Polypeptide permeable through outer membrane of gram-negative bacterium, antibacterial protein comprising said polypeptide, antibacterial agent or disinfectant, pharmaceutical composition, and antibacterial agent composition or disinfectant composition

Abstract

Provided are: a polypeptide of any one of the following items (i) to (iii); an antibacterial protein comprising the polypeptide; an antibacterial agent; a disinfectant; a pharmaceutical composition; an antibacterial agent composition; and a disinfectant composition. (i) A polypeptide comprising the amino acid sequence represented by SEQ ID NO:1; (ii) a polypeptide comprising an amino acid sequence having a structure such that one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO:1; or (iii) a polypeptide comprising an amino acid sequence having sequence identity of 80% or higher with the amino acid sequence represented by SEQ ID NO:1.

Description

Outer membrane permeable polypeptide of gram-negative bacteria, antibacterial protein containing said polypeptide, antibacterial agent or disinfectant, pharmaceutical composition, and antibacterial agent composition or disinfectant composition

[0001] The present invention relates to an outer membrane-permeable polypeptide of Gram-negative bacteria, an antibacterial protein containing said polypeptide, an antibacterial agent or disinfectant, a pharmaceutical composition, and an antibacterial agent composition or disinfectant composition.

[0002] In this specification, the term "polypeptide having permeability through the lipopolysaccharide-containing outer membrane of Gram-negative bacteria" may be abbreviated as "outer membrane-permeable polypeptide."

[0003] Pseudomonas aeruginosa is a pathogenic bacterium that is inherently drug-resistant and can also acquire drug resistance, causing intractable infections in humans and animals (such as cattle and dogs). It is also included in the WHO list of urgently needed drug-resistant bacteria. Today, there is a need to develop new antibacterial agents against Pseudomonas aeruginosa.

[0004] Research into small molecule compounds, which are traditional antibacterial drugs, is ongoing due to the development system. However, the library of small molecule compounds is limited, making it extremely difficult to discover antibacterial drugs with novel mechanisms of action. Due to this situation, antibacterial peptides, phages, and endolysins are currently being researched as completely different new modalities for antibacterial drugs.

[0005] Until now, research on endolysins has focused on natural phage endolysins.

[0006] US Pat. No. 5,629,999 discloses an endolysin protein against staphylococci.

[0007] Non-patent documents 1 to 3 disclose endolysin proteins of Gram-negative bacteria.

[0008] Gram-negative bacteria have an outer membrane in their cell walls, and natural endolysins alone cannot kill them. However, natural endolysins capable of killing such Gram-negative bacteria do not have the bactericidal effect necessary for the treatment of infectious diseases.

[0009] Patent Publication No. 2021-112152

[0010] Lai WCB, Chen X, Ho MKY, Xia J, Leung SSY. Bacteriophage-derived endolysins to target gram-negative bacteria. Int J Pharm. 589, 119833, 2020.De Maesschalck V, Gutierrez D, Paeshuyse J, Lavigne R, Briers Y. Advanced engineering of third-generation lysins and formulation strategies for clinical applications.Crit Rev Microbiol. 46(5), 548-564, 2020. Gontijo MTP, Jorge GP, Brocchi M. Current Status of Endolysin-Based Treatments against Gram-Negative Bacteria. Antibiotics (Basel). 10(10), 1143, 2021.

[0011] One object of the present invention is to provide an antibacterial agent or disinfectant that is effective against Gram-negative bacteria, including multidrug-resistant bacteria, particularly Pseudomonas aeruginosa.

[0012] Another object of the present invention is to provide a polypeptide capable of increasing the permeability of endolysin through the outer membrane of Gram-negative bacteria, and a polynucleotide encoding the same.

[0013] The present invention provides the following outer membrane-permeable polypeptides, antimicrobial proteins and polynucleotides encoding the same, antimicrobial agents or disinfectants, pharmaceutical compositions, and antimicrobial or disinfectant compositions. [1] Polypeptides having the ability to permeate the lipopolysaccharide-containing outer membrane of Gram-negative bacteria, which are any of the following (i) to (iii): (i) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 1: NSGTPKNVSRGTSSTKTTPKYKVKNGDNLTKIAKKHNTTVATLLKLNPGIKDPNMIRVGQTLNVTGSGGKTHKVKSGDTLSKIAVDNKTTVSKLMNLNPEITNPNHIKVGQTIRLSRKLRRLKRKIAHKVKKY (ii) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted, inserted and / or added, and which has the ability to permeate the lipopolysaccharide-containing outer membrane of Gram-negative bacteria, (iii) a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and which has the ability to permeate the lipopolysaccharide-containing outer membrane of Gram-negative bacteria.[2] Polynucleotides of (iv) to (vi) below: (iv) a polynucleotide consisting of a DNA sequence encoding the polypeptide of [1], (v) a polynucleotide consisting of the DNA sequence represented by SEQ ID NO: 2, SEQ ID NO: 2: AATTCTGGCACGCTAAGAACGTATCGCGCGGCACGTCATCCACGAAGACGACGCCTAAGTACAAGGTAAAGAACGGCGATAATCTCACCAAAATTGCCAAGAAACACAATACGACAGTGGCTACTTTATTAAAACTCAATCCAGGCATTAAAGATCCGAACATGATTCGTGTCGGTCAAACATTAAATGTGACAGGGAGCGGCGGTAAGACACACAAAGTAAAGAGTGGTGATACCTTGAGCAAGATTGCCGTGGATAATAAGACTACAGTTAGTAAGTTGATGAATTTGAATCCTGAGATCACAAATCCTAATCATATCAAAGTAGGGCAGACCATTCGCTTAAGCCGGAAGCTCCGTCGGCTTAAACGTAAGATCGCTCACAAGGTAAAAAAATAT (vi)

