Antibacterial capsid, therapeutic composition, bactericide, method for removing bacteria, method for killing bacteria, method for treating animals, method for introducing gene, method for adding bacterial function, method for producing antibacterial capsid, method for labeling antibacterial capsid, and method for intracellular delivery of antibacterial capsid

Abstract

Provided is an antibacterial capsid capable of efficiently killing parasitic bacteria in cells.　The antibacterial capsid is: a phage preparation for intracellular parasitic bacteria; or the like. The antibacterial capsid includes a capsid protein such as a phage that presents an intracellular localization signal on a surface. The intracellular localization signal may be a peptide that binds to a receptor for a nutrient necessary for an intracellular parasitic bacterium. The receptor may be a transferrin receptor or an oxLDL receptor. The intracellular parasitic bacterium may be an acid-fast bacterium that survives within the phagosome of a cell. The acid-fast bacterium includes any one of a bacterium belonging to the genus Mycobacterium, a bacterium belonging to the genus Mycolicibacillus, a bacterium belonging to the genus Mycolicibacter, a bacterium belonging to the genus Mycolicibacterium, and a bacterium belonging to the genus Mycobacteroides.

Description

Antibacterial capsid, therapeutic composition, disinfectant, method for removing bacteria, method for sterilization, method for treating animals, method for gene transfer, method for adding bacterial function, method for producing antibacterial capsid, method for labeling antibacterial capsid, and method for intracellular delivery of antibacterial capsid

[0001] In particular, the present invention relates to an antibacterial capsid, a therapeutic composition, a bactericide, a method for removing bacteria, a method for sterilization, a method for treating animals, a method for gene transfer, a method for adding bacterial functions, a method for producing an antibacterial capsid, a method for labeling an antibacterial capsid, and a method for intracellular delivery of an antibacterial capsid.

[0002] During bacterial infection, many bacteria are phagocytosed and killed by macrophages, but some bacteria can escape the macrophage's bactericidal mechanism and proliferate after being phagocytosed. A group of bacteria with this property is called intracellular parasitic bacteria. Intracellular parasitic bacteria (groups) include the genera Mycobacterium, Salmonella, Legionella, Listeria, and Yersinia. These bacteria evade the macrophage's bactericidal mechanism and become pathogenic within phagocytes such as macrophages by one or a combination of the following means: (1) proliferation, (2) escape from the phagosome into the cytoplasm, (3) inhibition of phagosome-lysosome fusion, and (4) production of bactericidal resistance factors. Intracellular parasitic bacteria often prolong infection by invading cells, hiding latent, and evading the host's immune system. Furthermore, many antibiotics, such as beta-lactams and aminoglycosides, have low intracellular internalization, limiting the types of antibiotics available for treating intracellular parasitic bacteria.

[0003] Meanwhile, the recent emergence and rapid spread of drug-resistant bacteria has made the use of conventional small-molecule antibiotics to treat bacterial infections increasingly difficult. Resistant bacteria have already emerged against all antibiotics. Furthermore, multidrug-resistant bacteria, resistant to three or more antibiotics simultaneously, are frequently reported, further complicating antibiotic treatment. In light of these circumstances, phage therapy, an antibacterial treatment using bacteriophages (hereafter simply referred to as "phages"), has attracted attention. Phage therapy utilizes the ability of phages to specifically infect and kill target bacteria to eliminate pathogenic bacteria. Because its bactericidal mechanism differs from that of conventional antibiotics, it exerts the same bactericidal effect on drug-resistant bacteria as on drug-susceptible bacteria. For this reason, research and development of phage therapy as an antibacterial treatment for drug-resistant bacterial infections has been actively pursued in recent years.

[0004] As an example of conventional phage therapy, Patent Document 1 discloses a purified, host-specific, non-toxic, broad-host-range, and virulent phage preparation that is effective in killing bacterial microorganisms in vivo.

[0005] Special Publication No. 2000-508322

[0006] Kolbe K et al., “Development and Optimization of Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis Reporter Front Microbiol, 2020, Dec 9;11:591866 Takaki K et al., “Evaluation of the pathology and treatment of Mycobacterium marinum infection in zebrafish”, Nat Protoc, 2013, Jun; 8(6), p. 1114-24

[0007] However, in conventional phage therapy such as that described in Patent Document 1, it was more difficult for phages to be introduced (invaded) into cells than for antibacterial drugs, and therefore the antibacterial therapeutic effect of phage therapy targeting intracellular parasitic bacteria was very limited.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to solve the above-mentioned problems.

[0009] The antibacterial capsid of the present invention is an antibacterial capsid for intracellular parasitic bacteria, and is characterized by comprising a capsid protein that displays an intracellular localization signal on its surface. The therapeutic composition of the present invention is characterized by comprising the antibacterial capsid. The bactericide of the present invention is characterized by comprising the antibacterial capsid. The bacteria removal method of the present invention is characterized by removing the intracellular parasitic bacteria with the antibacterial capsid. The sterilization method of the present invention is characterized by sterilizing the intracellular parasitic bacteria with the antibacterial capsid. The animal treatment method of the present invention is characterized by treating an animal with the antibacterial capsid. The gene transfer method of the present invention is characterized by introducing a foreign gene contained in the antibacterial capsid into the intracellular parasitic bacterium. The bacterial function addition method of the present invention is characterized by introducing a foreign gene contained in the antibacterial capsid into the intracellular parasitic bacterium to add a function to the intracellular parasitic bacterium. The antibacterial capsid production method of the present invention is a method for producing an antibacterial capsid for intracellular parasitic bacteria, and is characterized by displaying an intracellular localization signal on the surface of the capsid protein. The labeling method of the present invention is a method for labeling an antibacterial capsid for intracellular parasitic bacteria, characterized in that an intracellular localization signal is displayed on the surface of the capsid protein.The intracellular delivery method of the present invention is a method for delivering an antibacterial capsid for intracellular parasitic bacteria into a cell, characterized in that an intracellular localization signal is displayed on the surface of the capsid protein of the antibacterial capsid and the cell is infected.

[0010] According to the present invention, an antibacterial capsid having an enhanced antibacterial therapeutic effect against intracellular parasitic bacteria can be provided by incorporating a capsid protein that displays an intracellular localization signal on its surface.

[0011] FIG. 1 is a conceptual diagram of the construction of an intracellular parasitic bacteria-targeting antibacterial capsid according to an embodiment of the present invention. FIG. 2 is a conceptual diagram of the intracellular bactericidal mechanism of an intracellular parasitic bacteria-targeting antibacterial capsid according to an embodiment of the present invention. FIG. 3 is a conceptual diagram of the insertion of a peptide gene into the 3' end of a phage capsid gene according to Example 1 of the present invention. FIG. 4 is a diagram showing the results of labeling an acid-fast bacterium phage capsid with a receptor-binding peptide according to Example 1 of the present invention. FIG. 5 is a graph showing the results of measuring the cellular internalization of an intracellular parasitic bacteria-targeting antibacterial capsid according to Example 1 of the present invention. FIG. 6 is a conceptual diagram of the measurement of the cellular internalization of an intracellular parasitic bacteria-targeting antibacterial capsid according to Example 1 of the present invention. FIG. 7 is a graph showing the results of measuring the cellular internalization of an intracellular parasitic bacteria-targeting antibacterial capsid according to Example 1 of the present invention. FIG. 8 is a conceptual diagram of the measurement of the bactericidal activity of an intracellular parasitic bacteria-targeting antibacterial capsid according to Example 1 of the present invention against intracellular parasitic bacteria. 1 is a graph showing the measurement results of the bactericidal activity of an intracellular parasitic bacteria-targeting antibacterial capsid according to Example 1 of the present invention against intracellular parasitic bacteria. FIG. 2 is a conceptual diagram of the measurement of the bactericidal activity of an intracellular parasitic bacteria-targeting antibacterial capsid according to Example 1 of the present invention against intracellular parasitic bacteria. FIG. 3 is a conceptual diagram of labeling an intracellular parasitic bacteria-targeting antibacterial capsid with a receptor-binding peptide that does not involve genetic modification of the phage genome according to Example 2 of the present invention. FIG. 4 is a conceptual diagram of a binding test of an intracellular parasitic bacteria-targeting antibacterial capsid labeled with a streptavidin-binding peptide according to Example 2 of the present invention. FIG. 5 is a photograph showing the results of a binding test of an intracellular parasitic bacteria-targeting antibacterial capsid labeled with a streptavidin-binding peptide according to Example 2 of the present invention. FIG. 6 is a photograph of a control for a binding test of an intracellular parasitic bacteria-targeting antibacterial capsid labeled with a streptavidin-binding peptide according to Example 2 of the present invention. FIG. 10 is a conceptual diagram of the confocal microscope observation of the entry of an intracellular parasitic bacteria-targeting antibacterial capsid into a human cell according to Example 2 of the present invention.Fig. 1 is a photograph showing the results of confocal microscope observation of the penetration of an intracellular parasitic bacteria-targeting antibacterial capsid into a human cell according to Example 2 of the present invention. Fig. 2 is a conceptual diagram of the infection of an intracellular parasitic bacteria by an intracellular parasitic bacteria-targeting antibacterial capsid in a human cell according to Example 2 of the present invention. Fig. 3 is a conceptual diagram of the infection kinetics of an intracellular parasitic bacteria by an intracellular parasitic bacteria-targeting antibacterial capsid in a human cell according to Example 2 of the present invention. Fig. 4 is a photograph observing the infection kinetics of an intracellular parasitic bacteria by an intracellular parasitic bacteria-targeting antibacterial capsid in a human cell according to Example 2 of the present invention.

[0012] <Embodiment> Intracellular parasitic bacteria use the phagosomes of host cells such as macrophages as a cover to evade the immune system or resist anti-tuberculosis drugs, establishing persistent infection within the cells. For example, Mycobacterium tuberculosis, a type of intracellular parasitic bacteria, latently infects alveolar macrophages and foam cells differentiated from macrophages. However, because phages are even more difficult to introduce (penetrate) into cells than antibiotics, the antibacterial therapeutic effect of phage therapy against intracellular parasitic bacteria has been very limited.

[0013] Therefore, the inventors of the present invention aimed to develop a technology for highly efficient intracellular delivery of phages and efficient sterilization of intracellular parasitic bacteria. In this study, the inventors focused on the environment in which intracellular parasitic bacteria lurk. The substances available to intracellular parasitic bacteria are limited within the phagosome where intracellular parasitic bacteria lurk. Therefore, intracellular parasitic bacteria obtain necessary substances using the host cell's mechanism for intracellular uptake of substances. Therefore, the inventors came up with the idea of ​​using this cell-targeting mechanism to sterilize intracellular parasitic bacteria. After extensive experiments, the inventors confirmed that phages displaying intracellular localization signals on the capsid surface can target intracellular parasitic bacteria, thereby completing the present invention. Furthermore, the present inventors named this phage preparation targeting intracellular parasitic bacteria "Intracellular Bacterium Targeting Antimicrobial Capsid."

