KR102606251B1 - Nucleoside hydrolase inhibitors, compositions for inhibiting the toxicity of pathogenic microorganisms comprising the same, methods for inhibiting the toxicity of pathogenic microorganisms comprising the same and screening methods of nucleoside hydrolase inhibitors - Google Patents

Abstract

The present invention relates to a composition for inhibiting the toxicity of pathogenic microorganisms containing a nucleoside hydrolase inhibitor or a pharmaceutically acceptable salt thereof, a composition for preventing or treating pathogenic microbial infection, a composition for controlling plant diseases, and a treatment of the composition. It relates to a method for inhibiting the toxicity of pathogenic microorganisms, a method for controlling plant diseases, and a method for screening nucleoside hydrolase inhibitors, including the steps of:
The nucleoside hydrolase inhibitor according to the present invention can inhibit the toxicity of pathogens, that is, pathogenic microorganisms, or convert pathogenic bacteria into non-pathogenic symbiotic bacteria, and a composition for inhibiting the toxicity of pathogenic microorganisms containing the same. , a composition for preventing or treating pathogenic microbial infection, a composition for controlling plant diseases, a method for inhibiting the toxicity of pathogenic microorganisms including the step of treating the composition, and a method for controlling plant diseases, and the mechanism according to the present invention is a nucleoside It can be applied as a screening method for side hydrolase inhibitors.

Description

Nucleoside hydrolase inhibitors, compositions for inhibiting the toxicity of pathogenic microorganisms containing the same, methods for inhibiting the toxicity of pathogenic microorganisms containing the same, and screening methods for nucleoside hydrolase inhibitors {Nucleoside hydrolase inhibitors, compositions for inhibiting the toxicity of pathogenic microorganisms comprising the same, methods for inhibiting the toxicity of pathogenic microorganisms comprising the same and screening methods of nucleoside hydrolase inhibitors}

The present invention relates to a nucleoside hydrolase inhibitor, a composition for inhibiting the toxicity of pathogenic microorganisms containing the same, a composition for preventing or treating pathogenic microbial infection, a composition for controlling plant diseases, and a composition for controlling the toxicity of pathogenic microorganisms comprising the step of treating the composition. It relates to inhibition methods, plant disease control methods, and screening methods for nucleoside hydrolase inhibitors.

Gut microbial interactions are complex and evolutionarily conserved phenomena found in all metazoans. Although it is clear that the gut microbial community is deeply involved in a diverse range of host physiology, the detailed molecular mechanisms of host-microbe interactions that determine symbiosis or dysbiosis are largely unknown. This is partly because there are no genetic model systems that can look at both the host and the microbial sides.

In this regard, Drosophila provides a powerful genetic model of gut-microbe interactions for understanding symbiosis and dysbiosis/pathogenic interactions between the gut and microbes. Drosophila has a relatively simple commensal community dominated by the Lactobacillaceae and Acetobacteraceae families. These commensal bacteria influence many aspects of host physiology, such as immunity, development, metabolism, and behavior. In addition to these symbiotic gut-microbe interactions, the intestinal epithelium hosts naturally occurring opportunistic organisms such as Ecc15 ( Erwinia carotovora subspecies carotovora 15) and Pseudomonas entomophila , belonging to the genus Pectobacterium . May have pathogenic interactions with opportunistic pathogens.

During gut-microbe interactions, intestinal innate immunity is readily activated to control microorganisms. In Drosophila intestinal immunity, a member of the nicotinamide adenine dinucleotide phosphate oxidase family (dual oxidase, DUOX) plays a central role in combating invading pathogens by generating reactive oxygen species (ROS) against microorganisms. In addition to this ROS-based immunity, the immune deficiency (IMD) pathway is also involved in the intestinal antimicrobial response by producing antimicrobial peptides (AMP). Previous studies have shown that ROS-based immunity acts synergistically with AMP-based immunity during gut-microbe interactions. Both immune systems are specifically activated in response to microbial challenge. Microbe-associated molecular patterns (MAMPs) such as peptidoglycan (PGN) are known to activate the IMD pathway. For example, diaminopimelic acid (DAP)-type PGN, a common structure found in the membranes of Gram-negative bacteria, is recognized by PGN recognition proteins that initiate IMD pathway activation for AMP production.

However, DAP-type PGN is not a pathogen-specific molecular pattern because it is commonly found in both commensals such as Acetobacter and pathogens such as Ecc15. Because both bacteria can activate the IMD pathway, this pathway does not sufficiently explain the mechanism by which the intestinal epithelium distinguishes commensals from pathogens.

According to previous studies, DUOX is activated in response to Ecc15, but remains in a suspended state in response to Acetobacter. This indicates that DUOX-dependent ROS production is pathogen-specific. According to this study, bacterial-derived uracil serves as a ligand for DUOX, and pathogenic, but not commensal, bacteria release uracil. These data indicate that bacterial-derived uracil is a pathogen-specific signal used by the host to distinguish pathogens from commensals. Uracil can act as a ligand to initiate complex signaling pathways for DUOX activation in intestinal cells (Ha et al., Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in drosophila gut. Nature immunology 10 , 949-957., 2009; Lee et al., Inflammation-Modulated Metabolic Reprogramming Is Required for DUOX-Dependent Gut Immunity in Drosophila. Cell Host Microbe 23, 338-352., 2018; Lee et al., Bacterial uracil modulates Drosophila DUOX-dependent gut immunity via Hedgehog-induced signaling endosomes. Cell Host Microbe 17, 191-204., 2015). These DUOX-activated signaling pathways are targets of the PLCβ-Ca ²⁺ pathway, hedgehog-cadherin 99C pathway, and rapamycin-autophagy 1-mediated lipolytic pathway. Includes. In addition to its antibacterial role, DUOX-dependent ROS promotes pathogen removal by acting as a ligand for the reactive chemical receptor TrpA1 (Du et al., TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway. PLoS genetics 12., 2016).

Despite the importance of bacterial-derived uracil as a key ligand for DUOX-dependent immunity, it is currently unknown how and why pathogenic, but not commensal, bacteria release uracil.

Accordingly, the present inventors found that uracil and ribose are produced by the catabolism of bacterial nucleoside hydrolase (NH) using extracellular uridine in the intestinal lumen, and that uracil and ribose are produced by the catabolism of bacterial nucleoside hydrolase (NH) using extracellular uridine in the intestinal lumen, and are produced by pathogens. In contrast, commensal bacteria avoid DUOX activation by not producing uracil due to lack of functional NH activity, signaling essential for bacterial-cell communication and pathogen virulence in the transition from commensal to pathogen in vivo . By confirming the new role of ribose produced by NH, and based on this, it was confirmed that inhibiting nucleoside hydrolase can only inhibit pathogenicity (toxicity) without affecting the growth of pathogenic microorganisms, as well as novel The present invention was completed by discovering a nucleoside hydrolase inhibitor.

One object of the present invention is to provide a nucleoside hydrolase (NH) inhibitor.

Another object of the present invention is to provide a composition for inhibiting the toxicity of pathogenic microorganisms containing a nucleoside hydrolase inhibitor as an active ingredient.

Another object of the present invention is to provide a composition for preventing or treating pathogenic microbial infection containing the nucleoside hydrolase inhibitor as an active ingredient.

Another object of the present invention is to provide a composition for controlling plant diseases containing the nucleoside hydrolase inhibitor as an active ingredient.

Another object of the present invention is to provide a method for inhibiting the toxicity of pathogenic microorganisms, which includes treating an object other than humans with a composition containing the nucleoside hydrolase inhibitor as an active ingredient.

Another object of the present invention is to provide a method for controlling plant diseases, which includes treating an organism with a composition containing the nucleoside hydrolase inhibitor as an active ingredient.

Another object of the present invention is to provide a nucleoside hydrolase; Contacting the nucleoside hydrolase with a test substance; If the expression or activity of the nucleoside hydrolase is reduced in the nucleoside hydrolase in contact with the test substance and in the control group not in contact with the test substance, the test substance is treated with a nucleoside hydrolase inhibitor. To provide a screening method for nucleoside hydrolase inhibitors, including the step of selecting candidate substances.

Another object of the present invention is to provide a commensal bacteria of a pathogenic microorganism in which one or more genes selected from the group consisting of NH1, NH2, udp and deoD genes have been deleted.

Another object of the present invention is to provide an inhibitor of quorum sensing of pathogens, including a nucleoside hydrolase inhibitor.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.

One aspect of the present invention for achieving the above object provides a nucleoside hydrolase (NH) inhibitor.

The nucleoside hydrolase inhibitor of the present invention may be a compound represented by Formula 1 below, and may include a compound represented by Formula 1 below, or a pharmaceutically acceptable salt thereof, without limitation.

[Formula 1]

,

The R may be an unsubstituted or di(C ₁ -C ₂ alkyl)amino-substituted phenyl group.

The C ₁ -C ₂ alkyl may be, for example, -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₃ -CH ₃ , etc.

In the present invention, R is

, , ,

, , and It may be any one selected from the group consisting of, but is not limited thereto.

According to the above, the nucleoside hydrolase inhibitor of the present invention may include a compound represented by the following formulas 2 to 8, a compound represented by the following formulas 2 to 8, or a pharmaceutically acceptable compound thereof Salts may be included without limitation.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

In the present invention, Formula 2 is DMPAPIR ((2S,3S,4R,5R)-2-(4-(dimethylamino)phenyl)-5-(hydroxymethyl)pyrrolidine-3,4-diol, MW: 252.31), The formula 3 is DMMAPIR((2S,3S,4R,5R)-2-(3-(dimethylamino)phenyl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), and the formula 4 is DEPAPIR((2S, 3S,4R,5R)-2-(4-(diethylamino)phenyl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), Formula 5 is DEMAPIR((2S,3S,4R,5R)-2- (3-(diethylamino)phenyl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), Formula 6 is MEPAPIR((2S,3S,4R,5R)-2-(4-(methylethylamino)phenyl)- 5-(hydroxymethyl)pyrrolidine-3,4-diol), Formula 7 is MEMAPIR((2S,3S,4R,5R)-2-(3-(methylethylamino)phenyl)-5-(hydroxymethyl)pyrrolidine-3, 4-diol), Formula 8 may be named BnIR ((2S,3S,4R,5R)-2-benzyl-5-(hydroxymethyl)pyrrolidine-3,4-diol, MW: 223.27).

Among the intestinal microflora, most bacteria are harmless or beneficial (hereinafter referred to as “symbionts”), while others are conditionally pathogenic (especially when they become dominant) (hereinafter referred to as “pathobionts”). Accordingly, the intestinal epithelium faces an immunological dilemma and must control pathogens while maintaining a peaceful cohabitation with commensal bacteria.

The innate immune system, which consists of recognition of pathogens by microbe-associated molecular patterns (MAMPs) and subsequent activation of antimicrobial responses to antagonize pathogens, is well described in the mucosal immunity of other metazoans, including Drosophila. Given that all microorganisms without exception possess MAMPs, current knowledge of the innate immune system based on MAMP recognition explains how the gut distinguishes pathogens from commensals and how the gut activates pathogen-specific antibacterial responses. can not do. Previous studies have shown that pathogens and opportunistic commensals release uracil metabolites that serve as ligands for DUOX activation, whereas commensals avoid DUOX activation by not releasing uracil (Lee et al., Bacterial- derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila. Cell 153, 797-811., 2013).

These findings raise the intriguing possibility that microbe-contacting mucosal intestinal epithelium may recognize pathogens by sensing pathogen-specific metabolic activity that can produce pathogen-specific metabolites such as uracil, thereby driving microbial ROS production. . However, the existence of pathogen-specific metabolic activity has never been described, and it is difficult to explain why pathogens specifically release uracil.

Under this background, the present invention describes the nucleoside catabolism of “uridine-in and uracil-out” by bacterial nucleoside hydrolases using gut luminal uridine. confirming for the first time that "out" metabolic flux (Figure 7) is required to enhance inter-bacterial communications and bacterial pathogenesis in Drosophila , demonstrating that uridine-derived uracil is a dual oxidase ( A member of the nicotinamide adenine dinucleotide phosphate oxidase family, dual oxidase, DUOX) is required on the host side for dependent ROS (reactive oxygen species) production, while uridine-derived ribose is required for quorum sensing on the bacterial side. QS) and virulence gene expression. Deletion of the gene for bacterial nucleoside hydrolase is sufficient to block the transition from commensal to pathogen in vivo . Additionally, major commensal bacteria have It was discovered that there is no nucleoside catabolism required to achieve intestinal microbial symbiosis.As such, the present invention discovers a new role for the above-described bacterial nucleoside catabolism in symbiosis in other contexts of host-microbe interactions. It is significant in that it is the first to reveal the molecular mechanism of transition to pathogens.

In the present invention, the term "nucleoside hydrolase (NH)" is also called nucleoside N-ribohydrolases (NRHs; EC 3.2.2.-), and nucleoside It refers to a glycosidase that catalyzes the cleavage of the N-glycosidic bond at the side, enabling recycling of nucleobase and ribose. The process of recycling nucleosides and nucleobases is a method of conserving energy and is also called salvaging.

The nucleoside hydrolase inhibitor of the present invention may be characterized by inactivating the quorum sensing signal of pathogens.

In one embodiment of the invention, transcriptome analysis results show that the absence of uridine metabolism positively or negatively regulates a wide range of bacterial metabolic processes (Figure 4). Specifically, upregulation of primary metabolic activity and downregulation of quorum sensing processes are observed in ^Ecc15Δ4 , indicating the existence of an inverse correlation between primary metabolic activity and quorum sensing. In this regard, primary metabolic activity was higher in the bacterial quorum sensing-mutant strain than in the control strain, suggesting that quorum sensing functions as a metabolic brake. In line with this concept, we found similar metabolic patterns (i.e. upregulation of primary metabolic genes) between ^Ecc15Δ4 and quorum sensing-mutant bacteria, suggesting a link between uridine metabolism and quorum sensing.

Unlike uracil, ribose is not secreted from bacterial cells following uridine catabolism (Figure 1). RbsR, which has been identified as involved in nucleoside catabolism-dependent regulation of quorum sensing, is likely activated by uridine-induced ribose, which is required for full expression of important genes involved in quorum sensing activation, such as ExpR. (Figure 5). Downregulation of the ExpR/ExpI system in Ecc15 ^Δ4 may explain the low production of acyl homoserine lactone (AHL) (Figure 5). Communication between bacterial cells through quorum sensing plays a pivotal role in bacterial pathogenesis. Although the in vitro regulation of quorum sensing is relatively well established, its in vivo regulation within host tissues is less understood. Accordingly, the present invention revealed that nucleoside catabolism is a new in vivo quorum sensing regulatory system required for quorum sensing activation and subsequent toxic gene expression in the intestinal lumen (FIG. 6). The importance of nucleoside catabolism was further demonstrated by the confirmation that removal of uridine catabolism activity, as observed in Ecc15 ^Δ4, is sufficient to induce a phenotypic switch from a life-threatening pathogen to a growth-promoting commensal. It has been proven.

On the bacterial side, bacteria must recognize what external environment they are in (i.e., the intestinal epithelium in the case of enteric pathogens) to ensure successful infection. As we confirmed that uridine, which is abundant in the intestinal lumen, is easily utilized by bacterial nucleoside catabolism to induce ribose production and subsequent quorum sensing activation, we suggest that intestinal uridine may be an indicator for bacteria to recognize their biological environment. (Figure 7).

In one embodiment of the present invention, in order to confirm the inhibitory effect of DMPAPIR and BnIR, which are nucleoside hydrolase inhibitors of the present invention, on NH enzyme activity, the above compounds were added together with NH enzyme to measure the degree to which uridine, the substrate, is decomposed. As a result of the analysis, when only the enzyme and uridine were reacted (None), the uridine concentration decreased rapidly due to the activity of the enzyme, but when NH enzyme activity inhibitors were added at various concentrations, the enzyme activity increased in proportion to the concentration. It was confirmed that it was suppressed (Figures 13B-C). The minimum inhibitory concentration that can inhibit enzyme activity is measured to be ~1 μM for DMPAPIR (DM), ~100 μM for BnIR, and ~100 μM for PAPIR, making DMPAPIR approximately 100 times more effective. It was confirmed that the quality was excellent. In the case of BnIR, it was confirmed that the inhibitory function was similar to the existing PAPIR.

