A method for isolating and detecting newly formed extravesicles of intestinal bacteria

Abstract

This invention discloses a method for isolating and detecting newly formed bacterial exovesicles, comprising the following steps: (1) metabolic labeling of the microorganism under study with an azide-modified hydrochloric acid D-type amino acid metabolic marker, so that the newly formed bacterial exovesicles are coated with an azide group; (2) isolating and extracting bacterial exovesicles from a sample containing bacterial exovesicles; (3) adding a capture linker that can react with the azide group to the extracted bacterial exovesicles; (4) using a microfluidic chip to isolate, capture and detect the bacterial exovesicles obtained in step (3), and the obtained bacterial exovesicles are the newly formed bacterial exovesicles. The method of this invention can selectively capture newly formed bacterial exovesicles from a sample without affecting the expression of surface proteins of bacterial exovesicles, so as to perform downstream analysis of newly formed bacterial exovesicles of intestinal flora.

Description

Technical Field

[0001] This invention relates to a method for isolating and detecting newly formed extravesicles of intestinal bacteria. Background Technology

[0002] Intestinal bacterial extravesicles are spherical nanoparticles with a diameter of 20-400 nm secreted by intestinal bacteria. They are rich in cell membrane proteins, lipids, lipopolysaccharides, peptidoglycans, DNA, RNA, etc., which endows intestinal bacterial extravesicles with a variety of biological functions. For example, intestinal bacteria can achieve communication between bacteria and bacteria and between bacteria and hosts by releasing extravesicles. In addition, the concentration and phenotype of the secreted bacterial extravesicles also reflect information about their parent bacteria and the development of various diseases, such as inflammatory bowel disease, metabolic diseases (type 2 diabetes), colorectal cancer, liver diseases, etc. However, existing methods for isolating intestinal bacterial extravesicles have the following limitations, including: (1) the isolated intestinal bacterial extravesicles contain a large number of host-derived extracellular vesicles, and there is a lack of methods for selectively isolating the extravesicles derived from intestinal bacteria; (2) existing techniques for isolating intestinal bacterial extravesicles can only capture existing and newly generated total bacterial extravesicles indiscriminately, while the total bacterial extravesicles contain a large amount of "old" extravesicle information, which cannot truly, accurately and in real time reflect the dynamic changes of the organism. Therefore, there is an urgent need to develop a new method for isolating and detecting dynamically changing newly generated extravesicles of intestinal bacteria. Summary of the Invention

[0003] The main objective of this invention is to provide a method for isolating and detecting newly formed extravesicles of intestinal bacteria.

[0004] The technical solution of the present invention is as follows:

[0005] A method for isolating and detecting extravesicles of newly formed intestinal bacteria, comprising the following steps:

[0006] 1) An azide-modified D-amino acid hydrochloride metabolic marker is used to metabolically label the microorganism under study, resulting in newly formed bacterial exovesicles bearing azide groups; the azide-modified D-amino acid hydrochloride metabolic marker is an azide-modified D-alanine hydrochloride probe (DAA-N3). Its structure is as follows: .

[0007] 2) Isolate and extract bacterial exovesicles from samples containing bacterial exovesicles;

[0008] 3) Add a capture linker that can react with azide groups to the extracted bacterial exovesicles;

[0009] 4) The bacterial exovesicles obtained in step 3) are isolated and captured, and the resulting bacterial exovesicles are the newly formed bacterial exovesicles.

[0010] In this invention, the microorganisms to be studied include pure in vitro bacteria or in vivo microorganisms from animals. Further, the pure in vitro bacteria are Staphylococcus aureus (G+) and Pseudomonas aeruginosa (G-), and the in vivo microorganisms are the intestinal flora of mice.

[0011] Furthermore, in this invention, step 2) of the sample containing bacterial exovesicles includes at least one of bacterial culture medium and intestinal tissue.

[0012] Furthermore, in this invention, the capturing linker that can react with the azide group in step 3) is DBCO (dibenzocyclooctylene), alkynyl, and biotin modified with alkynes such as BCN.

[0013] Furthermore, in this invention, step 4) captures the reaction between biotin and streptavidin.

[0014] Furthermore, step 4) employs a microfluidic chip for capture.

[0015] Furthermore, the capture was achieved using a silicon sphere microfluidic chip.

[0016] Furthermore, the microfluidic chip is a silicon ball fishbone chip with mixing and separation functions.

[0017] Furthermore, the surface of the silicon ball fishbone chip is modified with streptavidin, which can capture biotin-modified new bacterial exovesicles.

[0018] Furthermore, it also includes step 5), which involves the detection and analysis of newly formed bacterial vesicles.

