WO2018008895A1 - Nano-vesicles derived from bacteria of genus propionibacterium and use thereof - Google Patents

Abstract

The present invention relates to vesicles derived from bacteria of the genus Propionibacterium and a use thereof. It was experimentally confirmed that the production of vesicles derived from bacteria of the genus Propionibacterium was increased in the body by a high-fat diet rather than a high-carbohydrate diet; the vesicles were significantly reduced in the blood of patients with cancers, such as breast cancer and liver cancer, inflammation diseases, such as asthma and atopic dermatitis, and metabolic diseases, such as diabetes and liver cirrhosis, compared with normal persons; and the vesicles inhibited the secretion of inflammatory mediators by pathogenic vesicles, inhibited the apoptosis of keratinocytes, and increased the expression of an androgen receptor in the body. The vesicles derived from bacteria of the genus Propionibacterium according to the present invention are expected to be advantageously used in a method for diagnosis or prediction of cancers, inflammatory diseases, endocrine diseases, or metabolic diseases, a pharmaceutical composition, a food, a cosmetic product, and the like.

Description

Nanovesicles from Bacteria of Propionibacterium and Their Uses

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.

In the 21st century, acute infectious diseases, which have been recognized as infectious diseases in the past, have become less important, while chronic diseases with immune dysfunctions caused by incompatibility between humans and microbiomes, especially cancer, cardiovascular diseases and chronic lung diseases. Metabolic diseases, neuro-psychiatric diseases, etc. are becoming a big problem in the 21st century.

The microorganisms symbiotic to the human body reaches 100 trillion times more than human cells, and the number of genes of microorganisms is known to be more than 100 times the number of human genes. Microbiota (microbiota or microbiome) 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.

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. 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).

Meanwhile, 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. . In addition, male hormone is known to play an important role in the prevention of osteoporosis, senescence through androgen receptor.

Accordingly, 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.

Specifically, 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. When the vesicles derived from propionibacterium were isolated in vitro and evaluated for therapeutic efficacy, anti-inflammatory and keratinocyte killing effects were shown. As a result of evaluating the action mechanism of the vesicles, the male hormone receptor As a result of increasing the expression, the present invention was completed.

Accordingly, 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.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, 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.

(a) extracting DNA from vesicles isolated from normal and patient derived samples;

(b) performing PCR on the extracted DNA by using a pair of vesicle detection primers derived from propionibacterium bacteria present in the 16S rDNA sequence; And

(c) Determining cancer, inflammatory diseases, endocrine diseases, or metabolic diseases when the content of the bacteria-derived vesicles in propionibacterium is lower than that of normal people through quantitative analysis of the PCR products.

In one embodiment of the invention, the patient derived sample may be blood or urine.

In another aspect, 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.

In one embodiment of the present invention, the bacteria in the Propionibacterium may be Propionibcerium acnes (Propionibacerium acnes).

In another embodiment of the present invention, the vesicles may have an average diameter of 10 to 1000 nm.

Of the present invention In another embodiment, the vesicles may be secreted naturally or artificially in bacteria of the genus Propionibacterium.

In another embodiment of the present invention, the patient-derived sample may be urine or blood.

In another embodiment of the invention, the composition may be an inhalant.

In another embodiment of the present invention, the composition may use a protein contained in the propionibacterium Acnes-derived vesicles.

In another embodiment of the present invention, the cancer may be liver cancer or breast cancer.

In another embodiment of the present invention, the inflammatory disease may be atopic dermatitis, asthma, diabetes, or cirrhosis of the liver.

In another embodiment of the present invention, the endocrine disease may be osteoporosis, senility (frailty), hair loss.

In another embodiment of the present invention, the metabolic disease may be diabetes or cirrhosis of the liver.

In another aspect, 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.

In another aspect, 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.

In another aspect, the present invention provides 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. In addition, 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. When the vesicles were pretreated with inflammatory cells, the effects of anti-inflammatory and immunomodulatory functions were observed. 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.

1 is a graphical illustration of a protocol for separating vesicles from high carbohydrate diet (RCD) and high fat diet (HFD) mouse feces and analyzing the metagenome.

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.

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.

11a shows the effect of E. coli vesicles (E. coli EV) on the secretion of inflammatory mediator IL-6 during the treatment of Escherichia coli vesicles (E. coli EV) after pretreatment with propionibacterium acnes-derived vesicles at various concentrations. The result of evaluating the impact.

FIG. 11B shows the secretion of TNF-α, 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.

13 is to evaluate the inhibitory effect of propionibacterium acnes-derived vesicles (P. acnes EV) on skin death by 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.

14 is a result of evaluating the expression pattern of the androgen receptor in prostate tissue by Western blot after administration of propionibacterium acnes-derived vesicles into the abdominal cavity 8 times for 4 weeks.

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.

Accordingly, the present invention provides an information providing method for diagnosing cancer, inflammatory disease, endocrine disease, or metabolic disease, comprising the following steps.

