A method for screening inhibitors targeting bacterial membrane protein folding machinery based on fluorescence complementation

Abstract

This invention discloses a method for screening inhibitors targeting bacterial membrane protein folding machines based on fluorescence complementarity, belonging to the field of biomedical detection and analysis. This invention provides a reporter vesicle system, comprising reporter vesicles and protein inclusion bodies. The reporter vesicles contain luminescent substrates (LgBiT and luciferase) and have a BamABCDE complex on their outer membrane. The protein inclusion bodies are folding proteins with a HiBiT tag attached to the passenger structure. This reporter vesicle system enables real-time, sensitive, and high-throughput monitoring of bacterial outer membrane protein folding processes in a vesicle-mimicking lipid membrane environment, and provides a method and system for rapidly and effectively screening inhibitors targeting the BamABCDE complex.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection and analysis, specifically relating to a method for screening targeted inhibitors of bacterial membrane protein folding machines based on fluorescence complementarity. Background Technology

[0002] The problem of drug resistance in Gram-negative bacteria is becoming increasingly serious, necessitating the development of novel antimicrobial drugs targeting new avenues. The proper folding and assembly of bacterial outer membrane proteins is crucial for their survival and pathogenicity. The BAM (β-barrel assembly machinery) complex, a key molecular machine responsible for the folding and insertion of outer membrane proteins, is considered a highly promising target for novel antimicrobial drugs.

[0003] Currently, screening methods for BAM inhibitors have many limitations:

[0004] (1) Traditional microbiological methods, such as the determination of minimum inhibitory concentration (MIC), have low throughput, are time-consuming, and cannot directly reflect the inhibitory effect on specific targets.

[0005] (2) In vitro biochemical methods: usually require purification of complex membrane protein complexes, which is a complicated process and is separated from the natural membrane environment, which may not be able to truly reflect the effect of the inhibitor under physiological conditions.

[0006] (3) Existing detection technology: There is a lack of convenient means to monitor the membrane protein folding process in real time, with high sensitivity and high throughput.

[0007] Therefore, there is an urgent need in this field for a technology that can directly and efficiently screen inhibitors targeting BAM complexes in a system that simulates a natural membrane environment. Summary of the Invention

[0008] Technical issues

[0009] This invention addresses the shortcomings of existing BAM inhibitor screening and detection methods by providing a method and system that enables real-time, sensitive, and high-throughput monitoring of bacterial outer membrane protein folding processes in a vesicle-mimicking lipid membrane environment, and based on this, rapidly and effectively screens inhibitors targeting the BAMABCDE complex.

[0010] Technical solution

[0011] The present invention first provides a reporter vesicle system, the reporter vesicle system comprising: a reporter vesicle and a protein inclusion body, wherein the reporter vesicle contains a luminescent substrate of LgBiT and luciferase and has a BamABCDE complex on its outer membrane; the protein inclusion body is a folded protein with a HiBiT tag attached to the front of the passenger domain.

[0012] In one embodiment of the invention, the protein to be folded is a bacterial outer membrane protein, such as EspP.

[0013] In one embodiment of the invention, the reporter vesicle system further includes the chaperone protein SurA.

[0014] This invention provides a method for preparing the reporter vesicle system, comprising the following steps:

[0015] Prepare a folded protein with a HiBiT tag attached, that is, construct the HiBiT tag before the passenger structure of the folded protein (e.g., EspP), which is provided in the form of an inclusion body;

[0016] Vesicle preparation, namely, the preparation of liposomes or protoplasmic vesicles containing two components: LgBiT and luciferase luminescent substrate.

[0017] To prepare reporter vesicles, purified BamABCDE complexes are embedded into the vesicle membrane via a detergent-mediated method, reconstructing their membrane environment-dependent folding activity to form fully functional reporter vesicles.

[0018] In one embodiment of the present invention, the specific steps for preparing vesicles may be:

[0019] (1) Dissolve the pre-concentrated chloroform in water. E . coli Polar Lipid mother liquor was dried with nitrogen gas to form a lipid film;

[0020] (2) The lipid membrane is resuspended in a buffer solution and sonicated to hydrate and clarify it to obtain a resuspension; the buffer solution may be 50 mM Hepes, 150 mM NaCl, pH 7.8;

[0021] (3) Add 100 μM of luciferase luminescent substrate Furimazine and 40 μM of LgBiT to the resuspension to obtain a mixture;

[0022] (4) The mixture was circulated through the polycarbonate membrane 21 times using a liposome extruder to obtain liposome vesicles with uniform particle size.

