Expression inhibitor and expression inhibition method for protein involved in bacterial-type fatty acid synthesis, and agent for altering fatty acid composition of organism and method for altering the same

Abstract

To provide a novel composition that enables inhibition of expression of a protein involved in bacterial-type fatty acid synthesis, wherein the inhibition of expression of a protein involved in bacterial-type fatty acid synthesis is achieved, and application of the composition to pathogenic microorganisms enables suppression of their growth or proliferation, thus contributing to prevention or amelioration of infectious diseases or other diseases in which microorganisms are involved in their onset or aggravation.SOLUTION: An expression inhibitor for a protein involved in bacterial-type fatty acid synthesis, comprising agaro-oligosaccharide as an active component.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent and method for inhibiting the expression of proteins involved in bacterial fatty acid synthesis, which use agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having 3,6-anhydro-L-galactose at the reducing end, as well as an agent and method for altering the fatty acid composition of an organism carrying a gene encoding a protein involved in bacterial fatty acid synthesis. [Background technology]

[0002] Fatty acids are metabolized into lipids in the body and play a variety of roles, including forming cell membranes, producing bioactive lipids, post-translational modification of proteins, and serving as an energy source. Fatty acid biosynthesis is carried out by acetyl-CoA carboxylase and fatty acid synthase (FAS).

[0003] Acetyl-CoA carboxylase is an enzyme that catalyzes the formation of malonyl-CoA from acetyl-CoA, the first reaction in fatty acid synthesis. It consists of biotin carboxylase (BC), biotin carboxyl transfer protein (BCCP), and carboxyl transferases (CTα, CTβ), and is rate-limiting in fatty acid synthesis. E. coli-type acetyl-CoA carboxylase exists as four polypeptides (gene names: accA (CTα), accB (BCCP), accC (BC), and accD (CTβ)). In contrast, animal-type acetyl-CoA carboxylase consists of a single polypeptide that contains all of the domains responsible for the above enzymatic functions.

[0004] FAS catalyzes the fatty acid synthesis pathway following the production of acetyl-CoA and malonyl-CoA. FAS can be broadly divided into type I and type II. Type I FAS, found in fungi, yeast, and animals, is a multifunctional enzyme in which multiple catalytic reactions are carried out by a single polypeptide. In contrast, type II FAS, found in bacteria such as Escherichia coli and Bacillus subtilis and plants, is a group of enzymes in which multiple catalytic reactions are carried out by separate enzymes. Type I FAS contains acyl group carrier proteins (ACPs), which act as carriers of fatty acids during this reaction, while type II FASs exist as separate polypeptides. Type II FASs consist of multiple proteins: ACP, malonyltransferase (FabD), β-ketoacyl-ACP synthase I / II / III (FabB, FabF, FabH), β-ketoacyl-ACP reductase (FabG), β-hydroxyacyl-ACP dehydratase (FabZ, FabA), and enoyl-ACP reductase I / II / III (FabI, FabK, FabL).

[0005] Thus, because the proteins involved in bacterial fatty acid synthesis are different from those in animals, compounds that inhibit the activity of these proteins may be useful as antibacterial agents. Therefore, efforts are being made to discover and utilize such compounds; for example, thiolactomycin has been reported as an antibiotic that inhibits FabD and FabF (Non-Patent Document 1), and fasamycins A and B have been reported as antibiotics that inhibit FabF (Non-Patent Document 2). Patent Document 1 also discloses FabK inhibitors as compounds that are useful in treating bacterial infections and have promising pharmaceutical potential.

[0006] On the other hand, as mentioned above, fatty acids are precursors of lipids, which are the main components of cell membranes. Since the production and maintenance of cell membrane function are thought to be directly linked to growth and the maintenance of biological functions, substances that alter the fatty acid composition inherent in bacteria may also be useful as antibacterial agents.

[0007] For example, arachidonic acid is an unsaturated long-chain fatty acid not generally present in bacteria, and it exhibits growth-inhibitory activity against Streptococcus pneumoniae. The mechanism of action is thought to be that arachidonic acid, which has a different length and double bond from the fatty acids conventionally contained in the pneumococcal cell membrane, is preferentially incorporated into the membrane, disrupting membrane functionality. It has also been reported that arachidonic acid is directly incorporated into the pneumococcal cell membrane, altering the fatty acid composition, and significantly reducing the expression of the fab gene cluster (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-091252 [Non-patent literature]

[0009] [Non-Patent Document 1] Akihiko Kawaguchi et al., Mechanism of action of the antibiotic thiolactomycin, Summary of the 1986 Research Report, [online], [Retrieved November 15, 2023], Internet <https: / / kaken.nii.ac.jp / ja / report / KAKENHI-PROJECT-60580127 / 605801271986kenkyu_seika_hokoku_gaiyo / #product_8> [Non-patent document 2] Akihiro Sugawara, A novel metagenomic-derived antibiotic that inhibits type II fatty acid synthase fabF, Pharmacia, p. 889, vol. 48, No. 9, 2012 [Non-patent document 3] Eijkelkamp BA, et al. (2018) Arachidonic Acid Stress impacts Pneumococcal Fatty Acid Homeostasis., Front. Microbiol. 9:813. doi: 10.3389 / fmicb.2018.00813 Summary of the Invention [Problem to be solved by the invention]

[0010] As described above, substances that inhibit the function of proteins involved in bacterial fatty acid synthesis or that alter fatty acid composition may be useful as antibacterial agents. They may also be useful as research reagents for bacterial lipid metabolism. Furthermore, because type II FAS is also present in plants, inhibiting its function or altering its fatty acid composition may potentially suppress plant growth. Therefore, these substances may also be useful as herbicides, for example. Therefore, the present invention aims to provide novel compositions that can inhibit the expression of proteins involved in bacterial fatty acid synthesis and novel compositions that alter the fatty acid composition of organisms that possess genes encoding these proteins.