(a) extracting DNA from vesicles isolated from normal and patient derived samples;

(b) performing PCR on the extracted DNA by using a pair of vesicle detection primers derived from propionibacterium bacteria present in the 16S rDNA sequence; And

(c) Determining cancer, inflammatory diseases, endocrine diseases, or metabolic diseases when the content of the bacteria-derived vesicles in propionibacterium is lower than that of normal people through quantitative analysis of the PCR products.

As used herein, the term "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. In the present invention, the cancer may preferably be liver cancer or breast cancer, but is not limited thereto.

As used herein, 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. In addition, 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. In the present invention, the inflammatory disease preferably means, but is not limited to, asthma or atopic dermatitis.

As used herein, the term "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. According to the present invention includes osteoporosis, senility, hair loss, and the like, and in the present invention, endocrine diseases preferably include, but are not limited to, osteoporosis, senility, or hair loss.

As used herein, the term "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.

As used herein, 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. In addition to proteins and nucleic acids, it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls. In the present invention, 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. In particular, 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. Technically, 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.

In the present invention, the patient-derived sample may be blood or urine, but is not limited thereto.

As another aspect of the present invention, 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.

As another aspect of the present invention, 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.

As another aspect of the present invention, 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.

As used herein, the term "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.

As used herein, the term "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.

As used herein, 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.

In the embodiment of the present invention, using a high-fat diet animal model, it was confirmed that the vesicle-derived bacteria of propionibacterium in the high-fat diet mouse stool is increased compared to the high-carbohydrate dietary stool (see Example 1).

In one embodiment of the present invention, 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).

In one embodiment of the present invention, 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).

In one embodiment of the present invention, 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).

In another embodiment of the present invention, based on the results of Examples 3 to 5 further study to analyze the properties of the propionibacterium acne species bacterial vesicle belonging to the genus propionibacterium bacteria, 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).

In yet another embodiment of the present invention, 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).

In another embodiment of the present invention, by culturing the propionibacterium Acnes strain and evaluating how the vesicles secreted from it affects the secretion of inflammatory mediators, as a result of the propionibacterium Acnes-derived vesicles in macrophages IL-6 secretion was significantly lower than that of Escherichia coli-derived vesicles (E. coli EV). In addition, after treating various concentrations of propionibacterium acnes-derived vesicles in macrophages, and treating E. coli vesicles, which cause inflammation and metabolic diseases, the secretion of inflammatory mediators was evaluated. And it was confirmed that the vesicles derived from Propionibacterium Acnes effectively inhibit TNF-α secretion (see Example 8).

In another embodiment of the present invention, in order to cultivate propionibacterium acnes strains and the vesicles secreted therefrom to evaluate the atopic dermatitis treatment effect, 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).

In another embodiment of the present invention, 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 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).

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  One. High Carb Diet  And bacteria in high fat mouse stool Germ-derived  parcel Metagenome  analysis

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. In order to remove foreign substances, 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. In addition, in order to separate the vesicles, the supernatant from which bacteria were removed was filtered using a 0.45 μm 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 ℃ 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). After converting the SFF file into a sequence file (.fasta) and a nucleotide quality score file, the credit rating of the lead is checked, and the window (20 bps) has an average base call accuracy of less than 99% (Phred score <20). It was. 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).

As a result of metagenome analysis of bacteria and bacteria-derived vesicles in the mouse stool administered with a high carbohydrate diet and a high fat diet for 12 weeks, as shown in FIG. 2, the distribution of the bacteria in the propionibacterium itself (Bacteria) is The high carbohydrate diet and the high fat diet were not different between the mice, but the distribution of propionibacterium-derived vesicles (EV) was found to be significantly increased in the feces of the high fat diet compared to the high carbohydrate mice.

Example  2. Analysis of Absorption, Distribution, and Excretion of Intestinal Bacteria and Bacterial-Derived Vesicles

In order to evaluate whether intestinal bacteria and bacterial-derived vesicles are absorbed systemically through the gastrointestinal tract, experiments were carried out in the following manner. Fluorescently labeled enterobacteriaceae and enteric bacteria-derived vesicles were administered to the gastrointestinal tract at doses of 50 μg, respectively, and the fluorescence was measured after 0, 5, 3, 6 and 12 hours.

As a result of observing the whole image of the mouse, as shown in FIG. 3A, 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.

After the intestinal bacteria and enteric bacteria-derived vesicles were absorbed systemically, in order to evaluate the infiltration into various organs, 50 μg of fluorescently labeled bacteria and bacteria-derived vesicles were administered as described above, followed by 12 hours. Blood, heart, liver, kidneys, spleen, fat and muscle were collected later.

As a result of fluorescence observation in the collected tissue, as shown in FIG. 3B, the bacteria-derived vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, muscle and kidney, but bacteria were not absorbed. there was.

