Novel polypeptides and antibiotics against Gram-negative bacterium comprising the same

Abstract

Provided are a novel polypeptide having endolysin activity, a fusion protein comprising the polypeptide and an antibiotic active protein, and an antibiotic use against a gram-negative pathogen of the polypeptide and/or fusion protein and/or a use for prevention and/or treatment of gram-negative pathogen infection and/or disease or symptoms related to gram negative pathogen infection.

Claims

1. A polypeptide, comprising: (1) the amino acid sequence of SEQ ID NO: 2, or (2) a mutation of at least one position selected from the group consisting of the 39th, 43th, 45th, 73th, 81th, 101th, and 113th in the amino acid sequence of SEQ ID NO: 2.

2. The polypeptide according to claim 1, wherein the mutation is at least one selected from the group consisting of the following 1) to 7): 1) a substitution of the amino acid corresponding to the 39th residue in the amino acid sequence of SEQ ID NO: 2 with serine, arginine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; 2) a substitution of the amino acid corresponding to the 43th residue in the amino acid sequence of SEQ ID NO: 2 with histidine, arginine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; 3) a substitution of the amino acid corresponding to the 45th residue in the amino acid sequence of SEQ ID NO: 2 with glutamic acid, arginine, histidine, lysine, aspartic acid, serine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; 4) a substitution of the amino acid corresponding to the 73th residue in the amino acid sequence of SEQ ID NO: 2 with valine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; 5) a substitution of the amino acid corresponding to the 81th residue in the amino acid sequence of SEQ ID NO: 2 with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; 6) a substitution of the amino acid corresponding to the 101th residue in the amino acid sequence of SEQ ID NO: 2 with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; and 7) a substitution of the amino acid corresponding to the 113th residue in the amino acid sequence of SEQ ID NO: 2 with valine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

3. The polypeptide according to claim 1, wherein the mutation comprises: 1) a substitution of the amino acid corresponding to the 39th residue in the amino acid sequence of SEQ ID NO: 2 with serine, 2) a substitution of the amino acid corresponding to the 43th residue in the amino acid sequence of SEQ ID NO: 2 with histidine, 3) a substitution of the amino acid corresponding to the 45th residue in the amino acid sequence of SEQ ID NO: 2 with glutamic acid, 4) a substitution of the amino acid corresponding to the 73th residue in the amino acid sequence of SEQ ID NO: 2 with valine, 5) a substitution of the amino acid corresponding to the 81th residue in the amino acid sequence of SEQ ID NO: 2 with serine, and 6) a substitution of the amino acid corresponding to the 101th residue in the amino acid sequence of SEQ ID NO: 2 with alanine.

4. The polypeptide according to claim 3, wherein a mutation further comprises a substitution of the amino acid corresponding to the 113th residue in the amino acid sequence of SEQ ID NO: 2 with valine.

5. The polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 6.

6. The polypeptide according to claim 1, further comprising Cecropin A at N-terminus or C-terminus.

7. The polypeptide according to claim 6, wherein the Cecropin A comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

8. The polypeptide according to claim 6, comprising the amino acid sequence of SEQ ID NO: 10.

9. The polypeptide according to claim 1, having antibiotic activity against gram-negative bacterium.

10. The polypeptide according to claim 6, having antibiotic activity against gram-negative bacterium.

11. A polynucleotide encoding the polypeptide of claim 1 or a fusion polypeptide comprising the polypeptide and Cecropin A.

12. A bacteriophage, comprising a polynucleotide encoding the polypeptide of claim 1 or a fusion polypeptide comprising the polypeptide and Cecropin A.

13. The bacteriophage according to claim 12, comprising the nucleic acid sequence of SEQ ID NO: 1.

14. A method for inhibiting growth of gram negative bacterium or killing gram negative bacterium, comprising administering a pharmaceutically effective dose of at least one selected from the group consisting of: the polypeptide of claim 1; a fusion polypeptide comprising the polypeptide and Cecropin A; a polynucleotide encoding the polypeptide or the fusion polypeptide, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the polynucleotide or the recombinant vector.

15. The method according to claim 14, further comprising administering a pharmaceutically effective dose of a polymyxin-based antibiotic.

16. The method according to claim 15, wherein the polymyxin-based antibiotic is polymyxin B, colistin or a combination thereof.

17. The method according to claim 14, wherein the gram-negative bacterium is at least one selected from the group consisting of Pseudomonas sp. bacterium, Acinetobacter sp. bacterium, Escherichia sp. bacterium, Enterobacter sp. bacterium and Klebsiella sp. bacterium.

18. A method for preventing or treating infection of gram negative bacterium or disease caused by gram negative bacterium, comprising administering a pharmaceutically effective dose of at least one selected from the group consisting of: the polypeptide of claim 1; a fusion polypeptide comprising the polypeptide and Cecropin A; a polynucleotide encoding the polypeptide or the fusion polypeptide, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the polynucleotide or the recombinant vector.

19. The method according to claim 18, further comprising administering a pharmaceutically effective dose of a polymyxin-based antibiotic.

20. The method according to claim 19, wherein the polymyxin-based antibiotic is polymyxin B, colistin or a combination thereof.

21. The method according to claim 18, 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; the gram negative bacterium is Acinetobacter sp. bacterium, and the disease caused by Acinetobacter sp. bacterium is skin infection, pneumonia, bacteremia or septicemia; or 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0247] FIG. 1 is a cleavage map of the expression vector expressing the endolysin (EC340) gene of the bacteriophage EC340-M-11-12 infecting Escherichia coli.