[0013]

[0014] A polynucleotide consisting of a DNA sequence having 80% or more sequence identity with the polynucleotide consisting of the DNA sequence of (iv) or (v), and encoding a polypeptide that has permeability to the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. [3] An antibacterial protein comprising an endolysin and the polypeptide of [1], wherein the polypeptide of claim 1 is located on the N-terminal or C-terminal side of the endolysin. [4] A polynucleotide encoding the antibacterial protein of [3]. [5] An antibacterial agent or disinfectant for Gram-negative bacteria, comprising the antibacterial protein of [3]. [6] The antibacterial agent or disinfectant of [5], wherein the Gram-negative bacterium is a multidrug-resistant bacterium or Pseudomonas aeruginosa. [7] A pharmaceutical composition comprising the antibacterial protein of [3]. [8] The pharmaceutical composition according to [7], wherein the disease targeted by Pseudomonas aeruginosa is one or more diseases selected from keratitis, dacryocystitis, endophthalmitis, eye infections due to cellulitis, sepsis, pneumonia, multidrug-resistant Gram-negative bacterial infections, opportunistic infections, and other diseases caused by Pseudomonas aeruginosa.[9] An antibacterial composition or disinfectant composition comprising the antibacterial protein of [3] and an outer membrane permeabilizing agent, wherein the outer membrane permeabilizing agent is ethylenediaminetetraacetic acid (EDTA) or a salt thereof.

[10] The antibacterial composition or disinfectant composition of [9], wherein the Gram-negative bacterium is a multidrug-resistant bacterium or Pseudomonas aeruginosa.

[11] A method for preventing a disease caused by infection with Gram-negative bacteria or killing Gram-negative bacteria, comprising administering to a human a pharmaceutical composition comprising the antibacterial protein of [3].

[12] A method for preventing a disease caused by infection with Gram-negative bacteria or killing Gram-negative bacteria, comprising administering to an animal a pharmaceutical composition comprising the antibacterial protein of [3].

[0014] According to the present invention, it is possible to provide an antibacterial agent, a disinfectant, an antibacterial composition and a disinfectant composition that are effective against Gram-negative bacteria including Pseudomonas aeruginosa, particularly against multidrug-resistant Gram-negative bacteria.

[0015] Viable cell count measurement results Viable cell count measurement results Morphological observation of bactericidal effect against Pseudomonas aeruginosa PAO1 strain Comparison of turbidity and morphological observation of Pseudomonas aeruginosa PAO1 strain Growth inhibitory effect against Pseudomonas aeruginosa Bactericidal effect against Pseudomonas aeruginosa Bactericidal effect against various bacteria Results of a 2-day observation of survival in a septic mouse model Method and results of a therapeutic experiment in Pseudomonas aeruginosa-infected mice Results of effectiveness study on a keratitis mouse model

[0016] As used herein, the singular forms (a, an, the, etc.) include both the singular and the plural unless otherwise specified herein or clearly contradictory in context. As used herein, the term "comprise" is a concept that encompasses "consist essentially of" and "consist of."

[0017] The antimicrobial protein of the present invention comprises an outer membrane-permeable polypeptide and an endolysin. The outer membrane-permeable polypeptide and the endolysin may be linked directly or via a suitable linker. The linker may be a polypeptide containing one amino acid or two or more amino acids.

[0018] As used herein, "amino acid" includes the 20 amino acids that constitute proteins (Gly, Ala, Met, Ser, Thr, Cys, Met, Asp, Asn, Glu, Gln, Leu, Ile, Val, His, Lys, Arg, Phe, Tyr, and Trp).

[0019] As used herein, "amino acid deletion" refers to the deletion of any amino acid selected from the amino acid sequence of an outer membrane-permeable polypeptide or endolysin. "Amino acid insertion" refers to the insertion of one to several amino acids at a site other than the N-terminus or C-terminus of the amino acid sequence of an outer membrane-permeable polypeptide or endolysin. "Amino acid addition" refers to the addition of one to several amino acids to the N-terminus or C-terminus of the amino acid sequence of an outer membrane-permeable polypeptide or endolysin. Examples of amino acid substitutions include conservative amino acid substitutions. "Conservative amino acid substitutions" refer to substitutions between amino acids with similar properties, such as polarity, electrical properties, and structural properties, such as hydrophobic amino acids, polar amino acids, acidic amino acids, basic amino acids, amino acids with branched side chains, and aromatic amino acids. Examples of hydrophobic (non-polar) amino acids include glycine, alanine, valine, leucine, isoleucine, and proline; examples of polar amino acids include serine, threonine, cysteine, methionine, asparagine, and glutamine; examples of acidic amino acids include aspartic acid and glutamic acid; examples of basic amino acids include lysine, arginine, and histidine; examples of branched side chain amino acids include valine, isoleucine, and leucine; and examples of aromatic amino acids include phenylalanine, tyrosine, tryptophan, and histidine. Preferred conservative amino acid substitutions include substitutions between amino acids selected from valine, leucine, and isoleucine, phenylalanine, and tyrosine, lysine and arginine, alanine and valine, and asparagine and glutamine.

[0020] The above-mentioned deletion, substitution, insertion, and / or addition of amino acids can be achieved by modifying the gene encoding the antibacterial protein, outer membrane-permeable polypeptide, or endolysin by techniques known in the art. Mutations can be introduced into the gene by known techniques such as the Kunkel method or the gapped duplex method, or methods equivalent thereto. For example, mutations can be introduced using a site-directed mutagenesis kit (e.g., Mutant-K (manufactured by TAKARA Bio) or Mutant-G (manufactured by TAKARA Bio)) or the LA PCR in vitro Mutagenesis series kit (manufactured by TAKARA Bio).