Example 3 Analysis of Bacterial-Derived Vesicle Metagenome in Blood of Normal, Breast, and Liver Cancer Patients

In order to first separate the vesicles from the patient and normal blood into a 10 ml tube, 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㎛ 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. Once again, the bacteria and foreign substances were removed using a 0.22㎛ filter, and the supernatant was discarded using ultra-centrifugation for 3 hours at 150,000 xg and 4 ℃ using a Type 90ti rotor, and the lumped pellet was dissolved in physiological saline (PBS). .

Blood of 96 breast cancer patients and 191 blood of normal control group matching gender and age; The genes were extracted from the blood of the vesicles in the blood of the normal control group which matched the sex and age with the blood of 91 liver cancer patients.

After performing the metagenome analysis by the method of Example 1, the distribution of bacteria-derived vesicles in the Propionibacterium at the genus level, as shown in Figure 5a, compared to the blood of breast cancer patients compared to normal blood as shown in Figure 5a Bacterial-derived vesicles of Onibacterium were significantly reduced (normal vs breast cancer: 2.2% vs 0.5%; fold change: 0.23; p <0.000001).

In addition, as shown in Figure 5b, it was confirmed that compared to the normal blood, hepatic cancer-derived vesicles of propionibacterium significantly decreased in the blood of liver cancer patients (normal vs liver cancer: 1.7% vs 0.6%; fold change: 0.34, p = 0.000001).

Example 4 Analysis of Bacterial-Derived Vesicle Metagenome in Blood of Normal, Asthmatic, and Atopic Dermatitis Patients

In the method of Example 3, the genes were extracted from the vesicles present in the blood of 277 asthma patients and 246 blood of the normal control group. After performing the metagenome analysis by the method of Example 1, the distribution of the bacteria-derived vesicles of the genus Propionibacterium at the genus level, as shown in Figure 6a, compared to the blood of asthma patients as compared to normal blood as shown in Figure 6a Bacterial-derived vesicles in Onibacterium were significantly reduced (normal vs asthmatic: 1.8% vs 0.2%; fold change: 0.12, p <0.000001).

In the method of Example 3, genes were extracted from the vesicles present in the blood of 25 atopic dermatitis patients and 138 blood of the normal control group. After performing the metagenome analysis by the method of Example 1, the distribution of the bacteria-derived vesicles from the Propionibacterium at the genus level, as shown in Figure 6b, compared to the blood of atopic dermatitis patients as compared to normal blood Bacterial-derived vesicles of propionibacterium were significantly reduced (normal vs. atopic dermatitis: 0.7% vs 0.1%; fold change: 0.15, p = 0.000003).

Example 5 Analysis of Bacterial-Derived Vesicle Metagenome in Blood of Normal, Diabetic, and Liver Cirrhosis Patients

In the method of Example 3, genes were extracted from vesicles present in the blood of 73 diabetic patients and 146 blood of the normal control group. After performing the metagenome analysis by the method of Example 1, the distribution of bacteria-derived vesicles of the genus Propionibacterium at the genus level, as shown in Figure 7a, as compared to the blood of asthma patients compared to normal blood as shown in Figure 7a Bacterial-derived vesicles of Onibacterium were significantly reduced (normal vs. diabetic: 1.3% vs 0.01%; fold change: 0.01, p <0.000001).

In the method of Example 3, 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. After performing the metagenome analysis by the method of Example 1, the distribution of the bacteria-derived vesicles of the genus Propionibium at the genus level, as shown in Figure 7b, compared to the blood of liver cirrhosis patients compared to normal blood as shown in Figure 7b Bacterial-derived vesicles of Onibacterium were significantly reduced (normal vs liver cirrhosis patients: 1.4% vs 0.5%; fold change: 0.4, p = 0.00004).

Example 6. Isolation and Characterization of Vesicles in Propionibacterium Acnes Culture

Based on the results of Examples 3 to 5, 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 ℃ 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. for 15 minutes, filtered through a 0.45 μm filter, and 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 μm filter, and the filtered supernatant was ultracentrifuged at 150,000 g, 4 ° C. for 3 hours, and the pellet was suspended in DPBS. Next, density gradient centrifugation was performed using 10%, 40%, and 50% Optiprep solution (Axis-Shield PoC AS, Norway) .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.

As a result, as shown in Figure 8a, it was observed that the vesicles separated from the propionibacterium Acnes culture medium is spherical, the size is smaller than 200 nm, the vesicles through the dynamic light scattering measurement results shown in Figure 8b It was confirmed that the size of 37.8 ± 13.5 nm.

Example  7. Propionibacterium  Acnes-derived vesicle proteome analysis

To determine what proteins were included in the propionibacterium acnes-derived vesicles, proteome analysis was performed. To this end, trypsin digested peptides were obtained through FASP (Filter-aid sample preparation) digestion. 18 μg 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. After further mixing with UA solution (0.2 mL of 8 M urea in 0.1 M Tris-HCl, pH 8.5) and repeating the centrifugation filter three times, 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. Similarly, after mixing the UA solution and repeating the centrifugation-based filter three times, 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 μl of solution was repeated twice. The collected samples were dried with a SpeedVac concentrator, then dissolved in 180 μl 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 μm × 2 cm) packed with C18 3-μm 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 μm × 50 cm) packed with C18 2-μm 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.

