Method and kit for detecting membrane vesicle-producing bacteria

Abstract

To provide a method for detecting membrane vesicle-producing bacteria.SOLUTION: The present invention provides a method for detecting membrane vesicle-producing bacteria, the method including the steps of: bringing the membrane vesicle-producing bacteria into contact with a fluorescent probe that selectively labels a high-curvature lipid bilayer; and measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to methods and kits for detecting membrane vesicle-producing bacteria. [Background technology]

[0002] "Membrane vesicles" produced by bacteria outside the cells are biological microparticles with a diameter of 20-400 nm. It is known that various prokaryotes such as gram-negative bacteria, gram-positive bacteria, and archaea release membrane vesicles outside the cells (Non-Patent Document 1). For example, outer membrane vesicles released by gram-negative bacteria contain various substances derived from bacteria, such as DNA, RNA, lipopolysaccharide, proteins, and phospholipids. Bacterial membrane vesicles have the function of delivering immune activating substances of the bacteria of origin to host cells and activating immune responses. Bacterial membrane vesicles also function as a medium for information transmission substances between microorganisms.

[0003] In recent years, membrane vesicles have been attracting attention for their potential applications in biotechnology. For example, research aimed at a wide range of applications has been actively conducted, such as the medical application of bacterial membrane vesicles as a medium for vaccines or drug delivery systems (Non-Patent Document 2), the control of gene expression related to bacterial pathogenicity by intercellular signal transduction (Non-Patent Document 3), and the control of biofilm formation (Non-Patent Document 4).

[0004] The present inventors have developed a fluorescent probe that selectively labels lipid bilayers with high curvature (Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Brooke L Deatherage and Brad TCookson, "Membrane vesicle release in bacteria, eukaryotes, and archaea: aconserved yet underappreciated aspect of microbial life", Infect Immun. 80(6):1948-57(2012). [Non-Patent Document 2] Qiong Long et al., "Engineeredbacterial membrane vesicles are promising carriers for vaccine design and tumorimmunotherapy", Adv. Drug Deliv. Rev. 186:114321 (2022). [Non-Patent Document 3] Lauren M. Mashburn and MarvinWhiteley, "Membrane vesicles traffic signals and facilitate groupactivities in a prokaryote", Nature 437, 422-425 (2005). [Non-Patent Document 4] Jonathan P. Remis et al.,"Biofilms: structures that may facilitate cell-cell interactions", TheISME Journal 4, 1085-1087 (2010). [Non-Patent Document 5] Yusuke Sato et al., "Amphipathichelical peptide-based fluorogenic probes for a marker-free analysis of exosomesbased on membrane-curvature sensing", RSC Adv. 10, 38323-38327 (2020). [Non-Patent Document 6] Guillaume Drin and Bruno Antonny, "Amphipathic helices and membrane curvature", FEBS Letters584(9), 1840-1847 (2010). [Non-Patent Document 7] Manuel Gimenez-Andres et al., "The Many Faces of Amphipathic Helices", Biomolecules 8(3): 45(2018) [Non-Patent Document 8] Jensen MB et al., "Membranecurvature sensing by amphipathic helices: a single liposome study using α-synucleinand annexin B12", The Journal of Biological Chemistry 286(49):42603-42614(2011). [Non-Patent Document 9] Agnieszka Martyna et al., "Membraneremodeling by the M2 amphipathic helix drives influenza virus membrane scission", Scientific Reports 7, 44695 (2017). [Non-Patent Document 10] Kenichi Kawano et al., "Development of a Membrane Curvature-Sensing Peptide Based on a Structure-ActivityCorrelation Study", Chem. Pharm. Bull. 67(10), 1131-1138 (2019). [Non-Patent Document 11] Masafumi Tanaka et al., "Effect of amino acid distribution of amphipathic helical peptide derived from human apolipoprotein AI on membrane curvature sensing", FEBS Letters587(5), 510-515 (2013). [Non-Patent Document 12] Kotaro Takaki et al., "Multilamellarand Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestistolB Mutant", Appl. Environ. Microbiol. 86:e01131-20 (2020). Summary of the Invention [Problem to be solved by the invention]

[0006] In order to apply bacterial membrane vesicles to biotechnology, a technique for detecting membrane vesicle-producing bacteria is essential for mass production of membrane vesicles. Conventionally, the amount of membrane vesicles formed has been evaluated by culturing isolated bacteria in a liquid medium and quantifying the membrane vesicles in the culture supernatant using phospholipids, lipopolysaccharides, proteins, etc. as indicators. However, since there is heterogeneity in the membrane vesicle production ability of bacteria and the membrane vesicle production ability of isolated bacteria is not uniform, the conventional method can only obtain average information within a bacterial population, and there is a problem in that the membrane vesicle production ability of individual bacteria cannot be detected.

[0007] The present disclosure aims to provide methods and kits for detecting membrane vesicle-producing bacteria. [Means for solving the problem]

[0008] The present inventors have found that when membrane vesicle-producing bacteria are contacted with a fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature, the membrane vesicle-producing bacteria are labeled with the fluorescent probe. Furthermore, the present inventors have found that it is possible to detect membrane vesicle-producing bacteria and separate membrane vesicle-producing bacteria using the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature as an indicator, thereby completing the present disclosure.

[0009] The present disclosure relates, for example, to the following: [1] A method for detecting membrane vesicle-producing bacteria, comprising: Contacting the membrane vesicle-producing bacteria with a fluorescent probe that selectively labels a lipid bilayer membrane with high curvature; and Measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe. The method includes: [1-1] A method for evaluating the membrane vesicle-producing ability of a membrane vesicle-producing bacterium, comprising: Contacting the membrane vesicle-producing bacteria with a fluorescent probe that selectively labels a lipid bilayer membrane with high curvature; and Measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe. The method includes: [2] The fluorescent probe has the following formula (I): XY (I) In the formula (I), X represents a fluorescent group; Y represents an amphipathic peptide having 5 to 130 amino acid residues; The method according to [1] or [1-1]. [3] The method according to [2], wherein the amphipathic peptide having 5 to 130 amino acid residues is a peptide selected from the group consisting of the peptides shown in SEQ ID NOs: 1 to 6. [4] The fluorescent probe has the following structural formula: [ka] The method according to [1] or [1-1], wherein the compound is represented by the formula: [5] The above method is a method for isolating membrane vesicle-producing bacteria, After the step of measuring the fluorescence, a step of separating membrane vesicle-producing bacteria having a fluorescence intensity greater than a predetermined value obtained in the step of measuring the fluorescence is performed. The method according to [1] or [1-1], further comprising: [6] The method according to [5], wherein the step of measuring the fluorescence and the step of isolating the membrane vesicle-producing bacteria are carried out using a flow cytometer. A population of membrane vesicle-producing bacteria isolated using the methods described in [7][5] or [6]. [8] A kit for detecting membrane vesicle-producing bacteria, comprising: A fluorescent probe that selectively labels lipid bilayers with high curvature, and A package insert stating that the fluorescent probe is contacted with the membrane vesicle-producing bacteria and the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe is measured. Including the kit. [9] A kit for isolating membrane vesicle-producing bacteria, comprising: A fluorescent probe that selectively labels lipid bilayers with high curvature, and A package insert stating that membrane vesicle-producing bacteria whose fluorescence intensity is greater than a predetermined value when contacted with the fluorescent probe is to be isolated. Including the kit. Effect of the Invention

[0010] According to one embodiment of the present disclosure, a method and kit for detecting membrane vesicle-producing bacteria are provided.

