WO2021079435A1 - Composition bactériophage - Google Patents

Composition bactériophage Download PDF

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WO2021079435A1
WO2021079435A1 PCT/JP2019/041541 JP2019041541W WO2021079435A1 WO 2021079435 A1 WO2021079435 A1 WO 2021079435A1 JP 2019041541 W JP2019041541 W JP 2019041541W WO 2021079435 A1 WO2021079435 A1 WO 2021079435A1
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bacteriophage
amino acid
endolysin
acid sequence
composition
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PCT/JP2019/041541
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English (en)
Japanese (ja)
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英知 岩野
豪紀 樋口
田村　豊
優 臼井
純平 藤木
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Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社
学校法人酪農学園
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Priority to PCT/JP2019/041541 priority Critical patent/WO2021079435A1/fr
Priority to PCT/JP2020/004908 priority patent/WO2021079536A1/fr
Priority to PCT/JP2020/039901 priority patent/WO2021079986A1/fr
Publication of WO2021079435A1 publication Critical patent/WO2021079435A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention comprises a composition for lysing Staphylococcus aureus, which comprises bacteriophage and endolysin having the property of lysing Staphylococcus aureus as active ingredients, and prevention or prevention of diseases caused by Staphylococcus aureus. With respect to the composition for treatment.
  • Staphylococcus aureus (Staphylococcus aureus, hereinafter sometimes abbreviated as "S. aureus”) is a gram-positive coccus belonging to the genus Staphylococcus (genus Staphylococcus), and is a gram-positive coccus in the nasal cavity, pharynx, and skin of humans and animals. It is resident in the intestinal tract and is widely distributed in the natural world, causing various diseases in humans and animals.
  • S. Aureus has protein A on the cell wall, which has the ability to bind to IgG, and also produces coagulase. By blocking the phagocytosis of macrophages and the like, potin A and coagulase are used in S. cerevisiae. Prolongs infection of aureus to the host.
  • S. Aureus causes purulent diseases, sepsis, pneumonia, acute endocarditis, etc. by invading the body through the wound, and S. aureus.
  • the exotoxin (enterotoxin) produced by aureus is known to cause food poisoning in humans.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSA methicillin-resistant Staphylococcus aureus
  • S. Aureus is one of the causative bacteria of mastitis (mastitis), which is a disease that causes a decrease in milk yield and quality of dairy cows for cows and the like.
  • mastitis a disease that causes a decrease in milk yield and quality of dairy cows for cows and the like.
  • S. cerevisiae. aureus about 150 types of mastitis-causing bacteria are known, but the most important pathogen is S. cerevisiae. aureus.
  • S. Mastitis Staphylococcus aureus mastitis caused by aureus is generally treated with antibiotics or the like.
  • currently known antibiotics are S. cerevisiae.
  • treatment with antibiotics is expensive because it is not specific to aureus.
  • S. cerevisiae acquired drug resistance. Since aureus has been discovered, the development of new therapeutic techniques to replace antibiotics and the like is required.
  • Bacteriophage is a general term for viruses that infect bacteria, and when infected with a host bacterium, it proliferates within the bacterium. The proliferated daughter phage is released from the cell wall of the bacterium to the outside of the bacterium, and the bacterium is killed by lysis.
  • the bacteriophage has advantages that it does not affect the bacterial flora other than the host pathogen, that it is easy to grow, and that a large amount of bacteriophage can be prepared at low cost.
  • endolysin is a peptidoglycan hydrolase produced by bacteriophage and is known to cleave bacterial cell wall peptidoglycan. As described above, endolysin exhibits a lytic effect on bacteria by a mechanism different from that of antibiotics, and is also effective against bacteria showing high resistance to antibiotics and the like. So far, Staphylococcus aureus bacteriophage lysin such as Lys-phiK, Lys-GH15, and Lys-fiPort have been isolated and reported.
  • endolysin are multidomain enzymes composed of an N-terminal histidine-dependent amide hydrolase / peptidase (CHAP) domain, an amidase (AMID) domain, and a C-terminal SH3b cell wall binding domain. Lysis and the like on aureus have been reported (Non-Patent Document 1). In particular, Lys-phiK has been reported to have an antibacterial effect on staphylococci clinically isolated from cattle and humans (Non-Patent Document 2).
  • the present invention is described in S.I. Compositions for lysing aureus, and S. cerevisiae. It is an object of the present invention to provide a composition for preventing or treating a disease caused by aureus.
  • Non-Patent Document 1 As a bacteriophage having the property of lysing aureus, a bacteriophage specified by Accession No. NITE BP-693 was found (Patent Document 1), and further, for isolation and identification of endolysin (Lys-phiSA012) produced by the bacteriophage. It has been successful (Non-Patent Document 3).
