Recombinant phage and gene screening method

Abstract

A purpose of the present disclosure is to provide a recombinant phage that can disable an anti-phage defense system of bacteria, including drug-resistant bacteria, and can be infected with bacteria, and that can grow within the bacteria and can eventually kill the bacteria. In addition, another purpose of the present disclosure is to provide a gene screening method in which a factor for disabling the anti-phage defense system is encoded. A recombinant phage according to the present disclosure includes genes that encode a factor for disabling an anti-phage defense system and includes genes that encode a factor for disabling an anti-phage defense system, wherein the genes that encode the disabling factor are isolated by using a screening method including specific steps.

Description

Recombinant phage and gene screening method

[0001] The present invention relates to recombinant phages and genetic screening methods.

[0002] Antibiotics have been the most effective and important treatment for bacterial infections. However, the emergence and rapid spread of drug-resistant bacteria, which are resistant to existing antibiotics, have rendered existing antibiotics ineffective, and bacterial infections have once again become a global problem threatening human health. What makes this problem so serious is that drug-resistant bacteria are emerging at a rate far exceeding the development of new antibiotics, and the development of new antibiotics has stalled. In other words, it is difficult to solve the problem of drug-resistant bacteria with conventional antibiotic development strategies.

[0003] In recent years, antibacterial treatment (phage therapy) using bacteriophages (hereafter referred to as phages), which have a different bactericidal mechanism than conventional antibiotics, has attracted attention, and clinical trials are being conducted mainly in Europe and the United States. However, when phages are actually infected with drug-resistant bacteria derived from clinical isolates, there is a high probability that the phages will be repelled, making it difficult to extract phages that can strongly infect drug-resistant bacteria.

[0004] This is because drug-resistant bacteria possess some kind of anti-phage defense system, which is a bacterial immune system-like defense mechanism against phages, and examples of such systems include Argonaute (RNAi in nematodes, plants, and animals), CRISPR-Cas, Retron (Non-Patent Document 1), and restriction enzyme systems.

[0005] Only phages that can overcome these anti-phage defense systems can infect drug-resistant bacteria. In other words, for phage therapy to be successful, phages must have the ability to neutralize the anti-phage defense systems of drug-resistant bacteria. For this reason, until now, phages that can neutralize the anti-phage defense systems of drug-resistant bacteria have been extracted each time and used in phage therapy. However, this method relies on chance, and it is not easy to extract phages that are effective against the diverse range of drug-resistant bacteria, and it requires a great deal of effort.

[0006] Specifically, for example, a bactericidal composition comprising a carrier and at least one type of phage has been studied, characterized in that the phage is provided in the composition at a concentration high enough to induce lysis from without by the phage (Patent Document 1).

[0007] However, when using the bactericidal composition, a high concentration of the composition must be added to the pathogenic bacteria that are the main target of bacterial sterilization. Furthermore, this method is also dependent on chance, as only phages that accidentally break through the bacteria's anti-phage defense system will grow inside the bacteria. Therefore, it is not easy to obtain phages that are effective against the diverse range of drug-resistant bacteria.

[0008] Japan Special Table No. 2011-501740

[0009] Adi Millman et al. , Cell, 2020, 183(6), p. 1551-1561

[0010] Therefore, an object of the present disclosure is to provide a recombinant phage that can disable the anti-phage defense system of bacteria, including drug-resistant bacteria, infect the bacteria, grow within the bacteria, and ultimately kill the bacteria. Another object of the present disclosure is to provide a method for screening for genes that encode factors that disable the anti-phage defense system.

[0011] The present inventors conducted extensive research to solve the above-mentioned problems and discovered that a genetic screening method using a library in which each gene constituting the anti-phage defense system has been deleted can be used to obtain recombinant phages containing genes encoding genes that disable the anti-phage defense system, thereby completing the present invention.

[0012] The present invention has the following configurations: 1. A recombinant phage comprising a gene encoding an invalidating factor of an anti-phage defense system. 2. The recombinant phage according to 1 above, wherein the anti-phage defense system is at least one selected from Brex type I, AVAST type III, SIR2+HerA, DUF4297+HerA, hhe, Druantia type I, tmn, Retron Ec67, DRT Type II, DRT Type III, DRT type IV, MzaABCDE, upx, and Retron Ec67, Ec78, Ec48, and Ec83. 3. The recombinant phage according to 1 above, wherein the gene encoding the invalidating factor is either (A) or (B) below: (A) a gene comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 27; (B) a gene encoding a protein having 80% or more identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 27 and having the activity of a disabling factor of an anti-phage defense system; 4. A recombinant phage comprising a gene encoding a disabling factor of an anti-phage defense system, wherein the gene encoding the disabling factor is a gene isolated by a screening method comprising the following steps (a1) to (a4): (a1) preparing a phage gene knockout library in which each gene is deleted; (a2) preparing bacteria that express the anti-phage defense system; (a3) ​​infecting the bacteria prepared in (a2) with the phage gene knockout library prepared in (a1) and measuring the infectivity by spot assay; and (a4) isolating the gene as encoding the disabling factor of the anti-phage defense system when the infectivity measured in (a3) ​​is 1 / 100 or less of that when wild-type bacteria are infected. 5. The recombinant phage according to any one of 1 to 4 above, which is a lytic phage. 6. An antibacterial agent or bacterial test agent comprising the recombinant phage according to 5 above. 7. A composition comprising the antibacterial agent or bacterial test agent according to 6 above. 8. A sterilization method for killing target bacteria, which comprises contacting the target bacteria with the composition according to 7 above. 9. The sterilization method according to 8 above, wherein the target bacteria are pathogenic bacteria.10. The sterilization method according to 9, wherein the pathogenic bacterium is at least one selected from Campylobacter jejuni, Vibrio cholerae, Escherichia coli O157, Legionella pneumophila, Yersinia pestis, Salmonella, and Shigella. 11. A method for screening for a gene encoding a neutralizing factor of an anti-phage defense system, comprising the following steps (b1) to (b4): (b1) preparing a phage library in which each gene has been deleted; (b2) preparing bacteria that express the anti-phage defense system; (b3) infecting the bacteria prepared in (b2) with the phage gene knockout library prepared in (b1) and measuring infectivity by spot assay; and (b4) isolating the gene as encoding the neutralizing factor of the anti-phage defense system when the infectivity measured in (b3) is 1 / 100 or less of that when a wild-type bacterium is infected.

[0013] The recombinant phages disclosed herein can neutralize the bacterial anti-phage defense system that protects against phage infection, and because they have a bactericidal mechanism different from that of conventional antibacterial drugs, they can kill drug-resistant bacteria.

