POLYPEPTIDE, FUSION POLYPEPTIDE, AND ANTIBIOTIC AGAINST GRAM-NEGATIVE BACTERIA COMPRISING SAME

Abstract

Provided are a novel polypeptide, a fusion polypeptide comprising the polypeptide, and a use thereof as an antibiotic. More specifically, provided are a novel polypeptide derived from a bacteriophage, a novel fusion polypeptide comprising cecropin A, and an antibiotic against Gram-negative bacteria comprising the polypeptide and/or the fusion polypeptide.

Claims

1. A polypeptide, comprising the amino acid of SEQ ID NO: 1 or SEQ ID NO: 6.

2. The polypeptide according to claim 1, wherein the polypeptide has endolysin activity.

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. A bacteriophage, comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

8. The bacteriophage according to claim 7, wherein the polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 comprises the nucleic acid sequence of SEQ ID NO: 2.

9. The bacteriophage according to claim 7, wherein the bacteriophage has antibiotic activity against Pseudomonas sp. bacterium.

10. The polypeptide according to claim 1, further comprising Cecropin A at N-terminus or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 6.

11. The polypeptide according to claim 10, wherein Cecropin A is represented by the amino acid sequence of SEQ ID NO: 8.

12. The polypeptide according to claim 10, wherein the Cecropin A and polypeptide of SEQ ID NO: 1 or SEQ ID NO: 6 are linked in order from the N-terminus.

13. The polypeptide according to claim 10, wherein the Cecropin A and polypeptide of SEQ ID NO: 1 or SEQ ID NO: 6 are linked by a peptide linker.

14. The polypeptide according to claim 10, represented by the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 13.

15. A polynucleotide encoding the polypeptide according to claim 1, or a fusion polypeptide comprising Cecropin A at N-terminus or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 6.

16. The polynucleotide according to claim 15, represented by the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 14.

17. (canceled)

18. (canceled)

19. A method for inhibiting growth of gram negative bacterium or killing gram negative bacterium, or preventing or treating infection of gram negative bacterium or disease caused by gram negative bacterium, administering a pharmaceutically effective dose of one or more kinds selected from the group consisting of the polypeptide of claim 1, a fusion polypeptide comprising Cecropin A at N-terminus or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 6, a polynucleotide encoding the polypeptide, a polynucleotide encoding the fusion polypeptide; a recombinant vector comprising a polynucleotide encoding the polypeptide, a recombinant vector comprising a polynucleotide encoding the fusion polypeptide; and a recombinant cell comprising the polynucleotide or recombinant vector, into a subject in need of inhibiting growth of gram negative bacterium or killing gram negative bacterium, or preventing or treating infection of gram negative bacterium or disease caused by gram negative bacterium.

20. The method according to claim 19, further administering a pharmaceutically effective dose of a polymyxin-based antibiotic.

21. The method according to claim 20, wherein the polymyxin-based antibiotic is polymyxin B, colistin, or a combination thereof.

22. (canceled)

23. The method according to claim 19, wherein the gram negative bacterium is one or more kinds selected from the group consisting of Pseudomonas sp. bacterium, Acinetobacter sp. bacterium, Escherichia sp. bacterium, Enterobacter sp. bacterium, and Klebsiella sp. bacterium.

24. The method according to claim 19, wherein the gram negative bacterium is one or more kinds selected from the group consisting of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae.

25. (canceled)

26. (canceled)

27. (canceled)

28. The method according to claim 19, wherein the gram negative bacterium is Pseudomonas sp. bacterium, and the disease caused by Pseudomonas sp. bacterium is skin infection, bedsore, pneumonia, bacteremia, septicemia, endocarditis, meningitis, otitis externa, otitis media, keratitis, osteomyelitis, enteritis, peritonitis or cystic fibrosis.

29. The method according to claim 19, wherein the gram negative bacterium is Acinetobacter sp. bacterium, and the disease caused by Acinetobacter sp. bacterium is skin infection, pneumonia, bacteremia or septicemia.

30. The method according to claim 19, wherein the gram negative bacterium is Escherichia sp. bacterium, and the disease caused by Escherichia sp. bacterium is enteritis, Crohn's disease, ulcerative colitis, bacillary dysentery, urinary tract infection, skin infection, bacteremia or septicemia.

31. (canceled)

32. (canceled)

33. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0131] FIG. 1 is a cleavage map of the expression vector which expresses the endolysin (LNT101) gene of bacteriophage PBPA90 infecting Pseudomonas aeruginosa.

[0132] FIG. 2 shows the process of purifying endolysin (LNT101) derived from bacteriophage PBPA90.

[0133] FIG. 3 is a graph showing the antibiotic effect against Pseudomonas aeruginosa in vitro of endolysin (LNT101) derived from bacteriophage PBPA90.

[0134] FIG. 4 is a graph showing the antibiotic effect against Pseudomonas aeruginosa in vivo of endolysin (LNT101) derived from bacteriophage PBPA90.

[0135] FIG. 5 shows the amino acid sequences of endolysin (LNT102) (SEQ ID NO: 6) and endolysin (LNT101) (SEQ ID NO: 1) according to one example in comparison.

[0136] FIG. 6 is a cleavage map of the expression vector pBT7-LNT102 for expression of endolysin (LNT102).

[0137] FIG. 7 shows the result of SDS-PAGE of reactants obtained in the process of purification of endolysin (LNT102).

[0138] FIG. 8 is a graph showing the killing ability against various gram negative bacterium of endolysin (LNT102) and endolysin (LNT101).

[0139] FIGS. 9 and 10 are graphs showing the killing ability against gram negative bacterium depending on the concentration and treatment time of endolysin (LNT102).

[0140] FIG. 11 is a schematic diagram of the fusion polypeptide (LNT103) according to one example.

[0141] FIG. 12 is an image showing the result of SDS-PAGE of the reactants obtained in the process of purification of the fusion polypeptide (LNT103) according to one example.

[0142] FIG. 13 is a graph showing the outer membrane permeabilization activity against various gram negative bacterium of the endolysin (LNT101), endolysin variant (LNT102) and fusion polypeptide (LNT103).

[0143] FIG. 14 is a graph showing the killing ability against various gram negative bacterium of the endolysin (LNT101), endolysin variant (LNT102) and fusion polypeptide (LNT103).

[0144] FIG. 15 is a graph showing the killing ability against various gram negative bacterium of the fusion polypeptides LNT103 and CecA-LNT101.

[0145] FIG. 16 is a graph showing the killing ability against gram negative bacterium of the fusion polypeptide (LNT103) according to one example and Cecropin A in comparison.

[0146] FIG. 17 is a graph showing the killing ability against gram negative bacterium depending on the concentration and treatment time of the fusion polypeptide according to one example (LNT103).

[0147] FIGS. 18 and 19 are graphs showing the evaluation result of cytotoxicity and hemolysis assay of the fusion polypeptide according to one example (LNT103).

[0148] FIG. 20 is a graph showing the viability when treating the fusion polypeptide LNT103 in an Acinetobacter baumannii systemic infection mouse model compared to treatment of colistin.

MODE FOR INVENTION

[0149] The novel endolysin and/or bacteriophage expressing the same provided in the present description exhibit excellent growth inhibitory ability and killing ability against Pseudomonas sp. bacterium, for example, Pseudomonas aeruginosa, and thereby, they can be usefully used as an antibiotic against Pseudomonas sp. bacterium, for example, Pseudomonas aeruginosa.

Example 1: Preparation of LNT101 Endolysin and Antibiotic Activity Test

1.1. Separation of Bacteriophage Capable of Killing Pseudomonas aeruginosa

1.1.1. Culture Condition of Strain

[0150] Pseudomonas aeruginosa (PR01957) was used as a host, and cultured with shaking in a LB (Luria-Bertani) medium under the condition of 37° C.

1.1.2. Separation of Bacteriophage

[0151] In order to select a bacteriophage infected by Pseudomonas aeruginosa, samples were collected from Gwacheon sewage treatment plant, Gwacheon-si, Gyeonggi-do, Korea. The collected samples and Pseudomonas aeruginosa were cultured at 37° C. for 3 hours, and then centrifuged at 500 rpm for 20 minutes and the supernatant was collected. Subsequently, the supernatant was filtered with a 0.45 μm filter, and then double agar layer plaque assay was performed.