Mass spectrometry (MS / MS) spectra obtained in three iterations was searched in the Propionibacterium Acnes Uniprot database (Reviewed Nov. 2015) using the SEQUEST engine tool (Bioworks-Browser rev. 3.1 coupled with percolator validation). It was. Up to two missed cleavage sites were included in the full tryptic specficity, and the mass tolerances of precursor ions and fragment ions were set to 10 ppm and 0.6 Da. Modification of carbamidomethyl cysteine was fixed and various modifications of methionine oxidation were allowed. Peptides were selected with a blocking value of FDR = 0.01.

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. In addition, based on the ratio of protein distribution by biological function, the metabolic process was analyzed as 15.9%, mass transport 7.5%, protein processing 5.2%, translation 4.8%, pathogenicity 4%. The propionibacterium acnes-derived vesicles are important for metabolic processes and mass transport of propionibacterium acnes. It is expected to play a role.

From the functions of the 252 proteins identified by the proteome analysis results, 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.

Among the proteins in vesicles derived from Propionibacterium acnes, 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. In addition, many of the proteins in the propionibacterium acnes-derived vesicles have functions related to transcription, translation, protein transport, and protein folding. From this, the 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.

In addition, from the proteins that bind bacterial peptidoglycan layers such as transglycosylase, beta-lactamase, multimodular transpeptidase-transglycosylase, FtsI, or penicillin-resistant proteins, 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 (PPA2127) 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. Can form biological niches using propionibacterium acnes-derived vesicles.

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, It can be expected that 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.

NlpC / P60 endopeptidase family protein, basic membrane protein, 60 kDa chaperonin, and 10 kDa chaperonin are essential proteins for the survival of propionibacterium acnes. Has been reported. From these facts, it can be seen that 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.

