Method for recognizing AX-responsive intestinal bacteria by combining metabolic marker with flow cytometry sorting

Abstract

The invention discloses a method for recognizing AX-responding intestinal bacteria by combining metabolic labeling with flow cytometry sorting. A D-type amino acid fluorescent probe (FDAA) is used for sequential labeling, the activity change of bacteria is quantitatively analyzed in combination with a flow cytometry sorting technology, meanwhile, double-labeled bacteria are sorted according to the change of fluorescence intensity, and target bacteria responding to dietary fibers are further identified by using a high-throughput sequencing technology. The method can be used for screening low-abundance target bacteria in a flora system and microorganisms which cannot be cultured at present. The invention provides a novel chemical biological method which is used for analyzing the complex interaction between the dietary fiber in the intestinal tract and the intestinal flora, and has a very high application value for realizing accurate quantitative analysis of the intestinal flora metabolism state caused by the specific dietary fiber.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the targeted screening of gut microbes that respond to arabinoxylan using D-amino acid metabolic probes, flow cytometry sorting, and microbial sequencing technologies. Background Technology

[0002] The information disclosed in the background section of this invention is merely intended to enhance the understanding of the overall background of the invention, and is not necessarily required to be fully understood. However, this is considered an admission or implication in any form that such information constitutes existing knowledge known to those skilled in the art. technology.

[0003] Arabicinoxylan (AX), a major hemicellulose component in the cell walls of cereals, herbs, and other plants, is characterized by a β-1,4-d-xylanosyl skeletal structure with α-l-arabinofuranosyl side chains. Phenolic compounds bound to AX, such as ferulic acid, exhibit significant anti-inflammatory and antioxidant activities. Studies have shown that AX intake is significantly associated with lowering cholesterol levels, promoting bile acid production, regulating immune function, and alleviating obesity-related metabolic dysfunction. Therefore, a better understanding of the interaction between dietary fiber and gut microbiota, and elucidating how the microbial community utilizes AX to generate bioactive metabolites and identify relevant key gut microbiota, is of significant scientific importance for achieving precision nutritional intervention based on microbiome information.

[0004] Currently, in vitro culture models, high-throughput sequencing, and isotope labeling analysis are mainly used to identify bacteria that degrade dietary fiber. While these techniques have some role in screening key bacteria for dietary fiber, they also have limitations. High-throughput sequencing is often used to analyze changes in the host gut microbiota composition after dietary fiber intervention and to identify differentially expressed microorganisms before and after intervention; however, it struggles to accurately identify key bacterial groups that respond to dietary fiber. Stable isotope probe technology can identify strains involved in fiber degradation, but the experimental procedures are complex and costly. Fluorescence in situ hybridization identifies and locates bacteria in the gut using fluorescent labels, but it may affect the natural metabolism of the gut microbiota. In vitro simulation systems study the gut microbiota's ability to metabolize dietary fiber by simulating the gut environment, but they often overlook the complex in vivo environment. Therefore, a precise method for in vivo screening of key bacteria responding to dietary fiber is still lacking.

[0005] Bacterial cell walls consist of a membrane layer and a rigid peptidoglycan (PGN) scaffold. Peptidoglycan is a network-like macromolecule composed of cross-linked peptides, providing mechanical strength to the cell wall, resisting osmotic and tumescent pressures, and determining the bacterial morphology throughout its life cycle. As a major component of the bacterial cell wall, peptidoglycan plays a crucial role in bacterial growth, division, and protection against the external environment. D-amino acids (DAAs) are important components of bacterial cell wall peptidoglycan; unlike natural amino acids, they are difficult to be degraded by enzymes in vivo. D-amino acid fluorescent probes, as a novel type of molecular probe, can be covalently linked to peptidoglycan through the action of endogenous bacterial transpeptidase. Due to their high biocompatibility and peptidoglycan specificity, these fluorescent probes have become a powerful tool for labeling peptidoglycan.