[0011] As a result of extensive research, the present inventors have found that agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having 3,6-anhydro-L-galactose at the reducing end can suppress the expression of proteins involved in fatty acid synthesis in bacteria, change the fatty acid composition of bacteria, and further suppress bacterial cell count. Based on these findings, the present inventors have completed the following inventions.

[0012] (1) The protein expression inhibitor according to the present invention is an agent for inhibiting the expression of a protein involved in bacterial fatty acid synthesis, and in a first embodiment thereof, contains agarooligosaccharides as active ingredients.

[0013] (2) In the present invention, the protein involved in bacterial fatty acid synthesis may be one or more selected from acetyl-CoA carboxylase, fatty acid synthase, and acyl group transport protein.

[0014] (3) The agent for changing fatty acid composition according to the present invention is an agent for changing the fatty acid composition of an organism having a gene encoding a protein involved in bacterial fatty acid synthesis, and in a first embodiment, the agent contains agarooligosaccharides as an active ingredient.

[0015] (4) The protein expression inhibitor and fatty acid composition altering agent of the present invention (hereinafter, these agents may be collectively referred to as "the agent") may be used as an antibacterial agent.

[0016] (5) In the present invention, the agarooligosaccharide may contain agarobiose.

[0017] (6) A second embodiment of the protein expression inhibitor according to the present invention comprises 3,6-anhydro-L-galactose and / or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end as an active ingredient.

[0018] (7) A second aspect of the agent for changing fatty acid composition according to the present invention contains, as an active ingredient, 3,6-anhydro-L-galactose and / or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end.

[0019] (8) The method of the present invention for inhibiting the expression of a protein involved in bacterial fatty acid synthesis comprises the step of contacting an organism carrying a gene encoding the protein with agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having agarooligosaccharides at their reducing ends.

[0020] (9) The method of the present invention for changing the fatty acid composition is a method for changing the fatty acid composition of an organism having a gene encoding a protein involved in bacterial fatty acid synthesis, and includes a step of contacting the organism with agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having agarooligosaccharides at their reducing ends.

[0021] In the present invention, the method for inhibiting protein expression and the method for changing fatty acid composition may be excluding medical procedures. [Effects of the Invention]

[0022] According to the present invention, it is possible to suppress the expression of a protein involved in bacterial fatty acid synthesis. It is also possible to change the fatty acid composition of an organism that possesses a gene encoding this protein. Therefore, when applied to pathogenic microorganisms, this can suppress their growth and proliferation, thereby contributing to the prevention and amelioration of diseases such as infectious diseases in which microorganisms are involved in the onset or worsening of the disease. Furthermore, when used as a research reagent, it can contribute to research on lipid metabolism, cell membrane function, and the like. Furthermore, when applied to areas where plant growth is undesirable, it can suppress the growth of weeds and the like, contributing to the management of living environments and crops. [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a volcano plot of gene expression ratios in Ruminococcus gnavus cultured in the presence / absence of agaro-oligosaccharides. [Figure 2] 1 is a table showing predicted functions of genes whose expression ratios were significantly reduced in the presence of agarooligosaccharides. [Figure 3] 1 is a bar graph showing the expression levels (relative expression ratios measured by quantitative PCR) of the fabF gene and the fabH gene in Ruminococcus gnavus and Fusobacterium nucleatum cultured in the presence (AOS medium) or absence (free medium) of agarooligosaccharides. [Figure 4] 1 is a bar graph showing the content of various fatty acids in Ruminococcus gnavus cultured in the presence (AOS medium) or absence (free medium) of agarooligosaccharides. [Figure 5] 1 is a bar graph showing the absorbance (OD660) of the culture medium in which Ruminococcus gnavus was cultured at different concentrations of agarooligosaccharides. [Figure 6] 1 is a bar graph showing the absorbance (OD660) of the culture medium in which Ruminococcus gnavus was cultured in the presence of agarooligosaccharides and disaccharides to octasaccharides. [Figure 7] 1 is a bar graph showing the absorbance (OD660) of the culture medium in which Fusobacterium nucleatum was cultured at varying concentrations of agarooligosaccharides. [Figure 8] 1 is a bar graph showing the absorbance (OD660) of the culture medium in which Fusobacterium nucleatum was cultured in the presence of agarooligosaccharides, disaccharides, or tetrasaccharides. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be further described below.

[0025] The term "protein involved in bacterial fatty acid synthesis" (sometimes abbreviated as "this protein" in the present invention) refers to a bacterial type of a series of proteins involved in fatty acid synthesis in vivo. Here, the bacterial type refers to a type possessed by bacteria such as Escherichia coli (with some exceptions such as coryneform bacteria).

