Peptide having antimicrobial activity against pathogens and antimicrobial peptide composition comprising the same
10150795 ยท 2018-12-11
Assignee
Inventors
Cpc classification
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
An antimicrobial peptide and an antimicrobial peptide composition comprising the same are provided. The antimicrobial peptide and the antimicrobial peptide composition have remarkably high antibacterial activity against gram-positive (+) and gram-negative () bacteria, compared to wild-type LPcin-I having an antimicrobial ability, which consists of a sequence of 23 amino acids. Also, the antimicrobial peptide can be useful in being easily synthesized and saving production costs since the antimicrobial peptide has a smaller number of amino acids, compared to the wild-type LPcin-I.
Claims
1. A peptide represented by the following sequence [General Formula I] having antimicrobial activity against pathogens: TABLE-US-00006 [GeneralFormulaI] [(N-terminus)-NKVKEWX.sup.1KX.sup.2LKX.sup.3X.sup.4F X.sup.5-(C-terminus)] wherein X.sup.1 is I or W, X.sup.2 is Y when X.sup.1 is I, and X.sup.2 is W when X.sup.1 is W; X.sup.3 is S or K; X.sup.4 is L or K; and X.sup.5 is S or K, wherein the sequence of the peptide is not NKVKEWIKYLKSLFS (SEQ ID NO: 1).
2. The peptide of claim 1, wherein the peptide is a peptide from which X.sup.3 X.sup.4 F X.sup.5 or F X.sup.5 in General Formula I is deleted.
3. The peptide of claim 1, wherein the peptide consists of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 10.
4. An antimicrobial peptide composition comprising the antimicrobial peptide of any one of claims 1 to 3 as an active ingredient.
5. The antimicrobial peptide composition of claim 4, wherein the antimicrobial activity is antimicrobial activity against one or more bacteria selected from the group consisting of Staphylococcus aureus, Salmonella spp., Listeria innocua, Pseudomonas aeruginosa, and Escherichia coli.
6. A peptide represented by the following sequence [General Formula I] having antimicrobial activity against pathogens: TABLE-US-00007 [GeneralFormulaI] [(N-terminus)- NKVKEWX.sup.1KX.sup.2LKX.sup.3X.sup.4FX.sup.5-(C-terminus)] wherein X.sup.1 is I or W, X.sup.2 is Y when X.sup.1 is I, and X.sup.2 is W when X.sup.1 is W; X.sup.3 is S or K; X.sup.4 is L or K; and X.sup.5 is S or K, and wherein the peptide is a peptide from which X.sup.3 X.sup.4 F X.sup.5 or F X.sup.5 in General Formula I is deleted.
Description
DESCRIPTION OF DRAWINGS
(1)
(2)
BEST MODE
(3) Hereinafter, the present invention will be described in further detail.
(4) As described above, lactophoricin (LPcin-I) present in cow milk is an cationic and amphipathic peptide that consists of 23 amino acid residues, and corresponds to a carboxyl terminal 113 to 135 region of PP3. LPcin-I inhibits the growth of both gram-positive and gram-negative bacteria, but does not have a hemolytic action at a concentration of 200 M or less. However, to commercialize the LPcin-I known to have an antimicrobial ability, there is an urgent demand to develop technology in which antimicrobial peptides consisting of shorter amino acid sequences are prepared so as to exhibit a higher antimicrobial ability than the wild-type LPcin-1 and reduce production costs.
(5) Therefore, to solve one of the above problems regarding to one exemplary embodiment of the present invention, the present invention provides a peptide represented by the following sequence [General Formula I] having antimicrobial activity against pathogens, thereby promoting a solution to the above problems. Accordingly, an antimicrobial peptide of the present invention and an antimicrobial peptide composition including the antimicrobial peptide have remarkably high antibacterial activity against gram-positive (+) and gram-negative () bacteria, compared to the wild-type LPcin-I which has an antimicrobial ability and consists of a sequence of 23 amino acids. Also, the antimicrobial peptide may be useful in being easily synthesized and saving production costs since the antimicrobial peptide has a smaller number of amino acids, compared to the wild-type LPcin-I:
(6) TABLE-US-00002 [GeneralFormulaI] [(N-terminus)-NKVKEWX.sup.1KX.sup.2LKX.sup.3X.sup.4 FX.sup.5-(C-terminus)]
(7) wherein X.sup.1 is I or W, X.sup.2 is Y when X.sup.1 is I, and X.sup.2 is W when X.sup.1 is W;
(8) X.sup.3 is S or K;
(9) X.sup.4 is L or K; and
(10) X.sup.5 is S or K.