[0014] [Antibacterial Capsid, Antibacterial Capsid Labeling Method, and Intracellular Delivery Method] The antibacterial capsid of this embodiment is an antibacterial capsid for intracellular parasitic bacteria, and is characterized by comprising a capsid protein that displays an intracellular localization signal on its surface. Specifically, the antibacterial capsid of this embodiment is produced by displaying an intracellular localization signal, and is an antibacterial preparation that can target and kill intracellular parasitic bacteria.

[0015] Here, the intracellular parasitic bacteria according to this embodiment are a group of bacteria that have the property of escaping the bactericidal mechanism of macrophages and proliferating after being phagocytosed by such cells during the infection process. These intracellular parasitic bacteria include bacteria of the genera Mycobacterium, Salmonella, Legionella, Listeria, and Yersinia. Of these intracellular parasitic bacteria, the antibacterial capsid according to this embodiment may target acid-fast bacteria that survive within the phagosome of host cells. Although these acid-fast bacteria are classified into tuberculosis bacteria and nontuberculous acid-fast bacteria, both can be targeted in this embodiment. Specifically, the acid-fast bacterium may include any of the genera Mycobacterium, Mycolicibacillus, Mycolicibacter, Mycolicibacterium, and Mycobacteroides.

[0016] The host cells that the intracellular parasitic bacteria of this embodiment parasitize may be, for example, immune-related cells of mammals, such as macrophages and monocytes. In addition, the host cells may be cells other than immune-related cells that parasitize within the phagosome. Furthermore, the host cells of this embodiment do not have to be mammalian cells, but may be established cell lines, special cultured cells, or the like, and may not be normal cells. For example, the host cells of this embodiment may be tumor cells such as cancer cells, hybridomas, disease model cells, genetically modified cells, or other cells that have been modified to more easily allow intracellular parasitic bacteria to parasitize within the phagosome.

[0017] The intracellular localization signal of this embodiment may be a substance that binds to a protein expressed on the surface of a host cell infected with the bacterium and translocates the antibacterial capsid into the cell. Specifically, the intracellular localization signal of this embodiment may be a label that confers host cell targeting on the surface of the phage capsid (shell) and enables highly efficient intracellular delivery. More specifically, the intracellular localization signal may be a substance, such as a peptide, that binds to a receptor on the surface of the host cell (on the cell membrane). This receptor may be a receptor that corresponds to the mechanism by which intracellular parasitic bacteria take up nutrients into the phagosome. Specifically, this receptor may be a transferrin receptor (hereinafter also referred to as "TfR") that is translocated into the phagosome, or an oxidized low-density lipoprotein (hereinafter referred to as "oxLDL") receptor. That is, when a peptide or the like displayed on the capsid surface binds to a receptor, the antibacterial capsid according to this embodiment is taken up into the host cell and transferred into the phagosome.

[0018] Therefore, the peptide displayed on the capsid surface according to this embodiment may be, for example, a peptide that binds to a receptor for nutrients required by the intracellular parasitic bacterium. That is, the intracellular parasitic bacterium takes in nutrients from outside the host cell it parasitizes. These nutrients may be, for example, metal ions such as iron ions, or nutrient sources such as oxLDL. In this case, the peptide may be, for example, a peptide that binds to the transferrin receptor (TfR), which transports iron ions, or a peptide that binds to the oxLDL receptor. By binding the peptide to the receptor in this way, the antibacterial capsid according to this embodiment is taken up into the cell together with the receptor and translocated into the phagosome. As a result, the antibacterial capsid reaches the bacteria lurking inside the host cell and exerts a bactericidal effect.

[0019] An overview of the antibacterial capsid according to this embodiment will be specifically described with reference to FIG. 1A . In this embodiment, the above-mentioned peptide is presented at the C-terminus of the capsid protein of an acid-fast bacterium phage. In this embodiment, a transferrin receptor-binding antibacterial capsid was constructed in which a peptide that binds to the transferrin receptor (TfR) was labeled on the capsid surface. Alternatively, an oxLDL receptor-binding antibacterial capsid was constructed in which a peptide that binds to the oxLDL receptor was presented on the capsid surface. As this oxLDL receptor, one corresponding to CD36 or LOX-1 was prepared.

[0020] Figure 1B shows the intracellular bactericidal mechanism of the antibacterial capsid targeting intracellular bacteria. As described above, intracellular bacteria possess a mechanism for ingesting nutrients from outside the host cell during intracellular latent infection. While maintaining a latent infection within the cell, intracellular bacteria ingest oxLDL as a nutrient source (carbon source) and transferrin as an iron source into the phagosome where the intracellular bacteria reside. When oxLDL is ingested, oxLDL bound to oxLDL receptors (CD36, LOX-1) is delivered to the latent phagosome via the early endosomal pathway. Meanwhile, the transferrin and iron complex reaches the phagosome via the transferrin receptor (TfR) and the recycling endosomal pathway. Therefore, after the peptide that serves as the intracellular localization signal of the antibacterial capsid of this embodiment binds to the oxLDL receptor or transferrin receptor, it is actively taken up into the phagosome where the tuberculosis bacteria are present, thereby infecting intracellular parasitic bacteria that are parasitic within the cells and efficiently killing them.

[0021] In summary, the antimicrobial capsid labeling method according to this embodiment is a method for labeling an antimicrobial capsid for intracellular parasitic bacteria, characterized in that an intracellular localization signal is displayed on the surface of the capsid protein. Furthermore, the antimicrobial capsid intracellular delivery method according to this embodiment is a method for delivering an antimicrobial capsid for intracellular parasitic bacteria into a cell, characterized in that an intracellular localization signal is displayed on the surface of the capsid protein of the antimicrobial capsid, and the antimicrobial capsid infects a host cell.

[0022] [Method for Producing Antibacterial Capsid] Next, a method for constructing (producing) an antibacterial capsid according to this embodiment will be described. The method for producing an antibacterial capsid for intracellular parasitic bacteria according to this embodiment is characterized by producing a recombinant phage or phage-like structure that displays an intracellular localization signal on the surface of the capsid protein.

[0023] Specifically, the antibacterial capsid according to this embodiment can be constructed as a genetically engineered phage in which a gene sequence encoding a peptide with host cell tropism is inserted at the 3' end of the gene encoding the capsid protein, which is the shell portion of a phage that infects intracellular parasitic bacteria, such as TM4 or D29. In this case, for example, when amplifying the capsid protein gene by a technique such as PCR (Polymerase Chain Reaction), the gene sequence encoding the peptide chain can be inserted into the downstream region using primers that include a gene sequence encoding the peptide chain and an overlap region of approximately 10 to 50 bp. In other words, the phage capsid can be labeled with a peptide that binds to a receptor for nutrients required by intracellular parasitic bacteria as an intracellular localization signal.

[0024] When constructing the antibacterial capsid of this embodiment, a linker that facilitates display of the peptide on the surface of the capsid protein may be inserted between the capsid protein and the intracellular localization signal. This linker may have a peptide sequence that does not change the folding structure of the capsid protein and allows the peptide that binds to the receptor of this embodiment to be displayed on the capsid surface. Specifically, the linker of this embodiment may be, for example, a GS linker having the sequence GGGGS. The intracellular localization signal of this embodiment may also be displayed on a phage structure other than the capsid protein. Specifically, this includes proteins such as portions of the capsid other than the head, the phage tail, and phage tail fibers, as well as phage nucleic acids.

[0025] Furthermore, for the gene sequence encoding the peptide chain, it is also preferable to extend the length of both ends of the homology arms by 100 bp or more to lengthen the region where homologous recombination occurs, thereby increasing the frequency of homologous recombination.

[0026] Additionally, the antibacterial capsid of this embodiment may contain not only the gene for the phage capsid protein that displays the intracellular localization signal, but also other foreign genes, such as a bactericidal gene, a biofilm-degrading gene, an antigen-presenting gene, and a transgene, or any combination thereof.

[0027] Among these, the bactericidal gene of this embodiment may be a gene that renders the antibacterial capsid unable to survive within the host cell. Specifically, for example, genes that inhibit bactericidal resistance factors may include genes that block channels that alkaline the phagosome to prevent digestion, genes that stop the expression or translation of such channels using antisense or ribozymes, genes that damage the cell wall or cell membrane to facilitate degradation within the phagosome, genes that inhibit the repair of damage to bacteria caused by reactive oxygen species or acids, genes that prevent escape from the phagosome into the cytoplasm, genes that facilitate fusion of the phagosome with the lysosome, genes involved in the transition of the bacteria to a dormant state, and genes that increase or impair the bacterial metabolism to render it unable to survive within the host cell.

[0028] Furthermore, the bactericidal gene according to this embodiment may produce a secreted bactericidal product. Specifically, the bactericidal gene according to this embodiment can produce a secreted bactericidal product that kills surrounding bacteria. That is, by incorporating a bactericidal gene that produces a secreted bactericidal product into the antibacterial capsid according to this embodiment, it is possible to kill the target bacterium and the bacterial group surrounding the target bacterium (mass sterilization). For example, this bactericidal gene may be a bacteriocin gene cluster (colicin E1 gene cluster) that produces and secretes bacteriocin. Bacteriocins are antibacterial proteins or peptides produced by bacteria that are secreted outside the bacterial body and exhibit antibacterial activity against related bacteria. Therefore, by incorporating a bacteriocin gene cluster into the antibacterial capsid, it is possible to kill the target bacterium and the bacterial group surrounding the target bacterium, thereby achieving a significant antibacterial effect.

[0029] Alternatively, the bactericidal gene according to this embodiment may be used as a bactericide by introducing CRISPR-Casl3a into bacterial cells. Specifically, CRISPR-Casl3a may be designed to include a target sequence (target gene recognition sequence) that recognizes a resistance gene sequence or the like, and DNA encoding this may be loaded onto a phage.

[0030] Furthermore, the biofilm decomposition gene according to this embodiment may be, for example, dextranase, protease, DNase, etc., which cleaves or decomposes the molecular structure of a biofilm composed of bacterial polysaccharides, peptidoglycans, proteins, nucleic acids, etc. The gene product of this biofilm decomposition gene can decompose a bacterial biofilm, making it impossible for the bacteria to survive and / or allowing the penetration of an antibacterial agent, etc.

