CN117679444A - Bacteroides thetaiotaomicron and application of MCM enzyme thereof in maintaining intestinal barrier steady state and improving intestinal inflammation - Google Patents

Abstract

The invention discloses an application of intestinal symbiotic bacteroides thetaiotaomicron and MCM enzyme thereof in maintaining intestinal barrier steady state and improving intestinal inflammation, and experiments prove that the genetic function of MCM and the physiological effects of the MCM in stimulating the differentiation of host intestinal epithelial goblet cells, increasing the number and enhancing the mucous secretion function in the process of interaction with a host are achieved. This effect provides a reduction in the pathological manifestations of DSS (sodium dextran sulfate) chemically induced colitis, while human group letter analysis also demonstrates the prevalence of MCM enzymes (methylmalonyl-coakutase, methylmalonyl-coa mutase) in the human intestinal flora and the negative correlation with human inflammatory bowel disease IBD. Therefore, the targeted intestinal symbiotic bacteria MCM regulates and controls the biological level of propionic acid so as to maintain the intestinal homeostasis of a host, and is a new thought for preventing and treating human inflammatory bowel diseases in the future.

Description

Bacteroides thetaiotaomicron and application of MCM enzyme thereof in maintaining intestinal barrier steady state and improving intestinal inflammation

Technical Field

The invention belongs to the technical field of biological medicines, relates to the field of intestinal probiotics, and in particular relates to related researches and applications of bacteroides enteroides and MCM enzymes thereof in charge of propionic acid synthesis so as to maintain intestinal homeostasis and improve inflammation.

Background

In recent years, with the development of multiple-mathematics technology, the study of intestinal flora gradually shifts from initial association analysis to causally-oriented molecular mechanism study. The existing studies have fully demonstrated that metabolites from the intestinal flora are critical messenger molecules for regulating host metabolic health. Among the numerous metabolites of bacterial origin, the propionic acid in short chain fatty acids has a remarkable effect, and propionic acid has an effect receptor in numerous organs of the body, and its health-benefit effects include mucosal immunomodulation, anti-inflammatory effect and metabolic regulation. Thus, modulation of propionic acid levels in the colon of a host is a potential intervention to maintain host immune and metabolic homeostasis. The intestinal symbiotic bacteroides is used as main propionic acid producing bacteria in human intestinal tracts, propionic acid can be synthesized through a succinic acid pathway, and the intestinal symbiotic bacteroides is a potential symbiotic candidate bacteria for researching and regulating a human intestinal propionic acid pool.

Although there is an overview of the bacterial species in the intestine that synthesize propionic acid, there is currently no known choice but to which genes and enzymes in the species are responsible for propionic acid biosynthesis. In addition, the effect of propionic acid on the physiological state of a host is mainly studied by adopting a form of correlation analysis of the whole level or exogenous addition supplement, and the investigation of propionic acid generated by a bacterial source through specific regulation is still unknown at the aspect of intestinal bacteria-host interaction angle.

Disclosure of Invention

Based on the limitation of the current research, the invention takes Bacteroides Thetaiotaomicron (BT) as a research chassis, which is a kind of Bacteroides representative strain with definite genome information sequencing, and solves two scientific problems: (1) The key enzyme and the coding gene which are responsible for the biosynthesis of propionic acid in BT bacteria are illustrated; (2) The effect of BT bacteria producing propionic acid on host physiological status, especially its direct effect on intestinal barrier homeostasis, is demonstrated.

The technical scheme of the invention is as follows: firstly, through a BT fungus in-vitro culture experiment, the BT fungus generates propionate under in-vitro culture conditions; through further functional genome analysis, the key enzyme methylmalonyl-coenzyme CoA mutase (MCM) responsible for propionic acid synthesis and the coding gene BT2090-2091 thereof are found from the BT strain succinic acid pathway, and the key synthetic gene operon scpA-mutA in BT strain is further identified, and the maintenance existence of scpA-mutA in bacteroides intestinal bacteria is proved.