[0152] Briefly describing the analysis method, the culture solution of the host bacterium, Pseudomonas aeruginosa and bacteriophage was mixed by 0.1 M.O.I. to the top agar 5 ml, and poured to an agar plate, and cultured at 37° C. for 24 hours to obtain a plaque. Through repeated performance of the process, the purified pure bacteriophage was obtained, and this bacteriophage was named bacteriophage PBPA90.

1.2: Separation and Analysis of Genome of Bacteriophage PBPA90

[0153] Sequencing for genome of the bacteriophage PBPA90 obtained in the Example 1.1 was conducted. After culturing Pseudomonas aeruginosa in a LB medium of 200 ml to OD.sub.600=0.5, here, it was lysed by infection with the filtered bacteriophage 10.sup.9 pfu/ml or 0.1 M.O.I. After that, sodium chloride was added so that the final concentration was 1 M here, and then it was left at 4° C. for 1 hour. Subsequently, after centrifuging at 11,000× g for 10 minutes, PEG (Polyethylene glycol 8000) was added to precipitates by 10%(w/v), and it was left at 4° C. for 1 hour. In succession, after centrifuging at 11,000× g for 10 minutes, the supernatant was removed, and the precipitates were suspended with SM buffer solution [100 mM NaCl, 10 mM MgSO.sub.4 (heptahydrate), 50 mM Tris-HCl, pH 7.5]. Chloroform was added at a ratio of 1:1 here, and voltexing was conducted, and then it was centrifuged at 3,000× g for 15 minutes to obtain the supernatant.

[0154] 40%(w/v) glycerol 3 ml was added to a polycarbonate test tube, and then, 5%(w/v) glycerol 4 ml was added so as not to be mixed. Subsequently, the supernatant was removed, and then precipitates were resuspended with SM buffer solution to obtain bacteriophage genome DNA. The bacteriophage genome DNA was separated using a phage DNA isolation kit (Norgen biotek corp.) according to the manufacturer's manual. The sequence for genome was analyzed using the genome sample separated as above (LAS, Illumina MiSeq platform).

[0155] The finally analyzed bacteriophage PBPA90 genome had the total nucleic acid sequence length of 304,052 bp, and had a 44% GC content. The full-length nucleic acid sequence of the bacteriophage PBPA90 genome was represented in SEQ ID NO: 5. The similarity with the conventionally known bacteriophage genome sequence was investigated using BLAST on Web based on the genome nucleic acid sequence information. As the result of BLAST investigation, it was confirmed that the genome sequence of the bacteriophage PBPA90 had low sequence similarity to Pseudomonas bacteriophage KTN4 (GenBank accession No.: KU521356.1) (query coverage: 4%, identity: 97.34%). Based on this fact, it was confirmed that the bacteriophage PBPA90 is a new bacteriophage which is not conventionally known.

1.3: Cloning and Purification of LNT101 Endolysin

[0156] Through ORF search for the genomic sequence of the analyzed bacteriophage PBPA90 (SEQ ID NO: 5), the ORF of 780 bp at positions 180,029-180,806 (SEQ ID NO: 2) was estimated to be an endolysin gene, and the bacteriophage PBPA90-derived endolysin and a gene encoding this were named LNT101 endolysin and LNT101 gene, respectively.

[0157] Using Primer (F: 5′-aaggatccatgggtactgtactcaaacgtggc-3′ (SEQ ID NO: 3), R: 5′-aactcgagtgcccgatgtttcgaaactttatcttc-3′ (SEQ ID NO: 4)), for the genome of the bacteriophage PBPA90, PCR (polymerase chain reaction) was performed to obtain LNT101 gene (nucleic acid sequence with a 780 bp length at positions 180,029-180,806 in SEQ ID NO: 5). The amino acid sequence of LNT101 endolysin encoded by the LNT101 gene was represented in SEQ ID NO: 1 (259 aa). The PCR was performed under the following condition: step 1: 94° C., 5 minutes; step 2: 94° C., 30 seconds; step 3: 52° C., 45 seconds; step 4: 72° C., 1 minute; step 5: repeating steps 2-4 30 times; step 6: 72° C., 10 minutes. The obtained PCT products were cloned with BamHI/XhoI site of pET-21a vector (Novagen) having N-terminal 6× His-tag, to prepare an expression vector for LNT101 endolysin expression (pET-LNT101 plasmid). The prepared expression vector pET-LNT101 was schematically shown in FIG. 1.

[0158] The prepared pET-LNT101 plasmid was transformed in E. coli BL21-pLysS strain (Novagen), and then was cultured in LB broth (1% Tryptone, 0.5%(w/v) Yeast extract, 0.5%(w/v) NaCl) to OD.sub.600=0.5. After that, 1 mM IPTG (Isopropyl β-d-1-thiogalactopyranosid) was added, and then was cultured with shaking at 37° C. for 4 hours. After cell harvest, it was resuspended with lysis buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF and 1 mg/ml lysozyme were added, and it was left on ice for 30 minutes. Cells were lysed by sonication, and centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. This was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. After that, it was washed with wash buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 30 mM imidazole), and then it was eluted with elution buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 300 mM imidazole), to purify LNT101 protein (including 6× His tag).

[0159] The purity of the LNT101 protein was confirmed by 15% SDS-PAGE and the concentration of the LNT101 protein was measured by Bradford assay. The result of confirming each reactant obtained in the purification process by SDS-PAGE was shown in FIG. 2. The molecular weight of the purified LNT101 protein confirmed by SDS-PAGE was about 28kDa. The amino acid sequence of the LNT101 protein was shown in SEQ ID NO: 1.

1.4. Target Bacteria Spectrum Investigation of Bacteriophage PBPA90 and Endolysin LNT101 Derived Therefrom

[0160] In the present example, in order to investigate a target bacteria spectrum of the bacteriophage PBPA90 selected in the Examples 1.1 and 1.2 and endolysin LNT101 separated and purifying in the Example 1.3, the antibacterial activity against gram negative bacterium such as Pseudomonas aeruginosa ATCC13388, ATCC9027, ATCC10145, ATCC15692, ATCC15522, ATCC25619, ATCC27853, CCARM2134, CCARM2200, CCARM2029, CCARM2144, CCARM2298, CCARM2326, PR01957, E. coli ATCC8739, Enterobacter cloacae CCARM0252, Klebsiella pneumoniae KCTC2261, Klebsiella aerogenes CCARM16006, Campylobacter jejuni KCTC5327, Cronobacter sakazakii KCTC2949, Salmonella typhimurium ATCC14028, Salmonella enteritidis ATCC13076 was tested.

[0161] The target bacteria spectrum of the bacteriophage PBPA90 and endolysin LNT101 was confirmed by a spot test. For the spot test, bacterium sterilized in 4 ml top agar (1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, 0.7% agar) of 1×10.sup.11 CFU/200 μl were added and poured to a LB plate. After hardening the top agar, the bacteriophage PBPA90 10 μl (1×10.sup.8 PFU/ml) or LNT101 10 μl (2 mg/ml) was spotted, and it was incubated at 37° C. for 18 hours.

[0162] Production of the halo zone formed as the result of the incubation was confirmed, and the result was shown in Table 1 below:

TABLE-US-00001 TABLE 1 Host LNT101 P. aeruginosa ATCC 13388 ◯ ATCC 9027 ◯ ATCC 10145 ◯ ATCC 15692 ◯ ATCC 15522 ◯ ATCC 25619 ◯ ATCC 27853 ◯ CCARM 2134 ◯ CCARM 2200 ◯ PR1957 ◯ CCARM 2029 ◯ CCARM 2144 ◯ CCARM 2298 ◯ CCARM 2326 ◯ E. coli ATCC 8739 ◯ E. cloacae CCARM 0252 ◯ K. pneumoniae KCTC 2261 ◯ K. aerugenes CCARM 16006 ◯ C. jejuni KCTC 5327 ◯ C. sakazakii KCTC 2949 ◯ S. typhimurium ATCC 14028 ◯ S. enteritidis ATCC 13076 ◯ (In the Table 1, ‘◯’ means production of the halo zone)

[0163] As shown in Table 1, the endolysin LNT101 formed the halo zone in all the tested gram negative bacterium, which were Pseudomonas aeruginosa, E, coli, Enterobacter cloacae, Klebsiella pneumoniae, Campylobacter, Cronobacter, and Salmonella strains. This result shows that the endolysin LNT101 has the degradation ability for peptidoglycans of various gram negative bacterium as aforementioned and has a broad target bacterium spectrum.