[0248] FIG. 2 shows the purification process of the endolysin (EC340) derived from the bacteriophage EC340-M-11-12.

[0249] FIG. 3 is a graph showing the antibiotic effect against Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa in vitro of the endolysin (EC340) derived from the bacteriophage EC340-M-11-12.

[0250] FIG. 4a and FIG. 4b show the composition and enzymatic activity of the endolysin used in the present application.

[0251] FIG. 4a shows the domain structure of EC340, mtEC340 and LNT113. It has a predicted lysozyme domain without a cell wall binding domain (CBD). The EC340 of the E. coli phage PBEC131 received several substitutions of amino acids (mtEC340). The positions of the substituted amino acids are indicated. In the mtEC340, cecropin A was fused to the N-terminus, and it was named LNT113.

[0252] FIG. 4b shows the analysis result of the purified endolysin, EC340 (19.5 kDa), mtEC340 (18.2 kDa) and LNT113 (23.2 kDa) in the SDS-PAGE and follow-up zymogram analysis.

[0253] FIG. 5a to FIG. 5d show the antibacterial activity of endolysin.

[0254] FIG. 5a to FIG. 5d show the antibacterial activity of endolysin (2 μM) against E. coli strain (FIG. 5a, FIG. 5c; ATCC 8739, ATCC 25922, CCARM 1A746, CCARM 1B684) and K. pneumoniae strain (FIG. 5b, FIG. 5d; ATCC 700603, KCTC 2208, CCARM 10143, F85) in 20 mM Tris-HCl buffer solution (pH 7.5) for 2 hours. Tris buffer was used as a negative control group (Control). The dotted line indicates the detection limit. In addition to the t-test, two-way ANOVA was performed, and it was indicated by a horizontal bar above a vertical bar (* p<0.05, **p<0.01, ***p<0.001).sub..

[0255] FIG. 6a to FIG. 6e show the cell penetration and antibacterial activity of LNT113.

[0256] FIG. 6a: The outer membrane permeability of the gram-negative E. coli ATCC 8739 was determined by NPN analysis. CecA-EGFP, Cecropin A fused to EGFP; cecA+mtEC340, cecropin A and mtEC340 were added to the mixture of 2 μM, respectively; PMB, polymyxin B.

[0257] FIG. 6b shows the enhanced antibacterial activity against E. coli ATCC8739 of LNT113 compared to Cecropin A and/or mtEC340. All were treated at the final concentration of 0.5 mM.

[0258] FIG. 6c shows the antibacterial activity of LNT113 (0.1, 0.3, 0.9 or 2.7 μM) at various concentrations against E. coli ATCC 8739.

[0259] FIG. 6d shows that the survival rate of the bacterium (E. coli ATCC8739) was reduced after adding endolysin (0.5 μM) at different lengths of time. The dotted line indicates the detection limit.

[0260] FIG. 6e shows the antibacterial activity of LNT113 against MCR-1 positive E. coli FORC81. Cells were treated with LNT113 or colistin at a concentration of 0.2 or 2 μM for 2 hours. The dotted line indicates the detection limit. The asterisk shows a statistical difference with the control group (*p<0.05, **p<0.01, ***p<0.001).

[0261] FIG. 7a to FIG. 7b show the cytotoxicity and hemolytic activity of LNT113.

[0262] FIG. 7a shows the analysis of the cytotoxicity of LNT113 in the Huh7 cell line. Cells were incubated with LNT113 at a different concentration for 24 hours or 48 hours. As a positive control group of cytotoxicity, Triton X-100 was used.

[0263] FIG. 7b shows the hemolytic activity of LNT113. The hemolytic activity was confirmed by culturing red blood cells with PBS or LNT113 at 37° C. for 1 hour and measuring the absorbance of the supernatant at 570 nm. As a positive control group of the hemolytic activity, Triton X-100 0.1% was used.

[0264] FIG. 8 shows the synergistic effect of LTN113 when treated in combination with colistin against three different E. coli strains. The isobologram. FIC index (FICI) of LNT113 and colistin shows the sum of FIC (Fractional Inhibitory Concentration) of LNT113 and colistin.

[0265] FIG. 9 shows the amino acid sequence alignment of putative endolysin used for site-directed mutagenesis of EC340 (CLUSTAL omega) (multiple sequence alignment of EC340 endolysin-like protein using CLUSTAL omega (version 1.2.4)).

[0266] FIG. 10a and FIG. 10b show the antibacterial activity of endolysin against E. coli ATCC8739 (FIG. 10a) and K. pneumoniae KCTC2208 (FIG. 10b). Exponentially grown bacterial cells were cultured with 2 μM endolysin in two different buffer solutions (20 mM Tris-HCl, pH 7.5 or 20 mM HEPES, pH 7.5) in presence or absence of 150 mM NaCl at 37° C. for 2 hours. The dotted line indicates the detection limit. In addition to the t-test, two-way ANOVA was performed, and it was indicated by a horizontal bar above a vertical bar (* p<0.05, **p<0.01, ***p<0.001).

MODE FOR INVENTION

[0267] Hereinafter, the present invention will be described in detail by examples.

[0268] The following examples are intended to illustrate the present invention only, and are not construed as limiting the present invention.

[0269] Material and Method

Preparation Example 1. Bacteriophage EC340-M-11-12 Isolation and EC340 Endolysin Purification

[0270] 1.1. Culturing Condition of Strain Escherichia coli (ATCC 8739) was used as a host, and was cultured with shaking in an LB (Luria-Bertani) medium under the condition of 37° C.