[0021] The number of amino acids in the linker is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 1, 2, 3, 4, 5, or 6, and even more preferably 1, 2, 3, or 4. The outer membrane-permeable polypeptide and the endolysin are preferably directly linked. Furthermore, the outer membrane-permeable polypeptide is preferably located on the C-terminal side of the endolysin.

[0022] (1) Outer membrane-permeable polypeptides and polynucleotides encoding them Outer membrane-permeable polypeptides are the following polypeptides (i) to (iii): (i) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1; (ii) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted, inserted, and / or added, and which has the ability to permeate the lipopolysaccharide-containing outer membrane of Gram-negative bacteria; (iii) a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and which has the ability to permeate the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. The polypeptide represented by SEQ ID NO: 1 is a polypeptide in which the following D domain and S domain are linked, with the D domain located at the N-terminus and the S domain located at the C-terminus of the polypeptide (DS). A polypeptide having an S domain at the N-terminus and a D domain at the C-terminus (SD) has a significantly weaker effect on enhancing the antibacterial activity of endolysin against Gram-negative bacteria than DS. Amino acid sequence of D domain (SEQ ID NO: 3): NSGTPKNVSRGTSSTKTTPKYKVKNGDNLTKIAKKHNTTVATLLKLNPGIKDPNMIRVGQTLNVTGSGGKTHKVKSGDTLSKIAVDNKTTVSKLMNLNPEITNPNHIKVGQTIRLS Amino acid sequence of S domain (SEQ ID NO: 4): RKLRRLKRKIAHKVKKY A particularly preferred outer membrane permeable polypeptide is that represented by SEQ ID NO: 1.

[0023] The outer membrane-permeable polypeptide may have one or several (e.g., 9, 8, 7, 6, 5, 4, 3, or 2) amino acids deleted, substituted, inserted, and / or added from the polypeptide represented by SEQ ID NO: 1, as long as it has the ability to permeate the lipopolysaccharide-containing outer membrane of Gram-negative bacteria.

[0024] Furthermore, the outer membrane-permeable polypeptide may have 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The sequence identity value is calculated using software (e.g., FASTA, DANASYS, and BLAST) that calculates identity between multiple amino acid sequences with default settings.

[0025] In one preferred embodiment of the present invention, the polynucleotide is a polynucleotide encoding an outer membrane permeable polypeptide.

[0026] Preferred polynucleotides encoding outer membrane-permeable polypeptides of the present invention are: (iv) a polynucleotide consisting of a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (v) a polynucleotide consisting of the DNA sequence shown in SEQ ID NO: 2; and (vi) a polynucleotide consisting of a DNA sequence that has 80% or more sequence identity with the polynucleotide consisting of the DNA sequence of (iv) or (v), and that encodes a polypeptide that has the ability to permeate the lipopolysaccharide-containing outer membrane of Gram-negative bacteria.

[0027] The polynucleotide encoding the outer membrane-permeable polypeptide may have 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to a polynucleotide consisting of a DNA sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide set forth in SEQ ID NO: 2. The sequence identity value is calculated using software (e.g., FASTA, DANASYS, and BLAST) that calculates identity between multiple amino acid sequences with default settings.

[0028] (2) Endolysin Endolysin is a protein having peptidoglycan-degrading activity. As used herein, the term "peptidoglycan-degrading activity" refers to the activity of binding to or recognizing peptidoglycan in the cell walls of Gram-negative bacteria, including Pseudomonas aeruginosa, and cleaving it to specifically cause bacteriolysis.

[0029] As the endolysin, known endolysins are widely used, including, for example, endolysins derived from phages of Gram-negative bacteria. Specific endolysins include a polypeptide called ply_pitti26 isolated from the lytic phage pitti26 and used for treating staphylococcal infections ( JP 2010-536354 A), an endolysin from S. aureus phage φ2638a ( JP Patent No. 6261086 A), an endolysin derived from phage phi MR11 ( J Infect Dis. 196(8):1237-47, (2007)), an endolysin derived from Pseudomonas aeruginosa phage PAJU2 ( Virus Research. 139(1), 131-134, 2009), and an endolysin derived from phage S25-3 ( Viruses, 11(9):769, 2019), two types of endolysins isolated from bacteriophage S6 (JP 2021-112152 A), those disclosed in the endolysins described in Non-Patent Documents 1 to 3, an endolysin derived from Bacillus amyloliquefaciens phage (FEBS Letter. 500(1-2), 56-59, 2001), and Artilysins (mBio. 2014 Jul 1;5(4):e01379-14. doi: 10.1128 / mBio.01379-14).

[0030] The endolysin contained in the antibacterial protein of the present invention may be a modified protein thereof as long as it has the above-mentioned peptidoglycan-degrading activity. Examples of modified proteins include proteins consisting of an amino acid sequence obtained by deleting, substituting, inserting, and / or adding one or several (e.g., 9, 8, 7, 6, 5, 4, 3, or 2) amino acids in the amino acid sequence of a known endolysin and having peptidoglycan-degrading activity, and proteins consisting of an amino acid sequence that shares 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of a known endolysin and having peptidoglycan-degrading activity. The sequence identity value indicates a value calculated using software that calculates identity between multiple amino acid sequences (e.g., FASTA, DANASYS, and BLAST) with default settings.

[0031] (3) Antibacterial Protein and Production Method Thereof The antibacterial protein of the present invention comprises an outer membrane-permeable polypeptide and an endolysin. A preferred antibacterial protein has an endolysin at the N-terminus and an outer membrane-permeable polypeptide at the C-terminus. The antibacterial protein can be tagged with a tag such as a Strep tag, His tag, Flag tag, Xpress tag, Avi tag, calmodulin tag, polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, X tag, SBP tag, Sof tag, V5 tag, CBP, GST, MBP, GFP, thioredoxin tag, or a combination thereof. A 6xHis tag is preferred.