[0014] Panel a of Figure 1 shows the results of infection experiments using all defense system libraries except for Druantia. Panel b of Figure 1 shows the results of analysis of each ORF using four independent synthetic phage strains. * indicates a phage gene for which anti-phage defense activity was confirmed in the complementation experiment of Figure 2. Panel c of Figure 1 shows the results of comparing the EOP of the ΦDruSM1 wild-type and ΦDruSM1 ORF71 deletion mutant of E. coli A17 strain. Panel d of Figure 1 shows the EOP ratio (A17 / DH10B) of the ΦDruSM1 ORF71 deletion mutant. Figure 2 shows the results of preparing bacteria with plasmid complementation of candidate genes infected with each deletion mutant phage and measuring the EOP. Figure 3 shows the results of comparing the EOP of KSA8 phage with anti-Druantia (ORF71) ＤｒｕＳＭ１Figure 4 shows the results of analyzing the bactericidal activity of phages carrying a gene encoding a factor that inhibits the Tmn defense system by spot assays. Phages carrying a gene encoding a factor that inhibits the Tmn defense system were prepared, and the bactericidal activity of the phages against bacteria carrying Durantia was analyzed by spot assays. Figure 4 shows the identified phage-derived factors that disable the anti-phage defense system, as well as the amino acid length and similar domain information of the factors. Figure 5 shows a diagram of the identified phage-derived factors that disable the anti-phage defense system. Figure 6(a) shows the results of analyzing the bactericidal activity of phages carrying a gene encoding a factor that inhibits the Tmn defense system against bacteria carrying Tmn. Figure 6(b) shows a schematic diagram comparing the genomes of each phage. Figure 6(c) shows the results of analyzing the bactericidal activity of phages carrying a gene encoding a factor that inhibits the Tmn defense system against bacteria carrying Tmn.

[0015] Hereinafter, the present invention will be described based on the embodiments, but the present invention is not limited to these embodiments. In this specification, bacteriophage is also simply referred to as "phage".

[0016] <Recombinant Phage> The recombinant phage of this embodiment contains a gene encoding a factor that neutralizes the anti-phage defense system. The factor that neutralizes the anti-phage defense system refers to a protein that has the activity of neutralizing the anti-phage defense system.

[0017] Whether or not a bacterium has the activity to neutralize an anti-phage defense system can be determined, for example, by the following methods 1) and 2): 1) Bacteria having an anti-phage defense system are infected with a recombinant phage in which nucleotides containing a gene encoding a target protein have been introduced into the phage genome, and it is examined whether or not infection can be established; 2) Nucleotides containing a gene encoding a target protein are expressed in advance in bacteria having an anti-phage defense system using a plasmid or the like, and the bacteria are infected with a phage genome lacking the nucleotides containing the gene, and it is examined whether or not infection can be established.

[0018] In this specification, the "anti-phage defense system" of bacteria is a general term for the mechanism by which bacteria protect themselves from phages (viruses that infect bacteria). When a phage infects a bacterium, it lyses the bacterium, releasing progeny phages that also kill surrounding bacteria, resulting in the demise of the colony. If the target bacterium has an anti-phage defense system, even if the phage infects the bacterium, an abortive infection will occur, causing the bacterium to die (commit suicide) before the phage can mature, thereby maintaining the survival of the colony.

[0019] Anti-phage defense systems include, for example, BREX, AVAST, SIR2, DUF, Durantia, Retron, DRT, Mza, upx, CRISPR-Cas, restriction-modification enzymes, and Argonaute.

[0020] Specific examples of anti-phage defense systems include Brex type I, AVAST type III, SIR2+HerA, DUF4297+HerA, hhe, Druantia type I, tmn, AVAST type II, Retron Ec67, DRT Type II, DRT Type III, DRT type IV, MzaABCDE, upx, and Retron Ec67, Ec78, Ec48, and Ec83.

[0021] In the recombinant phage of this embodiment, the gene encoding the disabling factor of the anti-phage defense system may be any of the following (A) to (C): (A) a gene comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 27; (B) a gene encoding a protein having 80% or more identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 27 and having disabling factor activity in the anti-phage defense system; (C) a gene comprising a nucleotide sequence encoding a protein described in the following [1] to [3]: [1] a gene comprising a nucleotide sequence encoding an amino acid sequence set forth in any one of SEQ ID NOs: 28 to 54; [2] a mutant protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, or added in the amino acid sequence set forth in any one of SEQ ID NOs: 28 to 54, and having disabling factor activity in the anti-phage defense system; [3] a homologous protein having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 28 to 54 and having disabling factor activity in the anti-phage defense system.

[0022] The gene (B) has an identity of 80% or more to the base sequence shown in any one of (B) SEQ ID NOs: 1 to 27, and preferably has an identity of 85% or more, 90% or more, 95% or more, more preferably 97% or more, even more preferably 98% or more, and most preferably 99% or more, in the following order.

[0023] The mutant protein of (C)[2] above refers to a protein obtained by artificially deleting or substituting amino acid residues in the original protein, or by inserting or adding amino acid residues into the protein.

[0024] In the mutant protein of (C)[2], the deletion, substitution, insertion, or addition of amino acids may mean the deletion, substitution, insertion, or addition of 1 to 10 amino acids at any position within the same sequence. The number of amino acids deleted, substituted, inserted, or added is 1 to 10, preferably 1 to 8, and most preferably 1 to 5.

[0025] The amino acids to be deleted, substituted, inserted, or added may be naturally occurring or non-naturally occurring. Naturally occurring amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0026] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3-hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

[0027] A gene containing a nucleotide sequence encoding the mutant protein of (C)[2] above can be obtained, for example, by subjecting a DNA consisting of the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 27 as a template to error-prone PCR or the like. Alternatively, the DNA of [2] above can also be obtained by PCR [Gene, 77, 51 (1989)] using a pair of PCR primers each having a nucleotide sequence at its 5' end that is designed to introduce a desired mutation (deletion, substitution, insertion, or addition).

[0028] The homologous protein of (C) [3] above is a protein that is similar in structure and function to the original protein, and therefore the gene encoding the protein is thought to have the same evolutionary origin as the gene encoding the original protein, and is a protein found in organisms existing in nature. In this embodiment, the homologous protein includes the protein of [3] above.

[0029] The homologous protein of (C)[3] is a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence shown in any one of SEQ ID NOs: 28 to 54. The identity is preferably 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, in the following order, and more preferably 99% or more.

[0030] In this embodiment, the identity of amino acid sequences and nucleotide sequences can be determined using the algorithm BLAST by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing nucleotide sequences using BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0031] The gene containing the base sequence of (B) and the gene containing the base sequence encoding the homologous protein of (C) [3] can be obtained, for example, by searching various gene sequence databases for base sequences that have 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, and more preferably 99% or more identity to the base sequence shown in any one of SEQ ID NOs: 1 to 27, or by searching various protein sequence databases for amino acid sequences that have 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, and more preferably 99% or more identity to the amino acid sequence shown in any one of SEQ ID NOs: 28 to 54, and by using probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search, and a microorganism containing the DNA, in a manner similar to the method for obtaining the DNA described above.

[0032] In (A) above, the nucleotide sequences shown in SEQ ID NOs: 1 to 27 encode the amino acid sequences shown in SEQ ID NOs: 28 to 54, respectively. For the nucleotide sequences shown in SEQ ID NOs: 1 to 27, the amino acid sequences of the disabling factors encoded by the nucleotide sequences, gene names, and targeted anti-phage defense systems are shown in Tables 1 to 3 below.