[0031] Furthermore, the antigen-presenting gene of this embodiment may be a gene that makes the antigen more easily recognized by the immune system or presents an antigen that serves as a target for attack. This antigen does not necessarily have to be an antigen against the intracellular parasitic bacterium that the antibacterial capsid infects, but may also be an antigen against other pathogenic organisms, viruses, animal tumors, etc. present in the focus of the disease. In other words, the antibacterial capsid of this embodiment can be used in the field of phage vaccines, where the DNA sequence introduced into the capsid can be used to carry the DNA of pathogenic bacteria or other bacteria, or to display antigens on the surface of the phage capsid. Furthermore, by carrying this DNA into intracellular parasitic bacteria that are taken up by cancer cells or cells of genetic diseases, the antibacterial capsid can also be used in the treatment of cancer or genetic diseases.

[0032] Furthermore, the foreign gene according to this embodiment may be a gene to be introduced into a bacterium. This gene to be introduced can be any of a variety of genes for adding functions to the bacterium that the antibacterial capsid infects. For example, genes involved in the synthesis or secretion of proteins or various substances can be added as this additional function. Furthermore, as described above, multiple genes can be introduced into the antibacterial capsid. This gene to be introduced can be, for example, a drug gene, a drug resistance gene, a gene encoding a protein related to attenuation or strengthening of the bacterium, a toxin gene, a gene for a specific metabolite, an enzyme gene for producing a specific metabolite, a gene for identifying the bacterium, a reporter gene used in transformation, a restriction enzyme used in genetic recombination or a sequence containing a sticky end, a repeat sequence, or any other "gene" that indicates a genetic type in a broad sense.

[0033] Furthermore, the exogenous gene according to this embodiment may be one that causes drug resistance to be lost or generated by nucleic acid acquisition and / or nucleic acid mutation. Specifically, since resistant bacteria can arise from the acquisition of foreign DNA or RNA (nucleic acid) and / or nucleic acid mutation, an exogenous gene that suppresses this nucleic acid acquisition and / or nucleic acid mutation can enhance the antibacterial effect. In this embodiment, it is preferable to use an exogenous gene product that is acid-resistant and difficult to inactivate even within the phagosome of the host cell.

[0034] In this way, by introducing a foreign gene contained in the antibacterial capsid according to this embodiment into an intracellular parasitic bacterium, it is possible to make it function as a method for introducing genes into intracellular parasitic bacteria. Alternatively, it is also possible to introduce a foreign gene contained in the antibacterial capsid according to this embodiment into an intracellular parasitic bacterium to add function to the intracellular parasitic bacterium.

[0035] The antibacterial capsid of this embodiment may be configured to carry multiple foreign genes. That is, the antibacterial capsid of this embodiment can contain multiple foreign genes within a single phage. In this case, multiple foreign genes may be included within the capsid to the extent that they can be included within the capsid. These multiple foreign genes may be arranged consecutively or at multiple sites within the sequence, or may be configured to include multiple foreign genes. For example, by carrying multiple bactericidal genes so that it can be applied to multiple types of bacteria, one antibacterial capsid can be applied to multiple resistant bacteria. Furthermore, in the case of multidrug-resistant bacteria that have multiple resistance genes, it is possible to carry multiple genes corresponding to all of these genes to improve antibacterial efficiency. Furthermore, the antibacterial effect can be further enhanced by carrying any combination of the above-mentioned bactericidal genes, biofilm degradation genes, antigen presentation genes, and transfection genes into the antibacterial capsid.

[0036] Here, by simultaneously screening multiple samples of phages into which a peptide-encoding gene sequence and a foreign gene have been introduced, it is possible to more reliably obtain a successfully recombined antibacterial capsid. After this homologous recombination occurs, the phage DNA can then be constructed into the antibacterial capsid of this embodiment using a phage DNA construction method commonly known to those skilled in the art. A specific example of this method is described in the Examples below. Meanwhile, the produced antibacterial capsid has the same infection mode as wild-type phage and can be transferred into the phagosome of target bacteria and infect intracellular parasitic bacteria. In other words, the antibacterial capsid can exert biological activity derived from its own DNA, including amplification within the target intracellular parasitic bacteria.

[0037] Furthermore, as a method for constructing (producing) an antibacterial capsid according to this embodiment, an antibacterial capsid for intracellular parasitic bacteria can be produced by introducing and expressing a gene for an acid-fast bacterium phage capsid containing an intracellular localization signal sequence into a parent strain of bacteria, and then infecting the bacterium with a phage that does not contain an intracellular localization signal sequence. In this case, a phage capsid gene, in which a gene sequence encoding a peptide with host cell tropism is inserted at the 3' end of the gene for the capsid protein, which is the shell portion of a phage that infects intracellular parasitic bacteria, such as TM4 or D29, can be cloned downstream of a high-expression promoter and introduced into the chromosome or extrachromosomal plasmid of a parent strain of acid-fast bacteria such as M. smegmatis or Escherichia coli. Then, when a parent strain is infected with a phage that does not contain an intracellular localization signal sequence in its genome, the capsid of the phage is replaced with an acid-fast bacterium phage capsid containing an intracellular localization signal sequence and assembled. This makes it possible to produce an antibacterial capsid according to this embodiment that is similar to the genetically engineered phage described above. This "phage that does not contain a sequence for an intracellular localization signal" may be a so-called "wild-type" phage that has not been genetically engineered, or a phage that contains the above-described foreign gene. Furthermore, a genetically engineered phage that contains the same intracellular localization signal may be introduced into the parent strain described above.

[0038] [Method for Removing Bacteria, Method for Sterilization, and Method for Gene Transfer] The method for removing bacteria according to this embodiment is characterized by using the antibacterial capsid described above to remove intracellular parasitic bacteria. As a method for this removal, intracellular parasitic bacteria can be sterilized using the antibacterial capsid. Here, the antibacterial capsid according to this embodiment is a phage preparation that targets intracellular parasitic bacteria, and therefore can selectively sterilize intracellular parasitic bacteria. Furthermore, by introducing a foreign gene or the like into the capsid as described above, gene transfer can also be performed using the antibacterial capsid according to this embodiment.

[0039] [Therapeutic Composition] The therapeutic composition (medicine for intracellular parasitic bacterial infection, medical composition, therapeutic composition) according to this embodiment is characterized by containing the antibacterial capsid described above. That is, the antibacterial capsid according to this embodiment can also be used in therapeutic compositions containing the antibacterial capsid. Alternatively, the bacterial agent according to this embodiment contains the antibacterial capsid described above. By administering the antibacterial capsid according to this embodiment for therapeutic purposes, it can be used in antibacterial therapy to kill intracellular parasitic bacteria. Specifically, the antibacterial capsid can realize an antibacterial therapy to kill intracellular parasitic bacteria. This enables antibacterial treatment of intracellular parasitic bacterial infections that are difficult to treat with antibacterial drugs, including infections caused by drug-resistant bacteria. Various formulations similar to those used in conventional antibacterial therapy using phages can be used to produce this therapeutic composition.

[0040] Furthermore, the therapeutic composition according to the embodiment of the present invention can be administered together with any pharmaceutically acceptable carrier (for example, physiological saline, isotonic solutions containing glucose or other auxiliary agents, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc., and suitable solubilizing agents, such as alcohol, specifically ethanol, polyalcohols, such as propylene glycol and polyethylene glycol, nonionic surfactants, such as polysorbate 80™ and HCO-50, but are not limited to these). Furthermore, the composition may further contain a suitable excipient, etc.

[0041] The therapeutic composition of this embodiment may also contain a suitable pharmaceutically acceptable carrier to prepare a pharmaceutically acceptable carrier. This carrier may include a biocompatible material such as silicone, collagen, or gelatin. Alternatively, it may be various emulsions. Furthermore, it may contain one or more formulation additives selected from, for example, diluents, flavorings, preservatives, excipients, disintegrants, lubricants, binders, emulsifiers, plasticizers, etc.

[0042] The therapeutic composition according to the present invention may be formulated using a pharmaceutically acceptable carrier known in the art in a dosage form suitable for intravenous or inhalation administration. The route of administration of the pharmaceutical composition according to the present invention is not particularly limited, and oral or parenteral administration is possible. Parenteral administration may be, for example, intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal administration, or direct administration, instillation, or application to bacterial flora or an infected site. The composition may also be administered into the lungs by inhalation or the like. This administration method may be performed in the same manner as conventional phage therapy.

[0043] Furthermore, when using the therapeutic composition according to an embodiment of the present invention for the above-described treatment, the administration interval and dosage can be appropriately selected and changed depending on various conditions, such as the state of the disease and the condition of the subject. The single dose and number of administrations of the therapeutic composition according to an embodiment of the present invention can be appropriately selected and changed depending on the purpose of administration and various conditions, such as the age and weight of the patient, the symptoms, and the severity of the disease. The number and duration of administration may be a single dose, or may be administered once to several times a day for several months, and the condition of the disease may be monitored, followed by re-administration or repeated administration depending on the condition.

[0044] The therapeutic composition according to an embodiment of the present invention can be used in combination with other compositions. The composition of the present invention may be administered simultaneously with other compositions, or may be administered at intervals, with no particular restriction on the order of administration. In an embodiment of the present invention, the period during which the disease is improved or alleviated is not particularly limited, and may be temporary, or may be for a certain period of time.

[0045] Furthermore, therapeutic compositions according to embodiments of the present invention can be used to treat organisms, parts of the body of organisms, or parts thereof extracted or excreted from organisms. Such organisms are not particularly limited and may include, for example, animals, plants, fungi, and the like. Examples of such animals include humans, livestock species, pet animals, and wild animals. Therefore, therapeutic compositions according to embodiments of the present invention can also be used in animal therapy. In other words, the antibacterial capsids according to the present embodiments can also be used in animal therapy methods targeting humans (Homo sapiens) and various non-human animals.

[0046] Specifically, the animal treatment method of this embodiment can treat bacterial infections in humans or non-human animals using a therapeutic composition containing an antimicrobial capsid. The animal may be, for example, a mammal possessing immune cells such as macrophages and monocytes, as described above. Alternatively, the animal is not particularly limited and broadly includes vertebrates and invertebrates, including animals in which intracellular parasitic bacteria reside within the phagosome. Vertebrates include fish, amphibians, reptiles, birds, and mammals. Specifically, mammals may include rodents, such as mice, rats, ferrets, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, cows, horses, or non-human primates. Wild animals include, in addition to mammals, fish, birds including poultry, and reptiles. Crustaceans, including shrimp and insects, and other invertebrates, such as squid, are also broadly included. That is, the therapeutic composition according to the embodiment of the present invention can be used not only for the treatment of humans but also for the treatment of animals, the promotion of growth in livestock, and the like.