[0006] D-amino acid fluorescent probes possess fluorescent labeling properties, allowing them to be combined with other molecular markers for multiplex labeling experiments. This provides significant flexibility in bacterial biology research. For example, they enable real-time imaging of bacterial cell wall synthesis, three-dimensional imaging of the gut microbiota, and revealing the dynamic changes in the cell wall at different stages of bacterial invasion. Furthermore, these probes have been successfully applied in various research areas, including bacterial morphology analysis, peptidoglycan growth model studies, peptidoglycan-enzyme correlation studies, in vitro peptidoglycan synthase activity detection, and antibiotic inhibition assays. However, whether these probes can be applied to in vivo animal screening for fiber-utilizing bacteria remains unclear. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method for identifying AX-responsive gut bacteria using metabolic markers combined with flow cytometry sorting, solving the difficulty in accurately screening responsive bacteria in studies of arabinoxylan functional activity. This method is rapid, simple, sensitive, accurate, efficient, and intuitive, providing new technical support for the development of arabinoxylan as a regulator of animal intestinal function.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for identifying AX-responsive gut bacteria by combining metabolic markers with flow cytometry sorting includes the following steps: a) Feeding and intervention of model animals: Select mammalian models and randomly divide them into control group, low-dose arabinoxylan (AX) group and high-dose AX group, and feed them diets containing 0%, 5% and 10% AX for a total of 14 days; b) Metabolic markers: At the end of the intervention, after fasting for 6 hours, the model animals in each group were gavaged with a concentration of 1 mM of FAM-DAA probe (carboxyfluorescein-D-type amino acid probe) and mouse feed was added at the same time. After 6 hours, the Cy5-DAA probe (cyanogen sulfate 5-D-type amino acid probe) was gavaged again. c) Sample processing and flow cytometry sorting: Cecal contents were collected, suspended in PBS, filtered through a 40 μm cell sieve, and washed by centrifugation. Double-positive bacteria (simultaneously expressing FAM and Cy5 fluorescence) were identified by flow cytometry under 488 nm (excitation FAM) and 633 nm (excitation Cy5) light. Target bacterial groups were screened based on forward scattering (FSC) and side scattering (SSC) parameters. d) Target bacterial identification and validation: 16S rRNA sequencing was performed on the sorted double-positive bacterial groups. Combined with metabolic markers and microbial data before and after sorting, short-chain fatty acid (SCFA) levels, and expression levels of colonic G protein-coupled receptors (such as GPR41 / 43 / 109A) and antimicrobial peptide genes (such as DEFB3 / 14 / 50), gut bacteria that respond to AX were screened. Their ability to utilize AX was further validated through in vitro culture.

[0009] The method described, In step a), the model animals were 5-week-old SPF-grade male C57BL / 6 mice, with 5 mice in each group; at the end of the intervention, mouse feces were collected for 16S rRNA sequencing. In step b), the dose of the probe administered to each mouse via gavage is 200 μL; In step c), before flow cytometry sorting, the fluorescently labeled bacteria are observed using a laser scanning confocal microscope: carboxyfluorescein (FAM) is excited with a 488 nm laser, and cyanogen sulfate 5 (Cy5) is excited with a 639 nm laser. The in vitro culture verification in step d) includes: adding AX and microcrystalline cellulose as carbon sources at a ratio of 2% to a culture medium that does not contain glucose and starch, co-culturing with the screened bacteria for 24 hours, and measuring the changes in OD600nm absorbance, pH, SCFA and reducing sugar at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h.

[0010] The flow cytometry sorting operation in step c) includes: taking cecal contents into 5 mL PBS, filtering the minced tissue and contents through a 40 μm cell sieve, centrifuging the filtrate to obtain a white bacterial precipitate, washing three times with 1.5 mL PBS, centrifuging again, and resuspending in PBS. The centrifugation parameters are 4 ℃, 12,000 g, 2 min; the parameters include: FSC range set to 50-1000 (channels), SSC range set to 30-800 (channels), FAM fluorescence threshold set to 200-1000 (channels), and Cy5 fluorescence threshold set to 150-900 (channels) to ensure that only double-positive bacteria are collected.

[0011] The FAM-DAA probe and Cy5-DAA probe are D-alanine derivatives, covalently labeled with carboxyfluorescein and cyanogen sulfate, respectively.