Gene Name Accession Protein Name GO-Biological Process GO-Cellular Component ftsY W4U385_PROAA Signal recognition particle receptor FtsY SRP-dependent cotranslational protein targeting to membrane Cytoplasm, Intrinsic component of plasma membrane PAST3_05111 A0A085B3F8_PROAA Uncharacterized protein - integral component of membrane JCM18909_1125 W4TIQ5_PROAA Uroporphyrinogen III decarboxylase porphyrin-containing compound biosynthetic process - HMPREF9570_01608 F1UIQ1_PROAA Uncharacterized protein - integral component of membrane HMPREF9344_01072 A0A0E1YHC7_PROAA Uncharacterized protein - - JCM18918_946 W4U3Y9_PROAA L-proline glycine betaine ABC transport system permease protein ProW - - pdxT W4U4U3_PROAA Pyridoxal 5'-phosphate synthase subunit PdxT glutamine catabolic process, pyridoxal phosphate biosynthetic process, vitamin B6 biosynthetic process - JCM18920_273 W4UDV8_PROAA Uncharacterized protein - integral component of membrane HMPREF9570_01578 F1UIW0_PROAA Uncharacterized protein - integral component of membrane HMPREF9570_00330 F1UEX6_PROAA ABC transporter, solute-binding protein - - HMPREF9206_0523 D1YFC9_PROAA Uncharacterized protein - integral component of membrane JCM18918_3895 W4U9Z7_PROAA Dolichol-phosphate mannosyltransferase - - JCM18916_1395 W4TTY6_PROAA Glutamine synthetase type I Nitrogen compound metabolic process - JCM18918_383 W4U0P5_PROAA Dolichol-phosphate mannosyltransferase - - JCM18909_3341 W4TNE4_PROAA Uncharacterized protein - integral component of membrane HMPREF9344_00075 A0A0E1YK98_PROAA Succinate CoA transferase acetyl-CoA metabolic process - PAST3_07564 A0A085B206_PROAA Fructose-bisphosphate aldolase glycolytic process - HMPREF9570_01325 F1UI30_PROAA Uncharacterized protein - integral component of membrane HMPREF9344_01711 A0A0E1YH92_PROAA ErfK / YbiS / YcfS / YnhG - - HMPREF1162_1003 F9NXK4_PROAA Pyridine nucleotide-disulfide oxidoreductase - - HMPREF9578_01543 E6D3Y4_PROAA ErfK / YbiS / YcfS / YnhG - - HMPREF9344_01854 A0A0E1YI81_PROAA Uncharacterized protein - integral component of membrane JCM18918_3903 W4UAZ1_PROAA 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acetyltransferase - - JCM18920_1123 W4UEG5_PROAA 2,4-dienoyl-CoA reductase - - JCM18916_1756 W4TVT1_PROAA Uncharacterized protein photosynthesis, light reaction plasma membrane light-harvesting complex JCM18909_4054 W4TQJ4_PROAA Uncharacterized protein - integral component of membrane PAST3_01596 A0A085B4Q8_PROAA Putative glycosyl transferase - - JCM18916_2713 W4TXW7_PROAA Uncharacterized protein - integral component of membrane HMPREF9344_01464 A0A0E1YGY4_PROAA Aminotransferase, class I / II - - rpoB R4L3U6_PROAA DNA-directed RNA polymerase (Fragment) transcription, DNA-templated - sdhA D4HEX0_PROAS Succinate dehydrogenase flavoprotein subunit - - JCM18918_3293 W4UA75_PROAA Uncharacterized protein - integral component of membrane JCM18920_3008 W4UKV6_PROAA DNA-directed RNA polymerase Transcription, DNA-templated - rpsJ F1UKL8_PROAA 30S ribosomal protein S10 translation ribosome HMPREF1162_2086 F9NXA8_PROAA Peptidase C1-like protein - - JCM18916_3283 W4TYC7_PROAA Uncharacterized protein - integral component of membrane JCM18916_839 W4TSZ0_PROAA Elongation factor Ts - small ribosomal subunit coxB D4HCM6_PROAS Cytochrome c oxidase, subunit II electron transport chain integral component of membrane JCM18916_1312 W4TUQ7_PROAA Uncharacterized protein - integral component of membrane rplL RL7_PROAC 50S ribosomal protein L7 / L12 translation ribosome cbiN A0A085B001_PROAA Cobalt transport protein CbiN cobalamin biosynthetic process integral component of membrane, plasma membrane JCM18920_2186 W4UIY5_PROAA Outer membrane protein A - Cell outer membrane, Integral component of membrane rplQ F1UKD4_PROAA 50S ribosomal protein L17 translation ribosome rplS W4UI67_PROAA 50S ribosomal protein L19 translation ribosome JCM18909_475 W4TF81_PROAA Methylmalonyl-CoA mutase - - JCM18909_2328 W4TLS3_PROAA LSU ribosomal protein L5p ribosome translation JCM18916_3017 W4TXR1_PROAA cAMP factor - - JCM18909_473 W4TH18_PROAA Methylmalonyl-CoA mutase - - PPA2127 E2ICJ6_PROAA Putative uncharacterized protein (Fragment) - - JCM18918_1025 W4U2V0_PROAA Signal peptidase I protein processing involved in protein targeting to mitochondrion integral component of membrane JCM18920_1269 W4UFS4_PROAA Methylmalonyl-CoA: pyruvate transcarboxylase - - HMPREF0675_5348 D4HBJ7_PROAS SH3 domain protein - - N / A Q50E66_PROAA CAMP factor 2 - - HMPREF1162_1035 F9NXN7_PROAA Tricorn protease homolog - cytoplasm HMPREF9344_00824 A0A0E1YKT1_PROAA Hyaluronate lyase Carbohydrate metabolic process Extracellular region N / A Q5QCW8_PROAA Camp2 - - HMPREF9344_01393 A0A0E1YGP6_PROAA NlpC / P60 family protein - - HMPREF9344_01070 A0A0E1YTB5_PROAA Tricorn protease homolog - cytoplasm PAST3_10318 A0A085B0H7_PROAA Beta-N-acetylhexosaminidase Carbohydrate metabolic process - JCM18920_1344 W4UFZ3_PROAA cAMP factor - - JCM18918_2370 W4U6W5_PROAA Peptidase E - - hflB A0A0E1YKG2_PROAA ATP-dependent zinc metalloprotease FtsH protein catabolic process integral component of membrane, plasma membrane HMPREF9344_00204 A0A0E1YLS2_PROAA SH3 domain protein - - PAST3_08146 A0A085B1J8_PROAA Serine acetyltransferase cysteine biosynthetic process from serine cytoplasm HMPREF1162_1127 F9NUI0_PROAA Bacterial SH3 domain protein - - HMPREF9578_02632 E6DAK7_PROAA Periplasmic binding protein - - rpsE A0A085B5C6_PROAA 30S ribosomal protein S5 Translation Small ribosomal subunit HMPREF9344_00841 A0A0E1YLV6_PROAA ABC transporter, ATP-binding protein - - HMPREF9570_02049 F1UK58_PROAA ABC