[0032] The antibacterial protein, antibacterial agent, disinfectant, antibacterial composition and disinfectant composition of the present invention are effective against gram-negative bacteria, particularly multidrug-resistant gram-negative bacteria. Gram-negative bacteria include Pseudomonas aeruginosa, Escherichia coli, enterohemorrhagic Escherichia coli (EHEC), Enterobacteriaceae, Vibrio, Campylobacter, Salmonella, Legionella, cholera, Haemophilus influenzae, Klebsiella, Enterobacter, Serratia, pertussis, Yersinia, Pseudomonas, and Helicobacter pylori. More specifically, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter spp., Serratia marcescens, Acinetobacter spp., Proteus spp., Klebsiella oxytoca, Bacteroides spp. including Bacteroides fragilis, Citrobacter spp., Haemophilus influenzae, Burkholderia cepacia, and Stenotrophomonas Examples include maltophilia, Chryseobacterium spp., and Salmonella enterica.

[0033] Multidrug-resistant Gram-negative bacteria include multidrug-resistant Acinetobacter (MDRA), multidrug-resistant Pseudomonas aeruginosa (MDRP), and carbapenem-resistant Enterobacteriaceae.

[0034] The antibacterial proteins of the present invention can be produced by genetic engineering methods using a polynucleotide containing a gene encoding the antibacterial protein. For example, RNA is prepared by in vitro transcription from a recombinant vector containing a polynucleotide containing a gene encoding the antibacterial protein, and then in vitro translation is performed using this as a template. Alternatively, a polynucleotide containing a gene encoding the antibacterial protein is operably linked to and integrated into a suitable vector, which is then introduced into a host cell to produce a transformed cell, and the target antibacterial protein is then expressed from the transformed cell. Here, "expression" includes any step involved in protein production, including transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0035] The vector can be appropriately selected and used depending on the host cell into which it is to be introduced. The vector also contains a polynucleotide containing a gene encoding the antimicrobial protein of the present invention operably linked to an appropriate promoter, and preferably contains a transcription termination signal, i.e., a terminator region, downstream of the polynucleotide. It can also contain a selection marker gene (e.g., a drug resistance gene or a gene complementing an auxotrophic mutation) for selecting transformants. It can also contain a sequence encoding a tag sequence useful for separating and purifying the expressed protein. The vector can also be integrated into the genome of the host cell.

[0036] The vector can be introduced into the host cell by a known transformation method such as the competent cell method, the protoplast method, or the calcium phosphate co-precipitation method.

[0037] The host cells into which the vector is introduced and used to express the recombinant protein may be any cells capable of expressing the vector, including commonly used and known microorganisms such as bacteria, yeast, fungi, and mammalian cells. Examples of bacteria include gram-positive bacteria such as Bacillus or Streptomyces. The recombinant cells can be cultured by a known method suitable for the host cell.

[0038] The expressed protein can be purified by one or more of the following known methods for purifying proteins or peptides: bacterial cells collected from the culture supernatant by centrifugation or the like are disrupted with ultrasound or glass beads, and solid matter such as cell debris is removed by centrifugation or the like to prepare a crude enzyme solution; the protein can then be purified using one or more of the following known methods for purifying proteins or peptides: ammonium sulfate precipitation, precipitation with organic solvents (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric focusing chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using a substrate or antibody, reverse phase column chromatography, chromatography such as HPLC, and filtration processes such as microfiltration, ultrafiltration, and reverse osmosis.

[0039] The antibacterial protein of the present invention can also be produced by chemical synthesis based on its amino acid sequence. When chemically synthesizing the antibacterial protein, it can be carried out by known chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

[0040] The present invention also provides the antimicrobial protein, antimicrobial agent or disinfectant, pharmaceutical composition, and antimicrobial composition or disinfectant composition of the present invention for use as human medicines. The antimicrobial agent, disinfectant, antimicrobial composition, and disinfectant composition of the present invention may contain a "pharmaceutically acceptable carrier." Pharmaceutically acceptable carriers for use in mammals, including humans, include any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the Japanese Pharmacopoeia, the U.S. Food and Drug Administration, etc. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and saccharose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, tallow and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, water for injection, saline solution, preferably physiological saline, Ringer's solution, ethyl alcohol, phosphate buffer solution, and the like.

[0041] The antimicrobial agents, disinfectants, antimicrobial compositions and disinfectant compositions of the present invention are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial) administration. In a preferred embodiment, the antimicrobial agents and antimicrobial compositions contemplated herein may be injected intravenously into a mammalian subject, such as a human, in a single dose.

[0042] Preferred embodiments of the antibacterial agent, disinfectant, antibacterial composition, and disinfectant composition of the present invention include use for diseases such as pneumonia, bacteremia, sepsis, tetanus, salmonella infection, meningitis, scarlet fever, grassland fever, sexually transmitted bacterial diseases, surgical site infections, necrotizing fasciitis, Escherichia coli infection, bacterial skin diseases, Campylobacter infection, cholera, gastritis, peptic ulcer, diarrhea, dysentery, food poisoning, typhoid fever, Legionnaires' disease, opportunistic infections, urinary tract infections, and whooping cough, or use for disinfecting hands, medical instruments, or medical materials. For example, they can be used for disinfecting medical instruments and peripheral instruments in the eyes, urinary tract, or respiratory system, where Pseudomonas aeruginosa is likely to be present.