[0011] According to one embodiment of the present disclosure, a method and kit for isolating membrane vesicle-producing bacteria are provided.

[0012] According to the method and kit of an embodiment of the present disclosure, it is possible to detect and evaluate membrane vesicle-producing bacteria at the single cell level of bacteria. Then, it is possible to separate membrane vesicle-producing bacteria with high membrane vesicle production ability based on the detection. In addition, since a wide variety of bacteria exist in a mixed state in the real environment, it is possible to quickly detect bacteria that actively release membrane vesicles from the bacterial group.

[0013] According to the method and kit according to an embodiment of the present disclosure, it is possible to detect or separate membrane vesicle-producing bacteria while the membrane vesicle-producing bacteria remain alive.

[0014] According to a method and kit according to an embodiment of the present disclosure, it is possible to detect membrane vesicle-producing bacteria or separate membrane vesicle-producing bacteria while minimizing the influence on the growth of the membrane vesicle-producing bacteria. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows fluorescence images obtained in Example 1 when the PAO1 strain of Pseudomonas aeruginosa or its DpqsA strain was contacted with the high curvature membrane recognition probe ApoC-NR or the control probe ApoCmut-NR. [Diagram 2] FIG. 13 shows fluorescent images obtained in Example 2 when the wild-type Buttiauxella agrestis JCM1090T strain or its membrane vesicle-overforming DtolB strain was contacted with the high-curvature membrane-recognizing probe ApoC-NR or the control probe ApoCmut-NR. [Diagram 3] FIG. 3 is a diagram showing bright-field images of the DtolB strain of Buttiauxella agrestis JCM1090 strain grown in contact with the high-curvature membrane recognition probe ApoC-NR or the control probe ApoCmut-NR in Example 3. [Figure 4] FIG. 4 shows the results of analysis of the B. agrestis JCM1090 strain or its DtolB strain labeled with the high curvature membrane recognition probe ApoC-NR in Example 4 using a flow cytometer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Although the embodiments of the present disclosure will be described below, the present disclosure is not limited to the following embodiments.

[0017] A first aspect of the present disclosure is a method for detecting membrane vesicle-producing bacteria, the method comprising a step of contacting membrane vesicle-producing bacteria with a fluorescent probe that selectively labels a lipid bilayer membrane with high curvature (contact step), and a step of measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe (fluorescence measurement step). In the present disclosure, "detecting membrane vesicle-producing bacteria" includes detecting the presence of membrane vesicle-producing bacteria, and detecting (measuring) membrane vesicle-producing bacteria to evaluate bacteria that are already known to be membrane vesicle-producing bacteria.

[0018] <Membrane vesicle-producing bacteria> Membrane vesicles are biological particles with a diameter of about 20-400 nm, whose surface is covered with a lipid bilayer membrane, and are released from various prokaryotes such as gram-negative bacteria, gram-positive bacteria, and archaea. The molecular biological mechanism of membrane vesicle release from prokaryotes is complex and diverse, and many parts remain unknown. When membrane vesicles are released from prokaryotes, the lipid bilayer membrane on the cell surface of the prokaryote partially protrudes, and the base of the protruding part narrows, and finally the protruding part separates from the lipid bilayer membrane on the cell surface of the prokaryote, releasing the membrane vesicle. Therefore, during the formation of membrane vesicles, the protruding part of the lipid bilayer membrane on the cell surface of the prokaryote becomes highly curvatured.

[0019] In the present disclosure, membrane vesicle-producing bacteria refers to bacteria that produce membrane vesicles. The type of bacteria is not particularly limited as long as it produces membrane vesicles, and may be, for example, either a gram-positive or gram-negative bacteria, or either aerobic or anaerobic bacteria. The membrane vesicle-producing bacteria may be, for example, a membrane vesicle-producing bacteria belonging to the phylum Proteobacteria, a membrane vesicle-producing bacteria belonging to the class Gammaproteobacteria, or a membrane vesicle-producing bacteria belonging to the order Enterobacteriales or the order Pseudomonadales. Examples of membrane vesicle-producing bacteria include Escherichia coli, Pseudomonas aeruginosa, Helicobacter pylori, Salmonella, Shigella, Yersinia, Neisseria, Vibrio, Campylobacter, Acinetobacter, Serratia, Shewanella, Myxococcus xanthus, Mycobacterium tuberculosis, periodontal disease bacteria (such as Porphylomonas gingivalis), and lactic acid bacteria (such as Lactobacillus).

[0020] <Highly curvature membrane recognition probe> In the present disclosure, a fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature (hereinafter, also referred to as a "high curvature membrane recognition probe") refers to a compound that has binding ability to a lipid bilayer membrane with a high curvature and can detect its binding to a lipid bilayer membrane with a high curvature using fluorescence as an indicator. Examples of the high curvature membrane recognition probe include a conjugate molecule of a binding site that selectively binds to a lipid bilayer membrane with a high curvature and a fluorescent dye, and a conjugate molecule of a binding site that binds to a lipid bilayer membrane and a fluorescent dye whose fluorescence intensity increases when the curvature of the lipid bilayer membrane to which the binding site is bound increases. The high curvature membrane recognition probe according to the present disclosure may be, for example, a compound that has binding ability to a lipid bilayer membrane with a high curvature and does not have binding ability to a lipid bilayer membrane with a low curvature, and can detect its binding to a lipid bilayer membrane with a high curvature using fluorescence as an indicator. The high curvature membrane recognition probe according to the present disclosure may be, for example, a fluorescent probe that selectively labels an anionic lipid bilayer membrane with a high curvature. The high curvature membrane recognition probe according to the present disclosure may be, for example, a fluorescent probe that selectively labels a phospholipid bilayer membrane with high curvature.In these cases, the lipid bilayer membrane with high curvature may be, for example, a lipid bilayer membrane with a curvature radius of 170 nm or less, 140 nm or less, 120 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, or 60 nm or less.In these cases, the high curvature membrane recognition probe may be, for example, a fluorescent probe that has a fluorescence intensity of 2 times or more, 3 times or more, 5 times or more, 10 times or more, or 20 times or more when bound to a lipid bilayer membrane with high curvature, compared to when it exists in a solution. The fluorescence intensity may be, for example, the fluorescence intensity measured by a fluorometer for an aqueous solution of a high curvature membrane recognition probe or an aqueous solution containing a high curvature membrane recognition probe and a lipid bilayer membrane of high curvature (e.g., a liposome), and the concentration of the high curvature membrane recognition probe in the measurement may be, for example, 1.0 to 10 μM, and the concentration of the liposome in the measurement may be, for example, 100 μM to 1.00 mM.