(11) The amino acids in the sequence used in the present invention are represented by the following abbreviations according to the IUPAC-IUB nomenclature:
(12) Alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).
(13) In General Formula I of the present invention, X.sup.1 may be I, W, or a non-polar amino acid, X.sup.2 is Y when X.sup.1 is I and X.sup.2 is W when X.sup.1 is W.
(14) Meanwhile, in the present invention, non-polar amino acids include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C), and proline (P). Polar amino acids include serine (S), threonine (T), tyrosine (Y), asparagine (N), and glutamine (Q). Acidic amino acids include aspartic acid (D), and glutamic acid (E), and basic amino acids include lysine (K), arginine (R), and histidine (H).
(15) Meanwhile, according to one preferred embodiment of the present invention, the peptide may be a peptide in which X.sup.3 X.sup.4 F X.sup.5 or F X.sup.5 is deleted from the C-terminus thereof. Specifically, antimicrobial peptides set forth in SEQ ID NOs: 9 to 11 according to the present invention are antimicrobial peptides from which F X.sup.5 is deleted, and an antimicrobial peptide set forth in SEQ ID NO: 12 is an antimicrobial peptide from which X.sup.3 X.sup.4 F X.sup.5 is deleted. Such antimicrobial peptides set forth in SEQ ID NOs: 1 to 12 exhibit remarkably excellent antibacterial activity, compared to the wild-type LPcin-I (SEQ ID NO: 13) (see Experimental Example 2).
(16) According to one preferred embodiment of the present invention, the peptide has any one amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO: 1 to 10, and thus may be very advantageous for reducing production costs and exhibiting excellent antibacterial activity (see Experimental Example 1). Most preferably, the antimicrobial peptides exhibiting excellent antibacterial activity in the present invention may be YK5 (SEQ ID NO: 3), YK8 (SEQ ID NO: 6), and YK11 (SEQ ID NO: 9) peptides.
(17) The antimicrobial peptides YK5, YK8 and YK11 of the present invention are more economical because the antimicrobial peptides may effectively inhibit the five above-mentioned strains when present at a lower concentration, compared to YK3. Also, YK11 has an economical advantage in that YK11 may be commercially produced because YK11 has an amino acid sequence which is shorter by 2 mers than that of the YK3.
(18) Meanwhile, according to one preferred embodiment of the present invention, the LPcin-I peptide set forth in SEQ ID NO: 13 and consisting of 23 amino acids may be prepared using a conventional peptide synthesis method, specifically, may be prepared using an automated peptide synthesizer or prepared by constructing a recombinant expression vector and purifying the recombinant expression vector. Korean Patent Application No. 2008-130593 which relates to a method of synthesizing an LPcin-I antimicrobial peptide using the recombinant expression vector is incorporated herein by reference in its entirety.
(19) Also, the LPcin-I peptide may be synthesized by a Merrifield's liquid-solid method using 9-fluorenylmethoxycarbonyl (Fmoc) as a group for protecting an amino group (Merrifield, R B., J. Am. Chem. Soc., 85, 2149, 1963).
