antimicrobial polypeptide of the present invention is an antimicrobial polypeptide consisting of SEQ ID NO: 2, and is named 'NGT-A002'. NGT-A002 is a sequence discovered by discovering an antimicrobial polypeptide containing it and then searching for a region exhibiting antimicrobial activity. NGT-A002 may be encoded by the nucleic acid sequence of SEQ ID NO: 9, but the scope of the present invention is not limited thereto.

[0069] The antimicrobial polypeptide of the present invention is designated as 'NGT-A007', which is an antimicrobial polypeptide having sequence number 7. NGT-A007 is derived from the antimicrobial polypeptide NGT-A002, which includes it, and contains various amino acid mutations. NGT-A007 may be encoded by the nucleic acid sequence of sequence number 14, but the scope of the present invention is not limited thereto.

[0070] According to another aspect of the present invention, the present invention provides an antimicrobial polypeptide represented by the following structural formula:

[0071] [constitutional formula]

[0072] N'- WG X1GK X2KY X3PQ (X4) n GKQKYLKKARKYHRK -C' (SEQ ID NO: 15)

[0073] In the above formula,

[0074] The above N' is the N-terminus of the antimicrobial polypeptide,

[0075] The above C' is the C-terminus of the antimicrobial polypeptide,

[0076] wherein X1 is KYY, RVV or RII,

[0077] The above X2 is IKKYIQ, ILKYLY, VKQYVQ or IKQIIQ,

[0078] The above X3 is K or I,

[0079] The above n is 0 or 1,

[0080] If the above n is 1, X4 is S.

[0081] In a specific embodiment of the present invention, the antimicrobial polypeptide may be represented by one or more sequences selected from the group consisting of SEQ ID NOs: 3 to 6.

[0082] When X1 is KYY, X2 is IKKYIQ, X3 is K, and X4 is S (n=1), the corresponding antimicrobial polypeptide is represented by the amino acid sequence of SEQ ID NO: 3 and is named NGT-A003. The NGT-A003 may be encoded by the nucleic acid sequence of SEQ ID NO: 10, but the scope of the present invention is not limited thereto.

[0083] When X1 is KYY, X2 is ILKYLY, X3 is I, and X4 is not included (n=0), the antimicrobial polypeptide is represented by the amino acid sequence of SEQ ID NO: 4 and is named NGT-A004. The NGT-A004 may be encoded by the nucleic acid sequence of SEQ ID NO: 11, but the scope of the present invention is not limited thereto.

[0084] When X1 is RVV, X2 is VKQYVQ, X3 is K, and X4 is not included (n=0), the antimicrobial polypeptide is represented by the amino acid sequence of SEQ ID NO: 5 and is named NGT-A005. The NGT-A005 may be encoded by the nucleic acid sequence of SEQ ID NO: 12, but the scope of the present invention is not limited thereto.

[0085] When X1 is RII, X2 is IKQIIQ, X3 is K, and X4 is not included (n=0), the antimicrobial polypeptide is represented by the amino acid sequence of SEQ ID NO: 6 and is named NGT-A006. The NGT-A006 may be encoded by the nucleic acid sequence of SEQ ID NO: 13, but the scope of the present invention is not limited thereto.

[0086] In a specific embodiment of the present invention, the antimicrobial polypeptide of the present invention may be composed of an amino acid sequence represented by SEQ ID NO: 2, and includes a functional equivalent of the antimicrobial polypeptide.

[0087] The above "functional equivalent" refers to a peptide that has at least 80%, preferably 90%, and more preferably 95% sequence homology (i.e., identity) with the amino acid sequence represented by SEQ ID NO: 2 as a result of addition, substitution, or deletion of amino acids, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology, and exhibits substantially the same physiological activity as the antibacterial polypeptide. In addition, the E antibacterial polypeptide of the present invention includes not only a protein having its native amino acid sequence, but also an amino acid sequence variant thereof, within the scope of the present invention. An antimicrobial polypeptide variant refers to a protein having a sequence that differs from the native amino acid sequence of the antimicrobial polypeptide by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in the art. The antimicrobial polypeptide or variant thereof can be extracted from nature, synthesized (Merrifleld, J. Amer. Chem. Soc. 85:2149-2156, 1963), or produced by genetic recombination methods based on a DNA sequence (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd ed., 1989).

[0088] Furthermore, the sequence homology can be determined by standard methods commonly used to compare similar portions of amino acid sequences that constitute peptides. Computer programs such as BLAST or FASTA align two or more proteins so that the amino acids constituting each protein optimally match (along the full-length sequence of one or both sequences or along predicted portions of one or both sequences). These programs provide a default opening penalty and a default gap penalty, and provide a scoring matrix such as PAM250 (a standard scoring matrix; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3, 1978) that can be used in conjunction with the computer program. For example, sequence homology expressed as a percentage can be calculated as follows: multiply the total number of identical matches by 100 and then divide by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0089] As used above, “substantially homogeneous physiological activity” means antibacterial activity.

[0090] Additionally, the scope of the above functional equivalents includes derivatives in which the basic framework and antibacterial activity of the antibacterial polypeptide of the present invention are maintained while some of the chemical structures of the constituent amino acids are modified. For example, this includes structural modifications to alter the stability, storability, volatility, or solubility of the protein.

[0091] In a specific example of the present invention, it is preferable that the antimicrobial polypeptide exhibits antimicrobial activity against at least one selected from the group consisting of gram-negative bacteria, gram-positive bacteria, antibiotic-resistant bacteria, and fungi.