[0021] In a preferred embodiment, the high curvature membrane recognition probe has the following formula (I): XY (I) In formula (I), X represents a fluorescent group, and Y represents an amphipathic peptide having 5 to 130 amino acid residues.

[0022] X in formula (I) is not particularly limited as long as it is a fluorescent group, and may be, for example, a fluorescent group obtained by removing one hydrogen atom from a fluorescent organic compound. The fluorescent organic compound may be, for example, an organic fluorescent dye or its derivative or linker-added body. The organic fluorescent dye is not particularly limited, and may be, for example, fluorescein, tetramethylrhodamine, coumarin, cyanine derivatives, etc. From the viewpoint of increasing the fluorescence intensity and improving the detection sensitivity, the fluorescence quantum yield of the organic fluorescent dye in a solution (for example, a 1:1 mixed solution of water-dioxane) may be, for example, 0.00001 or more, 0.0001 or more, 0.001 or more, 0.01 or more, 0.03 or more, or 0.10 or more. Furthermore, from the viewpoint of suppressing overlap with fluorescence derived from bacteria (autofluorescence) and improving detection sensitivity, the maximum fluorescence wavelength of the organic fluorescent dye may be, for example, 300 nm or more, 350 nm or more, 400 nm or more, 420 nm or more, 440 nm or more, 450 nm or more, 460 nm or more, 470 nm or more, 480 nm or more, 490 nm or more, or 500 nm or more, or may be 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 650 nm or less, or 600 nm or less.

[0023] In a preferred embodiment, the organic fluorescent dye is an environmentally responsive fluorescent dye. In other words, in a preferred embodiment, X in formula (I) is a fluorescent group obtained by removing one hydrogen atom from an environmentally responsive organic fluorescent dye, or a fluorescent group obtained by removing one hydrogen atom from an environmentally responsive organic fluorescent dye or its derivative or linker-added compound. In the present disclosure, an environmentally responsive fluorescent dye means a fluorescent dye whose fluorescence intensity when excited with excitation light of a specific wavelength in a hydrophobic environment is greater than that when excited with excitation light of the same wavelength in a hydrophilic environment. The environmentally responsive fluorescent dye may be, for example, a fluorescent dye whose fluorescence intensity when excited with excitation light of a specific wavelength in a state dissolved in an organic solvent such as tetrahydrofuran, n-heptane, n-hexane, cyclohexane, toluene, xylene, dimethylformamide, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, or acetonitrile is at least 2 times, 3 times, 5 times, 10 times, 20 times, 50 times, or 100 times greater than the fluorescence intensity when excited with excitation light of the same wavelength in a state dissolved in an aqueous solvent, ethanol, or methanol. More specifically, the environmentally responsive fluorescent dye may be, for example, a fluorescent dye whose fluorescence intensity when excited with excitation light of a specific wavelength in a 0.1 μg / mL solution of n-heptane is at least 20 times greater than the fluorescence intensity when excited with excitation light of the same wavelength in a 0.1 μg / mL solution of ethanol. If the above organic fluorescent dye is an environmentally responsive fluorescent dye, when the fluorescent probe binds to a lipid bilayer membrane with a high curvature, the fluorescence intensity increases in the lipid-soluble environment on the bacterial surface, making it easier to detect membrane vesicle-producing bacteria using the fluorescence intensity as an indicator, and also making it easier to detect membrane vesicle-producing bacteria without performing a washing operation.

[0024] Examples of environmentally responsive fluorescent dyes include dansyl dyes and derivatives thereof, Dapoxyl dyes and derivatives thereof, and benzophenoxazine dyes and derivatives thereof. Specific examples of environmentally responsive fluorescent dyes include allylnaphthalenesulfonic acids such as 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-anilinonaphthalene-6-sulfonic acid (MANS), and 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS); dimethylaminonaphthalenesulfonic acid; benzofurazan derivatives such as nitrobenzofurazan (NBD); Dapoxyl dye (Benzenesulfonic acid, 4-[5-[4-(dimethylamino)phenyl]-2-oxazolyl); Dapoxyl sulfonyl chloride, Dapoxylsuccinimidyl ester, Dapoxyl 3-sulfonamidopropionic acid, Dapoxyl (2-bromoacetamidoethyl) sulfonamide, and Dapoxyl(2-aminoethyl) Examples include dapoxyl derivatives such as sulfonamide; dansyl dyes such as dansyl chloride, dansylsulfonamide, dansylaminoethyl-3-phosphate, 1-dansylsulfonamide-3-N,N-dimethylaminopropane, dansylcholine, dansylgalactoside, dansyllysine, and dansylphosphatidylethanolamine; and benzophenoxazine derivatives such as Nile Red and Nile Blue.

[0025] Y in formula (I) is an amphipathic peptide having 5 to 130 amino acid residues. An amphipathic peptide is a peptide that forms an α-helix as a secondary structure, and has many hydrophobic residues among the residues exposed on a part of the surface of the α-helix, and many polar residues among the residues exposed on the surface of the α-helix excluding the part of the surface. Such amphipathic peptides are known to be easily bound to lipid bilayer membranes. The number of amino acid residues of the amphipathic peptide in the present disclosure may be 5 to 130, for example, 7 to 100, 9 to 80, 10 to 70, 11 to 60, 12 to 60, or 13 to 45. The polar residue may be, for example, an α-amino acid that does not have a ring structure in the side chain, at least a portion of which is positively and / or negatively charged under physiological conditions at pH 7.4, and / or an α-amino acid that has a hydrophilic residue (e.g., an amino group, a carboxyl group, a hydroxyl group, a carbamoyl group) in the side chain, and more specifically, may be a residue derived from aspartic acid (D), glutamic acid (E), lysine (K), histidine (H), arginine (R), threonine (T), serine (S), asparagine (N) or glutamine (Q). The hydrophobic residue may be, for example, a residue that does not belong to the polar residues and is derived from an α-amino acid (including proline) having three or more carbon atoms in the side chain, and more specifically, may be, for example, a residue derived from valine (V), proline (P), leucine (L), isoleucine (I), phenylalanine (F), methionine (M), tryptophan (W) or tyrosine (Y). The term "rich in hydrophobic residues" in the residues exposed on a portion of the surface may mean, for example, that the proportion of hydrophobic residues in the residues exposed on the portion of the surface is 50% or more. The term "rich in polar residues" in the residues exposed on the surface of the α-helix excluding the portion of the surface may mean, for example, that the proportion of polar residues in the residues exposed on the surface of the α-helix excluding the portion of the surface is 50% or more.In one embodiment, the amphipathic peptide may be a peptide that forms an α-helix as a secondary structure, in which the proportion of hydrophobic residues among residues exposed on a portion of the surface of the α-helix is ​​50% or more, the proportion of polar residues among residues exposed on the surface of the α-helix excluding that portion of the surface is 50% or more, and the proportion of hydrophobic residues among residues exposed on the surface of the α-helix excluding that portion of the surface is 30% or less. The amphipathic peptide according to the present disclosure is preferably a peptide consisting of residues derived from L-amino acids.