[0043] Another preferred embodiment of the present invention is for diseases in the field of ophthalmology, and the present invention can be used in the form of eye drops, eye ointments, etc. to treat keratitis, corneal ulcers, blepharitis, conjunctivitis, hordeolum, blepharoadenitis, dacryocystitis, and to prevent bacterial infections before and after ophthalmic surgery including cataract surgery, as a contact lens preservative, etc.

[0044] The antimicrobial proteins of the present invention can also be used for animals (veterinary purposes). For veterinary use, the antimicrobial proteins, antimicrobial agents or disinfectants, pharmaceutical compositions, and antimicrobial or disinfectant compositions of the present invention are typically administered as acceptable formulations according to normal veterinary practice, and a veterinarian can determine the dosage regimen and administration route most suitable for a specific animal. However, even for veterinary use, they may contain the aforementioned "pharmaceutically acceptable carriers," use the aforementioned administration routes, and be used for the aforementioned purposes of disinfection or prevention or treatment of ophthalmic diseases.

[0045] As used herein, the term "animal" includes mammals, non-mammals, birds, etc., and preferably, the animals of the present invention are livestock, pet animals, laboratory animals, farm animals, etc. More specifically, animals of the present invention include, but are not limited to, cows, horses, donkeys, camels, alpacas, pigs, goats, sheep, dogs, cats, chinchillas, rabbits, mink, ferrets, guinea pigs, mice, rats, hedgehogs, weasels, kangaroos, chimpanzees, gorillas, orangutans, cynomolgus monkeys, baboons, chickens, pigeons, turkeys, pheasants, ostriches, guinea fowl, ducks, geese, etc.

[0046] Furthermore, when used in veterinary medicine, the antibacterial protein of the present invention can be used for the prevention or treatment of typical infectious diseases in animals. Examples of such typical infectious diseases in animals include those described in Journal of the Japanese Society of Veterinary Medicine, 29, 429-433 (1976). More specifically, animal infectious diseases that can be treated with the antibacterial protein of the present invention include, but are not limited to, acute or chronic mastitis, genital infection, sepsis, and sole ulcers (foot disease) in cattle; endometritis, genital infection, and pulmonary suppuration in horses; bronchopneumonia, fibrous pleurisy, and hemorrhagic enteritis in pigs; otitis externa, genital infection, urethritis, endocarditis, pneumonia, sepsis, and eye inflammation in dogs; and otitis externa, genital infection, urethritis, endocarditis, pneumonia, sepsis, and eye inflammation in cats.

[0047] In yet another aspect, the present invention provides the use of an antibacterial protein of the present invention in the manufacture of a human or veterinary medicament for the prevention or treatment of a medical condition caused by a Gram-negative bacterial infection in a human or animal.

[0048] The present invention will be described in more detail below with reference to the following examples. Example 1 A protein was created by fusing endolysin from Pseudomonas aeruginosa phage PAJU2 (YP_002284408; 138 aa; SEQ ID NO: 5), a partial sequence of endolysin [Bacillus amyloliquefaciens phage Morita2001] (aa 143-258 of AAK40280; SEQ ID NO: 6), and a peptide SMAP17 (RKLRRLKRKIAHKVKKY) obtained by mutating a partial sequence of ovine antimicrobial peptide SMAP29 (aa 1-17 of AAA85470; SEQ ID NO: 7).

[0049] The base sequences used are as follows:

[0050]

[0051] The fusion genes DS and SD were cloned, and 27 was cloned to enable expression of a protein fused with 27 and DS or SD. Restriction enzyme sites are located at the ends of each gene.

[0052] Cloned genes pBluescript SK(-) SalI-DS-XbaI pBluescript SK(-) SalI-SD-XbaI pBluescript SK(-) SacI-DS-XhoI pBluescript SK(-) SacI-SD-XhoI pColdII XhoI-27-EcoRI

[0053] SalI-DS-XbaI, SalI-SD-XbaI, SacI-DS-XhoI, and SacI-SD-XhoI were introduced into pColdII XhoI-27-EcoRI to prepare the following fusion protein expression plasmids.

[0054]

[0055] [Protein Expression] The above plasmid was introduced into the BL21 strain, and the protein expression protocol was as follows.

[0056] Expression Method: 1. Pick a single colony from LB medium containing 100 μg / ml ampicillin, inoculate it into LB medium containing 100 μg / ml ampicillin, and grow overnight at 37°C with shaking. 2. Add the overnight culture to the culture medium (LB + 100 μg / ml ampicillin). When the OD600 reaches 1.0, chill the culture on ice and leave for 30 minutes. *It takes time to reach 1.0 OD, so for a 1L culture, approximately 40 ml of overnight culture is needed. *Takara's protocol calls for an OD600 of 0.4-0.5, but in our protein expression studies, we found that expression levels were highest at an OD of 1.0. 3. Add IPTG to a concentration of 1.0 mM, and grow at 15°C for 24 hours with shaking.

[0057] Purification Method: 1. After the culture was completed, the cells were centrifuged (10,000 × g, 10 minutes, 4°C) to obtain a pellet. 2. 25 mL of 100 mM phosphate buffer (300 mM NaCl, pH 7.8) was added to the bacterial pellet, and the mixture was homogenized on ice for 5 minutes (5 seconds on / 5 seconds off) using a Q700 ultrasonic homogenizer (Waken B-Tech Co., Ltd., Kyoto, Japan). (25 mL of phosphate buffer per 250 mL of culture medium.) 3. After sonication, the mixture was centrifuged (8,000 × g, 20 minutes, 4°C) to collect the supernatant. HIGH Density COBALT (Protenova Co., Ltd., Kagawa, Japan) equilibrated with 100 mM phosphate buffer (300 mM NaCl, pH 7.8) was added to the resulting supernatant, and the mixture was allowed to bind overnight at 4°C while rotating. 4. The protein-bound HIGH Density COBALT was allowed to fall freely, and the excess turbid liquid (supernatant) was discarded. 5. 25 ml of 100 mM phosphate buffer (300 mM NaCl, pH 7.8) was added, and the HIGH Density COBALT was washed for 10 minutes with rotation at 4°C. 6. The protein-bound HIGH Density COBALT was then allowed to fall freely, and the excess phosphate buffer (supernatant) was discarded. 7. Steps 5 and 6 were repeated twice. 8. 500 μl of phosphate buffer containing imidazole at a final concentration of 500 mM was added, and the eluted protein was collected.