Accordingly, it can be seen that the nucleoside hydrolase inhibitor of the present invention has a significantly superior nucleoside hydrolase inhibitory effect compared to existing nucleoside hydrolase inhibitors.

Another aspect of the present invention provides a composition for inhibiting the toxicity of pathogenic microorganisms, comprising the nucleoside hydrolase inhibitor of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

The terms used here are the same as described above.

The nucleoside hydrolase inhibitor of the present invention may be a compound represented by Formula 1, and may include a compound represented by Formula 1, or a pharmaceutically acceptable salt thereof, without limitation. Specifically, it may include compounds represented by Formulas 2 to 8, and may include compounds represented by Formulas 2 to 8, or pharmaceutically acceptable salts thereof, without limitation.

The nucleoside hydrolase inhibitor of the present invention can suppress the toxicity of pathogenic microorganisms or convert pathogenic bacteria into non-pathogenic commensal bacteria by inhibiting nucleoside hydrolase enzymes.

The pathogenic microorganism may be a microorganism expressing a nucleoside hydrolase, and is not particularly limited as long as it is an intestinal microorganism expressing a nucleoside hydrolase, but for example, Pectobacterium or Pseudomonas It may be a microorganism of the genus ( Pseudomonas ). The microorganisms of the Pectobacterium genus are Ecc15 ( Erwinia carotovora subspecies carotovora 15), Pectobacterium atrosepticum, Pectobacterium carotovorum , and Pectobacterium wasabi. wasabiae ), Erwinia amylovora , Erwinia pyrifoliae , etc., for example, Ecc15, etc., and the Pseudomonas genus microorganism is Pseudomonas aeruginosa , P. aeruginosa , PAO1, It may be Pseudomonas syringae, Pseudomonas tolaasii , Pseudomonas agarici , etc., and for example, it may be PAO1, but is not limited thereto.

The composition includes any of i) a nucleoside hydrolase inhibitor or a pharmaceutically acceptable salt thereof, ii) an agent that inhibits the expression of a nucleoside hydrolase or a gene encoding it, or iii) a combination thereof. It may contain one or more active ingredients.

Agents that inhibit the nucleoside hydrolase include oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, ligands, and PNA (Peptides) that specifically bind to the nucleoside hydrolase. nucleic acid) or aptamer; Agents that inhibit the expression of the gene encoding the nucleoside hydrolase include small interference RNA (siRNA), short hairpin RNA (shRNA), or miRNA that specifically binds to the gene encoding the nucleoside hydrolase. It may be (microRNA), but is not limited thereto.

As used herein, the term “antibody” is a term known in the art and refers to a specific protein molecule directed to an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the nucleoside hydrolase of the present invention, and such an antibody is cloned into the marker gene by cloning each gene into an expression vector according to a conventional method. The protein encoded by can be obtained and manufactured from the obtained protein by conventional methods. This also includes partial peptides that can be made from the above proteins. The form of the antibody of the present invention is not particularly limited, and as long as it is a polyclonal antibody, monoclonal antibody, or has antigen binding properties, a portion thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included.

Antibodies of the invention include intact forms with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule. Functional fragments of antibody molecules refer to fragments that possess at least an antigen-binding function and include Fab, F(ab'), F(ab') 2, and Fv.

In the present invention, “siRNA” refers to a small RNA fragment of 21-25 nucleotides in size, produced by cutting double-stranded RNA with a dicer, and specifically binds to mRNA with a complementary sequence and inhibits its expression. . For the purpose of the present invention, it means specifically binding to mRNA and suppressing the expression of the gene. siRNA can be synthesized chemically or enzymatically. The method for producing siRNA is not particularly limited, and methods known in the art can be used.

The siRNA used in the present invention is either a complete form with polynucleotide pairing itself, that is, a form that is introduced into cells through two transformation processes by directly synthesizing siRNA in a test tube, or a form that is administered in vivo to have this form. Forms in which single-stranded oligonucleotide fragments and their reverse complements can be derived from single-stranded polynucleotides separated by a spacer, for example, siRNA expression vectors or PCR-derived siRNAs constructed so that siRNA is expressed in cells. The expression cassette may be introduced into the cell through transformation or infection. The decision on how to prepare and introduce siRNA into cells or animals may depend on the purpose and cell biological function of the target gene product.

In the present invention, "shRNA" is a single-stranded RNA with a length of 45 to 70 nucleotides, which is synthesized by oligo DNA linking 3-10 base linkers between the sense and complementary nonsense of the target gene siRNA base sequence. When expressed by cloning into a plasmid vector or by inserting shRNA into retroviruses such as lentivirus and adenovirus, shRNA (short hairpin RNA) with a looped hairpin structure is created and enters the cell. This means that it is converted into siRNA by Dicer within it and shows RNAi effect. Expression constructs/vectors containing shRNA (small hairpin RNA) can be prepared by methods known in the art.

In the present invention, "miRNA (microRNA)" refers to an untranslated RNA of approximately 22 nt that acts as a post-transcriptional repressor through base binding to the 3' untranslated region (UTR) region of mRNA. There is no particular limitation on the method for producing miRNA, and methods known in the art can be used.

In one embodiment of the present invention, it was confirmed that the nucleoside hydrolase inhibitor DMPAPIR of the present invention reduces the activity of elastase, a representative virulence factor of the pathogen PAO1, in proportion to the elastase treatment concentration (Figure 13H).

The composition may be in the form of any one or more selected from the group consisting of liquids, granules, powders, emulsions, oils, wetting agents, and coating agents, and any suitable excipients commonly used in compositions for inhibiting the toxicity of pathogenic microorganisms may be added. These excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

The content of the active ingredient included in the composition of the present invention is not limited as long as the composition has an effect of inhibiting pathogenic microbial toxicity, but the content is 0.0001 to 99.9% by weight, more specifically 0.01 to 80% by weight, based on the total weight of the final composition. may be included.

The composition for inhibiting pathogenic microbial toxicity may be provided as a quasi-drug composition.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. It can be included.

The “pharmaceutically acceptable carrier” may refer to a carrier, excipient, or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the injected compound. Specifically, it may refer to a non-natural carrier. occurring carrier). The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used individually or in combination of two or more types.

The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, It can be prepared by mixing gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.

The quasi-drug composition of the present invention may include, but is not limited to, disinfectant cleaner, shower foam, ointment, wet tissue, coating agent, etc., and the formulation method, dosage, usage method, and components of the quasi-drug are commonly known in the technical field. It can be appropriately selected from the techniques.

Another aspect of the present invention provides a composition for preventing or treating pathogenic microbial infection, comprising the nucleoside hydrolase inhibitor of the present invention, or a pharmaceutically acceptable salt thereof, as an active ingredient.

The terms used here are the same as described above.

The nucleoside hydrolase inhibitor of the present invention may be a compound represented by Formula 1, and may include a compound represented by Formula 1, or a pharmaceutically acceptable salt thereof, without limitation. Specifically, it may include compounds represented by Formulas 2 to 8, and may include compounds represented by Formulas 2 to 8, or pharmaceutically acceptable salts thereof, without limitation.

The nucleoside hydrolase inhibitor of the present invention can suppress the toxicity of pathogenic microorganisms or convert pathogenic bacteria into non-pathogenic commensal bacteria by inhibiting nucleoside hydrolase enzymes.

The pathogenic microorganism may be a microorganism expressing a nucleoside hydrolase, and is not particularly limited as long as it is an intestinal microorganism expressing a nucleoside hydrolase, but may be, for example, a microorganism of the genus Pectobacterium or Pseudomonas. You can. The microorganisms of the Pectobacterium genus may be Ecc15, Pectobacterium atrosepticum, Pectobacterium carotovorum, Pectobacterium wasabiae, Erwinia amylobora, Erwinia pyrifoliae, etc. For example, it may be Ecc15, and the Pseudomonas genus microorganism may be PAO1, Pseudomonas syringae, Pseudomonas tolaasi, Pseudomonas agarisi, etc., and an example may be PAO1, etc., but is not limited thereto.

In one embodiment of the present invention, in order to confirm the inhibitory effect of DMPAPIR and BnIR, which are nucleoside hydrolase inhibitors of the present invention, on NH enzyme activity, the above compounds were added together with NH enzyme to measure the degree to which uridine, the substrate, is decomposed. As a result of the analysis, when only the enzyme and uridine were reacted (None), the uridine concentration decreased rapidly due to the activity of the enzyme, but when NH enzyme activity inhibitors were added at various concentrations, the enzyme activity increased in proportion to the concentration. It was confirmed that it was suppressed (Figures 13B-C). The minimum inhibitory concentration that can inhibit enzyme activity is measured to be ~1 μM for DMPAPIR (DM), ~100 μM for BnIR, and ~100 μM for PAPIR, making DMPAPIR approximately 100 times more effective. It was confirmed that the quality was excellent. In the case of BnIR, it was confirmed that the inhibitory function was similar to the existing PAPIR.

In another embodiment of the present invention, it was confirmed that the nucleoside hydrolase inhibitor DMPAPIR of the present invention reduces the activity of elastase, a representative virulence factor of the pathogen PAO1, in proportion to the elastase treatment concentration ( Figure 13H).

As used herein, the term "prevention" refers to all actions that inhibit or delay the onset of pathogenic microbial infection by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to pathogenic microbial infection by administration of the pharmaceutical composition. It refers to any action that improves or changes the symptoms of a suspected or diseased entity.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions. Specifically, the pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. You can.

In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, Examples include calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract and its fractions with at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups, and may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to the commonly used simple diluents such as water and liquid paraffin. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The content of the active ingredient included in the pharmaceutical composition of the present invention is not limited as long as the pharmaceutical composition has a preventive or therapeutic effect against pathogenic microbial infection, but is 0.0001 to 99.9% by weight, more specifically 0.01%, based on the total weight of the final composition. It may be included in an amount of from 80% by weight.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat or prevent a disease with a reasonable benefit/risk ratio applicable to medical treatment or prevention. It refers to the amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the administration time, administration route, and excretion rate of the composition of the present invention used. Treatment period , may be determined according to factors including the composition of the present invention and the drugs used in combination or simultaneous use, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects.

The dosage of the pharmaceutical composition of the present invention may be, for example, 0.1 to 500 mg/kg of body weight per day to animals, including humans, but is not limited thereto. The frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or divided into multiple doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but can be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, and intrarectal administration, depending on the purpose. It can be administered through Additionally, the composition can be administered by any device that allows the active agent to move to the target site, tissue or cell.

Another aspect of the present invention provides a composition for controlling plant diseases comprising the nucleoside hydrolase inhibitor of the present invention, or a pharmaceutically acceptable salt thereof, as an active ingredient.

The terms used here are the same as described above.

The nucleoside hydrolase inhibitor of the present invention, or a pharmaceutically acceptable salt thereof, may be a compound represented by Formula 1, and may be a compound represented by Formula 1, or a pharmaceutically acceptable salt thereof, without limitation. It can be included. Specifically, it may include compounds represented by Formulas 2 to 8, and may include compounds represented by Formulas 2 to 8, or pharmaceutically acceptable salts thereof, without limitation.

The nucleoside hydrolase inhibitor of the present invention inhibits the toxicity of pathogenic microorganisms that cause plant diseases through nucleoside hydrolase inhibition, or converts pathogenic bacteria that cause plant diseases into non-pathogenic symbiotic bacteria. You can.

The plant disease may be caused by microbial infection.

The microorganism causing the plant disease may be a microorganism expressing a nucleoside hydrolase, but is not particularly limited thereto, and may be, for example, a microorganism of the genus Pectobacterium or Pseudomonas . The microorganisms of the Pectobacterium genus are, for example , Pectobacterium carotovorum subsp. brasilliense (Pcb), Pectobacterium carotovorum subsp. carotovorum 15 ( Pectobacterium carotovorum subsp. carotovorum 15) , Pcc 15), Pectobacterium chrysanthemi (Pch), Pectobacterium atrosepticum , Pectobacterium betavasulorum (Pbe), etc., Microorganisms of the Pseudomonas genus may be, for example, Pseudomonas syrigae (Pst), but are not limited thereto.

The composition may further include antibiotics.

Conventionally, in order to control plant diseases, administration or spraying of large amounts of antibiotics was unavoidable. However, since the composition for controlling plant diseases of the present invention contains a nucleoside hydrolase inhibitor as an active ingredient, the concentration or dose of antibiotics can be adjusted to the existing concentration or There are economic and environmental advantages in that it can be used at lower capacity.

The antibiotic may be used without limitation as long as it is effective in controlling plant diseases. For example, it may be neomycin, ribostamycin, streptomycin, etc., but is not limited thereto.

In one embodiment of the present invention, the nucleoside hydrolase inhibitors of the present invention, DMPAPIR and BnIR, are plant pathogens , Pectobacterium carotovorum subsp. brasilliense (Pcb), Pectobactere. Pectobacterium carotovorum subsp. carotovorum15 , Pcc 15), Pectobacterium chrysanthemi (Pch), and Pectobacterium atrosepticum , Pectobacterium The incidence of plant diseases caused by Pectobacterium betavasulorum (Pbe) was significantly reduced (Figure 13).

The composition may be in the form of any one or more selected from the group consisting of liquids, granules, powders, emulsions, oils, wetters, and coating agents, and may further contain any suitable excipients commonly used in compositions for the purpose of controlling plant diseases. These excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

The content of the active ingredient contained in the composition of the present invention is not limited as long as the composition has a plant disease control effect, but is 0.0001 to 99.9% by weight, more specifically 0.01 to 80% by weight, based on the total weight of the final composition. may be included.

Another aspect of the present invention provides a method for inhibiting the toxicity of pathogenic microorganisms, comprising treating an object other than a human with the composition for preventing or treating pathogenic microbial infection of the present invention.

The terms used here are the same as described above.

In the above method, the composition of the present invention may be in the form of any one or more selected from the group consisting of liquid, granule, powder, emulsion, oil, wettable powder, and coating agent, and the method is not particularly limited and the form of the composition is not particularly limited. It can be appropriately selected and performed according to.

In the above control method, the composition of the present invention can be administered alone or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and the treatment (spray) amount or treatment time interval may vary depending on the individual and can be easily determined by a person skilled in the art. there is.

Another aspect of the present invention provides a method of treating pathogenic microbial infection, comprising administering the composition for preventing or treating pathogenic microbial infection of the present invention to an entity other than a human.

The terms used here are the same as described above.

Another aspect of the present invention provides a method for controlling plant diseases, comprising treating an object with the composition for controlling plant diseases of the present invention.

The terms used here are the same as described above.

In the above control method, the composition of the present invention may be in the form of one or more selected from the group consisting of liquid, granule, powder, emulsion, oil, wettable powder, and coating agent, and the control method is not particularly limited and the composition It can be appropriately selected and performed depending on the type. Specifically, the control method may be any one or more selected from the group consisting of foliage treatment, soil treatment, soaking treatment, branch treatment, and farming equipment treatment, but is not limited thereto.

In the above control method, the composition of the present invention can be treated alone or administered in combination with other control agents, can be administered sequentially or simultaneously with conventional control agents, and can be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects. The treatment (spraying) amount or treatment time interval may vary depending on the plant and can be easily determined by a person skilled in the art. there is. The preferred treatment amount of the composition of the present invention can be appropriately adjusted by a person skilled in the art in consideration of the growth degree of the plant, the cultivation environment, the degree of occurrence of plant virus diseases, etc., but is not limited thereto.

The composition of the present invention may be generally applied to plants at a concentration of 0.5 to 1000 μM (1 mM) per plant, but is not limited thereto. When manufacturing, storing, and transporting the composition of the present invention, the concentration is generally 1 to 100,000 times the concentration when treated with plants, specifically 100 to 50,000 times the concentration, and more specifically 500 to 10,000 times the concentration. It can be manufactured, stored, and transported in any concentration, but is not limited to this.