The gene knockout mutant strain is obtained by a gene editing technology, and the biological function of MCM enzyme in charge of propionic acid synthesis is clarified by in vitro culture or in vivo field planting respectively, and the BT knockout mutant strain with the MCM enzyme deletion cannot produce propionic acid. Further, it was found that BT MCM enzyme-mediated propionic acid synthesis promotes differentiation of host intestinal epithelial goblet cells, thereby increasing the number thereof and enhancing the mucus secretion function thereof. The DSS induced mouse acute colitis model is utilized to further prove that the BT MCM has the effect of promoting the host goblet cells, so that the BT MCM has the effect of relieving the protection of the DSS induced mouse colitis, and has a certain clinical application prospect.

According to the above study, the specific technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides an application of bacteroides intestinal symbiotic or a propionic acid synthetase promoter thereof in preparing a medicine for maintaining intestinal barrier steady state or improving intestinal inflammation.

Preferably, the intestinal symbiotic bacteroides is bacteroides thetaiotaomicron; the propionic acid synthase is methylmalonyl-coenzyme CoA mutase (MCM); intestinal inflammation includes acute colitis or inflammatory bowel disease.

Further preferably, the intestinal symbiotic bacteroides or propionic acid synthase promoter is selected from any one of the following cases:

(a) An intestinal canal symbiotic Bacteroides thetaiotaomicron active thallus;

(b) Substances that promote the proliferation or activity of bacteroides thetaiotaomicron;

(c) An exogenous MCM enzyme active agent or a homolog active agent;

(d) An active agent of exogenous nucleic acid molecules encoding MCM enzymes;

(e) And a substance for promoting the secretion of MCM enzyme by the intestinal symbiotic bacteroides.

Further preferably, the agent for maintaining intestinal barrier homeostasis or improving intestinal inflammation is an agent for increasing the number of goblet cells in the colon of a host, promoting the expansion and differentiation of goblet cells.

Experiments prove that the MCM functional enzyme activity is necessary for synthesizing propionic acid in intestinal symbiotic bacteria BT whether in vitro or in vivo; intestinal colonization by BT bacteria has the potential to increase intestinal epithelial barrier function, significantly increase host colonic goblet cell numbers and promote their differentiation and maturation, while protecting colonic epithelium from chemical damage to improve host colitis and inflammatory bowel disease, which require the presence of BT MCM functional enzymes.

Accordingly, in a second aspect the present invention provides a propionic acid synthase recombinant vector comprising an expression vector and a nucleic acid molecule encoding a methylmalonyl-CoA mutase, such as BT2090-2091, inserted into the expression vector.

Preferably, the expression vector is a plasmid vector, a cosmid vector, a phage vector, or a viral vector selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, coxsackievirus, herpes simplex virus, measles virus, newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus, and vesicular stomatitis virus.

In a third aspect, the invention provides an application of the propionic acid synthetase recombinant vector in preparation of a medicament for maintaining intestinal barrier steady state or improving intestinal inflammation.

In a fourth aspect of the invention, there is provided a pharmaceutical composition for maintaining intestinal barrier homeostasis or improving intestinal inflammation, comprising an active ingredient together with a pharmaceutically acceptable excipient, carrier or diluent. Wherein the active component is the intestinal canal symbiotic bacteroides or the propionic acid synthetase promoter or the propionic acid synthetase recombinant vector of the intestinal canal symbiotic bacteroides.

Compared with the prior art, the invention has the beneficial effects that:

the invention identifies and obtains a key enzyme MCM in intestinal symbiotic bacteria body responsible for synthesizing important dietary metabolite propionic acid, and proves the genetic function of the MCM and the physiological effects of stimulating the differentiation of host intestinal epithelial goblet cells, increasing the number and enhancing the mucous secretion function in the process of interacting with the host. This effect may provide a reduction in the pathological characterization of DSS chemically induced colitis, while human group biography also demonstrates the prevalence of MCM enzymes in the human intestinal flora and the negative correlation with human inflammatory bowel disease IBD. Therefore, the targeted intestinal symbiotic bacteria MCM regulates and controls the biological level of propionic acid so as to maintain the intestinal homeostasis of a host, and is a new thought for preventing and treating human inflammatory bowel diseases in the future.