(20) Also, according to one preferred embodiment of the present invention, the antimicrobial peptides having the amino acid sequences set forth in SEQ ID NOs: 1 to 10 may be prepared using conventional methods like the preparation of the antimicrobial peptide set forth in SEQ ID NO: 13. The antimicrobial peptides of the present invention thus prepared have high antimicrobial activity against gram-positive (+) and gram-negative () strains. In particular, the antimicrobial peptides have high antimicrobial activity against representative strains such as two gram-positive bacteria (Listeria innocua MC2 KCTC 3658 and Staphylococcus aureus ATCC 6538) and three gram-negative bacteria (Pseudomonas aeruginosa ATCC27853, Salmonella ATCC 19430, and Escherichia coli KCTC 1682), but the present invention is not limited thereto.
(21) In addition, the present invention relates to an antimicrobial composition including the antimicrobial peptide as an active ingredient. The antimicrobial composition including the antimicrobial peptide of the present invention as an active ingredient may be effectively used for additives such as antimicrobial and antifungal agents, food preservatives, cosmetic preservatives, vulneraries, eye drops, and pharmaceutical preservatives.
(22) For clinical administration, the antimicrobial peptide of the present invention may be parenterally administered, and may be used in the form of a general pharmaceutical preparation. The antimicrobial peptide of the present invention may be actually administered in the form of various parenteral formulations, and may be formulated using a diluent or excipient typically used in the art, such as a filler, a bulking agent, a binder, a wetting agent, a disintegrating agent, a surfactant, etc., when prepared into formulations. The preparations for parenteral administration may include a sterile aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, a lyophilized preparation, a suppository, etc. Vegetable oil such as propylene glycol, polyethylene glycol, and olive oil, an injectable ester such as ethyl oleate, and the like may be used as the non-aqueous solvent and the suspending agent. Witepsol, Macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, the like may be used as a base of the suppository.
(23) Also, the antimicrobial peptide of the present invention may be used in combination with various carriers commercially available as drugs such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, or other stabilizers may be used as drugs for enhancing stability or absorptivity.
(24) The antimicrobial peptide of the present invention may be administered at an effective dose of 0.1 to 3 mg/kg, preferably 0.5 to 1 mg/kg, and may be administered once to three times a day.
(25) The antimicrobial composition including the antimicrobial peptide of the present invention as an active ingredient may be administered to patients in the form of a bolus or at a single dose through infusion for a relatively short period of time, and may be administered according to the fractionated treatment protocol in which multiple doses are administered for a long period of time.
(26) A concentration of the administered antimicrobial peptide according to the present invention may be determined in consideration of various factors such the age and health condition of a patient, and the like as well as a route of administration of a drug and the number of treatments. Therefore, a person having ordinary skill in the art may easily determine a proper effective dose when considering these factors.
(27) Also, according to one preferred embodiment of the present invention, a feed composition including the antimicrobial peptide of the present invention as an active ingredient is provided. In this case, the feed composition may be included at an effective dose of 0.01 to 100 mg, based on 1 kg of the feed, and may be administered once to three times a day.
(28) Hereinafter, the present invention will be described in further detail with reference to examples. However, it will be apparent to those skilled in the art that the following examples are just preferred examples for the purpose of illustration only and are not intended to limit the scope of the invention.
MODE FOR INVENTION
Example 1
(29) Preparation of Novel Antimicrobial Peptide
(30) Peptides having sequences listed in the following Table 1 were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, US), and the synthesized peptides were purely separated using preparative reversed-phase high-performance liquid chromatography (Shimadzu Prominence HPLC using Shiseido Capcell Pak C18 columns).
(31) In the following Table 1, SEQ ID NO: 11 represents a sequence of a wild-type LPcin-I antimicrobial peptide having 23 amino acids, SEQ ID NOs: 1 to 6 represent sequences of the antimicrobial peptides which consist of 15 amino acids, and SEQ ID NOs: 7 to 9 represent sequences of the antimicrobial peptides which consist of 13 amino acids. Also, SEQ ID NO: 10 represents a sequence of the antimicrobial peptide which consists of 11 amino acids.