  • the present invention has been completed based on such findings, and has the following configuration.
  • ⁇ 1> Containing at least one bacteriophage selected from the group consisting of the bacteriophage specified by accession number NITE BP-693 and the bacteriophage specified by accession number NITE BP-694, and endolysin as active ingredients.
  • the composition (a) for lysing Staphylococcus aureus, wherein the endolysin is at least one protein selected from the group consisting of the following (a) to (c), is set to SEQ ID NO: 1.
  • Protein consisting of the above amino acid sequence (b) Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 (c) One or more in the amino acid sequence shown in SEQ ID NO: 1.
  • composition (a) for preventing or treating a disease caused by Staphylococcus aureus wherein the endolysin is at least one protein selected from the group consisting of the following (a) to (c).
  • the endolysin is at least one protein selected from the group consisting of the following (a) to (c).
  • Protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (b) Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 (c) Amino acid set forth in SEQ ID NO: 1.
  • the above-mentioned diseases are skin infections, urinary tract infections, blood infections, bone / joint infections, genital infections, breast infections, soft tissue infections, ear and nose infections, etc. due to Staphylococcus aureus.
  • the composition according to ⁇ 2> which is a wound infection, a respiratory infection, an eye infection, a central nervous system infection or a food poisoning.
  • the composition according to ⁇ 2> or ⁇ 3> which is an injectable composition, an oral composition, or an external composition.
  • ⁇ 5> The composition according to ⁇ 2> or ⁇ 3>, which is a composition for external use and is administered to a subject by spraying, dipping, spreading or dropping.
  • S. It is possible to lyse aureus and, by extension, prevent or treat diseases caused by the bacteria.
  • bacteriophage and endolysin which are the active ingredients in the present invention, can be used in combination to obtain S. cerevisiae. It exhibits high lytic activity against aureus and can efficiently sterilize the bacteria. Therefore, for example, mammals and birds have S.I.
  • Effective preventive and therapeutic agents for various infectious diseases epidermal infection, food poisoning, pneumonia, endocarditis, meningitis, arthritis, sepsis, mastitis, etc.
  • infectious diseases epidermal infection, food poisoning, pneumonia, endocarditis, meningitis, arthritis, sepsis, mastitis, etc.
  • Bacteriophage if a corresponding host exists, infects that host and proliferates explosively. However, bacteriophage cannot grow alone in the absence of a host. Therefore, if the particular host is completely lysed and killed by the bacteriophage, the bacteriophage cannot grow either. In addition, it does not affect other bacteria or the like by infecting them. Even in endolysin, if a specific bacterium does not exist, it does not affect other bacteria or the like. Therefore, using the composition of the present invention, S.I. After the lysis treatment of aureus, it is not necessary to further perform any treatment other than bacteriophage. From the above points, the present invention also has an advantage that it is safe and easy to use without adversely affecting the environment and other bacteria.
  • the amount of bacteriophage and endolysin added is MOI: 0.00001 to 10 for bacteriophage and uniformly 50 ⁇ g / mL for endolysin, as shown in “ ⁇ 0.00001 + Lys50” to “ ⁇ 10 + Lys50” in the figure. .. Further, “Lys50” in the figure indicates a case where only 50 ⁇ g / mL endolysin was added. It is a graph which shows the result of having analyzed the combined effect of bacteriophage ⁇ SA012 and endolysin Lyas-phiSA012 in the lytic activity against Staphylococcus aureus.
  • the vertical axis in the figure shows the absorbance of the Staphylococcus aureus culture solution at 660 nm, and the horizontal axis shows the elapsed time since the addition of the bacteriophage and endolysin (1.5625 ⁇ g / mL). (Unit: hour).
  • the amount of bacteriophage and endolysin added is MOI: 0.00001 to 10 for bacteriophage and uniformly 1 for endolysin, as shown in " ⁇ 0.00001 + Lys1.5625" to " ⁇ 10 + Lys1.5625" in the figure. It is .5625 ⁇ g / mL.
  • “Lys 1.5625” in the figure indicates a case where only 1.5625 ⁇ g / mL endolysin was added. It is a figure which shows the combined effect of bacteriophage ⁇ SA012 and endolysin Lyas-phiSA012 in the lytic activity against Staphylococcus aureus.
  • the horizontal axis shows the addition concentration of endolysin
  • the vertical axis shows the inoculation concentration of bacteriophage.
  • the combination shown in dark gray indicates that the absorbance of the Staphylococcus aureus culture medium at 660 nm 24 hours after the addition of the bacteriophage and / or endolysin is less than 0.2.