[0023] In one embodiment of the present invention, the specific steps for preparing the reporter vesicles may be:

[0024] (1) Add a detergent to the vesicles and incubate them at low temperature to loosen their membrane structure;

[0025] (2) Add BamABCDE complex and incubate at low temperature to form lipid-protein-detergent mixed micelles;

[0026] (3) The detergent is removed by dilution and centrifugation, the precipitate is collected and resuspended to obtain the report vesicle.

[0027] This invention provides a method for screening bacterial outer membrane protein folding inhibitors using the reporter vesicle system. The method utilizes the reporter vesicle system to monitor the folding process of bacterial outer membrane proteins in real time, and based on this, performs high-throughput screening for novel inhibitors targeting the bacterial membrane protein folding machinery. The method includes the following steps:

[0028] (1) Add the candidate bacterial outer membrane protein folding inhibitor to be tested to the BamABCDE complex and incubate;

[0029] (2) A reporter vesicle system was constructed using the BamABCDE complex after incubation with a candidate bacterial outer membrane protein folding inhibitor;

[0030] (3) Add the chaperone protein SurA to the reporter vesicle system;

[0031] (4) Incubate at a suitable temperature to initiate the folding reaction;

[0032] (5) Use an ELISA reader, fluorescence microscope or other optical detection equipment to detect the fluorescence signal of the reaction system in real time or at the endpoint. The fluorescence signal will only be triggered when the unfolded protein in the reporter vesicle system is correctly folded and inserted into the vesicle membrane; if the added candidate bacterial outer membrane protein folding inhibitor inhibits the function of the BamABCDE complex in the reporter vesicle system, the folding process will be blocked and the fluorescence signal will weaken or disappear.

[0033] This invention constructs a reporter vesicle system that couples the protein (e.g., bacterial outer membrane protein) folding process with the generation of a fluorescence signal. Its working principle is as follows:

[0034] (1) Without inhibitor (positive control): The unfolded EspP substrate interacts with the BAM folding machinery on the vesicle and folds. During folding, its passenger structure is removed, exposing the HiBiT tag at its tip. This HiBiT tag can enter the reporter vesicle and bind to LgBiT pre-existing within the reporter vesicle to form an active NanLuc luciferase. This enzyme immediately catalyzes the luminescence of the Furimazine substrate within the reporter vesicle, producing a detectable fluorescent signal.

[0035] (2) When folding inhibitors are present: If the compound to be tested can effectively inhibit the function of the BAM folding machine, the folding process of EspP is blocked. The HiBiT tag cannot be exposed and bind to LgBiT, so it cannot form an active luciferase, resulting in a significant reduction or complete absence of fluorescence signal.

[0036] By comparing the fluorescence signal intensity of the compound-added group, the control group without the compound (positive control), and the known inhibitor group (negative control), it can be determined whether the compound is a potential BAM inhibitor.

[0037] Beneficial effects

[0038] Compared with the prior art, the present invention has the following significant advantages:

[0039] (1) Direct targeting: Directly detects the effect on the function of membrane protein folding machinery, rather than the indirect inhibition of bacterial growth, with a higher hit rate.

[0040] (2) Real-time sensitivity: Based on NanoLuc fluorescence complementary technology, it has a high signal-to-background ratio and excellent sensitivity, enabling real-time dynamic monitoring.

[0041] (3) High throughput compatibility: The entire detection process can be carried out in 96-well or 384-well plates, which is very suitable for screening large-scale compound libraries.

[0042] (4) High physiological relevance: The screening is carried out in artificial vesicles, which simplifies the system and simulates the natural membrane environment to the greatest extent, thus improving the reliability of the screening results.

[0043] (5) High versatility: By changing the substrate protein (such as other substrate proteins for BAM) and the corresponding folding machinery, this method can be easily extended to screening inhibitors of other types of membrane protein assembly machinery. Attached Figure Description

[0044] Figure 1 The results of SDS-PAGE for the expression and purification of the BAM complex in Example 1 are shown.

[0045] Figure 2 The results of SDS-PAGE for the expression and purification of EspP inclusion bodies in Example 1 are shown.

[0046] Figure 3 The results of SDS-PAGE for LgBiT expression purification in Example 1 are shown.