[0026] Specific examples of the present protein include the proteins shown in Table 1 below. [Table 1]

[0027] The function of this protein in fatty acid synthesis is illustrated below. First, malonyl-CoA is synthesized by acetyl-CoA carboxylase. Malonyl-CoA is then converted to malonyl-ACP by malonyltransferase via conjugation with ACP. The following four steps proceed sequentially to form a cycle, and long-chain saturated fatty acids are biosynthesized through multiple cycles. The first step of the cycle is a condensation reaction catalyzed by β-ketoacyl-ACP synthase. In the first cycle, β-ketoacyl-ACP synthase III condenses malonyl-ACP with acetyl-CoA. In subsequent cycles, β-ketoacyl-ACP synthase I or II condenses malonyl-ACP with acyl-ACP. In the second step, β-ketoacyl-ACP reductase reduces β-ketoacyl-ACP. In the third step, β-hydroxyacyl-ACP is dehydrated by β-hydroxyacyl-ACP dehydratase to produce trans-2-enoyl-ACP. In the fourth step, it is reduced by enoyl-ACP reductase to give acyl-ACP, adding two carbon atoms per cycle to finally give palmitoyl-ACP (16C).

[0028] "Suppressing the expression of the present protein" means reducing the transcription level of the gene encoding the present protein, reducing the amount of the present protein, or reducing the activity of the present protein in a living organism.

[0029] In the Examples described below, inhibition of expression of the present protein by agarooligosaccharides was confirmed in Ruminococcus gnavus and Fusobacterium nucleatum, and Table 1 shows examples of the DNA sequences of the genes encoding the present protein in these bacterial species. However, as shown in the Examples, agarooligosaccharides inhibit expression of the present protein in organisms of different genera, even when the sequence identity of the gene is relatively low, at about 42%. This indicates that agarooligosaccharides can inhibit expression of the present protein in a wide range of organisms, not just in the genera Ruminococcus and Fusobacterium.

[0030] Examples of organisms that carry the gene encoding the present protein include bacteria and plants.

[0031] "Fatty acid composition of an organism" refers to the types of fatty acids and / or the amount or content ratio of each fatty acid (the ratio of the amount of each fatty acid to the total amount of fatty acids) contained in the whole organism or parts such as the cell membrane.

[0032] "Changing the fatty acid composition" means that when the active ingredients of the present invention (agarooligosaccharides, 3,6-anhydro-L-galactose and / or oligosaccharides having 3,6-anhydro-L-galactose at the reducing end) are used, the fatty acid composition is made different from that when these ingredients are not used.

[0033] An "antibacterial agent" refers to a composition that has the activity of inhibiting the growth and proliferation (bacterial count) of microorganisms (antibacterial activity). This agent exerts its antibacterial activity by suppressing the expression of this protein or by changing the fatty acid composition of an organism that possesses the gene encoding this protein. In other words, this agent can exert its antibacterial activity against bacteria as well as microorganisms that possess proteins involved in bacterial fatty acid synthesis or their genes. Therefore, this agent can be used as an antibacterial agent.

[0034] The present invention uses agarooligosaccharides as active ingredients. Agarooligosaccharides are oligosaccharides having 3,6-anhydro-L-galactose at the reducing end. Therefore, the active ingredient of the present invention may be 3,6-anhydro-L-galactose or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end. In this case, the number of sugars in the oligosaccharide may be, for example, 2 to 8 sugars, 2 to 10 sugars, or 2 to 12 sugars.

[0035] Agarooligosaccharides are even-numbered oligosaccharides consisting of repeating units of agarobiose, a disaccharide composed of D-galactose and 3,6-anhydro-L-galactose. Examples of agarooligosaccharides include the smallest unit, agarobiose (disaccharide), agarotetraose (tetrasaccharide), agarohexaose (hexasaccharide), agarooctaose (octasaccharide), and agarodecaose (decasaccharide). In the present invention, agarooligosaccharides contain at least one of these oligosaccharides, and may consist of one type or two or more types.

[0036] As shown in the Examples below, among agarooligosaccharides, agarobiose and agarotetraose exhibit particularly high growth inhibitory effects against bacteria. Therefore, it is preferable that the agarooligosaccharide contains agarobiose. In this case, the agarooligosaccharide may consist solely of agarobiose, or may contain agarooligosaccharides other than agarobiose. In this case, the agarobiose content in the agarooligosaccharide may be, for example, 1 to 100% by mass, 10 to 100% by mass, 20 to 100% by mass, 30 to 100% by mass, 40 to 100% by mass, or 50 to 100% by mass.

[0037] Agaroligosaccharides can be commercially available agarooligosaccharides (agar oligosaccharides), or can be produced by conventional methods. A typical method for producing agarooligosaccharides is, for example, a method of hydrolyzing agar. Hydrolysis can be performed using either an acid or an enzyme.

[0038] Examples of acid decomposition methods include those using solid acids as described in Japanese Patent No. 4796697, mineral acids such as sulfuric acid and hydrochloric acid, and organic acids such as acetic acid and citric acid, but any method can be used. Acid decomposition can produce an even-numbered sugar having 3,6-anhydro-L-galactopyranose at the reducing end.