[0019] Compared with the prior art, this technical solution has the following advantages:

[0020] The specific labeling and isolation detection of newly formed outer membrane vesicles of intestinal bacteria face numerous challenges. Since the main components of bacterial cell walls are peptidoglycan and lipopolysaccharide, and they lack eukaryotic cell-specific glycosylation modification pathways (such as the sialic acid metabolism pathway), conventional glycosylation labeling strategies (such as labeling with the non-natural sugar Ac4ManNAz) are difficult to apply directly. To address this challenge, this invention proposes a metabolic labeling method based on azide-modified D-alanine (DAA-N3). The inventors discovered that DAA-N3 can be efficiently integrated into the bacterial peptidoglycan synthesis pathway without modifying mammalian cells, thus achieving highly efficient and specific labeling of most bacteria both in vitro and in vivo. More importantly, this invention also found that this label can be stably delivered to bacterial secreted outer membrane vesicles, providing a specific recognition site for subsequent isolation and purification. By combining click chemistry technology, this method effectively solves the key problem of host-derived vesicle contamination in traditional methods; simultaneously, by integrating a microfluidic detection platform, the detection sensitivity of bacterial-derived outer membrane vesicles is significantly improved. Ultimately, the technical challenge mentioned in the background technology of "low abundance of isolated intestinal bacterial extracellular vesicles and a large number of host-derived extracellular vesicles" was solved, providing a reliable tool for the study of intestinal bacterial extracellular vesicles.

[0021] The method of this invention can selectively capture newly formed bacterial exovesicles from a sample without affecting the expression of surface proteins on bacterial exovesicles, so as to perform more effective analysis of newly formed bacterial exovesicles. Attached Figure Description

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0024] Figure 2 In Figure a, flow cytometry plots of DAA-N3-labeled S. aureus, P. aeruginosa, and THP-1 cells are shown, and in Figure b, the statistical proportion of DAA-N3-labeled S. aureus, P. aeruginosa, and THP-1 cells are shown.

[0025] Figure 3 Morphological images of bacterial vesicles from different sources.

[0026] Figure 4 This shows the modification status of azide groups on the surface of bacterial exovesicles.

[0027] Figure 5 SEM image of intestinal bacterial vesicles captured on a fishbone chip.

[0028] Figure 6The image shows the fluorescence signal of the external vesicles of intestinal bacteria captured on the fishbone chip (left) and the statistical value of the average fluorescence intensity of the external vesicles of intestinal bacteria captured on the fishbone chip (right). Detailed Implementation

[0029] Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0030] Using an azide-modified D-alanine hydrochloride (DAA-N3, purchased from MCE), a probe was used for metabolic labeling in mice, successfully attaching the azide group (-N3) to newly generated intestinal bacterial vesicles secreted by the gut microbiota. After co-incubating the intestinal bacterial vesicles with DBCO-PEG4-Biotin, the newly generated intestinal bacterial vesicles, due to their azide group, underwent a click chemistry reaction with DBCO, thus being modified with biotin. Finally, the newly generated intestinal bacterial vesicles with the biotin group were captured using a streptavidin (SA)-modified fishbone microarray. This method can efficiently and selectively isolate newly generated intestinal bacterial vesicles, enabling effective analysis of newly formed bacterial vesicles.

[0031] Example 1

[0032] 1. Extraction of bacterial extravesicles in vitro

[0033] 1) In vitro bacterial culture

[0034] Staphylococcus aureus and Pseudomonas aeruginosa were purchased from the U.S. Standard Microbial Bank. Single colonies were picked and activated overnight in 5 mL of LB liquid medium, then inoculated at 1% into 40 mL of fresh LB liquid medium containing 1 mM DAA-N3. After incubation at 37°C and 180 rpm for 24 h, the bacteria were used for extraction of exovesicles.

[0035] 2) Extraction of bacterial exovesicles

[0036] First, the bacterial culture was centrifuged at 2500 g for 30 minutes to remove bacterial cells, and the supernatant was collected. Next, the supernatant was filtered through a 0.22 µm membrane (Sangon Biotech, F513165-0001), and then concentrated by ultrafiltration (3280 g, 20 minutes) using an ultrafiltration tube (Merck Millipore, UFC910096). Finally, the concentrate was ultracentrifuged at 100,000 g for 2 hours, and the precipitated bacterial exovesicles were resuspended in PBS and stored at -80°C. All centrifugation steps were performed at 4°C.

[0037] 2. Extraction of extravesicles from intestinal bacteria

[0038] 1) Administer 200 µL of 20 mM DAA-N3 to 6-8 week old male C57 / BL6 mice by gavage for 6-9 hours for metabolic labeling.

