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WO2018008895A1 - Nanovésicules dérivées de bactéries du genre propionibacterium et leur utilisation - Google Patents

Nanovésicules dérivées de bactéries du genre propionibacterium et leur utilisation Download PDF

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WO2018008895A1
WO2018008895A1 PCT/KR2017/006889 KR2017006889W WO2018008895A1 WO 2018008895 A1 WO2018008895 A1 WO 2018008895A1 KR 2017006889 W KR2017006889 W KR 2017006889W WO 2018008895 A1 WO2018008895 A1 WO 2018008895A1
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Prior art keywords
vesicles
derived
propionibacterium
diseases
cancer
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English (en)
Korean (ko)
Inventor
김윤근
반창일
전진성
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POSTECH Academy Industry Foundation
MD Healthcare Inc
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POSTECH Academy Industry Foundation
MD Healthcare Inc
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Priority claimed from KR1020170081782A external-priority patent/KR101923969B1/ko
Application filed by POSTECH Academy Industry Foundation, MD Healthcare Inc filed Critical POSTECH Academy Industry Foundation
Priority to EP17824451.3A priority Critical patent/EP3483284B1/fr
Priority to US16/315,683 priority patent/US11193173B2/en
Priority to CN201780042554.XA priority patent/CN109715832B/zh
Priority to JP2019500448A priority patent/JP6808011B2/ja
Publication of WO2018008895A1 publication Critical patent/WO2018008895A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to nano-vesicles derived from the bacteria of the genus Propionibacterium, and more specifically, to a method for diagnosing cancer, inflammatory diseases, endocrine diseases, or metabolic diseases using nano-vesicles derived from propionium bacteria. And a prophylactic or therapeutic composition comprising the vesicles.
  • microbiota refers to a microbial community including bacteria, archaea and eukarya that exist in a given settlement.Intestinal microbiota is an important role in human physiology. It is known to have a great effect on human health and disease through interaction with human cells.
  • the symbiotic bacteria secrete nanometer-sized vesicles to exchange information about genes and proteins in other cells.
  • the mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through, so that the symbiotic bacteria cannot pass through the mucosa, but bacterial-derived vesicles are usually less than 100 nanometers in size. It passes freely through the mucous membrane and is absorbed by our body.
  • Pathogenic bacteria-derived vesicles absorbed by our bodies are inflammatory diseases such as inflammatory skin diseases such as atopic dermatitis, inflammatory respiratory diseases such as chronic rhinitis, asthma and chronic obstructive pulmonary disease (COPD), inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. It is found to be an important factor in the etiology of. In addition, it has attracted attention as it has recently been found that there is a close relationship with the occurrence of metabolic diseases such as diabetes, obesity, lung cancer, gastric cancer, colon cancer and the like.
  • COPD chronic obstructive pulmonary disease
  • the bacterium of Propionibacterium is an anaerobic Gram-positive bacillus that has a characteristic of synthesizing propionic acid through transcarboxylase enzyme.
  • the bacterium is a bacterium that coexists with animals including humans, and is known to coexist not only with the skin but also with the gastrointestinal tract. In most cases, the bacterium does not cause disease, but it is Propionibacerium acnes (Acne). It is known to use fatty acids present in sebum secreted by sebaceous glands as an energy source and the most important causative bacteria for skin diseases such as acne.
  • propionibacterium Bacteria in the propionibacterium are industrially used in the synthesis of vitamin B12, tetrapyrrole compounds and propionic acid, probiotics, and cheese production. To date, it has not been reported that bacteria in Propionibacterium secrete extracellular vesicles, and propionibacterium, preferably propionibacterium acnes, is a homologous species of propionibacterium acnes. Studies have been conducted only on the treatment of allergies, influenza vaccines, and digestive tract dysfunction (see Japanese Patent Application Laid-Open No. JP 2016-028033 and Korean Patent Registration No. KR 10-1459166).
  • the androgen receptor is an intracellular receptor that binds to male hormones such as testosterone and dihydrotestosterone, and plays an important role in the expression and maintenance of male phenotypes such as muscle mass, bone density, and hair growth.
  • male hormone is known to play an important role in the prevention of osteoporosis, senescence through androgen receptor.
  • the inventors of the present invention the vesicles derived from the propionibacterium bacteria, cancer, chronic inflammatory diseases, endocrine diseases, The present invention has been completed based on the fact that it is reduced in the blood of patients with metabolic diseases.
  • the present inventors confirmed that the vesicles can be used as a composition for preventing or treating cancer, inflammatory diseases, endocrine diseases, or metabolic diseases by first separating the vesicles from the bacteria of propionibacterium and confirming their properties.
  • the contents of bacterial vesicles derived from propionibacterium were significantly higher in cancer patients such as liver cancer, breast cancer, and atopic dermatitis, asthma, diabetes mellitus, cirrhosis, etc.
  • cancer patients such as liver cancer, breast cancer, and atopic dermatitis, asthma, diabetes mellitus, cirrhosis, etc.