(32) TABLE-US-00003 TABLE1 SEQ Peptidenames Aminoacidsequences IDNOs YK3(15-mer) NKVKEWIKYLKSLFS 1 YK4(15-mer) NKVKEWWKWLKSLFS 2 YK5(15-mer) NKVKEWIKYLKSLFK 3 YK6(15-mer) NKVKEWIKYLKSKFS 4 YK7(15-mer) NKVKEWWKWLKSLFK 5 YK8(15-mer) NKVKEWIKYLKSKFK 6 YK9(13-mer) NKVKEWWKWLKSL 7 YK10(13-mer) NKVKEWIKYLKKL 8 YK11(13-mer) NKVKEWWKWLKKL 9 YK12(11-mer) NKVKEWWKWLK 10 LPcin-I(23-mer) NTVKETIKYLKSLFSHAFEVVKT 11
Example 2
(33) Experiment for Measurement of Antibacterial Activity
(34) The antimicrobial activities of the 11 peptides of SEQ ID NOs: 1 to 11 synthesized in Example 1 were measured. Specifically, the antimicrobial activity against microbes was measured using brain heart infusion agar (Bacto). The measurement was based on an agar disc diffusion test performed on five microbes including two gram-positive bacteria (Listeria innocua MC2 KCTC 3658 and Staphylococcus aureus ATCC 6538) and three gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Salmonella ATCC 19430, and Escherichia coli KCTC 1682). Bacteria cultured overnight at 37 C. in 5 ml of a 3.7% brain heart infusion medium were prepared as the inoculum. A final concentration of a suspension was determined by adjusting a turbidity level of the suspension to a turbidity level (0.05) of a turbidity standard solution (110.sup.8 CFU/mL) using a spectrophotometer at 600 mu. 20 ml of brain heart infusion agar was plated on a cell culture plate having a diameter of 90 mm. 30 l of a bacterial suspension was inoculated onto an agar plate using a sterile spreader. The inoculated agar plate was dried at room temperature for 30 minutes. Sterile 6 mm-thick filter paper (Whatman No. 1) was placed on a surface of the agar plate, and each of the antimicrobial peptides was dissolved in sterile water to a concentration of 10 mM, and 20 l of the resulting solution was inoculated onto the filter paper. The solution was pre-diffused at room temperature for 30 minutes, and the plates were cultured at 37 C. for 24 hours. After 24 hours, the sensitivities of the antimicrobial peptides with respect to bacterial colonies were determined as a colony size and clearance in regions in which the growth of bacteria was inhibited by measuring growth inhibition diameters of the bacterial colonies around the filter paper. The sensitivity was obtained by repeating the measurement twice.
(35)
Example 3
(36) Cytotoxicity Experiment
(37) A cytotoxicity experiment was performed by a test method using a Cyto X cell viability assay kit (LPS SOLUTION). A 96-well cell culture plate was purchased from Corning, and mammalian cell lines were purchased from the American Type Culture Collection (ATCC). Also, the CytoX cell viability assay kit was purchased from LPS SOLUTION.
(38) As an experimental method, stored frozen cells were thawed, cultured in a DMEM or RP 11640 medium supplemented with 10% FBS, and then sub-cultured at intervals of 2 to 3 days until a density of the cells reached 80 to 90%. For this experiment, the cells were detached by treating the cells with trypsin-EDTA, and then divided into wells of a 96-well plate so that the number of the cells amounted to 10,000 cells. Thereafter, the cells were cultured at 37 C. for 24 hours in a CO.sub.2 incubator. Each of the prepared peptides was diluted with DMSO to concentrations of 10, 1, 0.1, 0.01, and 0.001 mM, and then diluted at 1:100 by adding 1 l of DMSO or each of the prepared peptides to each well. Then, the cells were cultured at 37 C. for 24 hours in a CO.sub.2 incubator. To measure cytotoxicity, 10 l of each of the peptides prepared using the Cyto X cell viability assay kit (LPS SOLUTION) was added to each well. The cells were cultured at 37 C. for 1 to 4 hours in a CO.sub.2 incubator, and an optical density was measured at 450 nm using a microplate reader. Then, the measured optical density was calculated as a percentage of a value measured for DMSO, and a graph was plotted using GraphPad Prism 5. Then, an IC.sub.50 value was calculated. The IC.sub.50 values of the four peptides (YK3, YK5, YK8, and YK11) in various mammalian cell lines are as listed in the following Table 2. When each of the IC.sub.50 values of the four peptides in each cell line was greater than or equal to 10 M, the peptides were judged to have no general cytotoxicity. As a result, it was confirmed that the four peptides were safe with respect to general cytotoxicity.