[0047] Furthermore, a sterilization method according to an embodiment of the present invention is characterized in that the antibacterial capsid removes (kills or sterilizes) intracellular parasitic bacteria targeted by the antibacterial capsid. Thus, a composition containing the antibacterial capsid according to this embodiment can be used as a bactericide (growth inhibitor). This bactericide can also be incorporated into an appropriate carrier, solution, or the like, as described above. That is, a composition containing the antibacterial capsid according to this embodiment may be provided as a disinfectant by being incorporated into various antibacterial products, such as detergents, hand soaps, mouthwashes, masks, napkins, and air purifier filters. It may also be configured to be sprayed in humid environments, such as for preventing or controlling infections caused by Legionella pneumophila and nontuberculous mycobacteria. Furthermore, a composition containing the antibacterial capsid according to this embodiment can also be used to remove bacterial peptidoglycan, biofilms, and the like. In this case, as described above, it is preferable to incorporate a biofilm-degrading gene as the exogenous gene of the antibacterial capsid. By applying, spraying, or otherwise distributing the bactericide according to this embodiment, it is possible to remove and inhibit the growth of bacteria from lesions, bacterial flora, and the environment. That is, it is possible to reduce the number of bacteria present in the bacterial flora of humans, animals, and / or the environment, and also to kill intracellular parasitic bacteria that have been transferred into the phagosome.

[0048] Furthermore, the bacteria removal method according to the present invention can also remove bacteria present inside or outside of food. Specifically, a food product containing the antibacterial capsid can be provided. That is, the antibacterial capsid according to the present invention can remove bacteria from food. This is because the antibacterial capsid according to the present invention does not infect humans even if obtained. It is also possible to provide food from which bacteria have been removed in this manner. This makes it possible to provide safe food from which, for example, toxin-producing food poisoning bacteria have been reliably removed. The antibacterial capsid according to the present invention can be incorporated into any cultivated, farmed, harvested, or other food, including various vegetables, meat, seafood, processed foods, dairy products, and the like. Furthermore, the antibacterial capsid according to the present invention can also be used in corrosion prevention methods that kill target bacteria and prevent corrosion of food and non-food items.

[0049] Furthermore, compositions containing the antibacterial capsids according to the present embodiments may be used to eliminate bacteria that infect plants. That is, they can be used as pesticides for eliminating plant pathogenic bacteria by directly spraying them on herbaceous plants or woody plants, by mixing them into soil, or by adding them to fertilizers.

[0050] [Diagnostic Method and Test Kit] The antibacterial capsid of this embodiment selectively kills intracellular parasitic bacteria, enabling them to be easily detected. That is, the antibacterial capsid of this embodiment may also be used for determining, testing, and diagnosing infection.

[0051] Specifically, for example, pulmonary macrophages or the like recovered from a patient are simultaneously cultured with the antibacterial capsid of this embodiment, and cytokines such as interferon gamma (hereinafter referred to as "IFN-γ"), interleukin-12 (hereinafter referred to as "IL-12"), interleukin-10 (hereinafter referred to as "IL-10"), and transforming growth factor-β (hereinafter referred to as "TGF-β"), which are infection indicators, can be measured to easily determine (test) whether or not the patient is infected with intracellular parasitic bacteria. Of these cytokines, IFN-γ is an important cytokine produced by T cells and NK cells. During infection with mycobacteria such as Mycobacterium tuberculosis, increased IFN-γ contributes to macrophage activation and control of Mycobacterium tuberculosis. IL-12 is a cytokine that plays an important role during infection and promotes IFN-γ production. During mycobacterial infection, increased IL-12 production induces an IFN-γ response. IL-10 is a cytokine that regulates inflammatory responses and is produced by regulatory T cells and macrophages. During tuberculosis infection, IL-10 production increases, suppressing inflammation while controlling the immune response. TGF-β is a cytokine involved in cellular functions such as cell proliferation and fibroblast activation. During mycobacterial infection, TGF-β increases and may be involved in the processes of fibrosis and tissue repair.

[0052] When testing for statistical significance from the measured amounts of the above-mentioned cytokines, the production amounts are compared with those of samples derived from healthy individuals, and a statistical test is performed to determine, for example, whether or not there is 5% significance (p<0.05). This test can be performed using methods such as T-tests, F-tests, and chi-square tests, as appropriate, depending on the amount and nature of the data. If the test results in statistical significance, the patient can be diagnosed as being infected with intracellular parasitic bacteria. Conversely, if the result is not statistically significant, the patient can be diagnosed as not being infected with intracellular parasitic bacteria. This diagnostic method makes it possible to determine with high reliability whether or not a patient is infected with intracellular parasitic bacteria, even in the early stages of onset. This can provide important information for determining a treatment plan.

[0053] As such, the antibacterial capsid of this embodiment kills acid-fast bacteria but not other species of bacteria, and can therefore be used to identify bacterial species using phages without culturing acid-fast bacteria. As described above, the bacteria identified and / or tested by this bacterial discrimination method may be bacteria present in the bacterial flora of humans, animals, food, or the environment, and / or genetically modified bacteria. It is also possible to provide a bacteria discrimination kit containing the antibacterial capsid of this embodiment.

[0054] A test kit for intracellular parasitic bacterial infection according to an embodiment of the present invention is characterized by including the above-described antibacterial capsid. Furthermore, the test kit according to this embodiment may include a program for determining, data processing, and visualizing test results on a computer in order to analyze the diagnostic results, as well as an apparatus and system equipped with the computer. Such apparatus and systems can be developed using techniques and methods commonly known to those skilled in the art. Such apparatus and systems enable high-throughput testing, facilitating patient diagnosis.

[0055] The above-described configuration can achieve the following effects. Intracellular parasitic bacteria evade the bactericidal mechanisms of macrophages and other phagocytes by one or a combination of methods, such as proliferation, escape from the phagosome into the cytoplasm, inhibition of phagosome-lysosome fusion, or production of bactericidal resistance factors, thereby becoming pathogenic. In addition, many antibiotics have poor intracellular penetration, resulting in poor therapeutic efficacy. Antibacterial treatment of intracellular parasitic bacteria is a medical challenge. In particular, the emergence and rapid spread of drug-resistant bacteria in recent years has made the antibacterial treatment of bacterial infections with conventional antibiotics even more difficult. Specifically, among diseases caused by intracellular parasitic bacteria, tuberculosis is an infectious disease that ranks among the top 10 causes of death worldwide. Ten million people develop tuberculosis annually, and 1.4 million die from it. It is estimated that the causative bacterium, Mycobacterium tuberculosis, latently infects 2 billion people, or one-quarter of the world's population. Development of new antituberculosis drugs and vaccines is underway worldwide, but tuberculosis is still far from being eradicated, and the establishment of innovative tuberculosis treatments is essential. In addition, infections caused by mycobacteria other than Mycobacterium tuberculosis are called nontuberculous mycobacterial diseases. In recent years, the number of patients with nontuberculous mycobacterial diseases in Japan has exceeded the number of tuberculosis patients, posing a clinical problem. Although the clinical course is slow, no effective treatment has been established. Antituberculous drugs have been used as alternative treatments for nontuberculous mycobacterial diseases, but their therapeutic efficacy has been limited. However, many antibiotics have poor intracellular penetration and are ineffective against intracellular parasitic bacteria. Therefore, antibacterial treatment against intracellular parasitic bacteria has been a medical challenge.

[0056] In this context, phage therapy, an antibacterial treatment using phages, has attracted attention. Phage therapy utilizes the characteristics of phages to specifically infect and kill target bacteria. Because its bactericidal mechanism differs from that of conventional antibiotics, it exerts a bactericidal effect on drug-resistant bacteria as well as on drug-susceptible bacteria. Therefore, in recent years, research and development of phage therapy as an antibacterial treatment for drug-resistant bacterial infections has been conducted. However, because phages have very low intracellular migration and are more difficult to introduce (penetrate) into cells than antibiotics, the effectiveness of phage therapy against intracellular parasitic bacteria has been limited. In fact, in the past, direct administration of phages to cultured macrophage-type cells infected with Mycobacterium tuberculosis or nontuberculous mycobacteria (M. avium) barely killed the intracellular M. tuberculosis or M. avium.

[0057] In contrast, the antibacterial capsid of this embodiment includes a capsid protein that displays an intracellular localization signal on the phage capsid surface, thereby enabling highly efficient delivery of the phage into cells. In particular, by displaying a peptide that binds to a receptor used by the phage to take up nutrients into the phagosome as the intracellular localization signal, the phage can be reliably transported into the phagosome. This enables the antibacterial capsid to effectively and efficiently kill intracellular parasitic bacteria as an intracellular parasitic bacteria targeting agent. As a result, an antibacterial formulation targeting intracellular parasitic bacteria can be realized, thereby improving the therapeutic efficacy of phage therapy against intracellular parasitic bacteria, including mycobacteria.

[0058] In fact, as shown in the Examples below, mycobacterial phages engineered to bind to host cell receptors can efficiently reach and kill mycobacteria localized inside the phagosome. Experiments using actual cultured cells confirmed that when antimicrobial capsids were administered to cultured human macrophage cells THP-1, the amount of capsid taken up into the cells increased by 2.6 to 63 times compared to control phages. Furthermore, when cultured human macrophage-like cells or monocyte-like cells were infected with BCG and then administered an antimicrobial capsid targeting intracellular parasitic bacteria, the intracellular parasitic BCG was killed by 2.8 to 7.1 times more than the control phage.

[0059] In the method for producing an antibacterial capsid according to this embodiment, a peptide tag is added to the capsid protein by directly adding a gene encoding the peptide to the phage capsid gene. Furthermore, by using a linker to connect the peptide and the phage capsid, a cellular localization signal can be displayed on the capsid surface without preventing capsid assembly.

[0060] Furthermore, by configuring a production method in which a gene for an acid-fast bacterium phage capsid containing an intracellular localization signal sequence is introduced into a parent strain, and a phage not containing an intracellular localization signal sequence is introduced into the parent strain, it is possible to incorporate an intracellular localization signal into an existing phage and construct an antibacterial capsid targeting intracellular parasitic bacteria without genetic modification of the phage genome of acid-fast bacteria, etc. By using an existing phage in this way, it is possible to make the existing phage target intracellular parasitic bacteria, thereby reducing the effort required for genetic modification.