  • the vesicles derived from propionibacterium were isolated in vitro and evaluated for therapeutic efficacy, anti-inflammatory and keratinocyte killing effects were shown.
  • the male hormone receptor As a result of increasing the expression, the present invention was completed.
  • an object of the present invention is to provide a method for providing information for the diagnosis of cancer, inflammatory diseases, endocrine diseases, or metabolic diseases.
  • Another object of the present invention is to provide a composition for preventing, ameliorating or treating cancer, inflammatory disease, endocrine disease, or metabolic disease, which comprises a bacterium-derived vesicle belonging to propionibacterium as an active ingredient.
  • the present invention provides a method for providing information for the diagnosis of cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, including the following steps.
  • the patient derived sample may be blood or urine.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, including the vesicles derived from propionibacterium bacteria as an active ingredient.
  • the bacteria in the Propionibacterium may be Propionibcerium acnes (Propionibacerium acnes).
  • the vesicles may have an average diameter of 10 to 1000 nm.
  • the vesicles may be secreted naturally or artificially in bacteria of the genus Propionibacterium.
  • the patient-derived sample may be urine or blood.
  • the composition may be an inhalant.
  • the composition may use a protein contained in the propionibacterium Acnes-derived vesicles.
  • the cancer may be liver cancer or breast cancer.
  • the inflammatory disease may be atopic dermatitis, asthma, diabetes, or cirrhosis of the liver.
  • the endocrine disease may be osteoporosis, senility (frailty), hair loss.
  • the metabolic disease may be diabetes or cirrhosis of the liver.
  • the present invention provides a food composition for preventing or improving cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, including the vesicles derived from propionibacterium bacteria as an active ingredient.
  • the present invention provides a cosmetic composition for improving cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, including the vesicles derived from the propionibacterium bacteria as an active ingredient.
  • the present invention also provides a method for preventing or treating cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, comprising administering to a subject a pharmaceutical composition comprising a bacterium-derived vesicle belonging to propionibacterium as an active ingredient. do.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a bacterium-derived vesicle of propionibacterium as an active ingredient for the prevention or treatment of cancer, inflammatory diseases, endocrine diseases, or metabolic diseases.
  • the present inventors confirmed that the activity of the bacteria in the propionibacterium is increased by fat by increasing the content of the vesicle-derived bacteria in the propionibacterium in the high fat diet-derived stool sample compared to the high-carbohydrate mice.
  • Bacterial-derived vesicles in the blood of patients have significantly reduced bacterial vesicles in propionibacterium in the blood of patients with liver cancer, breast cancer, asthma, atopic dermatitis, diabetes mellitus, and liver cirrhosis. It was confirmed.
  • propionibacterium acnes a species of bacteria in the propionibacterium, was cultured in vitro to isolate the vesicles, and when administered to mice, it was experimentally confirmed to increase the expression of the androgen receptor in the prostate tissue.
  • the vesicle-derived vesicles of propionibacterium according to the present invention are a cancer, inflammatory disease, endocrine disease, or metabolic disease. It is expected to be useful for diagnostic or predictive methods, pharmaceutical compositions, foods, and cosmetics.
  • RCD carbohydrate diet
  • HFD high fat diet
  • Figure 2 is a high-carbohydrate diet (RCD) and 12 weeks high-fat diet (HFD) mouse and vesicles from the stool to separate the results of the meta-genomic analysis in each.
  • RCD high-carbohydrate diet
  • HFD high-fat diet
  • Figure 3a is a photograph of the distribution of bacteria and vesicles over time after the oral administration of enteric bacteria and bacteria-derived vesicles (EV) to the mouse.
  • Figure 3b is a 12 hours after oral administration, blood, kidneys, and various organs were extracted to evaluate the distribution pattern of bacteria and vesicles in the body.
  • Figure 4 is a schematic diagram of a method for analyzing bacteria-derived vesicle metagenome in human derivatives.
  • Figure 5a is a result of comparing the distribution of bacteria-derived vesicles in propionibacterium after the analysis of bacteria-derived vesicles metagenome present in liver cancer patients and normal blood matched age and sex.
  • Figure 5b is a result of comparing the distribution of bacteria-derived vesicles in propionibacterium after the analysis of bacteria-derived vesicles metagenomics present in breast cancer patients and blood of age and sex matched normal.
  • Figure 6a is a result of comparing the distribution of bacteria-derived vesicles in propionibacterium after the analysis of bacteria-derived vesicles metagenome present in normal asthma patients and blood of age and sex matched.
  • Figure 6b is a result of comparing the distribution of bacteria-derived vesicles in propionibacterium after performing a bacterial-derived vesicle metagenome analysis present in atopic dermatitis patients and normal blood matched with age and sex.
  • Figure 7a is a result of comparing the distribution of bacteria-derived vesicles in propionibacterium after the analysis of bacteria-derived vesicles metagenomics present in diabetic patients and blood of age and sex matched normal.
  • Figure 7b is a result of comparing the distribution of bacteria-derived vesicles in propionibacterium after the analysis of bacterial-derived vesicles metagenomics present in liver cirrhosis patients and blood of normal age and sex matched.