[0039] Enzymatic degradation methods include degradation with α-agarase and degradation with β-agarase. As with acid degradation, α-agarase can be used to obtain even-numbered sugars having 3,6-anhydro-L-galactopyranose at the reducing end. Degradation with α-agarase can be carried out, for example, by the method described in Japanese Patent Application Publication No. H2-65789.

[0040] The agar hydrolysate may be used as agarooligosaccharides directly, or may be purified or pH-adjusted before use. Purification methods include filtration using filter paper or activated carbon. The agarooligosaccharide solution obtained by hydrolysis may be used in liquid form, or, if necessary, may be powdered by vacuum freeze-drying or other methods.

[0041] Agar is a dehydrated and dried mucilage extracted from red seaweeds such as Gelidium and Gracilaria, and contains the polysaccharides agarose and agaropectin as its main components. In addition to agar, substances containing agarose and agaropectin can also be used as raw materials for producing agarooligosaccharides. Specific examples of such substances include solutions obtained by hot water extraction of red algae from the Gelidaceae, Gracilaria, and Gracilaria families, which are the raw materials for agar. Examples of red algae from the Gelidaceae family include Acanthus nigricans, Acanthus nigricans, Acanthus nigricans, Acanthus obscurus, and Acanthus japonica. Examples of red algae from the Gracilaria family include Gracilaria gracilaria and Gracilaria sieboldii. Examples of red algae from the Gracilaria family include Acanthus gistus and Acanthus sieboldii. These red algae can be used alone or in combination of two or more.

[0042] The sugar composition of agarooligosaccharides can be confirmed by liquid chromatography, including high performance liquid chromatography, as shown in the Examples below. This allows agarooligosaccharides with a desired number of sugars, such as agarobiose only, agarotetraose only, or agarohexaose only, to be fractionated and used after adjusting the sugar composition of the agarooligosaccharides.

[0043] An example of the sugar composition confirmed by HPLC for agaro-oligosaccharides prepared by decomposing agar with concentrated sulfuric acid is shown below (Shirai I, Sakai T, Shiba K, Uzuhashi Y, Karasawa K. Agaro-oligosaccharides prevent myostatin hyperexpression and myosin heavy chain protein degradation in C2C12 myotubes induced by tumor necrosis factor-α. Cell Bio. 2018;7(2):23-34.). Disaccharide (agarobiose): 41.8 Tetrasaccharide (agarotetraose): 41.0 Hexasaccharide (agarohexaose): 14.5 8 sugars (agarooctaose): 2.7

[0044] 3,6-Anhydro-L-galactose can be prepared using commercially available reagents or by standard methods. Examples of such methods include the method described in Japanese Patent No. 4007760. Specifically, 450 μL of a 100 mM aqueous solution of agarobiose was mixed with 50 μL of 10x phosphate-buffered saline and 50 μL of 10 units / μL β-galactosidase phosphate-buffered saline, and the resulting mixture was allowed to react at 37°C for 1 hour. 5 mL of a 1:1 mixture of 1-butanol and ethanol was added to the reaction mixture, and the mixture was centrifuged to precipitate insoluble matter. The resulting supernatant was subjected to column chromatography using a silica gel column, and the mixture was compressed at 0.3 kg / cm using a compressor with a 5:5:1 mixture of 1-butanol, ethanol, and water as the eluent. 2 The mixture is pressurized to 100°C and separated. By separating the fractions so that each fraction is 7 ml, a liquid containing highly purified 3,6-anhydro-L-galactose can be obtained, for example, in fractions 14 to 17. These fractions can be collected and evaporated to dryness under reduced pressure to obtain 3,6-anhydro-L-galactose.

[0045] The active ingredients of the present invention (agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having 3,6-anhydro-L-galactose at the reducing end) can be used in a form that allows them to come into direct or indirect contact with organisms carrying the gene encoding the present protein. That is, examples of usage modes include adding, pasting, applying, spraying, etc., so that the active ingredients reach areas where organisms carrying the gene encoding the present protein are thought to live.

[0046] More specific modes of use can be appropriately determined depending on the subject of use and the purpose of use. For example, if used as a research reagent, the active ingredient may be added to the culture medium of the organism. Furthermore, if used to inhibit plant growth, the active ingredient may be sprayed or the like on the area where the plant grows. Furthermore, if used to prevent bacterial infection on the body surface of a human or animal, the active ingredient may be applied, coated, sprayed, or the like to the relevant area on the body surface. Furthermore, if used to prevent bacterial infection or inhibit the growth of harmful bacteria in the body of a human or animal, the active ingredient may be administered orally or via a tube to the human or animal. Furthermore, if used to maintain the hygienic condition of industrial products such as daily necessities or food, the active ingredient may be applied, sprayed, or the like to the surface of the product.

[0047] The active ingredient of the present invention may be used as it is in the form of a pharmaceutical product, a quasi-drug, a reagent, other medicine, food or drink, a supplement, etc., or may be used as a raw material for these products by blending it with other ingredients. These products can be produced by methods known to those skilled in the art using the active ingredient as a raw material.

[0048] The content of the active ingredient of the present invention in the product can be appropriately set depending on the form and use of the product. Specific examples of the content include 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 35% by mass or less, 30% by mass or less, 10% by mass or less, and 5% by mass or less.