[0058] After dialysis against PBS, the protein concentration was measured, and the enzyme activity was measured according to the following procedure.

[0059] [Preparation of bacteria] 0.25 ml of P. aeruginosa PAO1 strain, which had been cultured overnight, was added to 7 ml of LB broth (18 mm test tube), and cultured with shaking at 37°C. ↓ OD was measured using a Taitec digital colorimeter, simple OD monitor miniphoto518R, for 2 to 3 hours. 660 When the OD reaches around 0.6, collect the sample. ↓ Wash once with PBS (centrifuge, discard the supernatant, suspend in PBS, centrifuge, add PBS). ↓ Use a Taitec digital colorimeter, simple OD monitor miniphoto518R, to measure the OD. 660 Adjust the bacterial turbidity with PBS to around 0.2.

[0060] [Protocol for measuring viable cell count] The following was mixed in the wells of a 96-well plate and cultured with shaking at 37°C for 30 minutes, and 10 μL of the stock solution and serially diluted solutions were dropped into square Petri dishes.

[0061] Examination of the bactericidal effect of protein alone (Figure 1) Protein treatment: 50μL bacterial solution + 25μL protein (final 0.1 mg / ml) + 25μL PBS Control: 50μL bacterial solution + 50μL PBS Examination of the bactericidal effect of protein when EDTA is added (Figure 1) Protein treatment: 50μL bacterial solution + 25μL protein (final 0.025mg / ml) + 25μL EDTA (final 0.5mM) Control: 50μL bacterial solution + 25μL PBS + 25μL EDTA (final 0.5mM) From these results, the most active "27xDS" was selected.

[0062] Next, there is a linker between 27 and DS in 27xDS, and the effect of the presence or absence of this linker was examined.

[0063] The method was the same as above. The results are shown in Figure 2. Based on the above, "27DS" was selected as a candidate molecule.

[0064] 27xDS and expressed protein sequence of 27DS >27xDS (SEQ ID NO: 8) HMELGTLEMRTSQRGIDLIKSFEGLRLSAYQDSVGVWTIGYGTTRGVTRYMTITVEQAERMLSNDIQRFEPELDRLAKVPLNQNQWDALMSFVYNLGAANLASSTLLKLLNKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFLEPLSKLVDNSGTPKNVSRGTSSTKTTPKYKVKNGDNLTKIAKKHNTTVATLLKLNPGIKDPNMIRVGQTLNVTGSGGKTHKVKSGDTLSKIAVDNKTTVSKLMNLNPEITNPNHIKVGQTIRLSRKLRRLKRKIAHKVKKYSRHHHHHH (54 + 62 = 116) >27DS (SEQ ID NO: 9) HMMRTSQRGIDLIKSFEGLRLSAYQDSVGVWTIGYGTTRGVTRYMTITVEQAERMLSNDIQRFEPELDRLAKVPLNQNQWDALMSFVYNLGAANLASSTLLKLLNKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFL EPLSNSGTPKNVSRGTSSTKTTPKYKVKNGDNLTKIAKKHNTTVATLLKLNPGIKDPNMIRVGQTLNVTGSGGKTHKVKSGDTLSKIAVDNKTTVSKLMNLNPEITNPNHIKVGQTIRLSRKLRRLKRKIAHKVKKYHHHHHH

[0065] 27DS gene sequence >27DS (sequence number 10) ATGATGCGTACATCCCAACGAGGCATCGACCTCATCAAATCCTTCGAGGGCCTGCGCCTGTCCGCTTACCAGGAGACTCGGTGGGTGTCTGGGACCATAGGTTACGGCACCACTCGGGCGTCACCCGCTACATGACGATCACCGTCGAGCAGGCCGAGCGGATGCTGTCGAACGACATTCAGCGCTTCGAGCCAGAGCTAGACAGGGCTGGCGGAAGGGTGCCACTGAAGCGAGCTGGTCCGGAAGGGTGCCACTGAAGCGAGCTGGATGCCCTGGATGAGCTTCGTGTACAACCTGGGCGCGGCCAATCTGGCGTCGTCCAGCTGCTCAAGCTGCTGAACAAGGGTGACTACCAGGGAGCGACCAGCAGTTCCCGCGCTGGGTGAATGCGGGCGGTGAATGCGGCGGTGAAGCGCTTGGATGGTGTGAAGCGTCGAGCGCGAGCTGGCTGTTCCTGGAG CACTATCGAATTCTGGCACGCCTAAGAACGTATCCGCGGGCACGTCATCCACGAAGACGACGCCTAAGTACAAGGTAAAGAACGGCGATAATCTCACCAAAATTGCCAAGAAACACAATACGACAGTGGCTACTTTATTAAAACTCAATCCAGGCATTAAAGATCCGAACATGATTCGTGTCGGTCAAACATTAAATGTGACAGGGAGCGGCGGG TAAGACACCAAAGTAAAGAGTGGTGATACCTTGAGCAAGATTGCCGTGGATAATAAGACTACAGTTAGTAAGTTGATGAATTTGAATCCTGAGATCACAAATCCTAATCATATCAAAGTAGGGCAGACCATTCGCTTAAGCCGGAAGCTCCGTCGGCTTAAACGTAAGATCGCTCACAAGGTAAAAAAAATCATCATCATCATCATCATCATTAG