[0012] The colonic G protein-coupled receptors include GPR41 / 43 / 109A and the antimicrobial peptide genes include DEFB3 / 14 / 50.

[0013] Compared with the prior art, the present invention has the following advantages: This invention utilizes time-series labeling with dual fluorescent probes (FAM / Cy5) to differentiate metabolic states before and after intervention, enabling dynamic activity screening. Building upon metabolic labeling, this invention integrates omics data to form a correlation verification system of "metabolic activity-functional genes-microbiota structure," providing more comprehensive validation of microbiota function. This invention can rapidly and effectively identify responsive bacteria in microbiota samples, eliminating the complex and cumbersome steps of bioinformatics and cultinomycology for target bacterial isolation and identification, and can accurately measure low-abundance target bacteria in the microbiota system. This invention can also accurately and intuitively present the degree of response of intestinal fiber-utilizing bacteria to arabinoxylan at the microbiota level. Furthermore, this technology provides valuable reference for nutrient utilization in the gut. The method used is low-cost and has a certain degree of scalability, effectively improving the operability of research results. Attached Figure Description

[0014] Figure 1 This is a flowchart of the present invention.

[0015] Figure 2 These are the results of mouse gut microbiota before DAA probe labeling.

[0016] Figure 3 This represents the level of short-chain fatty acids before DAA metabolic labeling.

[0017] Figure 4 This is a laser confocal image after DAA metabolic labeling.

[0018] Figure 5 These are the gut microbiota results after DAA metabolic labeling.

[0019] Figure 6 It is the expression of G protein-coupled receptor and antimicrobial peptide genes.

[0020] Figure 7 It is a correlation analysis of gene expression of microorganisms, SCFA, G protein-coupled receptors, and antimicrobial peptides.

[0021] Figure 8 This involves in vitro culture to verify the utilization of arabinoxylan by responding bacteria.

[0022] Figure 9 It is a characteristic of differential microbial data before and after DAA labeling. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional experimental methods. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0024] Referring to the following detailed description and exemplary embodiments, it should be understood that this application is not limited to the description in the specification. Or the details or methods shown in the accompanying drawings. The experimental procedure design of the present invention is as follows: Figure 1 As shown.

[0025] 1. Experimental materials Specified pathogens free (SPF) grade C57BL / 6 male mice were purchased from Shanghai Slack Co., Ltd. (Shanghai, China). D-amino acid fluorescent probes: carboxyfluorescein-amino-D-alanine (FADA) and Sulfo-Cyanine5-amino-D-alanine (CY5ADA) were both purchased from China Peptide Co., Ltd. The feed formulation was adjusted based on AIN-93.

[0026] Table 1. Feed Formulation

[0027] 2. Experimental Methods (1) Feeding and grouping of experimental animals Fifteen 5-week-old SPF-grade male C57BL / 6 mice were housed in an SPF environment at 24℃. After a 7-day pre-feeding period, they were randomly divided into three groups of five mice each, and fed diets containing 0%, 5%, and 10% arabinoxylan for 14 consecutive days. Fecal samples were collected on day 14 for 16S RNA sequencing. After a 6-hour fast, each mouse was administered 200 μL of 1 mM FADA via gavage, followed by the addition of different levels of arabinoxylan-containing diets. Six hours later, 200 μL of 1 mM Cy5ADA was administered via gavage. Cecal contents, colonic tissue, and contents were collected.

[0028] (2) Measure the levels of short-chain fatty acids (SCFA), G protein-coupled receptors, and antimicrobial peptide genes. Dissolve 0.5 g of fecal sample in 500 μL of methanol and let stand for 5-10 min. Adjust the pH of the mixture with 1% sulfuric acid and let stand for another 5-10 min. Then centrifuge at 5,000 g for 20 min at 4 °C, transfer the supernatant to a new centrifuge tube, and centrifuge at 5,000 g for 1 min at 4 °C. Then take 200 μL for gas chromatography to detect SCFA content. Use the RT-qPCR primers for the genes listed in the table below, employing 2... -ΔΔCT The method was used to relatively quantify the expression of G protein-coupled receptor and antimicrobial peptide genes.