Drawings

FIG. 1 shows the conserved presence of scpA-mutA in Enterobacter sp;

FIG. 2 shows that propionic acid production was not detected in either the culture supernatant or the bacterial lysate of the MCM-deficient mutant strain;

FIG. 3 shows growth curves for BT wild type (BT WT), scpA-mutA mutant (BT. DELTA. ScpA-mutA) and BT. DELTA. ScpA-mutA complementation strain;

FIG. 4 shows propionic acid levels in the growth cycle of BT wild type (BT WT), scpA-mutA mutant (BT. DELTA. ScpA-mutA) and BT. DELTA. ScpA-mutA anaplerotic strains;

FIG. 5 shows that there was no significant difference in the cases of the respective implantation of BT WT, BT delta scpA-mutA into C57B6J mice pretreated with antibiotics;

FIG. 6 shows a comparison of propionic acid levels in mice after intragastrical BT WT, BT delta scpA-mutA;

FIG. 7 shows differentially expressed genes of mouse colon epithelial cells in two groups colonised by BT WT or BT delta scpA-mutA strains, associated with defense;

FIG. 8 shows Gene Ontolog enrichment analysis of differentially expressed genes, the enrichment pathway being associated with intestinal homeostasis;

FIG. 9 shows an increase in goblet cell occupancy of BT WT colonized mice over BT delta scpA-mutA colonized mice;

FIG. 10 shows the results of a goblet cell staining analysis of BT WT-plated mice versus BT delta scpA-mutA-plated mice;

FIG. 11 shows that mRNA expression levels of muc2 and spdef (important transcription factors related to goblet cell differentiation) in colon tissue of mice are significantly increased in BT WT colonized groups;

FIG. 12 shows that either culture supernatants or propionic acid of BT WT-colonized mice induce up-regulation of expression of transcription factors such as klf4, spdef, etc. and their target genes, specific marker genes (muc 2, muc1 and fcgbp) of goblet cells in cell lines, whereas BT.DELTA.scpA-mutA-colonized mice lose this function;

FIG. 13 shows the induction process of the acute colitis model;

figure 14 shows body weight curves for mice from different experimental groups;

FIG. 15 shows survival curves for mice from different experimental groups;

fig. 16 shows colon length for mice of different experimental groups;

FIG. 17 shows the results of HE histopathological staining and goblet cell number and mucus layer staining of different experimental groups of mice;

figure 18 shows that BT MCM genes are inversely related to human IBD.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.

EXAMPLE 1MCM functional enzyme Activity is required for propionic acid Synthesis in gut commensal BT

1. MCM enzyme identification

The theta iotaomicron strain was inoculated in brain heart infusion supplement medium BHIS (brain heart infusion (BHI) conventional culture liquid with addition of 5 mg/L heme (Meiluno) and 0.5 mg/L hemeVitamin K3 formation) liquid medium or culture was performed on BHIS agar plates under anaerobic conditions at 37 ℃. The inoculated liquid culture medium or flat solid culture medium is placed in an anaerobic incubator (Don Whitley Scientific Products) for growth, and the gas component contains 20% CO ₂ 、5％H ₂ And 75% N ₂ . If necessary, the following use concentration antibiotics were added to the medium: 50. Mu.g/ml ampicillin, 200. Mu.g/ml gentamicin and 25. Mu.g/ml erythromycin.

By functional genomic analysis, the key enzyme methylmalonyl-CoA mutase (MCM) responsible for propionic acid synthesis was found from the BT bacterial succinate pathway, and its key synthetic gene operon scpA-mutA in BT bacteria was identified and the conserved presence of scpA-mutA in bacteroides enterobacteria was demonstrated (fig. 1).