[0049] The dosage (intake) of the active ingredient of the present invention when applied to living bodies such as humans and animals can also be appropriately determined depending on the subject of administration, the form of the product, and the purpose. Specific examples of dosages for adults include 0.0125 mg / kg body weight or more, 0.025 mg / kg body weight or more, 0.05 mg / kg body weight or more, 0.1 mg / kg body weight or more, 1000 mg / kg body weight or less, 800 mg / kg body weight or less, 600 mg / kg body weight or less, 400 mg / kg body weight or less, and 200 mg / kg body weight or less per day.

[0050] The present invention will be described below based on examples, but the technical scope of the present invention is not limited to the features shown in these examples. [Example]

[0051] <Test Method> (1) Preparation of agarooligosaccharides 50 g of agar ("Ultra Agar AX-30" manufactured by Ina Food Industry Co., Ltd.) was added to 1000 g of purified water and heated to dissolve, after which 2 g of concentrated sulfuric acid was added and stirred at 90°C for 3 hours. The pH was adjusted to 3.5 with sodium hydroxide, and the mixture was treated with activated carbon. The filtrate was then filtered through a filter paper and the filtrate was collected. This was then further filtered through a filter with a pore size of 0.1 μm, and the filtrate was powdered by vacuum freeze-drying to obtain agarooligosaccharide powder.

[0052] (2) Preparation of disaccharides to octasaccharides The agarooligosaccharides prepared by test method (1) were subjected to recycle size exclusion chromatography to separate fractions containing disaccharides, tetrasaccharides, hexasaccharides, and octasaccharides. Recycle size exclusion chromatography was performed under the following conditions. <Conditions for recycling size exclusion chromatography> System: LaboACE LC-7080 Plus (Japan Analytical Industry) Column: JAIGEL-W252 / W253 (Japan Analytical Industry) Mobile phase: Aqueous solution containing 0.005% (v / v) acetic acid and 10% (v / v) ethanol Flow rate: 3.5mL / min

[0053] The components contained in each fraction were confirmed using high-performance liquid chromatography (Prominence® HPLC system (Shimadzu Corporation)). The HPLC measurement conditions were as follows: two columns (TSKgel® α-2500, Tosoh Corporation) connected in series, elution with H2O as solvent, a flow rate of 0.3 ml / min, and a temperature of 60°C, and detection was by RI (differential refractive index).

[0054] Each fraction was dried to obtain agarobiose, agarotetraose, agarohexaose, and agarooctaose. In this example, agarobiose, agarotetraose, agarohexaose, and agarooctaose may be referred to as "disaccharide," "tetrasaccharide," "hexasaccharide," and "octasaccharide," respectively.

[0055] (3) Strains For the genus Ruminococcus, Ruminococcus gnavus JCM6515, the type strain of Ruminococcus gnavus, was used. For the genus Fusobacterium, Fusobacterium nucleatum JCM8532, the type strain of Fusobacterium nucleatum, was used. Both strains were obtained from the Microbial Materials Development Laboratory, BioResource Research Center, RIKEN (JCM). Both strains were cultured anaerobically using the anaerobic culture kit "Anelopack" (Mitsubishi Gas Chemical).

[0056] (4) Culture medium The culture medium was 1 L of Brain-Heart infusion medium (*1) supplemented with 5 g of yeast extract, 5 g of K2HPO4, 8 g of glucose, 0.5 g of L-cysteine ​​hydrochloride, 1 g of Tween 80, 0.005 g of hemin, 0.002 g of vitamin K1, 0.001 g of resazurin sodium salt, 0.025 g of acetic acid, and 0.01 g of MgSO2·7H2O. *1 Composition of Brain-Heart infusion medium (per 1L): Brain-heart infusion, 5g / L yeast extract, 5g K2HPO4, 8g glucose, 0.5g L-cysteine ​​hydrochloride, 1g Tween 80, 0.005g hemin, 0.002g vitamin K1, 1mg resazurin, 50mL salt solution (*2). *2 Composition of salt solution (per liter): 5g sodium acetate, 2g ammonium citrate, 0.2g MgSO2·7H2O, 0.05g MnSO4·H2O, pH 6.8.

[0057] (5) Statistical analysis The results of the bacterial counts measured by the turbidimetric method and the quantitative PCR results were analyzed using the Kruskal-Wallis test and unpaired t-test, respectively, using the statistical analysis software GraphPad Prism version 9.5.1 (GraphPad Software). In this test, a P value of <0.05 was considered significant and is indicated by an *.

[0058] Example 1: Inhibitory effect on expression of proteins involved in fatty acid synthesis: Comprehensive analysis of gene expression levels (1) Cultivation of Ruminococcus gnavus Culture medium containing 0.1% (w / w) glucose was designated "free medium." Culture medium containing 0.1% (w / w) glucose and 0.1% (w / w) agarooligosaccharides was designated "AOS medium." Ruminococcus gnavus was inoculated into 3.0 mL of AOS medium or free medium and anaerobically cultured at 37°C. This was used as the seed broth. 100 μL of the seed broth prepared in AOS medium was added to 3.0 mL of fresh AOS medium and cultured under the same conditions for 72 hours (final optical density (OD660) = 0.04). 10 μL of the seed broth prepared in free medium was added to 3.0 mL of fresh free medium and cultured under the same conditions for 48 hours (final optical density (OD660) = 0.21).