[0047] Figure 4 The results are obtained from SDS-PAGE detection and proteinase K digestion verification of the reconstructed BAM complex liposomes in Example 2.

[0048] Figure 5This is a fluorescence kinetic curve for detecting BAM-folded EspP in Example 2. In the figure, the upper curve represents the BAM group, i.e., reporter vesicles containing the BamABCDE complex, and the lower curve represents the control group, i.e., blank vesicles.

[0049] Figure 6 This is a fluorescence kinetic curve for detecting BAM-folded EspP pretreated with JB-95 in Example 3. In the figure, the curve with the highest relative fluorescence intensity at 1200s is the BAM group, i.e., the untreated BamABCDE complex folded EspP, followed by BAM+JB95, i.e., the BamABCDE complex folded EspP pretreated with JB-95, and the lowest is the negative control. Detailed Implementation

[0050] The sources of the main materials and reagents used in the following examples:

[0051] Phanta Max Super-Fidelity DNA Polymerase and ClonExpress II One Step Cloning Kit were purchased from Nanjing Vazyme Biotechnology Co., Ltd.; DpnI restriction enzyme was purchased from New England Biotechnology Co., Ltd. (USA); plasmid miniprep kit was purchased from Beijing TIANGEN Biochemical Technology Co., Ltd.; SDS-PAGE gel rapid preparation kit was purchased from Shanghai Epizyme Biotechnology Co., Ltd.; LB premixed medium was purchased from BD Biosciences (USA); imported detergent DDM was purchased from Anatrace (USA); ampicillin / kanamycin was purchased from Shanghai Sangon Biotech Co., Ltd.; furimazine fluorescent substrate was purchased from Selleck Chemicals (USA); protein marker and desalted pre-packed gravity column were purchased from BIO-RAD (USA); and protein concentration tubes with different molecular weight cutoffs were purchased from Millipore (USA).

[0052] the term

[0053] Vesicles are tiny spherical structures formed by phospholipid bilayers, primarily used for transporting and storing substances inside and outside cells.

[0054] LgBiT is a large subunit of NanoLuc luciferase. When it binds to the HiBiT tag, the two spontaneously complement and reconstruct to form an active NanoLuc luciferase, which catalyzes the substrate (furan luciferin) to emit a high-intensity bioluminescent signal.

[0055] The HiBiT tag is a peptide tag consisting of 11 amino acids that can be linked to the target protein and rapidly bind to the luciferase subunit LgBiT to produce a highly active luciferase.

[0056] The BamABCDE complex is a protein complex located on the outer membrane of Gram-negative bacteria, known as the BAM folding machine. Its core function is to recognize, fold, and correctly insert and assemble newly synthesized β-barrel proteins into the outer membrane. It consists of five core protein subunits: BamA, BamB, BamC, BamD, and BamE, where BamA is the core catalytic subunit, and the others are accessory lipoproteins.

[0057] Inclusion bodies are dense, water-insoluble particles that form within cells and are primarily composed of misfolded or unfolded protein aggregates.

[0058] EspP is an important bacterial outer membrane protease secreted by pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli and enteropathogenic Escherichia coli.

[0059] Furimazine is a synthetic imidazopyrazinone compound that can serve as a substrate for NanoLuc luciferase (Nluc).

[0060] SurA, a chaperone protein, is a key peptidyl prolyl cis-trans isomerase and a universal molecular chaperone in the periplasm of Gram-negative bacteria. Its core function is to recognize, protect, and deliver newly synthesized, unfolded outer membrane proteins across the hydrophobic, crowded, and protease-rich periplasmic space, precisely delivering them to the BAM protein assembly machinery of the outer membrane.

[0061] Example 1: Construction of a report vesicle system for reporting bacterial outer membrane protein folding

[0062] S1: Expression vector construction

[0063] ① The expression vectors pTrc99a-BamABCDE (8×His) for BamABCDE of Escherichia coli K12 strain; pET28a-His-sumo-TEV-SurA for the chaperone protein SurA; and pET28a-EspP and pET28a-HiBiT-EspP for the EspP folding substrate were all constructed and preserved in our laboratory. The LgBiT expression vector pET28a-His-LgBiT was commercially synthesized. The amino acid sequences of each protein are shown in Table 2.