2. MCM enzyme function identification

The scpA and mutA gene operons were deleted tracelessly in the BT VPI-5482 strain using an optimized set of genetic manipulation systems applied to the Enterobacter strains to produce mutant strains deficient in MCM. Detection after in vitro culture showed that no propionic acid production was detected in either the culture supernatant or the cell lysate (FIG. 2).

Further, a anaplerotic strain having a scpA-mutA gene deletion mutant was constructed. By measuring the growth curves of the three strains BT wild type (BT WT), scpA-mutA mutant (BT. DELTA. ScpA-mutA) and BT. DELTA. ScpA-mutA strains, no significant difference in the growth ability was found between the three strains (FIG. 3). Furthermore, we measured Short Chain Fatty Acids (SCFAs) in the lysates of the three strain cells and in the culture supernatant using targeted metabonomics analysis, found that essentially no propionic acid production was detected in BT Δscpa-mutA, whereas BT WT strains detected higher propionic acid levels, and that propionic acid levels gradually accumulated in the culture supernatant with prolonged culture time (fig. 4). Importantly, the repacking of the scpA-mutA gene cluster in the BT.DELTA.scpA-mutA strain restored to a large extent the ability of the mutant to produce propionic acid.

Next, with PBS as a negative control, BT WT, BT Δscpa-mutA were separately transplanted into C57B6J mice pretreated with antibiotics, and in the absence of significant differences in engraftment (fig. 5), propionic acid levels in the faeces of mice perfused with BT WT increased significantly four days after engraftment, while BT Δscpa-mutA lavage groups maintained lower propionic acid levels similar to PBS groups (fig. 6). The above results indicate that MCM functional enzyme activity is required for propionic acid synthesis in gut commensal BT, either in vitro or in vivo.

Example 2MCM functional enzyme promotes BT WT to increase intestinal epithelial barrier function

To further elucidate the role of BT MCM-mediated propionic acid biosynthetic pathways in the intestinal interaction with the host, we colonized BT WT and BT Δscpa-mutA strains into the intestinal tract of sterile mice with free feeding of high fiber diet containing 10% (w/w) pea fibers for 9 weeks. In the absence of significant differences in colonisation, BT WT gavage significantly increased the propionic acid content in GF mice, whereas little propionate was detected in the faeces of BT Δscpa-mutA colonised GF mice and control GF mice with gavage PBS.

To further explore the alterations of the host colon epithelium, we isolated colon tissues from three groups of mice and performed single cell RNA sequencing. From single cell sequencing results, a series of genes related to the body defense process, including muc2, clca1, fcgbp and the like, were found to be significantly up-regulated in the colon of BT WT colonized mice compared to the other two groups (fig. 7); meanwhile, gene otolog enrichment analysis of differentially expressed genes further revealed that colonic epithelium of BT WT colonized group mice was significantly upregulated in biological processes associated with cell attachment assembly, cell adhesion regulation, etc. (fig. 8). Together, these data indicate that intestinal colonization by BT WT has the potential to increase intestinal epithelial barrier function, which requires the presence of BT MCM functional enzymes.

Example 3 participation of MCM functional enzymes in BT WT to promote goblet cell expansion, differentiation and maturation

Dimensionality reduction clustering was performed on intestinal epithelial cells to assess the effect of BT MCM-mediated propionic acid production on intestinal epithelial cell composition. We found that the goblet cell fraction was significantly higher in BT WT colonized groups than in the other two groups (fig. 9), this change suggests that BT bacterial colonization significantly increased host colon goblet cell numbers and that this phenomenon is specifically dependent on BT MCM activity. In addition, staining results of colon tissue sections of mice, including alcian blue/periodate schiff (AB/PAS) staining and immunofluorescent staining for goblet-specific markers (MUC 2 and AGR 2), further confirmed that the proportion of goblet cells in BT WT colonized groups was significantly higher than in the other two groups (fig. 10). Furthermore, qPCR results also showed that mRNA expression levels of muc2 and spdef (important transcription factors related to goblet cell differentiation) in colon tissue of mice were significantly increased in BT WT colonization groups (fig. 11). These results indicate that BT promotes goblet cell expansion and its intestinal epithelial barrier function, an effect that requires the presence of BT MCM activity.