[0271] 1.2. Isolation of Bacteriophage

[0272] In order to select a bacteriophage infecting Escherichia coli, samples were collected from Gwacheon Sewage Treatment Plant in Gwacheon-si, Gyeonggi-do, Korea. The collected samples and Escherichia coli were cultured with shaking at 37° C. for 3 hours, and then centrifuged by 500 rpm for 20 minutes to collect the supernatant. After that, the supernatant was filtered with a 0.45 μm filter, and then double agar layer plaque assay was performed.

[0273] Briefly describing the assay, the culture solution of the host bacterium Escherichia coli and bacteriophage was mixed with 0.1 M.O.I. to the top agar 5 ml, and poured into an agar plate, and cultured at 37° C. for 24 hours to obtain plaques. It was possible to secure the purified pure bacteriophage through repeated performance of the process, and this bacteriophage was named bacteriophage EC340-M-11-12.

[0274] 1.3. Genome Isolation and Analysis of Bacteriophage EC340-M-11-12

[0275] Sequencing for genome of the bacteriophage EC340-M-11-12 obtained in Preparation example 1.2. above was conducted. After culturing Escherichia coli in an LB medium of 200 ml to OD.sub.600=0.5, herein, it was lysed by infection with the filtered bacteriophage 10.sup.9 pfu/ml or 0.1 M.O.I., and then, sodium chloride was added so that the final concentration was to be 1 M, and then was left at 4° C. for 1 hour. Then, after centrifuging at 11,000×g for 10 minutes, PEG (Polyethylene glycol 8000) was added to the precipitate at 10% (w/v), and was placed at 4° C. for 1 hour. After that, it was centrifuged at 11,000×g for 10 minutes, and then the supernatant was removed, and the precipitate was suspended with SM buffer solution [100 mM NaCl, 10 mM MgSO.sub.4 (heptahydrate), 50 mM Tris-HCl, pH 7.5]. Herein, chloroform was added at a ratio of 1:1 and vortexed, and then centrifuged at 3,000×g for 15 minutes to obtain a supernatant.

[0276] 3 ml of 40% (w/v) glycerol was added in a polycarbonate test tube, and then, 4 ml of 5% (w/v) glycerol was added without mixing. Herein, the prepared supernatant was added and centrifuged at 11,000×g at 4° C. for 1 hour. After that, the supernatant was removed, and then the precipitate was resuspended with SM buffer solution to obtain bacteriophage genome DNA. The bacteriophage genome DNA was isolated using a phage DNA isolation kit (Norgen biotek corp.) according to the manufacturer's manual. The nucleotide sequence for genome was analyzed using the genome sample isolated as above (LAS, Illumina MiSeq platform).

[0277] The finally analyzed bacteriophage EC340-M-11-12genome has a total nucleic acid sequence length of 42,751 bp. The full-length nucleic acid sequence of the bacteriophage EC340-M-11-12 genome was shown in SEQ ID NO: 1. Based on the genome nucleic acid sequence information, using BLAST on Web, the similarity with the conventionally known bacteriophage genome sequence was investigated. As the result of BLAST investigation, it was confirmed that the genome sequence of the bacteriophage EC340-M-11-12 had the sequence homology of query coverage: 80%, identity: 93.45% to the Escherichia bacteriophage vB_EcoS-Golestan (GenBank accession No.: NC_042084.1). Based on this fact, it was confirmed that the bacteriophage EC340-M-11-12 is a new bacteriophage which is not known conventionally.

[0278] 1.4. Cloning and Purification of EC340M Endolysin

[0279] Through ORF search for the genome sequence (SEQ ID NO: 1) of the bacteriophage EC340-M-11-12 analyzed, it was assumed that the ORF of 489 bp (SEQ ID NO: 3) was an endolysin gene, and the endolysin derived from the bacteriophage EC340-M-11-12 and a gene encoding the same were named EC340 endolysin and EC340 gene, respectively.

[0280] Using primers (F: 5′-AAGGATCCGTGTCTCGAAACATTAGCAACAATGGC-3′(SEQ ID NO: 4), R: 5′-AACTCGAGACCTTTCACCGCGCGCC-3′ (SEQ ID NO: 5)), for genome of the bacteriophage EC340-M-11-12, PCR (polymerase chain reaction) was performed to obtain the EC340 gene (nucleic acid sequence of 489 bp length in SEQ ID NO: 1). The amino acid sequence of the EC340 endolysin encoded by the EC340 gene was shown in SEQ ID NO: 2 (162 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 step 2-4 30 times; step 6: 72° C., 10 minutes. The obtained PCR product was cloned with BamHI/Xhol site of pET-21a vector with N-terminal 6× His-tag (Novagen), to prepare an expression vector for expressing the EC340 endolysin (pET-EC340 plasmid). The prepared expression vector pET-EC340 was schematically shown in FIG. 1.

[0281] The prepared pET-EC340 plasmid was transformed to E. coli BL21-pLysS strain (Novagen), and then cultured in LB broth (1% Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) by OD.sub.600=0.5. Then, 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 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. The 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, 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 EC340 protein (comprising 6×His tag).

[0282] The purity of the EC340 protein was confirmed by 15% SDS-PAGE, and the concentration of the EC340 protein was measured by Bradford assay. The result of confirming each reactant obtained during the purification process was shown in FIG. 2. The molecular weight of the purified EC340 protein confirmed through SDS-PAGE was about 19 kDa.