[0064] ② Construction of the pTrc99a-BamABCDE(8×His) vector: stored in the NCBI database Escherichia coli Using the K12 genome as a reference sequence, specific gene amplification primers were designed for amplification. bamA , bamB , bamC , bamD , bamE The gene was inserted, and an 8×His tag was introduced at the C-terminus of BamE. Linearization primers were designed using the pTrc99a vector sequence as a template. The high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase was used with preserved DNA from our laboratory. Escherichia coli Using K12 genomic DNA and the pTrc99a vector as templates, PCR amplification was performed to obtain the target gene fragment and the linearized vector. The target gene and the linearized vector were ligated using the ClonExpress II One Step Cloning Kit at a molar ratio of 3:1. The ligation product was transformed into DH5α competent cells and screened on LB agar plates containing 100 μg / mL ampicillin. After culturing at 37 °C for 12 h, single colonies were picked and expanded, and the bacterial culture was sent to the company for sequencing. Successfully sequenced engineered strains were used to extract plasmids. The final expression vector pTrc99a-BamABCDE (8×His) was obtained.

[0065] ③ Construction of the pET28a-His-sumo-TEV-SurA vector: stored in the NCBI database Escherichia coli Using the K12 genome as a reference sequence, specific amplification primers were designed for amplification. surA The gene was introduced, along with a TEV enzyme restriction site (ENLYFQG). Linearization primers were designed using the pET28a-His-sumo vector sequence as a template. The high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase was used with preserved DNA from our laboratory. Escherichia coli Using K12 genomic DNA and the pTrc99a vector as templates, PCR amplification was performed to obtain the target gene fragment and the linearized vector. The target gene and the linearized vector were ligated using the ClonExpress II One Step Cloning Kit at a molar ratio of 3:1. The ligation product was transformed into DH5α competent cells and screened on LB agar plates containing 50 μg / mL kanamycin. After culturing at 37 °C for 12 h, single colonies were picked and expanded, and the bacterial culture was sent to the company for sequencing. Successfully sequenced engineered strains were used to extract plasmids. The final expression vector pET28a-His-sumo-TEV-SurA was obtained.

[0066] ④ Construction of the pET28a-EspP vector: stored in the NCBI database Escherichia coli Using the O157:H7 plasmid as a reference sequence, specific amplification primers were designed to amplify the nucleotide sequence of the C-terminal β-barrel domain, 25 adjacent residues from the passenger domain, and 21 upstream linker residues of EspP. Linearization amplification primers were designed using the pET28a empty vector sequence as a template. PCR amplification was performed using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase with genomic DNA stored in our laboratory and the pTrc99a plasmid as a template to obtain the target gene fragment and the linearized vector. The target gene and the linearized vector were ligated using the ClonExpress II One Step Cloning Kit at a molar ratio of 3:1. The ligation product was transformed into DH5α competent cells and screened on LB plates containing 50 μg / mL kanamycin. After culturing at 37 °C for 12 h, single colonies were picked for expansion culture, and the bacterial culture was sent to a company for sequencing. Successfully sequenced engineered strains were used to extract plasmids. The final expression vector pET28a-EspP was obtained.

[0067] ⑤ Primers were designed according to the principle of site-directed mutagenesis. Using pET28a-EspP as a template, a short peptide tag of HiBiT (VSGWRLFKKIS) was inserted before the N-terminal passenger domain of EspP. The primer sequences are shown in Table 1 below.

[0068] Table 1 Primer Sequences

[0069]

[0070] Amplification was performed using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase with pET28a-EspP plasmid as a template. Unmutated templates in correctly identified point-mutated PCR products were removed using DpnI restriction enzyme and identified by agarose gel electrophoresis at 37 °C for 30 min. The digested products were then transformed into DH5α competent cells and screened on LB agar plates containing 50 μg / mL kanamycin. After culturing at 37 °C for 12 h, single colonies were picked and expanded, and the bacterial culture was sent to a company for sequencing. Successfully sequenced engineered strains were used to extract plasmids. The final expression vector pET28a-HiBiT-EspP was obtained.

[0071] Table 2. Amino acid sequences of related proteins

[0072]

[0073] S2: Expression and purification of the BamABCDE complex

[0074] ① The pTrc99a-BamABCDE (8×His) expression vector was transferred into competent expression states. E . coli C43 (DE3) strains were successfully transformed through screening for ampicillin resistance genes. Single colonies were picked and cultured in 100 mL of LB broth containing 100 μg / mL ampicillin for 12 h. 100 mL of the bacterial culture was then inoculated into 3 L of LB broth containing 100 μg / mL ampicillin and cultured at 37 °C and 220 rpm for 3 h. When the bacterial culture showed OD... 600 When the value approached 1.0, the culture conditions were changed to 20°C, 180 rpm, and IPTG was added to a final concentration of 0.2 mM to induce protein expression for 12 h. The bacterial pellet was collected by centrifugation at 4000 rpm for 12 min.