[0026] As an amphipathic peptide having 5 to 130 amino acid residues that can be used as Y in formula (I), for example, the amphipathic peptides described in Non-Patent Document 6 or Non-Patent Document 7 can be used. Specific α-helical peptides include ArfGAPl [199-223] (R. norvegicus), nucleoporin Nupl33 [245-267] (H. sapiens), α-synuclein [9-52] (H. sapiens, SEQ ID NO: 1, Non-Patent Document 8), DivIVA [24-41] (B. subtillis), dAmph [3-28] (D. melanogaster), endophilin Al [2-21] (R. norvegicus), Sarlp [1-23] (S. cervisiae), antimicrobial magainin 2 (X. laevis), the string-like N-terminus of GMAP-210 [1-38] (H. sapiens), the sterol transporter Keslp [7-29] (S. cerevisiae), SpoVM (B. subtilis), and Epsin Examples of peptides include peptides derived from the peptides [3-15] (H. sapiens, SEQ ID NO: 2, Non-Patent Document 6), Binl / amphiphysin II [8-33] (H. sapiens), BRAP / Bin2-derived H0-NBAR [16-34], myristoylated Arf 1 [1-17] (B. taurus), melittin (A. mellifera), Arf1 [1-18] (SEQ ID NO: 3, Non-Patent Document 9), SNX1 [281-298] (SEQ ID NO: 4, Non-Patent Document 10), and M2 [42-62 of A / Udorn / 72] (SEQ ID NO: 5, Non-Patent Document 9), or peptides based on these peptides (for example, a part of the peptide, or one to several, more specifically about one to five, amino acids of the peptide mutated). Furthermore, an example of an amphipathic peptide having 5 to 130 amino acid residues that can be used as Y in formula (I) is a peptide of 22 residues from the C-terminus of human apolipoprotein (apo) AI (apoA-I 220-241, SEQ ID NO: 6, Non-Patent Document 11).The amphipathic peptide having 5 to 130 amino acid residues that can be used as Y in formula (I) may be a peptide selected from the group consisting of the peptides shown in SEQ ID NO: 1 to 6, and more preferably the peptide shown in SEQ ID NO: 6.

[0027] α-synuclein [9-52]: DVFMKGLSKAKEGVVAAAEKTK (SEQ ID NO: 1) Epsin [3-15]: SSLRRQMKNIVHN (SEQ ID NO: 2) Arf1 [1-18]: MGNIFANLFKGLFGKKEM (SEQ ID NO: 3) SNX1 [281-298]: GAGLLKXFNKATDAVSKX (SEQ ID NO: 4) M2 [42-62 of A / Udorn / 72]:RLFFKSIYRFFEHGLKRG (SEQ ID NO:5) ApoA-I 220-241: PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 6)

[0028] The type of bond between X and Y in formula (I) is not particularly limited as long as it is a covalent bond, and may be, for example, an amide bond, an ester bond, a carbamate bond (-NH-C(=O)-O-) or a urea bond (-NH-C(=O)-NH-), preferably an amide bond, a carbamate bond or a urea bond between the main chain amino group of the α-amino acid at the N-terminus of Y and X, or an amide bond, an ester bond or a carbamate bond between the main chain carboxyl group of the α-amino acid at the C-terminus of Y and X, and more preferably an amide bond between the main chain amino group of the α-amino acid at the N-terminus of Y and X, or an amide bond or ester bond between the main chain carboxyl group of the α-amino acid at the C-terminus of Y and X.

[0029] In a preferred embodiment, the high curvature membrane recognition probe has the following structural formula: [ka] ApoC-NR is a compound represented by the formula (hereinafter, also referred to as "ApoC-NR"). ApoC-NR is a high curvature membrane recognition probe developed by the present inventors, and has a chemical structure in which a linker-added Nile Red, an environmentally responsive fluorescent dye, and the amphipathic peptide shown in SEQ ID NO: 6 are covalently bonded by an amide bond (Non-Patent Document 5).

[0030] Although not wishing to be bound by any theory, it is believed that the high curvature membrane recognition probe according to the present disclosure can label membrane vesicle-producing bacteria by binding to lipid packing defects present on the surface of membrane vesicle-producing bacteria or on the surface of membrane vesicles released by membrane vesicle-producing bacteria. It is known that amphipathic peptides having 5 to 130 amino acid residues have binding properties to lipid packing defects present on the surface of membrane vesicle-producing bacteria or on the surface of membrane vesicles released by membrane vesicle-producing bacteria (Non-Patent Documents 5 to 7). In addition, for example, when Apoc-NR is contacted with membrane vesicle-producing bacteria, the amphipathic peptide portion binds to the high curvature portion of the lipid bilayer membrane on the cell surface of the membrane vesicle-producing bacteria, and the fluorescence intensity of Nile Red increases in response to a lipid-soluble environment, which is believed to label the membrane vesicle-producing bacteria.

[0031] The high curvature membrane recognition probe can be synthesized by a method commonly used by those skilled in the art. For example, a fluorescent dye or its derivative or linker-added compound obtained by purchasing a commercially available product or synthesizing by a known method and an amphiphilic peptide obtained by outsourcing to an external company or by a method commonly used by those skilled in the art, such as Fmoc solid-phase synthesis, can be synthesized by amide condensation using a condensing agent such as HATU or PyBOP. In addition, when the high curvature membrane recognition probe is Apoc-NR, Apoc-NR can be synthesized according to the method described in Non-Patent Document 5.

[0032] <Contact process> In the contact step, the membrane vesicle-producing bacteria are contacted with the high curvature membrane recognition probe. The contact method in the contact step is not limited as long as it can contact the membrane vesicle-producing bacteria with the high curvature membrane recognition probe, but is usually performed by adding a solution of the high curvature membrane recognition probe to a pellet of membrane vesicle-producing bacteria or a suspension thereof, suspending the obtained membrane vesicle-producing bacteria, and incubating the aqueous solution containing the high curvature membrane recognition probe (hereinafter also referred to as "bacteria labeling solution").

[0033] A pellet of membrane vesicle-producing bacteria can be prepared by a method commonly used by those skilled in the art, for example, by centrifuging a liquid medium such as LB liquid medium, M9 medium, TB medium, or Hanahan liquid medium in which the membrane vesicle-producing bacteria is cultured to precipitate the membrane vesicle-producing bacteria to the bottom of the container, and then removing the supernatant.

[0034] A suspension of membrane vesicle-producing bacteria can be prepared by a method commonly used by those skilled in the art. For example, a liquid medium in which membrane vesicle-producing bacteria are cultured may be used as a suspension of the membrane vesicle-producing bacteria. Alternatively, an aqueous solution (buffer) such as PBS or HBSS, whose pH and osmotic pressure are adjusted to within the viable range of the membrane vesicle-producing bacteria, may be added to a pellet of membrane vesicle-producing bacteria prepared according to the method described above, and the pellet may be broken up by pipetting or tapping, etc., to suspend the membrane vesicle-producing bacteria.