[0061] In the above-described embodiment, acid-fast bacteria are used as the intracellular parasitic bacteria to be sterilized, but the method can also be applied to other intracellular parasitic bacteria. In other words, by changing the type of intracellular localization signal and the type of phage, the host cell and the intracellular parasitic bacteria to be sterilized can be changed. In this case, an appropriate phage can be selected for each type of intracellular parasitic bacteria. Here, as shown in the examples described below, it was possible to obtain an effect by adding a peptide to the capsid protein of the acid-fast bacterium phage D29, which has a different genetic background from the TM4 phage. Therefore, the method for producing an antibacterial capsid according to this embodiment can be similarly applied to other phages.

[0062] In the above-described embodiment, an example was described in which a peptide that binds to a receptor for nutrients required by intracellular parasitic bacteria was used as the intracellular localization signal. However, the intracellular localization signal is not limited to this, and other labels that translocate into the phagosome where intracellular parasitic bacteria reside can also be used. For example, a label corresponding to an intracellular organelle, such as a lysosome, that is translocated into the phagosome can also be used.

[0063] Furthermore, in the above-described embodiment, an example has been described in which an intracellular localization signal, specifically a peptide gene, is inserted into a capsid gene. However, a gene encoding a peptide may be inserted into a phage gene other than the capsid gene. Specifically, this phage gene other than the capsid gene may be a phage tail gene, a phage tail fiber gene, or the like. Furthermore, the intracellular localization signal may be displayed not only on the surface of the phage but also inside the phage. Furthermore, the intracellular localization signal may be directly attached to a nucleic acid, such as phage DNA or RNA, or a sugar chain, in addition to a phage protein.

[0064] In the above-described embodiment, an example was described in which a phage was used that introduced a peptide displayed on the capsid surface of an antimicrobial capsid. Furthermore, an example was described in which a bactericidal gene, a biofilm degradation gene, an antigen presentation gene, and a transgene were used as genes to be introduced into the genome of the antimicrobial capsid. However, any gene can be targeted for introduction. Furthermore, the foreign gene may include a gene whose expression is associated with, controlled by, or related to a drug resistance gene or a toxin gene. In addition, it may include other pathogenic genes related to pathogenicity and genes that suppress these genes.

[0065] Furthermore, the gene introduced into the antibacterial capsid of this embodiment may be a "gene" in the broad sense, such as a genetic mutation sequence, single base substitution, or repeat that is a target for cancer treatment or hereditary disease. In this case, the bacterium itself can be administered for treatment as a drug carrier. That is, a drug corresponding to the cell containing this foreign gene can be contained in the cytoplasm of the target bacterium, and then dispersed to tissues or around the target cells by bacteriolysis. In this case, it is also possible to provide the bacterium itself containing the prophage. The bacterium may also contain a prophage that is activated by a specific immune response.

[0066] In addition, the antibacterial capsid may be configured to be something other than a phage that replicates within bacteria. In this case, it may be a recombinant phage that does not replicate. Alternatively, the phage capsid may not contain nucleic acid but may contain a drug, toxin, or the like. In this case, the antibacterial capsid may be used to introduce a drug into cells, like a DDS (Drug Delivery System).

[0067] In the above-described embodiment, the antimicrobial capsid is used as a therapeutic composition for treating an infectious disease. However, this treatment can be used not only for the treatment of the infectious disease itself, but also for modifying the bacterial flora. Specifically, antigen-presenting genes can be used to induce immunity against bacteria in the bacterial flora that are required for treatment.

[0068] Furthermore, just as the FDA (U.S. Food and Drug Administration) has approved the addition of phages to food, the antibacterial capsid can also be used to disinfect food and prevent spoilage.

[0069] The antibacterial capsid according to the embodiment of the present invention can also be used in combination with other compositions. Furthermore, it can also be provided as a "cocktail" containing multiple types of antibacterial capsids. Furthermore, the composition of the present invention can be administered, sprayed, applied, etc. simultaneously with other compositions.

[0070] The antibacterial capsid according to the embodiment of the present invention will be described in more detail below as an example based on specific experiments, but this example is merely an example and is not intended to be limiting.

[0071] [Materials and Methods] (Bacteria and Phage Growth) For phage amplification and recombination, Mycolicibacterium smegmatis mc 2 155 was used. To evaluate the intracellular bactericidal activity of the phage, another acid-fast bacterium, Mycobacterium bovis BCG pTEC15, was used. The acid-fast bacterium was cultured at 37°C in either 7H9 / ADC / Gly / Tw(+) medium (Middlebrook 7H9 (Difco) supplemented with 10% ADC, 0.5% glycerol, and 0.05% Tween 80) or 7H10 / ADC / Gly medium (Millipore) supplemented with 10% ADC and 0.5% glycerol. Other special media and culture conditions are described in the individual experimental sections. Hygromycin (50 μg / mL) or kanamycin (20 μg / mL) was added to maintain the plasmid, if necessary. Escherichia coli DH5α was used to amplify the plasmid. E. coli was cultured in Luria broth (Difco) or Luria agar (Difco) at 37°C.

[0072] (Phage Amplification) In this example, TM4 and D29 were used as acid-fast bacterium phages.2 155 was used as a host. M. smegmatis mc cultured in 7H9 / ADC / Gly / Tw(+) for 1 to 4 days. 2 155 was centrifuged at 8,000 × g for 2 minutes at 300 μL per plate, and the supernatant was removed. 2 7H9 medium supplemented with 7H9 / ADC / Gly / Tw(-) / CaCl 2 The cell pellet was suspended in 300 μL of the cell suspension. 4 ~10 10 pfu) was added and left at room temperature for 5 to 30 minutes. 2 The medium was then layered on 7H10 / ADC / Gly medium. After overnight incubation at 37°C, 3 mL of SMM buffer was added dropwise onto the soft agar medium. The phage was extracted by thoroughly suspending the agar and SMM buffer using a conical rod, and the phage solution was transferred together with the agar medium into a 50 mL centrifuge tube. After centrifugation at 15,000 x g for 5 minutes, the supernatant was transferred to a new 50 mL centrifuge tube. The solution was filtered through a 0.4 μm nylon filter and used as the phage solution.

[0073] (Purification of phage) M. smegmatis mc cultured in 7H9 / ADC / Gly / Tw(+) for 1 to 4 days 2 155 was replaced with fresh medium the day before. 2 10 mL / 10 plates of 155 were centrifuged at 8,000 x g for 2 minutes, and the supernatant was removed. An equal volume (1 mL / plate) of 7H9 / ADC / Gly / Tw(-) was added to suspend the pellet. 1 mL / plate of the cell suspension was transferred to a 15 mL test tube, and phage solution (>10 10pfu) was added and left at room temperature for 5 to 30 minutes. Furthermore, 10 mL of 7H9 soft agar was added and layered on 7H10 / ADC / Gly medium. After overnight incubation at 37°C, 3 mL of SMM buffer was added dropwise onto the soft agar medium. The phage was extracted by thoroughly suspending the agar and SMM buffer using a conical rod, and the phage solution was transferred together with the agar medium to a 50 mL centrifuge tube. After centrifugation at 15,000 x g for 5 minutes, the supernatant was transferred to a new 50 mL centrifuge tube. 20 μL of DNase solution (Nippon Gene Co., Ltd.) and 20 μL of RNase solution (Nippon Gene Co., Ltd.) were added and the mixture was allowed to react at 37°C for 30 minutes or more. After centrifugation at 15,000 x g for 5 minutes, the supernatant was transferred to a new 50 mL centrifuge tube. The phage solution was then filtered through a 0.4 μm nylon filter. Add CsCl to the ultracentrifuge tube. 2 Buffer (CsCl 2 40g, dH 2 26.4 mL of HO, 3 mL of M-Tris solution) and dH 2 The solutions adjusted to 7:3, 6:4, and 5:5 ratios of 0 were layered on top of each other, and approximately 9 mL of the phage solution was added on top. The mixture was centrifuged at 30,000 rpm for 1 hour using a swing rotor. The phage fraction was extracted using a syringe. The collected solution was passed through a centrifugal ultrafiltration filter (Amicon Ultra 100K, Merck) to replace the solvent with SMM buffer, and the resulting solution was used as a purified phage solution.

[0074] (Extraction of phage DNA) Purified phage solution or phage solution (up to 10 10To 50 μL of the 50 μL (pfu / ml) PBS, 450 μL of TE buffer, 10 μL of 0.5 M EDTA, 10 μL of 10% SDS, and 10 μL of proteinase K solution were added and incubated at 56°C for 1-2 hours to remove proteins. Next, 500 μL of phenol / chloroform / isoamyl alcohol was added, mixed, and centrifuged at 21,500 × g for 5 minutes. The supernatant was transferred to a new 1.5 mL tube, and the phenol extraction was repeated three times. 500 μL of chloroform / isoamyl alcohol was then added, mixed, and centrifuged at 21,500 × g for 5 minutes. The supernatant was transferred to a new 1.5 mL tube, and the chloroform extraction was repeated twice. Finally, 1 mL of ethanol was added, and the mixture was left at -20°C for 1 hour. The mixture was then centrifuged at 21,500 × g for 10 minutes to precipitate the DNA. After washing twice with 80% ethanol, the mixture was centrifuged at 21,500×g for 1 minute, and the ethanol was completely removed with a pipette. Finally, 50 μL of TE buffer was added to dissolve the DNA.

[0075] (Recombination of phage capsid gene) A gene sequence encoding a peptide with host cell tropism was inserted into the 3' end of the capsid protein gene gp9 of phage TM4. This was done with reference to the BRED method.

[0076] The insertion of a peptide gene into the 3' end of this phage capsid gene is described with reference to Figure 2. A gene sequence encoding a short peptide chain was inserted into the 3' end of the capsid gene gp9 of phage TM4. Gene fragments 300 bp upstream and 300 bp downstream of the insertion site were amplified by PCR. Specifically, approximately 300 bp of each region upstream (primers J3508 and J3510) and downstream (primers J3509 and J4144: for GS-TfR6) of the 3' end of gp9 were amplified by PCR. The primer sequences are shown in Table 5 and the Sequence Listing below.

[0077] Here, when amplifying the downstream region by PCR, primers designed to overlap the ends of the two upstream and downstream PCR products by 20 bp were used. That is, these primers were primers that added a 20 bp overlap region to the gene sequence encoding the peptide chain. This resulted in the insertion of the gene sequence encoding the peptide chain into the downstream region. PRIME STAR MAX (manufactured by TaKaRa) was used as the PCR enzyme. A 20 μL PCR reaction solution was used. The thermal cycler reacted at 98°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 65°C for 5 seconds, and 72°C for 3 seconds. The PCR product was purified using agarose gel electrophoresis and the FastGene Gel / PCR Extraction Kit (manufactured by Nippon Genetics).