1.5. Investigation of Killing Ability Against Pseudomonas aeruginosa of Endolysin LNT101

[0164] In the present example, for Pseudomonas aeruginosa (ATCC 13388) confirmed against which the bacteriophage PBPA90 and endolysin LNT101 had the antibacterial activity in the Example 1.4, the bacterium killing ability of endolysin LNT101 was tested.

In Vitro Test

[0165] For this, the endolysin LNT101 at a concentration of 0.1, 0.5, 1.0 μM and Pseudomonas aeruginosa (ATCC 13388) 1Δ10.sup.6 CFU were added so that the final volume was 200 μl to reaction buffer (20 mM Tris-Cl, pH 7.5) and they were left at 37° C. for 1 hour. In 30 minutes, 1 hour and 2 hours, the number of colonies of Pseudomonas aeruginosa was confirmed, and the result was shown in FIG. 3. As shown in FIG. 3, it was confirmed that the endolysin LNT101 had the killing ability against Pseudomonas aeruginosa in all the tested treatment dose and time, and it was shown that this Pseudomonas aeruginosa killing ability was dependent on the treatment time and dose.

[0166] In addition, that the LNT101 endolysin had the antibacterial ability in various gram negative bacterium was confirmed by a CFU reduction test, and the result was shown in Table 2 below.

TABLE-US-00002 TABLE 2 Host LNT101 P. aeruginosa ATCC 13388 ◯ ATCC 15522 ◯ CCARM 2092 ◯ CCARM 2134 ◯ CCARM 2144 ◯ CCARM 2326 ◯ F141 ◯ PAO1 ◯ A. baumannii ATCC 2508 ◯ F4 ◯ F15 ◯ E. cloacae CCARM 0252 ◯ K. aerugenes CCARM 16006 ◯ (In the Table 2, ‘◯’ means reduction of 0.5 log CFU or more through the CFU reduction assay)

[0167] As shown in Table 2, it was confirmed that it had the bacterium killing effect in a dose dependent manner in all the test gram negative bacterium of 8 kinds of Pseudomonas aeruginosa, 3 kinds of Acinetobacter baumannii, 1 kind of Enterobacter cloacae, and 1 kind of Klebsiella aerogenes, including antibiotic-resistant bacterium.

In Vivo Test

[0168] The Pseudomonas aeruginosa killing ability of the endolysin LNT101 was also confirmed in vivo. As an animal model for the in vivo effectivity evaluation, Galleria mellonella was used. The Galleria mellonella model was divided into the healthy group (non-infection group), Pseudomonas aeruginosa infected group (drug non-administration group), 2 LNT101 administration groups in which LNT101 was administered into the infection model, Colistin administration group in which colistin was administered into the Pseudomonas aeruginosa infected model, and combination administration group in which LNT101 and colistin were administered in combination into the Pseudomonas aeruginosa infected model to progress an experiment, and 10 of Galleria mellonella per each group was used.

[0169] The Pseudomonas aeruginosa infected model was prepared by infecting Pseudomonas aeruginosa PA01 at the LD80 concentration, 50 CFU/larva, and LNT101 was administered at 0.6 μg/larva (3 mg/Kg) and 6 μg/larva (30 mg/Kg), respectively. Colistin was administered at 0.5 μg/larva (2.5 mg/Kg), and for administration in combination, Colistin at 0.5 μg/larva (2.5 mg/Kg) and LNT101 at 6 μg/larva (30 mg/Kg) were administered. As the result of observation for 72 hours, the viability of Galleria mellonella was shown in FIG. 4. As shown in FIG. 4, it was confirmed that the infection group was dead 100%, but in the LNT101 and colistin administration groups, the viability was increased. In particular, in the LNT101 6 μg/larva (30 mg/Kg) administration group, the viability of 30% was confirmed.

1.6. Synergy Effect of Pseudomonas aeruginosa Killing Ability of Endolysin LTN101 and Polymyxin Antibiotic

[0170] The synergy effect by treatment in combination of the polymyxin-based antibiotic having a mechanism acting on the cell membrane of bacterium and endolysin LNT101 was confirmed. Specifically, polymyxin B (32 μg/ml-0.03 μg/ml) and colistin (128 μg/ml-0.1 μg/ml) were diluted by ½ per well in a microplate, and then a LNT101 endolysin 1 μM combination treatment group and the same amount of PBS treatment group was made. In all wells, P. aeruginosa 1×10.sup.5 CFU/ml was treated by total 100 μl. After that, they were cultured at 37° C. for 18 hours. MIC (Minimum Inhibitory Concentration) means the concentration value of the minimum polymyxin-based antibiotic of the well where bacterium did not grow, and the MIC test was performed by broth microdilution technique.

[0171] The MIC change of the polymyxin-based antibiotic by combination treatment of the polymyxin-based antibiotic (polymyxin B, Colistin) and LNT101 endolysin at various concentrations was confirmed, and shown in Table 3 below.

TABLE-US-00003 TABLE 3 Polymyxin Polymyxin Colistin + B B + LNT101 Colistin LNT101 MIC of 4 μg/ml 2.7 μg/ml 8 μg/ml 4 μg/ml antibiotics

[0172] As shown in Table 3, it was confirmed that the MIC of the polymyxin-based antibiotics, polymyxin B and Colistin was reduced by 50%, in case of use in combination of the LNT101 endolysin.

Example 2. Production of LNT102 Endolysin and Antibiotic Activity Test

2.1. Discovery of a Novel Polypeptide Having Endolysin Activity

[0173] The endolysin LNT101 separated and purified in the Example 1.3 (SEQ ID NO: 1) was composed of PG_binding_1 domain (10-65 amino acid) and transglycosylase SLT domain (95-179 amino acid).

[0174] Eleven amino acids were substituted for LNT101 endolysin and other amino acid sites with the dominant amino acid sequence of the comparative protein, by comparing the amino acid sequence of endolysin LNT101 PG_binding_1 domain with glycoside hydrolase family 25 of Thermoanaerobacterium phage THSA-485A (accession no. YP_006546280.1), peptidoglycan binding protein of Serratia phage phiMAM1 (accession no. YP_007349105.1), Serratia phage 2050H1 peptidoglycan binding protein (accession no. ASZ78903.1), putative peptidoglycan binding protein of Serratia phage vB_SmaA_3M (accession no. AYP28388.1), putative peptidoglycan binding protein of Enwinia phage vB_EamM-Bue1 (accession no. AV022912.1), putative peptidoglycan binding protein of Pseudomonas phage Noxifer (accession no. YP_009609055.1), endolysin of Salmonella phage Mutine (accession no. AUG88272.1) and peptidoglycan binding protein of Salmonella phage bering (accession no. QIQ61961.1), which have a similar amino acid sequence, by BLASTp analysis.

[0175] Four amino acids were substituted for LNT101 endolysin and other amino acid sites with the dominant amino acid sequence of the comparative protein, by comparing the amino acid sequence of LNT101 endolysin transglycosylase SLT domain with tail fiber protein of Pseudomonas phage Noxifer (accession no. YP_009609112.1), hypothetical protein SL2_199 of Pseudomonas phage SL2 (accession no. YP_009619739.1), putative endolysin of Pseudomonas phage KTN4 (accession no. ANM44938.1), PHIKZ144 of Pseudomonas phage phiKZ (accession no. NP_803710.1), tail fiber protein of Pseudomonas phage Psa21 (accession no. QBJ02724.1) and hypothetical protein of Pseudomonas phage vB_PaeM_PS119XW (accession no. QEM41943.1), which have a similar amino acid sequence, by BLASTp analysis.