[0075] ② Resuspend the bacterial culture in 50 mL of Buffer A (containing 50 mM Hepes, 300 mM NaCl, 10% Glycerol, pH 7.8), add PMSF to a final concentration of 1 mM, and homogenize using a high-pressure homogenizer at 800-900 bar for 2 min.

[0076] ③ Centrifuge the disrupted bacterial cell solution at 35,000 rpm and 4 °C for 50 min to collect the membrane precipitate. Resuspend the precipitate in 50 mL of Buffer A containing 1% DDM (LABLEAD) and homogenize with a tissue homogenizer until no large particles remain. Extract at room temperature for 30 min. Centrifuge the extracted bacterial cell solution at 35,000 rpm and 4 °C for 30 min to collect the supernatant and filter it through a 0.45 μm filter membrane.

[0077] ④ Equilibrate the Ni-NTA packing material using 10 mL of Buffer A containing 10 mM imidazole and 0.03% DDM (Anatrace). Integrate the supernatant from step ③ with the Ni-NTA packing material at room temperature for 1 h. After collecting the flow-through, wash with 90 mL of Buffer A containing 30 mM imidazole and 0.03% DDM (Anatrace) (5 mL each time, incubated for 2 min before elution). Elute the target protein with 20 mL of Buffer A containing 300 mM imidazole and 0.03% DDM (Anatrace) (3 mL each time, incubated for 3 min before collection).

[0078] ⑤ Take the eluted target protein and replace it with a desalting column in Buffer B (50 mM Hepes, 150 mM NaCl, pH 7.8) containing 0.03% DDM (Anatrace), and adjust the protein concentration to 2-3 mg / mL.

[0079] ⑥ The target protein was subjected to SDS-PAGE electrophoresis and then stained with Coomassie Brilliant Blue to determine the purification status of the BamABCDE complex. The results are as follows: Figure 1 As shown, BamA has a molecular weight of 90.6 kDa, BamB has a molecular weight of 41.9 kDa, BamC has a molecular weight of 36.8 kDa, BamD has a molecular weight of 27.8 kDa, and BamE has a molecular weight of 12.3 kDa. The target bands were present at the corresponding size positions, and the protein purity was good.

[0080] S3: Expression and purification of the folded substrate EspP inclusion bodies

[0081] ① Transform the pET28a-HiBiT-EspP expression vector into competent expression cells. E . coli In BL21, strains successfully transformed through screening for kanamycin resistance genes were selected. Single colonies were picked and cultured in 100 mL of LB liquid medium containing 50 μg / mL kanamycin for 12 h. 100 mL of the bacterial culture was then inoculated into 2 L of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C and 220 rpm for 3 h. When the bacterial culture showed OD... 600 When the value approached 1.0, IPTG was added to a final concentration of 1 mM to induce protein expression for 3 h. The bacterial pellet was collected by centrifugation at 4000 rpm for 12 min.

[0082] ② Resuspend the bacterial culture in 50 mL of Buffer B (containing 50 mM Hepes, 150 mM NaCl, pH 7.8), add PMSF to a final concentration of 1 mM, and homogenize using a high-pressure homogenizer at 800-900 bar for 2 min.

[0083] ③ Centrifuge the broken bacterial solution at 4000 rpm and 4 °C for 30 min to collect the inclusion body precipitate.

[0084] ④ Add 15 mL of Buffer B to resuspend the inclusion body precipitate, centrifuge at 4000 rpm and 4 °C for 10 min, and repeat the washing twice.

[0085] ⑤ Dissolve the inclusion body precipitate obtained in the previous step in 10 mL of buffer B containing 8 M urea at room temperature for 1 h.

[0086] ⑥ Transfer the dissolved liquid, after it has dissolved to a 2 mL Eppendorf tube, centrifuge at 12,000 rpm for 20 min, and filter the supernatant through a 0.22 μm filter membrane to remove impurities. Adjust the final concentration of inclusion bodies to approximately 5 mg / mL, aliquot, and flash freeze in liquid nitrogen, then store at -80 °C.