[0029] Table 2. PCR primer sequences

[0030] (3) DAA metabolic sequence markers combined with flow cytometry to screen target gut microbiota Cecal contents were collected in 5 mL PBS. The minced tissue and contents were filtered through a 40 μm cell sieve. The filtrate was centrifuged to obtain a white bacterial precipitate, washed three times with 1.5 mL PBS, centrifuged again, and resuspended in PBS. Centrifugation parameters were 4 ℃, 12,000 g, 2 min. The bacterial suspension was then subjected to fluorescence imaging using a laser scanning confocal microscope: carboxyfluorescein (FAM) was excited with a 488 nm laser, and cyanogen sulfate 5 (Cy5) was excited with a 639 nm laser to observe the fluorescently labeled bacteria. Flow cytometry sorting was then performed: the bacterial suspension was sorted using a BD FACS II flow cytometer. The bacterial suspension was classified as green fluorescent protein positive and red fluorescent protein positive under 488 nm and 633 nm excitation light. Forward scattering (FSC) and side scattering (SSC) ranges were selected based on the size and particle size of the target fluorescently labeled bacteria. A range of fluorescent intensity for the collected bacteria was set to ensure that only double-positive bacteria could be sorted; at least 5 × 10⁶ bacteria were sorted from each sample. 6 Two double-positive bacteria were sorted, and the bacterial culture was sequenced using 16S RNA.

[0031] (4) Microbial sequencing Microbial genomic DNA was extracted from the test samples (feces / sorted bacterial culture); PCR amplification was performed using specific primers targeting the variable region of the 16S rRNA gene (such as the V3-V4 region), and a sequencing library was constructed; high-throughput paired-end sequencing of the amplified products was performed on the Illumina platform; quality control, chimera filtering, operational taxonomic unit clustering, and species annotation were performed on the original sequences to finally obtain microbial community composition data.

[0032] (5) Co-culture of Bacteroides acidogeneticus and arabinoxylan in vitro The in vitro experimental medium used was a modified Gifu anaerobic medium (without glucose and starch), with vitamin K1 and heme added as required by the formula. The control group and AX group were supplemented with 2% MCC and AX as carbon sources, respectively. Before use, the cultured bacterial suspension was inoculated into the medium and incubated in an anaerobic incubator for 24 hours. Growth curve determination: 1% bacterial suspension was inoculated into the medium, and 200 μL was taken into 96-well plates at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, and the absorbance at OD600 nm was measured using a microplate reader. pH determination: The culture medium at 24 h was taken, mixed well, and the pH value of the bacterial suspension was measured using a pH meter. Reducing sugar determination: 2 mL of culture medium was taken into centrifuge tubes at 0, 6, 12, 18, and 24 h, and centrifuged at 4,000 g for 5 min at 4 ℃. Take 1 mL of the supernatant into a new centrifuge tube, add 2 mL of DNS reagent, boil at 100 °C for 5 min, cool to room temperature, and take 200 μL to detect the absorbance at 540 nm.

[0033] 3. Experimental Results (1) Changes in gut microbiota in mice fed diets with different levels of arabinoxylan Compared with the control group, the Chao1 index of the gut microbiota in the 5% AX group was significantly increased (p < 0.05). β-diversity analysis, PCoA and NMDS results all showed significant differences in the gut microbiota structure between the 5% AX and 10% AX groups and the control group (R² = 0.906, p = 0.001). Bacteroides acidifaciens The highest abundance was observed in the 5% AX and 10% AX groups; furthermore, Uncultured_ bacterium_g_Prevotellaceae_Ga6A1_group and Bacteroides vulgatus The abundance of [the species] also showed a clear upward trend.

[0034] (2) Stability verification of D-amino acid metabolism probe Results of mouse gut microbiota before DAA probe labeling: Figure 2 As shown, the levels of short-chain fatty acids before DAA metabolic labeling are as follows: Figure 3 As shown, the laser confocal microscopy after DAA metabolic labeling is as follows: Figure 4 As shown, the gut microbiota results after DAA metabolic labeling are as follows: Figure 5 As shown.