Another aspect of the present invention includes providing a nucleoside hydrolase; Contacting the nucleoside hydrolase with a test substance; If the expression or activity of the nucleoside hydrolase is reduced in the nucleoside hydrolase in contact with the test substance and in the control group not in contact with the test substance, the test substance is treated with a nucleoside hydrolase inhibitor. Provided is a screening method for nucleoside hydrolase inhibitors, including the step of selecting candidate substances.

The terms used here are the same as described above.

The nucleoside hydrolase inhibitor candidate may prevent uridine from being decomposed by inhibiting the activity of nucleoside hydrolase that decomposes uridine.

Another aspect of the present invention provides a composition for converting pathogenic microorganisms into commensal bacteria, including an agent that inhibits the expression of a nucleoside hydrolase or a gene encoding the same.

The terms used here are the same as described above.

The agent that inhibits the nucleoside hydrolase is an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA, or aptamer that specifically binds to the nucleoside hydrolase; The agent that inhibits the expression of the gene encoding the nucleoside hydrolase may be siRNA, shRNA, or miRNA that specifically binds to the gene encoding the nucleoside hydrolase, but is not limited thereto.

Another aspect of the present invention provides a symbiotic bacterium of a pathogenic microorganism in which one or more genes selected from the group consisting of NH1, NH2, udp, and deoD genes are deleted in the pathogenic microorganism.

The terms used here are the same as described above.

In a pathogenic microorganism, when the gene encoding the nucleoside hydrolase of the present invention, including any one or more of the NH1, NH2, udp and/or deoD genes, is deleted or its activity is suppressed, the pathogenic microorganism, that is, , pathogenic bacteria can be converted into non-pathogenic commensal bacteria, as described above.

The NH1, NH2, udp and deoD genes have, contain, consist of, or consist essentially of the base sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. of) can.

Specifically, the NH1, NH2, udp and deoD genes have at least 70%, at least 75%, at least 80%, and at least 85% homology or identity with the sequences of SEQ ID NO: 1 or SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. It has or includes a nucleotide sequence that is 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100%, or SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4. A base sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the sequence of It may consist of or necessarily consist of, but is not limited to this.

In addition, if it is a base sequence that has such homology or identity and shows efficacy corresponding to the NH1, NH2, udp and deoD genes, a variant having a base sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also present. It is obvious that it is included within the scope of the invention.

The NH1, NH2, udp, and deoD polypeptides encoded by the NH1, NH2, udp, and deoD genes include, consist of, or consist of the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. It may be mandatory, but is not limited to this.

The pathogenic microorganism may be a microorganism of the genus Pectobacterium or Pseudomonas, and may specifically be Ecc15, but is not limited thereto.

Another aspect of the present invention provides a quorum sensing inhibitor of pathogens, comprising the nucleoside hydrolase inhibitor of the present invention.

The terms used here are the same as described above.

The nucleoside hydrolase inhibitor of the present invention may be a compound represented by Formula 1, and may include a compound represented by Formula 1, or a pharmaceutically acceptable salt thereof, without limitation. Specifically, it may include compounds represented by Formulas 2 to 8, and may include compounds represented by Formulas 2 to 8, or pharmaceutically acceptable salts thereof, without limitation.

The nucleoside hydrolase inhibitor may inactivate the quorum sensing signal of the pathogen, as described above.

The nucleoside hydrolase inhibitor according to the present invention can inhibit the toxicity of pathogens, that is, pathogenic microorganisms, or convert pathogenic bacteria into non-pathogenic symbiotic bacteria, and a composition for inhibiting the toxicity of pathogenic microorganisms containing the same. , a composition for preventing or treating pathogenic microbial infection, a composition for controlling plant diseases, a method for inhibiting the toxicity of pathogenic microorganisms including the step of treating the composition, and a method for controlling plant diseases, and the mechanism according to the present invention is a nucleoside It can be applied as a screening method for side hydrolase inhibitors.

Figure 1 is a diagram showing that pathogens induce “uridine-in and uracil-out” metabolic fluxes. (A) Metabolomics profile after intestinal Ecc15 infection shown as a heat map. Flies were orally infected with sucrose solution in the absence or presence of Ecc15 for 2 h. As a result of metabolomics analysis of intestinal luminal fluid using CETOF-MS (Capillary Electrophoresis Time-of Flight Time Spectrometry), a total of 152 metabolites were detected. Each metabolite was classified into eight metabolic processes. The number of metabolites in each metabolic process is indicated, with colored bars representing the slope of the log _2- fold change in metabolite levels after intestinal infection compared with controls at each time point, and gray bars representing metabolites not detected in the assay. (B) Nucleotide metabolic processes are enriched by up- and down-regulated metabolites after intestinal infection. Bars represent -log ₁₀ ( p value), where the p value determined using DAVID software indicates the significance of processes enriched by up- and down-regulated metabolites. (C) Differential levels of up- and down-regulated metabolites involved in nucleotide metabolism shown as a heat map. Color bars represent the slope of the log _2- fold change in metabolite levels after intestinal infection compared to control levels at each time point. Uridine and uracil are indicated by arrows. (DE) Uracil is derived from extracellular uridine by Ecc15. Ecc15 was cultured in M9 minimal medium containing uridine (D) or isotopically labeled uridine (E). The positions of radioactive N and C are shown in red. Uridine, uracil, and ribose molecules in the culture supernatant were analyzed at different time points using LC-MS/MS analysis. Data are expressed as the mean ± SD of at least three experiments. ND, not detected.
Figure 2 is a diagram showing that pathogens induce “uridine-in and uracil-out” metabolic fluxes. (AB) Pathogenic members of the Acetobacteraceae secrete uracil for dual oxidase (DUOX; a member of the nicotinamide adenine dinucleotide phosphate oxidase family) activation. (A) Bacteria were cultured in mannitol medium containing ¹⁵ N labeled uridine for 8 hours, and ¹⁵ N labeled uracil in the culture supernatant was quantified using LC-MS/MS analysis. (B) Intestinal DUOX-dependent ROS production was measured using R19S dye after oral ingestion of the indicated bacteria (approximately 1 × ¹⁰ CFU) for 1.5 h. Data were expressed as mean ± SD. ND, not detected; NS, not significant; *** P <0.001 (analyzed using analysis of variance (ANOVA)). (C) This is the result of heat map analysis of genes related to the nucleotide metabolism pathway using whole genome information. Genes from different bacteria were compared with the corresponding genes from Gluconobacter morbifer ( G. morbifer ), and the percentage of amino acid sequence identity is indicated by a blue gradient bar. Red color indicates genes that are absent (i.e., less than 40% identity) in a given bacterial genome. Only four genes indicated by arrows (NH, NupC, AdeC and Cdd) were present in all lesion members but not in all commensal members. (D) Diagram of the nucleoside import and catabolism pathways for uracil production and release from lesion members.
Figure 3 is a diagram showing that bacterial NH activity causes intestinal dysbiosis. (AD) Pathology NH activity acts as a colitogenic factor responsible for intestinal pathology and early host death. The NH gene of the pathogen G. morbifer ^WT was deleted to produce G. morbifer ^△NH . Complementation of the G. morbifer ^△NH mutant was achieved by introducing the NH gene to create G. morbifer ^△NH-NH . In vitro uracil secretion capacity was determined using ¹⁵ N-labeled uridine (A), ROS production capacity (B), enterocyte apoptosis (C), and host survival (D). (EH) In Acetobacteraceae , the absence of nucleoside catabolism is required for gut-microbe symbiosis. To induce the ability to secrete uracil , A. asteurianus ^NupC_NH was created by introducing the NH and NupC genes of G. morbifer into A. pasteurianus ^WT . In vitro uracil secretion capacity was measured using ¹⁵ N-labeled uridine (E), ROS production capacity (F), enterocyte apoptosis (G), and host survival (H). Data (C, D, G, and H) were obtained using germ-free flies singly mated with the indicated bacterial strains and analyzed using ANOVA (A, B, F) or Welch's t-test (E). Values are expressed as the mean ± SD (*** P < 0.001) of at least three independent experiments. In (C and G), each dot represents an individual fly and the horizontal line represents the average value. *** P <0.001 (ANOVA). In (D and H), log-rank analysis of lifespan (Kaplan-Meier method) shows significant differences in survival rates between G. morbifer ^WT - and G. morbifer ^ΔNH -introduced GF flies. *** P <0.001. (D) and A. asteurianus ^WT- and A. pasteurianus ^NH_NupC- related GF flies (* P < 0.05) (H). ND, not detected; NS, not significant.
Figure 4 is a diagram showing that the nucleoside catabolism of Ecc15 is required for uracil secretion and regulation of bacterial metabolism. (A) In vitro uracil secretion from Ecc15 requires nucleoside catabolic activity. Ecc15 ^△4 was created by deleting the two NH genes and two NP genes of Ecc15 ^WT . Complementation of Ecc15 ^△4 was achieved by introducing the NH gene to create Ecc15 ^△4_NH . Bacteria were cultured in M9 minimal medium containing uridine for the indicated times, and uridine and uracil levels in the culture supernatants were quantified using LC-MS/MS analysis. (B) In vivo uracil secretion from Ecc15 requires nucleoside catabolic activity. Flies were orally infected with the indicated bacteria for 2 h. Intestinal luminal fluid was used to quantify uracil and uridine levels through LC-MS/MS analysis. (C) Nucleoside catabolic activity is required for ROS generation in vivo after intestinal infection. Intestinal DUOX-dependent ROS production was measured using R19S dye after oral ingestion of the indicated bacteria (approximately 1 × 10 ¹⁰ CFU) for 1.5 h. (D) In vivo uracil secretion is observed after intestinal infection by different pathogens. Flies were orally infected with different pathogens (approximately 2 Х 10 ⁹ CFU for Pseudomonas aeruginosa , P. aeruginosa and approximately 1 Х 10 ¹⁰ CFU for other bacteria) for 2 hours. Intestinal luminal fluid was analyzed to quantify uracil and uridine levels using LC-MS/MS analysis. (EF) Represents the relative proportion of 320 differentially expressed genes between Ecc15 and Ecc15 ^Δ4 according to the associated gene ontology biological processes (GOBP). GOBP terms at level 1 (E) and levels 2–4 (F) were used for general cellular and metabolic processes, respectively. (G) Biological processes enhanced by genes upregulated or downregulated in Ecc15 ^Δ4 . Bars represent -log ₁₀ ( p value) and p value in DAVID software indicates the significance of processes enriched by up- and down-regulated genes. (H) Heat map showing differential expression of up- and down-regulated genes involved in different metabolic processes. Color bars represent the slope of the log _2- fold change in mRNA expression levels of the Ecc15 ^Δ4 strain compared to the Ecc15 strain. In (BD), data were analyzed using ANOVA. Values are expressed as the mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001) of at least three independent experiments. ND, not detected; NS, not significant.
Figure 5 is a diagram showing that nucleoside catabolic activity is required for acyl homoserine lactone (AHL)-dependent quorum sensing (QS) activation and toxic gene expression in an RbsR-dependent manner. (A) Nucleoside catabolism is required for the production of AHL-type quorum sensing molecules. Culture supernatants of Ecc15 or Ecc15 ^Δ4 at different growth time points were incubated with E. coli pSB401 strain (bioluminescence-based AHL type QS biosensor), and bioluminescence was measured using a luminometer. For bioluminescence imaging, strain pSB401 was incubated with bacterial culture supernatant (obtained after 10.5 h of incubation) for 2 h. An Ecc15 mutant (Ecc15 ^ΔExpIR ) with a deletion of the AHL-synthesizing ExpI enzyme was used as a negative control. Data are expressed as the mean ± SD of at least three experiments. (B) This is the measurement result of 3-oxo-C6-HSL (N-(3-oxo-hexanoyl)-L-homoserine lactone) produced by bacteria. Culture supernatants of Ecc15 or Ecc15 ^Δ4 at different growth time points were used to quantify 3-oxo-C6-HSL levels by LC-MS/MS. Data are expressed as the mean ± SD of at least three experiments. (C) Nucleoside catabolism and ribose-regulated transcriptional regulators are required for expression of the global QS regulator. The expression levels of ExpR and VqsM genes were analyzed by qPCR analysis using Ecc15 ^WT , Ecc15 ^Δ4 and Ecc15 ^ΔExpIR . Target gene expression in Ecc15 ^WT was arbitrarily set to 1, and the results are expressed as relative expression levels. Bars represent the mean ± SD (*** P < 0.001) of at least three independent experiments. (D) RbsR is required for the production of AHL-type QS molecules. Culture supernatants of Ecc15 or Ecc15 ^Δ4 at various growth time points were incubated with E. coli pSB401 strain, and bioluminescence was measured using a luminometer. For bioluminescence imaging, strain pSB401 was incubated with bacterial culture supernatant (obtained after 10.5 h of incubation) for 2 h. An Ecc15 mutant (Ecc15 ^Δ4 ) lacking nucleoside catabolic activity was used as a control. Data are expressed as the mean ± SD of at least three experiments. (EF) Evf expression is under the control of nucleoside catabolism, RbsR, and ExpIR-dependent QS system. Expression levels of evf were analyzed using qPCR (E) and Pevf::GFP reporter activity (F). The target gene expression of Ecc15 ^WT was arbitrarily set to 1, and the results were expressed as relative expression levels. Bars represent the mean ± SD (*** P < 0.001) of at least three independent experiments.
Figure 6 is a diagram showing that nucleoside catabolic activity is required for conversion to bacterial QS and commensal pathogens in the intestine. (A) Nucleoside catabolic activity is required for QS activation in vivo in the intestinal lumen. Flies were orally infected with Ecc15 ^WT or Ecc15 ^Δ4 along with the bioluminescent reporter E. coli pSB401 strain. Bioluminescence imaging of live flies was obtained 1.5 h after infection. Each dot represents an individual fly and the horizontal line represents the average value. *** P <0.001 (ANOVA). Representative images are shown. (B) Nucleoside catabolic activity is required for virulence gene activation in vivo in the intestinal lumen. Ecc15 ^WT or Ecc15 ^Δ4 carrying the Pevf::GFP reporter were used for oral infection using larvae or Drosophila for 2 h. The fluorescence intensity of individual fields was measured through confocal microscopy. Each dot represents an individual animal and the horizontal line represents the average value. *** P <0.001 (ANOVA). Representative confocal images are shown. (C) Shows the effect of bacterial nucleoside catabolism on developmental rate. Bacteria of the indicated genotypes were added to germ-free embryos and the rate of pupa formation was monitored every 12 h. ^Ecc15Δ4 enhanced the rate of host development, similar to the growth-promoting commensal A. pomorum . (DE) Shows the effect of bacterial nucleoside catabolism on animal size. Bacteria of the indicated genotypes were added to germ-free embryos and larval size was measured 108 hours after oviposition. Each dot represents an individual animal and the horizontal line represents the average value. *** P <0.001 (ANOVA). NS, not significant. Representative images are shown (E).
Figure 7 is a diagram showing a model for the role of bacterial nucleoside catabolism in other gut-microbe interactions. Pathogens can use NupC and NH to drive the “uridine-in and uracil-out” metabolic flux. On the pathogen side, uridine-derived ribose activates RbsR for bacterial cell-cell communication (via QS activation) and QS-dependent pathogenesis. On the host side, host intestinal cells recognize the presence of pathogens by sensing uridine-derived uracil. Uracil serves as a ligand for DUOX activation and subsequent bactericidal ROS generation. A kind of arms race occurs between host defense (uracil-dependent microbial ROS) and pathogen virulence (ribose-dependent evf toxins). Pathogens can use NupC and NH to induce a “uridine-in and uracil-out” metabolic flux, which results in chronic DUOX activation, enterocyte damage, and premature host death. However, commensal members lacking nucleoside catabolism can interact with enterocytes without DUOX activation. All members of the Acetobacter family (pathogens or commensals) lack a QS system.
Figure 8 is a diagram showing that uridine in the intestinal lumen of Drosophila is highly likely to be derived from host intestinal cells. (A) Nucleoside levels in luminal fluid measured without intestinal infection. Concurrent (CV) flies (5 days old) were orally fed with 5% sucrose for 2 h before preparing intestinal lumen fluid. (B) Intestinal luminal uridine is not derived from gut bacteria or nutrition. CV or GF flies (3 days old) were fed either a standard cornmeal yeast diet or a Holy Dick diet lacking uridine for 10 days (i.e., 13 day old CV or GF flies were used for uridine measurements). Higher levels of uridine were detected in the intestinal lumen of GF flies compared to CV flies, indicating that uridine does not originate from gut bacteria. Additionally, no significant differences in uridine levels were observed between the standard diet and the Holidic diet, indicating that intestinal luminal uridine is not derived from the diet. In all experiments, intestinal luminal fluid from 30 midguts was used to quantify nucleoside levels by LC-MS/MS analysis. Data are expressed as the mean ± SD of at least three independent experiments.
Figure 9 shows the results of bioluminescence measurement by AI-2 QS molecules produced in bacteria, and no difference in LuxS-dependent AI-2 levels was observed between Ecc15 and Ecc15 ^△4 culture supernatants. Culture supernatants of Ecc15 or Ecc15 ^Δ4 at various growth time points were incubated with Vibrio harveyi MM32 strain (bioluminescence-based AI-2-type QS biosensor), and bioluminescence was measured using a luminometer. An Ecc15 mutant (Ecc15 ^ΔLuxS ) with deletion of the AI-2-synthesizing enzyme LuxS was used as a negative control. Data are expressed as the mean ± SD of at least three independent experiments.
Figure 10 is a diagram showing that genes involved in primary metabolism are significantly upregulated in Ecc15 ^Δ4 . The expression levels of 11 genes involved in primary metabolism were determined by qPCR analysis using Ecc15 ^WT , Ecc15 ^Δ4 , Ecc15 ^ΔRbsR and Ecc15 ^ΔExpIR . The primary metabolic genes are as follows: purH (Phosphoribosylaminoimidazolecarboxamide formyltransferase, GJS26_00239), pgm (Phosphoglucomutase, GJS26_01330), pgk (Phosphoglycerate kinase, GJS26_03794), adk (Pyruvate kinase, GJS26_01172), pyk ( Pyruvate kinase, GJS26_01172) nah (Nucleoside-diphosphate kinase, GJS_03171), carA (carbamoyl phosphate synthase A, GJS26_03753), nrdB (Ribonucleoside-diphosphate reductase, GJS_01196), udk (Uridine kinase, GJS_01409), add (Adenosine deaminase, CJS_02167) atpE ( GJS26_04373). Target gene expression in Ecc15 ^WT was arbitrarily set to 1, and the results are expressed as relative expression levels. Bars represent the mean ± SD of at least three independent experiments.
Figure 11 is a diagram showing that RbsR and ExpIR (but not luxs) are required for conversion to commensal pathogens in the intestine. (A) Effect of RbsR regulator and quorum sensing on developmental rate. Bacteria of the indicated genotypes were added to germ-free (GF) embryos and the percentage of pupa formation was monitored every 12 h. (B) Effect of RbsR regulator and quorum sensing on animal size. Bacteria of the indicated genotypes were added to GF embryos and larval size was measured 108 h after oviposition. Each dot represents an individual fly and the horizontal line represents the average value. *** P <0.001 (ANOVA). NS, not significant. Representative images are displayed. Conventional (CV) flies were used as controls. Deletion of ExpIR, but not LuxS, is sufficient to induce pathogen-community switching, indicating that ExpI-dependent quorum sensing is required for bacterial pathogenicity.
Figure 12 is a diagram showing the role of nucleoside catabolic activity on QS-dependent toxicity of P. aeruginosa PAO1. A single NH gene was identified in the genome of P. aeruginosa PAO1. To avoid the autolysis and autoaggregation behavior of P. aeruginosa , the pqsL-PAO1 strain was used (Hazan, et al., Auto Poisoning of the Respiratory Chain by a Quorum-Sensing-Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance. Current biology : CB 26, 195-206., 2016). PAO1 ^ΔNH was produced by deleting the NH gene. PAO1 ^△NH_NH was constructed by introducing the NH gene into the PAO1 ^△NH mutant. (AB) Indicates the activity of virulence factors. Elastase activity (A) and pyocyanin production (B) were measured in the presence and absence of NH activity. Elastase activity and pyocyanin production were measured using culture supernatants. (C) QS activity analysis results. The level of AHL with acyl chain length C4 in culture supernatants was measured using the lux-based E. coli AHL biosensor pSB536. (D) Host survival results after intestinal infection. Adult female flies (5-6 days old) were orally infected with the indicated bacteria. In (AB), values are expressed as the mean ± SD (*** P < 0.001, ** P < 0.005) of at least three independent experiments. In (D), log-rank analysis (Kaplan-Meier method) for lifespan shows a significant difference in survival (*** P < 0.001).
Figure 13 is a diagram showing the function of an NH (Nucleoside hydrolase) enzyme activity inhibitor of pathogens. (A) Structural formula of NH enzyme activity inhibitor. (B) The extent to which the NH enzyme decomposes the corresponding substrate, uridine, was measured using a spectrophotometric assay. When only the enzyme and uridine were added to react (None), the uridine concentration rapidly decreased due to the activity of the enzyme. On the other hand, when the inhibitor DMPAPIR was added at various concentrations, it was confirmed that enzyme activity was inhibited in proportion to the concentration. (C) The degree of inhibition of BnIR, a newly developed NH enzyme activity inhibitor, and PAPIR, an NH inhibitor of the protozoan parasite Leishmania reported in a previous study, was compared using spectrophotometry. The minimum inhibitory concentration that can inhibit the enzyme activity of each inhibitor was measured to be ~1 M for DMPAPIR, ~100 M for BnIR, and ~100 M for PAPIR. In the case of DMPAPIR, it can be confirmed that the effectiveness is about 100 times superior, and in the case of BnIR, the inhibitory function is similar to that of the existing PAPIR, but the process of producing the inhibitor is much easier than that of PAPIR, making it valuable as an effective inhibitor. . (D) Ecc15 ( Erwinia carotovora subsp. carotovora 15, belonging to the genus Pectobacterium ) is not only a pathogen of fruit flies, but also a plant pathogen that can cause disease in a wide range of plants. Since the genus Pectobacterium is known to cause various plant diseases, including soft rotting in plants, it was determined whether these plant diseases could be controlled by inhibiting NH enzyme activity. Potatoes were treated with less than 10 ⁶ CFU of wild-type bacteria (ECCWT) and mutant bacteria with NH enzyme activity removed (Ecc15 ^△4 ), respectively, and also treated with 0.1 mM and 1 mM DMPAPIR, and the degree of softness of the potatoes was measured after 48 hours (rotting vol). As a result of measuring .), it was confirmed that when mutant bacteria (Ecc15 ^△4 ) or Ecc15 and inhibitors were treated together, soft rot caused by wild-type bacteria occurred at less than 10%, reducing the incidence. (E) It was confirmed whether DMPAPIR suppresses disease in several other species belonging to the genus Pectobacterium by inhibiting NH enzyme activity. Pectobacterium carotovorum subsp. brasilliense (Pcb), which can cause disease in a wide range of plants; Pectobacterium carotovorum subsp. carotovorum15 ; Pcc15) and Pectobacterium chrysanthemi (Pch), which are reported to cause diseases in ornamental plants and plant bulbs, and Pectobacterium atro, which causes blackleg of potatoes. The degree of soft rot in potatoes was reduced by DMPAPIR using the same method as (D) targeting Pectobacterium atrosepticum and Pbe, which are known to cause diseases in sugar beets. As a result of measuring whether the pathogenicity was determined, it was confirmed that the pathogenicity of various strains of different species belonging to the genus Pectobacterium could be greatly reduced through the corresponding inhibitor. (F) As a result of measuring the effect of BnIR, another NH enzyme activity inhibitor, on soft rot of potatoes by the same method as (D), it was confirmed that BnIR can also significantly inhibit soft rot of potatoes caused by Ecc15. (G) In Pseudomonas syrigae (Pst), another representative plant pathogen, it was confirmed whether NH enzyme activity has a significant effect on the virulence of the bacteria. Pseudomonas syringa has been reported to cause necrosis or gummosis in various plants. Leaves of Arabidopsis , a representative leaf plant model, are wounded and less than 2X10 ⁶ CFU of wild-type bacteria (Pst ^WT ) and mutant bacteria with NH enzyme activity removed (Pst ^△NH ) are introduced, respectively, or 0.1 mM or 1 mM. As a result of treating DMPAPIR with wild-type bacteria and observing the degree of necrosis of plant leaves after a week, it was found that when the mutant bacteria (Pst ^△NH ) and DMPAPIR were treated together, necrosis of plant leaves caused by wild-type bacteria was reduced. Confirmed. (H) It was confirmed whether the NH enzyme activity inhibitor also acts on the pathogen Pseudomonas aeruginosa (PAO1). Culture supernatant was obtained by culturing PAO1 bacteria with or without treatment with NH enzyme activity inhibitor, and the elastase activity of PAO1 was measured by reacting the supernatant with elastin-congo red. As a result, the representative toxicity of PAO1 was measured. It was confirmed that the activity of the factor, Elesatase, decreased in proportion to the treatment concentration of the inhibitor.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1. Confirmation of induction of “uridine-in and uracil-out” metabolic flow by pathogens