[0176] 15 substituted amino acid sequence parts were modified in the LNT101 gene sequence part in consideration of codon usage (SEQ ID NO: 7), and this was named LNT102 (SEQ ID NO: 6). The comparison of the sequences of LNT101 and LNT102 was shown in FIG. 5. The LNT102 was cloned to gene synthesis and C-terminal 6× histidine tag-attached pBT7-C-His vector (Bioneer). The expression vector pBT7-LNT102 of LNT102 prepared as such was schematically shown in FIG. 6.

[0177] The sequences of the described LNT102 and LNT101 were summarized in Table 4 below:

TABLE-US-00004 TABLE 4 Amino acid sequence (N.fwdarw.C) or SEQ ID nucleic acid sequence (5′.fwdarw.3′) NO: Endolysin MGTVLKRGDRGSAVEDLQMKLNVAGYNLSADGIFGGDTEKAVRDVQAGAGLVVDGKVG 6 LNT102 PKTLYAIAKSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGVDSRLLATFASIES AFDYTVKAKTSSATGWFQFLDATWDDMIKAYGSKYGIPKDPTRALRKDPRANALMGAE FIKGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGEVLFPKPAKANPS IFSNKGVPRTLAEIYKLFEDKVSKHRA Endolysin ATGGGTACTGTACTCAAACGTGGCGACCGCGGCTCTGCTGTGGAAGATCTACAAATGA 7 LNT102 AACTTAACGTCGCAGGATACAACCTGAGCGCTGACGGAATCTTCGGTGGAGATACAGA coding GAAAGCTGTTCGTGATGTGCAAGCTGGCGCGGGCTTGGTGGTTGACGGAAAAGTTGGA gene CCTAAAACTCTATATGCGATTGCCAAATCCGCTACTGTTCCTGCTAAATGGGAAGCTA TCCCTTTCCCAACAGCTAATAAATCTCGGTCGGCTGCAATGCCCACTCTGAATGCGGT TGGAGCAATGACTGGTGTGGATTCTCGGTTACTCGCTACATTCGCTTCCATTGAGTCT GCTTTTGATTACACTGTCAAAGCAAAAACATCTTCGGCTACTGGTTGGTTCCAGTTCC TTGATGCTACATGGGATGACATGATCAAAGCATATGGTTCCAAATACGGGATACCTAA AGATCCCACTAGGGCACTCCGTAAAGACCCACGTGCAAATGCATTAATGGGTGCAGAA TTCATTAAAGGAAATGCAGCTGTATTACGTCCAGTAATCAATCGCGAACCGAGTGATA CAGACTTGTATTTGGCACATTTCCTTGGTGCTGGCGGCGCTAAGAAATTCCTATCCGC AGATCAGAAAACTCTCGGTGAAGTTCTATTCCCGAAACCTGCTAAAGCAAACCCGTCG ATCTTTAGCAATAAAGGTGTACCACGTACCCTTGCAGAGATCTACAAGCTGTTCGAAG ATAAAGTTTCGAAACATCGGGCA Endolysin MGTVLKRGDRGSAVEDLQMKLRVAGYAVSADGIFGGDTEKAVRDFQASKALVVDGKVG 1 LNT101 PATLAELAKSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGTDSRLLATFASIES AFDYTVKASTSSATGWFQFLDATWDDMIKAHGSKYGIPKDPTRALRKDPRANALMGAE FLKGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGEVLFPKPAKANPS IFSNKGVPRTLAEIYKLFEDKVSKHRA

2.2. Purification of LNT102 Endolysin

[0178] After transforming the prepared pBT7-LNT102 plasmid into the E. coli BL21-Star(DE3) strain (Invitrogen), it was cultured in LB broth (1% Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) by OD.sub.600=0.5. After that, 1 mM IPTG (Isopropyl β-d-1-thiogalactopyranosid) was added and then cultured with shaking at 37° C. for 4 hours. After cell harvest, it was resuspending with lysis buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF and 1 mg/ml lysozyme were added and it was left on ice for 30 minutes. Cells were lysed by sonication, and they were centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. This was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. Then, after washing with wash buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 30 mM imidazole), it was eluted with elution buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 300 mM imidazole), to purify LNT102 protein (including 6× His tag).

[0179] The purity of the LNT102 protein was confirmed by 15% SDS-PAGE and the concentration of the LNT102 protein was measured by Bradford assay. The result of confirming each reactant obtained in the process of purification by SDS-PAGE was shown in FIG. 7. The molecular weight of the purified LNT102 protein confirmed by SDS-PAGE was about 28 kDa.

2.3. Investigation of Killing Ability Against Gram Negative Bacterium of Endolysin LNT102

[0180] In the present example, the bacterium killing ability and target bacterium spectrum of the endolysin LNT102 purified in Example 2.2 for various gram negative bacterium were confirmed.

In Vitro Test

[0181] For this, Endolysin LNT101 and LNT102 at a concentration of 2 μM and each 1×10.sup.6 CFU of Pseudomonas aeruginosa (PA01; ATCC 15692), Acinetobacter baumannii (ATCC 17978), Escherichia coli (ATCC 8739), Klebsiella pneumoniae (ATCC 13883), Enterobacter aerogenes (CCARM 16006), and Salmonella enteritidis (ATCC 13076) was added in reaction buffer (20 mM Tris-Cl, pH 7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours. In 2 hours, the number of colonies of each gram negative bacterium was confirmed, and the result was shown in FIG. 8 (in FIG. 8, PA90 represents endolysin LNT101 and mtPA90 represents endolysin LNT102, respectively). As shown in FIG. 8, it was confirmed that all the endolysin LNT101 and LNT102 had the excellent killing ability compared to the control group against all the tested gram negative bacterium, and in particular, LNT102 showed the more excellent killing ability against gram negative bacterium in the same dose-, compared to LNT101.

[0182] The endolysin LNT102 at a concentration of 0.1, 0.5, 2.5 μM, and each 1×10.sup.6 CFU of Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli was added in reaction buffer (20 mM Tris-Cl, pH 7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours. In 30 minutes, 1 hour and 2 hours, and the result was shown in FIGS. 9 and 10. As shown in FIGS. 9 and 10, it was confirmed that endolysin LNT102 had the killing ability against gram negative bacterium in all the tested treatment doses and time, and it was shown that this killing ability against gram negative bacterium was dependent on the treatment time and dose.

2.4. Synergy Effect of Killing Ability Against Gram Negative Bacteria in Case of Use in Combination of Endolysin LTN102 and Antibiotics

[0183] A synergy effect by combination treatment of a polymyxin-based antibiotic having a mechanism acting on the cell membrane of bacterium and endolysin LNT102 purified in Example 2.2 was confirmed. Specifically, after treating 4 μg/ml Polymyxin B to the 96 well microplate row A, serial dilution was performed by ½ in rows B-G. Polymyxin was not treated to the row H. After treating 8 μg/ml Colistin to the 96 well microplate row A, a combination treatment group in which LNT102 endolysin 1 μM was further treated and a PBS treatment group in the same amount were made. All wells were treated with Pseudomonas aeruginosa and Escherichia coli in an amount of 1×10.sup.5 CFU/ml, total 100 μl, respectively. After that, they were cultured at 37° C. for 18 hours. MIC (Minimum Inhibitory Concentration) means a concentration value of a minimum polymyxin-based antibiotic of a well in which bacterium do not grow, and the MIC test was performed by broth microdilution technique according to the standard test method of CLSI (Clinical and Laboratory Standards Institute) (Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacterium that grow aerobically; approved standard. 11th ed. Document M07. Wayne, Pa.: CLSI; 2018).

[0184] The MIC change of the polymyxin-based antibiotics by combination treatment of the polymyxin-base antibiotics (polymyxin B, Colistin) at various concentrations and LNT102 endolysin was measured and shown in Table 5 below.

TABLE-US-00005 TABLE 5 Polymyxin Polymyxin Colistin + Colistin + B + PBS B + LNT102 PBS LNT102 P. aeruginosa 0.5 μg/ml <0.06 μg/ml 2 μg/ml 0.5 μg/ml (ATCC 27853) A. baumannii 0.5 μg/ml 0.03 μg/ml 2 μg/ml 0.25 μg/ml (ATCC 19606) E. coli(ATCC 0.5 μg/ml 0.06 μg/ml 2 μg/ml 1 μg/ml 8739)

[0185] As shown in Table 5, it was confirmed that the MIC of the polymyxin-based antibiotics, polymyxin B and Colistin was reduced by 1/16 times at maximum, in case of use in combination of the LNT102 endolysin. This result shows that a significantly increased antibiotic effect can be obtained when a conventional antibiotic, for example, a polymyxin-based antibiotic and LNT102 endolysin are treated in combination.