[0059] (2) Total RNA extraction and RNA sequencing (RNA-seq) The culture medium from Example 1(1) was cooled on ice for 10 minutes and then centrifuged at 6,000 rpm at 4°C for 5 minutes to collect the bacterial cells. After adding 0.6 mL of RNA preservation solution (RNA later®, Thermo Fisher Scientific) to the precipitate (bacterial cells), total RNA was extracted from the bacterial cells as previously reported (Fujii T, et al. Ribotype-dependent growth inhibition and promotion by erythritol in Cutibacterium acnes. J Cosmet Dermatol. 2022;21(10):5049-57; doi: 10.1111 / jocd.14958.). RNA sequencing was performed on the resulting total RNA to obtain lead information.

[0060] The resulting read information was analyzed using the analysis tool iDEP (integrated Differential Expression and Pathway analysis).96 (http: / / bioinformatics.sdstate.edu / idep96 / ) to calculate the ratio (expression ratio) of the expression level of each gene in the culture medium containing AOS to that in the culture medium containing no AOS, and volcano plots were created. The results are shown in Figure 1.

[0061] As shown in the circled boxes in Figure 1, it was revealed that there were several genes whose expression ratios were significantly reduced in the culture medium grown on AOS medium. The functions of these gene products were predicted using PANNZER2 (http: / / ekhidna2.biocenter.helsinki.fi / sanspanz / ). The results are shown in Figure 2.

[0062] As shown in Figure 2, the 10 genes whose expression ratios were significantly decreased were those encoding acyl transfer protein (acpP), biotin carboxyl transfer protein (accB), biotin carboxylase (accC), enoyl-ACP reductase (fabK), β-ketoacyl-ACP reductase (fabG), malonyltransferase (fabD), β-hydroxyacyl-ACP dehydratase (fabZ), β-ketoacyl-ACP synthase II (fabF), carboxyltransferase (accD), and β-ketoacyl-ACP synthase III (fabH). All of these genes encode proteins involved in fatty acid synthesis. In other words, the expression levels of genes encoding a series of proteins involved in fatty acid synthesis were significantly reduced in Ruminococcus gnavus cultured in the presence of agarooligosaccharides. These results demonstrate that agarooligosaccharides can suppress the expression of proteins involved in fatty acid synthesis.

[0063] Example 2: Inhibitory effect on fab gene expression: quantitative PCR In the presence of agarooligosaccharides, the expression levels of genes encoding proteins involved in fatty acid synthesis were reduced, and the expression levels of the fabF and fabH genes were quantitatively confirmed in Ruminococcus gnavus and Fusobacterium nucleatum. The sequence identity between the fabF gene of Ruminococcus gnavus and that of Fusobacterium nucleatum is 42.7% (566 / 1325). The sequence identity between the fabH gene of Ruminococcus gnavus and that of Fusobacterium nucleatum is 59.8% (76 / 127).

[0064] (1) Cultivation of Ruminococcus and Fusobacterium Ruminococcus gnavus and Fusobacterium nucleatum were cultured by the method described in Example 1(1). However, for the main culture of Fusobacterium nucleatum, 80 μL of seed mother liquor was added to the AOS medium and cultured for 54 hours (final absorbance (OD660) = 0.11), and for the non-AOS medium, 10 μL of seed mother liquor was added and cultured for 24 hours (final absorbance (OD660) = 0.125).

[0065] (2) Total RNA extraction and reverse transcription Total RNA was extracted from the culture medium in Example 2(1) as previously reported (Panchal VV, et al. Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureus is associated with RNA polymerase alterations and fine tuning of gene expression. PLoS Pathog. 2020;16(7):e1008672; doi: 10.1371 / journal.ppat.1008672.). Using this as a template, reverse transcription was performed using the commercially available kit "The Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics)" and random primers to obtain complementary DNA (cDNA).

[0066] (3) Quantitative PCR Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) was used to design specific primers capable of amplifying the fabF gene (SEQ ID NO: 6) and fabH gene (SEQ ID NO: 7) of Ruminococcus gnavus and their homologs in Fusobacterium nucleatum (SEQ ID NO: 11, SEQ ID NO: 12). Additionally, as reference genes for correcting the amount of template RNA, the gyrB gene (encoding the β subunit of DNA gyrase) of Ruminococcus gnavus and the 16S rRNA gene of Fusobacterium nucleatum were designed, and specific primers capable of amplifying these genes were also prepared. The sequences of the specific primers are shown in Table 2. [Table 2]

[0067] Real-time quantitative PCR was performed using the cDNA from Example 2(2) as a template and the primers listed in Table 2. PCR reaction and detection were performed using a "QuantStudio 3" (Thermo Fisher Scientific). The reaction mixture was prepared using the "PowerTrack SYBR Green Master Mix" (Thermo Fisher Scientific) reagent according to the accompanying instructions. The reaction conditions were an initial 2 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C, 15 seconds at an annealing temperature (55°C or 58°C), and 15 seconds at 72°C, followed by 1 minute at 72°C. After the reaction, melting curve analysis was performed to confirm the specificity of the amplification reaction using each primer. The expression levels of fabF and fabH were comparatively quantified using the ΔΔCt method. The expression level in the culture medium containing AOS was calculated as a ratio (relative expression ratio) of the expression level in the culture medium containing no fabF and fabH. The results are shown in Figure 3.