To determine whether uridine and uracil metabolic flux is induced by pathogens, the Drosophila intestine was infected with the Drosophila opportunistic pathogen Ecc15 ( Erwinia carotovora subspecies carotovora 15), and then the luminal fluid of the Drosophila midgut was subjected to TOF analysis. Capillary electrophoresis (CE) coupled with -MS (Time-of Flight Time Spectrometry) was performed.

Specifically, Drosophila was reared at 25°C in Bloomington Drosophila stock center's standard cornmeal medium. Drosophila melanogaster White Mutant w ¹¹¹⁸ , Drosophila melanogaster w ¹¹¹⁸ ) were used as control animals.

Ecc15 strain contains 100 μg/ml of ampicillin, 30 μg/ml of kanamycin, 25 μg/ml of apramycin, 20 μg/ml of tetracycline, and 50 μg/ml of rifampicin. and cultured at 30°C in LB or minimal M9 medium containing 35 μg/ml of chloramphenicol.

To infect the Drosophila intestine, Drosophila (5-6 days old) were orally infected with a sucrose solution containing up to ¹⁰ CFU of Ecc15 for 2 hours, and up to 150 dissected female midguts were inoculated before and after oral infection. Gut luminal fluids were obtained from the midguts. The dissected midgut was cut into 4-5 pieces and further incubated in ice-cold phosphate buffered saline (PBS) solution for 10 minutes to obtain diffused luminal fluid. The diffused luminal fluid was centrifuged at 4,000 rpm for 1 minute and filtered through a 0.2 μm cellulose acetate filter (ADVANTEC) for metabolomics analysis (Human Metabolome Technologies Inc.). Metabolites were collected from two biological replicates per sample: gut luminal fluids from non-infected gut (NI luminal fluid) and gut luminal fluids from infected gut (I luminal fluid). )) was measured with an Agilent CETOF-MS system (Capillary Electrophoresis Time-of Flight Time Spectrometry, Agilent Technologies Inc.) in positive and negative ion modes. Peaks detected in CETOF-MS analysis were extracted using automatic integration software (MasterHands version 2.16.0.15 developed by Keio University) to obtain peak information including m/z, migration time (MT), and peak area. did. The abundance of 158 metabolites detected in NI and I luminal fluids was calculated by peak area. Differentially expressed (DE) metabolites between NI and I luminal fluid were then identified as metabolites with absolute log ₂ -fold-changes > 1 (2-fold) or those detected only in NI or I luminal fluid. To identify metabolite groups (e.g., nucleotides and amino acids) enriched by DE, enrichment analysis was performed using Fisher's exact test and groups with a p -value less than 0.05 were selected.

As a result, among the intestinal luminal metabolome after Ecc15 infection, the most significant changes were observed in metabolites belonging to the nucleotide metabolic pathway (Figures 1A-B). Additionally, after Ecc15 infection, the levels of nucleosides (e.g., uridine), which are uracil molecules attached to the ribose ring, were significantly decreased, while the levels of nucleobases (e.g., uracil) were significantly increased. (Figure 1C).

These results suggest that uridine in the gut lumen is rapidly taken up by bacterial cells (i.e., uridine-in), leading to catabolism of uridine to ribose and uracil, which are then absorbed by these cells. This suggests that it is secreted by (i.e., uracil-out).

Next, to further investigate uridine-in and uracil-out metabolic fluxes, uracil and uridine levels in Ecc15 culture supernatants were analyzed by LC-MS coupled to tandem mass spectrometry. It was measured using /MS((liquid chromatography-tandem mass spectrometry)). Specifically, Ecc15 was cultured in minimal medium containing uridine, and uridine, uracil, and D-ribose obtained from culture supernatant or intestinal luminal fluid obtained from 30 midguts were electrosprayed. The analysis was performed using an Agilent 6460 Triple Quadrupole mass spectrometer (Agilent Technologies, USA). Separation was performed on an XSelect® HSS T3 2.5 μm (2.1 x 100 mm, 2.5 μm particle size, Island Waters) column.

For analysis of uridine or uracil, samples were mixed with 1 μL of internal standard (5-bromouracil at 500 μg/mL in water) and vortexed for 5 min in ethyl acetate. (ethyl acetate): isopropyl alcohol (isopropyl alcohol) 9: 1 (v/v) mixture was extracted with 1 mL. Each sample was centrifuged at 13,000 rpm for 10 min at 4 °C. An 800 μL aliquot of the upper organic layer was evaporated to dryness under nitrogen gas at 36°C. The residue was dissolved in 50 μL of water containing 0.2% acetic acid and vortexed for 5 min. The supernatant of each sample and standard solution was injected into the LC-MS/MS system. The column temperature was maintained at 30°C and the injection volume was set at 1 μL. The mobile phase (A) was water containing 0.2% acetic acid, and the mobile phase (B) was acetonitrile containing 0.2% acetic acid. Flow rate was 0.2 mL/min in gradient mode: 0-5 min, 0% (B); 10 min, 15% (B); 10.1-15 min, was 0% (B).

For ribose analysis, samples were mixed with 50 μL of internal standard (6-13C-fucose at 500 ng/mL in water) and then 5 mM 3 mixed in 100 μL of methanol. -Amino-9-ethylcarbazole (3-amino-9-ethylcarbazole, AEC), 50 μL of 10 mM NaCNBH ₃ solution, and 10 μL of acetic acid were mixed in that order. The reaction mixture was incubated at 60 °C for 60 min, and each tube was cooled on ice for 1 min before adding 300 μL of water and 300 μL of dichloromethane-hexane (2:1, v/v) solvent. was added. Each sample was vortexed for 5 minutes and centrifuged at 10,000 rpm for 5 minutes. A 100 μL aliquot of the upper aqueous phase was injected into the LC-MS/MS system. The column temperature was maintained at 40°C and the injection volume was set at 1 μL. The mobile phase (A) was water containing 0.1% formic acid, and the mobile phase (B) was acetonitrile containing 0.1% formic acid. Flow rate was 0.2 mL/min in gradient mode: 0 min, 25% (B); 8 min, 100% (B); and 8.1-15 min, 25% (B). Total run time was 15 minutes.

As a result, it was confirmed that the level of uracil in the culture supernatant gradually increased after culturing Ecc15, while uridine completely disappeared from the culture supernatant (Figure 1D).

Next, to investigate whether the excreted uracil derives directly from the absorbed extracellular uridine, the isotopes carrying the isotope at the nucleobase moiety ( ¹⁵ N-uridine) or the ribose moiety ( ¹³ C-uridine) were used. Ecc15 was cultured in mannitol medium containing radioactive uridine ( ¹⁵ N-uridine, ¹³ C-uridine, ¹⁵ N-uracil, and ¹³ C-ribose) for 8 hours. The levels of ¹⁵ N-uridine, ¹³ C-uridine, ¹⁵ N-uracil and ¹³ C-ribose in the culture supernatant were then quantified using LC-MS/MS analysis.

As a result, the levels of ¹⁵ N-uridine and ¹³ C-uridine gradually decreased in the culture supernatant over a 4-hour culture period (Figure 1E). On the other hand, the ¹⁵ N-uracil content in the culture supernatant gradually increased over the same period, confirming that uracil secreted from Ecc15 cells was actually derived from absorbed ¹⁵ N-uridine. In contrast to the nucleobase moiety, ¹³ C-ribose was not detectable in the culture supernatant (Figure 1E), confirming that the ribose moiety is not secreted after cleavage of uridine in Ecc15 cells.