[0035] Laser confocal microscopy analysis confirmed that: 1) all microorganisms in the experimental groups (Con, 5% AX, and 10% AX) could be effectively labeled; and 2) the fluorescence signal was specifically localized to the bacterial cell wall region. These results not only verified the reliability of the D-amino acid metabolic probe labeling technology, but also confirmed that this method can efficiently screen functional bacteria that utilize AX functions.

[0036] (3) Analysis of the composition of double-labeled bacteria after flow cytometry sorting Compared with the control group, the Chao1 index of the bacterial communities in the 5% AX and 10% AX groups showed an increasing trend. β-diversity analysis of the AX and Con groups, as well as PCoA and NMDS results, both showed significant differences in the bacterial community structure between the 5% AX, 10% AX, and control groups (R² = 0.396, p = 0.006). At the genus level, compared with the control group, the 5% AX group... Bacteroidetes and unclassified_Bacteroidales_UCG_001 Increased abundance; 10% AX group Bacteroidetes and Lactobacillus The abundance of [a specific substance] was significantly increased (p<0.05). Wilcoxon rank-sum test was performed on the bacterial community after flow cytometry sorting. Compared with the control group, in the 5% AX group, [the following information was found to be missing from the original text]. Bacteroides acidifaciens and Lachnospiraceae bacterium strain 1XD8_59 The abundance of [a substance] increased significantly.

[0037] (4) Effects of arabinoxylan on the expression levels of SCFAs, G protein-coupled receptors and antimicrobial peptide-related genes G protein-coupled receptor and antimicrobial peptide gene expression, such as Figure 6 As shown in the figure, AX treatment significantly increased the content of SCFAs in feces of mice. Compared with the control group, the content of total SCFAs, acetic acid, propionic acid, and valerate was significantly increased in the 5% AX group (p < 0.05). Compared with the control group, the content of total SCFAs, acetic acid, and propionic acid was significantly increased in the 10% AX group (p < 0.05). AX significantly upregulated the gene expression level of GPRs in the colon. Compared with the control group, the gene expression levels of GPR41 and GPR109A were significantly increased in the 5% AX group (p < 0.05). Compared with the control group, the gene expression levels of GPR41 and GPR43 were significantly increased in the 10% AX group (p < 0.01). In addition, the gene expression level of GPR43 was significantly increased in the 10% AX group compared with the 5% AX group. AX significantly upregulated the expression level of antimicrobial peptide-related genes in the colon. Compared with the control group, the expression levels of DEFB50 and DEFB14 genes were significantly increased in the 5% AX group (p < 0.05); the expression levels of DEFB50, DEFB14 and DEFB3 genes were significantly increased in the 10% AX group (p < 0.05).

[0038] (5) Correlation analysis of microorganisms, SCFAs and gene levels Correlation analysis of gene expression of microorganisms, SCFA, G protein-coupled receptors, and antimicrobial peptides, such as Figure 7 As shown. Bacteroides acidifaciens and Bacteroides vulgatus It was significantly correlated with acetic acid and propionic acid levels (p < 0.05). Lachnospiraceae bacterium strain 1xd8_59 This showed a significant correlation with acetic acid levels (p < 0.05). Furthermore, Uncultured bacterium _f_Lachnospiraceae bacterium Significant correlations were observed between GPR41 and the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid (p < 0.01). Further analysis revealed that GPR41 gene expression was significantly correlated with acetic acid and propionic acid levels (p < 0.05), while GPR43 gene expression was significantly correlated with propionic acid and isovaleric acid levels (p < 0.05).

[0039] (6) Characteristics of Bacteroides acidogenic metabolizing arabinoxylan in vitro In vitro culture verification of the utilization of arabinoxylan by responding bacteria, such as Figure 8 As shown in the figure. Compared with the control group, the pH value of the AX group decreased significantly (p < 0.05). The growth curves of BA in AX and glucose (GLU) showed that BA could utilize AX and grow well in the medium with AX as the carbon source. The reducing sugar determination results showed that the control group, using MCC as the carbon source, contained almost no reducing sugar, and the result approached 0 within 24 hours. In the AX group, using AX as the carbon source, the reducing sugar content first increased and then decreased with time, indicating that BA can metabolize and utilize AX to produce reducing sugar.