Preparation Example 2. Bacterium Strain and Culturing Condition

[0283] The strain used in the present research was acquired from American Type Culture Collection (ATCC, USA), Korean Collection for Type Cultures (KCTC, Korea) and Culture Collection of Antimicrobial Resistant Microbes (CCRM, Korea). The F strain and uropathogenic E. coli (UPEC) strain were clinically isolated and provided by professor KwanSoo Ko (Sungkyunkwan University, College of Medicine). The adherent invasive E. coli (Adherent invasive E. coli (AIEC)) collection (ECOR) strain was provided by professor Christel Neut (Rahmouni et al., 2018). The MCR-1 positive E. coli FORC81 strain was provided by professor Sangryoul Rye (Seoul National University) (Kim et al., 2019). All the strains used in this work were grown in an LB (lysogeny broth) or CAA medium (5 g/l casamino acid, 5.2 mM K2HPO4 and 1 mM MgSO4) at 37° C.

Preparation Example 3. Bacteriophage and Endolysin

[0284] The bacteriophage PBEC131 used in the following example was same as obtained in Preparation example 1.

[0285] The gene expressing putative endolysin (SEQ ID NO: 3) was obtained from the bacteriophage PBEC131, and the putative lysozyme-like superfamily domain was found by BLASTp.

Preparation Example 4. Molecular Cloning

[0286] The gene encoding the putative endolysin of the bacteriophage PBEC131 (SEQ ID NO: 3) was cloned into pET21a+ (Novagen, USA) using BamHI and XhoI restriction sites and named EC340 gene. The EC340 polypeptide was represented by the amino acid sequence of SEQ ID NO: 2.

[0287] The mutant EC340 (mtEC340; SEQ ID NO: 6) was produced by substituting 7 amino acids in the amino acid sequence of SEQ ID NO: 2 (FIG. 4a; H39S, D43H, T45E, A73V, A81S, T101A, and I113V).

[0288] LNT113 (SEQ ID NO: 10) was constructed by fusing the antibacterial peptide Cecropin A (NCBI PRF 0708214A; SEQ ID NO: 8) with a (GGGGS).sub.3 linker at the N-terminus of the mutant EC340 (mtEC340; SEQ ID NO: 6). (In other words, in the following example, the fusion polypeptide comprising Cecropin A and the mutant polypeptide (mtEC340) was named LNT113.)

[0289] All the components used in the examples of the present application have a C-terminal hexa-histidine tag (HHHHHH) for affinity purification. In addition, a gene encoding an enriched green fluorescent protein (EGFP; GenBank accession no. AAB02576.1) was cloned with a gene encoding cecropin A (CecA) at the 5′ end into the pET21a+ vector.

TABLE-US-00002 Used sequences: Recombinant mtEC340 having the His(6H) tag [methionine(Met) + mtEC340 (SEQ ID NO: 6) + His tag(HHHHHH)] (SEQ ID NO: 7) (SEQ ID NO: 7) MVSRNISNNGIKFTAAFEGFRGTAYRATPNEKYLTIGYGSYGPHVEPGK TITPGQGLLLLNRDMAKAVAAVDAVAHHSLTQSQFDAVCDLVYNAGAGV IAAATGTGKALRSGDVATLRAKLALFINQNGKPLLGLRRRTAGRLALFD GKPWQEAEAIGRAVKGLEHHHHHH Recombinant fusion protein LNT113 [methionine(Met) + Cecropin A + GGGGSx3 linker +  mtEC340 + His tag(HHHHHH)] (SEQ ID NO: 11) (SEQ ID NO: 11) MKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKGGGGSGGGGSG GGGSVSRNISNNGIKFTAAFEGFRGTAYRATPNEKYLTIGYGSYGPHVE PGKTITPGQGLLLLNRDMAKAVAAVDAVAHHSLTQSQFDAVCDLVYNAG AGVIAAATGTGKALRSGDVATLRAKLALFINQNGKPLLGLRRRTAGRLA LFDGKPWQEAEAIGRAVKGLEHHHHHH

Preparation Example 5. Recombinant Protein Purification

[0290] For protein expression, an Endolysin expression vector was introduced to E. coli BL21 (DE3) Star (Invitrogen, USA). For expressing a plasmid retaining a gene encoding Cecropin A (CecA) fusion EGFP, it was introduced into E. coli BL21(DE3) pLysS (Novagen). After growing cells in an LB broth to an exponential phase (OD600=0.4-0.5), they were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG; Duchefa, The Netherlands) at a concentration of 0.5 mM at 25° C. for 5 hours. Bacterial cells were harvested by centrifugation and pellets were washed with phosphate buffered saline (PBS). Cells were resuspended with dissolution buffer solution (20 mM Tris-HCl, pH 7.5, 300 mM NaCl and 20 mM imidazole) and destroyed by ultrasonication (Sonics, USA) for 5 minutes. The supernatant was obtained from the cell lysate by centrifugation at 15,000×g for 30 minutes. The extract was loaded in a Ni-NTA affinity chromatography column using FPLC (AKTA go, Cytiva, UK). Protein was eluted using a linear gradient of 20 to 500 mM imidazole. The protein was then loaded onto a HiTrap SP HP column (Cytiva) for cation exchange chromatography, and eluted with a linear gradient of NaCl from 0 to 1 M in 20 mM Tris-HCl, pH 7.5. The protein was then dialyzed against storage buffer solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl buffer solution (pH 7.5)).