transporter, solute-binding protein - - JCM18909_3059 W4TNL8_PROAA ABC transporter ATP-binding protein - - rpsD F1UKD2_PROAA 30S ribosomal protein S4 translation small ribosomal subunit JCM18916_3143 W4TY03_PROAA ABC transporter ATP-binding protein - - HMPREF9578_00861 E6D6Z0_PROAA Sugar-binding domain protein - - rpsF A0A085B274_PROAA 30S ribosomal protein S6 translation ribosome JCM18920_3587 W4ULS0_PROAA ABC transporter - - PA4687 Q06WK8_PROAA HtaA-like surface protein - - atpA A0A085B090_PROAA ATP synthase subunit alpha ATP hydrolysis coupled proton transport, plasma membrane ATP synthesis coupled proton transport - rplN F9NX47_PROAA 50S ribosomal protein L14 translation large ribosomal subunit HMPREF9570_00519 F1UFZ3_PROAA Periplasmic binding protein - - HMPREF0675_4199 D4HDU5_PROAS Beta-lactamase - - JCM18916_557 W4TRS6_PROAA ATP synthase gamma chain ATP synthesis coupled proton transport - JCM18916_980 W4TSU8_PROAA Membrane protein - - HMPREF0675_4855 D4H9R7_PROAS Triacylglycerol lipase - - JCM18918_412 W4U2L4_PROAA ATP synthase beta chain ATP synthesis coupled proton transport - JCM18920_1413 W4UG55_PROAA ATP synthase delta chain ATP synthesis coupled proton transport membrane JCM18909_3148 W4TN95_PROAA Enoyl- [acyl-carrier-protein] reductase - - tmRNAPropi_acnes_SK137 V6BXX0_PROAA Proteolysis tag peptide encoded by tmRNA Propi_acnes_SK137 (Fragment) - - HMPREF9570_01661 F1UJ13_PROAA Kinase domain protein - - JCM18916_1386 W4TUE1_PROAA PTS system phosphoenolpyruvate-dependent sugar phosphotransferase system integral component of membrane, plasma membrane HMPREF9570_00339 F1UEY5_PROAA Uncharacterized protein - - pgk A0A085B4M9_PROAA Phosphoglycerate kinase glycolytic process cytoplasm JCM18918_1238 W4U4T1_PROAA MoxR-like ATPases - - JCM18918_4037 W4UAB5_PROAA PTS system phosphoenolpyruvate-dependent sugar phosphotransferase system - JCM18918_3273 W4U8C3_PROAA Inosine-5'-monophosphate dehydrogenase - - purL W4THC8_PROAA Phosphoribosylformylglycinamidine synthase subunit PurL de novo 'IMP biosynthetic process cytoplasm JCM18918_1333 W4U3P8_PROAA Cytochrome c oxidase polypeptide I - - JCM18918_303 W4U2A8_PROAA Protein YceG like - - sucC A0A085B1K8_PROAA Succinyl-CoA ligase [ADP-forming] subunit beta tricarboxylic acid cycle - tatA A0A085B0L5_PROAA Sec-independent protein translocase protein TatA Protein secretion, Protein transport by the Tat complex Integral component of plasma membrane, TAT protein transport complex PAST3_07599 A0A085B213_PROAA Biotin carboxyl carrier protein of methylmalonyl-CoA: Pyruvate transcarboxylase - - JCM18916_723 W4TR26_PROAA Lysine-tRNA ligase lysyl-tRNA aminoacylation cytoplasm rho W4U2H4_PROAA Transcription termination factor Rho DNA-templated transcription, termination, regulation of transcription, DNA-templated - JCM18909_1988 W4TKY5_PROAA Cation diffusion facilitator family transporter - - HMPREF9570_01974 F1UJY3_PROAA Uncharacterized protein - integral component of membrane PAST3_09633 A0A085B110_PROAA ABC-type transporter, periplasmic component - - JCM18916_3759 W4TZ18_PROAA Preprotein translocase subunit SecE Protein secretion, protein targeting Integral component of membrane, Intracellular glyQS A0A085B438_PROAA Glycine-tRNA ligase glycyl-tRNA aminoacylation cytoplasm HMPREF9206_1837 D1YCA6_PROAA Tat pathway signal sequence domain protein - - HMPREF9344_02504 A0A0E1YDI6_PROAA Lon protease (S16) C-terminal proteolytic domain protein protein catabolic process integral component of membrane secD D4HDW9_PROAS Protein translocase subunit SecD Intracellular protein transmembrane transport, proteintargeting, protein transport by the seccomplex Integral component of membrane, Intracellular, Plasma membrane JCM18916_470 W4TSE3_PROAA Enzyme of poly-gamma-glutamate biosynthesis - - groL A0A085B1J1_PROAA 60 kDa chaperonin Protein refolding cytoplasm HMPREF9570_00463 F1UFN7_PROAA ABC transporter, solute-binding protein - - groL E6D9F5_PROAA 60 kDa chaperonin Protein refolding cytoplasm groS CH10_PROAC 10 kDa chaperonin protein folding cytoplasm JCM18920_241 W4UCN3_PROAA PspA / IM30 family protein - - secA A0A0E1YDL8_PROAA Protein translocase subunit SecA intracellular protein transmembrane transport, protein import, protein targeting cytoplasm, plasma membrane pstS F1UIJ3_PROAA Phosphate-binding protein PstS phosphate ion transmembrane transport - JCM18916_1981 W4TVZ8_PROAA Heme ABC transporter - - HMPREF9578_01591 E6D431_PROAA FHA domain protein - - HMPREF9344_02598 A0A0E1YF67_PROAA Peptidyl-prolyl cis-trans isomerase Protein folding - JCM18918_4160 W4UBK0_PROAA Cell envelope-related function transcriptional attenuator - - JCM18920_274 W4UC57_PROAA Peptidyl-prolyl cis-trans isomerase protein folding - JCM18916_1704 W4TTJ3_PROAA ABC transporter ATP-binding protein - - JCM18909_251 W4TFA1_PROAA Peptidyl-prolyl cis-trans isomerase protein folding - JCM18909_2781 W4TM00_PROAA Argininosuccinate synthase Arginine biosynthetic process cytoplasm JCM18918_2061 W4U6R2_PROAA Integral membrane protein - integral component of membrane JCM18909_1400 W4THK0_PROAA NADH-ubiquinone oxidoreductase - - JCM18918_1420 W4U3H8_PROAA Arginine deiminase arginine catabolic process - HMPREF9570_00243 F1UES3_PROAA Uncharacterized protein - - JCM18916_2258 W4TX28_PROAA Cell division protein FtsI Cell division - pepA AMPA_PROAC Probable cytosol aminopeptidase - cytoplasm JCM18916_3790 W4TZQ4_PROAA Uncharacterized protein - - JCM18916_1932 W4TU75_PROAA Cell division protein FtsI Cell division - metN D4H9G7_PROAS Methionine import ATP-binding protein MetN - - JCM18916_3473 W4TZX4_PROAA Uncharacterized protein - -