[0035] The solution of the high curvature membrane recognition probe can be prepared by a method commonly performed by those skilled in the art. For example, when the solution of the high curvature membrane recognition probe is added to a pellet of membrane vesicle-producing bacteria, the solution of the high curvature membrane recognition probe may be an aqueous solution in which the high curvature membrane recognition probe or its salt or its solvate is dissolved in an aqueous solution (buffer) such as PBS or HBSS, whose pH and osmotic pressure are adjusted to within the viable range of the membrane vesicle-producing bacteria. In addition, for example, when the solution of the high curvature membrane recognition probe is added to a suspension of membrane vesicle-producing bacteria, the solution of the high curvature membrane recognition probe may be, for example, a solution in which the high curvature membrane recognition probe or its salt or its solvate is dissolved in an organic solvent such as dimethyl sulfoxide (DMSO) or ethanol, or an aqueous solution in which the high curvature membrane recognition probe or its salt or its solvate is dissolved in pure water, physiological saline, PBS, HBSS, or other aqueous solution. In addition, when the high curvature membrane recognition probe is added to the suspension of membrane vesicle-producing bacteria, the high curvature membrane recognition probe may be added in the form of a solid such as a high curvature membrane recognition probe or its salt or a solvate thereof, rather than in the form of a solution. As described above, in the contact step, the high curvature membrane recognition probe that is brought into contact with the membrane vesicle-producing bacteria may be added in the form of a salt, or a solvate of a compound or salt. In addition, in the contact step, the high curvature membrane recognition probe that is brought into contact with the membrane vesicle-producing bacteria may be brought into contact with the membrane vesicle-producing bacteria in the form of a salt, or a solvate of a compound or salt.

[0036] The concentration of membrane vesicle-producing bacteria in the bacterial labeling solution is not particularly limited.

[0037] The concentration of the high curvature membrane recognition probe in the bacterial labeling solution is not particularly limited as long as it is a concentration at which the fluorescence of the labeled membrane vesicle-producing bacteria can be detected, and may be, for example, 1 nM to 10 mM, 10 nM to 1 mM, 100 nM to 100 μM, 300 nM to 30 μM, 500 nM to 20 μM, or 1 μM to 10 μM.

[0038] The bacterial labeling solution may contain other components in addition to the membrane vesicle-producing bacteria and the high curvature membrane recognition probe, such as a buffer, a pH adjuster, an osmotic pressure adjuster (e.g., sodium chloride), an enzyme extract, peptone, natural amino acids, vitamins and monosaccharides (e.g., D-glucose), and antibiotics.

[0039] The pH of the bacterial labeling solution is not particularly limited as long as it is within a range in which the high curvature membrane recognition probe can label membrane vesicle-producing bacteria, but from the viewpoint of increasing the survival rate and / or labeling efficiency of the membrane vesicle-producing bacteria, it may be, for example, 4.0 to 10.0, 5.0 to 9.0, 5.5 to 8.5, or 6.0 to 8.0.

[0040] The temperature of the bacterial labeling solution is not particularly limited as long as it is within a range in which the high curvature membrane recognition probe can label membrane vesicle-producing bacteria, but from the viewpoint of increasing the survival rate of the membrane vesicle-producing bacteria, it may be kept, for example, at 10 to 50°C, 15 to 45°C, 20 to 42°C, or 25 to 40°C.

[0041] The choice of aerobic or anaerobic conditions in the contact step can be freely made by a person skilled in the art depending on the type of membrane vesicle-producing bacteria, but from the viewpoint of increasing the survival rate of the membrane vesicle-producing bacteria, if the membrane vesicle-producing bacteria is an aerobic bacterium, the contact step may be carried out under aerobic conditions such as an aerated environment or an environment inside an incubator with adjusted humidity, oxygen concentration and / or carbon dioxide concentration, and if the membrane vesicle-producing bacteria is an anaerobic bacterium, the contact step may be carried out under anaerobic conditions such as a sealed environment.

[0042] In the contact step, the time for which the high curvature membrane recognition probe is contacted with the membrane vesicle-producing bacteria is not particularly limited as long as it is a time that allows the high curvature membrane recognition probe to label the membrane vesicle-producing bacteria, and may be, for example, from 1 second to 24 hours, from 10 seconds to 3 hours, from 1 minute to 1 hour, or from 3 minutes to 30 minutes.

[0043] <Fluorescence measurement process> In the fluorescence measurement step, the fluorescence of the membrane vesicle-producing bacteria labeled with the high curvature membrane recognition probe in the contact step is measured. The excitation wavelength and the fluorescence wavelength when measuring the fluorescence can be appropriately determined by a person skilled in the art depending on the structure of the high curvature membrane recognition probe.

[0044] The measurement of the fluorescence of the membrane vesicle-producing bacteria in the fluorescence measurement step can be carried out using equipment commonly used by those skilled in the art, for example, a fluorescence microscope capable of measuring fluorescence, such as a transmission fluorescence microscope, an epifluorescence microscope or a confocal microscope, a flow cytometer, a microwell plate reader or a fluorometer.

[0045] In a preferred embodiment, the fluorescence measurement step may include measuring the fluorescence of each membrane vesicle-producing bacterium. In other words, in a preferred embodiment, the fluorescence measurement step may include measuring the fluorescence of the membrane vesicle-producing bacterium at the single cell level. When the fluorescence measurement step includes measuring the fluorescence of each membrane vesicle-producing bacterium, it is possible to determine whether each membrane vesicle-producing bacterium is a membrane vesicle-producing bacterium, or to evaluate the membrane vesicle production ability of each membrane vesicle-producing bacterium. In this case, the measurement of the fluorescence of the membrane vesicle-producing bacterium in the fluorescence measurement step can be performed by a method that is usually performed by a person skilled in the art when measuring fluorescence at the single cell level, and can be performed using, for example, a fluorescence microscope such as a transmission type fluorescence microscope, an epifluorescence microscope, or a confocal microscope capable of measuring fluorescence, or a flow cytometer.