  • Figure 8a is a result of culturing propionibacterium acnes in vitro and separating the vesicles from the culture medium and observed the shape of the vesicles with an electron microscope.
  • Figure 8b is the result of measuring the size of the vesicles separated from propionibacterium Acnes culture medium by dynamic light scattering method.
  • Figure 9a shows the analysis of the proteome contained in the propionibacterium Acnes-derived vesicles are shown to classify the identified proteins as cell components.
  • Figure 9b shows the results of analyzing the proteome contained in the propionibacterium Acnes-derived vesicles are shown to classify the identified proteins as a function.
  • Figure 10a is a result of measuring the degree of apoptosis after treatment with a propionibacterium Acnes-derived vesicles macrophages.
  • Figure 10b is the result of measuring the secretion amount of IL-6, an inflammation medium after the propionibacterium Acnes-derived vesicles treated with macrophages.
  • E. coli EV E. coli vesicles
  • IL-6 Escherichia coli vesicles
  • propionibacterium acnes-derived vesicles at various concentrations. The result of evaluating the impact.
  • FIG. 11B shows the secretion of TNF- ⁇ , an inflammatory mediator of Escherichia coli vesicles, when E. coli EVs, which are pathogenic vesicles, are pretreated with various concentrations of vesicle vesicles derived from propionibacterium acnes in mouse macrophage lines. This is the result of evaluating the impact.
  • 12A shows the results of evaluating the degree of killing keratinocytes caused by Staphylococcus aureus-derived vesicles after treating S. aureus EV vesicles at various concentrations in the skin keratinocytes.
  • 12B is a result of evaluating the degree of killing keratinocytes by propionibacterium acnes-derived vesicles after treatment with various concentrations of propionibacterium acnes-derived vesicles (P. acnes EV) in the skin keratinocytes.
  • P. acnes EV propionibacterium acnes-derived vesicles
  • propionibacterium acnes-derived vesicles P. acnes EV
  • S. aureus EV vesicles pathogenic vesicles of skin diseases, various concentrations It is the result of measuring the apoptosis of keratinocytes by pretreatment of the propionibacterium acne-derived vesicles with keratinocytes, followed by treatment with high concentrations of Staphylococcus aureus-derived vesicles.
  • the present invention relates to vesicles derived from bacteria of the genus Propionibacterium and uses thereof.
  • the present inventors conducted metagenome analysis of the contents of bacteria derived from propionibacterium in the samples derived from patients with endocrine-metabolic diseases such as liver cancer, breast cancer and other inflammatory diseases such as atopic dermatitis and asthma, diabetes and liver cirrhosis, compared to normal people. It was confirmed that this is significantly reduced, the present invention was completed based on this.
  • the present invention provides an information providing method for diagnosing cancer, inflammatory disease, endocrine disease, or metabolic disease, comprising the following steps.
  • diagnosis in the broad sense means to determine the actual condition of the patient in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. Diagnosis in the present invention is to determine the onset of cancer, inflammatory diseases, or metabolic diseases and the level of the disease.
  • Cancer the disease to be diagnosed according to the present invention, refers to a malignant tumor that grows rapidly while infiltrating surrounding tissues and spreads or metastasizes to various parts of the body and threatens life.
  • Cells the smallest unit of the body, divide and grow normally under the control function of the cells themselves, and die off at the end of their life or damage to maintain the balance of the overall number, but for a number of reasons If a problem occurs in the cell's own regulatory function, abnormal cells that normally must die will overgrow, invading surrounding tissues and organs, forming a mass, and destroying or modifying existing structures.
  • the cancer may preferably be liver cancer or breast cancer, but is not limited thereto.
  • the term "inflammatory disease” refers to a disease caused by an inflammatory response in a mammalian body. Representative examples include respiratory inflammatory diseases such as asthma, chronic obstructive pulmonary disease and rhinitis. ; Skin inflammatory diseases such as atopic dermatitis, psoriasis, acne and contact dermatitis; Gastrointestinal inflammatory diseases such as gastritis, peptic ulcer, and inflammatory bowel disease; And complications thereof.
  • the inflammatory disease is used in the sense including cancer associated with the inflammatory response, in addition to the general inflammatory disease, and includes, for example, lung cancer, stomach cancer, colon cancer and the like.
  • the inflammatory disease preferably means, but is not limited to, asthma or atopic dermatitis.
  • endocrine disease refers to the occurrence of disorders caused by excess or deficiency of hormones in the body of a mammal, for example, in the reduction of breast cancer and male hormones caused by the excess of female hormones.
  • endocrine diseases preferably include, but are not limited to, osteoporosis, senility, or hair loss.
  • metabolic disease refers to a disease caused by metabolic disorders and complications thereof in a mammal's body, for example, hyperlipidemia due to lipid metabolism abnormality, diabetes due to carbohydrate metabolism abnormality Or cirrhosis such as metabolic complications, and in the present invention, metabolic diseases preferably include, but are not limited to, diabetes or cirrhosis.