These results suggested that pathogenic Ecc15 can take up extracellular uridine and catabolize it into ribose and uracil. Additionally, uracil, but not ribose, was secreted from bacterial cells.

Next, to confirm the origin of Drosophila intestinal lumen uridine, we compared the intestinal lumen uridine of CV (conventional) flies and germ free (GF) flies. GF flies were constructed as previously described (Ryu, et al., Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319, 777-782., 2008). To construct GF flies, approximately ^2x107 bacterial cells were washed twice with sterile PBS and administered into a sterile food vial containing GF embryos. GF flies were reared on standard autoclaved cornmeal medium or holidic medium (Piper, et al., A holidic medium for Drosophila melanogaster. Nat Methods 11, 100-105., 2014).

As a result, higher levels of uridine were detected in the intestinal lumen of GF flies compared to CV flies, indicating that uridine does not originate from intestinal bacteria. Additionally, no significant differences in uridine levels were observed between the standard diet and the Holidic diet, indicating that intestinal luminal uridine is not derived from the diet (Figure 8).

Example 2. Is there a relationship between “uridine-in and uracil-out” metabolic flux and dual oxidase (DUOX; a member of the nicotinamide adenine dinucleotide phosphate oxidase family) activation by pathogens? check

In the NCBI database for microbial genomes, genomic information is currently available for approximately 100 species of the Acetobacteraceae family.

Ten bacterial species from the Acetobacter family for which these genomes were identified were selected to investigate the relationship between bacterial uridine-in and uracil-out metabolic fluxes and intestinal DUOX activation. Specifically, to measure DUOX-dependent reactive oxygen species (ROS) production in vivo, the R2 region of the intestine was stained with hypochlorous acid (HOCl)-specific rhodamine-based R19S dye as previously described (Lee, et al. . Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila. Cell 153, 797-811., 2013). DUOX activity was expressed as percentage of R19S positive fields.

As a result, it was confirmed that 7 species of bacteria showed uridine-in and uracil-out metabolic flows, while 3 species did not (Figure 2A). Importantly, all seven bacterial species representing the uridine-in and uracil-out metabolic fluxes induced DUOX-dependent ROS production in the gut (Figure 2B).

These results indicate that the uridine-in and uracil-out metabolic fluxes of bacteria are related to their ability to induce DUOX activation in the intestine. Based on these results, these Acetobacteriaceae members can be classified into two distinct groups, i.e., uridine-in and uracil-out metabolic fluxes, which result in DUOX activation, depending on their effects on the host intestine. It can be divided into a group of opportunistic commensal microorganisms (pathobionts) and a group of commensal microorganisms that do not have DUOX activation because they do not have this metabolic flow.

Example 3. Comparative genome analysis between pathogens and commensal members of the Acetobacter family

To understand the molecular mechanisms of pathogen-specific uridine-in and uracil-out metabolic fluxes, we performed a comparative genomic analysis focusing on nucleoside metabolism. Specifically, the amino acid sequences of Gluconobacter morbifer ( G. morbifer ) for 52 proteins involved in the nucleotide metabolism pathway were collected from the NCBI protein sequence database. To assess the conservation of the above proteins in other bacteria, the sequence of G. morbifer was mapped to the protein sequences of nine other Aceatobacteraceae members using the BLASTP program, and the protein with the lowest E-value in the nine bacteria was determined. was selected as a potential ortholog. For each ortholog, percent identity was calculated as the number of identical amino acids divided by the total number of amino acids. Acetobacteriaceae strains were cultured at 30°C in mannitol medium.

As a result, nucleoside metabolic pathways, especially pyrimidine utilization and purine/pyrimidine conversion, differed between the commensal and pathogen groups (Figure 2C). In particular, nucleoside hydrolase (NH, converts nucleosides to nucleosides and ribose), nucleoside permease (NupC, transports nucleosides into cells), and adenine deaminases. Four genes, adenine deaminase (AdeC, which converts adenine to hypoxanthine) and cytidine deaminase (Cdd, which converts cytidine to uridine), are found in pathogen members. It was present only in (Figure 2C).

Based on this, it can be seen that pathogen-specific NupC and NH genes provide a mechanism by which pathogens can induce uridine-in and uracil-out metabolic fluxes for DUOX activation (Figure 2D ). The comparative genome analysis results may explain why commensal bacteria lacking the NupC and NH genes were unable to import extracellular uridine and produce uracil from uridine.

Example 4. Confirmation of NH activity participation in uracil out of pathogens

To confirm the direct participation of NH activity in uracil-out of pathogens, mutants of the pathogen G. morbifer ( G. morbifer ^△NH , Gmo ^△NH ) lacking NH activity were generated by site-directed mutagenesis. Produced. Specifically, in-frame deletion mutants of G. morbifer were constructed using homologous recombination. The disruption plasmid was demethylated using dam-/dcm- E. coli (NEB, C2925i) and introduced into G. morbifer by electroporation. Deletion of the target gene was confirmed by PCR and sequencing analysis. For complementation of G. morbifer ^ΔNH , the NH gene from Pseudomonas aeruginosa ( P. aeruginosa ) was placed under the control of the constitutive PntpII promoter in the pCM62 vector to construct pCM62-PntpII::NH. Plasmid pCM62-PntpII::NH was demethylated and introduced into G. morbifer ^△NH to produce G. morbifer ^△NH_NH .

As a result of examining uracil out from bacterial cells, it was confirmed that uracil out observed in the wild type parent strain ( G. morbifer ^WT , Gmo ^WT ) was completely abolished in Gmo ^ΔNH (Figure 3A). Additionally, when the NH gene was reintroduced ( G. morbifer ^△NH_NH , Gmo ^△NH_NH ), the impaired uracil-out ability of Gmo ^△NH was restored (Figure 3A). Unlike Gmo ^WT or Gmo ^△NH_NH , Gmo △NH not only did not confirm uracil out, but also could not ^induce ROS production in the intestine (Figure 3B).

These results indicate that bacterial NH activity is required for uracil out and subsequent DUOX-dependent ROS generation.

Example 5. Determination of relationship between NH activity of pathogens and gut pathology and colitogenic factors

To investigate whether the NH activity of the pathogen is a direct cause of intestinal pathology, we compared the rate of intestinal cell damage between Gmo ^WT or Gmo ^ΔNH and single-introduced GF flies, respectively.

Specifically, the midguts of 45-day-old female GF flies mono-introduced with Gmo ^WT or Gmo ^ΔNH , respectively, were dissected in PBS and analyzed for apoptosis using the Click-iT ^TM Plus TUNEL Assay Kit (Invitrogen Inc. C10618). was carried out. Apoptosis index was calculated as the percentage of the number of apoptotic cells relative to the total number of cells.

As a result, it was confirmed that the high intestinal cell apoptosis rate observed in Gmo ^WT single-introduced GF flies was almost absent in Gmo ^ΔNH single-introduced GF flies (Figure 3C). Consistent with the intestinal pathology results, Gmo ^WT mono-introduced GF flies had a shorter lifespan than Gmo ^ΔNH mono-introduced GF flies (Figure 3D). Therefore, it was confirmed that removal of NH activity was sufficient to induce bacterial phenotypic transition from pathogen to commensal. Additionally, reintroduction of NH activity into the mutant pathogen (Gmo ^ΔNH_NH ) was sufficient to restore all host pathology, such as enterocyte damage and high mortality, similar to levels observed in Gmo ^WT single-introduced flies (Figure 3C- D).

These results suggested that the NH activity of the pathogen acts as a colitogenic factor causing intestinal pathology and early host death.

Example 6. Determination of relationship between gut-microbe symbiosis and nucleoside catabolism of Acetobacter family

Based on the results of the above examples, we suggest that the absence of key genes involved in uridine catabolism (e.g. NH and NupC ) as observed in commensal bacteria can interact peacefully with the host intestine by avoiding chronic DUOX activation. Assuming, and to test this hypothesis, strains ( A. pasteurianus ^NH_NupC , Apa ^NH_NupC ) were created in which NH and NupC genes were introduced into the symbiotic bacteria of Acetobacter pasteurianus ( A. pasteurianus ). Specifically, to produce A. pasteurianus ^NH_NupC , A. pasteurianus was cultured in YPGD medium (containing 0.5% glycerol, 0.5% polypeptone, 0.5% glucose, and 0.5% yeast extract), and then cultured in Escherichia coli ( E. . coli) was transformed into pCM62-PntpII::NH_NupC by triparental mating using HB101/pKR2013 (Matsutani, et al., J Biotechnol 165, 109-119., 2013). Afterwards, bacteria were selected on YPGD medium supplemented with tetracycline (20 μg/ml) and acetic acid (0.1% (v/v)). Using the constructed strain, DUOX-dependent ROS production was analyzed in the same manner as in Example 4, and intestinal cell apoptosis was analyzed in the same manner as in Example 5.

As a result, it was confirmed that, unlike the wild-type parent strain (Apa ^WT ), Apa ^NH_NupC can induce uridine-in and uracil-out metabolic fluxes and subsequent DUOX-dependent ROS generation (Figure 3E -F).

Additionally, GF flies mono-introduced with Apa ^NH_NupC had significantly increased enterocyte apoptosis, showing a higher mortality rate than GF flies mono-introduced with Apa ^WT (Figure 3G-H).

These results confirm that ectopic expression of NH and NupC in commensals is sufficient to establish uracil-producing capacity, which leads to a bacterial phenotypic switch from commensals to pathogens, and also shows that NH-NupC is sufficient to establish uracil-producing capacity. The absence of cleoside catabolism appears to be required for gut-microbe symbiosis by avoiding chronic DUOX activation.

Example 7. Confirmation of NH-dependence of intestinal luminal uridine catabolism by pathogens

In addition to the gut microbiota, the Drosophila intestine naturally interacts with opportunistic pathogens such as Ecc15. On the basis that Ecc15 can induce uridine-in and uracil-out metabolic fluxes in vitro (Figure 1D-E), to investigate the role of uridine catabolism in pathogenic bacteria; First, genes involved in nucleoside catabolism were investigated. Because the genome information of Ecc15 is not yet available, draft genome sequence information was established through a whole-genome shotgun strategy (based on DDBJ/EMBL/GenBank WNLC00000000).

As a result of analysis of the Ecc15 genome, it was confirmed that the bacterium possesses two NH genes (NH1 and NH2 genes). In addition to these genes, Ecc15 also contains two nucleoside phosphorylase (NP) genes (udp, deoD genes), which can convert nucleosides into nucleobases and ribose 1-phosphate. ) was held.

Accordingly, a quadruple mutant Ecc15 (Ecc15 ^△4 ) lacking both the two NH genes and the two NP genes was constructed using the pDM4 suicide vector (Milton, et al., Journal of bacteriology 178, 1310-1319., 1996). Ecc15 ^△4 is Uridine-in and uracil-out metabolic fluxes could not be induced (Figure 4A), suggesting that NH and NP activities are required for catabolism of extracellular uridine.

To investigate whether uridine-in and uracil-out metabolic fluxes operate in vivo, i.e., when pathogens are introduced into the intestinal lumen, Ecc15 ^Δ4 and Ecc15 △ ^{4_NH, in which the NH gene was introduced into the Ecc15 △ 4} mutant, was orally infected by the method of Example 1 to cause intestinal infection, and the levels of uracil and uridine in the luminal fluid before and after intestinal infection were measured. Ecc15 ^△4_NH was constructed by cloning the full-length NH gene into the pTac3 vector under the control of the lac promoter to create pTac3-Plac::NH, and introducing the pTac3-Plac::NH into the Ecc15 ^△4 strain.

As a result, in the absence of intestinal infection, uridine levels were high, uracil was barely detectable, and the uracil/uridine ratio in the intestinal lumen was very low (Figure 4B). On the other hand, after intestinal infection, uracil levels increased significantly as uridine levels decreased significantly, resulting in a high uracil/uridine ratio (Figure 4B). These results demonstrated that Ecc15 exports uracil from the intestinal lumen in vivo.

However, when Ecc15 ^Δ4 was used for intestinal infection, the uracil/uridine ratio was not further increased (Figure 4B).

Additionally, when Ecc15 ^△4_NH was used for intestinal infection, the low uracil/uridine ratio observed during Ecc15 ^△4 infection was largely restored, resulting in a high uracil/uridine ratio (Figure 4B).

These results demonstrated that the NH activity of the Ecc15 pathogen induces nucleoside catabolism in vivo by using luminal uridine to produce and secrete uracil into the intestinal lumen.

Next, Ecc15 ^△4 , Ecc15 ^△4_NH and control group DUOX-dependent ROS production was confirmed using the pathogen in the same manner as in Example 4. As a result, it was confirmed that Ecc15 ^Δ4 could not induce DUOX-dependent ROS generation (Figure 4C), and it was confirmed that nucleoside catabolism ability was related to DUOX-activation ability.

Additionally, other pathogens (Salmonella enterica ( Salmonella typhimurium , S. typhimurium ), Serratia marcescens ( S. marcescens ), Shigella flexneri ( S. flexneri ), E. coli O157, and P aeruginosa ) was introduced into the intestinal lumen, an infection-induced increase in the uracil/uridine ratio was observed (Figure 4D).

Taken together, the above results suggest that nucleoside-in and nucleobase-out metabolic flows occur through bacterial nucleoside catabolism but are absent in Drosophila commensals, and only in pathogens different from commensals. It can be seen that this is a common feature.

Example 8. Determination of the relationship between nucleoside catabolism and down-regulation of primary metabolic pathways and up-regulation of quorum sensing (QS)

Considering that nucleoside catabolism is a novel pathogen-specific metabolism that is absent in commensals, to investigate the role of nucleoside catabolism in the physiology of pathogenic bacteria, we used Ecc15 and Ecc15 ^Δ4 to obtain high-resolution RNA- Sequence (RNA-Seq) analysis was performed. Specifically, Ecc15 and Ecc15 ^Δ4 were grown to late-exponential phase (OD600 = ~ 1.5) in M9 medium supplemented with 1 mM uridine at 30°C. RNA was extracted using TRIzol (Invitrogen) and treated with 2U of TURBO DNase (Ambion) for 30 minutes at 37°C. Finally, RNA was purified and concentrated with the RNeasy MiniElute kit (Qiagen). The cDNA library was prepared with the Ribo-Zero Bacteria Truseq ^TM Stranded Total RNA H/M/R Prep Kit (Illumina) according to the manufacturer's protocol. The cDNA library was sequenced using Illumina Hi-Seq 2500. The Illumina CASAVA pipeline (version 1.8.2) was used for base-calling and cutadapt (version 2.6) was used to discard reads containing Illumina adapter sequences. The resulting reads were mapped to the Ecc15 contig sequence using the TopHat aligner (version 2.1.1) with default options. After alignment, we calculated mapped reads for gene function using HTSeq (version 0.6.1) and normalized the counts using library size and gene length to obtain estimated fragments per kilobase of transcript per million fragments mapped. per kilobase of transcript per million (FPKM) was calculated. The raw data of mRNA sequencing was deposited in the Gene Expression Omnibus database (GSE140194) on April 7, 2020.

Additionally, 'expressed' genes were identified as genes with FPKM > 1 in Ecc15 or Ecc15 ^Δ4 . For these expressed genes, the FPKM value was added to the FPKM value and the FPKM value was converted to log ₂ -FPKM. log ₂ -FPKM values were normalized using the quantile normalization method (Bolstad, BM, Irizarry, RA, Astrand, M., and Speed, TP (2003). A comparison of normalization methods for high density oligonucleotide Array data based on variance and bias. Bioinformatics 19, 185-193.). Afterwards, DEGs between Ecc15 and Ecc15 ^Δ4 were identified with absolute log ₂ -fold-changes > 0.58 (1.5-fold). To identify the cellular processes represented by DEGs, we performed an enrichment analysis of gene ontology biological processes (GOBPs) for genes using Fisher's exact test and selected GOBPs with p -values less than 0.05.