[0033] The recombinant phage of this embodiment contains a gene encoding a factor that disables the anti-phage defense system described above, thereby being able to inhibit the anti-phage defense system in the target bacterium, and allowing the recombinant phage to infect the target bacterium.

[0034] Methods for incorporating a gene encoding a factor that disables the anti-phage defense system into a phage include, for example, inserting the gene into the phage genome, inserting the gene during assembly of the phage genome in vitro or intracellularly and then rebooting the gene within the bacterial cell or in a test tube, and recombining the phage gene within the bacterial cell (Diana P. Pires et al., Microbiology and Molecular Biology Reviews, 2016, Vol. 80, No. 3).

[0035] In the phage genome, preferred sites for inserting a gene encoding a factor that disables the anti-phage defense system include, for example, a region that controls metabolism at the early stage of infection, a region that controls phage DNA amplification, and a capsid synthesis region that is expressed in high amounts.

[0036] The recombinant phage of this embodiment is preferably a recombinant phage modified to enhance expression of a gene encoding a neutralizing factor of the anti-phage defense system. Methods for enhancing expression of the gene include, for example, having multiple copies of the gene present in the phage genome, substituting a stronger expression regulatory sequence such as a promoter for the gene, optimizing codons, enhancing enzyme activity, etc. Examples of such promoters include rRNA promoters, housekeeping gene promoters, and LexA promoters.

[0037] <Method for screening genes encoding invalidating factors of an anti-phage defense system> One aspect of this embodiment is a recombinant phage containing a gene encoding a invalidating factor of an anti-phage defense system, wherein the gene encoding the invalidating factor is a gene isolated by a screening method comprising the following steps (a1) to (a4): (a1) preparing a phage gene knockout library in which each gene is deleted; (a2) preparing bacteria that express the anti-phage defense system; (a3) ​​infecting the bacteria prepared in (a2) with the phage gene knockout library prepared in (a1) and measuring infectivity by spot assay; and (a4) isolating the gene as encoding the invalidating factor of the anti-phage defense system if the infectivity measured in (a3) ​​is 1 / 100 or less of that measured when wild-type bacteria are infected with the phage gene knockout library. Each step is described below.

[0038] (a1) Step of Preparing a Phage Gene Knockout Library in Which Each Gene Has Been Deleted Step (a1) is a step of recovering phages from a sample such as sewage, wastewater, etc., and preparing a phage gene knockout library in which each gene has been deleted in the phages. Specifically, for example, the sample is concentrated by a concentration method (e.g., centrifugation, filter, PEG concentration method, etc.), and the concentrated sample is centrifuged to recover the phages.

[0039] The phage genomes prepared from the recovered phages are amplified by PCR, and circular DNA is constructed using primers that remove each gene. This artificially synthesized circular DNA is then introduced into host cells (e.g., Escherichia coli) by electroporation to construct a phage gene knockout library.

[0040] (a2) Step of preparing bacteria expressing the anti-phage defense system Step (a2) is a step of preparing bacteria to be infected with the phage gene knockout library prepared in step (a1). Specifically, for example, bacteria expressing the anti-phage defense system are grown in an appropriate medium and then collected.

[0041] (a3) A step of infecting the bacteria prepared in (a2) with the phage gene knockout library prepared in (a1) and measuring the infectivity by spot assay. Step (a3) ​​is a step of infecting the bacteria prepared in step (a2) with the phage gene knockout library prepared in step (a1) and measuring the infectivity by spot assay, for example, by the following procedure. i) Spreading bacteria onto plate: The bacteria prepared in step (a2) are uniformly spread on a flat plate coated with an agar medium. ii) Phage spot creation: A solution containing the phage gene knockout library prepared in step (a1) is placed on the plate as a small drop (spot). The spots can be created at different dilution rates to adjust the concentration and amount of phage. iii) Incubation: After the phage spots are placed on the plate, the plate is incubated (cultured) at an appropriate temperature. During this period, phages from the phage gene knockout library infect the bacteria, forming infection sites. iv) Measurement of infectivity: After incubation, the plate is observed to check for growth or lysis of bacteria around the phage spots. If the phage is infected, the bacteria around the spots will lyse and a clear zone (plaque) will be formed. The number or size of the clear zones will be evaluated to quantitatively analyze the infectivity of the phage.

[0042] (a4) A step of identifying a phage whose infectivity measured in (a3) ​​is 1 / 100 or less of that when infected with wild-type bacteria, and isolating the gene deleted from the phage as a gene encoding a neutralizing factor of the anti-phage defense system.In step (a4), if the infectivity of the phage quantitatively analyzed in step (a3) ​​iv) is 1 / 100 or less of that when infected with wild-type bacteria, the gene deleted from the phage is isolated as a gene encoding a neutralizing factor of the anti-phage defense system.

[0043] The infectivity of the phage is 1 / 100-fold or less, more preferably 1 / 10-fold or less, compared to that when it infects wild-type bacteria.

[0044] <Gene Screening Method> Another aspect of this embodiment includes a method for screening for genes encoding disabling factors of an anti-phage defense system, comprising the following steps (b1) to (b4): (b1) preparing a phage gene knockout library in which each gene is deleted; (b2) preparing bacteria that express the anti-phage defense system; (b3) infecting the bacteria prepared in (b2) with the phage gene knockout library prepared in (b1) and measuring the infectivity by spot assay; (b4) isolating the gene as encoding a disabling factor of the anti-phage defense system when the infectivity measured in (b3) is 1 / 100 or less of that measured in infection with wild-type bacteria. Steps (b1) to (b4) are the same as steps (a1) to (a4) above.

[0045] According to the gene screening method of this embodiment, genes encoding factors that disable the anti-phage defense system can be screened efficiently.

[0046] <<Phage>> In this embodiment, examples of phages include lytic phages and temperate phages, with lytic phages being preferred. A lytic phage infects a host bacterium, grows inside the bacterium, and then lyses the bacterium from the inside, releasing the grown phages outside the bacterium. At this time, the bacterium is killed. Unlike temperate phages, lytic phages grow inside the bacterium without going through a lysogenic cycle. Therefore, there is no risk of incorporating the genomic DNA of the host bacterium and acquiring toxicity to the host cell, and they do not become part of the host genome, making them preferable from the perspective of safety.

[0047] Examples of lytic phages include Myoviridae phages (genus T4-like viruses, genus P1-like viruses, genus P2-like viruses, genus Mu-like viruses, genus SPO1-like viruses, and genus phiH-like viruses), Siphoviridae phages (genus λ-like viruses, genus γ-like viruses, genus T1-like viruses, genus T5-like viruses, genus c2-like viruses, genus L5-like viruses, genus PsiM1-like viruses, genus phiC31-like viruses, and genus N15-like viruses), and Podoviridae phages (genus T7-like viruses). genus, phi29-like viruses, P22-like viruses, N4-like viruses), Tectiviridae phages (genus Tectivirus), Corticoviridae phages (genus Corticovirus), Lipothrixviridae phages (genus Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus), Plasmavirus Plasmaviridae phages (genus Plasmavirus), Rudiviridae phages (genus Rudivir), Fuselloviridae phages (genus Fusellovirus), Inoviridae phages (genus Inovirus, genus Plectrovirus, genus M13-like virus, genus fd-like virus), Microviridae phages (genus Microvirus, genus Spiromicrovirus, Bde genus Allovirus, genus Chlamydiavirus), Leviviridae phages (genus Levivirus, genus Allolevivirus), Cystoviridae phages (genus Cystovirus), Ampullaviridae phages, Bicaudaviridae phages, Clavaviridae phages, Globuloviridae phages,Examples include phages of the genus Guttavirus.