[0092] Preferably, the Gram-negative bacteria may be at least one selected from the group consisting of Scherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes, Klebsiella pneuminuse, Neisseria gonorrhoeae, Yersinia pestis, Haemophilus influenzae, Fusobacterium nucleatum, Aeromonas hydrophila, Citrobacter freundii, Proteus mirabilis, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Tenacibaculum maritimum, Edwardsiella tarda, Legionella pneumophila, Leptospira interrogans, and Bordetella pertussis. In addition, the above Gram-positive bacteria may be at least one selected from the group consisting of Enterococcus hirae, Streptococcus mutans, Cutibacterium acnes, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus pseudintermedius, Bacillus anthracis, Bacillus cereus, Mycobacterium chelonae, Mycobacterium marinum, Clostridium perfringens, and Listeria monocytogenes.In addition, the antibiotic-resistant bacteria may be at least one selected from the group consisting of antibiotic-resistant Staphylococcus aureus, antibiotic-resistant Enterococci, antibiotic-resistant Escherichia coli, antibiotic-resistant Klebsiella pneumoniae, antibiotic-resistant Acinetobacter baumannii, antibiotic-resistant Escherichia coli, antibiotic-resistant Enterobacter cloacae and antibiotic-resistant Enterobacteriaceae, and more preferably, the antibiotic-resistant bacteria are Methicillin-resistant Staphylococcus aureus, Vancomycin-Resistant Enterococci, Vancomycin-Resistant Escherichia coli, Ampicillin-resistant recombinant Escherichia coli, Tetracycline-resistant Escherichia coli, Carbapenem-resistant Escherichia coli, Carbapenem-resistant Enterobacter cloacae, Carbapenem-resistant Klebsiella pneumoniae, Carbapenem-resistant It may be one or more species selected from the group consisting of Acinetobacter baumannii and Carbapenem-resistantEnterobacteriaceae.The fungi include Malassezia furfur, Malassezia pachydermatis, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, Candida albicans, Aspergillus fumigatus, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Geotrichum candidum, Mucor circinelloides, Mucor It may be one or more species selected from the group consisting of racemosus, Mucor indicus, Sporothrix schenckii, Cryptococcus neoformans, Blastomyces dermatitidis, Saprolegnia parasitica and Saprolegnia diclina.

[0093] The antimicrobial polypeptide of the present invention exhibits excellent antimicrobial activity against various pathogenic microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and fungi, and was confirmed to have a low minimum inhibitory concentration, particularly against multidrug-resistant bacteria (K. pneumoniae, E. faecium). This indicates that the antimicrobial polypeptide not only exhibits dual-mechanism antimicrobial activity through cell membrane disruption and gDNA aggregation, but also contributes to the restoration of antibiotic susceptibility through a synergistic effect when used in combination with antibiotics. Therefore, the antimicrobial polypeptide of the present invention can be usefully utilized in various fields, such as pharmaceutical compositions, food compositions, and cosmetic compositions.

[0094]

[0095] According to another aspect of the present invention, the present invention provides a composition comprising the antimicrobial polypeptide. Preferably, the composition is an antimicrobial composition, an antimicrobial pharmaceutical composition, an antimicrobial food composition, an antimicrobial cosmetic composition, or an antimicrobial feed composition.

[0096] When the composition of the present invention is an antibacterial composition, the antibacterial composition of the present invention means a composition that exhibits a growth inhibition or killing effect on microorganisms such as bacteria, fungi, and viruses, and specifically includes a preparation or formulation containing an antibacterial polypeptide having antibacterial activity as an active ingredient.

[0097] The above composition may be an external preparation such as an ointment, cream, gel, lotion, spray, patch, or liquid depending on the form of the formulation, and, if necessary, may also be manufactured into a pharmaceutical composition such as an oral preparation or injection. In addition, the present composition may be applied in the form of a medicine, a quasi-drug, a functional cosmetic, or a hygiene product.

[0098] The antimicrobial polypeptides described herein can be formulated alone or in combination with other antibiotics, bactericides, preservatives, skin soothing agents, thickeners, emulsifiers, fat-soluble agents, etc., and the content, concentration, and formulation within the composition can be variously adjusted depending on the intended use and application site.

[0099] When the composition of the present invention is used as a pharmaceutical composition, the antibacterial pharmaceutical composition may be a composition for preventing or treating a bacterial infectious disease. Examples of the bacterial infectious disease include skin or soft tissue infections (e.g., impetigo, cellulitis, folliculitis, surgical site infection), respiratory infections (e.g., pneumonia, bronchitis, pharyngitis, tonsillitis), urogenital infections (e.g., cystitis, urinary tract infection, pyelonephritis, prostatitis), digestive infections (e.g., bacterial enteritis, food poisoning), otolaryngological infections (e.g., otitis media, sinusitis), bone and joint infections (e.g., osteomyelitis, arthritis), and systemic infections (e.g., sepsis, meningitis). It can also be used to treat infections caused by resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), carbapenem-resistant Enterobacteriaceae (CRE), and multidrug-resistant Klebsiella pneumoniae (MDR-KP).

[0100] The pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0101] The composition of the present invention may contain one or more known effective ingredients having an antibacterial effect together with an antibacterial polypeptide.

[0102] The composition of the present invention may further include a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0103] The composition of the present invention can be administered in various oral or parenteral dosage forms during actual clinical administration. When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. It is preferable to use suitable formulations known in the art as disclosed in the literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA).

[0104] The above solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, the above liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

[0105] The above-mentioned parenteral administration formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0106] Pharmaceutically acceptable salts of the above compounds include salts of acidic or basic groups that may be present in the compounds, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxyl groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and the like, and can be prepared by methods for preparing salts known in the art.

[0107] The dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary knowledge in the relevant technical field can easily determine and prescribe an effective dosage for the desired treatment.

[0108] The dosage of the pharmaceutical composition of the present invention is preferably administered at a concentration of, for example, 0.05 to 5 mg / kg, more preferably 0.1 to 0.4 mg / kg, even more preferably 0.2 to 0.35 mg / kg, and even more preferably 0.25 mg / kg, but the dosage does not limit the scope of the present invention in any way.

[0109] The route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in their method, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.

[0110] The pharmaceutical composition may be administered orally or parenterally. Parenteral administration methods include, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

[0111] The pharmaceutical composition of the present invention can be used alone for antibacterial activity or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0112] The antimicrobial polypeptide of the present invention can be used in combination with existing antimicrobial agents. For example, it can be administered in combination with beta-lactam antibiotics (e.g., penicillin, ampicillin, cephalosporin), aminoglycoside antibiotics (e.g., gentamicin, amikacin), quinolone antibiotics (e.g., ciprofloxacin, levofloxacin), tetracycline antibiotics (e.g., doxycycline), macrolide antibiotics (e.g., erythromycin, clarithromycin), glycopeptide antibiotics (e.g., vancomycin), polymyxin antibiotics (e.g., colistin), etc. Through such combination, an increase in antimicrobial effect or an effect of overcoming antibiotic resistance can be expected.