Example  8. Propionibacterium  Anti-inflammatory Effects of Acnes-derived Vesicles

To investigate the effects of propionibacterium acnes-derived vesicles on inflammatory mediator secretion in inflammatory cells, propionibacterium acnes- derived vesicles ( P. acnes) 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 ㎍ / ml) and then secreted by inflammatory mediators (IL-6, TNF-α, etc.) through apoptosis and ELISA (R & D system, USA), respectively. Was measured. More specifically, 1 × 10 ⁵ Raw 264.7 cells were dispensed into 24-well cell culture plates, and then cultured in DMEM complete medium for 24 hours. Since apoptosis was performed by MTT assay (Sigman, USA). In addition, in order to evaluate the secretion ability of the mediator media, propionibacterium Acnes-derived vesicles were mixed and treated with fresh DMEM complete medium, and then cultured in a 37 ℃ 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.

For ELISA analysis, the Capture antibody was diluted in PBS, 50 μl was dispensed into 96-well polystyrene plates according to the working concentration, and then reacted overnight at 4 ° C. After washing twice with 100 μl of PBST (PBS containing 0.05% tween-20) solution, dispense 100 μl of RD (PBST containing 1% BSA) solution and block for 1 hour at room temperature. After washing twice with 100 μl, 50 μl of the sample and standard were dispensed according to the concentration and reacted at room temperature for 2 hours. After washing twice with 100 μl of PBST again, the detection antibody was diluted in RD and 50 μl was dispensed at the action concentration for 2 hours at room temperature. After washing twice with 100 μl of PBST again, Strpetavidin-HRP was diluted to 1/200 in RD and 50 μl divided and reacted at room temperature for 30 minutes. Finally, after washing three times with 100 μl of PBST, 50 μl of a solution of 1: 1 mixed TMB substrate and 0.04% hydrogen peroxide solution was dispensed and waited for color development. 50 μl of sulfuric acid solution was dispensed to stop the reaction and the absorbance was measured at 450 nm using Synergy ™ HT multi-detection microplate reader (BioTek, USA).

As a result, as shown in FIG. 10A, apoptosis of inflammatory cells was not induced regardless of the treatment concentration of the propionibacterium acnes-derived vesicles.

In addition, as shown in Figure 10b, it was confirmed that the secretion of macrophage inflammatory mediators by propionibacterium Acnes-derived vesicle treatment is significantly reduced compared to E. coli EV vesicles, which are pathogenic vesicles.

The results show that propionibacterium acnes-derived vesicles are safer when absorbed into the body.

Based on the results, in order to evaluate the anti-inflammatory effects of propionibacterium acnes-derived vesicles, various concentrations of propionibacterium acne-derived vesicles were treated with E. coli-derived vesicles (1 μg / ml). After 12 hours of administration, inflammatory cytokine secretion by E. coli-derived vesicles was measured by ELISA.

As a result, as shown in Fig. 11a and 11b, when pre-treated with propionibacterium Acnes-derived vesicles, it was confirmed that IL-6 and TNF-α secretion by E. coli-derived vesicles is significantly suppressed. This means that 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

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.

As a result, as shown in Fig. 12a and 12b, it was confirmed that the killing of keratinocytes by propionibacterium Acnes-derived vesicles is significantly reduced compared to the vesicles derived from Staphylococcus aureus.