[0048] Examples of temperate phages include, but are not limited to, those of the Phietavirus family, the Lambdavirus family, and the Punavirus family.

[0049] <Method for Preparing Recombinant Phage> The recombinant phage of this embodiment can be prepared by a method including the following steps (1) to (4): (1) preparing the following a) and b): a) a phage genome; b) nucleotides containing a gene encoding a factor that disables the anti-phage defense system; (2) inserting the nucleotides of b) into the a) phage genome to obtain a recombinant phage genome; (3) obtaining a recombinant phage into which the recombinant phage genome has been introduced; and (4) recovering the recombinant phage. Each step will be described below.

[0050] <<Step (1)>> Step (1) is a step of preparing a) a phage genome and b) a nucleotide sequence containing a gene encoding a neutralizing factor of the anti-phage defense system.

[0051] a) Phage genomes include genomes derived from the phages mentioned above in the section "Phages".

[0052] b) Examples of nucleotides containing a gene encoding a disabling factor of the anti-phage defense system include the nucleotides containing the genes described in (A) to (C) above, as described above in the section <Recombinant Phage>.

[0053] <<Step (2)>> Step (2) is a step of inserting the b) nucleotide into the a) phage genome prepared in step (1) to obtain a recombinant phage genome. Methods for inserting the b) nucleotide into the a) phage genome include the methods described above in the section <Recombinant Phage>.

[0054] <<Step (3)>> Step (3) is a step of obtaining a recombinant phage into which the recombinant phage genome obtained in step (2) has been introduced. The recombinant phage can be obtained by introducing the recombinant phage genome obtained in step (2) into a bacterium to obtain a phage-synthesizing bacterium and allowing the phage-synthesizing bacterium to produce the recombinant phage.

[0055] Methods for introducing a phage genome into bacteria include, for example, the competent cell method, electroporation, etc., and may be carried out by conventional methods. Examples of bacteria into which a recombinant phage genome can be introduced include common bacteria such as Escherichia coli.

[0056] <<Step (4)>> Step (4) is a step of recovering recombinant phages from the phage-synthesizing bacteria obtained in step (3). Specifically, the recombinant phages are recovered by lysing the recombinant phages from the phage-synthesizing bacteria and / or releasing the phages extracellularly. Any known method can be used to recover the recombinant phages.

[0057] <Antibacterial Agent or Bacterial Test Agent and Composition Comprising the Antibacterial Agent or Bacterial Test Agent> The recombinant phage according to this embodiment exhibits excellent infectivity against bacteria having an anti-phage defense system, and is therefore useful as an active ingredient of an antibacterial agent. An "antibacterial agent" refers to an agent that has the action or effect of inhibiting bacterial growth (bacteriostasis) or the action or effect of killing bacteria (sterilization). The antibacterial agent or bacterial test agent according to this embodiment is provided for use in various applications as a composition containing it. Both the antibacterial agent or bacterial test agent and the composition according to this embodiment may be in a dry state or may be dissolved in a solution.

[0058] The concentration of the recombinant phage in the antibacterial agent of this embodiment can be appropriately selected and changed depending on various conditions such as the target bacterium and the intended use. ６ ~10 １０ PFU / ml is preferred, and 10 ８ PFU / ml to 10 １０ PFU / ml.

[0059] <<Target Bacteria>> The target bacteria are not particularly limited, and there are no particular restrictions on their shape (generally broadly classified into spherical, rod-shaped, and spiral shapes), Gram stainability (Gram-positive or Gram-negative), oxygen requirement (aerobic, anaerobic, or facultative anaerobic), mode of existence, etc.

[0060] Examples of target bacteria include Escherichia coli, Shigella bacteria (S. dysenteriae, S. frexneri, S. sonnei, etc.), Salmonella bacteria (S. typh, S. paratyphi-A, S. schottmülleri, S. typhimurium, S. enteritidis, etc.), Enterobacter bacteria (E. aerogenes, E. cl), oacae, etc.), Klebsiella (e.g., K. pneumoniae, K. oxytoca, etc.), Proteus (e.g., P. mirabilis, P. vulgaris, etc.), Yersinia (e.g., Y. pestis, Y. enterocolitica, etc.), Vibrio (e.g., V. cholerae, V. parahaem, etc.), lyticus, etc.), Haemophilus bacteria (e.g., H. influenzae, H. parainfluenzae, H. ducreyi, etc.), Pseudomonas bacteria (e.g., P. aeruginosa, P. cepacia, P. putida, etc.), Acinetobacter bacteria (e.g., A. calcoaceticus, A. baumann, etc.), ii, A. lwoffii, etc.), Legionella (e.g., L. pneumophila, etc.), Bordetella (e.g., B. pertussis, B. parapertussis, B. bronchiseptica, etc.), Brucella (e.g., B. melitensis, B. abortus, B. suis, etc.), Francisella tularensis), Bacteroides (e.g., B. fragilis, B. melaninogenicus, etc.), Neisseria (e.g., N. gonorrhoeae, N. meningitidis, etc.), Staphylococcus (e.g., S. aureus,S. epidermidis, S. saprophyticus, etc.), Streptococcus bacteria (e.g., S. pyogenes, S. agalactiae) , S. viridans, S. pneumoniae, etc.), Enterococcus (e.g., E. faecalis, E. faecalis) Bacillus (e.g. B. subtilis, B. anthracis, B. cere) us, etc.), Clostridium (e.g., C. difficile, C. botulinum, C. perfrin) gens, C. tetani, etc.), Corynebacterium (e.g., C. diphtheriae, etc.), Mycobacterium (e.g., M. tuberculosis, M. bovis, M. leprae, M. avium, M. intracellulare, M. kansasii, M. ulcerans, etc.), Mycoplasma, Borrelia (e.g., B. recurrentis, B. burgdorferi, etc.), Treponema pallidum palidum), Campylobacter bacteria (e.g., C. coli, C. jejuni, C. fetus, etc.), Helicobacter bacteria (e.g., H. pylori, H. heilmannii, etc.), Rickettsia bacteria (e.g., R. prowazekil, R. mooseri, R. tsutsugamushi, etc.), Chlamydia bacteria (e.g., C. trachomatis, C. psittaci, etc.), and Listeria bacteria (e.g., L. monocytogenes, etc.).