[0078] Using these two PCR products (DNA fragments) as templates, PCR was performed with primers J3508 and J3509 to prepare a single DNA fragment. This DNA fragment was approximately 600 bp long and contained a central gene sequence encoding each peptide and 18 bp to 36 bp of the inserted peptide gene. This DNA fragment and phage DNA were introduced into the M. smegmatis pJV53 strain, resulting in genetic recombination within M. smegmatis cells, allowing the phage to restart, thereby constructing a phage having a phage capsid into which a desired peptide had been inserted.

[0079] The PCR enzyme used to create this DNA fragment was PRIME STAR MAX (manufactured by TaKaRa). A 100 μL PCR reaction solution was used. The thermal cycler was run at 98°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 65°C for 5 seconds, and 72°C for 10 seconds. The PCR product was purified using agarose gel electrophoresis and a FastGene Gel / PCR Extraction Kit (manufactured by Nippon Genetics).

[0080] The competent cells for genetic recombination are M. smegmatis mc with improved recombination efficiency. 2 155 pJV53 strain was used. M. smegmatis mc 2155 pJV53 was cultured in 7H9 / ADC / Gly / Tw(+) / KM20 medium at 37°C for 3 days. 1 mL of this culture was added to 10 mL of 7H9 / ADC / Gly / Tw(+) / KM20 medium and further cultured overnight. The next day, 210 μL of the culture was subcultured into 210 mL of 7H9 induction medium (7H9 / 0.2% glycerol / 0.2% succinate / 0.05% Tween 80 / KM20) and cultured for approximately 15 hours (OD 600 <0.4). Then, 2.1 mL of 20% acetamide was added to induce the recombinant enzyme, and the culture was continued at 37°C. When the turbidity of the bacterial solution reached OD 600 The culture was terminated when the pH reached 0.8-1.0, and the culture medium was transferred to a 50 mL centrifuge tube and cooled on ice for 30 minutes. The tube was centrifuged at 3,000 x g for 10 minutes using a swing-out rotor. The supernatant was removed, and the pellet was washed with 50 mL of ice-chilled 10% glycerol / 10% Tween 80 buffer. After centrifugation at 3,000 x g for 10 minutes, the supernatant was removed, and the pellet was washed with 25 mL of the same buffer. Finally, the pellet was suspended in 4 mL of chilled 10% glycerol stock solution, dispensed into 100 μL aliquots, and stored at -80°C.

[0081] Before use, the competent cells were thawed on ice. Next, 100-500 ng of phage DNA and 200-1000 ng of the PCR product for recombination were mixed and left on ice for 10 minutes. The entire mixture was then transferred to an electroporation cuvette and electroporated at 1,500 mV using an in vitro high-energy gene transfer device (ELEPO 21, manufactured by Nepa Gene). The reaction solution was suspended in 1 mL of 7H9 / ADC / Gly / Tw(-) / CaCl2 medium, transferred to a 15 mL tube, and cultured at 37°C for approximately 2 hours. Then, overnight-cultured M. smegmatis mc suspended in 7H9 / ADC / Gly / Tw(-) / CaCl2 medium was added. 2 300 μL of 155 was added to 3 mL of 7H9 soft agar, mixed, and layered on 7H10 / ADC / Gly medium, followed by incubation at 37° C. for 1 to 2 days.

[0082] Plaques containing recombined phages were selected by PCR. PCR was performed using primers J3877 (for CD36) and J3878 (for LOX-1) designed on the inserted gene sequence and primer J3688 designed on the TM4 phage located outside the region of the recombination PCR product. These PCR products were approximately 400 bp. Because recombination efficiency was very low, 24 to 96 plaques were confirmed by PCR at a time. 100 μL of SMM buffer was dispensed into a 96-well plate. Plaques were collected with the tip of a 200 μL pipette tip and suspended in SM buffer in the 96-well plate. 5 μL of a mixture of primers and PCR enzyme (KOD ONE, Toyobo) was dispensed into a 96-well PCR plate, and 5 μL of the plaque suspension was added and mixed. The thermal cycler was run at 95°C for 3 minutes, followed by 35 cycles of 98°C for 10 seconds and 68°C for 10 seconds. The presence or absence of recombination was confirmed by agarose gel electrophoresis.

[0083] To purify the recombinant phage, the plaque suspension was 2 Double to 10 6 After overnight incubation, the M. smegmatis mc was suspended in 7H9 / ADC / Gly / Tw(-) / CaCl2 medium. 2 300 μL of 155 was mixed with 3 mL of 7H9 soft agar and layered on 7H10 / ADC / Gly medium. Recombination was then confirmed by PCR. This phage purification was repeated two to three times. Furthermore, the gene sequence of the recombined region was confirmed using a DNA sequencer using the Sanger method. As with TM4, a gene sequence encoding a peptide with host cell tropism was inserted at the 3' end of the major capsid protein gene gp17 of the mycobacterial phage D29.

[0084] (Measurement of the amount of antimicrobial capsid taken up into human cells) Monocyte-type THP-1 cells were stimulated with PMA for 2 days to induce differentiation into macrophages. The cells were washed with PBS (phosphate-buffered saline) and then cultured in a normal medium for 1 day. Thereafter, the cells were washed and cultured at 1.2 × 10 6 Serum-free medium was added so that the cells / plate was 1 x 10 10PFU / mL was added and cultured for 2 hours. After washing the cells three times, they were treated with citrate buffer for 3 minutes to inactivate extracellular phages. After washing the cells three times and removing the citrate buffer, they were trypsinized, and the cell pellet was lysed by treating it with 0.5 mL of 0.1% Triton X for 5 minutes. The cell lysate was diluted with 1.5 mL of PBS and stored on ice until plaque counting (total volume 1 mL). After overnight culture, M. smegmatis mc was suspended in 7H9 / ADC / Gly / Tw(-) / CaCl2 medium. 2 To 300 μL of 155, 100 μL, 10 μL, 1 μL, 0.1 μL, and 0.01 μL of cell lysate were added, respectively, and left at room temperature for 15 minutes. 2 mL of 7H9 soft agar was added and mixed, and the mixture was layered on 7H10 / ADC / Gly medium. After culturing at 37°C for 1 to 2 days, the number of plaques formed was counted.

[0085] (Verification of the bactericidal effect of antibacterial capsids on intracellular parasitic bacteria) First, monocyte-type THP-1 cells were stimulated with PMA for two days to induce differentiation into macrophages. After washing the cells with PBS, they were cultured in a normal medium for one day. After washing the cells once, serum-free medium was added to adjust the cell density to 0.5 × 10 5 Next, logarithmically growing M. bovis BCG pTEC15 (expressing green fluorescent protein mWasabi) cultured in 7H9 / ADC / Gly / Tw(+) medium was passed through a 5 μm filter and then centrifuged to form a pellet. The pellet was suspended in the same medium and the OD 600 The concentration was adjusted to 0.2. 600 / mL = 2 x 10 7 cfu / mL. 6 Macrophage-type cells were infected with M. bovis BCG pTEC15 at 1 cfu / plate. Four hours after infection, the cells were washed with PBS and immediately added with fresh medium containing 30 μg / mL kanamycin. The cells were cultured overnight to kill extracellular M. bovis BCG. The next day, the medium was replaced to remove the kanamycin, and the cells were then conditioned for 2 hours in medium without antibiotics. Then, phage (1 × 10) cells were added to fresh medium. 11PFU / plate) and cultured for 24 hours. The cells were washed twice and treated with citrate buffer for 3 minutes to inactivate extracellular phages. After further washing and removing the citrate buffer, the cells were treated with trypsin, and the cell pellet was lysed by incubating with 0.5 mL of 0.1% Triton X / 0.05% Tween 80 for 5 minutes. The cell lysate was diluted with 1.5 mL of PBS and stored on ice (total volume: 1 mL) until experiments to count colonies were performed. 100 μL and 10 μL of the cell lysate were plated on 7H10 / ADC / Gly / Tw(+) medium and cultured at 37°C for 7-14 days, after which the formed colonies were counted.

[0086] [Results] (Infectious host range of the phages used) In this example, the acid-fast bacterium phages TM4 and D29 were used. TM4 was isolated in the United States in 1984, and D29 in 1954. The properties and genome structures of both phages have been analyzed. The base sequence registration numbers of the phages used in this example are shown in Table 1 below.

[0087]

[0088] TM4 and D29 infect many tuberculosis bacteria and nontuberculous mycobacteria. The host ranges of infection of both phages are shown in Table 2 below.

[0089]

[0090] In Table 2, "◯" indicates an acid-fast bacterial species that the present inventors have experimentally confirmed to cause infection, and "ND" indicates a bacterial species that has been known to cause infection in the literature.

[0091] (Intracellular localization signal) Mycobacteria, including tuberculosis, harbor intracellularly within macrophages and foam cells differentiated from monocytes. These monocytic cells express CD36 and transferrin receptor (TfR) on their surface. Furthermore, LOX-1 is expressed on the surface of foam cells. Mycobacteria utilize TfR to deliver transferrin (iron ions) from outside the infected cell to the phagosome within the cell in which they reside. Similarly, they use CD36 and LOX-1 to deliver oxLDL as a nutrient source into the cell. In this example, we utilized this delivery mechanism to label the surface of the phage capsid with peptide tags that bind to TfR, CD36, and LOX-1, and used these as intracellular localization signals. This enabled the construction of an "intracellular parasitic bacteria-targeting" antibacterial capsid that would deliver phages into cells with high efficiency and effectively kill intracellular parasitic bacteria.

[0092] In this example, peptides that bind to TfR or oxLDL receptor (CD36 or LOX-1) were used as the intracellular localization signals. The intracellular localization signals used in this example are shown in Table 3 below.

[0093]

[0094] In Table 3, the TfR-binding peptide is 7 amino acids long, and the oxLDL receptor-binding peptide is 6 to 7 amino acids long. To facilitate the display of these amino acids on the surface of the capsid, a cell-entering capsid was also constructed in which a GS linker was inserted between the capsid protein and the intracellular localization signal.

[0095] (Construction of an antibacterial capsid targeting intracellular parasitic bacteria) Genetic modification of the mycobacterial phage was performed with reference to the BRED method. This method uses M. smegmatis pJV53, which expresses a protein with high homologous recombination efficiency, as a host, and simultaneously introduces a linear DNA fragment with 100-bp homology arms at both ends and phage DNA, resulting in highly efficient phage gene recombination. In E. coli, this method is known as the "Red / ET homologous recombination method."