Based on the above results, in order to evaluate the inhibitory effect of propionibacterium acne vesicles on the killing of skin keratinocytes by Staphylococcus aureus vesicles, various concentrations of propionibacterium acnes-derived vesicles were yellow on keratinocytes. After administration of Staphylococcus aureus-derived vesicles (100 μg / ml concentration) 12 hours before treatment, MTT assay (Sigman, USA) was performed.

As a result, as shown in Figure 13, when pre-treated with propionibacterium Acnes vesicles, it was confirmed that keratinocyte death caused by Staphylococcus aureus vesicles is suppressed. This means that skin diseases induced by pathogenic vesicles, such as Staphylococcus aureus-derived vesicles, can effectively suppress the vesicles derived from Propionibacterium acnes.

Example  10. Androgens  For receptor expression Propionibacterium  Effect of Acnes-derived Parcels

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. We tried to find out the treatment mechanism in detail. To this end, as shown in Fig. 14, 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 μg of propionibacterium acnes-derived vesicles (PaEV), and 1 μg of peptidoglycan (PGN), a well-known antigen of PBS or propionibacterium acnes-derived vesicles, 1 μg 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.

For Western blot analysis, 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 μg 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. After washing three times with TBS-T for 30 minutes, 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 β-actin were used as controls.

As a result, as shown in Fig. 14, androgen receptors were injected into the prostate tissue when the propionibacterium acnes-derived vesicles were administered, unlike the well-known antigens peptidoglycan (PGN) and lipoteichoic acid (LTA). Overexpression was confirmed. Through a phenomenon in which atopic dermatitis improves during puberty when the androgen receptor is stimulated and the androgen receptor expression is increased for breast cancer treatment, propionibacterium acne vesicles increase the expression of androgen receptor and show therapeutic effect. It can be seen that one. In addition, the overexpression of the androgen receptor can be expected to be made of protein components other than non-protein components such as PGN and LTA in propionibacterium acnes-derived vesicles.

The description of the present invention set forth above is for illustrative purposes, and one of ordinary skill in the art may understand that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

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.

Claims (20)

A method of providing information for diagnosing cancer, inflammatory disease, endocrine disease, or metabolic disease, comprising the following steps: (a) extracting DNA from vesicles isolated from normal and patient derived samples; (b) performing PCR on the extracted DNA by using a pair of vesicle detection primers derived from propionibacterium bacteria present in the 16S rDNA sequence; And (c) Determining cancer, inflammatory diseases, endocrine diseases, or metabolic diseases when the content of the bacteria-derived vesicles in propionibacterium is lower than that of normal people through quantitative analysis of the PCR products. The method of claim 1, The patient-derived sample is characterized in that the blood or urine, information providing method. The method of claim 1, The cancer is characterized in that the liver cancer or breast cancer, information providing method. The method of claim 1, The inflammatory disease is atopic dermatitis or asthma, characterized in that the information providing method. The method of claim 1, The endocrine disease is characterized in that osteoporosis, senility, or hair loss, information providing method. The method of claim 1, The metabolic disease is characterized in that diabetes or cirrhosis, information providing method. A pharmaceutical composition for preventing or treating cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, comprising a vesicle-derived bacterium from Propionibacterium as an active ingredient. The method of claim 7, wherein The bacterium in Propionibacterium is Propionibcerium acnes acnes ), characterized in that the pharmaceutical Composition. The method of claim 7, wherein The vesicles are characterized in that the average diameter of 10 to 1000 nm, Composition. The method of claim 7, wherein The vesicles are characterized in that they are secreted naturally or artificially from bacteria of the genus Propionibacterium, Composition. The method of claim 7, wherein The cancer is characterized in that the breast cancer or liver cancer, pharmaceutical Composition. The method of claim 7, wherein The inflammatory disease is characterized in that atopic dermatitis or asthma, pharmaceutical Composition. The method of claim 7, wherein The endocrine disease is characterized in that osteoporosis, senility, or hair loss, pharmaceutical composition. The method of claim 7, wherein The metabolic disease is characterized in that diabetes or liver cirrhosis, pharmaceutical Composition. The method of claim 7, wherein The composition, characterized in that the inhalant. The method of claim 8, The composition is characterized in that using the protein contained in the propionibacterium Acnes-derived vesicles, pharmaceutical composition. A method of preventing or treating cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, comprising administering to a subject a pharmaceutical composition comprising a bacterium-derived vesicle from Propionibacterium as an active ingredient. Preventive or therapeutic use of cancer, inflammatory diseases, endocrine diseases, or metabolic diseases of the pharmaceutical composition comprising a bacterium derived from propionibacterium as an active ingredient. Food composition for the prevention or improvement of cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, comprising the vesicles derived from the propionibacterium genus as an active ingredient. A cosmetic composition for improving cancer, inflammatory diseases, endocrine diseases, or metabolic diseases, comprising vesicle-derived vesicles of propionibacterium.

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