  • the combinations shown and shown in light gray indicate that the staphylococcus aureus culture medium 24 hours after the addition of bacteriophage and / or endolysin has an absorbance of 0.2 or greater at 660 nm. ..
  • the present invention relates to the prevention or treatment of diseases caused by Staphylococcus aureus by using bacteriophage and endolysin, which will be described later, in combination to lyse Staphylococcus aureus.
  • Staphylococcus aureus which is the subject of the present invention, is a gram-positive bacterium belonging to the genus Staphylococcus (genus Staphylococcus). Further, "lysing" means that the cell membrane and cell wall of the bacterium are destroyed by the bacteriophage and endolysin, the cytoplasm is released (eluted) extracellularly, and the bacterium is killed.
  • Bacteriophage One of the bacteriophages used in the present invention is the Patented Microorganism Depositary Center, Biotechnology Headquarters, National Institute of Technology and Evaluation, dated December 25, 2008 (postal code 292-0818, Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan). It is a bacteriophage deposited under the accession number NITE BP-693 on foot 2-5-8). The present inventors have named this bacteriophage " ⁇ SA012".
  • the other bacteriophage according to the present invention was dated December 25, 2008, National Institute of Technology and Evaluation, Biotechnology Headquarters, Patented Microbial Deposit Center (post code 292-0818, Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan). It is a bacteriophage deposited under the accession number NITE BP-694 on foot 2-5-8). The present inventors have named this bacteriophage " ⁇ SA039".
  • the bacteriophage according to the present invention can be obtained by making a request for sale to the above depository institution where these are deposited.
  • sewage running water of ordinary households, wastewater collected from sewage treatment facilities, etc., or S.I. Obtained from biological samples such as blood, nasal cavity, pharynx, skin, and intestinal tract derived from animals infected with aureus by, for example, separation and purification by a conventional method such as the plaque assay / separation method described in Patent Document 1. You can also do it.
  • a sample such as waste water is centrifuged at 10,000 to 15,000 rpm for about 10 to 15 minutes to remove impurities, and then, for example, PEG6000 and NaCl are added to precipitate phage particles, and then further 10,000 to 10000.
  • Bacteriophage is collected by a conventional method of centrifuging at 15,000 rpm for about 10 to 15 minutes.
  • the detected single plaque on the soft agar medium is collected, and for example, a density gradient centrifugation such as a conventional plaque formation method, filtration, or cesium chloride (CsCl) density gradient centrifugation is performed.
  • Bacteriophage can be isolated by conventional methods of isolation. Further, the bacteriophage may be purified by a known method such as ultracentrifugation or ultrafiltration. Further, the bacteriophage according to the present invention can be grown by a general bacteriophage growth method such as the plate lysate method described in Patent Document 1.
  • the mode of the bacteriophage contained in the composition of the present invention is not particularly limited, and examples thereof include isolated and / or purified bacteriophage.
  • End Lysin The endolysin according to the present invention is described in S.I. It is a peptidoglycan hydrolase that has an activity of cleaving cell wall peptidoglycan of aureus and exhibits a lytic activity against the bacterium. It is a multidomain enzyme composed of an N-terminal histidine-dependent amide hydrolase / peptidase (CHAP) domain, an amidase (AMID) domain, and a C-terminal SH3b cell wall-binding domain, and more specifically, "SEQ ID NO: 1". A protein consisting of the amino acid sequence described in the above.
  • CHAP N-terminal histidine-dependent amide hydrolase / peptidase
  • AMID amidase
  • SEQ ID NO: 1 A protein consisting of the amino acid sequence described in the above.
  • Lys-phiSA012 (amino acid sequence described in SEQ ID NO: 1, amino acid sequence described in GenBank Accession No. BAO47098.1) is a genomic DNA of phiSA012 (DNA sequence described in GenBank Accession No. AB903967.1). It is encoded by 27339-28826 (ORF51).
  • the endolysin according to the present invention has the lytic activity, from the amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1. Protein is also included.
  • plural generally means 100 amino acids or less (for example, 90 amino acids or less, 80 amino acids or less, 70 amino acids or less), preferably 60 amino acids or less (for example, within 90 amino acids) in the entire amino acid sequence of endolysin according to the present invention. 50 amino acids or less, 40 amino acids or less), more preferably 30 amino acids or less (for example, 20 amino acids or less, 10 amino acids or less), particularly preferably several amino acids or less (for example, 5 amino acids or less, 3 amino acids or less, 2 amino acids or less) Is.
  • the introduction of mutations into the amino acid sequence can be performed, for example, through the introduction of mutations into the DNA sequence encoding the amino acid sequence.