[0068] As shown in Figure 3, the relative expression ratios of Ruminococcus gnavus culture medium grown on AOS medium were 0.03667 for fabF and 0.06667 for fabH, both significantly lower than 1. The relative expression ratios of Fusobacterium nucleatum culture medium grown on AOS medium were also 0.07333 for fabF and 0.07333 for fabH, both significantly lower than 1. In other words, the expression levels of the fabF and fabH genes in these bacteria were significantly reduced when cultured in the presence of agarooligosaccharides. These results demonstrate that agarooligosaccharides can suppress the expression of proteins involved in fatty acid synthesis.

[0069] Example 3: Effect of varying fatty acid composition Since it was revealed that agarooligosaccharides suppress the expression of proteins involved in fatty acid synthesis, the fatty acid composition of Ruminococcus gnavus cultured in the presence of agarooligosaccharides was examined.

[0070] Ruminococcus gnavus was cultured in AOS medium and free medium according to the method described in Example 1(1), and the cells were collected by centrifugation and lyophilized. 7±2 mg of lyophilized cells were placed in a 4 mL vial to prepare a sample. The fatty acids in the sample were methyl-esterified using a fatty acid methylation kit (product number 06482-04, Nacalai Tesque), and the methylated fatty acids were purified using a methylated fatty acid purification kit (product number 06483-94, Nacalai Tesque). One mL of the purified methylated fatty acids was transferred to a new vial and subjected to gas chromatography-mass spectrometry (GC / MS) under the conditions shown in Table 3 to measure the type and amount of fatty acids. The fatty acid content in the sample was expressed as a percentage, with the total amount of fatty acids being 100% (w / w). The results are shown in Figure 4. [Table 3]

[0071] As shown in Figure 4, the myristic acid (C14:0) content was 19.5% in the culture medium without myristic acid, while it was 7.2% in the AOS medium. Similarly, the palmitic acid (C16:0) content was 42.8% in the culture medium without myristic acid, while it was 23.6% in the AOS medium. In other words, the content of medium-chain saturated fatty acids was significantly reduced in the culture medium grown in AOS medium.

[0072] On the other hand, caprylic acid (C8:0) was 0.1% in the absence medium and 0.4% in the AOS medium. Capric acid (C10:0) was 4.1% in the absence medium and 13.3% in the AOS medium. Lauric acid (C12:0) was 7.0% in the absence medium and 13.1% in the AOS medium. Stearic acid (C18:0) was 4.4% in the absence medium and 10.6% in the AOS medium. Oleic acid (C18:1) was 18.2% in the absence medium and 26.8% in the AOS medium. Thus, the contents of relatively short-chain saturated fatty acids and long-chain fatty acids were significantly increased in the culture medium grown on AOS medium.

[0073] These results revealed that agarooligosaccharides alter the fatty acid composition of bacteria.

[0074] Example 4: Growth inhibitory effect of Ruminococcus gnavus Since it was revealed that agarooligosaccharides suppress the expression of proteins involved in fatty acid synthesis and alter the fatty acid composition of the body, we examined the growth of Ruminococcus gnavus cultured in the presence of agarooligosaccharides. While Ruminococcus gnavus is found in extremely low numbers in the intestines of healthy individuals, its abundance has been reported to increase in the intestines of patients with inflammatory bowel disease, heart failure, coronary artery disease, and other diseases. It has also been reported that it produces inflammatory polysaccharides and induces the secretion of inflammatory cytokines from host dendritic cells, making it an undesirable bacterium, at least for humans.

[0075] (1) Cultivation in the presence of agarooligosaccharides Ruminococcus gnavus was inoculated into the culture medium and cultured anaerobically at 37°C for 37 hours. This was used as the seed broth. Agarose solutions were prepared by adding agarooligosaccharides to a 20% (w / w) aqueous glucose solution to final concentrations of 0, 0.1, and 0.2% by mass. The culture medium was dispensed into deep well plates (AxyGen Scientific, CA, USA) at 470 μL per well, and 2.5 μL of the sugar solution was dispensed into each well. Then, 25 μL of the seed broth was inoculated into each well and cultured for 23 hours under the same conditions (final concentrations of agarooligosaccharides in the culture medium for the main culture were 0, 0.1, and 0.2% by mass).

[0076] (2) Measurement of bacterial count by turbidity method 20 μL of the culture medium from the main culture was taken and diluted 10-fold by adding 180 μL of water. The absorbance (OD660) of the diluted culture medium was measured using a microplate reader (Wako SUNRISE Rainbow) (N=8). The results are shown in Figure 5.

[0077] As shown in Figure 5, the absorbance (OD660) was 1.27 at an agarooligosaccharide concentration of 0% by mass, but 0.77 and 0.09 at 0.1% and 0.2% by mass, respectively. The higher the final concentration of agarooligosaccharide in the medium, the smaller the absorbance (OD660). In other words, agarooligosaccharides suppressed the number of Ruminococcus gnavus bacteria in the medium in a concentration-dependent manner. These results demonstrated that agarooligosaccharides can suppress the growth of Ruminococcus spp.