[0061] The target bacterium is preferably a pathogenic bacterium, such as Campylobacter jejuni, Vibrio cholerae, Escherichia coli O157, Legionella pneumophila, Yersinia pestis, Salmonella, or Shigella.

[0062] Representative uses of the composition of this embodiment include a pharmaceutical (therapeutic, preventive, or bacterial testing) composition, a disinfectant composition, a cleaning composition, and an agricultural chemical composition. (i) Pharmaceutical Composition The pharmaceutical composition of this embodiment is used for treating or preventing bacterial infections or for bacterial testing (phage typing). The pharmaceutical composition of this embodiment can exert a therapeutic or preventive effect against bacterial infections. The therapeutic or preventive effect here includes (1) preventing bacterial infection, (2) preventing, suppressing, or delaying the onset of bacterial infection, (3) alleviating (alleviating) symptoms characteristic of bacterial infections or accompanying symptoms, and (4) preventing, suppressing, or delaying the worsening of symptoms characteristic of bacterial infections or accompanying symptoms. Note that the therapeutic effect and the preventive effect are concepts that partially overlap.

[0063] Pharmaceutical compositions can be formulated according to conventional methods. The pharmaceutical compositions of this embodiment may contain a suitable pharmaceutically acceptable carrier to prepare a pharmaceutically acceptable carrier. The carrier may include a biocompatible material such as silicone, collagen, or gelatin. Alternatively, the carrier may be various emulsions. Furthermore, the pharmaceutical composition may contain one or more formulation additives selected from, for example, diluents, flavorings, preservatives, excipients, disintegrants, lubricants, binders, emulsifiers, plasticizers, and the like.

[0064] The dosage form when formulated is not particularly limited. Examples of dosage forms include tablets, powders, fine granules, granules, capsules, syrups, injections, topical preparations (ointments, creams, lotions, liquids, gels, poultices, plasters, tapes, aerosols, etc.), and suppositories. One aspect of the pharmaceutical composition according to this embodiment can be formulated into a dosage form suitable for oral administration using a pharmaceutically acceptable carrier well known in the art.

[0065] The administration route of the pharmaceutical composition according to this embodiment is not particularly limited, and it can be administered orally or parenterally. Parenteral administration can be, for example, intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal administration, or direct administration to bacterial flora or an infected site. This administration method may be performed in the same manner as conventional phage therapy.

[0066] Furthermore, when using the pharmaceutical composition according to this embodiment for the above-mentioned 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.

[0067] The single dose and frequency of administration of the pharmaceutical composition of this embodiment can be selected and changed as appropriate depending on the purpose of administration, as well as various conditions such as the age and weight of the patient, and the severity of the symptoms and disease.

[0068] The number and duration of administration may be one time only, or may be one to several times a day for several weeks, and the state of the disease may be monitored, and administration may be repeated or repeated depending on the state. The concentration of the recombinant phage in the pharmaceutical composition according to this embodiment can be appropriately selected and changed depending on various conditions such as the state of the disease and the condition of the subject, but is usually 10 ６ ~10 １０ PFU / ml is preferred, and 10 ８ PFU / ml to 10 １０ PFU / ml.

[0069] The pharmaceutical composition according to this embodiment can be used in combination with other pharmaceutical compositions, etc. The pharmaceutical composition according to this embodiment may be administered simultaneously with the other pharmaceutical compositions, or may be administered at intervals, with no particular restriction on the order of administration.

[0070] Furthermore, in the pharmaceutical composition of this embodiment, the period during which the disease is improved or alleviated is not particularly limited, but may be temporary improvement or alleviation, or improvement or alleviation for a certain period of time.

[0071] Furthermore, the pharmaceutical composition of this embodiment can be used to treat an organism, a portion of the organism's body, or a portion thereof extracted or excreted from the organism. The organism is not particularly limited and may be, for example, an animal, a plant, or a fungus. Examples of the animal include humans, domestic animals, and wild animals. Therefore, the pharmaceutical composition of this embodiment can also be used in animal therapy. In other words, the recombinant phage of this embodiment can also be used in animal therapy methods for various animals other than humans.

[0072] (ii) Disinfecting composition, cleaning composition The disinfecting composition or cleaning composition according to this embodiment is used, for example, to disinfect or clean rooms (including hospital rooms), kitchens, toilets, washrooms, bathrooms, etc., disinfect or clean tableware, cutlery (e.g., knives, forks, spoons, etc.), cooking utensils (e.g., kitchen knives, knives, pots, mixers, microwave ovens, ovens, etc.), medical instruments and devices, and disinfect or clean hands, fingertips, the oral cavity, etc. The disinfectant or cleaning agent of this embodiment is, for example, in a liquid form (e.g., spray, lotion), gel form, or solid form (e.g., powder), and is applied by coating, spraying, sprinkling, etc. The disinfecting composition or sterilizing composition of this embodiment may be supported or attached to a carrier (e.g., sheet-like) made of natural fibers, synthetic fibers, etc., to form products used for wiping purposes, infection prevention masks, etc.

[0073] Antibacterial or disinfecting ingredients such as benzalkonium chloride, cetylpyridinium chloride, phenoxyethanol, isopropylmethylphenol, chlorhexidine gluconate, pH adjusters, surfactants, adsorbents, carriers, etc. may be added to the disinfecting composition or cleaning composition of this embodiment. The concentration of the recombinant phage in the disinfecting composition and cleaning composition of this embodiment can be appropriately selected and changed depending on various conditions such as the state of the disease and the condition of the subject, but is usually 10 ６ ~10 １０ PFU / ml is preferred, and 10 ８ PFU / ml to 10 １０ PFU / ml.

[0074] (iii) Pesticide Composition The pesticide composition of the present embodiment is effective against various crops widely cultivated in agriculture and horticulture, for example, cultivated monocotyledonous plants such as rice, wheat, and corn, and cultivated dicotyledonous plants such as kidney bean, soybean, tomato, potato, eggplant, and cucumber.

[0075] The pesticide composition of the present embodiment is usually used by mixing with various carriers such as liquid, solid, gaseous, etc. A surfactant or other pesticide adjuvant may be further added to the pesticide composition of the present embodiment, if necessary.

[0076] Examples of formulation forms include emulsifiable concentrates, wettable powders, dry flowables, flowables, water-soluble formulations, granules, microgranules, granules, powders, paints, spray formulations, aerosol formulations, microcapsule formulations, fumigation formulations, and fumigation formulations.

[0077] The concentration of the recombinant phage in the pesticide composition according to this embodiment can be appropriately selected and changed depending on various conditions such as the target situation. ６ ~10 １０ PFU / ml is preferred, and 10 ８ PFU / ml to 10 １０ PFU / ml.

[0078] <Sterilization Method> The method for sterilizing the target bacteria of this embodiment simply involves contacting the antibacterial agent or composition of this embodiment with the target bacteria. If the target bacteria are in a state of forming colonies on a solid medium or the like, the antibacterial agent or composition in powder or solution form can be sprayed onto the colonies. Furthermore, if the target bacteria are in a state of being present in a solution, the amount of powder or the concentration of the liquid can be adjusted and added to achieve the required concentration. Examples of target bacteria include the bacteria listed above in the section <<Target Bacteria>>.