  • Mutation introduction into such a DNA sequence is carried out by a known method such as the Kunkel method or the Gapped duplex method, or a method similar thereto, for example, a mutation introduction kit using a site-specific mutagenesis method (for example, Mutant-K (for example, Mutant-K). It can be carried out using (manufactured by TAKARA), Mutant-G (manufactured by TAKARA), etc., or by using the LA PCR in vitro Mutagenesis series kit of TAKARA.
  • bacterium for obtaining such a homologous gene include a bacterium belonging to the Staphylococcus family (Staphylococcus family), preferably a bacterium belonging to the Staphylococcus genus (Staphylococcus genus).
  • Methods for obtaining homologous genes include hybridization techniques (Southern, EM, J. Mol. Biol., 98: 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, RK. , Et al. Science, 230: 1350-1354, 1985, Saiki, RK et al. Science, 239: 487-491, 1988) and the like.
  • PCR polymerase chain reaction
  • a hybridization reaction is usually carried out under stringent conditions.
  • the stringent hybridization condition is, for example, about "1XSSC, 0.1% SDS, 37 ° C.”, and the stricter condition is about "0.5XSSC, 0.1% SDS, 42 ° C.”.
  • the protein encoded by the acquired homologous gene usually has high identity with the amino acid sequence shown in SEQ ID NO: 1.
  • High identity means, for example, 80% or more, preferably 85% or more, more preferably 90% or more (for example, 91% or more, 92% or more, 93% or more, 94% or more), still more preferably 95% or more (for example, 91% or more, 92% or more, 93% or more, 94% or more).
  • 96% or more, 97% or more, 98% or more, 99% or more sequence identity for example, 96% or more, 97% or more, 98% or more, 99% or more sequence identity.
  • Sequence identity can be determined by using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information (basic local alignment search tool of the US National Center for Biological Information)) (for example, default, that is, default parameter). Can be determined (using).
  • the endolysin according to the present invention also contains a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 as long as it has the lytic activity.
  • the protein variants, homologues, etc. consisting of the amino acid sequence shown in SEQ ID NO: 1 are S.I. Whether or not it has lytic activity against aureus can be determined, for example, by performing a lytic activity test as shown in Examples described later. Specifically, S. When the mutant or the like is added to the culture solution of aureus, a change in the absorbance in the culture solution is detected, and a decrease in the absorbance is observed after the addition, the mutant or the like is referred to as S. It can be determined that it has lytic activity against aureus. Those skilled in the art can appropriately set various conditions in such a test.
  • the DNA encoding the endolysin is placed on an appropriate vector, and a host cell such as Escherichia coli, animal cells, insect cells, plant cells, or a cell-free protein is used. It can be expressed as a recombinant protein by introducing it into a synthetic system (for example, reticulated erythrocyte extract, wheat germ extract). Furthermore, the recombinant protein expressed in a host cell or the like can be purified by a known peptide purification method.
  • the endolysin is used in a form in which a functional protein such as glutathione-S-transferase (GST) protein or His-tag protein is fused.
  • GST glutathione-S-transferase
  • Examples thereof include a method of synthesizing and purifying by binding to a GST-affinitive resin and a metal chelate resin (Smith, MC et al., J. Biol. Chem.
  • the endolysin according to the present invention can also be prepared by chemically synthesizing it with a commercially available polypeptide synthesizer based on its amino acid sequence. Further, the endolysin according to the present invention is S. lysin infected with ⁇ SA012. It can also be prepared from aureus culture or the like by the above-mentioned known peptide purification method.
  • composition for lysis The present invention comprises the above-mentioned bacteriophage and endolysin as active ingredients.
  • a composition for lysing aureus (hereinafter, also referred to as “composition for lysis”) is provided. More specifically It contains at least one bacteriophage selected from the group consisting of the bacteriophage specified by accession number NITE BP-693 and the bacteriophage specified by accession number NITE BP-694, and endolysin as an active ingredient.
  • the amino acid according to SEQ ID NO: 1 of the composition (a) for lysing Staphylococcus aureus, wherein the endolysin is at least one protein selected from the group consisting of the following (a) to (c).
  • Protein consisting of sequence (b) Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 (c) One or more amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1.
  • the present invention provides a protein consisting of an amino acid sequence that is deleted, added, and / or inserted.
  • composition of the present invention can be used in combination, and as a result, S.I.
  • aureus When aureus is lysed, the bacteriophage and endolysin according to the present invention can also be present separately in two or more compositions.
  • the bacteriophage according to the present invention contained as an active ingredient in the composition of the present invention is as described above, but the concentration thereof is not particularly limited and can be appropriately adjusted by those skilled in the art according to the mode of use thereof. it can.