[0046] In one embodiment of the detection of membrane vesicle-producing bacteria, for example, bacteria whose fluorescence is detected in the fluorescence measurement step may be evaluated (detected) as membrane vesicle-producing bacteria. Also, for example, bacteria whose fluorescence intensity is high in the fluorescence measurement step may be evaluated (detected) as membrane vesicle-producing bacteria. In this case, bacteria whose fluorescence intensity is higher than a predetermined value may be evaluated (detected) as membrane vesicle-producing bacteria, and the predetermined value may be, for example, the upper limit of an 80% confidence interval, an 85% confidence interval, a 90% confidence interval, a 95% confidence interval, a 97% confidence interval, a 99% interval, or a 99.5% confidence interval when the distribution of the fluorescence intensity of the bacteria that is not contacted with the high curvature membrane recognition probe and / or the bacteria that is contacted with the control probe that does not have binding ability to the membrane vesicle-producing bacteria is fitted as following a normal distribution, and may be, for example, the upper limit of a 95% confidence interval. In addition, the predetermined value may be, for example, the upper limit of an 80% confidence interval, an 85% confidence interval, a 90% confidence interval, a 95% confidence interval, a 97% confidence interval, a 99% interval or a 99.5% confidence interval when the distribution of fluorescence intensity of a mutant strain in which a gene mutation is introduced into the membrane vesicle-producing bacterium and the membrane vesicle production ability is reduced and / or a bacterium that does not have the ability to produce membrane vesicles and that is contacted with a high curvature membrane recognition probe is fitted as following a normal distribution, for example, the upper limit of a 95% confidence interval. In addition, the predetermined value may be, for example, a value determined so that when a population of bacteria including membrane vesicle-producing bacteria is contacted with a high curvature membrane recognition probe, 30%, 25%, 20%, 15%, 10%, 5%, 3% or 1% of the bacteria in the whole bacteria are evaluated (detected) as membrane vesicle-producing bacteria. In these cases, the indicator of the fluorescence intensity for each bacterium may be, instead of the measured value of the fluorescence intensity for each bacterium, the value obtained by dividing the measured value by the volume of each bacterium or an indicator thereof (e.g., forward scatter (FSC)).

[0047] In one embodiment of the detection of membrane vesicle-producing bacteria, the membrane vesicle-producing ability of the membrane vesicle-producing bacteria may be evaluated, for example, using the fluorescence intensity in the fluorescence measurement step as an index. In other words, the method according to the first aspect of the present disclosure may be, in one embodiment, a method for evaluating the membrane vesicle-producing ability of the membrane vesicle-producing bacteria, which includes a step of contacting the membrane vesicle-producing bacteria with a fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature, and a step of measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe. In this case, for example, bacteria with high fluorescence intensity in the fluorescence measurement step may be evaluated (detected) as membrane vesicle-producing bacteria with high membrane vesicle production ability. In this case, bacteria with a fluorescence intensity greater than a predetermined value may be evaluated (detected) as membrane vesicle-producing bacteria with high membrane vesicle production ability, and the predetermined value may be a value determined so that, for example, when a population of membrane vesicle-producing bacteria or a population of bacteria containing membrane vesicle-producing bacteria is contacted with a high curvature membrane recognition probe, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 3% or 1% of the bacteria in the total bacteria are evaluated (detected) as membrane vesicle-producing bacteria with high membrane vesicle production ability. In addition, the predetermined value may be, for example, the upper limit value of an 80% confidence interval, an 85% confidence interval, a 90% confidence interval, a 95% confidence interval, a 97% confidence interval, a 99% confidence interval or a 99.5% confidence interval when the distribution of the fluorescence intensity of the bacteria not contacted with the high curvature membrane recognition probe and / or the bacteria contacted with a control probe that does not have binding ability to membrane vesicle-producing bacteria is fitted as following a normal distribution, and may be, for example, the upper limit value of the 95% confidence interval. In addition, the predetermined value may be, for example, the upper limit of an 80% confidence interval, an 85% confidence interval, a 90% confidence interval, a 95% confidence interval, a 97% confidence interval, a 99% interval, or a 99.5% confidence interval when the distribution of fluorescence intensity of a mutant strain in which a gene mutation has been introduced into the membrane vesicle-producing bacterium and the membrane vesicle production ability has been reduced and / or a bacterium that has not been contacted with the high curvature membrane recognition probe and has not been able to produce membrane vesicles is fitted as following a normal distribution, and may be, for example, the upper limit of a 95% confidence interval. In addition, the indicator of the fluorescence intensity of each bacterium in these cases may be, instead of the measured value of the fluorescence intensity of each bacterium, a value obtained by dividing the measured value by the volume of each bacterium or an indicator thereof (for example, forward scatter (FSC)).A bacterium having a fluorescence intensity greater than a predetermined value has a high ability to produce membrane vesicles, which may mean, for example, that the number of membrane vesicles produced is 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, or 10 times or more, compared to a bacterium having a fluorescence intensity equal to or less than a predetermined value.

[0048] In the present disclosure, when the fluorescence of each membrane vesicle-producing bacterium is measured, the fluorescence intensity of the membrane vesicle-producing bacterium labeled with a fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature is large, and in addition to the case where the curvature of the protruding part of the lipid bilayer membrane on the cell surface of the membrane vesicle-producing bacterium is large, the membrane vesicles released from the membrane vesicle-producing bacterium may remain on the cell surface of the membrane vesicle-producing bacterium or in its vicinity. In other words, in the detection method of membrane vesicle-producing bacterium according to the first aspect of the present disclosure, the subject of the fluorescence measurement includes, in addition to the membrane vesicle-producing bacterium itself, membrane vesicles having a lipid bilayer membrane structurally integrated with the lipid bilayer membrane on the cell surface of the membrane vesicle-producing bacterium, and membrane vesicles that are structurally separated from the lipid bilayer membrane of the membrane vesicle-producing bacterium in part or in whole but are present in spatial proximity to the membrane vesicle-producing bacterium, and for example, when the intensity of the fluorescence emitted by these is large, it may be evaluated that the measured bacterium is a membrane vesicle-producing bacterium, or that the measured membrane vesicle-producing bacterium has a high membrane vesicle production ability.

[0049] <Preparative process> In one embodiment, the method according to the first aspect of the present disclosure may be a method for separating membrane vesicle-producing bacteria, which includes a step (separation step) of separating membrane vesicle-producing bacteria having a fluorescence intensity greater than a predetermined value obtained in the fluorescence measurement step after the fluorescence measurement step. In other words, one embodiment of the first aspect of the present disclosure may be a method for separating membrane vesicle-producing bacteria, which includes a step of contacting membrane vesicle-producing bacteria with a fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature, a step of measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe, and a step of separating membrane vesicle-producing bacteria having a fluorescence intensity greater than a predetermined value obtained in the fluorescence measurement step. In a preferred embodiment, the method according to the first aspect of the present disclosure may be a method for separating membrane vesicle-producing bacteria having a high membrane vesicle production ability, which includes a step of separating membrane vesicle-producing bacteria having a fluorescence intensity greater than a predetermined value obtained in the fluorescence measurement step after the fluorescence measurement step as membrane vesicle-producing bacteria having a high membrane vesicle production ability.

[0050] In a preferred embodiment, the fluorescence measurement step and the sorting step in the sorting method are performed using a flow cytometer. That is, in a preferred embodiment, the fluorescence measurement step and the sorting step in the sorting method are performed by cell sorting using a flow cytometry method. In this case, the fluorescence measurement step and the sorting step are usually performed for each bacterium, that is, the fluorescence measurement step and the sorting step for one bacterium are usually repeated the same number of times as the number of bacteria, and may include, for example, a fluorescence measurement step (first fluorescence measurement step) and a sorting step (first sorting step) for a first bacterium, followed by a fluorescence measurement step (second fluorescence measurement step) and a sorting step (second sorting step) for a second bacterium.