[0087] ⑦ After SDS-PAGE electrophoresis, the target protein was stained with Coomassie Brilliant Blue to verify the purity and band position of the inclusion bodies. For example... Figure 2 As shown, the molecular weight of EspP is 38 kDa, and there is a target band at the corresponding size position. The purity of EspP inclusion bodies is also good.

[0088] S4: Expression and purification of the luciferase subunit LgBiT

[0089] ① Transform the pET28a-His-LgBiT expression vector into competent expression cells. E . coli In BL21, strains successfully transformed through screening for kanamycin resistance genes were selected. Single colonies were picked and cultured in 100 mL of LB liquid medium containing 50 μg / mL kanamycin for 12 h. 100 mL of the bacterial culture was then inoculated into 2 L of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C and 220 rpm for 3 h. When the bacterial culture showed OD... 600 When the value approached 1.0, the culture conditions were changed to 20°C and 180 rpm, and IPTG was added to a final concentration of 1 mM to induce protein expression for 16 h. The bacterial pellet was collected by centrifugation at 4000 rpm for 12 min.

[0090] ② Resuspend the bacterial culture in 50 mL of Buffer B (containing 50 mM Hepes, 150 mM NaCl, pH 7.8), add PMSF to a final concentration of 1 mM, and homogenize using a high-pressure homogenizer at 800-900 bar for 2 min.

[0091] ③ Centrifuge the broken bacterial solution at 12000 rpm and 4 °C for 30 min, collect the supernatant, and filter it with a 0.45 μm filter membrane to remove impurities.

[0092] ④ Equilibrate the Ni-NTA packing material with 10 mL of Buffer B containing 10 mM imidazole. Bind the supernatant from step ③ to the Ni-NTA packing material at 4 °C for 2 h. After flow-through, wash with 200 mL of Buffer B containing 10 mM imidazole. Elute the target protein with 30 mL of Buffer B containing 250 mM imidazole (3 mL each time, collected after standing for 3 min).

[0093] ⑤ Take the eluted target protein and replace it with Buffer B (50 mM Hepes, 150 mM NaCl, pH 7.8) using a desalting column, and adjust the protein concentration to approximately 8 mg / mL.

[0094] ⑥ The purity and band position of the target protein were verified by SDS-PAGE electrophoresis followed by Coomassie Brilliant Blue staining. For example... Figure 3 As shown, LgBiT has a molecular weight of 18 kDa, exhibits the target band at the corresponding size position, and has good protein purity.

[0095] Example 2: Method for detecting bacterial extracellular protein folding using a report vesicle system

[0096] S1: Constructing BamABCDE liposomes

[0097] ① According to protein and E . coli Phospholipids ( E . coli Calculate the required amount of phospholipids using a 1:100 molar ratio (Polar Lipid Extract), take out the phospholipid stock solution that has been dissolved in chloroform, and slowly dry the solvent in a fume hood using a nitrogen dryer to form a dry lipid film.

[0098] ② The lipid membrane obtained in ① was resuspended using buffer B (50 mM Hepes, 150 mM NaCl, pH 7.8), and sonicated for 20 min to clarify it, yielding a resuspension. Furimazine substrate (final concentration 100 μM) and luciferase subunit LgBiT (final concentration 40 μM) were added to the resuspension to obtain a mixture. A liposome extruder was prepared in advance, with the gasket and polycarbonate membrane (0.4 μm) installed. The mixture was circulated and extruded 21 times to obtain uniformly sized liposome vesicles encapsulating Furimazine and LgBiT.

[0099] ③ Add Buffer B containing 50 mg / mL DDM (Anatrace) to the liposomes obtained in ② at a mass ratio of 1:0.8 of liposomes to detergent, and incubate on ice for 1 h to loosen the liposome structure.

[0100] ④ Add the BamABCDE complex to the loose liposomes obtained in ③ according to the specified ratio, and incubate on ice for 1 h. Dilute the total solution volume to 25 mL using Buffer B, and centrifuge at 300,000 g, 4 °C for 30 min. Resuspend the liposome precipitate in 300 μL of Buffer B to obtain reporter vesicles.

[0101] Repeat step ④, but use Buffer B instead of the BamABCDE complex to prepare blank vesicles without the BamABCDE complex as a control.