  • it when it is used in a liquid dosage form (liquid antibacterial agent, etc.) described later, it is preferably 1 ⁇ 10 3 to 1 ⁇ 10 13 PFU / ml (PFU: plaque forming unit plaque forming unit). It is more preferably 1 ⁇ 10 5 to 1 ⁇ 10 11 PFU / ml, and even more preferably 1 ⁇ 10 8 to 1 ⁇ 10 10 PFU / ml.
  • the bacteriophage contained in the composition of the present invention may be one kind (strain, strain), and may further contain other bacteriophages other than the bacteriophage according to the present invention.
  • the other bacteriophage is not particularly limited, and examples thereof include bacteriophage against bacteria different from the staphylococcus according to the present invention (for example, S. hyicus, S. chromogenes). Also. It may be not only lytic but also lysogenic bacteriophage.
  • the endolysin according to the present invention contained as an active ingredient in the composition of the present invention is as described above, but the concentration thereof is not particularly limited and can be appropriately adjusted by those skilled in the art according to the mode of use thereof. it can.
  • it when used in a liquid dosage form (liquid antibacterial agent, etc.) described later, it is preferably 0.16 to 500 ⁇ g / ml, more preferably 0.16 to 50 ⁇ g / ml1. It is more preferably .6 to 50 ⁇ g / ml.
  • the endolysin contained in the composition of the present invention may be one kind or may further contain other endolysin other than the endolysin according to the present invention.
  • the other endolysin is not particularly limited, and examples thereof include Lys-phiK, Lys-GH15, and Lys-phiWater (see Non-Patent Documents 1 and 2).
  • the composition for lysis of the present invention is S.I. Since aureus can be lysed, it can take the form of a pharmaceutical composition.
  • the pharmaceutical composition S. Pharmaceutical compositions for preventing or treating diseases caused by aureus (pharmaceuticals such as therapeutic agents, preventive agents, symptomatological agents, etc.), S.A. Examples thereof include pharmaceutical compositions for killing or killing aureus (bactericidal agents and the like, for example, pharmaceuticals such as disinfectants, quasi-drugs such as medicated soap).
  • the lytic composition of the present invention is not limited to the pharmaceutical composition, but in the form of food, S.I. It can also be used for the prevention or amelioration of diseases caused by aureus.
  • Such food and drink compositions include, for example, foods for specified health use, foods with nutritional function and foods with functional claims, as well as animal feeds, nutritional supplements, health supplements, nutritionally prepared foods, and sick people. It can be a food product or a food additive.
  • composition for lysis of the present invention is S.I. Compositions for reducing the number of aureus (sterilizers, etc.), S. cerevisiae. It can take the form of a composition (antibacterial agent, etc.) for suppressing the growth of aureus.
  • the lytic compositions of the present invention are used for research purposes (eg, in vitro and in vivo experiments). It may also take the form of a composition (reagent or the like) for lysing aureus.
  • the form of the lytic composition of the present invention can be appropriately changed depending on the target (administration target, etc.), and for example, solid, semi-solid, and liquid are adopted.
  • the lytic composition of the present invention when used as a pharmaceutical composition, a disinfectant, an antibacterial agent, etc., it is prepared in a form (formulation form, dosage form) suitable for the subject.
  • the dosage form include an oral composition (internal composition), an external composition, an injectable composition (including a breast injection composition and the like), and more specifically, as an oral composition.
  • Tablets chewable tablets, effervescent tablets, troches, drops, capsules (hard capsules, soft capsules), large pills, granules, powders, pills, granules, powders, dry syrups, soaking agents / decoctions , Solid agents such as liposome preparations, semi-solid agents such as licking agents, jelly agents and chewing gum agents, liquid agents such as syrup agents, drink agents, suspending agents, oil agents, emulsions and alcoholic preparations.
  • Solid agents such as liposome preparations, semi-solid agents such as licking agents, jelly agents and chewing gum agents, liquid agents such as syrup agents, drink agents, suspending agents, oil agents, emulsions and alcoholic preparations.
  • the external composition includes solid preparations such as suppositories, poultices and plasters, ointments, creams, mousses, sheet preparations, resin preparations, gel preparations (Nazar gel preparations, etc.), inhaler preparations and the like.
  • Solid preparations such as suppositories, poultices and plasters, ointments, creams, mousses, sheet preparations, resin preparations, gel preparations (Nazar gel preparations, etc.), inhaler preparations and the like.
  • the liquid agent may take a form such as a woven fabric, a knitted fabric, a non-woven fabric or the like contained by a method such as immersion.
  • the disinfectant, the antibacterial agent and the like may also take the form of a wettable powder, a flowable agent, a heat transpiration agent, a fuming agent, a smoking agent, a ULV agent, a pelleting agent and the like.
  • auxiliary component medium or carrier (solid carrier, liquid carrier, gaseous carrier, etc.).