[0039] 2) The intestinal flora of mice was obtained according to the method described in the literature. In short, the mice were euthanized by neck, the cecum was removed and placed in 2 mL of PBS, and the cecum tissue was minced with scissors; the contents of the cecum were then filtered through a 70 µm cell sieve, and the collected filtrate was the intestinal bacterial culture.

[0040] 3) Centrifuge the above intestinal bacterial culture at 12,000 rpm for 3 minutes to remove intestinal bacteria, collect the supernatant, filter the supernatant through a 0.22 µm filter membrane, and finally centrifuge the filtrate at 100,000 g for 2 hours. The precipitate obtained is the intestinal bacterial vesicle, which is resuspended in PBS and stored in a -80°C freezer.

[0041] 3. Chip fabrication and modification

[0042] 1) A microchannel mold was fabricated on a silicon wafer using SU-8 photoresist, with channel height controlled by spin coating (15–35 μm). PDMS prepolymer (a 10:1 mixture of matrix and curing agent) was then poured onto the mold and cured at 70°C for 4 h. After peeling, holes were punched to form the inlet and outlet ports of the microfluidic chip. The patterned PDMS chip was then sealed to a glass substrate, and a silica gel suspension (10% w / v) was injected into a reservoir to fill the microchannels via capillary action. Solvent evaporation drove the colloidal self-assembly, forming an ordered nanoporous structure. Finally, the patterned chip was removed, and the self-assembled nano-herringbone structure was aligned with the PDMS detection chip under a microscope and sealed.

[0043] 2) Add 5% (v / v) (3-mercaptopropyl)trimethoxysilane (MPTS) to the chip inlet and react at room temperature for 1 h. Then, rinse the chip with 70% ethanol, add 0.5 mg / mL NY-maleimide butyryl-oxysuccinimide ester (GMBS) to the inlet, and react at room temperature for 30 min. After rinsing with PBS, introduce 50 μg / mL streptavidin (SA) to the inlet and react at room temperature for 1 h. Finally, block the chip with 5% BSA at room temperature for 2 h and store at 4°C for later use.

[0044] 4. Sample characterization

[0045] Verify the azide group modification status on the surface of bacterial exovesicles.

[0046] 1) Coating bacterial exovesicles with 4% latex aldehyde beads: Mix bacterial exovesicles with 4% latex aldehyde beads at a ratio of 5 µg : 2 µL and incubate at room temperature for 15 min; then add 500 µL of filtered PBS and incubate at room temperature for 3-4 h (or overnight at 4°C); after incubation, add 55 µL of 20% BSA containing 1 M Gly and continue incubating at room temperature for 30 min, then centrifuge at 6500 rpm for 3 minutes, remove the supernatant, wash twice with 0.5% BSA and then resuspend in 0.5% BSA.

[0047] 2) Characterization of DiO membrane dye and DBCO-Cy3 dye: Take 100 µL of the above latex aldehyde beads coated with bacterial exovesicles, add 1 µL of 1 mM DiO dye and 1 µL of 1 mM DBCO-Cy3 dye to make the final concentration of both 10 µM, incubate at 37℃ for 1 h, wash twice with 0.5% BSA, and finally perform confocal imaging.

[0048] To verify whether azide-modified bacterial exovesicles can be captured by streptavidin-modified fishbone microarrays after reacting with DBCO-PEG4-Biotin.

[0049] 5 µg of bacterial exovesicles were incubated with 10 µM DBCO-PEG4-Biotin in 20 µL of PBS containing 0.5% BSA at 37°C for 1 hour, followed by incubation with 5 µM DiO dye at 37°C in the dark for 30 min. After incubation, the bacterial exovesicles were washed three times with 8000 g using a 100 kDa ultrafiltration tube. The labeled bacterial exovesicles were then introduced into a streptavidin-modified fishbone chip and incubated at 37°C for 30-60 minutes. Uncaptured bacterial exovesicles were washed away with PBS containing 0.5% BSA. Finally, fluorescence signals were acquired using a fluorescence microscope, and the fluorescence signals on the chip were processed and statistically analyzed using ImageJ software to obtain the fluorescence intensity of different groups.

[0050] Results analysis:

[0051] To verify that DAA-N3 can only specifically label pure bacteria, a Gram-positive bacterium, *Staphylococcus aureus*, a Gram-negative bacterium, *P. aeruginosa*, and a human mononuclear macrophage (THP-1) were selected for DAA-N3 metabolic labeling. Three hours later, a DBCO-Cy3 probe was used to induce a click chemical reaction with the bacteria, and the results were characterized by flow cytometry. Figure 2As can be seen, compared with the blank control group, both bacterial strains showed a shift in PE fluorescence signal in the DAA-N3 group. The shift was significantly greater in Gram-positive bacteria than in Gram-negative bacteria. This may be because Gram-negative bacteria have an outer membrane that prevents the DBCO-Cy3 probe from entering the peptidoglycan layer and undergoing a click chemical reaction with DAA-N3, resulting in a lower degree of PE signal shift. For THP-1 cells, there was no significant difference in the shift between the groups without and with DAA-N3 metabolic labeling, indicating that DAA-N3 cannot achieve metabolic labeling of THP-1 cells. These results suggest that the DAA-N3 probe can only specifically label bacteria and not animal cells.