  • the term "nanovesicle” or “vesicle” refers to a structure of nanoscale membranes secreted by various bacteria.
  • Gram-negative bacteria-derived vesicles or outer membrane vesicles (OMVs) contain toxic proteins, bacterial DNA and RNA as well as lipopolysaccharides, and gram-positive bacteria-derived vesicles.
  • OMVs outer membrane vesicles
  • the nano-vesicles or vesicles are naturally secreted or artificially produced by the bacteria of the Propionibacterium, spherical form, has an average diameter of 10 to 1000 nm.
  • the vesicles were centrifuged, ultra-fast centrifugation, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by filter, gels containing culture medium containing bacteria of propionibacterium.
  • the separation can be carried out using one or more methods selected from the group consisting of filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
  • the term "metagenome” used in the present invention also referred to as "gunoelectric”, refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated region such as soil, animal intestine, mainly culture It is used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms that are not.
  • the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species.
  • rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
  • the patient-derived sample may be blood or urine, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, including the vesicles derived from propionibacterium bacteria as an active ingredient.
  • the present invention provides a food composition for preventing or ameliorating cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, comprising as an active ingredient a bacterium derived from propionibium bacteria.
  • the present invention provides a cosmetic composition for improving cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, comprising as an active ingredient a bacterium derived from propionibium bacteria.
  • prevention means any action that inhibits or delays the onset of cancer, inflammatory disease, endocrine disease, or metabolic disease by administration of a food, inhalant, or pharmaceutical composition according to the present invention. do.
  • treatment means any action that improves or advantageously changes the symptoms for cancer, inflammatory disease, endocrine disease, or metabolic disease by administration of the pharmaceutical composition according to the present invention.
  • the term " improvement" means at least a parameter related to a condition in which cancer, inflammatory disease, endocrine disease, or metabolic disease is treated by administration of a food or cosmetic composition according to the present invention, for example, the degree of symptoms It means all acts of diminishing.
  • bacterial metagenome analysis was performed using vesicles isolated from blood of breast cancer patients, liver cancer patients, and normal persons. As a result, it was confirmed that the vesicles derived from propionibacterium were significantly reduced in the blood of breast cancer and liver cancer patients compared to normal blood (see Example 3).
  • bacterial metagenome analysis was performed using vesicles isolated from blood of asthma patients, atopic dermatitis patients, and normal persons. As a result, it was confirmed that the vesicles derived from propionibacterium were significantly reduced in the blood of asthma and atopic dermatitis patients compared to normal blood (see Example 4).
  • bacterial metagenome analysis was performed using vesicles isolated from blood of diabetic patients, cirrhosis patients, and normal persons. As a result, compared with normal blood, it was confirmed that the vesicles derived from propionibacterium were significantly reduced in blood of diabetic and cirrhosis patients (see Example 5).
  • the vesicles It was confirmed that the average diameter is less than 200 nm, preferably a circle size of 37.8 ⁇ 13.5 nm (see Example 6).
  • 252 proteins present in propionibacterium acnes-derived vesicles were identified by proteome analysis, and are classified and shown by cell constituents and protein functions (see Example 7).
  • Staphylococcus aureus EV vesicles are a major causative factor of atopic dermatitis ).
  • the treatment of keratinocytes caused by Staphylococcus aureus vesicles is inhibited by the propionibacterium acne-derived vesicles. (See Example 9).
  • one of the therapeutic mechanisms for endocrine diseases of propionibacterium acnes-derived vesicles to evaluate the effect on male hormone receptor expression the propionibacterium acne-derived vesicles in the mouse
  • the propionibacterium acne-derived vesicles in the mouse As a result of extracting and analyzing prostate tissue after administration, it was confirmed that when the vesicles were administered, the expression of androgen receptor was increased in the prostate tissue (see Example 10).
  • Stool was collected according to the method shown in FIG. 1 in a normal mouse fed a high-carbohydrate diet (RCD) for 12 weeks and a mouse fed a high fat diet (HFD) for 2 months and inducing obesity. It was.
  • the weight (grams) of the collected mouse feces was then measured and dispersed in PBS.
  • the supernatant was separated by centrifugation for 5 minutes at 500 ⁇ g, 5 minutes at 3,000 ⁇ g, and 5 minutes at 4,350 rpm, followed by ultrafast centrifugation at 10,000 ⁇ g for 20 minutes.
  • the bacteria contained in the feces were isolated.
  • the supernatant from which bacteria were removed was filtered using a 0.45 ⁇ m filter, followed by protein quantification. Thereafter, 0.8 M and 2.5 M sucrose cushions were made, and ultra-high centrifugation was repeatedly performed twice at 2,800 ° C. for 2 hours at 4 ° C., and ultra-high centrifugation was performed at 4,200 ° C. for 2 hours at 40 ° C. to obtain stool-derived vesicles. And the resulting vesicles were dispersed and stored in PBS.