[0051] <Application> The method according to the first aspect of the present disclosure can be used, for example, for the detection, evaluation, and / or separation of membrane vesicle-producing bacteria. In addition, since a wide variety of bacteria exist in a mixed state in the real environment, it is also possible to quickly detect, evaluate, and / or separate bacteria that actively release membrane vesicles from among the bacterial group. For example, membrane vesicle-producing bacteria obtained by the method according to the first aspect of the present disclosure, membrane vesicle-producing bacteria evaluated to have high membrane vesicle production ability, or separated membrane vesicle-producing bacteria can be used for the production of membrane vesicles to be used as carriers for drug delivery systems or vaccines, for example. In addition, by introducing an active ingredient or a precursor thereof of a drug or vaccine into these membrane vesicle-producing bacteria, it is possible to produce membrane vesicles containing the active ingredient or a precursor thereof, that is, to produce membrane vesicles that can be used as a therapeutic agent or vaccine. In addition, it is possible that these membrane vesicle-producing bacteria can be used as activated sludge in the activated sludge method. Furthermore, by detecting membrane vesicle-producing bacteria from a complex microbial system using the detection method of membrane vesicle-producing bacteria relating to the first aspect and analyzing them phylogenetically, it is expected that a library of membrane vesicles and / or membrane vesicle-producing bacteria containing membrane vesicles with various characteristics and systematically organized can be constructed.

[0052] <Other aspects> Another aspect of the present disclosure is a kit for detecting membrane vesicle-producing bacteria, comprising a fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature, and a package insert that describes contacting the membrane vesicle-producing bacteria with the fluorescent probe and measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe. In this case, the fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature (high curvature membrane recognition probe) may be the same as that used in the first aspect of the present disclosure. The high curvature membrane recognition probe included in the kit may be in the form of a salt, or may be in the form of a solvate of the high curvature membrane recognition probe or its salt. In addition, the description in the package insert may be a description corresponding to the method for detecting membrane vesicle-producing bacteria according to the first aspect of the present disclosure.

[0053] Another aspect of the present disclosure is a kit for separating membrane vesicle-producing bacteria, the kit including a fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature, and a package insert that describes separating membrane vesicle-producing bacteria whose fluorescence intensity is greater than a predetermined value when contacted with the fluorescent probe. In this case, the fluorescent probe that selectively labels a lipid bilayer membrane with a high curvature (high curvature membrane recognition probe) can be the same as that used in the first aspect of the present disclosure. The high curvature membrane recognition probe included in the kit may be in the form of a salt, or may be in the form of a solvate of the high curvature membrane recognition probe or its salt. In addition, the description in the package insert may be a description corresponding to the method for separating membrane vesicle-producing bacteria according to the first aspect of the present disclosure. In a preferred embodiment, another aspect of the present disclosure is a kit for isolating membrane vesicle-producing bacteria with high membrane vesicle production ability, the kit including a fluorescent probe that selectively labels a lipid bilayer membrane with high curvature, and a package insert that describes that membrane vesicle-producing bacteria that have a fluorescence intensity greater than a predetermined value when contacted with the fluorescent probe are isolated as membrane vesicle-producing bacteria with high membrane vesicle production ability. In this case, the description in the package insert may correspond to the method for isolating membrane vesicle-producing bacteria with high membrane vesicle production ability according to the first aspect of the present disclosure. EXAMPLES

[0054] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to the following examples.

[0055] [Example 1: Labeling of membrane vesicle-producing bacteria (Pseudomonas aeruginosa PAO1 strain) using a high-curvature membrane recognition probe] The relationship between the membrane vesicle formation ability and the fluorescent labeling using a high curvature membrane recognition probe was verified using the wild-type Pseudomonas aeruginosa PAO1 strain and its membrane vesicle formation-decreased strain DpqsA strain. The DpqsA strain is a mutant strain that does not produce Pseudomonas quinolone signal (PQS), a signal substance that promotes the formation of membrane vesicles. PQS is released outside the cell and then inserted into the cell surface, curving the outer cell membrane and inducing membrane vesicle formation (Non-Patent Document 3). Therefore, the DpqsA strain has a significantly reduced membrane vesicle production ability compared to the PAO1 strain. The Pseudomonas aeruginosa PAO1 strain and DpqsA strain used in the following examples were bacterial strains described in Non-Patent Document 3.

[0056] The PAO1 strain or its DpqsA strain of Pseudomonas aeruginosa was inoculated into 5 mL of LB liquid medium in a test tube and pre-cultured by shaking at 30 ° C. The pre-culture solution was inoculated into 5 mL of LB liquid medium so that the turbidity at 600 nm was 0.1, and cultured with shaking at 30 ° C. and 200 rpm for 6 hours. The culture solution was placed in a 1.5 mL tube and centrifuged at 6,000 × g, 5 min in a centrifuge. The supernatant was discarded, and the pelleted bacteria were resuspended in PBS, and a high curvature membrane recognition probe ApoC-NR (Non-Patent Document 5) or a control probe ApoCmut-NR (the 11th leucine of ApoC-NR is replaced with proline, so it does not recognize high curvature membranes. Non-Patent Document 5; the amino acid sequence of the peptide portion is described as SEQ ID NO: 7) was added to a concentration of 2 μM, and the reaction was allowed to proceed at room temperature for 5 minutes. The suspension of labeled bacteria was dropped onto a glass slide, covered with a cover slip, and observed under a fluorescence microscope (Olympus IX-73, excitation wavelength 530-550 nm, emission wavelength 575-625 nm) using a 100x objective lens.

[0057] The results are shown in Figure 1. According to Figure 1, when the high curvature membrane recognition probe ApoC-NR was contacted with the wild-type PAO1 strain, which has high membrane vesicle production ability, fluorescence was observed from the bacteria, whereas when the wild-type PAO1 strain was contacted with the control probe ApoCmut-NR, and when the membrane vesicle formation-reduced strain DpqsA strain was contacted with ApoC-NR, fluorescence from the bacteria was not observed. From this, it became clear that membrane vesicle-producing bacteria can be detected by contacting the high curvature membrane recognition probe with membrane vesicle-producing bacteria and measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the probe.

[0058] [Example 2: Membrane vesicle-producing bacteria (Buttiauxella agrestis JCM1090) using a high curvature membrane recognition probe T strain (DtolB strain) labeling] Wild-type Buttiauxella agrestis JCM1090 T Using the Buttiauxella agrestis JCM1090 strain and its membrane vesicle over-forming strain, the DtolB strain, the relationship between membrane vesicle formation ability and fluorescent labeling using a high curvature membrane recognition probe was examined. The inventors have found that the DtolB strain has about 15 times the membrane vesicle formation ability compared to the wild-type strain, and that the released membrane vesicles have a wide variety of shapes, including small, multi-membrane vesicles, and multivesicular membrane vesicles (Non-Patent Document 12). Furthermore, it has been confirmed that the cell morphology is changed in the DtolB strain, and fine protrusions are formed on the cell surface. The Buttiauxella agrestis JCM1090 strain used in the following examples T The strain used was provided by the RIKEN Bioresource Center. T The DtolB strain used was the strain described in Non-Patent Document 12.