Example 3: Preparation of Fusion Polypeptide

3.1. Preparation of Polypeptide (LNT101 and LNT102 Endolysin)

[0186] Endolysin LNT101 (SEQ ID NO: 1) is composed of PG_binding_1 domain (10-65 amino acid) and transglycosylase SLT domain(95-179 amino acid). The endolysin LNT101 variant of SEQ ID NO: 6 (hereinafter, named endolysin LNT102) was prepared by adding 15 amino acid mutations in the endolysin LNT101 (SEQ ID NO: 1).

[0187] The coding gene of the endolysin LNT102 (SEQ ID NO: 7) was inserted to pBT7 plasmid to prepare pBT7-LNT102 plasmid for endolysin LNT102 expression. The prepared pBT7-LNT102 plasmid was transformed into E. coli BL21-Star(DE3) strain (Invitrogen), it was cultured in LB broth (1% Tryptone, 0.5%(w/v) Yeast extract, 0.5%(w/v) NaCl) by OD.sub.600=0.5. After that, 1 mM IPTG (Isopropyl β-d-1-thiogalactopyranosid) was added, and then it was cultured with shaking at 37° C. for 4 hours. After cell harvest, it was resuspended with lysis buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF and 1 mg/ml lysozyme were added and it was left on ice for 30 minutes. Cells were lysed by sonication, and it was centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. This was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. Then, after washing with wash buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 30 mM imidazole), it was eluted with elution buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 300 mM imidazole), to purify the LNT102 protein (including 6Δ His tag).

[0188] The purity of the LNT102 protein was confirmed by 15% SDS-PAGE, and the concentration of the LNT102 protein was measured by Bradford assay. As the result of confirming each reactant obtained during the process of purification by SDS-PAGE, the molecular weight of the purified LNT102 protein was about 31 kDa.

3.2. Preparation of Fusion Polypeptide

[0189] A fusion polypeptide in which Cecropin A was fused in the prepared LNT102 protein was prepared. Specifically, a polynucleotide (SEQ ID NO: 11) encoding a fusion polypeptide (named LNT103) (SEQ ID NO: 10) comprising [Cecropin A (SEQ ID NO: 8)]-[linker (GSGSGS) (SEQ ID NO: 12)]-[endolysin (named LNT102) (SEQ ID NO: 6)] from the N-terminus in order was inserted into pET 21a (Novagen) plasmid to prepare plasmid pET-LNT103 for LNT103 expression. After transforming the prepared pET-LNT103 plasmid into E. coli BL21-Star(DE3) strain (Invitrogen), it was cultured in LB broth (1% (w/v) Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) by OD.sub.600=0.5.

[0190] After that, after adding 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside), it was cultured with shaking at 25° C. for 6 hours. After cell harvest, it was resuspended with lysis buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF (phenylmethylsulfonyl fluoride) and 1 mg/ml lysozyme were added, and it was left on ice for 30 minutes. Cells were lysed by sonication, and it was centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. The obtained supernatant was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. Then, after washing with wash buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 30 mM imidazole), it was eluted with elution buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 300 mM imidazole). It was purified/confirmed by 15% SDS-PAGE and the concentration was measured by Bradford assay.

[0191] The obtained SDS-PAGE result was shown in FIG. 12. As confirmed in FIG. 12, it was confirmed that the molecular weight of the purified fusion polypeptide LNT103 (SEQ ID NO: 10) was 34 kDa.

[0192] In addition, by the same method as the above method, a fusion polypeptide in which Cecropin A was fused to endolysin LNT101 (SEQ ID NO: 1) (CecA-LNT101; SEQ ID NO: 13) was produced. For the produced CecA-LNT101, SDS-PAGE was performed by the aforementioned method, to confirm that the molecular weight of CecA-LNT101 was 34 kDa.

[0193] On the other hand, for convenience of the test, the first amino acid M of the purified fusion polypeptide LNT103 (SEQ ID NO: 10) and CecA-LNT101(SEQ ID NO: 13) was substituted with MAS (Met-Ala-Ser), and it was produced in a form in which an extra sequence and a His tag were added to the C-terminus (SEQ ID NO: 15 or SEQ ID NO: 16), and in the following test, SEQ ID NO: 15 was used as LNT103, and SEQ ID NO: 16 was used as CecA-LNT101, respectively.

[0194] The amino acid sequences and nucleic acid sequences of the polypeptide and their coding genes described in the present example were summarized in Table 6 below:

TABLE-US-00006 TABLE 6 Amino acid sequence (N.fwdarw.C) or SEQ ID nucleic acid sequence (5′.fwdarw.3′) NO: Cecropin A KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK 8 Cecropin A AAATGGAAACTGTTTAAAAAAATTGAAAAAGTGGGCCAGAACATTCGCGATGGCA 9 coding gene TTATTAAAGCGGGCCCGGCGGTGGCGGTGGTGGGCCAGGCGACCCAGATTGCGAA A linker GSGSGS 12 Endolysin MGTVLKRGDRGSAVEDLQMKLNVAGYNLSADGIFGGDTEKAVRDVQAGAGLVVDG 6 LNT102 KVGPKTLYAIAKSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGVDSRLLAT FASIESAFDYTVKAKTSSATGWFQFLDATWDDMIKAYGSKYGIPKDPTRALRKDP RANALMGAEFIKGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGE VLFPKPAKANPSIFSNKGVPRTLAEIYKLFEDKVSKHRA Endolysin ATGGGTACTGTACTCAAACGTGGCGACCGCGGCTCTGCTGTGGAAGATCTACAAA 7 LNT102 TGAAACTTAACGTCGCAGGATACAACCTGAGCGCTGACGGAATCTTCGGTGGAGA coding gene TACAGAGAAAGCTGTTCGTGATGTGCAAGCTGGCGCGGGCTTGGTGGTTGACGGA AAAGTTGGACCTAAAACTCTATATGCGATTGCCAAATCCGCTACTGTTCCTGCTA AATGGGAAGCTATCCCTTTCCCAACAGCTAATAAATCTCGGTCGGCTGCAATGCC CACTCTGAATGCGGTTGGAGCAATGACTGGTGTGGATTCTCGGTTACTCGCTACA TTCGCTTCCATTGAGTCTGCTTTTGATTACACTGTCAAAGCAAAAACATCTTCGG CTACTGGTTGGTTCCAGTTCCTTGATGCTACATGGGATGACATGATCAAAGCATA TGGTTCCAAATACGGGATACCTAAAGATCCCACTAGGGCACTCCGTAAAGACCCA CGTGCAAATGCATTAATGGGTGCAGAATTCATTAAAGGAAATGCAGCTGTATTAC GTCCAGTAATCAATCGCGAACCGAGTGATACAGACTTGTATTTGGCACATTTCCT TGGTGCTGGCGGCGCTAAGAAATTCCTATCCGCAGATCAGAAAACTCTCGGTGAA GTTCTATTCCCGAAACCTGCTAAAGCAAACCCGTCGATCTTTAGCAATAAAGGTG TACCACGTACCCTTGCAGAGATCTACAAGCTGTTCGAAGATAAAGTTTCGAAACA TCGGGCATAG Fusion MKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKGSGSGSMGTVLKRGDRG 10 Polypeptide SAVEDLQMKLNVAGYNLSADGIFGGDTEKAVRDVQAGAGLVVDGKVGPKTLYAIA LNT103 KSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGVDSRLLATFASIESAFDYT VKAKTSSATGWFQFLDATWDDMIKAYGSKYGIPKDPTRALRKDPRANALMGAEFI KGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGEVLFPKPAKANP SIFSNKGVPRTLAEIYKLFEDKVSKHRA Fusion ATGAAATGGAAACTGTTTAAAAAAATTGAAAAAGTGGGCCAGAACATTCGCGATG 11 Polypeptide GCATTATTAAAGCGGGCCCGGCGGTGGCGGTGGTGGGCCAGGCGACCCAGATTGC LNT103 GAAAGGCAGCGGCTCGGGTAGTATGGGTACTGTACTCAAACGTGGCGACCGCGGC coding gene TCTGCTGTGGAAGATCTACAAATGAAACTTAACGTCGCAGGATACAACCTGAGCG CTGACGGAATCTTCGGTGGAGATACAGAGAAAGCTGTTCGTGATGTGCAAGCTGG CGCGGGCTTGGTGGTTGACGGAAAAGTTGGACCTAAAACTCTATATGCGATTGCC AAATCCGCTACTGTTCCTGCTAAATGGGAAGCTATCCCTTTCCCAACAGCTAATA AATCTCGGTCGGCTGCAATGCCCACTCTGAATGCGGTTGGAGCAATGACTGGTGT GGATTCTCGGTTACTCGCTACATTCGCTTCCATTGAGTCTGCTTTTGATTACACT GTCAAAGCAAAAACATCTTCGGCTACTGGTTGGTTCCAGTTCCTTGATGCTACAT GGGATGACATGATCAAAGCATATGGTTCCAAATACGGGATACCTAAAGATCCCAC TAGGGCACTCCGTAAAGACCCACGTGCAAATGCATTAATGGGTGCAGAATTCATT AAAGGAAATGCAGCTGTATTACGTCCAGTAATCAATCGCGAACCGAGTGATACAG ACTTGTATTTGGCACATTTCCTTGGTGCTGGCGGCGCTAAGAAATTCCTATCCGC AGATCAGAAAACTCTCGGTGAAGTTCTATTCCCGAAACCTGCTAAAGCAAACCCG TCGATCTTTAGCAATAAAGGTGTACCACGTACCCTTGCAGAGATCTACAAGCTGT TCGAAGATAAAGTTTCGAAACATCGGGCATAG Endolysin MGTVLKRGDRGSAVEDLQMKLRVAGYAVSADGIFGGDTEKAVRDFQASKALVVDG 1 LNT101 KVGPATLAELAKSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGTDSRLLAT FASIESAFDYTVKASTSSATGWFQFLDATWDDMIKAHGSKYGIPKDPTRALRKDP RANALMGAEFLKGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGE VLFPKPAKANPSIFSNKGVPRTLAEIYKLFEDKVSKHRA Endolysin ATGGGTACTGTACTCAAACGTGGCGACCGCGGCTCTGCTGTGGAAGATCTACAAA 2 LNT101 TGAAACTTCGAGTCGCAGGATACGCAGTTAGCGCTGACGGAATCTTCGGTGGAGA coding gene TACAGAGAAAGCTGTTCGTGATTTCCAAGCTTCTAAAGCTTTGGTGGTTGACGGA AAAGTTGGACCTGCTACTCTAGCTGAACTAGCCAAATCCGCTACTGTTCCTGCTA AATGGGAAGCTATCCCTTTCCCAACAGCTAATAAATCTCGGTCGGCTGCAATGCC CACTCTGAATGCGGTTGGAGCAATGACTGGTACCGATTCTCGGTTACTCGCTACA TTCGCTTCCATTGAGTCTGCTTTTGATTACACTGTCAAAGCATCCACATCTTCGG CTACTGGTTGGTTCCAGTTCCTTGATGCTACATGGGATGACATGATCAAAGCACA TGGTTCCAAATACGGGATACCTAAAGATCCCACTAGGGCACTCCGTAAAGACCCA CGTGCAAATGCATTAATGGGTGCAGAATTCCTTAAAGGAAATGCAGCTGTATTAC GTCCAGTAATCAATCGCGAACCGAGTGATACAGACTTGTATTTGGCACATTTCCT TGGTGCTGGCGGCGCTAAGAAATTCCTATCCGCAGATCAGAAAACTCTCGGTGAA GTTCTATTCCCGAAACCTGCTAAAGCAAACCCGTCGATCTTTAGCAATAAAGGTG TACCACGTACCCTTGCAGAGATCTACAAGCTGTTCGAAGATAAAGTTTCGAAACA TCGGGCATAG Fusion MKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKGSGSGSMGTVLKRGDRG 13 Polypeptide SAVEDLQMKLRVAGYAVSADGIFGGDTEKAVRDFQASKALVVDGKVGPATLAELA CecA-LNT101 KSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGTDSRLLATFASIESAFDYT VKASTSSATGWFQFLDATWDDMIKAHGSKYGIPKDPTRALRKDPRANALMGAEFL KGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGEVLFPKPAKANP SIFSNKGVPRTLAEIYKLFEDKVSKHRA Fusion ATGAAATGGAAACTGTTTAAAAAAATTGAAAAAGTGGGCCAGAACATTCGCGATG 14 Polypeptide GCATTATTAAAGCGGGCCCGGCGGTGGCGGTGGTGGGCCAGGCGACCCAGATTGC CecA-LNT101 GAAAGGCAGCGGCTCGGGTAGTATGGGTACTGTACTCAAACGTGGCGACCGCGGC coding gene TCTGCTGTGGAAGATCTACAAATGAAACTTCGAGTCGCAGGATACGCAGTTAGCG CTGACGGAATCTTCGGTGGAGATACAGAGAAAGCTGTTCGTGATTTCCAAGCTTC TAAAGCTTTGGTGGTTGACGGAAAAGTTGGACCTGCTACTCTAGCTGAACTAGCC AAATCCGCTACTGTTCCTGCTAAATGGGAAGCTATCCCTTTCCCAACAGCTAATA AATCTCGGTCGGCTGCAATGCCCACTCTGAATGCGGTTGGAGCAATGACTGGTAC CGATTCTCGGTTACTCGCTACATTCGCTTCCATTGAGTCTGCTTTTGATTACACT GTCAAAGCATCCACATCTTCGGCTACTGGTTGGTTCCAGTTCCTTGATGCTACAT GGGATGACATGATCAAAGCACATGGTTCCAAATACGGGATACCTAAAGATCCCAC TAGGGCACTCCGTAAAGACCCACGTGCAAATGCATTAATGGGTGCAGAATTCCTT AAAGGAAATGCAGCTGTATTACGTCCAGTAATCAATCGCGAACCGAGTGATACAG ACTTGTATTTGGCACATTTCCTTGGTGCTGGCGGCGCTAAGAAATTCCTATCCGC AGATCAGAAAACTCTCGGTGAAGTTCTATTCCCGAAACCTGCTAAAGCAAACCCG TCGATCTTTAGCAATAAAGGTGTACCACGTACCCTTGCAGAGATCTACAAGCTGT TCGAAGATAAAGTTTCGAAACATCGGGCATAG Fusion MASKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKGSGSGSMGTVLKRGD 15 Polypeptide RGSAVEDLQMKLNVAGYNLSADGIFGGDTEKAVRDVQAGAGLVVDGKVGPKTLYA LNT103 IAKSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGVDSRLLATFASIESAFD with MAS, YTVKAKTSSATGWFQFLDATWDDMIKAYGSKYGIPKDPTRALRKDPRANALMGAE His tag, FIKGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGEVLFPKPAKA and extra NPSIFSNKGVPRTLAEIYKLFEDKVSKHRALEHHHHHH sequence Fusion MASKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKGSGSGSMGTVLKRGD 16 Polypeptide RGSAVEDLQMKLRVAGYAVSADGIFGGDTEKAVRDFQASKALVVDGKVGPATLAE CecA- LAKSATVPAKWEAIPFPTANKSRSAAMPTLNAVGAMTGTDSRLLATFASIESAFD LNT101 with YTVKASTSSATGWFQFLDATWDDMIKAHGSKYGIPKDPTRALRKDPRANALMGAE MAS, His FLKGNAAVLRPVINREPSDTDLYLAHFLGAGGAKKFLSADQKTLGEVLFPKPAKA tag, and NPSIFSNKGVPRTLAEIYKLFEDKVSKHRALEHHHHHH extra sequence

3.3. Evaluation of Outer Membrane Permeabilization Activity of Fusion Polypeptide LNT103

[0195] In order to evaluate the outer membrane permeabilization activity of the gram negative bacterium of the fusion polypeptide prepared in the Example 3.2, NPN uptake assay was performed. As representative gram negative bacterium, Pseudomonas aeruginosa and Acinetobacter baumannii were used for the test.