  • the bacteria and vesicles separated by the above method were boiled at 100 ° C. to let the DNA inside the lipids out and then cooled on ice for 5 minutes. And centrifuged at 10,000 x g, 4 °C 30 minutes to remove the remaining suspended solids and collected only the supernatant. The amount of DNA was quantified using Nanodrop. Thereafter, PCR was performed with the 16s rRNA primer shown in Table 1 to confirm whether the bacteria-derived DNA exists in the extracted DNA, and it was confirmed that the bacteria-derived gene was present in the extracted gene.
  • DNA extracted by the above method was amplified using the above 16S rDNA primers, followed by sequencing (Illumina MiSeq sequencer), and the results were outputted in a Standard Flowgram Format (SFF) file, using GS FLX software (v2.9).
  • SFF Standard Flowgram Format
  • GS FLX Standard Flowgram Format
  • Clustering is performed according to sequence similarity using UCLUST and USEARCH for Operational Taxonomy Unit (OTU) analysis, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity Clustering is based on the phylum, class, order, family, and genus levels of each OTU, and BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) is used to identify bacteria with greater than 97% sequence similarity at the genus level. Was profiled (QIIME).
  • OTU Operational Taxonomy Unit
  • the bacteria were not absorbed systemically, but in the case of the bacteria-derived vesicles, they were absorbed systemically 5 minutes after administration, and the bladder was fluorescence at 30 minutes of administration. Observed strongly, the vesicles were found to be excreted by the urinary system. In addition, the vesicles were found to exist in the body until 12 hours of administration.
  • the bacteria-derived vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, muscle and kidney, but bacteria were not absorbed. there was.
  • the blood was allowed to settle by centrifugation (3,500 x g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing bacteria and foreign substances using a 0.22 ⁇ m filter, it was transferred to centripreigugal filters (50 kD) and centrifuged at 1500 x g and 4 ° C for 15 minutes to discard materials smaller than 50 kD and concentrated to 10 ml.
  • centripreigugal filters 50 kD
  • the genes were extracted from the vesicles present in the blood of 277 asthma patients and 246 blood of the normal control group.
  • Bacterial-derived vesicles in Onibacterium were significantly reduced (normal vs asthmatic: 1.8% vs 0.2%; fold change: 0.12, p ⁇ 0.000001).
  • the blood was extracted from the vesicles present in the blood of the blood of 100 patients with liver cirrhosis and 100 of the normal control group.
  • propionibacterium acnes strains were cultured and their vesicles were separated and analyzed.
  • Propionic sludge tumefaciens arc Ness (P. acnes) after the the 6919 strain absorbance (OD600) at 37 °C anaerobic chamber cultured in BHI (brain heart infusion) culture medium until a 1.0 to 1.5 were sub-culture. Since no P. acnes The media supernatant of 6919 was recovered, centrifuged at 10,000 g, 4 ° C.
  • the filtered supernatant was purified by ultrafiltration using a QuixStand benchtop system (GE Healthcare, UK) using a 100 kDa hollow filter membrane. Concentrated to mL volume. Thereafter, the concentrated supernatant was once again filtered with a 0.22 ⁇ m filter, and the filtered supernatant was ultracentrifuged at 150,000 g, 4 ° C. for 3 hours, and the pellet was suspended in DPBS.
  • Optiprep solution was prepared using HEPES-buffered saline (20 mM HEPES) to prepare low density solution. , 150 mM NaCl, pH 7.4) was used. After centrifugation for 2 hours at 200,000 g, 4 ° C conditions, the ultracentrifugation was further performed for 3 hours at 150,000 g, 4 ° C each solution fractionated in the same volume of 1 mL from the upper layer. Thereafter, the protein was quantified by BCA assay, and the following experiments were performed on the obtained vesicles.
  • the vesicles were separated from the culture medium of propionibacterium acnes cultured according to the above method, and the shape and size were evaluated by electron microscopy.
  • proteome analysis was performed. To this end, trypsin digested peptides were obtained through FASP (Filter-aid sample preparation) digestion. 18 ⁇ g of protein extracted from propionibacterium acne-derived vesicles was mixed with a reducing solution (4% SDS and 0.1 M DTT in 0.1 M Tris-HCl, pH 7.6) and reacted at 37 ° C. for 45 minutes. After boiling for 7 minutes, centrifugation-based filter was performed at 16 ° C for 40 minutes at 14,000 g.
  • FASP Ferter-aid sample preparation
  • UA solution 0.2 mL of 8 M urea in 0.1 M Tris-HCl, pH 8.5
  • 0.1 ml of 55 mM IAA solution was added and the reaction was performed at room temperature for 20 minutes. And centrifuged for 40 minutes.
  • the sample was diluted in 0.2 ml of 100 mM TEAB solution and centrifuged twice for further solution exchange for trypsin treatment.
  • the filter is placed in a 1.5 ml tube and 100 mM TEAB solution containing high purity trypsin is digested for 12 hours at 37 ° C in the filter, followed by centrifugation at 16 ° C for 20 minutes at 14,000 g for 100 mM TEAB. The process of adding 75 ⁇ l of solution was repeated twice.