(39) In particular, when a CHO-K1 cell line was treated with each of the four peptides, the YK3 peptide had an IC.sub.50 value at 56.2 M, and the YK5, YK8 and YK11 peptides had a respective IC.sub.50 value at 57.0, 71.9 and 86.8 M, which were higher than the IC.sub.50 value of the YK3 peptide. That is, it was confirmed that the YK5, YK8 and YK11 peptides exhibited lower cytotoxicity than the YK3 peptide.
(40) TABLE-US-00004 TABLE 2 Measurement results of cytotoxicity IC.sub.50 (M) Compounds VERO HFL-1 L929 NIH3T3 CHO-K1 YK3 99.1 94.0 >100 76.0 56.2 YK5 >100 >100 >100 >100 57.0 YK8 >100 >100 >100 >100 71.9 YK11 83.7 >100 >100 96.6 86.8
Example 4
(41) Experiment for Measuring Minimal Inhibitory Concentration
(42) An antimicrobial assay was performed by measuring a minimal inhibitory concentration (MIC) value using a standard broth microdilution method. The antimicrobial activity of each of the peptides was measured using two gram-positive bacteria (Listeria innocua MC2 KCTC 3658 and Staphylococcus aureus ATCC 6538) and three gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Salmonella ATCC 19430, and Escherichia coli KCTC 1682). 50 l of each of the five bacterial strains were prepared, mixed with 5 ml of 3.7% BHI, and then cultured overnight under conditions of 37 C. and 240 rpm. Thereafter, 50 l of each of the cultured bacterial strains was mixed with 5 ml of 3.7% BHI, and cultured for 2 hours under conditions of 37 C. and 240 rpm. Each of the cultured bacterial strains was diluted with a 0.037% BHI solution to a concentration of 110.sup.8 CFU/ml.
(43) A dissolved synthetic peptide was diluted twofold, stepwise, with BHIB in a 96 well-plate. 5 l of each of the strains prepared through dilution was mixed with 100 l of the synthetic peptide at various concentrations in a 96-well plate (a microreader plate), and incubated at 37 C. for 12 hours while stirring. After the incubation, a change in optical density at 600 nm was measured using Microplate Reader Multiskan FC (Thermo Scientific, Waltham, Mass., USA), and a range of the MIC was set as the lowest concentration of a peptide in which the growth of bacteria starts to be inhibited. In addition to the MIC, a half maximal inhibitory concentration (IC.sub.50) at which the growth of bacteria reaches 50% compared to the control was determined. As a solvent control, a mixture obtained by mixing 100 l of a BHIB solution having no peptide with 5 l of a bacterial strain was tested in the same manner as in the experimental groups. In this case, this experiment was repeatedly performed in triplicate for the accuracy of the experiment.
(44) As a result, it was revealed that all the peptides had antibacterial activity against the pathogens used in this experiment, as shown in
(45) TABLE-US-00005 TABLE3 Experimentalpeptideinformation Molecular SEQ Peptide Aminoacidsequence weight IDNO 15-mers YK3 NKVKEWIKYLKSLFS 1883.2 1 YK5 NKVKEWIKYLKSLFK 1883.2 3 YK8 NKVKEWIKYLKSKFK 1939.2 6 13-mer YK11 NKVKEWWKWLKKL 1786.2 9
INDUSTRIAL APPLICABILITY
(46) As described above, the antimicrobial peptide prepared in the present invention has a remarkable antibacterial effect against both of gram-positive and gram-negative bacteria and is not harmful to human bodies, and thus can be effectively used for feed additives, food preservatives, cosmetic and pharmaceutical preservatives, wound healing promoters, vulneraries, mouthwash, and eye drops.