As a result, we found that the genes shown in Figures 4E-H were dramatically up- and down-regulated in the absence of nucleoside catabolism. Functional enrichment analysis showed that the absence of nucleoside catabolism enhanced the “metabolic process” (65.2%) the most among the different biological processes (Figure 4E). In particular, cellular amino acid metabolic processes (28.2%), nucleobase-containing small molecule metabolic processes (22.0%), and carbohydrate metabolic processes (19.2%) were most frequently hyperactivated in Ecc15 ^Δ4 compared to Ecc15 (Figure 4F-H ). However, QS processing was downregulated in Ecc15 ^Δ4 (Figure 4H). That is, genes involved in primary metabolism were significantly upregulated in ^Ecc15Δ4 with downregulated QS.

These results suggested that nucleoside catabolism is required for down-regulation of primary metabolic activity and up-regulation of QS processes, and that QS activation is required to maintain appropriate levels of core metabolism.

Example 9. Determination of the relationship between complete production of acyl homoserine lactone-type quorum sensing molecules and nucleoside catabolism

Based on the results of the above examples, nucleoside catabolic activity may be required for QS, and impairment of QS activity caused by the absence of nucleoside catabolic activity may be responsible for the hyperactivated primary metabolic processes observed in Ecc15 ^Δ4. It was assumed that could be derived.

To confirm this hypothesis, we directly measured the QS molecular levels of Ecc15 ^Δ4 and Ecc15. Ecc15 produces two types of QS molecules: acyl homoserine lactone (AHL) produced by ExpI and autoinducer-2 (AI-2) produced by LuxS enzyme. It is known (Anal Bioanal Chem 387, 415-423., 2007). To measure these molecules, bioluminescent-based QS biosensors, especially the lux-based biosensor, E. coli pSB401 (Winson, et al., Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett 163, 185-192., 1998) and Vibrio harveyi ( V. harveyi ) MM32 strain (Miller, et al., Salmonella typhimurium recognizes a chemically Determine the levels of AHL and AI-2 by emitting bioluminescence upon recognition of AHL and AI-2, respectively, using a distinct form of the bacterial quorum-sensing signal AI-2. Mol Cell 15, 677-687, 2004) did. V. harveyi MM32 was cultured in AB broth for 30 min (Bassler, BL, Wright, M., and Silverman, MR (1994). Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi. Molecular microbiology 12, 403-412 .) was cultured. Bioluminescence was measured using a MicroLumat Plus LB 96V microplate luminometer (Berthold Technologies, UK). Data were expressed as relative light units (RLU) of the luminescence value of each sample. E. coli pSB401 was incubated with bacterial culture supernatant for 2 h and bioluminescence imaging was performed using a NightOWL II LB 983 imaging system (Berthold Technologies, UK).

As a result, the level of ExpI-dependent AHL was significantly higher in Ecc15 culture supernatant than in Ecc15 ^Δ4 culture supernatant (Figure 5A). However, no differences were seen in LuxS-dependent AI-2 levels between Ecc15 and Ecc15 ^Δ4 culture supernatants.

Next, an Ecc15 mutant (Ecc15 ^△LuxS ) in which AI-2-synthesizing enzyme LuxS was deleted was used as a negative control, and bioluminescence by AI-2 QS molecules produced in bacteria was measured. Ecc15 ^△LuxS was constructed using the pDM4 suicide vector.

As a result, no difference in LuxS-dependent AI-2 levels was observed between Ecc15 and Ecc15 ^Δ4 culture supernatants (Figure 9).

These results indicated that Ecc15 ^Δ4 was impaired in the production of AHL-type QS molecules, but not in the production of AI-2-type QS molecules.

Next, Ecc15 is known to produce 3-oxo-C6-HSL (N-(3-oxo-hexanoyl)-L-homoserine lactone), a major AHL-type (Barnard and Salmond, Quorum sensing in Erwinia species. Anal Bioanal Chem 387, 415-423., 2007), 3-oxo-C6-HSL was quantitatively analyzed using LC-MS/MS. Specifically, Ecc15 and Ecc15 ^Δ4 were cultured in M9 medium supplemented with 1 mM uridine. Bacterial cells were centrifuged at 6,000 rpm for 5 minutes and then quantitative analysis of 3-oxo-C6-HSL was performed using 500 μl of culture supernatant. To extract 3-oxo-C6-HSL from bacterial culture supernatant, after liquid-liquid extraction, 500 μL of standard solution and filtered bacterial culture supernatant were taken to contain 5 μL of C7-HSL (contained at 100 pg/mL in water). and then extracted with 1 mL of ethyl acetate. Only 800 μL of the upper organic layer was taken and evaporated to dryness under nitrogen gas at 40°C. The residue was dissolved in 250 μL of water containing 0.1% formic acid. After sonicating for 2 min and vortexing for 5 min, the supernatant was analyzed using an Agilent 6460 triple quadrupole mass spectrometer with electrospray interface (Agilent Technologies, USA). Separation was performed on an XSelect® HSS T3 2.5 μm (2.1 x 100 mm, 2.5 μm particle size, Island Waters) column while maintaining the column temperature at 30 °C. The mobile phase (A) was water containing 0.1% formic acid, and the mobile phase (B) was acetonitrile containing 0.1% formic acid. The chromatographic method using this maintains an initial mobile phase composition of 10% (B) for 1 min, increases linearly to 90% (B) in 6 min, and then finally returns to 10% (B) in 7.5 min and continues for 7.5 min. Equilibration was performed. Total run time was 15 minutes. The retention times of 3-oxo-C6-HSL and C7-HSL (used as internal standards) were 5.87 and 7.62, respectively. Qualitative and quantitative analysis was performed by Agilent Mass Hunter software.

As a result, 3-oxo-C6-HSL was significantly damaged in Ecc15 ^Δ4 (Figure 5B), suggesting that nucleoside catabolism is required for complete production of AHL-type QS molecules.

Example 10. Determination of the relationship between ribose produced through nucleoside catabolism and quorum sensing activity

Since Ecc15 ^Δ4 shows reduced AHL production, we investigated how nucleoside catabolism affects AHL production and AHL-dependent QS signaling.

Expression of two global regulators of QS, ExpR (a QS-dependent transcriptional regulator acting downstream of Exp) and VqsM (a novel AraC-type regulator of QS signaling and virulence), was regulated by Ecc15. Analyzed by qPCR analysis using ^WT , Ecc15 ^Δ4 , and Ecc15 ^ΔRbsR . Specifically, bacteria were cultured in M9 medium supplemented with 1 mM uridine, and fluorescence real-time PCR was performed to quantify gene expression using SYBR Green (Bioline). The rpoB gene was used as an internal control. Ecc15 ^△RbsR is It was constructed using the pDM4 suicide vector.

As a result, expression of ExpR and VqsM was significantly impaired in Ecc15 ^Δ4 (Figures 4H and 5C). Considering that the ExpR-AHL complex induces QS pathway activation, including AHL production, the reduced AHL production observed in Ecc15 ^Δ4 may be due to reduced expression of ExpR.

These results indicated that nucleoside catabolism is required for full activation of the global QS regulator.

Next, 11 genes involved in primary metabolism (purH (Phosphoribosylaminoimidazolecarboxamide formyltransferase, GJS26_00239), pgm (Phosphoglucomutase, GJS26_01330), pgk (Phosphoglycerate kinase, GJS26_03794), adk (Pyruvate kinase, GJS26_01172) , pyk (Pyruvate kinase, GJS26_01172 ) nA(Nucleoside-diphosphate kinase, GJS_03171), carA(carbamoyl phosphate synthase A, GJS26_03753), nrdB(Ribonucleoside-diphosphate reductase, GJS_01196), udk(Uridine kinase, GJS_01409), add(Adenosine deaminase, CJS_02167) at pE(GJS26_04373 )) was determined by qPCR analysis using Ecc15 ^WT , Ecc15 ^Δ4 , Ecc15 ^ΔRbsR , and Ecc15 ^ΔExpIR . Ecc15 ^ΔExpIR was constructed using the pDM4 suicide vector. Target gene expression in Ecc15 ^WT was arbitrarily set to 1, and expressed as the relative expression level.

As a result, as shown in Figure 10, the expression of 11 genes related to primary metabolism was significantly increased in Ecc15 ^△4 , Ecc15 ^△RbsR , and Ecc15 ^△ExpIR compared to Ecc15 ^WT .

Next, the mechanism by which nucleoside catabolism affects the expression of key QS regulators was investigated. Because nucleoside catabolic activity can generate intracellular ribose from extracellular uridine, we hypothesized that RbsR, a ribose-regulated transcriptional regulator, is involved in the transcriptional regulation of the QS regulator. To confirm this hypothesis, Ecc15 ^ΔRbsR was constructed and expression of ExpR and VqsM was examined in the absence of RbsR. Ecc15 ^ΔRbsR was constructed using the pDM4 suicide vector.

As a result, as observed in Ecc15 ^Δ4 , Expr and VqsM expression was significantly reduced in Ecc15 ^ΔRbsR (Figure 5C), indicating that RbsR is required for the full expression of these QS regulators. Consistent with this, as observed in Ecc15 ^Δ4 , AHL-dependent bioluminescence activity was confirmed to be greatly reduced in Ecc15 ^ΔRbsR (Figure 5D).

These results suggested that ribose generated by nucleoside catabolic activity upregulates QS regulators and induces QS mechanism activity in an RbsR-dependent manner.

Example 11. Determination of the relationship between nucleoside catabolism and complete expression of pathogen virulence genes

Ecc15 is an entomopathogen that can persist in the fruit fly intestine. Erwinia virulence factor ( evf ) is known to act as a major virulence gene (Basset et al., A single gene that promotes interaction of a phytopathogenic bacterium with its insect vector, Drosophila melanogaster.EMBO Rep 4, 205 -209., 2003), causing bacterial pathogenesis.

On this basis, to investigate whether the evf gene is under the control of a specific QS pathway, AHL-type QS molecules and AI-2-type QS were identified in bacteria carrying mutations in the ExpI pathway or the LuxS pathway (Ecc15 ^ΔExpIR or Ecc15 ^ΔLuxS ). The molecule could not be produced. A reporter plasmid carrying GFP under the control of the evf promoter region (Pevf::GFP) was introduced into these QS mutants. Specifically, the evf promoter (608 bp) fused to GFP was cloned into the pTac vector to create pTac-Pevf::gfp. To analyze evf gene expression in vitro and in vivo, pTac-Pevf::gfp was introduced into Ecc15 ^WT , Ecc15 ^△4 , Ecc15 ^△RbsR , Ecc15 ^△ExpIR , and Ecc15 ^△LuxS . Bacteria were cultured in M9 medium supplemented with 1mM uridine, and GFP signals were analyzed using a SPECTRAL Ami X molecular imager (Spectral Instruments Imaging).

To analyze bacterial evf gene expression in the Drosophila intestine, larval or adult flies were orally infected with 10 ¹⁰ CFU or less of bacteria (Ecc15 ^WT or Ecc15 ^Δ4 ) carrying a Pevf::GFP reporter for 2 h. Whole intestines were dissected in PBS and fixed in formalin. Confocal fluorescence images of each sheet were analyzed using a Zeiss LSM 700 confocal microscope. GFP levels in the midgut R2 region were quantified using ZEN software.

As a result, it was confirmed that Pevf::GFP reporter activity was severely downregulated in Ecc15 ^ΔExpIR but was normally expressed in Ecc15 ^ΔLuxS (Figure 5F).

These results demonstrated that expression of key virulence genes is mainly under the control of the AHL-type QS system.

Next, considering that nucleoside catabolism acts upstream of the AHL-dependent QS pathway, we investigated whether nucleoside catabolism is required for full activation of the AHL-dependent evf gene in vitro.

As a result, Pevf::GFP reporter activity and evf gene expression were severely impaired in Ecc15 ^Δ4 and Ecc15 ^ΔRbsR compared to Ecc15 (Figure 5E-F).

These results suggested that nucleoside catabolic activity and subsequent RbsR activity are required for QS-dependent virulence gene expression.

Example 12. Determination of the relationship between nucleoside catabolism and quorum sensing-dependent toxicity in vivo

To investigate whether QS activation controlled by nucleoside catabolism actually occurs in vivo, i.e. within the intestinal lumen, we examined AHL production after intestinal infection. To visualize AHL production in the intestine, oral Ecc15 infection was performed with the QS biosensor E. coli pSB401 strain. Specifically, oral infection experiments were performed using Ecc15 ^WT , Ecc15 ^Δ4 , and E. coli pSB401 in mid-exponential phase (OD600 = ~ 0.5). Female flies (5-6 days old) were orally infected with Ecc15 ^WT or Ecc15 ^Δ4 (up to ^5x109 CFU) with E. coli pSB401 (up to ^5x109 CFU) for 1.5 hours. Bioluminescence imaging of live animals was obtained using a NightOWL II LB 983 imaging system (Berthold Technologies, UK).

As a result, high levels of AHL-dependent bioluminescence were detected in the intestinal lumen after Ecc15 ingestion (Figure 6A). In contrast, AHL-dependent bioluminescence was barely detectable after Ecc15 ^Δ4 ingestion (Figure 6A), suggesting that QS activation within the intestinal lumen is dependent on nucleoside catabolism.

Next, to confirm QS-dependent evf expression in bacteria when Ecc15 entered the intestinal lumen, we constructed two Pevf::GFP reporter bacteria in meta-exponential phase (Ecc15-Pevf::GFP and Ecc15 ^Δ4- Pevf::GFP).

At this growth time point, Pevf::GFP expression levels were basal and similar between the two reporter bacteria. However, when the two bacteria were introduced into the intestinal lumen, a significant difference appeared in the Pevf::GFP expression of Ecc15-Pevf::GFP and Ecc15 ^Δ4 -Pevf::GFP (Figure 6B). In Ecc15-Pevf::GFP bacteria, intense GFP expression was observed mostly in the anterior part of the midgut, but only weak GFP expression was observed in Ecc15 ^Δ4 -Pevf::GFP bacteria (Figure 6B).

These results demonstrated that the nucleoside catabolism of Ecc15 is required for QS and virulence gene expression during in vivo interactions between the gut and microorganisms.

Example 13. Confirmation of induction of conversion from pathogenic bacteria to commensal bacteria by removal of bacterial nucleoside catabolism

Intestinal commensals such as Acetobacter affect various features of host physiology such as development and metabolism, which can be confirmed through the host's survival, development rate, and growth under conditions such as protein malnutrition.

GF flies were created by adding up to 2x10 ⁷ CFU of A. pomorum , Ecc15, or Ecc15 mutants to GF embryos. Pupa formation was monitored every 12 h. Body size of GF larvae was measured at a development point of 108 h after oviposition. For larval development experiments, a protein-poor diet containing low yeast (1.5% (v/v) yeast) and deficient in bokinin and propionic acid was used. Adult female flies (5-6 days old) were used for in vivo ROS measurements, metabolite analysis, and confocal image analysis.

Additionally, to measure lifespan, GF flies were single introduced with G. morbifer ^△NH , G. morbifer ^△4NH_NH or A. pasteurianus ^NH_NupC . G. morbifer and A. pasteurianus carrying a mock vector (pCM62 carrying a tetracycline resistance marker) were used as controls. GF flies were transferred to fresh sterile medium supplemented with 20 μg/ml tetracycline every 4–5 days. Survival was monitored over time in at least three independent cohorts, each consisting of approximately 25 flies. GF flies or GF flies related to specific bacterial strains were reared on standard autoclaved cornmeal medium or Holidig medium.

As a result, under conditions such as protein malnutrition, GF flies can survive, but their developmental rate and growth are delayed (Figure 6C-E). This developmental defect is completely rescued in the presence of normal microbiota (i.e., CV flies) or A. pomorum (i.e., A. pomorum -introduced GF flies) (Figure 6C-E). However, when Ecc15 was abundant in GF flies, Ecc15-transduced GF flies displayed more dramatic pathological changes than GF flies, including growth arrest and severe larval mortality (>50% mortality) (Figure 6C).

Additionally, the appearance of host pathology upon reintroduction of the NH gene into Ecc15 ^Δ4 (Figure 6C-E) demonstrated that nucleoside catabolism is required for pathogen virulence.