  • the collected samples were dried with a SpeedVac concentrator, then dissolved in 180 ⁇ l of 5% ACN dissolved solution (0.1% formic acid in distilled water solvent) and decontaminated with a C-18 rotary column (Thermo Scientific, USA), followed by vacuum drying. I was.
  • the dried samples were analyzed with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Germany) in conjunction with EASY nLC1000 (Thermo Fisher Scientific, Germany) using the nano-LC-ESI-MS / MS method. Trypsin-cut peptides were loaded into a trap column (75 ⁇ m ⁇ 2 cm) packed with C18 3- ⁇ m resin and then subjected to a speed of 300 nl / min for 85 minutes using a 5% -40% linear gradient of B solvent. Eluted. Eluted peptides were separated by an analytical column (75 ⁇ m ⁇ 50 cm) packed with C18 2- ⁇ m resin, followed by electrospraying at 2.0 kV to the nano ESI source.
  • the Q Exactive mass spectrometer was operated in a top 10 data-dependent method. Higher-energy collisional dissociation separation is achieved by setting the normalized collisional energy to 30%, the dynamic exclusion time to 30 seconds, and the precursor isolation window to 2. The most abundant precursor ions were selected using a survey scan (m / z; 400-2,000). Survey MS scans were obtained with a resolution of 70,000 using an Orbitrap analyzer with the HCD Spectra resolution set to 17,500.
  • propionibacterium acnes-derived vesicles As a result, as shown in FIGS. 9A and 9B, among proteins analyzed in propionibacterium acnes-derived vesicles, highly reliable proteins detected two or more times in three repetitions were selected, resulting in a total of 252 proteins. Found. More than half the properties of the proteins analyzed were not well known because of the lack of proteomic studies of propionibacterium acnes. The distribution ratio of the analyzed proteins by cell component was 27.4% for membrane-related protein, 7.1% for cytoplasmic protein, 1.2% for extracellular protein and 4.4% for liposome protein. This means that propionibacterium acnes-derived vesicles, like commonly known bacterial vesicles, are wrapped in membranes and contain both cytoplasmic and extracellular proteins.
  • propionibacterium acnes-derived vesicles are important for metabolic processes and mass transport of propionibacterium acnes. It is expected to play a role.
  • propionibacterium acnes-derived vesicles exhibit prominent features such as biochemical lifestyle, survival, cell adhesion, pathogenicity and inflammatory properties of propionibacterium acnes. It can be seen that.
  • oxidative phosphorylation cytochrome c oxidase, Rieske domain protein
  • nitrogen fixation bacterial cytochrome ubiquinol oxidase, cytochrome d ubiquinol oxidase
  • oxygen-free breathing succinate dehydrogenases, methylmalonyl-CoA mutase
  • ATP-binding cassettes-related proteins are present, consistent with the fact that Propionibacterium Acnes has an aerobic and anaerobic nonkinetic lifestyle.
  • proteins in the propionibacterium acnes-derived vesicles have functions related to transcription, translation, protein transport, and protein folding.
  • propionibacterium acnes-derived vesicles function as intercellular transport of biochemical processes and energy metabolism-related substances or proteins, and are large transporters that transport nutrients on behalf of the non-motile propionibacterium acnes. It can be expected to function as.
  • propionibacterium acnes is present in various environments. It can be expected that propionibacterium acnes-derived vesicles will be used to survive and display antibiotic resistance at the site of infection.
  • Putative uncharacterized protein which binds to dermatan sulfate of host and shows immunity
  • Putative glycosyl-transferase which binds to epidermal cells of host and forms biofilm
  • Glyceraldehyde- which shows pathogenicity by attaching to cell
  • Propionibacterium acnes from proteins that play a key role in attaching cells or forming biofilms, such as 3-phosphate dehydrogenase (GAPDH), a polysaccharide deacetylase that binds to epidermal cells and escapes the host's immune response from lysozymes.
  • GPDH 3-phosphate dehydrogenase
  • Hyaluronate lyase which induces immune response in inflammatory cells by decomposing host's extracellular matrix
  • Endoglycoceramidase which breaks down glycosphingolipid on cell surface to prevent signal transduction
  • Endo-beta-N-acetylglucosaminidase H which metabolizes host cell-derived substrate
  • propionibacterium acnes-derived vesicles can be efficiently transferred into cells from proteins such as Christie-Atkins-Munch-Peterson (CAMP) factors, which invade the host and puncture cells.
  • CAMP Christie-Atkins-Munch-Peterson
  • NlpC / P60 endopeptidase family protein, basic membrane protein, 60 kDa chaperonin, and 10 kDa chaperonin are essential proteins for the survival of propionibacterium acnes.
  • propionibacterium acnes derived protein can be used for immunomodulation through propionibacterium acnes derived vesicles by stimulating the host's immune response.
  • Proteins identified through propionibacterium acnes derived vesicle proteome analysis are shown in Table 2 below.