[0113] In particular, in order to compensate for the low effectiveness of existing antibiotics administered alone against resistant strains, a synergistic effect or sensitivity recovery effect can be induced by co-administering the antimicrobial polypeptide of the present invention.

[0114] When the composition of the present invention is an antibacterial food composition, the food composition of the present invention means a food having an antibacterial effect, and must be harmless to the human body when taken for a long period of time.

[0115] There are no specific restrictions on the types of foods mentioned above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, including all health foods in the conventional sense.

[0116] In an embodiment of the present invention, the food composition of the present invention may be a food additive. The food additive may be used by adding the antimicrobial polypeptide directly or in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of active ingredients mixed may be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment).

[0117] In an embodiment of the present invention, the food composition of the present invention may be a health beverage composition. In addition to the antimicrobial polypeptide, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, similar to conventional beverages. The natural carbohydrates described above may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

[0118] In addition to the above, the composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may include fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0119] When the composition of the present invention is an antibacterial cosmetic composition, it may include conventional cosmetic base ingredients in addition to the antibacterial polypeptide, for example, conventional ingredients such as stabilizers, solubilizers, pigments, and fragrances, and carriers. In addition, it may be manufactured into any formulation conventionally manufactured in the cosmetic industry, and, without particular limitation, may have the formulation of, for example, a toner, essence, lotion, paste, surfactant-containing cleanser, cream, pack, gel, ointment, powder, patch, or spray.

[0120] When the formulation of the above cosmetic composition is a paste, cream or gel, at least one selected from the group consisting of animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc and zinc oxide may be used as a carrier component, and when the formulation is a toner or essence, a solvent, a solubilizer or an emulsifier may be used as a carrier component, and for example, at least one selected from the group consisting of water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil, glycerol aliphatic ester, polyethylene glycol, and fatty acid ester of sorbitan may be used.

[0121] In addition, when the formulation of the cosmetic composition is a powder or spray, at least one selected from the group consisting of lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powder may be used as a carrier component, and particularly, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included. In addition, when the formulation of the cosmetic composition is a surfactant-containing cleansing, at least one selected from the group consisting of aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, and ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0122] At this time, it is preferable that the antibacterial polypeptide in the cosmetic be added in an amount of 0.01 to 50% by weight of the total weight, preferably 0.01 to 30% by weight.

[0123] In addition, the present invention provides a composition for feed addition comprising the antimicrobial polypeptide.

[0124] Antibiotics used in feed additives in livestock and fisheries are used for disease prevention. However, administering antibiotics for preventive purposes is problematic because it increases the risk of developing resistant bacteria and can transmit antibiotic residues from livestock to humans. If antibiotics are absorbed into the human body through meat, they can induce antibiotic resistance and contribute to the spread of disease. Furthermore, the wide variety of antibiotics mixed into feed increases the risk of developing multidrug-resistant bacteria. Therefore, the antimicrobial polypeptide of the present invention can be utilized as a novel feed additive antibiotic that is more environmentally friendly and solves the problems arising from the use of existing antibiotics.

[0125] In addition, the present invention can provide an antibacterial feed composition, and the feed of the present invention can be manufactured by separately preparing an antibacterial polypeptide in the form of a feed additive and mixing it into the feed, or by directly adding it during the manufacture of the feed. The antibacterial polypeptide in the feed of the present invention can be in a liquid or dry state, and is preferably in the form of a dried powder. The drying method can be ventilation drying, natural drying, spray drying, and freeze drying, but is not limited thereto. The antibacterial polypeptide of the present invention can be mixed in a powder form at a component ratio of 0.05 to 10 wt%, preferably 0.1 to 2 wt%, of the feed weight. In addition, the feed may further include conventional additives that can increase the preservative property of the feed in addition to the antibacterial polypeptide of the present invention.

[0126] The feed additive composition of the present invention may further include other non-pathogenic microorganisms. Microorganisms that may be added include Bacillus subtilis capable of producing protein-decomposing enzymes, lipolytic enzymes, and sugar-converting enzymes; Lactobacillus sp. having physiological activity and organic matter-decomposing ability under anaerobic conditions such as the stomach of a cow; Aspergillus oryzae and Saccharomyces cerevisiae, which show effects of increasing animal weight, increasing milk production, and increasing the digestibility and absorption rate of feed.

[0127] Feeds containing the antimicrobial polypeptide of the present invention include, but are not limited to, plant-based feeds such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, starches, grain by-products, etc., and animal-based feeds such as proteins, inorganic materials, oils, minerals, single-cell proteins, zooplankton, and leftover food.

[0128] The feed additive composition of the present invention may include a binder, an emulsifier, a preservative, etc. added to prevent quality deterioration, and may include an amino acid agent, a vitamin agent, an enzyme agent, a probiotic agent, a flavoring agent, a non-protein nitrogen compound, a silicate agent, a buffer agent, a coloring agent, an extractant, an oligosaccharide, etc. added to the feed to increase utility, and may additionally include a feed mixer, etc.

[0129]

[0130] According to another aspect of the present invention, there is provided a method for treating a microbial infection, comprising administering an antimicrobial polypeptide to a subject in need thereof. The antimicrobial polypeptide may be (i) an antimicrobial polypeptide comprising a peptide represented by the amino acid sequence of SEQ ID NO: 2 or 7; or (ii) an antimicrobial polypeptide represented by the following structural formula:

[0131] [constitutional formula]

[0132] N'- WG X1GK X2KY X3PQ (X4) n GKQKYLKKARKYHRK -C' (SEQ ID NO: 15)

[0133] In the above formula,

[0134] The above N' is the N-terminus of the antimicrobial polypeptide,

[0135] The above C' is the C-terminus of the antimicrobial polypeptide,

[0136] wherein X1 is KYY, RVV or RII,

[0137] The above X2 is IKKYIQ, ILKYLY, VKQYVQ or IKQIIQ,

[0138] The above X3 is K or I,

[0139] The above n is 0 or 1,

[0140] If the above n is 1, X4 is S.