Preparation Example 6. Zymogram Assay

[0291] Zymogram assay was performed with some modifications of the previously described method (Khakhum et al., 2016). In other words, an overnight culture of E. coli ATCC 8739 was harvested, washed with phosphate buffered solution (PBS) once, and then harvested by centrifugation at 4,000×g for 15 minutes. Then, the pellets were resuspended in 3 ml of deionized water. The cells were autoclaved and added to a 15% SDS-PAGE gel prior to polymerization. Then, 3 μg of purified endolysin (EC340, mtEC340 or LNT113) was mixed with 2× sample buffer (0.5 mM Tris-HCl, pH 6.8, 20% glycerol, 0.2% bromophenol blue) and loaded on SDS-PAGE. After electrophoresis, the gel was washed with deionized water for 1 hour and incubated in reaction buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5) at a room temperature. The enzymatic activity of endolysin was observed in a clear area of the gel containing E. coli lysate.

Preparation Example 7. Antibacterial Activity Analysis

[0292] The antibacterial activity of the purified protein was tested for E. coli and various K. pneumoniae strains. The bacterium were grown to an exponential phase (OD600=0.5) and harvested by centrifugation at 12,000×g for 3 minutes. Then, the pellets were washed with reaction buffer solution (20 mM Tris-HCl, pH 7.5) and diluted to about 10.sup.6 cells/ml in the buffer solution. Then, 100 μl bacterial suspension was mixed with the purified endolysin of 100 μl in the reaction buffer solution and incubated at 37° C. for 2 hours. Finally, the mixture was diluted with PBS and loaded on an LB plate. After culturing at 37° C. overnight, the bacterial colonies were counted. All the analysis was performed in triplicate.

[0293] The same reaction was performed for the selected subject bacterium in 20 mM HEPES-NaOH, 150 mM NaCl [pH 7.4], and the unique antibacterial activity of Tris and dependence of endolysin for turgor pressure were excluded (FIG. 10a and FIG. 10b). FIG. 10 shows the effect of NaCl for the antibacterial activity of endolysin for E. coli ATCC 8739(A) (FIG. 10a) and K. pneumoniae KCTC 2208(B) (FIG. 10b). The bacterial cells grown geometrically were cultured with 2 μM endolysin in low tonicity buffers (20 mM Tris-HCl, pH 7.5 or 20 mM HEPES, pH 7.5) or high tonicity buffers (buffer solution comprising 150 mM NaCl) at 37° C. for 2 hours. The cells treated with storage buffer solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl buffer solution (pH 7.5)) were used as a negative control group. The dotted line represents the detection limit (1 Log CFU/ml).

Preparation Example 8. 1-N-Phenylnaphthylamine (NPN) Absorption Analysis

[0294] NPN absorption analysis was performed by the previously described method (Helander and Mattila-Sandholm, 2000). In other words, E. coli ATCC 8739 grown to an exponential phase (OD600=0.4) was washed and resuspended with buffer solution (5 mM HEPES, pH 7.2) to about 10.sup.8 cells/ml. 40 μM NPN (Sigma-Aldrich) 50 μl was mixed with the purified endolysin or Cecropin A (AbClon, Korea) 50 μl so that the final concentration was 2 μM in a 96-well black plate. Outer membrane permeation agents, 1 mM EDTA (Duchefa) and 2 μM Polymyxin B (Sigma-Aldrich) were used as a positive control group. Then, cell suspension 100 μl was added to each well, and cultured at 37° C. for 5 minutes. Buffer alone, buffer+NPN, cell suspension+buffer and cell suspension+buffer+NPN were used as a negative control group. Fluorescence was measured at excitation (350 nm) and emission (420 nm) using a microplate reader (SpectraMax iD3, Molecular Devices, USA). An NPN absorption factor was calculated by dividing the fluorescence value after background removal (the value that the value without NPN subtracted from the fluorescence value of the cell mixture with the sample) with the fluorescence value of the cell suspension with buffer subtracted from the fluorescence value of buffer without NPN.

Preparation Example 9. Hemolysis Analysis

[0295] Red blood cells (Sheep RBC) of sheep were used for the in vitro hemolytic activity test. 1 ml red blood cells (MB Cell, Korea) were diluted with 9 ml PBS. Then, 180 μl RBC solution was added to 20 μl LNT113 (final concentration, 2-128 μg/ml), PBS (negative control group) or 0.1% Triton X-100 (positive control group) and cultured at 37° C. for 30 minutes. The mixture was centrifuged at 500×g for 5 minutes, and the supernatant was transferred to a 96-well microplate. Absorbance was measured at 570 nm. The hemolysis rate was calculated using Equation 1 below.

[00001]$\begin{array}{cc}\%\mathrm{Hemolysis}=\left\{\frac{\left[\mathrm{Abs}\left(\mathrm{Sample}\right)-\mathrm{Abs}\left(\mathrm{negativecontrol}\right)\right]}{\left[\mathrm{Abs}\left(\mathrm{positivecontrl}\right)-\mathrm{Abs}\left(\mathrm{negativecontrol}\right)\right]}\right\}\times 100& \left[\mathrm{Equation}1\right]\end{array}$

Preparation Example 10. In Vitro Cytotoxicity Analysis

[0296] The human hepatocellular carcinoma cell line Huh7 (obtained from Korea Cell Line Bank) was used for cytotoxicity analysis. First, 1×10.sup.4 Huh7 cells were inoculated in a 96-well plate. After 24 hours, LNT113 (final concentration, 125 or 250 μg/ml), 1% Triton X-100 (positive control group) or PBS (negative control group) was added to the cells and cultured for 24 hours or 48 hours. Then, tetrazolium salt solution 10 μl of WST-8 Cell Viability Assay Kit (Dyne Bio, Korea) was added to each well. Production of formazan was measured at 450 nm after culturing in a 5% CO2 incubator at 37° C. for 1 hour.