[0052] Figure 3 The results showed that extravesicles derived from Staphylococcus aureus and Pseudomonas aeruginosa in vitro, as well as those derived from in vivo mouse intestinal flora, were successfully extracted. The morphology and size of the azide-modified bacterial extravesicles were not different from the control group, exhibiting typical goblet-shaped vesicle structures with a size of approximately 100 nm. This indicates that DAA-N3 metabolic treatment does not affect the morphology and size of extravesicles secreted by Staphylococcus aureus, Pseudomonas aeruginosa, or in vivo mouse intestinal flora.

[0053] Figure 4 The results showed that vesicles derived from Staphylococcus aureus and Pseudomonas aeruginosa in vitro, or from intestinal flora in mice, were successfully labeled with azide groups.

[0054] Figure 5 The results showed that azide-modified intestinal bacterial vesicles treated with DBCO-PEG4-Biotin could be successfully captured by streptavidin-modified silica ball fishbone chip, indicating that this chip platform combined with an azide-modified hydrochloric acid D-amino acid metabolic labeling strategy can be used to capture newly formed bacterial vesicles.

[0055] Figure 6 The results showed that the fluorescence intensity of the azide-modified intestinal bacterial vesicles after pre-staining with DiO membrane dye was higher than that of the control group without azide treatment, further proving that the azide-modified intestinal bacterial vesicles could be successfully captured by the streptavidin-modified fishbone chip, indicating that the capture system can successfully separate and analyze newly generated intestinal bacterial vesicles.

[0056] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for isolating and detecting newly formed extravesicles of mouse intestinal bacteria, for non-disease diagnosis and treatment purposes, comprising the following steps: 1) Azide-modified hydrochloric acid D-type amino acid metabolic markers are used to metabolically label the microorganisms under study, so that the exovesicles of newly formed bacteria are equipped with azide groups; The azido-modified D-amino acid metabolic marker for hydrochloride is the azido-modified D-alanine hydrochloride probe DAA-N3, as shown in the following chemical formula. ； 2) Isolate and extract bacterial exovesicles from samples containing bacterial exovesicles; 3) Add a capture linker that can react with azide groups to the extracted bacterial exovesicles; 4) The bacterial extravesicles obtained in step 3) are isolated and captured, and the obtained bacterial extravesicles are the new intestinal bacterial extravesicles. Steps 1) and 2) are as follows: a. Administer 200 µL of 20 mM DAA-N3 to 6-8 week old male C57 / BL6 mice by gavage for 6-9 hours for metabolic labeling; b. The mouse was euthanized by neck, the cecum was removed and placed in 2 mL PBS, and the cecum tissue was cut into small pieces with scissors; the contents of the cecum were then filtered through a 70 µm cell sieve, and the collected filtrate was the intestinal bacterial culture. c. Centrifuge the above intestinal bacterial culture at 12000 rpm for 3 minutes to remove intestinal bacteria, collect the supernatant, filter the supernatant through a 0.22 µm filter membrane, and finally centrifuge the filtrate at 100,000g for 2 hours. The precipitate obtained is the intestinal bacterial vesicle, which is resuspended in PBS and stored in a -80℃ freezer. Step 3) The capture linker that can react with the azide group is at least one of DBCO and BCN-modified biotin; Step 4) Capture the reaction between biotin and streptavidin; In step 4), a microfluidic chip is used to separate and capture the bacterial exovesicles from step 3).

2. The method for isolating and detecting newly formed extravesicles of mouse intestinal bacteria according to claim 1, characterized in that: The capture was performed using a silicon sphere microfluidic chip.

3. The method for isolating and detecting newly formed extravesicles of mouse intestinal bacteria according to claim 1, characterized in that: The microfluidic chip is a silicon ball fishbone chip with mixing and separation functions.

4. The method for isolating and detecting newly formed extravesicles of mouse intestinal bacteria according to claim 3, characterized in that: The surface of the silicon ball fishbone chip is modified with streptavidin, which can capture biotin-modified new bacterial exovesicles.

5. A method for isolating and detecting newly formed extravesicles of mouse intestinal bacteria according to any one of claims 1-4, characterized in that: It also includes step 5), which involves the detection and analysis of the extravesicular vesicles of newly formed intestinal bacteria.

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