[0141] In a specific embodiment of the present invention, the subject may be, but is not limited to, a subject expected to develop a microbial infection; a subject that has developed the disease; or a subject that has been judged to have been cured.

[0142] In a specific embodiment of the present invention, the microbial infection is preferably a disease caused by a microbial infection. Specifically, the disease includes pneumonia, urinary tract infection, skin and soft tissue infection, fungal infection, or various other infectious diseases caused by pathogenic microorganisms. Furthermore, the disease includes diseases caused by multidrug-resistant bacteria, including MRSA, VRE, and CRE.

[0143]

[0144] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0145] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0146]

[0147] Example 1. Preparation of recombinant antimicrobial polypeptides

[0148] 1-1. Gene synthesis and expression vector cloning for the production of recombinant antimicrobial polypeptide NGT-A001.

[0149] In this example, a recombinant polypeptide NGT-A001 (SEQ ID NO: 1) was designed.

[0150] Based on the above NGT-A001, the core sequence of antibacterial activity, NGT-A002 (SEQ ID NO: 2), was derived through 3D modeling and helical wheel prediction.

[0151] To improve activity, NGT-A002 was modified, and as a result, NGT-A003 to NGT-A007 were designed.

[0152]

[0153] The design was performed by adding NdeI-XhoI restriction enzyme sequences to the 5' and 3' ends, respectively, and the above genes were cloned into the pET-22b(+) vector treated with NdeI and XhoI restriction enzymes. The completed vectors were named pET-NGT-A001, pET-NGT-A002, pET-NGT-A003, pET-NGT-A004, pET-NGT-A005, pET-NGT-A006, and pET-NGT-A007. The completed vectors were designated as 'pET-NGT-A001 ~ pET-NGT-A007', and their vector maps are shown in Figures 1a and 1b.

[0154]

[0155] In addition, the characteristics of the produced antibacterial polypeptides NGT-A001 to NGT-A007 are shown in Table 1.

[0156] Peptide length (aa)Molecular weight (da)Hydrophobicity (%)Net chargeNGT-A001 (SEQ ID NO: 1)19622,666.8827.41+39NGT-A002 (SEQ ID NO: 2)334,361.2311.76+14NGT-A003 (SEQ ID NO: 3)344,452.3820.0+13NGT-A004 (SEQ ID NO: 4)334,370.3226.47+11NGT-A005 (SEQ ID NO: 5)334,237.1326.47+12NGT-A006 (SEQ ID NO: 6)334,243.2229.41+12NGT-A007 (SEQ ID NO: 7)354,346.3447.22+7

[0157]

[0158] 1-2. Strain cultivation

[0159] For vector construction in Figure 1, Escherichia coli DH5a strain was used, and for antimicrobial polypeptide expression, Escherichia coli BL21 (DE3) strain was used. E. coli was cultured in TB (terrific broth) medium at 37°C and 200 rpm, and the culture medium contained 100 ug / mL of ampicillin. For protein expression, IPTG (isopropyl-β-D-thiogalactopyranoside) was added, and the temperature was incubated at 20–39°C as needed.

[0160]

[0161] 1-3. Protein expression and cell fractionation

[0162] The recombinant vector produced in Example 1-1 was transformed into E. coli BL21 (DE3). The transformed E. coli was cultured at 37, 28, and 20°C at 200 rpm. The cell concentration was OD 600 When it reached 0.6 to 0.8, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added and cultured for 12 hours.

[0163] OD based on final volume of 10 mL after culture 600 The solution was diluted to an M of 1.0 and centrifuged at 4°C and 4,500×g for 10 minutes to harvest the cells. The harvested cells were resuspended using lysis buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0). The resuspended cells were disrupted by ultrasonication in ice-water. The lysate was centrifuged at 4°C and 10,000×g for 10 minutes to obtain the supernatant and pellet. The obtained supernatant was named the soluble fraction (S), and the pellet was resuspended in the same volume of lysis buffer and named the insoluble fraction (IS).

[0164]

[0165] 1-4. Purification of recombinant antimicrobial polypeptides

[0166] Ni-NTA resin (QIAGEN, Germany) was added to the soluble fraction obtained from the above soluble fraction (S) and reacted. In addition, after washing with wash buffer (50 mM sodium phosphate, 300 mM NaCl, 60 mM imidazole, pH 8.0) to minimize nonspecific binding, the protein was purified using elution buffer (50 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, pH 8.0). To confirm each purified product (P), SDS-PAGE was performed. For the insoluble fraction (IS) corresponding to NGT-A001, the protein was purified through an SP column after acetic acid extraction.

[0167] In addition, a large amount of antimicrobial polypeptide powder was produced by purifying in bulk using FPLC (fast protein liquid chromatography) under the conditions described above, dialyzing (0.1% acetic acid), and then freeze-drying.

[0168]

[0169] The recombinant antimicrobial polypeptides NGT-A001 to NGT-A007 obtained in this example are represented by the amino acid sequences of SEQ ID NOs: 1 to 7, respectively. The nucleic acid sequences and amino acid sequences of the recombinant polypeptides NGT-A001 to NGT-A007 are shown in Table 2.

[0170]

[0171]

[0172] Example 2. Expression analysis of recombinant antimicrobial polypeptides

[0173] The cell fractions of Example 1 were separated using SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The separated proteins were then stained with Coomassie blue to analyze the expression patterns of antimicrobial polypeptides NGT-A001 to NGT-A007. The expression patterns of the recombinant antimicrobial polypeptide NGT-A001 of Example 1 and the results of purification in E. coli BL21 (DE3) strain are shown in Fig. 2a, and the expression patterns by temperature are shown in Fig. 2b. In addition, the expression patterns of antimicrobial polypeptides NGT-A002 to NGT-A007 are shown in Fig. 2c.