[0096] The present inventors have attempted to add a small 10 aa tag (Strep-tag II) to the C-terminus of the capsid protein GP9 of phage TM4 using the BRED method, but failed. The reason for this failure was thought to be that, although recombination occurs, it occurs at a low frequency, making it difficult to purify the recombined phage. Therefore, in this example, we succeeded in inserting a peptide into the C-terminus of the phage capsid by (1) extending the length of both ends of the homology arms to 300 bp, thereby lengthening the region where homologous recombination occurs, (2) increasing the frequency of homologous recombination, (3) simplifying the screening method for recombinant phage, and (4) simultaneously screening multiple samples.

[0097] Figure 3A shows the external appearance of this capsid, and Figure 3B shows the results of its construction. Mycobacterial phage TM4 has the gp9 gene, which encodes the capsid protein GP9. Predictive analysis of the protein's three-dimensional structure suggested that the C-terminus of GP9 is located on the surface of the capsid. Therefore, a transferrin receptor-binding peptide (hereinafter referred to as "TfRp") (1), a CD36-binding peptide (2) as an oxLDL receptor-binding peptide, or a LOX-1-binding peptide (3) was inserted immediately before the termination codon of gp9. Furthermore, capsids were constructed in which a GS linker (GGGGS) was inserted between the host receptor-binding peptide and the capsid protein.

[0098] (Efficiency of Intracellular Parasitic Bacteria-Targeting Antibacterial Capsid Entering Human Cells) In this example, a peptide of 3 to 40 amino acid residues that binds to receptor molecules such as CD36 and LOX-1 on the surface of host cells infected by mycobacteria was displayed on the capsid of an mycobacterial phage to construct an intracellular parasitic bacteria-targeting antibacterial capsid that targets intracellular parasitic bacteria. Table 4 below shows this antibacterial capsid and the sequence added to the C-terminus.

[0099]

[0100] Figures 4A to 4D illustrate the results of measuring the amount of uptake into macrophages and foam cells to examine the intracellular penetration efficiency of this intracellular parasitic bacteria-targeting antimicrobial capsid. Figure 4A is a conceptual diagram showing the measurement of the amount of antimicrobial capsid that penetrated into macrophages, after administering TfRp-presenting antimicrobial capsids to cultured macrophage cells obtained by inducing differentiation of THP-1 cells into macrophage cells. Figure 4B shows the results of measuring the amount of antimicrobial capsid that penetrated into macrophage cells. The vertical axis of the graph shows the measured value of pfu (Plaque Forming Unit) of antimicrobial capsid that penetrated into the cells. "TM4-WT" shows the measurement result of the wild-type mycobacterial phage TM4, which is the control. "TM4-GS-TfRp" shows the measurement result of the TfRp-presenting antimicrobial capsid of this example. As a result, the intracellular internalization rate of the TfRp-displaying antibacterial capsid of this example was increased by approximately 63 times compared to that of the wild-type phage.

[0101] Figure 4C is a conceptual diagram showing the administration of oxLDL receptor-binding antibacterial capsids to cultured foam cells, which were THP-1 cells induced to differentiate into follicular cells, and the amount of antibacterial capsid that penetrated into the cells was measured. Specifically, human cultured THP-1 cells were differentiated into foam cells, and antibacterial capsids displaying CD36-binding peptide (CD36p) and LOX-1-binding peptide (LOX1p) on the capsid surface were administered. Figure 4D shows the results of measuring the amount of oxLDL receptor-binding antibacterial capsid that penetrated into foam cells. The vertical axis of the graph indicates pfu of antibacterial capsid that penetrated into the cells. "TM4-WT" indicates the measurement results for the control wild-type mycobacterial phage TM4. "TM4-CD36p" indicates the measurement results of a CD36-binding antibacterial capsid in which a CD36-binding peptide was displayed on a TM4 phage capsid. "TM4-LOX1p1" indicates the measurement results of a LOX-1-binding antibacterial capsid in which a LOX-1-binding peptide 1 was displayed on a TM4 phage capsid. "TM4-GS-LOX1p1" indicates the measurement results of a LOX-1-binding antibacterial capsid in which a LOX-1-binding peptide 1 was displayed on a TM4 phage capsid via a GS linker. "TM4-GS-LOX1p2" indicates the measurement results of a LOX-1-binding antibacterial capsid in which a LOX-1-binding peptide 2 was displayed on a TM4 phage capsid via a GS linker. As a result, the pfu increased by 2.6 to 6.9 times compared to the wild-type phage. In other words, by displaying an intracellular localization signal on the surface of the phage capsid, the phage particles could be taken up into human cells with high efficiency.

[0102] (Bactericidal effect of intracellular parasitic bacteria-targeting antibacterial capsid on intracellular parasitic bacteria) Mycobacterium tuberculosis latently infects the phagosome of the host cell. BCG is an acid-fast bacterium with low pathogenicity, but it has an intracellular latency mechanism similar to that of Mycobacterium tuberculosis. Therefore, THP-1 cells were induced to differentiate into macrophage-type cells and then treated with the TfRp antibacterial capsid described above, or uninduced THP-1 cells were treated with the monocyte-type cultured cell oxLDL receptor-binding antibacterial capsid.

[0103] The results are shown in Figures 5A to 5D. Figure 5A is a conceptual diagram of measuring the bactericidal effect of TfRp antibacterial capsid on intracellular parasitic bacteria. After infecting macrophage-type cultured cells with BCG, transferrin-binding antibacterial capsid was administered, and the bactericidal effect was measured by colony forming unit (cfu). Figure 5B shows the results of measuring the number of BCG surviving within the cells after infection with TfRp antibacterial capsid. The vertical axis of the graph shows the measured cfu, which is the number of BCG bacteria within the cells. "No bacteria" shows the measurement results for the control BCG and those without administration of antibacterial capsid. "BCG + No phage" shows the measurement results for the control without administration of antibacterial capsid after BCG infection. "BCG + TM4-WT" indicates the measurement results when wild-type TM4 phage was administered after BCG infection. "BCG + TM4-GS-TfRp" indicates the measurement results when TfRp antibacterial capsid was administered after BCG infection. As a result, the number of intracellular BCGs was reduced to below the detection limit. In other words, compared to the wild-type phage, the intracellular parasitic bacteria-targeting antibacterial capsid significantly killed intracellular parasitic BCG.

[0104] Figure 5C is a conceptual diagram showing the measurement of the bactericidal effect of oxLDL receptor-binding antimicrobial capsids on intracellular parasitic bacteria. After infecting monocyte-type cultured cells with BCG, transferrin-binding antimicrobial capsids were administered, and the bactericidal effect was measured in terms of colony counts (cfu). Figure 5D shows the results of measuring the number of surviving BCG cells in cells after infection with oxLDL receptor-binding antimicrobial capsids. The vertical axis of the graph shows the measured cfu, which is the number of BCG bacteria in the cells. "BCG + No phage" shows the measurement results for a control group infected with BCG but not administered with antimicrobial capsid. "BCG + TM4-WT" shows the measurement results for a group infected with BCG and then administered with wild-type TM4 phage. "BCG + TM4-CD36p" shows the measurement results for a group infected with BCG and then administered with CD36-binding antimicrobial capsid. "BCG + TM4-LOX1p" shows the measurement results of cells infected with BCG and then administered with LOX-1-binding antibacterial capsids. When undifferentiated monocytic THP-1 cells were administered with CD36-binding antibacterial capsids and LOX-1-binding antibacterial capsids, significant bactericidal effects were observed, 7.1-fold and 2.8-fold, respectively, compared to the wild-type phage.

[0105] [Materials and Methods] (Construction of Phage Capsid-Overexpressing M. smegmatis and Phage Capsid Labeling Using a Capsid Gene-Expressing Strain) The fluorescent protein gene (mWasabi) of Plasmid L5 attB::Pleft*mWasabi described in Non-Patent Document 1 was replaced with the capsid gene gp9 of TM4. First, the gene region of Plasmid L5 attB::Pleft*mWasabi excluding the mWasabi gene and the gp9 gene similar to that described in Example 1 above were amplified by PCR. These PCR products were purified and then ligated using NEBuilder (NEB) to construct the plasmid Plasmid L5 attB::Pleft*gp9. Next, this constructed plasmid and pBSint encoding the integrase gene that integrates the plasmid into the chromosome were transfected into M. smegmatis mc 2The plasmid was introduced into M. smegmatis mc155 by electroporation. Colonies grown in kanamycin-containing 7H10 / ADC / Gly medium were purified. Furthermore, a strain was prepared in which a base sequence encoding an arbitrary peptide tag was integrated into the gp9 gene of Plasmid L5 attB::Pleft*gp9. 2 155 L5 attB::Pleft* gp9 was infected with TM4 phage and the phage was amplified, thereby labeling the capsid protein of the phage capsid with a peptide tag without recombining the genome of TM4 phage.

[0106] (Streptavidin binding assay) Plasmid L5 attB::Pleft* gp9-StAv (without linker), Plasmid L5 attB::Pleft* gp9-GS-StAv (with GS linker), and Plasmid L5 attB::Pleft* gp9-wGS-StAv (with two GS linkers) were constructed by linking a streptavidin-binding peptide (WSHPQFEK) and the above-mentioned GS linker to the 3' end of the gp9 gene of Plasmid L5 attB::Pleft* gp9. 2 155 by electroporation to generate M. smegmatis strains expressing streptavidin-binding peptide-labeled capsids. These were infected with TM4 phage to obtain phages whose phage capsids were labeled with streptavidin-binding peptides. These phages were purified in the same manner as in Example 1 above. 5 μL of Streptavidin FG beads (manufactured by Tamagawa Seiki Co., Ltd.) were added to 20 μL of phage solution and allowed to react at room temperature for 1 hour. After washing six times with SM buffer, the phage was suspended in 200 μL of SM buffer. A 10-fold dilution series was then prepared. Overnight-cultured M. smegmatis mc was suspended in 7H9 / ADC / Gly / Tw(-) / CaCl2 medium. 2300 L of 155 was mixed with 3 mL of 7H9 soft agar and layered onto 7H10 / ADC / Gly medium. 2 μL of a dilution series of phage was then inoculated onto the medium. The medium was then cultured at 37°C for 1-2 days, and the number of plaques was determined.

[0107] (Observation of antibacterial capsid uptake into human cells using a confocal microscope) Transferrin receptor-binding antibacterial capsid and TM4 phage solution (1 × 10 10 PFU / mL), 0.1 μL of SYBR-Safe (Thermo Fisher Scientific) was added and allowed to react for 4 hours at 4°C. Unbound SYBR-Safe was removed using an Amicon Ultra-4 centrifugal filter (Merck), and the buffer was replaced with D-PBS(-). 5 × 10 THP-1 cells 4 3 x 10 cells 8 PFU of phage was added, and the cells were cultured for 24 hours at 37° C. Nuclei were stained with Nuc Blue (Invitrogen) for 15 to 30 minutes and then observed under a confocal microscope.