Next, to assess whether QS activation induced by nucleoside catabolism is involved in this pathogenesis, Ecc15 ^Δ4 or Ecc15 ^ΔRbsR single-introduced GF embryos were generated.

As a result, Ecc15 ^Δ4 or Ecc15 ^ΔRbsR single-introduced GF flies showed completely normal larval development compared to commensal ( A. pomorum )-introduced GF flies (Figure 6C-E).

Next, bacteria of the Ecc15 ^WT , Ecc15 ^Δ4 , Ecc15 ^ΔExpIR and Ecc15 ^ΔLuxS genotypes were added to GF embryos and the percentage of pupa formation was monitored every 12 h.

As a result, as shown in Figure 11A-B, Ecc15 ^△RbsR , Ecc15 ^△ExpIR single-introduced GF flies showed completely normal larval development, and Ecc15 ^△LuxS single-introduced cells showed no changes in the host as if the wild type was single-introduced. The etiology can be confirmed.

The above results show that deletion of ExpIR, but not LuxS, is sufficient to induce pathogen-community switching, indicating that ExpI-dependent quorum sensing is required for bacterial pathogenicity.

These results also suggested that removing bacterial nucleoside catabolic activity from pathogens is sufficient to induce the transition from pathogens to commensals in the Drosophila intestine.

Next, the role of nucleoside catabolic activity on QS-dependent toxicity of P. aeruginosa (PAO1) was confirmed. A single NH gene was identified in the genome of P. aeruginosa PAO1. To avoid the autolysis and autoaggregation behavior of P. aeruginosa , the pqsL-PAO1 strain was used (Hazan, et al., Auto Poisoning of the Respiratory Chain by a Quorum-Sensing-Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance. Current biology : CB 26, 195-206., 2016). PAO1 ^ΔNH was constructed by deleting the NH gene. PAO1 ^△NH_NH was created by introducing the NH gene into the PAO1 ^△NH mutant.

To confirm the activity of virulence factors, the amount of pyocyanin and the activity of elastase were measured.

Specifically, the amount of pyocyanin was measured as previously described (Smith, et al., Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem Biol 10, 81-89., 2003). Specifically, Pseudomonas aeruginosa was grown in LB medium at 37°C for 12 hours, and 5 ml of culture supernatant was extracted with 3 ml of chloroform and then re-extracted with 1 ml of 0.2N HCl. The absorbance of the extracted solution was measured at 380 nm.

Elastase B activity was measured as previously described ( Smith et al., 2003 ). Specifically, P. aeruginosa was cultured in M9 medium supplemented with 1 mM uridine at 37°C for 8.5 hours, the culture supernatant was filtered, and incubated at 200°C at 37°C with elastin-congo red solution. After culturing with agitation at rpm for 12 hours, elastase activity was measured at 495 nm.

The level of AHL with acyl chain length C4 was measured in culture supernatants of PAO1 using the lux-based E. coli AHL biosensor pSB536.

Additionally, adult female flies (5-6 days old) were orally infected with the indicated bacteria and analyzed for host survival.

As a result, as shown in Figure 12, quorum sensing activation is regulated by NH enzyme activity in the pathogen P. aeruginosa , and as a result, the activity of elastase B, a representative virulence factor dependent on the quorum sensing of P. aeruginosa, and the production of the toxin pyocyanin It was demonstrated that the mortality rate of the host was significantly reduced in PAO ^ΔNH .

Example 14. Assaying the effect of inhibitors of NH enzyme activity of pathogens

To confirm whether the compound shown in Figure 13A has an inhibitory effect on NH enzyme activity, the degree to which NH enzyme decomposes uridine, a substrate, upon addition of the inhibitor was analyzed.

Specifically, NH enzyme and uridine (None, negative control), NH enzyme and uridine together with NH enzyme activity inhibitor DMPAPIR (DM) 0.5, 1, 2.5, 10, 100 μM, BnIR 10, 50, 100 μM; Alternatively, as a positive control, 10, 50, and 100 μM PAPIR, an NH inhibitor of the protozoan parasite Leishmania reported in a previous study, was added and NH enzyme activity was measured through a spectrophotometric assay.

As a result, when only the enzyme and uridine were reacted (None), the uridine concentration decreased rapidly due to the activity of the enzyme, but when NH enzyme activity inhibitors were added at various concentrations, the enzyme activity was inhibited in proportion to the concentration. This was confirmed (Figure 13B-C). The minimum inhibitory concentration that can inhibit enzyme activity is measured to be ~1 μM for DMPAPIR (DM), ~100 μM for BnIR, and ~100 μM for PAPIR, making DMPAPIR approximately 100 times more effective. It was confirmed that the quality was excellent. In the case of BnIR, the inhibitory function is similar to that of existing PAPIR, but the process of producing the inhibitor is much easier than that of PAPIR, making it valuable as an effective inhibitor.

Example 15. Assaying the effect of inhibitors of NH enzyme activity of pathogens

Ecc15, belonging to the genus Pectobacterium, is not only a pathogen of fruit flies but also a plant pathogen that can cause disease in a wide range of plants. Since the genus Pectobacterium is known to cause various plant diseases including soft rotting in plants, we checked whether these plant diseases could be controlled by inhibiting NH enzyme activity.

Specifically, potatoes were treated with less than 10 ⁶ CFU of wild type bacteria (ECCWT) and mutant bacteria with NH enzyme activity removed (Ecc15 ^△4 ), respectively, and also treated with 0.1 mM (100 μM) and 1 mM (1000 μM) of DMPAPIR. The softness of the potatoes (rotting vol.) was measured after 48 hours.

As a result, it was confirmed that when Ecc15 ^Δ4 or Ecc15 and DMPAPIR were treated together, the incidence of soft rot caused by wild-type bacteria was reduced to less than 10% (Figure 13D).

Next, it was confirmed whether DMPAPIR suppresses disease in several other species belonging to the genus Pectobacterium by inhibiting NH enzyme activity. Pectobacterium carotovorum subsp. brasilliense (Pcb), which can cause disease in a wide range of plants, Pectobacterium carotovorum subsp. carotovorum 15 ( Pectobacterium carotovorum subsp. carotovorum15 , Pcc 15) and Pectobacterium chrysanthemi (Pch), which has been reported to cause disease in ornamental plants and plant bulbs, and Pectobacterium Art, which causes blackleg of potatoes. Measure whether the degree of soft rot in potatoes is reduced by DMPAPIR using the same method as above targeting Pectobacterium atrosepticum and Pbe, which are known to cause diseases in sugar beets. did.

As a result, it was confirmed that the pathogenicity of several different species of strains belonging to the genus Pectobacterium was significantly reduced by DMPAPIR (Figure 13E). In addition, it was confirmed that the incidence of soft rot was significantly reduced by BnIR (Figure 13F).

In Pseudomonas syrigae (Pst), another representative plant pathogen that has been reported to cause necrosis or gummosis in various plants, it was confirmed whether NH enzyme activity has a significant effect on the virulence of the fungus. Specifically, the leaves of Arabidopsis , a representative leaf plant model, were wounded and less than 2X10 ⁶ CFU of wild-type bacteria (Pst ^WT ) and mutant bacteria with NH enzyme activity removed (Pst ^△NH ) were introduced, respectively, or 0.1 mM , 1 mM DMPAPIR (DM) was simultaneously treated with wild-type bacteria and the degree of necrosis of plant leaves was observed after one week.

As a result, it was confirmed that plant leaf necrosis caused by wild-type bacteria was reduced when the mutant bacteria (Pst ^ΔNH ) and DMPAPIR (DM) were treated together (Figure 13G).

To confirm whether the NH enzyme activity inhibitor also acts on the pathogen P. aeruginosa (PAO1), PAO1 was cultured with or without DMPAPIR (DM) treatment to obtain culture supernatant, and the supernatant and elastin-congo red were used. was reacted to measure the elastase activity of PAO1.

As a result, it was confirmed that the activity of elastase, a representative virulence factor of PAO1, decreased in proportion to the treatment concentration of the NH enzyme activity inhibitor (Figure 13H).

From these results, the nucleoside hydrolase inhibitor according to the present invention acts on pathogens of the genus Pectobacterium and Pseudomonas to effectively inhibit the activity of nucleoside hydrolase, resulting in quorum sensing-dependent It was proven that pathogenesis was suppressed.

In addition, the nucleoside hydrolase inhibitor according to the present invention can suppress the toxicity of pathogens, that is, pathogenic microorganisms, or convert pathogenic pathogens into non-pathogenic commensal bacteria, thereby suppressing the toxicity of pathogenic microorganisms containing them. It can be applied as a composition for use, a composition for preventing or treating pathogenic microbial infection, a composition for controlling plant diseases, a method for inhibiting the toxicity of pathogenic microorganisms including the step of treating the composition, and a method for controlling plant diseases, and the mechanism according to the present invention is It can be applied as a screening method for nucleoside hydrolase inhibitors.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