[0174] As shown in Figures 2a and b, when NGT-A001 (approximately 22.5 kDa) was expressed in E. coli BL21 (DE3) (1 mM IPTG, 37°C), it was confirmed that NGT-A001 was expressed as an insoluble fraction (IS). In addition, it was confirmed that as the expression temperature decreased, the soluble fraction (S) increased and the insoluble fraction (IS) decreased along with the total expression, and the final OD decreased rapidly.

[0175] As shown in Fig. 2c, expression of antimicrobial polypeptides NGT-A002 to NGT-A007 in E. coli was confirmed, and expression of NGT-A002, NGT-A003, NGT-A004, and NGT-A006 was confirmed. In addition, NGT-A002, NGT-A003, NGT-A004, and NGT-A006 were confirmed to be mainly water-soluble fractions (S).

[0176] The above results indicate that the recombinant antimicrobial polypeptide NGT-A001 of Example 1 does not significantly affect the growth of E. coli because it is expressed as an insoluble fraction (IS), but when expression is induced in a low-temperature expression environment with high expression intensity, the soluble fraction (S) can affect cell growth, indicating that it has antimicrobial activity and that it can be obtained in large quantities because the insoluble fraction is well expressed. In addition, the soluble fraction (S) suggests that it is an antimicrobially active form, and the insoluble fraction (IS) suggests that it is an antimicrobially inactive form.

[0177] This suggests that even small amounts of unexpressed NGT-A005 and NGT-A007, when expressed in E. coliBL21(DE3) under high-intensity IPTG-induced conditions, significantly impact vital activity. This suggests that NGT-A005 and NGT-A007 may exhibit even stronger antibacterial activity, and could be usefully synthesized using a peptide synthesizer.

[0178]

[0179] Example 3. Evaluation of antibacterial activity of recombinant antibacterial polypeptides

[0180] To evaluate antibacterial activity, a topical application standard (ASTM-E-2315) was prepared using the recombinant antibacterial polypeptide NGT-A001 of Example 1. The prepared topical application formulation was requested to the Korea Testing & Research Institute for antibacterial activity evaluation. Specifically, Gram-positive bacteria (Methicillin-resistant S. aureus (MRSA) NCCP 14752, E. hirae ATCC 10541, S. mutans ATCC 25175), Gram-negative bacteria (E. coli ATCC 8739, K. pneumoniae ATCC 4352, P. aeruginosa ATCC 15442, S. typhimurium ATCC 14028) and fungi (M. furfur ATCC 14521, C. albicans ATCC 10231) were used for the antibacterial activity evaluation. After pre-cultivating the bacteria on a medium suitable for each strain, the bacterial concentration was 1.00 X 10 8 CFU / mL to 10.0 X 10 8 It was diluted to CFU / mL. 0.05 mL of the test bacterial solution was inoculated into 5 mL of 44.4 uM NGT-A001 dissolved in PBS, mixed, and incubated at room temperature for 5 minutes. Then, it was mixed with agar medium cooled to 50°C and solidified. Distilled water was added instead of the sample for the positive control, and sterile saline solution was added for the negative control. The solidified medium was incubated at 35°C for 24–48 hours. The results were observed using a colony counter, and the bacterial reduction rate (%) for each antibacterial activity evaluation was calculated by the following formula.

[0181]

[0182] Fungal reduction rate (%) = {(C t -S t ) / C t}X100

[0183]

[0184] The results of the antibacterial activity evaluation against Gram-positive bacteria, Gram-negative bacteria, and fungi are shown in Figure 3.

[0185] As shown in Fig. 3, NGT-A001 has antibacterial activity against gram-negative bacteria, gram-positive bacteria, and fungi at physiological salt concentrations, and is further effective against resistant bacteria such as MRSA.

[0186]

[0187] Example 4. Measurement of minimum inhibitory concentration (MIC) of recombinant antimicrobial polypeptide

[0188] 4-1. Selection of the optimal solvent for antimicrobial activity of antimicrobial polypeptides

[0189] It is known that the antibacterial activity of antibacterial polypeptides is basically due to the amphipathic α-helix structure, which consists of positively charged amino acids and hydrophobic amino acids as essential elements, and this structure is known to be mainly affected by salt concentration, temperature, pH, etc.

[0190] In this example, considering the amino acid and structure of NGT-A001, the MIC was measured using E. coli DH5α as the target strain to select the solvent conditions that exhibit the optimal antibacterial activity. Due to the characteristics of the sample, the medium was M9 medium, and E. coli DH5α was pre-cultured at 37°C for 5 to 6 hours, and then 0.5 McFarland (1X10 8A strain stock solution was prepared by diluting the strain stock solution to 10 CFU / mL. The strain stock solution was diluted 1 / 100 in M9 medium, and then mixed 1:1 with the NGT-A001 solution corresponding to each solvent and concentration, and incubated at 35°C for 18 to 20 hours. After that, the absorbance of each well was measured at 600 nm using an Agilent microplate reader, and the lowest concentration that completely inhibited the growth of the microorganism was determined as the minimum inhibitory concentration. The minimum inhibitory concentration was determined as the average value obtained from three independent experiments. The minimum inhibitory concentration of the antimicrobial polypeptide NGT-A001 according to the missolvent condition is shown in Table 3.

[0191] SolventMIC(uM)NGT-A001Distilled water>20Normal Saline>200.8% Ascorbic acid>1560mM KCl1510-60mM NaCl7.7PBS(137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4)6.650mM NaCl, 30mM KCl4.4

[0192]

[0193] As shown in Table 3, the MIC values ​​of the recombinant antimicrobial polypeptide NGT-A001 tended to vary depending on salt concentration. In particular, the antimicrobial activity of the recombinant antimicrobial polypeptide NGT-A001 was confirmed to increase in the presence of 50 mM NaCl and 30 mM KCl.