[0066] Example 2: Examination of the in vitro bactericidal effect of "27DS" [Bacteria preparation] 0.25 ml of P. aeruginosa PAO1 strain, cultured overnight, was added to 7 ml of LB broth (18 mm test tube), and cultured with shaking at 37°C. ↓ OD was measured using a Taitec digital colorimeter, simple OD monitor miniphoto518R. 660 When the OD reaches around 0.6, collect the cells. Wash once with PBS (centrifuge, discard the supernatant, suspend in PBS, centrifuge, add PBS). Measure the OD using a 96-well absorbance measuring instrument (ThermoFisher Scientific Multi-scan FC). 595 Adjust the bacterial turbidity with PBS to 0.5 to 1.0.

[0067] 2.1 Morphological observation of bactericidal effect against Pseudomonas aeruginosa PAO1 strain The following were mixed in the wells of a 96-well plate. Examination of the bactericidal effect of 27DS alone: ​​27DS treatment: 50 μL of bacterial solution + 25 μL of 27DS (final 0.1 mg / ml) + 25 μL of PBS. Control: 50 μL of bacterial solution + 50 μL of PBS. Examination of the bactericidal effect of 27DS when EDTA was added: 27DS treatment: 50 μL of bacterial solution + 25 μL of 27DS (final 0.025 mg / ml) + 25 μL of EDTA (final 0.5 mM). Control: 50 μL of bacterial solution + 25 μL of PBS + 25 μL of EDTA (final 0.5 mM).

[0068] The cells were incubated with shaking at 37°C for 30 minutes, and then gluteraldehyde (2%) was added to stop the reaction. After centrifugation, the cells were coated with 1% agarose and washed with 5% sucrose in PBS. They were fixed with 1.5% osmium tetroxide in 0.1 mol / L phosphate buffer containing 5% sucrose for 1 hour at 4°C. After dehydration using a series of ethanol concentrations, the fixative was replaced with propylene oxide, and the samples were embedded in epoxy resin. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (Figure 3).

[0069] As a result, 27DS treatment resulted in bacterial swelling. These results demonstrated that 27DS has the effect of destroying the cell wall. Furthermore, EDTA treatment tended to cause greater changes in the morphology of the bacteria. EDTA has the effect of changing the structure of LPS, which is thought to enhance the bactericidal activity of 27DS. These results are consistent with the results of in vitro tests of the bactericidal effect of 27DS.

[0070] 2.2. In Vitro Validation Results 1: Observation of Turbidity and Morphology of PAO1 Strain in PBS. The following mixtures were mixed in the wells of a 96-well plate: 27DS treatment: 50 μL of bacterial solution + 25 μL of 27DS (final 0.1 mg / ml) + 25 μL of PBS; control: 50 μL of bacterial solution + 50 μL of PBS. The cultures were incubated with shaking at 37°C for 0 to 180 minutes. After each incubation, the turbidity of the samples was measured at 595 nm and the time course was compared with that of the control. While the turbidity of the control remained nearly constant throughout the experimental period, the turbidity of the 27DS-treated samples increased from 0 to 60 minutes and then decreased thereafter, suggesting that growth of PAO1 strain was inhibited. Furthermore, the cultures were treated as described in 2.1 and observed under a transmission electron microscope. This indicated that 27DS treatment induced morphological changes in the bacterial cells (Figure 4).

[0071] 2.3. In Vitro Validation Results 2: Effect of 27DS on PAO1 Strain During Culture. 0.15 ml of overnight cultured P. aeruginosa PAO1 strain was added to 10 ml of LB broth (18 mm test tube) and incubated with shaking at 37°C. At logarithmic growth phase (60 min incubation, OD600 = 0.2), 0.1 ml of PBS (control) and 0.1 ml of 27DS (5 mg / ml) were added to one culture and the other. Culture was then continued for 320 min. After 320 min of incubation, the OD600 value for the PBS-added control was approximately 1.0, whereas the OD600 value for the 27DS-added sample remained at approximately 0.6, demonstrating inhibition of PAO1 growth (Figure 5).

[0072] 2.4. Examination of Bactericidal Effect on Various Bacteria [Protocol for Measuring Viable Bacteria Count] The following mixtures were mixed in the wells of a 96-well plate. Examination of the Bactericidal Effect of 27DS Alone: ​​27DS Treatment: 50 μL of Bacterial Solution + 25 μL of 27DS (final 0.1 mg / ml) + 25 μL of PBS. Control: 50 μL of Bacterial Solution + 50 μL of PBS. Examination of the Bactericidal Effect of 27DS with Added EDTA: 27DS Treatment: 50 μL of Bacterial Solution + 25 μL of 27DS (final 0.1 mg / ml) + 25 μL of EDTA (final 0.5 mM). Control: 50 μL of Bacterial Solution + 25 μL of PBS + 25 μL of EDTA (final 0.5 mM). The mixtures were shaken at 37°C for 30 minutes, and 10 μL of the stock solution and serially diluted versions were dropped into square Petri dishes. Culture was then performed.

[0073] The number of bacteria was expressed as a ratio (Figures 6 and 7). Bacterial proportion (%) = bacterial concentration (treatment group; cfu / mL) / bacterial concentration (PBS group; cfu / mL). 27DS alone can kill Pseudomonas aeruginosa, including multidrug-resistant Pseudomonas aeruginosa. 27DS + EDTA showed a very high bactericidal effect.