Thereafter, to determine how BT MCM-mediated propionic acid biosynthesis promotes an increase in goblet cell number, we further performed in vitro experiments using a commonly used goblet cell model LS174T cell line, examined changes in expression of several transcription factors critical to intestinal goblet cell development, differentiation, maturation, including spdef, klf4, etc., and found that either BT WT culture supernatants or propionic acid induced upregulation of expression of klf4, spdef, etc., and their target genes, and goblet cell specific marker genes (muc 2, muc1, and fcgbp), whereas BT Δscpa-mutA culture supernatants lost this induction (fig. 12). These data further demonstrate that BT MCM-mediated propionate biosynthesis induces goblet cell expansion primarily by promoting cell differentiation.

Mechanically, propionate exerts its function primarily through binding to the G protein-coupled receptors GPR43 and GPR41 or through inhibition of HDAC. To determine the molecular mechanisms by which propionate regulates goblet cell differentiation, we treated LS174T cell lines with GPR41 agonist AR420626, GPR43 agonist 4-CMTB and a class of classical HDAC inhibitors trichostatin A (TSA), respectively, found that only AR420626 was able to enhance the expression of the relevant transcription factors and klf4 and spdef target genes in LS174T cells, mimicking the effects of BT WT supernatant and propionate, whereas 4-CMTB and TSA treatments had little effect. To further confirm the effect of GPR41, we cultured WT and Gpr41 by isolation ^-/- The intestinal organoids of mice were stimulated with BT culture supernatant or propionate, and BT WT supernatant was foundOr 1mM propionic acid stimulation can induce differentiation of intestinal organoids and expression of MUC2 in organoids, whereas BT.DELTA.scpA-mutA supernatant does not. These results confirm the promoting effect of BT MCM and propionate on goblet cell differentiation, importantly, the induction effect is in Gpr41 ^-/- The intestinal organoids of mice were greatly attenuated. Taken together, BT MCM activity-mediated propionate biosynthesis promotes goblet cell differentiation at least in part through the GPR41 signaling pathway.

Example 4 acute colitis animal model

In view of the above-mentioned functions of BT MCM enzyme, we further propose whether it is possible to provide BT with the ability to alleviate diseases associated with intestinal mucosal barrier. We induced an acute colitis model by simultaneous supplementation of antibiotic-pretreated SPF C57B6J mice with propionate drinking water, respectively, with gastric PBS, BT WT strain, BT Δscpa-mutA strain, or gastric BT Δscpa-mutA strain, followed by administration of dextran sulfate sodium salt (DSS) drinking water to each group of mice on this basis (fig. 13).

In experiments we found that the body weight of PBS group or BT Δscpa-mutA gavage group showed a significant trend of decrease, whereas the body weight of mice in BT WT gavage group or BT Δscpa-mutA gavage while supplementing propionate drinking group was significantly more gradual (fig. 14); the survival rate analysis results of the experimental end points show that the survival rate of the BT WT gastric lavage group or the BT delta scpA-mutA gastric lavage group and the drinking water group supplemented with propionate is obviously higher than that of the PBS group or the BT delta scpA-mutA gastric lavage group (figure 15); at the same time, the results of the colon length of mice also suggest that the colon length of the PBS group or BT DeltatpA-mutA intragastric group mice is significantly shortened, while the colon length of the BT WT intragastric group or BT DeltatpA-mutA intragastric group while supplementing the propionate drinking group is less shortened (FIG. 16). Furthermore, by colonic histologic analysis, BT WT gavage or BT Δscpa-mutA gavage mice with the propionate-supplemented drinking group exhibited a lesser degree of inflammatory pathology, colonic histopathological scores were relatively lower than DSS control mice, while BT Δscpa-mutA gavage mice did not exhibit any remission phenotype. Importantly, colon AB/PAS, MUC2 and AGR2 staining further revealed: the colon goblet cell number and mucus content of BT WT gavage or BT Δscpa-mutA gavage mice supplemented with propionate drinking group were significantly higher than those of PBS group and BT Δscpa-mutA gavage group mice (fig. 17). Taken together, these data indicate that BT WT strains largely protect colonic epithelium from chemical damage and thereby improve host colitis, and that this protection relies on the presence of BT MCM enzymes that mediate propionic acid synthesis.