  • propionibacterium acnes-derived vesicles were applied to raw macrophage cell lines, Raw 264.7 cells. EV) was treated at various concentrations (0.001, 0.01, 0.1, 1, 10 ⁇ g / ml) and then secreted by inflammatory mediators (IL-6, TNF- ⁇ , etc.) through apoptosis and ELISA (R & D system, USA), respectively.
  • IL-6 inflammatory mediators
  • apoptosis and ELISA R & D system, USA
  • propionibacterium Acnes-derived vesicles were mixed and treated with fresh DMEM complete medium, and then cultured in a 37 °C incubator for 6 hours to 24 hours to obtain a culture supernatant. .
  • the culture supernatant was collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, the supernatant was collected and stored at 4 ° C., followed by ELISA analysis.
  • the Capture antibody was diluted in PBS, 50 ⁇ l was dispensed into 96-well polystyrene plates according to the working concentration, and then reacted overnight at 4 ° C. After washing twice with 100 ⁇ l of PBST (PBS containing 0.05% tween-20) solution, dispense 100 ⁇ l of RD (PBST containing 1% BSA) solution and block for 1 hour at room temperature. After washing twice with 100 ⁇ l, 50 ⁇ l of the sample and standard were dispensed according to the concentration and reacted at room temperature for 2 hours.
  • PBST PBS containing 0.05% tween-20
  • the detection antibody was diluted in RD and 50 ⁇ l was dispensed at the action concentration for 2 hours at room temperature.
  • Strpetavidin-HRP was diluted to 1/200 in RD and 50 ⁇ l divided and reacted at room temperature for 30 minutes.
  • 50 ⁇ l of a solution of 1: 1 mixed TMB substrate and 0.04% hydrogen peroxide solution was dispensed and waited for color development.
  • 50 ⁇ l of sulfuric acid solution was dispensed to stop the reaction and the absorbance was measured at 450 nm using Synergy TM HT multi-detection microplate reader (BioTek, USA).
  • propionibacterium acnes-derived vesicles can effectively suppress inflammatory diseases such as asthma and metabolic diseases such as diabetes induced by pathogenic vesicles such as E. coli-derived vesicles.
  • Example 9 Skin dead skin cells caused by Staphylococcus aureus-derived vesicles Propionibacterium Acnes-derived parcels Anti-killing effect
  • Example 8 In order to evaluate the inhibitory effect of Propionibacterium acnes-derived vesicles on the killing of skin epithelial cells by Staphylococcus aureus-derived vesicles, the same pretreatment as in Example 8 was used using a keratinocyte line (HaCaT cell) instead of macrophages. After the procedure, the Staphylococcus aureus-derived vesicles and Propionibacterium acnes-derived vesicles were treated for 24 hours, and then MTT assay (Sigman, USA) was performed.
  • HaCaT cell keratinocyte line
  • Examples 3 and 5 confirmed that the vesicles derived from propionibacterium were significantly reduced in the blood of breast cancer and atopic dermatitis patients compared to normal people, and further, for the atopic dermatitis and breast cancer of the vesicles.
  • propionibacterium acnes-derived vesicles were treated in 6-week-old C57BL / 6 male mice to evaluate androgen receptor expression in mouse prostate tissue.
  • Each experimental group consisted of 1 ⁇ g of propionibacterium acnes-derived vesicles (PaEV), and 1 ⁇ g of peptidoglycan (PGN), a well-known antigen of PBS or propionibacterium acnes-derived vesicles, 1 ⁇ g of lipoteichoic acid (LTA) was injected intraperitoneally twice a week for a total of 4 weeks. Three days after the last injection, the prostate tissue was removed, ground, and supernatant was obtained, and the expression pattern of androgen receptor (AR) was confirmed by western blot.
  • PaEV propionibacterium acnes-derived vesicles
  • PPN peptidoglycan
  • LTA lipoteichoic acid
  • the extracted mouse prostate tissue was transferred to a 1.5 ml tube and placed in liquid nitrogen for rapid cooling. The tissue was then added to Tissuelyser II (Qiagen, Germany) with lysis solution and beads. . The ground tissue solution was transferred to a new 1.5 ml tube and centrifuged at 17,000 g for 15 minutes at 4 ° C. to obtain supernatant twice. Supernatant proteins were quantified using BCA assay. 150 ⁇ g of protein sample was mixed with loading solution and subjected to SDS-PAGE at 160 V for 1 hour in 4% stacking gel and 7.5% seperating gel, and then the PA gel was 400 mA 50 minutes with activated PVDF membrane for 5 minutes in 100% ethanol.
  • the transfer was carried out in an ice bucket. Tranferd PVDF membrane was shaken incubated for 40 minutes at room temperature in RD solution (TBS-T containing 5% skim milk). After washing three times with TBS-T for 30 minutes, the primary antibody against androgen receptor (androgen receptor) was diluted in 1/4000 RD and shaken incubation for 1 hour and half at room temperature. After washing three times with TBS-T for 30 minutes, the secondary antibody was diluted in RD to 1/4000 and shaken incubation for 1 hour at room temperature.