Preparation Example 11. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MB C) Measurement

[0297] The MIC of the LNT113 or antibiotic was determined using modification of the broth microdilution method in a 96-well, round bottom, microplate according to the previously described method (Heselpoth et al., 2019). Exponentially grown cells were diluted to a concentration of 10.sup.6 CFU/ml in a CAA medium, and then cultured with recombinant LNT113 (1-64 μg/ml) or antibiotics at 35° C. for 20 hours. The MIC value was determined as the lowest antibacterial concentration that completely inhibited bacterial growth. The MBC was defined as the lowest concentration of the antibacterial agent with no growth on a plate. All analyses were performed in duplicate.

Preparation Example 12. Checkerboard Assay

[0298] Checkerboard assay was performed using a serial dilution method as previously described (Thummeepak et al., 2016). LNT113 was vertically diluted 2-fold and the antibiotic was serially diluted horizontally in a 96-well plate. Bacterium at a concentration of 10.sup.6 CFU/ml were added to each well in a CAA medium. After incubating at 35° C. for 20 hours, the MIC was visually confirmed for non-growing cells. The fractional inhibitory concentration (FIC) for the LNT113 or antibiotic was calculated by dividing the MIC of two drugs by combining it with the MIC of each drug alone. In order to confirm interaction of the two drugs, the FIC index (FICI), which is the sum of the FICs of each drug, was used. The FICI was considered as a synergistic effect ≤0.5, an additive >0.5 ˜≤1, an independent effect (indifference)>1.0 ˜≤2, and an antagonistic effect (antagonism) >2.

Preparation Example 13. Statistical Analysis

[0299] Prism version 9 (GraphPad software) was used for all statistical analysis. For in vitro research, all experiments were performed in triplicate and the result was provided as mean±standard error of mean (SEM). Differences between each data set were compared using two-way Anova with Tukey's multiple comparison test.

[0300] Experimental Result

Example 1. Investigation of Killing Activity of Endolysin EC340 Against Gram-Negative Bacterium

[0301] In the present example, in order to confirm the antibacterial activity of endolysin EC340 against Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (ATCC 13388) and Escherichia coli (ATCC 8739), the bacterial killing activity was tested.

[0302] In Vitro Test

[0303] For this, endolysin EC340 at a concentration of 2 μM and each test bacterium were added to reaction buffer (20 mM Tris-Cl, pH 7.5) so that the final concentration was to be 200 μl, and were left at 37° C. for 2 hours. After 2 hours, the number of colonies of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli was confirmed, and the result was shown in FIG. 3. As shown in FIG. 3, it was confirmed that endolysin EC340 had the killing activity against Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli.

Example 2. Bacteriophage PBEC131-Derived Endolysin and Modification Thereof

[0304] The ORF consisting of 162 amino acids of bacteriophage PBEC131 was annotated with putative endolysin. This endolysin, designated EC340 (SEQ ID NO: 2), has a phage-associated lysozyme (muramidase) domain (pfam00959) (FIG. 4a). This gene was cloned into an expression vector and expressed, and the enzymatic activity was observed by performing zymogram assay with the purified endolysin (See Preparation example 6) (EC340 of FIG. 4b).

[0305] To enhance the activity of endolysin, BLASTp was performed with 8 proteins having a sequence very similar to EC340 (GenBank accession no. QQNO11705.1, QN011629.1, QN011777.1, QBJ02951.1, HAM5207786.1, YP_009168880.1, QIG59335.1, and YP_009113200.1) (FIG. 9). In addition, computer-aided modeling of proteins was conducted at https://swissmodel.expasy.org/. In particular, it was thought that changes in amino acids H39S, A73V, T101A and 1113V exposed to the outside could lead to better transmembrane by increasing hydrophobicity of proteins. D43H and T45E alterations are in the externally exposed hinge region, where a switch of charged amino acids may result in a more sterically stable property for the corresponding region. The change in A81S located in the third helix region of proteins may not result in noticeable changes in properties of proteins. Mutation was introduced at 7 amino acid positions in the enzymatic active domain of EC340 (FIG. 4a; H39S, D43H, T45E, A73V, A81S, T101A, and I113V), and some changes were made to GRAVY (Grand average of hydropathicity index; index used to indicate a hydrophobicity value of a peptide). In other words, the mutant polypeptide mtEC340 represented by the amino acid sequence of SEQ ID NO: 10 is a polypeptide in which H39S, D43H, T45E, A73V, A81S, T101A, and I113V mutations are introduced, in EC340 represented by the amino acid sequence of SEQ ID NO: 2.

[0306] The mtEC340 mutation showed the increased antibacterial activity by up to 1 log at maximum (FIG. 5a to FIG. 5d) when tested against various strains of E. coli or K. pneumoniae including type strains, drug-resistant strains and clinically isolated strains (resistance profiles are shown in Table 1 below). Higher antibacterial efficacy against endolysin was observed in E. coli than K. pneumoniae. This is consistent with the previous report that the capsule thickness of K. pneumoniae is 16 times or more than that seen in E. coli.