[0074] 27DS treatment showed bactericidal activity against Enterobacter cloacae complex NDM1, Escherichia coli NDM2, Escherichia coli NDM4, Salmonella Typhimurium IID1000, Citrobacter freundii NIH10018-68, Escherichia coli DH5α, and Acinetobacter calcoaceticus IAM1517. Treatment with 27DS + EDTA showed high bactericidal activity.

[0075] 27DS treatment and 27DS + EDTA treatment showed weak bactericidal activity against Klebsiella pneumonia (Klebsiella pneumoniae NDM3, Klebsiella pneumoniae IID5209).

[0076] It does not have bactericidal activity against Enterococcus faecalis EF24 or Staphylococcus aureus SA27.

[0077] Example 3: Efficacy of "27DS" in an Infected Mouse Model 3.1. Efficacy in a Septic Mouse Model Pseudomonas aeruginosa PAO1 was cultured in LB medium, raised to mid-log phase, and washed three times with saline. 6.8 × 10 Pseudomonas aeruginosa PAO1 cells suspended in saline were collected. 6 Bacteria / mL were prepared.

[0078] 0.2 mL of P. aeruginosa + 0.2 mL of PBS, 0.2 mL of P. aeruginosa + 0.2 mL of EDTA (2 mM), 0.2 mL of P. aeruginosa + 0.2 mL of 27DS (0.2 mg / mL), and 0.2 mL of P. aeruginosa + 0.2 mL of 27DS (0.2 mg / mL) + EDTA (2 mM) were mixed, and 0.4 mL of each mixture was intraperitoneally administered to six mice in each group. (27DS 0.1 mg / mL, EDTA 1 mM) Mice were observed for survival over two days. The results are shown in Figure 8.

[0079] Next, we conducted a therapeutic experiment in mice infected with Pseudomonas aeruginosa. After inoculation, 0.2 mL of PBS or the following drugs were administered 0, 2, and 6 hours later. The method and results are shown in Figure 9.

[0080] 27DS (0.2 mg / mL) EDTA (2 mM) 27DS (0.2 mg / mL) + EDTA (2 mM)

[0081] 3.2. Efficacy study on a mouse keratitis model P. aeruginosa PAO1 was cultured in LB medium, raised to mid-log phase, and washed three times with saline.

[0082] General anesthesia (3-type mixture) 5 x 10 4Three lines were incised into the eye, and 5 μl of bacteria was instilled. Immediately after instillation, the mouse was awake with antisedant. 30 minutes later, the mouse was held and 10 μl of the antibacterial enzyme 27DS (0.58 mg / ml) + EDTA (10 mM) was instilled into the eye (one time). 24 hours later, the sacrificed eye was removed, crushed in 500 μl of PBS, and diluted. 15 μl of the bacterial solution was then added dropwise to a rectangular selective medium and cultured for 24 hours. 125 μl was removed for measurement of neutrophil myeloperoxidase (MPO) and measured undiluted. The results are shown in Figure 10. 27DS + EDTA was found to be effective against keratitis.

[0083] Example 4: Low incidence of resistant bacteria to 27DS 27DS was subcultured, and the minimum inhibitory concentration (MIC) was measured before and after culture. The measurement method was in accordance with the CLSI (Clinical & Laboratory Standards Institute) Guidelines. Even after 20 subcultures, no change was observed in the MIC value of the bacterial strain, suggesting that resistant bacteria to 27DS are unlikely to emerge.

Claims

1. A polypeptide having lipopolysaccharide-containing outer membrane permeability to Gram-negative bacteria as described in (i) or (ii) below, characterized in that it enhances antibacterial activity against Gram-negative bacteria in the presence of ethylenediaminetetraacetic acid (EDTA) or a salt thereof: (i) A polypeptide consisting of the amino acid sequence represented by Sequence ID No. 1, (ii) A polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence represented by Sequence ID No.

1.

2. The following polynucleotides (iv) or (v): (iv) A polynucleotide comprising a DNA sequence encoding the polypeptide described in claim 1, (v) A polynucleotide consisting of the DNA sequence represented by Sequence ID No.

2.

3. An antimicrobial protein comprising endolysin and the polypeptide described in claim 1, wherein the polypeptide described in claim 1 is present at the N-terminal or C-terminal end of the endolysin.

4. A polynucleotide encoding an antimicrobial protein as described in claim 3.

5. An antimicrobial agent or disinfectant for Gram-negative bacteria comprising the antimicrobial protein described in claim 3.

6. The antibacterial agent or disinfectant according to claim 5, wherein the Gram-negative bacteria are multidrug-resistant bacteria or Pseudomonas aeruginosa.

7. A pharmaceutical composition comprising the antimicrobial protein described in claim 3.

8. The pharmaceutical composition according to claim 7, wherein the disease targeted by Pseudomonas aeruginosa is one or more diseases selected from keratitis, dacryocystitis, endophthalmitis, cellulitis, ocular infections, sepsis, pneumonia, multidrug-resistant Gram-negative bacterial infections, opportunistic infections, or other diseases caused by Pseudomonas aeruginosa.

9. An antimicrobial composition or disinfectant composition comprising the antimicrobial protein described in claim 3 and an outer membrane permeable agent, wherein the outer membrane permeable agent is ethylenediaminetetraacetic acid (EDTA) or a salt thereof.

10. The antimicrobial agent composition or disinfectant composition according to claim 9, wherein the Gram-negative bacteria are multidrug-resistant bacteria or Pseudomonas aeruginosa.

11. A method for preventing a disease caused by infection with Gram-negative bacteria or killing Gram-negative bacteria, comprising administering to a human being a pharmaceutical composition containing the antimicrobial protein described in claim 3.

12. A method for preventing a disease caused by infection with Gram-negative bacteria or killing Gram-negative bacteria, comprising administering to an animal a pharmaceutical composition containing the antimicrobial protein described in claim 3.