  • auxiliary component include , Aqueous media such as buffers (phosphate buffers, etc.), physiological saline, distilled water, non-aqueous media such as polyethylene glycol, propylene glycol, ethyl oleate, surfactants, vegetable oils, solvents, bases, emulsifiers, Suspensions, matrix-forming agents, excipients, vehicles, thickeners, pressure-sensitive agents, spreading agents, binders, diluents, lubricants, protective agents, viscosity accelerators, wicking agents, stabilizers, Buffers, isotonic agents, chelating agents, disintegrants, pH adjusters, solubilizers, wetting agents, coloring agents, dyes, pigments, flavoring agents, fragrances, flavoring agents, sweeteners, flavoring agents, wetting agents, coloring agents, dyes, pigments, flavoring agents, fragrances, flavoring agents, sweet
  • the lytic composition of the present invention may further contain other drugs, or may be used in combination. When used in combination, they may be administered at the same time or before or after.
  • the drug can also be added to the bacteriophage and / or endolysin described above.
  • other agents include bacteriophage and endolysin S. cerevisiae according to the present invention. It is not particularly limited as long as it does not lose the bacteriolytic activity against aureus, but it is not particularly limited. Drugs commonly used for each application, such as agents, antifungal agents, and antiviral agents, can be appropriately selected and used according to the mode of use.
  • antibacterial agent examples include ⁇ -lactams, aminoglycosides, lincomycins, fosfomycins, tetracyclines, chloramphenicols, macrolides, ketolides, polypeptides, glycopeptides, and streptogramins. Systems, quinolones, and oxazolidinones are examples, but are not limited to these.
  • anti-inflammatory agent examples include steroid agents and non-steroidal agents.
  • examples of the "steroid agent” include corticosteroids, and more specifically, prednisolone, beclomethasone, betamethasone, fluticasone, dexamethasone, hydrocortisone, prednisolone, mometasone and derivatives thereof. Not limited.
  • examples of the "non-steroidal agent” include, but are not limited to, salicylic acid-based, propionic acid-based, acetic acid-based, oxime-based, pilin-based, non-pyrin-based compounds, and COX-2 inhibitors.
  • immunosuppressive agent examples include, but are not limited to, antimetabolites, alkylating agents, microtubule synthesis inhibitors, lymphocyte signal transduction inhibitors, cytokine inhibitors, and antibodies.
  • the lysis of aureus is not particularly limited, and the bacteriophage and endolysin according to the present invention, or a composition for lysis containing them as active ingredients, is to be subjected to the lysis treatment. It can be done by contacting the above.
  • the "target” is not particularly limited, and in addition to the animals exemplified in the following (treatment method, etc.), S.A. Things where aureus can exist, environment (for example, food, equipment and devices used for processing, cooking and storage of food, food processing factories, environment where food is exposed, breeding facilities for mammals (pigs, etc.), birds (for example) Breeding facilities for chickens, etc.) can be mentioned.
  • environment for example, food, equipment and devices used for processing, cooking and storage of food, food processing factories, environment where food is exposed, breeding facilities for mammals (pigs, etc.), birds (for example) Breeding facilities for chickens, etc.
  • the "contact” method is not particularly limited, and for example, the bacteriophage and endolysin according to the present invention can be brought into contact with the subject by spraying, dipping, spreading, dropping, etc., which will be described later.
  • the contact time with the subject is not particularly limited, and the bacteriophage and endolysin according to the present invention or the lytic composition of the present invention lyse Staphylococcus aureus in the subject, and the staphylococcus. It suffices if the time is sufficient to prevent the growth of the staphylococcus.
  • the lysis method of the present invention is to use the bacteriophage and the endolysin according to the present invention in combination, and the bacteriophage according to the present invention and the endolysin according to the present invention may be brought into contact with each other independently. That is, the bacteriophage according to the present invention and the endolysin according to the present invention may be brought into contact with each other at the same time or separately, and the contact order and the number of contacts may be different from each other.
  • the bacteriophage and endolysin according to the present invention or a pharmaceutical composition containing them as an active ingredient, is administered to the subject to obtain S. cerevisiae. It also provides a method of preventing or treating a disease caused by aureus.
  • the "target” for treatment, etc. is S.
  • aureus there is no particular limitation as long as aureus can be infected, and it may be a human or a non-human animal.
  • Non-human animals include livestock, pets and laboratory animals, more specifically pigs, cows, horses, sheep, goats, monkeys, rabbits, cats, dogs, minks, mice, rats, hamsters and guinea pigs. Examples include mammals such as guinea pigs and birds such as chickens.
  • the "disease caused by S. aureus” is not particularly limited, and examples thereof include Staphylococcus aureus infection, and more specifically, skin infection (dermatitis) caused by Staphylococcus aureus.