TABLE-US-00003 TABLE 1 MIC of various antibiotics against drug-resistant strains MIC (μg/ml) E. coli K. pneumoniae of antibiotics CCARM 1A746 CCARM 1B684 CCARM 10143 Ampicillin ≥128 ≥128 ≥128 Cephalothin ≥128 64 ≥128 Ciprofloxacin 128 64 ≤0.25 Gentamicin ≥128 128 ≥128 Tetracycline 128 ≥128 2 Cefotaxime 128 0.25 16 Trimethoprim- ≥128 — ≥32 sulfamethoxazole Streptomycin ≥128 ≥128 — Norfloxacin ≥128 32 — Source: http://knrrb.ccarm-bio.or.kr —: not determined

[0307] In other words, as could be confirmed in FIG. 5a to FIG. 5d, as the result of measuring the number of bacterium in case of treating nothing (control), treating EC340, treating mtEC340 and treating LNT113 to E. coli ATCC8739, E. coli ATCC25922, E. coli CCARM1A746, E. coli CCARM1B684, K. pneumoniae ATCC700603, K. pneumoniae KCTC2208, K. pneumoniae CCARM10143, and K. pneumoniae F85 strains, the excellent antibacterial activity was shown in all the EC340, mtEC340, and LNT113. It was confirmed that the mtEC340 and LNT113 showed more excellent antibacterial activity among them.

[0308] Furthermore, ATCC700603, one of K. pneumoniae strains used in the present experiment, was known to have a capsule structure. Thick capsules have the potential to impede endolysin access to the peptidoglycan cell wall. K. pneumoniae capsule polysaccharides have been reported to mediate resistance to antibacterial peptides. In order to further increase the activity, 37 amino acid sequences (SEQ ID NO: 8) of cecropin A, which is an antibacterial peptide, were fused to the N-terminus of mtEC340, thereby constructing endolysin LNT113 (SEQ ID NO: 10). This showed a maximum 4-log increase in activity when compared to mtEC340. No viable bacterial cells were detected after culturing for 2 hours for all the tested strains. Since the antibacterial activity of endolysin is potentially dependent on turgor pressure and Tris itself can act as an outer membrane permeation agent, additional antibacterial analysis was performed under various physiological conditions (Tris buffer comprising 150 mM NaCl or Hepes buffer comprising 150 mM NaCl (FIG. 10 a and FIG. 10b)). Unexpectedly, the antibacterial activity was higher in Hepes buffer than Tris buffer without NaCl. However, adding NaCl almost inactivated EC340 and mtEC340. However, as previously reported, the negative effect of salt presence was significantly reduced when cecropin A-fused endolysin (LNT113) was used.

Example 3. Antibacterial Efficacy Enhanced by Enhanced Cell Permeability of LNT113

[0309] In general, it is difficult for external endolysin to reach the peptidoglycan layer because of presence of an outer membrane in gram-negative bacterium. Fusion with cecropin A interacting with the outer membrane should increase membrane penetration.

[0310] Through NPN (1-N-phenylnaphthylamine) analysis (See Preparation example 8), the transmembrane ability of various members including cecropin A and/or endolysin was compared (FIG. 6a).

[0311] As could be confirmed in FIG. 6a (outer membrane permeability of E. coli ATCC 8739), it was observed that the membrane penetration was increased 1.8 times in the cells treated with LNT113 fused with cecropin A (LNT113 of FIG. 6a). It was also 1.3-fold higher than that of cecropin A alone (CecA of FIG. 6a). In addition, when mtEC340 and cecropin A were co-administered (CecA+mtEC340 in FIG. 6a), the membrane permeability was increased. Conversely, cecropin A fused to EGFP (CecA+EGFP of FIG. 6a) showed a limited ability to permeate the membrane when compared to LNT113, and this suggests that the partnership of AMP and endolysin showing a strong ability to permeate the membrane is necessary to achieve an additional effect.

[0312] The bactericidal efficacy of each construct shown in FIG. 6b was proportional to its ability to permeate the membrane as shown in FIG. 6a. In vitro, LNT113 exhibited the antibacterial activity according to dose and time (E. coli ATCC 8739 (FIG. 6c) and FIG. 6d). E. coli FORC81, a colistin-resistant strain, was tested for susceptibility to LNT113 (E. coli FORC81 (FIG. 6e)). A 1.2-log decrease was observed in colistin-treated cells at a concentration of 2 μM, whereas at the same concentration, LNT113 succeeded in reducing the amount of bacterial cells below the detection limit.

Example 4. Determination of Minimal Inhibitory Concentration (MIC)

[0313] E. coli is related to various disease of humans and animals. Pathogenic E. coli causes disease by forming colonies in various parts of the human body, such as urinary tract, kidney, bloodstream and the like. Through MIC measurement (See Preparation example 11), the antibacterial activity of LNT113 in various E. coli strains was confirmed (Table 2).

TABLE-US-00004 TABLE 2 MIC and MBC of LNT113 for various E. coli strains Strains MIC MBC Strains MIC MBC Strains MIC MBC ATCC 8739 8 8 UPEC 90 64 64 ECOR 1 8 8 ATCC 25922 64 64 UPEC 3038 16 16 ECOR 2 8 8 ATCC 51739 8 8 UPEC 3042 16 16 ECOR 9 4 4 CCARM 1A746 4 4 UPEC 3051 16 16 ECOR 15 32 32 CCARM 1G490  16 16 UPEC 3150 8 8 ECOR 35 16 16 F485 4 4 UPEC 3151 64 64 ECOR 36 >64 >64 F524 8 8 UPEC 3163 16 16 ECOR 43 16 16 F576 4 4 UPEC 3164 32 32 ECOR 45 4 4 F716 >64 >64 UPEC 3168 8 8 ECOR 52 64 >64 F852 8 16 UPEC 3181 8 8 ECOR 69 >64 >64 FORC81 8 8

[0314] The target strain included type strains, drug-resistant strains (CCRM and FORC81), clinically isolated strains (F), uropathogenic strains (UPEC) and adherent invasive strains (ECOR). The MIC was shown to be 4-64 μg/ml in most of the strains (LNT113 of 1 mM was 23.2 mg/ml and LNT113 of 1 mg/ml was 0.0431 mM). The MIC for endolysin EC340 or mtEC340 was >128 mg/ml (Table 3).