[0079] The present invention will be specifically described below using examples and comparative examples, but the present invention is not limited to these.

[0080] [Test Example 1] Analysis of the diversity of susceptibility to anti-phage defense systems in a phage library. The defensive activity and range of protection against each phage differ for each defense system. However, there are no reports that comprehensively describe the activity and range of these defense systems. Therefore, we created E. coli DH10B (pLG001-034) transformed with each defense system-expressing plasmid and investigated the infectivity of 262 phages to each transformed strain. Thirteen phages were collected from urban sewage using DH10B as the host bacterium.

[0081] Druantia type I, RAIDAR1, DRT type III, Sir2 + HerA, and DRT type II were defense systems that inhibited a wide range of phage infections by 99.7%, 62.3%, 54.6%, and 39.8%, respectively. Phage isolation from sewage using DH10B host bacteria expressing Druantia type I resulted in plaque formation rates ranging from 0% to 0.6% of those of the wild-type strain. For RADAR1 and DRT type III, recovery rates ranged from approximately 20% to 80%. Searching for phages that formed clear plaques on DH10B expressing Druantia type I resulted in the isolation of one phage from sewage, designated ΦDruSM1.

[0082] Whole genome sequencing revealed that ΦDruSM1 has a 60 kb genomic DNA and belongs to the Quenovirinae family of phages, a small Siphoviridae with a genome size of 56-61 kb. The phage library infected with DH10B also contained Quenovirinae phages designated KSA3, KSA8, KSS4, KSW4, and SHIN8 in the defense library (pLG001-034).

[0083] The defense patterns of Quenovirinae against the defense library (pLG001-034) were similar, and all tested phages were protected by the defense system consisting of the reverse transcriptase domain, RT-AbiP2, Retron-TIR, Retron-Ec67, Retron-Ec78, Retron-Ec86, DRT types IV, and mzaABCDE. Although the genomes and defense patterns of these six Quenovirinae phages were similar, only ΦDruSM1 escaped Druantia type I.

[0084] [Test Example 2] Construction of a Phage Gene Knockout Library Of the six genoviridae, only ΦDruSM1 was not inhibited by Druantia type I. From this, we hypothesized that ΦDruSM1 possesses a unique gene that overcomes Druantia type I. ΦDruSM1 has 105 open reading frames in its genome, but many of the genes have unknown functions, and the gene sequences of ΦDruSM1 and other genoviridae are slightly different, making it difficult to predict the genes responsible for evading Druantia type I. Therefore, we constructed a phage single-gene knockout mutant library using in vitro phage synthesis16,17 to identify genes involved in evading Druantia type I defense activity.

[0085] First, the phage genome was amplified by PCR and assembled to create circular DNA using primers that removed one gene. This artificially synthesized genome was electroporated into E. coli HST08 to reactivate the phage. The number of plaques generated by the reactivation of the synthesized phage was counted, and the efficiency of successful phage synthesis varied greatly depending on the gene being deleted. If no phage plaques were observed, it is possible that the gene to be deleted was essential for the phage.

[0086] As a result, 32 genes were predicted to be essential and 72 genes were predicted to be non-essential in ΦDruSM1. In particular, when genomes lacking genes involved in lysis, terminase, capsid, tail structure, and nucleotide metabolism were electroporated into HST08, few or no plaques were observed. However, most gene-deficient phages, excluding these genes, produced numerous plaques upon reactivation. Overall, 72 ORF knockout mutants were successfully constructed and used for further experiments.

[0087] [Test Example 3] Searching for genes related to anti-phage defense systems using a phage gene knockout library To search for anti-Druantia defense genes from ΦDruSM1, DH10B carrying Durantia type I was infected with the ΦDruSM1 gene knockout library, and the plaque efficiency (EOP) was measured. If the EOP of the gene-deficient phage was reduced, the deleted gene may be anti-Druantia. In an experiment comparing the susceptibility of ΦDruSM1 to bacteria carrying the defense system, ΦDruSM1 and other Kenoviridae phages showed different susceptibilities to defense systems other than Durantia. Therefore, infection experiments were performed with all defense system libraries other than Durantia. The results are shown in Figure 1(a).

[0088] Because defective phages can carry genetic mutations acquired during synthesis, four independent synthetic phage strains were analyzed for each ORF. The results are shown in Figure 1b. As shown in Figure 1b, a total of 19 defective phages were found in more than three of the four strains, with infection efficiency reduced by more than 100-fold. Seven defective phages were found with reductions of 10- to 100-fold or more. Four genes were identified with efficiency improved by more than 100-fold. Mutant strains with altered EOPs were confirmed by PCR.

[0089] As shown in Figure 1a and b, the phages with reduced infectivity against the Durantia type I strain were the deletion mutants of ORF71 and ORF65. No change in plaque size was observed in the deletion mutant of ORF65, but the EOP was 10 in the Durantia type I-carrying strain. －１ The 9 ORF deletion mutant was less infectious in Brex1 type 1 bacteria.

[0090] In particular, gene deletion phages in the "low activity, auxiliary metabolic genes, and host transfer" region, including ORFs 41-48, reduced infectivity against not only Brex1 but also the restricted type. Phages with reduced infectivity against bacteria harboring the AVAST type III defense system containing the DUF4297 and STAND domains were those lacking ORFs 55, 72, 83, and 84.

[0091] The mutant with the lowest infectivity was ORF84, with an EOP of 10 －４ All phages with reduced infectivity against AVAST type III formed small plaques. The phages with reduced infectivity against bacteria containing the qatABCD defense system, which has the ATPase, QueC, and TatD domains, were deletion mutants of ORF84 and ORF85, and had EOPs of 10 and 10, respectively. －２ and 10 －４ It was reduced.

[0092] The phage with reduced infectivity against bacteria carrying the hhe defense system, which contains DUF4011, a helicase, and a Vsr domain, is a deletion mutant of ORF65 and has an EOP of 10. －３ Deletion of PHORF69, located upstream of the ORF65 phage, also reduced the EOP for hhe-containing bacteria.

[0093] The deletion mutant of ORF58 reduced the EOP of SIR2+HerA, DUF4297+HerA, and ppl. Interestingly, the deletion mutant of ORF58 increased the EOP of bacteria with Retron-Ec86, Retron-Ec78, and DRT type II, which contain the reverse transcriptase domain.

[0094] The gene names, anti-phage defense systems (abbreviated as "target defense systems" in the tables), protein names, domain names, nucleotide sequences, and amino acid sequences of the anti-phage defense system-related genes are shown in Tables 1 to 4. The genes shown in Tables 1 to 3 are genes that inactivate the anti-phage defense system, and the genes shown in Table 4 are genes that activate the anti-phage defense system.

[0095]

[0096]

[0097]

[0098]

[0099] [Test Example 4] Effect of ORF71 on Escherichia coli clinical strains carrying Durantia To determine the effect of the anti-phage defense system on clinical isolates carrying the defense system, the effect of ORF71, an anti-Druantia type I candidate, was evaluated on Escherichia coli clinical isolate A17, which natively carries Durantia type I.