  • skin infection skin infection
  • dermatitis skin infection
  • epidermitis cystitis, pyoderma
  • urinary tract infections cystitis, urinary tract infections, etc.
  • blood infections catheter-related
  • endocarditis bone / joint infection
  • bone marrow / spondylitis eg, purulent myelitis, purulent spondylitis, etc.
  • Bone fragility sagging disease
  • arthritis bone / arthritis, etc.
  • genital infections vaginitis, uterine purulence, etc.
  • the method of "administration" of the bacteriophage and endolysin according to the present invention, or the pharmaceutical composition of the present invention containing them as an active ingredient is not particularly limited, and for example, transdermal administration, intradermal administration, subcutaneous administration, etc.
  • Intravascular injection intravenous administration, intraarterial administration
  • local administration for example, local injection such as breast injection
  • oral administration transmucosal (nasal injection, etc.) administration
  • intraperitoneal administration intraairway administration
  • rectal administration and muscle examples include oral administration and administration by infusion.
  • Such administration can be carried out by appropriately adopting the above-mentioned dosage form.
  • the dose is appropriately adjusted according to the type, age (week age), body weight, symptom, health condition, etc. of the subject (animal).
  • the pharmaceutical composition of the present invention when used as an external composition, as described above, although it depends on the type of the subject and the like, the external composition contains about 1 ⁇ 10 6 to 1 ⁇ 10 12 PFU / ml.
  • the bacteriophage according to the present invention and the endolysin according to the present invention of about 0.16 to 500 ⁇ g / ml can be contained and administered.
  • Examples of the method for administering the external composition include spraying, dipping, spreading, and dropping. More specifically, the immersion includes bathing of the target in the external composition or water containing the external composition.
  • Examples of spraying include spraying, spraying, spraying, showering, and dusting (dusting) an external composition. For spreading, apply or wipe the external composition, or apply the external composition to strips, plates, bands, collars, earmarks, rim bands, labeling devices, etc. It is also possible to attach and disperse the external composition to the animal when the objects (animals) to which the molded product is attached come into contact with each other.
  • Examples of the dropping include pore-on and spot-on on the external composition.
  • the external composition when applied or sprayed to a target, as described above, it depends on the type and condition of the target, but for example, 1 ⁇ 10 4 to 1 ⁇ 10 12 per 5 cm 2 of the affected area.
  • the present invention of about PFU (preferably about 1 ⁇ 10 5 to 1 ⁇ 10 12 PFU) and the present invention of about 0.16 to 500 ⁇ g / ml (preferably about 0.16 to 50 ⁇ g / ml).
  • a concomitant agent containing such endolysin may be applied or sprayed, but the present invention is not limited thereto.
  • the injection containing the bacteriophage of the present invention when it is locally injected, it varies depending on the type of the subject, the condition of the affected area, etc., but for example, about 1 ⁇ 10 4 to 1 ⁇ 10 12 PFU (preferably 1 ⁇ ).
  • the bacteriophage of the present invention varies depending on the type of subject, the condition of the affected area, and the like.
  • 1 ⁇ 10 4 to 1 ⁇ 10 12 PFU / Kg, and the endolysin according to the present invention is about 0.16 to 500 ⁇ g / ml (preferably about 0.16 to 50 ⁇ g / ml).
  • the therapeutic method of the present invention is to use the bacteriophage and endolysin according to the present invention in combination, and the bacteriophage according to the present invention and the endolysin according to the present invention may be administered independently. That is, the bacteriophage according to the present invention and the endolysin according to the present invention may be administered simultaneously or separately, and the order of administration and the number of administrations of each may be different.
  • Phage and endolysin have high host specificity and show lytic activity only to the host bacteria, and have almost no toxicity when administered to animals. Therefore, it is possible to administer a larger amount of bacteriophage depending on the type of subject, the condition of the affected area, and the like. For example, if the animal to be administered is a large animal, larger amounts of bacteriophage and endolysin can also be administered.
  • the present inventors have previously succeeded in isolating a bacteriophage ( ⁇ SA012) specified by accession number NITE BP-693 as a bacteriophage having a property of lysing Staphylococcus aureus.
  • a bacteriophage ⁇ SA012 specified by accession number NITE BP-693
  • SA003 strain Staphylococcus aureus isolated from bovine mastitis (mastitis) infected raw milk was evaluated in the test shown below.
  • Staphylococcus aureus Staphylococcus aureus (SA003) was separated by the method described in Patent Document 1. More specifically, raw milk of a dairy cow (Hokkaido) that developed mastitis was collected, spread in 100 ⁇ L in a brain-heart infusion agar (manufactured by Nihon Pharmaceutical Co., Ltd.), and cultured at 37 ° C. for 15 hours.