[0078] (3) Cultivation in the presence of disaccharides to octasaccharides Ruminococcus gnavus was cultured by the method described in Example 4(1) except that agarooligosaccharides were replaced with disaccharides, tetrasaccharides, hexasaccharides, or octasaccharides, and the number of bacteria was measured by the turbidity method described in Example 4(2) (N=2). The concentration of disaccharides to octasaccharides in the medium was 0.1% by mass. The results are shown in Figure 6.

[0079] As shown in Figure 6, the absorbance (OD660) was 0.03, 0.04, 0.02, and 0.06 when the disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide were added to the medium, respectively. These values ​​were significantly lower than the 1.37 obtained when the medium did not contain these sugars (glucose only). These results demonstrated that agarobiose, agarotetraose, agarohexaose, and agarooctaose can inhibit the growth of Ruminococcus. Furthermore, because all of these oligosaccharides contain 3,6-anhydro-L-galactose at their reducing ends, it was also demonstrated that 3,6-anhydro-L-galactose or oligosaccharides containing 3,6-anhydro-L-galactose at their reducing ends can inhibit the growth of Ruminococcus.

[0080] Example 5: Growth inhibitory effect on Fusobacterium nucleatum Since it was revealed that agarooligosaccharides suppress the expression of genes involved in fatty acid synthesis and change the fatty acid composition of the body, the growth rate of Fusobacterium nucleatum cultured in the presence of agarooligosaccharides was examined. Fusobacterium nucleatum is a pathogenic bacterium that is known to cause periodontal disease and has also been shown to be involved in the progression of colon cancer.

[0081] (1) Cultivation in the presence of agarooligosaccharides Ruminococcus gnavus was cultured in place of Fusobacterium nucleatum by the method described in Example 4(1), and the absorbance of the culture solution was measured by the turbidity method described in Example 4(2) (N=8). However, the concentration of agarooligosaccharides in the medium was 0.2% by mass, and the culture time for the main culture was 9 hours. The results are shown in Figure 7.

[0082] 7, the absorbance (OD660) was 0.96 when the agarooligosaccharide concentration in the medium was 0% by mass, while it was 0.40 when the agarooligosaccharide concentration was 0.2% by mass, which was a lower value when the medium contained agarooligosaccharides. These results demonstrate that agarooligosaccharides can inhibit the growth of Fusobacterium.

[0083] (2) Cultivation in the presence of disaccharides and tetrasaccharides Ruminococcus gnavus was replaced with Fusobacterium nucleatum, and agarooligosaccharides were replaced with disaccharides or tetrasaccharides. Fusobacterium nucleatum was cultured by the method described in Example 4(1), and the number of bacteria was measured by the turbidity method described in Example 4(2) (N=2). The concentration of the disaccharide and tetrasaccharide in the medium was 0.1% by mass. The results are shown in Figure 8.

[0084] As shown in Figure 8, the absorbance (OD660) was significantly lower when the medium contained a disaccharide and a tetrasaccharide, 0.04 and 0.27, respectively, compared to 1.23 when the medium did not contain these sugars (glucose only). In particular, agarobiose had the lowest absorbance (OD660). These results demonstrated that agarobiose and agarotetraose can inhibit the growth of Fusobacterium. Furthermore, because both of these oligosaccharides contain 3,6-anhydro-L-galactose at their reducing ends, it was also demonstrated that 3,6-anhydro-L-galactose or oligosaccharides containing it at their reducing ends can inhibit the growth of Fusobacterium.

Claims

1. An inhibitor of the expression of proteins involved in bacterial fatty acid synthesis, containing agarooligosaccharides as the active ingredient.

2. The agent according to claim 1, wherein the protein is one or more selected from acetyl-CoA carboxylase, fatty acid synthase, and acyl group transport protein.

3. An agent containing agarooligosaccharide as an active ingredient that alters the fatty acid composition of an organism that possesses a gene encoding a protein involved in bacterial fatty acid synthesis.

4. The agent according to any one of claims 1 to 3, which is used as an antibacterial agent.

5. The agent according to any one of claims 1 to 3, wherein the agarooligosaccharide is an agarooligosaccharide containing agarobiose.

6. An inhibitor of the expression of a protein involved in bacterial fatty acid synthesis, which comprises as an active ingredient 3,6-anhydro-L-galactose or an oligosaccharide having 3,6-anhydro-L-galactose at its reducing end.

7. An agent for altering the fatty acid composition of an organism having a gene encoding a protein involved in bacterial fatty acid synthesis, the agent comprising, as an active ingredient, 3,6-anhydro-L-galactose or an oligosaccharide having 3,6-anhydro-L-galactose at its reducing end.

8. A method for inhibiting the expression of a protein involved in bacterial fatty acid synthesis, the method comprising the step of contacting an agarooligosaccharide with an organism carrying a gene encoding said protein.

9. A method for altering the fatty acid composition of an organism that possesses a gene encoding a protein involved in bacterial-type fatty acid synthesis, the method comprising the step of contacting the organism with agarooligosaccharides.

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