[0100] An amino acid sequence alignment between Gao et al.'s defense library, Druantia type I, and E. coli A17 Druantia type I revealed high similarity, with similarities of 99.2% for DruA, 98.5% for DruB, 98.4% for DruC, 99.7% for DruD, and 99.4% for DruE. Figure 1c shows a comparison of the EOPs of the wild-type (WT) and ORF71-deleted mutants of E. coli A17. As shown in Figure 1c, only a single gene, ORF71, was found to affect infectivity in clinical isolates of E. coli carrying Druantia type I.

[0101] The EOP ratio (A17 / DH10B) of the ΦDruSM1 ORF71 deletion mutant is shown in Fig. 1(d). As shown in Fig. 1(d), the EOP ratio (A17 / DH10B) was reduced by more than 1,000-fold compared to the ΦDruSM1 wild-type. The plaque sizes of the ΦDruSM1 wild-type and ORF71 deletion mutant of E. coli strain A17 were reduced compared to DH10B, and the ORF71 deletion mutant plaques were smaller than those of the wild-type and could not be counted with the naked eye.

[0102] [Test Example 5] Identification of genes that inactivate the anti-phage defense system Candidates for defense system inhibitors were identified by screening using a gene deletion library (Figure 1a and b). To confirm that these genes act as defense system inhibitors, bacteria carrying plasmid complementation of the candidate genes infected with each deletion mutant phage were prepared, and EOP was measured. The results are shown in Figure 2.

[0103] Ectopic expression of ORF71 by ΦDruSM1 Coexpression of Brex1 type I with ORFs 38, 46, and 72 restored the infectivity of the ORF71 deletion mutant to the same plaque size as the wild type for Durantia type I. Deletion mutants of ORFs 41, 45, and 48 also reduced infectivity for Brex type I, but coexpression assays of these ORFs with Brex type I did not restore infectivity for Brex type I.

[0104] [Test Example 6] Identification of a gene that activates the anti-phage defense system Screening using a gene deletion library revealed that ORF58 deletion mutants had increased infectivity against bacteria carrying retron-Ec86, retron-Ec78, and DRT type 2 (Figure 1a and 1b). These findings suggested that ORF58 is the gene that activates retron-Ec86, retron-Ec78, and DRT type 2. To confirm activation of the defense system by ORF58, ORF58 and retron-Ec86 were coexpressed in the DH10B strain. In retron-carrying strains, induction of ORF58 expression resulted in cytotoxicity. In contrast, the combination of ORF58 with retron-Ec78 or DRT type 2 did not result in cytotoxicity.

[0105] Test Example 7 Prediction of Proteins that Inactivate / Activate the Anti-phage Defense System Function prediction of anti-defense genes or sensor genes was performed using HH pred. ORF46. The anti-Brex type I active ORF was predicted as "trimethylamine methyltransferase corrinoid protein." ORF55, the anti-AVAST3 type active ORF, was predicted as "ATP-dependent DNA ligase." ORF58, which has anti-SIR2+HerA and DUF4297+HerA activity and Reron Ec86 sensor activity, was predicted as "Mu-like prophage host-nuclease inhibitor protein Gam." ORF65, an anti-hhe active ORF, was predicted as "Transcriptional regulator protein (SplA)." ORF71, an anti-Druantia type I active ORF, was predicted as "Family of unknown function (DUF6614)." This protein family has been found in bacteria. ORF72, an anti-Brex type I active ORF, was predicted to be "KfrA_N; Plasmid replication region DNA-binding N-term." ORF83, an anti-AVAST type 3 active ORF, was predicted to be "Smf; Predicted Rossmann fold nucleotide-binding protein DprA / Smf involved in DNA uptake."

[0106] FIG. 3 shows the KSA8 phage anti-Druantia (ORF71 ＤｒｕＳＭ１) was prepared, and the bactericidal activity of the phage against bacteria carrying Durantia was analyzed by spot assay. As shown in Figure 3, when the phage was equipped with a gene encoding an inhibitor against Durantia type I, the bactericidal activity against bacteria carrying Durantia increased.

[0107] The phage-derived defense-related factors identified in this analysis are shown in Figures 4 and 5. Figure 4 shows a list of ORFs related to the defense system carried by ΦDruSM1 and the domains encoded by these ORFs. Figure 5 shows a schematic diagram illustrating the function of the defense system carried by ΦDruSM1.

[0108] Test Example 8 Analysis of Factors Inhibiting the Tmn Defense System Phages carrying genes encoding factors that inhibit the Tmn defense system were prepared, and the bactericidal activity of the phages against Tmn-carrying bacteria was analyzed by spot assay. The results are shown in Figure 6(a) and (c).

[0109] In Figure 6(a), the E. coli experimental strain DH10B, expressing (Tmn, ORF35 from ΦSM_S22) = (-,-), (+,-), (+,+), was infected in a spot test with six phages (ΦSM_A8, SM_W22, SM_A2, KS_S9, SM_S24, and Dru_SM5). When Tmn alone was expressed, infection of these six phages was strongly inhibited by Tmn, but co-expression of ORF35 restored infectivity. This indicates that ORF35 is a Tmn inhibitor.

[0110] Figure 6b shows a schematic diagram comparing the genomes of each phage. In Figure 6b, the ORF35 homologue (anti-Tmn) of ΦKS_S9 ＫＳ－Ｓ９ ) was knocked out, and instead, the Tmn inhibitor identified in a. (anti-Tmn ＳＭ－Ｓ２２ A phage (ΦKSS9_antitmn_1-3) carrying the nucleotide sequence 1-3 was synthesized.

[0111] In Figure 6(c), the wild-type phage of KS_S9 (KSS9_WT) and the ΦKSS9_antitmn_1-3 synthesized in (b) were spot-tested to infect Tmn-expressing and non-Tmn-expressing E. coli strains DH10B. KSS9_WT was strongly inhibited by Tmn, while ΦKSS9_antitmn_1-3 escaped Tmn infection.

[0112] As shown in Figure 6a and 6c, when a gene encoding a Tmn inhibitor was loaded onto a phage, the bactericidal activity against Tmn-carrying bacteria increased.

[0113] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2024-053820) filed on March 28, 2024, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0114] The recombinant phage of this embodiment and the recombinant phage containing the gene screened by the screening method of this embodiment are highly likely to be usable as an active ingredient of an antibacterial agent, and may also be used for editing bacterial flora, removing bacteria from food, as a disinfectant, or as a pesticide.