[0196] Pseudomonas aeruginosa (PA01; ATCC 15692) or Acinetobacter baumannii (ATCC 19606) was cultured by OD.sub.600=0.3 and centrifuged at 1,000 g for 10 minutes, and then it was resuspended with a ½ volume of 5 mM HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid) (pH 7.2). 40 μM NPN (1-N-phenylnaphthylamine) solution (40 μM NPN in 5 mM HEPES, pH7.2) 50 μl, the test substance (LNT101, LNT102 or LNT103) 50 μl, and strain suspension solution 100 μl (total 200 μl) were added to a microplate and reacted at 37° C. for 1 hour. Then, fluorescence was measured under the condition of excitation 350 nm and emission 420 nm with a microplate reader (Infinite M200 Pro, TECAN). As a positive control group, Cecropin A (SEQ ID NO: 8), EDTA, or polymyxin B was used, and as a negative control group, 10 μM NPN solution was used. All the experiments were performed in 3 sets.

[0197] The obtained result was shown in FIG. 13. In order to compare the outer membrane permeability activity of the LNT101, LNT102 and LNT103 against gram negative bacterium (Pseudomonas aeruginosa (PA01; ATCC 15692) and Acinetobacter baumannii (ATCC 19606)), the concentration of 2 μM which was the same concentration was treated, and 2 μM of Cecropin A and polymyxin B and 1 mM of EDTA were treated. The obtained result was shown in A and B of FIG. 13 as a fold change over control. As shown in A and B of FIG. 13, it was confirmed that the fusion polypeptide LNT103 had higher outer membrane permeabilization activity against gram negative bacterium than the polypeptides LNT101 and LNT102 at the same concentration, and it was confirmed that it was higher even than the positive control groups, cecropin A, EDTA and polymyxin B.

[0198] On the other hand, in order to confirm the difference in the outer membrane permeabilization activity against gram negative bacterium (Pseudomonas aeruginosa (PA01; ATCC 15692) and Acinetobacter baumannii (ATCC 19606)) according to the concentration of LNT103, LNT103 was treated in an amount of 0.29 μM, 0.88 μM, or (2.65 μM), and 2 μM of cecropin A and polymyxin B, and 1 mM of EDTA were treated. The obtained result was shown in C and D of FIG. 13 as a fold change over negative control. As shown in C and D of FIG. 13, it was confirmed that LNT103 had excellent outer membrane permeabilization activity against gram negative bacterium compared to the LNT101, LNT102, cecropin A and polymyxin B, and the outer membrane permeabilization activity against gram negative bacterium was increased in a concentration-dependent manner.

3.4. Investigation of Killing Ability Against Gram Negative Bacteria of Fusion Polypeptide LNT103

[0199] In order to confirm the killing ability against gram negative bacterium of the fusion polypeptide LNT103 prepared in Example 3.2, CFU reduction evaluation was performed for various gram negative bacterium. In order to perform the CFU reduction evaluation, 2 μM of each of LNT101, LNT102, and LNT103 prepared in Example 3, and 1×10.sup.6 CFU of each of Pseudomonas aeruginosa (PA01; ATCC 15692), Acinetobacter baumannii (ATCC 17978), Escherichia coli (ATCC 8739), Klebsiella pneumoniae (ATCC 13883), and Enterobacter aerogenes (CCARM 16006) were added to reaction buffer (20 mM Tris-Cl, pH7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours. After that, the number of the colonies of each gram negative bacterium was confirmed to compare and evaluate the antibiotic effect of each polypeptide. As a control group, the PBS treatment group (treating PBS in the same volume as the fusion polypeptide) was used.

[0200] The obtained result was shown in FIG. 14. As shown in FIG. 14, it was confirmed that the fusion polypeptide LNT103 showed the CFU reduction effect of 1.5˜5.5 log CFU/ml compared to LNT101 and LNT102. In particular, the CFU Counting result (log CFU/ml) against Acinetobacter baumannii (ATCC 19606, ATCC 17978), E. coli (ATCC 8739), Klebsiella pneumoniae (ATCC 2208) of the fusion polypeptide LNT103 killed all bacterium treated as 0.

[0201] In addition, the same test was performed by using the fusion polypeptide LNT103, and CecA-LNT101 at a concentration of 2 μM, and the result was shown in FIG. 15. For comparison, the same test was performed for groups treated with LNT102 or PBS. As shown in FIG. 15, all the groups treated with the fusion polypeptide LNT103, and CecA-LNT101 had excellent antibiotic activity than the groups treated with LNT102 or PBS, and in particular, the counting result (log CFU/ml) for all the tested bacterium was shown as 0 in case of treating LNT103, and CecA-LNT101 in an amount of 2 μM, respectively, and therefore, it was confirmed that they killed all the bacterium.

[0202] On the other hand, in order to compare the antibacterial activity in case that a protein other than endolysin (LNT102 or LNT101) was fused to Cecropin A, the killing ability against gram negative bacterium of the fusion polypeptide LNT103 and cecropin A-EGFP fusion protein (a protein in which EGFP (GenBank Accession No. AAB02572.1) having no endolysin activity by replacing LNT102 in LNT103) was evaluated. For this, the proteins were treated in an amount of 0.2 μM or 2 μM, respectively, to perform the CFU reduction test. The obtained result was shown in FIG. 16. As shown in FIG. 16, it was confirmed that the Cecropin A-EGFP fusion protein had no antibacterial activity, while the fusion polypeptide LNT103 had excellent antibacterial activity. In particular, it could be confirmed that the CFU Counting result for all the tested gram negative bacterium killed all bacterium treated with 0, when LNT103 2 μM was treated.

[0203] Furthermore, the killing ability against gram negative bacterium according to the concentration and/or treatment time of the fusion polypeptide LNT103 was tested. Specifically, the fusion polypeptide LNT103 at a concentration of 0.1, 0.3, 0.9, or 2.7 μM and 1×10.sup.6 CFU of Pseudomonas aeruginosa (PA01; ATCC 15692) or Acinetobacter baumannii (ATCC 19606) were added to reaction buffer (20 mM Tris-Cl, pH7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours, and then the number of the colonies was confirmed, and the result was shown in A of FIG. 17.

[0204] In addition, the fusion polypeptide LNT103 at a concentration of 2 μM and 1×10.sup.6 CFU of Pseudomonas aeruginosa (PA01; ATCC 15692) were added to reaction buffer (20 mM pH7.5) so that the final volume was 200 μl, and in 0, 20 minutes, 40 minutes and 60 minutes at 37° C., the number of the colonies was confirmed, and the result was shown in B of FIG. 17.

[0205] Moreover, the fusion polypeptide LNT103 at a concentration of 1 μM and 1×10.sup.6 CFU of Acinetobacter baumannii (ATCC 19606) were added to reaction buffer (20 mM pH7.5) so that the final volume was 200 μl, and in 0, 1 minute, 3 minutes, 5 minutes and 10 minutes at 37° C., the number of the colonies was confirmed, and the result was shown in C of FIG. 17.

[0206] As shown in A, B and C of FIG. 17, it was confirmed that the fusion polypeptide LNT103 had the killing ability against gram negative bacterium in a concentration and time-dependent manner (in A, B and C of FIG. 17, the part where the bar graph is not represented means that the log CFU/ml value is 0, meaning that all treated bacteria are killed).