[0040] (7) Characteristics of differentially expressed microbial data before and after DAA labeling Microbiological characteristics before and after DAA labeling, such as Figure 9 As shown, compared with the differentially expressed microorganism data before DAA labeling, most of the differentially expressed bacteria after DAA labeling were unclassified. Combined with the previous rank-sum test results, it was also found that the number of unculturable differentially expressed microorganisms increased. This indicates that this method improves the accuracy of screening for currently unculturable microorganisms in the microbial community system.

[0041] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, all of which fall within the protection scope of the present invention. The protection scope of the present invention is defined by the appended claims and any equivalent technical solutions.

Claims

1. A method of identifying gut bacteria that respond to AX by metabolic labeling combined with flow cytometric sorting, characterized in that, Comprising the following steps: a) feeding model animals and intervention: select mammalian models, randomly divided into control group, low-dose arabinoxylan (AX) group and high-dose AX group, respectively fed with 0%, 5% and 10% AX-containing daily ration for a total of 14 days; b) metabolic labeling: at the end of the intervention, each group of model animals was fasted for 6 hours and then given FAM-DAA probe (carboxyfluorescein-D-amino acid probe) with a concentration of 1 mM by gavage, while adding mouse feed, and then given Cy5-DAA probe (sulfur cyanide 5-D-amino acid probe) with a concentration of 1 mM by gavage 6 hours later; c) sample processing and flow sorting: collect cecal contents, suspend in PBS, filter through a 40 μm cell strainer, and wash by centrifugation, then identify double-positive bacteria by flow cytometry under 488 nm and 633 nm light, and screen target flora according to forward scatter (FSC) and side scatter (SSC) parameters; d) target bacteria identification and verification: 16S rRNA sequencing of the sorted double-positive bacteria, combined with metabolic labeling and pre- and post-sorting microbial data, short-chain fatty acid (SCFA) levels, and colon G protein-coupled receptor and antimicrobial peptide gene expression, to screen for AX-responsive intestinal bacteria; further verify their ability to utilize AX through in vitro culture.

2. The method of claim 1, wherein: In step a), the model animals are 5-week-old SPF C57BL / 6 male mice, 5 in each group; at the end of the intervention, collect mouse feces for 16S rRNA sequencing; In step b), the dose of probe given to each mouse by gavage is 200 μL; In step c), before flow sorting, use a laser scanning confocal microscope to observe the fluorescently labeled bacteria: excite carboxyfluorescein (FAM) with a 488 nm laser and excite sulfur cyanide 5 (Cy5) with a 639 nm laser; The in vitro culture verification of step d) includes: adding AX and microcrystalline cellulose as carbon sources to the culture medium without glucose and starch at a ratio of 2%, and co-culturing with the screened bacteria for 24 hours, and measuring the OD600nm absorbance, pH, SCFA, and reducing sugar changes at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 h.

3. The method according to claim 1 or 2, characterized in that: The flow sorting operation of step c) includes: taking the cecal contents in 5 mL PBS, filtering the cut tissue and contents through a 40 μm cell strainer, and obtaining white bacterial precipitate by centrifugation treatment of the filtrate, washing three times with 1.5 mL PBS, resuspending in PBS after centrifugation, and centrifuging at 4°C, 12,000g for 2 min; parameters include: FSC range set to 50-1000, SSC range set to 30-800, FAM fluorescence threshold set to 200-1000, and Cy5 fluorescence threshold set to 150-900, to ensure that only double-positive bacteria are collected.

4. The method of claim 1, wherein: The FAM-DAA probe and Cy5-DAA probe are D-type alanine derivatives, covalently labeled with carboxyfluorescein and sulfur cyanide 5, respectively.

5. The method of claim 1, wherein: The colon G protein-coupled receptors include GPR41 / 43 / 109A and the antimicrobial peptide genes include DEFB3 / 14 / 50.

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