[0115] SEQ ID NO: 1: Nucleotide sequence of DruSM1_ORF46 SEQ ID NO: 2: Nucleotide sequence of DruSM1_ORF55 SEQ ID NO: 3: Nucleotide sequence of DruSM1_ORF58 SEQ ID NO: 4: Nucleotide sequence of DruSM1_ORF65 SEQ ID NO: 5: Nucleotide sequence of DruSM1_ORF71 SEQ ID NO: 6: Nucleotide sequence of DruSM1_ORF72 SEQ ID NO: 7: Nucleotide sequence of DruSM1_ORF83 SEQ ID NO: 8: Nucleotide sequence of SM_S22_ORF35 SEQ ID NO: 9: Nucleotide sequence of T5j_ORF99 SEQ ID NO: 10: Nucleotide sequence of SM_S22_ORF50 SEQ ID NO: 11: Nucleotide sequence of SM_S22_ORF35 SEQ ID NO: 12: Nucleotide sequence of SM_S22_ORF38 SEQ ID NO: 13: Nucleotide sequence of SM_S22_ORF40 SEQ ID NO: 14: Nucleotide sequence of Shin31_ORF61 SEQ ID NO: 15: Nucleotide sequence of Shin31_ORF71 SEQ ID NO: 16: Nucleotide sequence of Rad_SP15 SEQ ID NO: 17: Nucleotide sequence of DruSM1_ORF32 SEQ ID NO: 18: Nucleotide sequence of DruSM1_ORF38 SEQ ID NO: 19: Nucleotide sequence of DruSM1_ORF41 SEQ ID NO: 20: Nucleotide sequence of DruSM1_ORF42 SEQ ID NO: 21: Nucleotide sequence of DruSM1_ORF45 SEQ ID NO: 22: Nucleotide sequence of DruSM1_ORF48 SEQ ID NO: 23: Nucleotide sequence of DruSM1_PH_ORF69 SEQ ID NO: 24: Nucleotide sequence of DruSM1_ORF68 SEQ ID NO: 25: Nucleotide sequence of DruSM1_ORF84 SEQ ID NO: 26: Nucleotide sequence of DruSM1_ORF85 SEQ ID NO: 27: Nucleotide sequence of SM_A8_ORF58 SEQ ID NO: 28: Amino acid sequence of DruSM1_ORF46 SEQ ID NO: 29: Amino acid sequence of DruSM1_ORF55 SEQ ID NO: 30: Amino acid sequence of DruSM1_ORF58 SEQ ID NO: 31: Amino acid sequence of DruSM1_ORF65 SEQ ID NO: 32: Amino acid sequence of DruSM1_ORF71 SEQ ID NO: 33: Amino acid sequence of DruSM1_ORF72 SEQ ID NO: 34: Amino acid sequence of DruSM1_ORF83 SEQ ID NO: 35: Amino acid sequence of SM_S22_ORF35 SEQ ID NO: 36: Amino acid sequence of T5j_ORF99 SEQ ID NO: 37: Amino acid sequence of SM_S22_ORF50 SEQ ID NO: 38: Amino acid sequence of SM_S22_ORF35 SEQ ID NO: 39: Amino acid sequence of SM_S22_ORF38SEQ ID NO: 40: Amino acid sequence of SM_S22_ORF40 SEQ ID NO: 41: Amino acid sequence of Shin31_ORF61 SEQ ID NO: 42: Amino acid sequence of Shin31_ORF71 SEQ ID NO: 43: Amino acid sequence of Rad_SP15 SEQ ID NO: 44: Amino acid sequence of DruSM1_ORF32 SEQ ID NO: 45: Amino acid sequence of DruSM1_ORF38 SEQ ID NO: 46: Amino acid sequence of DruSM1_ORF41 SEQ ID NO: 47: Amino acid sequence of DruSM1_ORF42 SEQ ID NO: 48: Amino acid sequence of DruSM1_ORF45 SEQ ID NO: 49: Amino acid sequence of DruSM1_ORF48 SEQ ID NO: 50: Amino acid sequence of DruSM1_PH_ORF69 SEQ ID NO: 51: Amino acid sequence of DruSM1_ORF68 SEQ ID NO: 52: Amino acid sequence of DruSM1_ORF84 SEQ ID NO: 53: Amino acid sequence of DruSM1_ORF85 SEQ ID NO: 54: Amino acid sequence of SM_A8_ORF58 SEQ ID NO: 55: Nucleotide sequence of ORF58 SEQ ID NO: 56: Nucleotide sequence of SM_S22_ORF6 SEQ ID NO: 57: Nucleotide sequence of SM_S22_ORF33 SEQ ID NO: 58: Nucleotide sequence of SM_S22_ORF34 SEQ ID NO: 59: Amino acid sequence of ORF58 SEQ ID NO: 60: Amino acid sequence of SM_S22_ORF6 SEQ ID NO: 61: Amino acid sequence of SM_S22_ORF33 SEQ ID NO: 62: Amino acid sequence of SM_S22_ORF34

Claims

1. Recombinant phage containing genes encoding neutralizing factors of the anti-phage defense system.

2. The recombinant phage according to claim 1, wherein the anti-phage defense system is at least one selected from Brex type I, AVAST type III, SIR2+HerA, DUF4297+HerA, hhe, Druantia type I, tmn, Retron Ec67, DRT Type II, DRT Type III, DRT type IV, MzaABCDE, upx, and Retron Ec67, Ec78, Ec48, and Ec83.

3. The recombinant phage according to claim 1, wherein the gene encoding the invalidating factor is either (A) or (B) below: (A) a gene comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 27; or (B) a gene encoding a protein having 80% or more identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 27 and having invalidating factor activity in the anti-phage defense system.

4. A recombinant phage comprising a gene encoding a disabling factor of an anti-phage defense system, wherein the gene encoding the disabling factor is a gene isolated by a screening method comprising the following steps (a1) to (a4): (a1) preparing a phage gene knockout library in which each gene has been deleted, (a2) preparing bacteria that express the anti-phage defense system, (a3) ​​infecting the bacteria prepared in (a2) with the phage gene knockout library prepared in (a1) and measuring the infectivity by spot assay, and (a4) isolating the gene as encoding the disabling factor of the anti-phage defense system when the infectivity measured in (a3) ​​is 1 / 100 or less of that measured when a wild-type bacterium is infected.

5. The recombinant phage according to any one of claims 1 to 4, which is a lytic phage.

6. An antibacterial agent or bacteriologic agent containing the recombinant phage according to claim 5.

7. A composition comprising the antibacterial agent or bacteriological test agent according to claim 6.

8. A method of killing target bacteria, comprising contacting the target bacteria with the composition of claim 7.

9. The sterilization method according to claim 8, wherein the target bacteria are pathogenic bacteria.

10. The sterilization method according to claim 9, wherein the pathogenic bacterium is at least one selected from Campylobacter jejuni, Vibrio cholerae, Escherichia coli O157, Legionella pneumophila, Yersinia pestis, Salmonella, and Shigella.

11. A method for screening for a gene encoding a factor that disables an anti-phage defense system, comprising the steps of: (b1) to (b4) below: (b1) preparing a phage library in which each gene has been deleted; (b2) preparing bacteria that express the anti-phage defense system; (b3) infecting the bacteria prepared in (b2) with the phage gene knockout library prepared in (b1) and measuring the infectivity by spot assay; and (b4) isolating the gene as encoding a factor that disables the anti-phage defense system when the infectivity measured in (b3) is 1 / 100 or less of that measured when a wild-type bacterium is infected.

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