  • the ECL femto substrate (Thermo Scientific, USA) was diluted in distilled water in 1/10 and sprayed onto the PVDF membrane to obtain Western images with the LAS 4000 (GE Healthcare, UK) instrument. Western images for ⁇ -actin were used as controls.
  • Bacterial-derived vesicles of propionibacterium according to the present invention increased the expression of androgen receptors in prostate tissue, and when the vesicles were pretreated in inflammatory cells, the effects of anti-inflammatory and immunomodulatory functions were observed.
  • the present invention is expected to be useful for diagnosing or predicting inflammatory diseases, endocrine diseases, or metabolic diseases, pharmaceutical compositions, foods, and cosmetics.

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Abstract

La présente invention concerne des vésicules dérivées de bactéries du genre Propionibacterium et leur utilisation. Il a été confirmé de manière expérimentale que la production de vésicules dérivées de bactéries du genre Propionibacterium était augmentée dans le corps par un régime riche en matières grasses plutôt qu'un régime riche en glucides ; les vésicules étaient significativement réduites dans le sang de patients atteints de cancers, tels que le cancer du sein et le cancer du foie, de maladies inflammatoires, telles que l'asthme et la dermatite atopique, et de maladies métaboliques, telles que le diabète et la cirrhose du foie, par rapport aux personnes normales ; et les vésicules inhibaient la sécrétion de médiateurs inflammatoires par des vésicules pathogènes, inhibaient l'apoptose des kératinocytes et augmentaient l'expression du récepteur des androgènes dans le corps. Les vésicules dérivées de bactéries du genre Propionibacterium selon la présente invention devraient être avantageusement utilisées dans un procédé de diagnostic ou de prédiction de cancers, de maladies inflammatoires, de maladies endocriniennes ou de maladies métaboliques ; dans une composition pharmaceutique, un aliment, un produit cosmétique et analogue.
PCT/KR2017/006889 2016-07-08 2017-06-29 Nanovésicules dérivées de bactéries du genre propionibacterium et leur utilisation Ceased WO2018008895A1 (fr)

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CN201780042554.XA CN109715832B (zh) 2016-07-08 2017-06-29 源自丙酸杆菌属的细菌的纳米囊泡及其用途
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CN110546278A (zh) * 2017-02-24 2019-12-06 Md保健株式会社 通过细菌宏基因组分析来诊断慢性阻塞性呼吸道疾病的方法
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CN112041465A (zh) * 2018-02-08 2020-12-04 Md保健株式会社 来源于乳球菌属细菌的纳米囊泡及其用途
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WO2018225945A1 (fr) * 2017-06-07 2018-12-13 주식회사 엠디헬스케어 Procédé de diagnostic de la dermatite atopique par analyse métagénomique microbienne
WO2019078434A1 (fr) * 2017-10-18 2019-04-25 주식회사 엠디헬스케어 Procédé de diagnostic du cancer de la tête et du cou par l'intermédiaire de l'analyse métagénomique bactérienne
WO2019078433A1 (fr) * 2017-10-18 2019-04-25 주식회사 엠디헬스케어 Procédé de diagnostic du syndrome métabolique par l'intermédiaire de l'analyse métagénomique bactérienne
US11898156B2 (en) 2018-01-12 2024-02-13 Md Healthcare Inc. Nano-vesicles derived from genus Morganella bacteria and use thereof
JP7191411B2 (ja) 2018-01-12 2022-12-19 エムディー ヘルスケア インコーポレイテッド フィーカリバクテリウム・プラウスニッツィイ由来のナノ小胞およびその用途
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WO2019139279A1 (fr) * 2018-01-12 2019-07-18 주식회사 엠디헬스케어 Nanovésicules issues de bactéries morganella et utilisations associées
JP2021510300A (ja) * 2018-01-12 2021-04-22 エムディー ヘルスケア インコーポレイテッドＭｄ Ｈｅａｌｔｈｃａｒｅ Ｉｎｃ． フィーカリバクテリウム・プラウスニッツィイ由来のナノ小胞およびその用途
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JP2022008864A (ja) * 2018-01-12 2022-01-14 エムディー ヘルスケア インコーポレイテッド フィーカリバクテリウム・プラウスニッツィイ由来のナノ小胞およびその用途
US11666607B2 (en) 2018-01-12 2023-06-06 Md Healthcare Inc. Nanovesicles derived from Faecalibacterium prausnitzii and uses thereof
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JP2020513799A (ja) * 2018-02-21 2020-05-21 エムディー ヘルスケア インコーポレイテッドＭｄ Ｈｅａｌｔｈｃａｒｅ Ｉｎｃ． クプリアウィドゥス属細菌由来のナノ小胞及びその使用
US11771742B2 (en) 2018-02-21 2023-10-03 Md Healthcare Inc. Nano-vesicles derived from genus cupriavidus bacteria and use thereof
EP3567125A4 (fr) * 2018-02-21 2020-09-02 MD Healthcare Inc. Nano-vésicules dérivées de bactéries de l'espèce cupriavidus et utilisation associée
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