[0315] In addition, the minimal bactericidal concentration (MBC) was same as MIC, suggesting that the action mode is sterilization. The MIC of LNT113 was confirmed for various gram-negative bacterium such as A. baumannii, P. aeruginosa, and K. pneumoniae (Table 4).

TABLE-US-00005 TABLE 3 MIC of endolysin combined with colistin in E. coli strain (μg/ml) ATOCB739 UPEC3150 FORC81 ATCC8738 ATCC8738 LNT113 Colistin LNT113 Colistin LNT113 Col istin EC340 Colistin mtEC340 Colistin 8 0 8 0 8 0 >128 0 >128 0 4 0.0625 4 0.0625 4 1 32 0.0625 32 0.125 2 0.5 2 0.5 2 4 16 0.125 16 0.125 1 1 1 2 <0.25 8 <2 0.25 <2 0.25 0 2 0 4 0 16 0 2 0 2

TABLE-US-00006 TABLE 4 MIC of LNT113 against various gram-negative bacterium (μg/ml) A. baumannii P. aeruginosa K. pneumoniae K. acrogenes Strains MIC Strains MIC Strains MIC Strains MIC ATCC 8 PA01 4 ATCC 16 CCARM 16 700603 16006 ATCC 16 ATCC 8 KCTO 8 CCARM 8 15522 2208 16008 CCARM 8 F102 16 CCARK 4 CCARM 8 10143 16010 F4 4 F125 4 P1O4 16 F276 F65 8 F141 16 Fil8 16 E. cloacae F66 8 F171 32 F144 16 ATCC 13047 16 F67 8 F388 32 CCARM 4 0252 CCARM 8 16003

[0316] The strain includes type strains, clinically isolated strains and drug-resistant strains. The MIC of the antibiotic for the drug-resistant strain used was described in Table 1 above. The MIC ranged from 4-32 μg/ml. In case of Salmonella typhimurium and Salmonella enteritidis, the MIC was >64 μg/ml.

Example 5. Cytotoxicity and Hemolytic Activity of LNT113

[0317] In order to confirm the cytotoxicity of LNT113, WST-1 analysis was performed for human liver cancer cell line Huh7 (See Preparation example 10). When LNT113 was treated by 250 or 125 μg/ml for 48 hours, reduction of the cell survival rate was not observed (FIG. 7a).

[0318] In order to measure the hemolytic activity, LNT113 was added to red blood cells at a concentration of 2-128 μg/ml. The hemolytic activity of LNT113 was not observed (FIG. 7b).

Example 6. Synergistic Effect of LNT113 and Various Antibiotics

[0319] In order to confirm the effect of binding LNT with various antibiotics, checkerboard assay was performed (See Preparation example 12) At first, the FICI (fractional inhibitory concentration index) was measured to observe the combination effect of LNT113 and colistin in the E. coli strain. The checkerboard analysis result represented by isobologram including FIC floating of LNT113 and colistin was shown in FIG. 8. The sum (FICI) of two values was 0.25 when colistin and LNT113 were treated in E. coli ATCC 8739. This shows the synergistic effect of two agents. In case of the UPEC 3150 strain, the FICI was 0.375 and the synergistic effect was showed. In case of E. coli FORC81, the FICI was 0.5 and another synergistic effect was shown. It is noteworthy that the MIC of colistin in the colistin-resistant E. coli FORC81 decreased from 16 mg/ml to 1 mg/ml when treated in combination with LNT113 of 4 mg/ml (Table 3).

[0320] The synergistic effect between the LNT113 and 8 different antibiotics was confirmed for 5 different E. coli strains by determining the FICI (Table 5).

TABLE-US-00007 TABLE 5 Fractional inhibitory concentration index (FICI) of LNT113 using various antibiotics E. coli strains ATCC ATCC UPEC CCARM Antibiotics 8739 51739 3150 1A746 FORC81 Colistin 0.25 0.5 0.38 0.63 0.5 Ceftazidime 2 2 1 2 1 Meropenem 0.63 0.75 1 1 2 Kanamycin 2 1 1 n.d.* 1 Tigecycline 2 0.63 0.63 2 2 Chloramphenicol 1 0.75 0.75 n.d.* 2 Azithromycin 1 0.75 0.75 1 2 Ciprofloxacin 0.75 0.75 0.75 1 0.56 (*not determined FICI: ≤0.5, synergy; >0.5-≤1.0, additive; >1.0 to ≤2, indifference; >2.0, antagonism)

[0321] While the synergistic effect with colistin was observed in 4 strains among 5 strains, an additive or indifferent effect was observed in all the other combinations.

[0322] In addition, it was observed that the MIC of colistin was significantly reduced, when there was the endolysin EC340 or mtEC340 having the MIC of >128 mg/ml (Table 3).

[0323] From the above description, those skilled in the rat to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the examples described above are illustrative and not restrictive in all respects. The scope of the present application should be construed as including all changed or modified forms derived from the meaning and scope of the claims to be described later and equivalent concepts thereof rather than the detailed description.