[0194]

[0195] 4-2. Measurement of antibacterial activity against multidrug-resistant bacteria

[0196] In this example, the MIC of NGT-A001 against multidrug-resistant bacteria K. pneumoniae NCCP 15782 and E. faecium NCCP NMS1867 was confirmed using the solvent conditions selected in Example 4-1. The MIC confirmation was performed in the same manner as in Example 4-1. The results of confirming the MIC of NGT-A001 against multidrug-resistant bacteria are shown in Table 4.

[0197] Minimum inhibitory concentration (MIC test) Sample K. pneumoniae NCCP 15782 E. faecium NCCP NMS1867 NGT-A001 0.55 uM 2.22 uM

[0198] As shown in Table 4, the minimum inhibitory concentration (MIC) of NGT-A001 against multidrug-resistant K. pneumoniae NCCP 15782 was confirmed to be 0.55 uM. In addition, the minimum inhibitory concentration (MIC) of NGT-A001 against E. fasecium NCCP NMS 1867 was confirmed to be 2.22 uM.

[0199]

[0200] In addition, the MICs of antimicrobial polypeptides NGT-A002, NGT-A003, NGT-A004, and NGT-A006 against multidrug-resistant K. pneumoniae NCCP 15782 were confirmed, and the results are shown in Table 5.

[0201] Minimum inhibitory concentration (MIC test) Sample K. pneumoniae NCCP 15782 NGT-A0026 uMNGT-A003 >22.4 uMNGT-A004 2.3 uMNGT-A006 23 uM

[0202] As shown in Table 4, the selected strains NGT-A002, NGT-A003, NGT-A004, and NGT-A006 were all confirmed to exhibit excellent antibacterial activity against the multidrug-resistant strain K. pneumoniae NCCP 15782. In particular, NGT-A004 had a very low MIC against the multidrug-resistant strain, indicating excellent antibacterial activity.

[0203]

[0204] 4-3. Measurement of the effect of combined antibiotic use against multidrug-resistant bacteria

[0205] In this example, we examined whether synergistic effects or recovery of antibiotic susceptibility were observed when NGT-A001 was used in combination with antibiotics to which the strain was resistant. The experiment was conducted in the same manner as Example 3. In this experiment, K. pneumoniae NCCP 15782, which is resistant to 13 antibiotics, was used, and 4 antibiotics to which the strain was resistant (ampicillin 100 ug / mL, kanamycin 100 ug / mL, chloramphenicol 100 ug / mL, and vancomycin 20 ug / mL) were used. The results of examining the combined antibiotic effect against multidrug-resistant bacteria are shown in Figure 4.

[0206] As shown in Fig. 4, the group treated with antibiotics alone that were resistant to the strain showed a bacterial inhibition rate of 2 to 10%. In addition, the group treated with 4.4 μM NGT-A001 showed a bacterial inhibition rate of 48.87%, and the group treated with 44 μM NGT-A001 showed a bacterial inhibition rate of 92.76%. The group treated with 4.4 μM NGT-A001 and resistant antibiotics (ampicillin, chloramphenicol, or vancomycin) in combination showed an improvement in bacterial inhibition rate of approximately 10 to 30% compared to the control group treated with 4.4 μM NGT-A001 alone.

[0207] Therefore, the NGT-A001 of the present invention not only restores sensitivity to resistant antibiotics, but also is effective in treating multidrug-resistant infections when used in combination with other antibiotics.

[0208]

[0209] Example 5. Dual antimicrobial activity of antimicrobial polypeptides against microorganisms

[0210] 5-1. Investigation of antibacterial activity using microbial cell membrane disruption

[0211] This example analyzed the effect of the above-mentioned antimicrobial polypeptide on the cell membrane of microorganisms. Specifically, after treatment with the antimicrobial polypeptide NGT-A001 for 1 minute, the antimicrobial effect was analyzed using a scanning electron microscope (SEM). For SEM analysis, E. coli DH5α was pre-cultured using TB medium and washed once with PBS. Subsequently, NGT-A001 was treated at 44.4 μM for 1 minute, and primary fixation was performed using a 2.5% formaldehyde, 2.5% glutaraldehyde in PBS solution at room temperature for 4 hours. After primary fixation, the cells were washed twice with PBS and then secondary fixation was performed with a 1% osmium tetroxide solution (room temperature, 2 hours). After secondary fixation, the sample was washed twice with PBS, dehydrated with 30, 50, 70, 95, and 100% ethanol for 10 minutes each step, and dried overnight. Before SEM analysis, carbon tape was attached to the sample holder, coated with Platinum for 60 seconds, and then analyzed by SEM. The results of the SEM analysis are shown in Fig. 5a.

[0212] As shown in Figure 5a, the control group was morphologically normal, whereas the NGT-A001-treated group showed abnormal morphology due to disruption of the cell membrane.

[0213] The above results suggest that NGT-A001 of the present invention inhibits normal life activities of microorganisms by inducing electrostatic instability through penetration or intercalation between cell membrane components.

[0214]

[0215] 5-2. Confirmation of gDNA aggregation using the Thioflavin T assay (ThT)

[0216] To determine whether the antibacterial activity of the antibacterial polypeptide of the present invention exhibits additional activity in addition to cell membrane disruption, a gDNA aggregation test using Thioflavin T was performed.

[0217] Specifically, E. coli DH5α was pre-cultured in TB medium and then disrupted by ultrasonication. The resulting homogenate was treated with Recombinant DNase I and RNase H, incubated at 37°C for 1 hour, and then freeze-dried to prepare E. coli protein. In addition, E. coli culture was obtained by pre-cultivation using the same method as above, and E. coli DNA was extracted using the Genomic DNA prep kit from Enzynomics. Finally, 2.2 uM NGT-A001, 500 ug / ml LE. coli protein, 50 ng / mL gDNA, and 20 uM ThT were mixed and reacted at 37°C for 10 minutes. Fluorescence was measured at the excitation wavelength (450 nm) and emission wavelength (485 nm) corresponding to ThT, and the results are shown in Fig. 5b.

[0218] Additionally, antimicrobial polypeptides NGT-A003, NGT-A004, and NGT-A006 were also analyzed for fluorescence using the same method, and the results are shown in Table 6.