[0108] (Observation of infection of target bacterial cells in human cells with antibacterial capsids using a confocal microscope) First, a TM4 phage carrying a reporter gene was prepared. An mScarlet gene cassette was inserted into the chromosome of the TM4 phage. Next, the reporter gene-carrying phage was transfected into M. smegmatis mc 2 155 L5 attB::Pleft* gp9-GS-TfR to synthesize antibacterial capsids tagged with TfR peptide. The target cells used were BCG pTEC15 described in Non-Patent Document 2. 5x10 THP-1 cells 4 The cells were infected with BCG pTEC15. After culturing at 37°C for 4 hours, kanamycin was added to remove extracellular BCG. After 24 hours, 3 x 10 8 PFU of phage was added, and the cells were further cultured at 37°C for 24 hours and observed under a confocal microscope.

[0109] [Results] (Construction of an intracellular parasitic bacteria-targeting antibacterial capsid without genetic modification of the phage genome) Figure 6 is a conceptual diagram of labeling an acid-fast bacterium phage capsid with a receptor-binding peptide without genetic modification of the phage genome. An M. smegmatis strain was constructed that constitutively expresses the capsid gene of the acid-fast bacterium phage TM4 at high levels. The TM4 capsid gene was cloned downstream of a high-expression promoter and then inserted into the chromosome of the M. smegmatis strain. In this case, modifying the TM4 capsid gene inserted into the M. smegmatis chromosome made it possible to introduce any peptide into the capsid gene. In this example, a phage capsid gene with an intracellular localization signal inserted at its 3' end was cloned downstream of a high-expression promoter and introduced into the chromosome of an M. smegmatis strain. By infecting the M. smegmatis strain thus prepared, which constitutively expresses a high level of the phage capsid gene, with the TM4 phage, it becomes possible to label the mycobacterial phage capsid with any peptide without genetic modification of the phage genome. In this example, we succeeded in labeling the same intracellular localization signal as in Example 1. That is, in this example, we were able to label the mycobacterial phage capsid with an intracellular localization signal without genetic modification of the phage chromosome, thereby constructing an intracellular parasitic bacterium-targeting antibacterial capsid without genetic modification of the phage genome.

[0110] (Streptavidin Binding Assay) Referring to FIG. 7A , to evaluate the efficiency of signal labeling of the capsid protein in this example, an M. smegmatis strain was constructed in which a streptavidin-binding peptide was introduced into the 3' end of the TM4 capsid gene, and the binding strength to streptavidin beads was analyzed. Specifically, a streptavidin-binding peptide was introduced into the TM4 capsid gene to construct an M. smegmatis strain that highly expresses the streptavidin-binding peptide-labeled capsid gene. Unmodified TM4 was introduced into this strain to obtain TM4 bearing a capsid protein labeled with the streptavidin-binding peptide. This TM4 was mixed with streptavidin beads, and the streptavidin-binding peptide-labeled phage capsid was reacted with the beads, followed by purification using a magnetic stand. In this example, 0 to 2 GS linkers were inserted between the streptavidin-binding peptide and the capsid gene, and the binding properties to streptavidin were compared.

[0111] Figure 7B shows the results of 10-fold dilutions of purified mycobacterial phage capsids labeled with streptavidin-binding peptides, spotted onto medium containing M. smegmatis, and observing streptavidin binding. Figure 7C shows the results of a control without streptavidin. TM4 phage capsids (gp9 GS-StAV, with GS linker) in which one GS linker was introduced between the capsid protein and the streptavidin-binding peptide and TM4 phage capsids (gp9 wGS-StAV, with two GS linkers) showed more than 10-fold increased binding to streptavidin beads compared to wild-type phage (gp9 WT) and those without GS linkers (gp9 StAV, without linker). In other words, any peptide could be labeled onto the mycobacterial phage capsid without genetic modification of the phage genome.

[0112] (Observation of the uptake of antimicrobial capsids into human cells using a confocal microscope) Next, the intracellular internalization of the antimicrobial capsids prepared in the same manner as in Example 1 was evaluated under a confocal microscope. Figure 8A shows a conceptual diagram of this observation. In this example, transferrin receptor-binding antimicrobial capsids and the DNA within the capsid of TM4 phage were stained with SYBR-Safe. THP-1 cells were infected with the stained antimicrobial capsids at an MOI of 600, and observed 24 hours later using a confocal microscope. The nuclei of THP-1 cells were stained with Nuc Blue (Invitrogen). The cells were observed 24 hours later using a confocal microscope.

[0113] Figure 8B shows the nucleus, phage, and merged photographs of the phage / capsid for the transferrin receptor (TfR)-binding antimicrobial capsid, wild-type TM4 phage, and control (No phage). Compared with the wild-type TM4 phage, more phage particles were present intracellularly in THP-1 cells infected with the transferrin receptor-binding antimicrobial capsid. This demonstrates that tagging the capsid surface with a transferrin receptor-binding peptide improves the efficiency of entry into THP-1 cells.

[0114] (Visualization of the infection dynamics of antibacterial capsids with intracellular parasitic bacteria within human cells) Next, to investigate whether antibacterial capsids actually infect intracellular parasitic bacteria within human cells, we constructed antibacterial capsids equipped with a reporter gene and observed their infection dynamics using a confocal microscope.

[0115] In this example, the mScaret gene, which serves as a reporter gene, was loaded into the genome of the TM4 phage. Next, M. smegmatis, which highly expresses the TM4 capsid gene into which the transferrin receptor-binding peptide had been introduced, was infected, thereby labeling the phage capsid with the transferrin receptor-binding peptide. Subsequently, THP-1 cells were infected with BCG expressing the fluorescent protein mWasabi, and after removing extracellular bacteria (BCG) with kanamycin, the cells were cultured for 24 hours. The cells were then administered with a reporter gene-loaded, transferrin receptor-binding peptide-labeled antimicrobial capsid, and observed over time using a confocal microscope.

[0116] The concept of a reporter gene-loaded antibacterial capsid is shown in Figure 9A. By loading the mScarlet gene into the phage genome, when the phage infects bacteria, mScarlet is expressed in the infected bacterial cells, resulting in red fluorescence.

[0117] Figure 9B is a schematic diagram of intracellular infection of mycobacterial cells with antimicrobial capsids. BCG pTEC15 expressing mWasabi was used. 5 x 10 THP-1 cells 4 The cells were infected with BCG pTEC15. After culturing at 37°C for 4 hours, kanamycin was added to remove extracellular BCG. After 24 hours, 3 x 10 8 PFU of phage was added, and the cells were further cultured at 37°C for 24 hours and observed under a confocal microscope.

[0118] Figure 9C shows the results of confocal microscopy observation of the infection kinetics of intracellular parasitic bacteria with antimicrobial capsids. 24 hours after administration of the transferrin receptor-binding peptide-labeled antimicrobial capsid, the intracellular BCG began to fluoresce red. This indicates that the antimicrobial capsid reached the intracellular parasitic mycobacteria and caused infection.

[0119] It goes without saying that the configurations and operations of the above-described embodiments are merely examples, and can be modified as appropriate within the scope of the present invention.

[0120] According to the present invention, by killing acid-fast bacteria using an antibacterial capsid for intracellular parasitic bacteria, it is expected that a pharmaceutical for the early treatment of intracellular parasitic bacterial infections can be provided. Furthermore, since metabolic products derived from humans and intracellular parasitic bacteria can also be applied to diagnosis using biomarkers specific to intracellular parasitic bacterial infections, the present invention is industrially applicable.

Claims

1. An antibacterial capsid for intracellular parasitic bacteria, comprising a capsid protein that displays an intracellular internalization signal on its surface.

2. The antibacterial capsid according to claim 1, wherein the intracellular localization signal is a peptide that binds to a receptor for nutrients required by intracellular parasitic bacteria.

3. The antibacterial capsid according to claim 2, characterized in that the receptor is a transferrin receptor or an oxLDL receptor.

4. The antibacterial capsid according to claim 2, characterized in that a linker that facilitates the presentation of the peptide on the surface of the capsid protein is inserted between the capsid protein and the intracellular localization signal.

5. The antibacterial capsid according to claim 1, characterized in that the intracellular parasitic bacterium is an acid-fast bacterium that survives within the phagosome of a cell.

6. The antibacterial capsid according to claim 5, characterized in that the acid-fast bacteria include any one of the genera Mycobacterium, Mycolicibacillus, Mycolicibacter, Mycolicibacterium, and Mycobacteroides.

7. A therapeutic composition comprising an antibacterial capsid according to any one of claims 1 to 6.

8. A bactericide comprising the antibacterial capsid according to any one of claims 1 to 6.

9. A method for eliminating intracellular parasitic bacteria, comprising eliminating the intracellular parasitic bacteria using the antibacterial capsid according to any one of claims 1 to 6.

10. A method for killing intracellular parasitic bacteria, comprising the step of killing the intracellular parasitic bacteria using the antibacterial capsid according to any one of claims 1 to 6.

11. A method for treating an animal, comprising treating the animal with an antibacterial capsid according to any one of claims 1 to 6.

12. A method for gene transfer, comprising introducing a foreign gene contained in the antibacterial capsid according to any one of claims 1 to 6 into said intracellular parasitic bacterium.

13. A method for adding a bacterial function, comprising introducing a foreign gene contained in the antibacterial capsid according to any one of claims 1 to 6 into the intracellular parasitic bacterium, thereby adding a function to the intracellular parasitic bacterium.

14. A method for producing an antibacterial capsid for intracellular parasitic bacteria, comprising displaying an intracellular localization signal on the surface of the capsid protein.

15. A method for producing an antibacterial capsid as described in claim 14, characterized in that an acid-fast bacterium phage capsid gene containing the intracellular localization signal sequence is introduced into a parent strain of bacteria, expressed, and the bacterium is infected with a phage that does not contain the intracellular localization signal sequence, thereby producing an antibacterial capsid for intracellular parasitic bacteria.

16. A method for labeling an antibacterial capsid for intracellular parasitic bacteria, comprising presenting an intracellular localization signal on the surface of the capsid protein.

17. A method for intracellular delivery of an antibacterial capsid for intracellular parasitic bacteria, comprising the steps of: presenting an intracellular localization signal on the surface of a capsid protein of the antibacterial capsid; and infecting the cell.

Images