[0059] In a test tube, 5 mL of LB liquid medium was added to the wild-type strain Buttiauxella agrestis JCM1090. TThe strain or its membrane vesicle over-forming strain DtolB strain was inoculated and pre-cultured by shaking at 30 ° C. The pre-culture solution was inoculated into 5 mL of LB liquid medium so that the turbidity at 600 nm was 0.01, and cultured at 30 ° C. and 200 rpm for 16 hours with shaking. The culture solution was placed in a 1.5 mL tube and centrifuged at 6,000 × g, 5 min in a centrifuge. The supernatant was discarded, and the pelleted bacteria were resuspended in PBS, and the high curvature membrane recognition probe ApoC-NR or the control probe ApoCmut-NR was added to a concentration of 2 μM, and reacted at room temperature for 5 minutes. The suspension of the labeled bacteria was dropped onto a slide glass, a cover glass was placed on it, and the mixture was observed using a 100x objective lens under a fluorescence microscope (Olympus IX-73, excitation wavelength 530-550 nm, fluorescence wavelength 575-625 nm).

[0060] The results are shown in FIG. 2. According to FIG. 2, when ApoC-NR was used, no fluorescence was observed from the bacteria in the wild type strain with low membrane vesicle production ability, whereas fluorescence was observed from the bacteria in the DtolB strain with excessive membrane vesicle formation ability. Furthermore, even when the control probe ApoCmut-NR was used, no fluorescence was observed from the DtolB strain. This shows that the detection of membrane vesicle-producing bacteria using the high curvature membrane recognition probe demonstrated in Example 1 is possible in membrane vesicle-producing bacteria not limited to the Pseudomonas aeruginosa PAO1 strain.

[0061] [Example 3: Verification of the effect of contacting a high curvature membrane recognition probe on bacterial growth] We examined whether contact with a high curvature membrane recognition probe affects bacterial growth.

[0062] The Buttiauxella agrestis JCM1090 DtolB strain was cultured in the same manner as in Example 2, washed with PBS, and then the high curvature membrane recognition probe ApoC-NR or the control probe ApoCmut-NR, or PBS was added to a concentration of 2 μM, and reacted at room temperature for 5 minutes. The sample after the reaction was serially diluted to 1 / 10, and 7.5 μL was dropped onto the LB agar medium. The agar medium was cultured overnight at 30 ° C., and a bright field image was obtained. The results are shown in FIG. 3. According to FIG. 3, even when the high curvature membrane recognition probe ApoC-NR was added, the Buttiauxella agrestis JCM1090 DtolB strain grew in the same way as when the control probe ApoCmut-NR or PBS group was added, so it was revealed that contact with the high curvature membrane recognition probe does not affect the growth of the bacteria. Therefore, it has been demonstrated that the method disclosed herein makes it possible to detect membrane vesicle-producing bacteria while keeping the membrane vesicle-producing bacteria alive and minimizing the effects on the growth of the membrane vesicle-producing bacteria.

[0063] [Example 4: Examination of the feasibility of isolating membrane vesicle-producing bacteria with high membrane vesicle production ability using a high curvature membrane recognition probe] We verified whether bacteria fluorescently labeled with a high curvature membrane labeling probe can be separated. As in Example 2, B. agrestis JCM1090 strain and its DtolB strain were cultured and labeled with the high curvature membrane recognition probe ApoC-NR. In addition, bacteria not labeled with ApoC-NR were also prepared as a control. These bacteria were analyzed using a flow cytometer (manufactured by Sony Corporation). The results are shown in FIG. 4. The results of the wild strain not labeled with a probe are shown in FIG. 4A. The results of the wild strain labeled with ApoC-NR are shown in FIG. 4B. The results of the DtolB strain not labeled with a probe are shown in FIG. 4C. The results of the DtolB strain labeled with ApoC-NR are shown in FIG. 4D. In FIG. 4A to FIG. 4D, the FSC (Forward Scatter) on the vertical axis indicates the size of the cells, and the Red Fluorescence on the horizontal axis indicates the fluorescence derived from ApoC-NR. According to these results, the DtolB strain (D) had many plots at positions of high fluorescence intensity compared to the wild-type strain (B) contacted with ApoC-NR, and membrane vesicle-producing bacteria with high membrane vesicle production ability could be selected by gating. This shows that the method disclosed herein makes it possible to detect membrane vesicle-producing bacteria at the single cell level of bacteria and evaluate their membrane vesicle production ability. In addition, it was shown that membrane vesicle-producing bacteria with high membrane vesicle production ability can be separated by cell sorting the bacteria contacted with the high curvature membrane labeling probe by flow cytometry.

Claims

1. A method for detecting membrane vesicle-producing bacteria, comprising: Contacting the membrane vesicle-producing bacteria with a fluorescent probe that selectively labels a lipid bilayer membrane with high curvature; and Measuring the fluorescence of the membrane vesicle-producing bacteria labeled with the fluorescent probe. The method includes:

2. The fluorescent probe has the following formula (I): X-Y... (I) In the formula (I), X represents a fluorescent group; Y represents an amphipathic peptide having 5 to 130 amino acid residues; The method of claim 1.

3. The method according to claim 2, wherein the amphipathic peptide having 5 to 130 amino acid residues is a peptide selected from the group consisting of peptides shown in SEQ ID NOs: 1 to 6.

4. The fluorescent probe has the following structural formula: 【Chemistry 1】 The method according to claim 1 , wherein the compound is represented by the formula:

5. The method is a method for isolating membrane vesicle-producing bacteria, a step of separating membrane vesicle-producing bacteria having a fluorescence intensity greater than a predetermined value obtained in the step of measuring the fluorescence after the step of measuring the fluorescence The method of any one of claims 1 to 4, further comprising:

6. The method according to claim 5 , wherein the steps of measuring the fluorescence and isolating the membrane vesicle-producing bacteria are carried out using a flow cytometer.

7. A kit for detecting membrane vesicle-producing bacteria, comprising: A fluorescent probe that selectively labels lipid bilayers with high curvature, and A package insert which describes that the fluorescent probe is contacted with a membrane vesicle-producing bacterium and the fluorescence of the membrane vesicle-producing bacterium labeled with the fluorescent probe is measured. Including the kit.

8. A kit for isolating membrane vesicle-producing bacteria, comprising: A fluorescent probe that selectively labels lipid bilayers with high curvature, and A package insert stating that membrane vesicle-producing bacteria having a fluorescence intensity greater than a predetermined value when contacted with the fluorescent probe are to be separated. Including the kit.

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