[0219] ThT aggregation assayFluorescent intensity(au)NGT-A00347,904NGT-A00432,720NGT-A00639,306

[0220] As shown in Fig. 5b and Table 5, NGT-A001, NGT-A003, NGT-A004, and NGT-A006 were confirmed to strongly interact with E. coligDNA and aggregate, respectively.

[0221] The above results imply that the rapid and potent antibacterial activity of the antibacterial polypeptide of the present invention is due to disruption of the microbial cell membrane and interaction with gDNA. In other words, the antibacterial polypeptide of the present invention exhibits microbial inhibition or death induction and resistance suppression activity through the above-described dual mechanisms, and thus can be utilized in the development of next-generation antibiotics.

[0222]

[0223] Example 6. Confirmation of biosafety of recombinant antimicrobial polypeptides

[0224] It is known that general antimicrobial peptides are accompanied by red blood cell hemolysis and cytotoxicity. Accordingly, in this example, the red blood cell hemolysis and cytotoxicity of the antimicrobial polypeptide NGT-A001 of the present invention were confirmed.

[0225]

[0226] 6-1. Measurement of antimicrobial polypeptide erythrocyte hemolytic activity (Hemolysis)

[0227] To confirm the hemolytic activity of erythrocytes, rat blood was collected by centrifugation at 1000 rpm at 4°C for 10 minutes, washed three times with PBS, and then mixed with 150 uL of 10% erythrocyte storage solution and 50 uL of various concentrations of NGT-A001 (2–500 uM) and incubated at 37°C for 60 minutes. Afterwards, the reaction solution was centrifuged, and 150 uL of the supernatant was dispensed into a 96-well plate, and the absorbance was measured (540 nm). The sample treated with only PBS was defined as 0%, and the absorbance of the sample treated with 1% Triton X-100 was defined as 100% hemolysis. The hemolytic activity (Hemolysis) of the antimicrobial polypeptide NGT-A001 was calculated using the following mathematical formula, and the results are shown in Fig. 6a.

[0228]

[0229] [Mathematical formula]

[0230] Erythrocyte hemolytic activity (%) = {(H p - H0) / H100 - H0}X100

[0231] ※ H p : Absorbance of samples treated with antimicrobial polypeptides

[0232] H0: Absorbance of sample treated with PBS only

[0233] H 100 : Absorbance of samples treated with 1% Triton X-100.

[0234]

[0235] As shown in Fig. 6a, the hemolytic activity of the NGT-A001 500 uM or less experimental group was 3 to 4% or less, which was much lower than that of the 1% Triton X-100 treatment group. In the field, it is generally known that a hemolytic reaction of 5% or less is biosafe, which means that the NGT-A001 of the present invention is biosafe.

[0236]

[0237] 6-2. Cytotoxicity of antimicrobial polypeptides against human skin keratinocyte cell lines (HaCaT cells)

[0238] To verify the toxicity of antimicrobial polypeptides to human cells, human skin keratinocyte cell line (HaCaT cells) was treated with antimicrobial polypeptides at each concentration, and then MTT assay was performed. HaCaT cells were cultured in a 24-well plate at 37°C in a medium containing 10% FBS (Fetal Bovine Serum) and 1% Penicillin-streptomycin in Dulbecco's Modified Eagle's Medium (DMEM) at 37°C in the presence of 5% CO2 at a density of 2 x 10 4The cells were cultured at a density of 10 cells / well for 24 hours, treated with antimicrobial polypeptides (0.3–11.1 μM), and cultured for 24 hours. 100 μL of MTT solution was added to each well and incubated at 37°C for 2 hours. After removing the culture medium, 500 μL of DMSO solution was added, shaken thoroughly, and measured at 570 nm and 630 nm. Each concentration condition was independently tested three times, and the average and standard deviation were calculated and used for analysis. Cell viability was calculated as follows.

[0239] Cell viability (%) = {(A s - A0) / (A 100 -A0)} X 100

[0240] ※ A s : Average absorbance of samples treated with antimicrobial polypeptides

[0241] A0: Average absorbance of DMSO

[0242] A 100 : Absorbance of the untreated group.

[0243] As shown in Figure 6b, NGT-A001 was confirmed to be non-toxic to HaCaT cells. Therefore, it can be seen that the antimicrobial polypeptide of the present invention can be developed as a relatively safe and effective treatment for multidrug-resistant infections.

[0244]

[0245] Example 7. Confirmation of antibacterial activity and cytotoxicity of ointment containing recombinant antibacterial polypeptide

[0246] In this example, antibacterial activity and cytotoxicity against HaCaT cells were confirmed by visual evaluation. Specifically, an ointment containing the antibacterial polypeptide NGT-A001 of the present invention was prepared and its antibacterial activity and cytotoxicity were confirmed. Specifically, an ointment was prepared by heating commercially available petrolatum for cosmetic use to 80°C and then mixing and dispersing the antibacterial polypeptide NGT-A001 at concentrations of 44 μM and 222.2 μM. The ointment containing 44 μM of the antibacterial polypeptide NGT-A001 was used for antibacterial activity testing, and the ointment containing 222.2 μM of the antibacterial polypeptide NGT-A001 was used for cytotoxicity testing. The antibacterial activity and cytotoxicity of the prepared ointment were visually evaluated. The control group was an ointment containing only PBS and petrolatum. The results of the antibacterial activity testing are shown in Table 7, and the results of the cytotoxicity testing are shown in Figure 7.

[0247] Test strain Test results (unit) Control (PBS) Control (PBS and Vaseline) Test E. coli DH5α CFU / mL 4.1 X 10 4 5.9 X 10 4 5 X 10 2 Bacterial inhibition rate (%)--98.7 compared to PBS--99.1 compared to Vaseline

[0248] As shown in Table 7, the ointment containing NGT-A001 44.4 uM was confirmed to have antibacterial activity that was up to 99.1% higher than that of the control group (PBS treatment group, Vaseline treatment group).

[0249] As shown in Fig. 7, the cytotoxicity of the ointment containing NGT-A001 222.2 uM was confirmed to be at a similar level (i.e., no cytotoxicity) to the control group (PBS treatment group, Vaseline treatment group).