[0207] In addition, MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) of the fusion polypeptide LNT103 were measured. The MIC and MBC were performed by broth microdilution technique according to the standard test method of CLSI (Clinical and Laboratory Standards Institute), and in the corresponding test method, it was performed by using CAA media (Casamino acid 5 g/L, K2HPO4 5.2 mM, MgSO4 1 mM) instead of MH broth (Casein acid hydrolysate 17.5 g/L, Beef extract 3.0 g/L, Starch 1.5 g/L. pH 7.3) (Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacterium that grow aerobically; approved standard. 11.sup.th ed. Document M07. Wayne, Pa.: CLSI; 2018). Specifically, the LNT103 was treated at a concentration of 64 μg/ml to a 96 well microplate row A, and the LNT103 was treated at a concentration where serial dilution was performed by ½ in rows B-G. To the row H, LNT103 was not treated. After that, the corresponding bacterium strain (type or QC, CCARM and clinical separation strain; See Tables 2-4) was treated in an amount of 5×10.sup.5 CFU/ml, total 100 μl in all wells. Then, it was cultured at 37° C. for 18 hours. MIC was determined to be the lowest concentration at which no colonies were identified by confirming the number of the colonies in each well. The obtained result was shown in Table 7 to Table 9 below:

TABLE-US-00007 TABLE 7 MIC MBC Species Strain (μg/ml) (μg/ml) A. baumannii ATCC 17978 8 8 ATCT 19606 8 8 KACC 13090 8 8 KACC 14233 8 8 CCARM 12001 8 8 CCARM 12015 16 16 CCARM 12026 8 8 CCARM 12035 8 8 CCARM 12202 8 8 F4 16 16 F15 8 8 F65 4 4 F66 4 4 F67 8 8 F68 4 4 F69 8 8 F70 16 16 F71 8 8 3680(Gyeongsang 8 8 National University) 643(Kyungpook 8 16 National University)

TABLE-US-00008 TABLE 8 MIC MBC Species Strain (μg/ml) (μg/ml) P. ATCT 13388 8 16 aeruginosa ATCC 9027 16 16 ATCC 10145 16 32 PAO1(ATCC 8 16 15692) ATCC 15522 16 16 CCARM 2134 8 16 CCARM 2144 32 32 F102 8 8 F141 8 8 F341 8 8 F388 8 8 F265 16 16

TABLE-US-00009 TABLE 9 MIC MBC Species Strain (μg/ml) (μg/ml) E. coli ATCC 8739 8 8 CCARM 1460 4 4 CCARM 1A746 8 8 CCARM 1B684 4 8 CCARM 1G448 4 4 FORC81 4 4 F340 16 16 F481 8 8 F485 4 8 F524 16 16 F716 16 32 F852 16 32 F859 32 32 F862 4 4 UPEC 3042 32 32 UPEC 3181 8 8

3.5. Synergy Effect of Killing Ability Against Gram Negative Bacteria According to Use in Combination of Fusion Polypeptide LNT103 and Polymyxin Antibiotic

[0208] The synergy effect of killing ability against gram negative bacterium by combination treatment of a polymyxin-based antibiotic having a mechanism acting on the cell membrane of bacterium and the fusion polypeptide LNT103 was confirmed. Specifically, colistin (polymyxin E) of 16 μl/ml was treated to a 96 well microplate column 1, and it was treated at a concentration where serial dilution was performed by ½ in columns 2-11. The LNT103 of 16 μg/ml was treated to row A, and it was treated at a concentration where serial dilution was performed by ½ in rows B-G. After that, Acinetobacter baumannii (ATCC 19606) was treated in an amount of 5×10.sup.5 CFU/ml, total 100 μl in all wells. Then, it was cultured at 37° C. for 18 hours, and the FIC (Fractional Inhibitory Concentration) index value was calculated by the following equation:

FIC index=FIC.sub.A+FIC.sub.B=(C.sub.A/MIC.sub.A)+(C.sub.B/MIC.sub.B)

[0209] (C.sub.A and C.sub.B are each concentration of substances treated in combination (polymyxin E and LNT103), and MIC.sub.A and MIC.sub.B are MIC of single drug,

[0210] FIC value: synergy effect <0.5; antagonism >4; additive 0.5-4)

[0211] As the result of the test, the FIC index value was shown as 0.375, and it was confirmed that there was a synergy effect between the two substances.

3.6. In Vitro Toxicity Evaluation of Fusion Polypeptide LNT103

[0212] In vitro toxicity of the fusion polypeptide LNT103 was evaluated by cell cytotoxicity assay (WST assay) using Huh-7 cell line and hemolysis assay using sheep blood (MB cell).

[0213] The process of progressing the cell cytotoxicity assay was as follows. The Huh07 cell culture solution was prepared and aliquoted in an amount of 1×10.sup.9 cells/well per well in a 96 well plate, and it was cultured in a CO.sub.2 incubator for 24 hours. Then, PBS as a negative control and 1%(w/v) triton X-100 as a positive control were treated, and as an experimental group, the fusion polypeptide LNT103 was treated at a concentration of 0.25, 0.5, or 1 mg/ml, and then it was left in a CO.sub.2 incubator for 24 hours and 48 hours. Then, the cell viability was measured using D-Plus™ CCK cell viability assay kit (Dongin LS), and the result was shown in FIG. 18. As shown in FIG. 18, when the fusion polypeptide LNT103 was treated at a concentration of 0.25 mg/ml, 0.5 mg/ml, or 1 mg/ml for 24 hours, compared to the negative control (PBS), the cell viability of 93%, 90%, 78% was shown, respectively, and when it was treated for 48 hours, compared to the negative control, the cell viability of 108%, 105%, 86% was shown, respectively. In other words, it was confirmed that it showed the cell viability of about 80% or more compared to the control group (PBS) in case of treating the fusion polypeptide LNT103, and thereby, it could be confirmed that the cytotoxicity was relatively low. By this result, it was confirmed that the IC50 of LNT103 was >1 mg/ml.

[0214] The process of progressing the hemolysis assay was as follows. After mixing 3 ml sheep blood and 14 ml PBS (pH 7.2), it was centrifuged at 1000 g, 4° C. for 5 minutes to remove the supernatant. After that, PBS was filled again as much as the removed volume and the above washing was progressed 4 times in total. Hemoglobin lysed through washing was removed, and after the last centrifugation, 400 μl of sunken RBC (red blood cell) was dissolved in 9.6 ml PBS to make 4%(v/v) blood solution. After mixing 50 μl of the sample and 50 μl of 4%(v/v) blood solution in a 96 well microplate, it was reacted at 37° C. for 1 hour. For an experimental group, the fusion polypeptide LNT103 was treated from 128 μg/ml to 2 μg/ml in a 2-fold dilution range (PBS, pH 7.2), and for a positive control group, 0.1%(w/v) Triton X-100 and for a negative control group, PBS were treated. After reacting for 1 hour, the microplate was centrifuged at 1000 g, 4° C. for 5 minutes, and 50 μl of the supernatant was collected and it was transferred to a new microplate and the absorbance was measured at 570 nm (Infinite M200 Pro, TECAN). The obtained result was shown in FIG. 19. As shown in FIG. 19, it was confirmed that the fusion polypeptide LNT103 did not show hemolytic activity at all the tested concentrations. By this result, it was confirmed that the MHC (Minimal hemolytic concentration) of LNT103 was >128 μg/ml.

3.7. In Vitro Effectivity Evaluation of Fusion Polypeptide LNT103

[0215] For in vivo effectivity evaluation of the fusion polypeptide LNT103, the viability was confirmed in an Acinetobacter baumannii ATCC 19606 systemic infection mouse model was confirmed.

[0216] The process of progressing an animal experiment was as follows. Mice were used when ICR male 4-week-old mice were purchased and a one-week adaptation period was passed, and the weight was 20˜21 g at 5-6 weeks of age. The Acinetobacter bacteria were mixed with 10% mucin at 1:1, and 0.5 ml of 2×10.sup.8 CFU/ml (5% mucin) of the bacterial solution was inoculated intraperitoneally into mice. In 1 hour and 4 hours after infection, LNT103 20 mpk and LNT103 100 mpk, and colistin 20 mpk as a comparative group were administered, respectively, through a subcutaneous injection. After that, the viability for 96 hours was confirmed, and the result was shown in FIG. 20. As confirmed in FIG. 20, the group in which the LNT103 was administered into the systemic infection mouse model showed higher viability compared to the non-administration group (control) and comparative group (colistin administration group), and the LNT103 showed high viability in a concentration-dependent manner, and therefore, it was confirmed that there was the effect in the corresponding mouse model.

[0217] From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be executed in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that Examples described above are illustrative and not restrictive in all respects. The scope of the present application should be construed as that all changed or modified forms derived from the meaning and scope of claims to be described below and equivalent concepts rather than the detailed description are included in the scope of the present application.