<110> Seoul National University R&DB Foundation <120> Nucleoside hydrolase inhibitors, compositions for inhibiting the toxicity of pathogenic microorganisms comprising the same, methods for inhibiting the toxicity of pathogenic microorganisms comprising the same and screening methods of nucleoside hydrolase inhibitors <130> KPA200031- KR-P1 <150> KR 10-2020-0019941 <151> 2020-02-18 <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 954 <212> DNA <213> Unknown <220> <223 > NH 1 NT <400> 1 atgagcgctt tacctattat tattgattgt gatccgggga tagatgacgc gattgcgtta 60 ttaagtgcat ttgtgtcacc agagttggat attcgcggta tttgcgcggt gtgtggtaac 120 cagcctgtgg aaaaaacggt gcgtaatgcg ctgcaaattg ttgagctggg tcagcgcacc 180 gatattcccg ttttcgccgg ctgccatcgg ccgctgttgc gtgatcctat tcatggccag 240 ttccatggcg aaaacggact gggccagacg gtgttgccta cgccgcaaaa acaggccgac 300 gcgcagcatg cggt gaattt tattattgcg cagtgcaaac aggcgatcgc tgacggcacg 360 ccgatcacgc tctgtacgtt agggccgtta accaatgtcg cgatggcgct gcgtatgtct 420 cctggcattg tcgacggtat tgcacgcatt gtgatgatgg gcggcgcata ccgggaagcg 480 ggcaaccgca gcctgacctc tgaattcaat atgatcgccg atccgca ggc ggccaaggtc 540 gtgttcgact cgtcgattgc catcgtcgcg ctaccgctgg atgtcacgca tcaggtgatt 600 ttaacgccgg aattggtggc acgtttcatt gcgctatccg gacgtatttc cgccccattg 660 ggtgaaatga tggcgttttg ggat cgcaat gacatccgcc gctatggctc acgcggcggc 720 ccgctacacg atccgctggt catcgcctgg gttctggcgc cacattgctt cacgacggaa 780 aaagccagcg tttacattga gcaggaaagc gagctgtgca tggggcagac cgttgctgac 840 tggtacggaa aaaccgatcg gcagccgaat gtggatgtgg tgacgggcgt tgatgccaaa 900 caggttgttg agctgtttgc tgacttactg agccgctatg gagagggtgt ctga 95 4 <210> 2 <211> 1005 <212> DNA <213> Unknown <220> <223> NH 2 NT <400> 2 atggttcgag aacgcataat tattgatact gaccccggtg ttgatgatgc aatagccatc 60 tggctggcat tggcctcgcc cgaactggac gtactcggga ttaccattgt ggctggtaat 120 gtgccgcttg ctgcgacgtt gccgaatgcc tgcaacgtgg tgggcgtgac aggcagaacc 180 g atgtgccta tttttgccgg tgcttcgcgt ccgctcatcc gcgatcaggt tttcggtaaa 240 tacgctcata tcggtaagtt ttcctctgac tgggtgccag aaagcacgct gtcgccggaa 300 gaggaacatg ccgtcgattt tctggtgcgg atgacgcgtc aggcag cggc cgataataac 360 ccgatcacca tttgttctat aggtccgatg acgaatctgg cattggcact gtgtttccat 420 cctgatgtcg cacgcggcat caaacagatt gtgtccatga gctgcgcctt taccgcgatg 480 gggaatcgcg taccgtgggc ggattttaac gtctatgccg atccgcatgc ggcggaaatc 540 gttttttctt ccggtgttcc cattgtcatc atgccgctgg atgtgacctt tcaggcactg 600 at tcagacgg aacaagttga tgccatcgaa cgtagcggcg gtgcgccggg taaggcgatg 660 gccgcgctgc tgcgtatgtt tgaccgtagc gaggttgagc gctttggccg tgaaggcggg 720 ccgattcatg atgcgaccgt cattgcatgg ctgttgaagc cggagct gtt taagtcaaaa 780 cgcgctcgta taggagtgga agtcgccggt aagatggcgg gttacgtgtt tgccgacttt 840 tatcacaaac tgggtgagcc ggagaatgcg ctgatcatgc gtgaaatcga cgaacaagga 900 ttcctcgggc tgattgccga tcgcctgcgt cgctatggcc ctgaagcaat aaatgatgtt 960 gaagcaataa atggttccga agcaatcaat cggtctgagg agcgc 1005 <210> 3 <211> 771 < 212> DNA <213> Unknown <220> <223> NP 1(udp) NT <400> 3 atgtctacat ctgatgtgtt ccatcttggt ctggtcaaga acgatttaca gggcgcgacg 60 ctggctattg tccccggcga tcctgagcgt gttgaaaaaa ttgctcgcct gatggataac 120 ccggtacatc tggcatcaca ccgtgaattt acgtcctggc gtgcagagtt ggacggcaag 180 gcagtgattg tttgctcgac cggtattggc ggcccgtcaa cgtcaatcgc ggtagaagaa 240 ttggcacagc tcggcatccg gactttcctg cgcgtcggca cgacgggggc gattcagtcc 300 ggcatcaacg tcggcgatgt gctagtgacc acagccgccg tccgtctgga cggggctagc 360 ctgcacttcg cgccaatgga attcccagcc gttgctgatt tcggctgtac caccgcgctg 420 gtggaagcgg cgaaggcctc gggtgcggcg ttgcatgtgg gcatcacggc gtcttccgat 480 acgttctacc ccggtcagga acgctatgac accttctctg gacgcgttgt ccgtcgcttc 540 cgcggctcga tggaagaatg gcagagcatg ggcgtgctga actatgaaat ggaatccgcc 600 acgctgttga ccatgtgcgc cagccagggg ctgaaagcgg gcatgatcgc gggcgtgatc 660 gtgaaccgta cccagcagga aatcccgaat gcggcgacca tgaaactggc cgaaaccacc 720 gtcatcaagg tgttgctgga cgcgactcgc cgtttgttag ct gctgaata a 771 <210> 4 <211> 720 <212 > DNA <213> Unknown <220> <223> NP 2(deoD) NT <400> 4 atggctacgc cacatattaa tgcagaaatg ggtgatttcg cggacgttgt actgatgccg 60 ggcgacccgc tgcgcgctaa gtacattgca gaaacctttc tggatgatgc ccgcgaag tg 120 aacaacgtgc gtggcatgtt agggttcacg gggacttaca aaggccgtaa aatttcggtc 180 atgggccacg gcatgggtat cccatcctgc tcaatttatg cgaaagaact gatcaccgaa 240 ttcggcgtga agaagatcat ccgtgtgggt tcctgcggtg cggtacgtga agatgttaag 300 ctgcgcgacg tggtaatcgg catgggtgcc tgtacggatt ctaaagtgaa ccgtatgcgt 360 ttcaaagatc acgactacgc ggcgattgct gatttcgata t ggtgcgtaa tgccgttgat 420 gctgccaaag cacgcgatgt ttctgtccgc gtcggtaaca tcttctccgc cgatctgttc 480 tacacgccag atccgcagat gttcgacgtg atggaaaaat acggcattct gggtgtggaa 540 atggaagcgg ccggtatcta tggcgtcgcg gcagagttcg gtgcgaaagc gctggccatc 600 tgtaccgttt ctgaccacat tcgtaccggt gcacaaacca cgtcagaaga gcgtcaaaac 660 acgttcaacg agatgatcga aatcgcgctg gaatccgttc tgctgggcga taacgagtaa 720 720 <210> 5 <211> 317 <212> PRT <213> Unknown <220> <223> NH 1 AA <400> 5 Met Ser Ala Leu Pro Ile Ile Ile Asp Cys Asp Pro Gly Ile Asp Asp 1 5 10 15 Ala Ile Ala Leu Leu Ser Ala Phe Val Ser Pro Glu Leu Asp Ile Arg 20 25 30 Gly Ile Cys Ala Val Cys Gly Asn Gln Pro Val Glu Lys Thr Val Arg 35 40 45 Asn Ala Leu Gln Ile Val Glu Leu Gly Gln Arg Thr Asp Ile Pro Val 50 55 60 Phe Ala Gly Cys His Arg Pro Leu Leu Arg Asp Pro Ile His Gly Gln 65 70 75 80 Phe His Gly Glu Asn Gly Leu Gly Gln Thr Val Leu Pro Thr Pro Gln 85 90 95 Lys Gln Ala Asp Ala Gln His Ala Val Asn Phe Ile Ile Ala Gln Cys 100 105 110 Lys Gln Ala Ile Ala Asp Gly Thr Pro Ile Thr Leu Cys Thr Leu Gly 115 120 125 Pro Leu Thr Asn Val Ala Met Ala Leu Arg Met Ser Pro Gly Ile Val 130 135 140 Asp Gly Ile Ala Arg Ile Val Met Met Gly Gly Ala Tyr Arg Glu Ala 145 150 155 160 Gly Asn Arg Ser Leu Thr Ser Glu Phe Asn Met Ile Ala Asp Pro Gln 165 170 175 Ala Ala Lys Val Val Phe Asp Ser Ser Ile Ala Ile Val Ala Leu Pro 180 185 190 Leu Asp Val Thr His Gln Val Ile Leu Thr Pro Glu Leu Val Ala Arg 195 200 205 Phe Ile Ala Leu Ser Gly Arg Ile Ser Ala Pro Leu Gly Glu Met Met 210 215 220 Ala Phe Trp Asp Arg Asn Asp Ile Arg Arg Tyr Gly Ser Arg Gly Gly 225 230 235 240 Pro Leu His Asp Pro Leu Val Ile Ala Trp Val Leu Ala Pro His Cys 245 250 255 Phe Thr Thr Glu Lys Ala Ser Val Tyr Ile Glu Gln Glu Ser Glu Leu 260 265 270 Cys Met Gly Gln Thr Val Ala Asp Trp Tyr Gly Lys Thr Asp Arg Gln 275 280 285 Pro Asn Val Asp Val Val Thr Gly Val Asp Ala Lys Gln Val Val Glu 290 295 300 Leu Phe Ala Asp Leu Leu Ser Arg Tyr Gly Glu Gly Val 305 310 315 <210> 6 <211> 335 <212> PRT <213> Unknown <220> <223> NH 2 AA <400> 6 Met Val Arg Glu Arg Ile Ile Ile Asp Thr Asp Pro Gly Val Asp Asp 1 5 10 15 Ala Ile Ala Ile Trp Leu Ala Leu Ala Ser Pro Glu Leu Asp Val Leu 20 25 30 Gly Ile Thr Ile Val Ala Gly Asn Val Pro Leu Ala Ala Thr Leu Pro 35 40 45 Asn Ala Cys Asn Val Val Gly Val Thr Gly Arg Thr Asp Val Pro Ile 50 55 60 Phe Ala Gly Ala Ser Arg Pro Leu Ile Arg Asp Gln Val Phe Gly Lys 65 70 75 80 Tyr Ala His Ile Gly Lys Phe Ser Ser Asp Trp Val Pro Glu Ser Thr 85 90 95 Leu Ser Pro Glu Glu Glu His Ala Val Asp Phe Leu Val Arg Met Thr 100 105 110 Arg Gln Ala Ala Ala Asp Asn Asn Pro Ile Thr Ile Cys Ser Ile Gly 115 120 125 Pro Met Thr Asn Leu Ala Leu Ala Leu Cys Phe His Pro Asp Val Ala 130 135 140 Arg Gly Ile Lys Gln Ile Val Ser Met Ser Cys Ala Phe Thr Ala Met 145 150 155 160 Gly Asp Arg Val Pro Trp Ala Asp Phe Asn Val Tyr Ala Asp Pro His 165 170 175 Ala Ala Glu Ile Val Phe Ser Ser Gly Val Pro Ile Val Ile Met Pro 180 185 190 Leu Asp Val Thr Phe Gln Ala Leu Ile Gln Thr Glu Gln Val Asp Ala 195 200 205 Ile Glu Arg Ser Gly Gly Ala Pro Gly Lys Ala Met Ala Ala Leu Leu 210 215 220 Arg Met Phe Asp Arg Ser Glu Val Glu Arg Phe Gly Arg Glu Gly Gly 225 230 235 240 Pro Ile His Asp Ala Thr Val Ile Ala Trp Leu Leu Lys Pro Glu Leu 245 250 255 Phe Lys Ser Lys Arg Ala Arg Ile Gly Val Glu Val Ala Gly Lys Met 260 265 270 Ala Gly Tyr Val Phe Ala Asp Phe Tyr His Lys Leu Gly Glu Pro Glu 275 280 285 Asn Ala Leu Ile Met Arg Glu Ile Asp Glu Gln Gly Phe Leu Gly Leu 290 295 300 Ile Ala Asp Arg Leu Arg Arg Tyr Gly Pro Glu Ala Ile Asn Asp Val 305 310 315 320 Glu Ala Ile Asn Gly Ser Glu Ala Ile Asn Arg Ser Glu Glu Arg 325 330 335 <210> 7 <211> 256 <212> PRT < 213> Unknown <220> <223> NP 1(udp) AA <400> 7 Met Ser Thr Ser Asp Val Phe His Leu Gly Leu Val Lys Asn Asp Leu 1 5 10 15 Gln Gly Ala Thr Leu Ala Ile Val Pro Gly Asp Pro Glu Arg Val Glu 20 25 30 Lys Ile Ala Arg Leu Met Asp Asn Pro Val His Leu Ala Ser His Arg 35 40 45 Glu Phe Thr Ser Trp Arg Ala Glu Leu Asp Gly Lys Ala Val Ile Val 50 55 60 Cys Ser Thr Gly Ile Gly Gly Pro Ser Thr Ser Ile Ala Val Glu Glu 65 70 75 80 Leu Ala Gln Leu Gly Ile Arg Thr Phe Leu Arg Val Gly Thr Thr Gly 85 90 95 Ala Ile Gln Ser Gly Ile Asn Val Gly Asp Val Leu Val Thr Thr Ala 100 105 110 Ala Val Arg Leu Asp Gly Ala Ser Leu His Phe Ala Pro Met Glu Phe 115 120 125 Pro Ala Val Ala Asp Phe Gly Cys Thr Thr Ala Leu Val Glu Ala Ala 130 135 140 Lys Ala Ser Gly Ala Ala Leu His Val Gly Ile Thr Ala Ser Ser Asp 145 150 155 160 Thr Phe Tyr Pro Gly Gln Glu Arg Tyr Asp Thr Phe Ser Gly Arg Val 165 170 175 Val Arg Arg Phe Arg Gly Ser Met Glu Glu Trp Gln Ser Met Gly Val 180 185 190 Leu Asn Tyr Glu Met Glu Ser Ala Thr Leu Leu Thr Met Cys Ala Ser 195 200 205 Gln Gly Leu Lys Ala Gly Met Ile Ala Gly Val Ile Val Asn Arg Thr 210 215 220 Gln Gln Glu Ile Pro Asn Ala Ala Thr Met Lys Leu Ala Glu Thr Thr 225 230 235 240 Val Ile Lys Val Leu Leu Asp Ala Thr Arg Arg Leu Leu Ala Ala Glu 245 250 255 <210> 8 <211> 239 <212> PRT <213> Unknown <220> <223> NP 2(deoD) AA <400> 8 Met Ala Thr Pro His Ile Asn Ala Glu Met Gly Asp Phe Ala Asp Val 1 5 10 15 Val Leu Met Pro Gly Asp Pro Leu Arg Ala Lys Tyr Ile Ala Glu Thr 20 25 30 Phe Leu Asp Asp Ala Arg Glu Val Asn Asn Val Arg Gly Met Leu Gly 35 40 45 Phe Thr Gly Thr Tyr Lys Gly Arg Lys Ile Ser Val Met Gly His Gly 50 55 60 Met Gly Ile Pro Ser Cys Ser Ile Tyr Ala Lys Glu Leu Ile Thr Glu 65 70 75 80 Phe Gly Val Lys Lys Ile Ile Arg Val Gly Ser Cys Gly Ala Val Arg 85 90 95 Glu Asp Val Lys Leu Arg Asp Val Val Ile Gly Met Gly Ala Cys Thr 100 105 110 Asp Ser Lys Val Asn Arg Met Arg Phe Lys Asp His Asp Tyr Ala Ala 115 120 125 Ile Ala Asp Phe Asp Met Val Arg Asn Ala Val Asp Ala Ala Lys Ala 130 135 140 Arg Asp Val Ser Val Arg Val Gly Asn Ile Phe Ser Ala Asp Leu Phe 145 150 155 160 Tyr Thr Pro Asp Pro Gln Met Phe Asp Val Met Glu Lys Tyr Gly Ile 165 170 175 Leu Gly Val Glu Met Glu Ala Ala Gly Ile Tyr Gly Val Ala Ala Glu 180 185 190 Phe Gly Ala Lys Ala Leu Ala Ile Cys Thr Val Ser Asp His Ile Arg 195 200 205 Thr Gly Ala Gln Thr Thr Ser Glu Glu Arg Gln Asn Thr Phe Asn Glu 210 215 220 Met Ile Glu Ile Ala Leu Glu Ser Val Leu Leu Gly Asp Asn Glu 225 230 235 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-rbsB-UP <400> 9 gctctggtgg tttctacgct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-rbsB-DN <400> 10 ggtcgggttg atcagcagaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-evf-UP <400> 11 attcaagatg tgtggatctg 20 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> ECC-evf- DN <400> 12 agtaagcttg gtaattgtaa tttgcttgg 29 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-expR-UP1 <400> 13 acacacgact acagcttgcc 20 <210> 14 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-expR-DN1 <400> 14 attgtcgccg tgatcgtgaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> ECC-vqsM-UP1 <400> 15 ccgtaaaagc acgcagtgtc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-vqsM-DN1 <400> 16 cgtcaggaaa ggacgcaaac 20 < 210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-purH-UP <400> 17 tgcacgttag aagatgcggt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-purH-DN <400> 18 tgatggtgct gtagtcgctg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-pgm-UP <400> 19 tttgggccgt aagctggtag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-pgm-DN <400> 20 gcactctctt caccaccgaa 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-pgk-UP <400> 21 ctgctgcctg tcgttgacta 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-pgk -DN <400> 22 agtacaacca gctcgccttc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-pyk-UP <400> 23 aactcgtgca gaggtgatgg 20 <210> 24 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-pyk-DN <400> 24 cgcctaaaca cactttcgcc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> ECC-atpE-UP <400> 25 cgctcttggg gtcctctttc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-atpE-DN <400> 26 atcgcgtaca gtgccatcaa 20 < 210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-adk-UP <400> 27 tttcggcctt tgcctctcaa 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-adk-DN <400> 28 agcagaacca gcgtaaggac 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-ndk-UP <400> 29 gctggaaggt gaaaacgctg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-ndk-DN <400> 30 tgaagctgtc cgcgtaatcc 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-carA-UP <400> 31 cgtgaaaaag gcgcacagaa 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-carA -DN <400> 32 ttttgccaga tccatgccct 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-nrdB-UP <400> 33 gcgaactgat ggaaggcaac 20 <210> 34 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-nrdB-DN <400> 34 caatctgcgc catttctggg 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> ECC-udk-UP <400> 35 agcatccgct tcgggtaaaa 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-udk-DN <400> 36 cttccatcgt caggtggctt 20 < 210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-add-UP <400> 37 gcggttcagg gcatagagat 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ECC-add-DN <400> 38 ttgctgtttt tcctgctcgc 20 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ECC-Pevf-F <400> 39 cataatcact cctattgtgg tgg 23 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ECC-Pevf-R <400> 40 gagaggtacc aatagcattc agcg 24 <210> 41 <211> 26 < 212> DNA <213> Artificial Sequence <220> <223> GFP-F2 <400> 41 ctttataagg aggaaaaaca tatgag 26 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GFP- Trrn-R2 <400> 42 ccaattcctg gcagtttatg g 21 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GM-NH-TG-F <400> 43 ggatgaccag aaggacgcct gacg 24 < 210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GM-NH-TG-R <400> 44 gtgtgatgct ccaaagtcat gaccg 25 <210> 45 <211> 24 <212> DNA < 213> Artificial Sequence <220> <223> GM_nupC_start_F <400> 45 gagtcatcgc atatggtcct gctg 24 <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GM_nupC_stop_R <400> 46 ccaccacgca gcatatgatc atcc 24 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAO-NH-TG2-F <400> 47 ctgatcatcg acaccgatcc 20 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PAO-NH-TG-R <400> 48 gagcaggccc tgcgccaact cg 22 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pntp2-F2 <400 > 49 gcaaaaagcg gccgcaaccg 20 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Pntp2-R2<400> 50 ctcatatgtt tttcctcc 18

Claims (19)

Nucleoside hydrolase (NH) inhibitor, represented by Formula 1:
[Formula 1]
,
The R is
, , ,
, and A nucleoside hydrolase inhibitor that is any one selected from the group consisting of.
delete The nucleoside hydrolase inhibitor according to claim 1, wherein the nucleoside hydrolase inhibitor inactivates the quorum sensing signal of pathogens.
A composition for inhibiting pathogenic microbial toxicity, comprising as an active ingredient a nucleoside hydrolase inhibitor represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]
,
The R is
, , ,
, and A composition for inhibiting the toxicity of pathogenic microorganisms, which is any one selected from the group consisting of.
A composition for preventing or treating pathogenic microbial infection, comprising as an active ingredient a nucleoside hydrolase inhibitor represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]
,
The R is
, , ,
, and A composition for preventing or treating pathogenic microbial infection, which is any one selected from the group consisting of.
The composition according to claim 4 or 5, wherein the pathogenic microorganism is a microorganism of the genus Pectobacterium or Pseudomonas .
A composition for controlling plant diseases, comprising as an active ingredient a nucleoside hydrolase inhibitor represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]
,
The R is
, , ,
, and A composition for controlling plant diseases, which is any one selected from the group consisting of.
The composition for controlling plant diseases according to claim 7, wherein the plant disease is caused by microorganisms of the genus Pectobacterium or Pseudomonas .
The method of claim 8, wherein the plant disease is Pectobacterium carotovorum subsp. brasilliense (Pcb), Pectobacterium carotovorum subsp. carotovorum 15 ( Pectobacterium carotovorum subsp. carotovorum15 , Pcc 15), Pectobacterium chrysanthemi (Pch), Pectobacterium atrosepticum , Pectobacterium betavasulorum (Pbe) and Pseudomonas syringae A composition for controlling plant diseases, which is caused by one or more microorganisms selected from the group consisting of ( Pseudomonas syrigae , Pst).
The composition for controlling plant diseases according to claim 7, wherein the composition further comprises an antibiotic.
A method for inhibiting the toxicity of pathogenic microorganisms, comprising the step of treating the composition of claim 4 to an object other than a human.
A method for controlling plant diseases, comprising treating the subject with the composition of claim 7.
delete delete A symbiotic bacterium of a pathogenic microorganism in which at least one gene selected from the group consisting of NH1, NH2, udp and deoD genes has been deleted in the pathogenic microorganism, wherein the pathogenic microorganism is Ecc15 ( Erwinia carotovora subspecies carotovora 15). of commensal bacteria.
The symbiotic bacterium of a pathogenic microorganism according to claim 15, wherein the NH1, NH2, udp and deoD genes consist of base sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.
delete delete A quorum sensing inhibitor of pathogens, comprising a nucleoside hydrolase inhibitor represented by Formula 1:
[Formula 1]
,
The R is
, , ,
, and A quorum sensing inhibitor of pathogens, which is any one selected from the group consisting of.