[0250] Therefore, when NGT-A001 is mixed and dispersed in a fat-soluble composition such as Vaseline and manufactured into a cream or ointment formulation, it can be used to develop highly safe anti-infection treatment agents and cosmetics at higher concentrations while maintaining antibacterial activity.

[0251]

[0252] In summary, the present inventors have confirmed that the recombinant antimicrobial polypeptide NGT-A001 and its variants (NGT-A002 to NGT-A007) exhibit excellent antimicrobial activity against various pathogenic microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and fungi, and particularly have low minimum inhibitory concentrations (MICs) even against multidrug-resistant bacteria (K. pneumoniae, E. faecium). This means that the polypeptides not only exhibit antimicrobial activity through a dual mechanism of cell membrane disruption and gDNA aggregation, but also can contribute to the restoration of antibiotic sensitivity through a synergistic effect when used in combination with antibiotics. Therefore, the antimicrobial polypeptides of the present invention can be usefully utilized in various fields such as pharmaceutical compositions, food compositions, and cosmetic compositions.

[0253]

[0254] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antimicrobial polypeptide comprising a peptide represented by the amino acid sequence of sequence number 2 or 7.

2. In the first paragraph, the antimicrobial polypeptide is represented by the amino acid sequence of sequence number 1.

3. An antimicrobial polypeptide represented by the following structural formula: [constitutional formula] N'- WG X1GK X2KY X3PQ (X4) n GKQKYLKKARKYHRK -C' (SEQ ID NO: 15) In the above formula, The above N' is the N-terminus of the antimicrobial polypeptide, The above C' is the C-terminus of the antimicrobial polypeptide, wherein X1 is KYY, RVV or RII, The above X2 is IKKYIQ, ILKYLY, VKQYVQ or IKQIIQ, The above X3 is K or I, The above n is 0 or 1, If the above n is 1, X4 is S.

4. In the third paragraph, the antimicrobial polypeptide is represented by one or more sequences selected from the group consisting of sequence numbers 3 to 6.

5. An antimicrobial polypeptide according to any one of claims 1 to 4, wherein the antimicrobial polypeptide exhibits antimicrobial activity against at least one selected from the group consisting of gram-negative bacteria, gram-positive bacteria, antibiotic-resistant bacteria, and fungi.

6. In the fifth paragraph, the gram-negative bacteria are at least one selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes, Klebsiella pneuminuse, Neisseria gonorrhoeae, Yersinia pestis, Haemophilus influenzae, Fusobacterium nucleatum, Aeromonas hydrophila, Citrobacter freundii, Proteus mirabilis, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Tenacibaculum maritimum, Edwardsiella tarda, Legionella pneumophila, Leptospira interrogans, and Bordetella pertussis.

7. An antimicrobial polypeptide according to claim 5, wherein the Gram-positive bacteria is at least one selected from the group consisting of Enterococcus hirae, Streptococcus mutans, Cutibacterium acnes, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus pseudintermedius, Bacillus anthracis, Bacillus cereus, Mycobacterium chelonae, Mycobacterium marinum, Clostridium perfringens, and Listeria monocytogenes.

8. In paragraph 5, the antibiotic-resistant bacteria is at least one selected from the group consisting of antibiotic-resistant Staphylococcus aureus, antibiotic-resistant Enterococci, antibiotic-resistant Escherichia coli, antibiotic-resistant Klebsiella pneumoniae, antibiotic-resistant Acinetobacter baumannii, antibiotic-resistant Escherichia coli, antibiotic-resistant Enterobacter cloacae, and antibiotic-resistant Enterobacteriaceae.

9. An antimicrobial polypeptide according to claim 8, wherein the antibiotic-resistant bacteria are at least one selected from the group consisting of Methicillin-resistant Staphylococcus aureus, Vancomycin-resistant Enterococci, Vancomycin-resistant Escherichia coli, Ampicillin-resistant recombinant Escherichia coli, Tetracycline-resistant Escherichia coli, Carbapenem-resistant Escherichia coli, Carbapenem-resistant Enterobacter cloacae, Carbapenem-resistant Klebsiella pneumoniae, Carbapenem-resistant Acinetobacter baumannii, and Carbapenem-resistant Enterobacteriaceae.

10. An antimicrobial polypeptide according to claim 5, wherein the fungus is at least one selected from the group consisting of Malassezia furfur, Malassezia pachydermatis, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, Candida albicans, Aspergillus fumigatus, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Geotrichum candidum, Mucor circinelloides, Mucor racemosus, Mucor indicus, Sporothrix schenckii, Cryptococcus neoformans, Blastomyces dermatitidis, Saprolegnia parasitica, and Saprolegnia diclina.

11. An antibacterial composition comprising an antibacterial polypeptide according to any one of claims 1 to 4.

12. An antibacterial pharmaceutical composition comprising an antibacterial polypeptide according to any one of claims 1 to 4.

13. An antibacterial food composition comprising an antibacterial polypeptide according to any one of claims 1 to 4.

14. An antibacterial cosmetic composition comprising an antibacterial polypeptide according to any one of claims 1 to 4.

15. An antimicrobial feed composition comprising an antimicrobial polypeptide according to any one of claims 1 to 4.

16. A method for treating a microbial infection, comprising the step of administering to a subject in need thereof an antimicrobial polypeptide comprising a peptide represented by the amino acid sequence of SEQ ID NO: 2 or 7.

17. A method for treating a microbial infection, comprising administering to an individual in need thereof an antimicrobial polypeptide represented by the following structural formula: [constitutional formula] N'- WG X1GK X2KY X3PQ (X4) n GKQKYLKKARKYHRK -C' (SEQ ID NO: 15) In the above formula, The above N' is the N-terminus of the antimicrobial polypeptide, The above C' is the C-terminus of the antimicrobial polypeptide, wherein X1 is KYY, RVV or RII, The above X2 is IKKYIQ, ILKYLY, VKQYVQ or IKQIIQ, The above X3 is K or I, The above n is 0 or 1, If the above n is 1, X4 is S.

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