  • Bacteria were collected from each of the resulting colonies and subjected to a normal coagulase test using a mannitol salt agar medium and a normal hemolysis test using a sheep blood agar medium. As a result of the above tests, one colony that was positive in both the coagulase test and the hemolysis test was selected and isolated, and stored as SA003 at -80 ° C.
  • SA003 at the time of use After culturing by a conventional method using a mannitol agar medium, the bacteria were caught, suspended in an LB medium, placed in an incubator (37 ° C.) and cultured for 9 hours, and the cells were enriched to obtain test bacteria.
  • the composition of the LB medium is as follows. In the case of LB liquid medium, it contains 0.5 w / v% of NaCl, 0.5 w / w% of yeast extract (Yeast extract), and 1 w / v% of polypeptone. In the case of LB agar medium, the above composition further contains 1.5% agar.
  • Endolysin Endolysin (Lys-phiSA012, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1) was prepared by the method described in Non-Patent Document 3. More specifically, first, the DNA encoding Lys-phiSA012 was amplified, and a plasmid for expressing the endolysin in Escherichia coli was constructed.
  • Lys012-1Fw 5'-TCCCCAGGATATTCCCATGGCTAGACTCAAGCAGA-3', SEQ ID NO: 2
  • Lys012-495Rv 5'-CGCTCGAGTCGACCCTACTTGAACTGACTGATCCGCTACTCACTGAACT
  • the DNA encoding Lys-phiSA012 was amplified by PCR using: 3).
  • the amplified fragment was purified and subcloned into pGEX-6P-2 (GE, Buckinghamshire, UK) using an In-Fusion HD cloning kit (Clontech, Palo Alto, CA, USA). Subcloning was performed on E.
  • Escherichia coli BL21 (DE3) was transformed using the obtained plasmid, and Lys was added by adding 0.1 mM isopropyl- ⁇ -thiogalactopyranoside (final concentration) (Nacalai Tesque, Kyoto, Japan). -Induced the expression of fiSA012. By inserting it into pGEX-6P-2, Lys-phiSA012 will be expressed in the form of a GST tag fused to its C-terminal side. Then, the Escherichia coli was incubated with a bioshaker BR-40LF (Taitec, Saitama, Japan) at 25 ° C. with shaking overnight, and after centrifugation at 2300 xg at 4 ° C.
  • a bioshaker BR-40LF Teitec, Saitama, Japan
  • the GST tag was excluded from the column by treating the column to which the GST tag fusion Lys-phiSA012 was bound with a protease for GST fusion protein cleavage (PreConfiguration Protein). Lys-phiSA012 was eluted. The protein thus purified was Lys-phiSA012, which was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was also measured by SDS-PAGE. The obtained Lys-phiSA012 was stored at ⁇ 30 ° C. until use.
  • FIG. 1A shows the single effect of the bacteriophage when the MOI was set to 0.00001 to 10
  • FIG. 1B shows the combined effect of the bacteriophage (MOI 0.00001 to 10) and endolysin 50 ⁇ g / ml.
  • FIG. 2 shows the combined effect of each combination of the inoculation amount of bacteriophage and each concentration of endolysin.
  • the combined use of bacteriophage ⁇ SA012 and endolysin, which show lytic activity against Staphylococcus aureus exerts synergistically high lytic activity as compared with each of them alone. Staphylococcus aureus can be sterilized efficiently.
  • the present invention is useful in eradication of Staphylococcus aureus, prevention or treatment of diseases caused by the bacteria, and the like.

Abstract

L'invention concerne une composition destinée à la lyse du staphylocoque doré ou au traitement ou à la prévention de maladies provoquées par le staphylocoque doré et contient, en tant que principes actifs, au moins un bactériophage choisi dans le groupe constitué par le bactériophage spécifié par n° de dépôt NITE BP-693 et le bactériophage spécifié par le n° de dépôt NITE BP-694 et une endolysine, qui est au moins une protéine choisie dans le groupe constitué par (a) une protéine présentant la séquence d'acides aminés décrite dans SEQ ID NO : 1, (b) des protéines présentant des séquences d'acides aminés possédant au moins 80 % d'identité avec la séquence d'acides aminés décrite dans SEQ ID NO : 1 et (c) des protéines présentant des séquences d'acides aminés obtenues par la réalisation d'au moins une substitution, délétion, addition et/ou insertion d'acide aminé dans la séquence d'acides aminés décrite dans SEQ ID NO : 1.
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Title
FUJIKI, J. ET AL.: "Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage", PHARMACEUTICALS, vol. 11, no. 1, 24 February 2018 (2018-02-24), pages 25, XP055819272 *

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