EXAMPLE 5BT MCM and inflammatory diseases

To further determine the presence of BT MCM in the human metagenome and its relationship to human intestinal inflammatory disease (IBD), we analyzed BT MCM homologs using metagenomic data from the curatedMetagnomicData database, selected adult IBD patients and age, sex and BMI matched control samples, and compared the relative abundance and frequency of BT MCM between the two groups. The results showed that both the abundance and the frequency of detection of BT MCM homologs in the intestinal metagenome of IBD patients were significantly reduced compared to the control group, indicating that BT MCM genes were inversely correlated with human IBD (figure 18).

In conclusion, the research identifies a key enzyme MCM mediating propionic acid biosynthesis in BT strain by using the models of BT mode strain, sterile mice, intestinal organoids, DSS enteritis mice and the like and utilizing the technologies of intestinal fungus gene editing, single cell sequencing and the like, and further explores the influence of MCM mediated propionic acid synthesis pathways on the intestinal barrier steady state of a host, possible molecular mechanisms and protective effects on the aspects of BT strain relief and restoration of colon inflammation of the host.

While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.

Claims (9)

1. Use of intestinal symbiotic bacteroides or propionic acid synthetase promoter in preparation of medicine for maintaining intestinal barrier steady state or improving intestinal inflammation is provided.

2. The use according to claim 1, characterized in that:

wherein the propionic acid synthetase is MCM enzyme;

the intestinal symbiotic bacteroides is bacteroides thetaiotaomicron.

3. The use according to claim 1, characterized in that:

wherein the intestinal inflammation comprises enteritis or inflammatory bowel disease.

4. The use according to claim 1, characterized in that:

wherein the intestinal tract symbiotic bacteroides or the propionic acid synthetase promoter is selected from any one of the following conditions:

(a) An intestinal canal symbiotic Bacteroides thetaiotaomicron active thallus;

(b) Substances that promote the proliferation or activity of bacteroides thetaiotaomicron;

(c) An exogenous MCM enzyme active agent or a homolog active agent;

(d) An active agent of exogenous nucleic acid molecules encoding MCM enzymes;

(e) And a substance for promoting the secretion of MCM enzyme by the intestinal symbiotic bacteroides.

5. The use according to claim 1, characterized in that:

wherein, the medicine for maintaining intestinal barrier steady state or improving intestinal inflammation is a medicine for increasing the number of host colon goblet cells and promoting goblet cell expansion and differentiation maturation.

6. A propionic acid synthase recombinant vector comprising an expression vector and a nucleic acid molecule encoding an MCM enzyme disposed on the expression vector.

7. The propionic acid synthase recombinant vector of claim 6, wherein:

wherein the expression vector is a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, coxsackie virus, herpes simplex virus, measles virus, newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus and vesicular stomatitis virus.

8. Use of the recombinant propionic acid synthase vector of claim 6, in the manufacture of a medicament for maintaining intestinal barrier homeostasis or ameliorating intestinal inflammation.

9. A pharmaceutical composition for maintaining intestinal barrier homeostasis or improving intestinal inflammation, characterized in that the pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable excipient, carrier or diluent,

wherein the active component is the intestinal canal symbiotic bacteroides or the propionic acid synthetase promoter thereof according to any one of claims 1 to 5 or the propionic acid synthetase recombinant vector according to any one of claims 6 to 7.