[0102] ⑤ The reporter vesicle samples were subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue to verify their purity and band positions. Simultaneously, proteinase K digestion was used to verify the correct orientation of the BamABCDE complex insertion into the liposomes. Proteinase K is a non-specific, highly active proteolytic enzyme. It cannot penetrate the intact lipid bilayer. Therefore, it can only degrade the protein fractions exposed in solution that are not protected by the membrane structure. Figure 4 As shown, without proteinase K treatment, bands of BamA, BamB, BamC, BamD, and BamE were present in the vesicles. After treatment with proteinase K, most of the correctly oriented BamA proteins in the vesicles remained, while the proteins in the periplasm were hydrolyzed by proteinase K, indicating that the protein vesicles constructed in this study were correctly oriented.

[0103] S2: Real-time dynamic detection of BAM folded ESPP

[0104] ① Prepare the reaction system under ice bath conditions. The system contains 30 μL of reporter vesicles obtained from S1, 40 μM of chaperone protein SurA, and 40 μM of EspP inclusion bodies. Transfer the reaction solution to pre-cooled 96-well black microplates. Set up three replicates for each group and set up a control group containing only blank vesicles.

[0105] ② Fluorescence kinetics were monitored using an ELISA reader. The detection time was set to 20 minutes, and 61 readings were performed. The detection wavelengths were set to 465 nm for excitation and 515 nm for emission, and the changes in fluorescence intensity over time were recorded in real time. The results are as follows: Figure 5 As shown, the reporter vesicle system containing the BamABCDE complex exhibited a rapid increase in fluorescence signal within minutes after the addition of the substrate EspP, indicating that EspP was successfully folded and inserted into the membrane, triggering the fluorescence signal. In contrast, the control group containing only blank vesicles (without the BAM complex) maintained a very low background fluorescence signal throughout the monitoring period, showing no significant change. This result confirms that the reporter vesicle system constructed in this invention can monitor the BAM-mediated bacterial outer membrane protein folding process in real time and specifically, with a high signal-to-background ratio and rapid response.

[0106] Example 3: A method for high-throughput screening of bacterial extracellular protein folding inhibitors using a reporter vesicle system.

[0107] ① JB-95 pretreatment of BamABCDE complex:

[0108] Taking the BAM inhibitor JB-95 as an example, the purified BamABCDE complex and the inhibitor JB-95 were mixed at a molar ratio of 1:2 in Buffer B (50 mM Hepes, 150 mM NaCl, pH 7.8) containing 0.03% DDM (Anatrace) and incubated at 4°C for 2 hours to ensure sufficient binding of the inhibitor to the target site. Simultaneously, a sample of the BamABCDE complex without the inhibitor was prepared and incubated under the same conditions as a positive control.

[0109] ② Referring to the method described in S1 of Example 2, corresponding reporter vesicles and blank vesicles were prepared using BamABCDE complex pretreated with JB-95, BamABCDE complex untreated with JB-95 (positive control), and an equal volume of Buffer B (negative control).

[0110] ③ Referring to the method described in S2 of Example 2, the efficiency of BamABCDE complex folding EspP pretreated with JB-95 was dynamically monitored in real time, while untreated BamABCDE complex and blank vesicles were set as controls. The results are as follows: Figure 6 As shown, compared with the positive control group without inhibitors, the fluorescence signal intensity of the BAM complex pretreated with JB-95 was significantly reduced, its kinetic curve rose slowly, and the final signal intensity was only slightly higher than that of the blank vesicle control group. This indicates that JB-95 effectively inhibited the folding activity of the BAM complex, leading to the obstruction of the folding and insertion process of the substrate EspP, and a reduction in fluorescence signal generation. This experiment, using known inhibitors as examples, verified that the reporter vesicle system can sensitively and reliably detect and distinguish inhibitors of the BAM folding mechanism, demonstrating its feasibility for high-throughput drug screening.

[0111] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A vesicle system, characterized in that, The vesicle system for reporting protein folding includes: a reporter vesicle and a protein inclusion body. The reporter vesicle is a vesicle containing a luminescent substrate of LgBiT and luciferase, and having a BamABCDE complex on its outer membrane. The protein inclusion body is a folding protein with a HiBiT tag attached to the front of the passenger structure. The protein to be folded is a bacterial outer membrane protein.

2. The vesicle system according to claim 1, characterized in that, The vesicle system also includes the chaperone protein SurA.

3. A method for preparing the vesicle system of claim 1, characterized in that, Includes the following steps: A: The HiBiT tag is constructed before the passenger structure of the protein to be folded, which is provided in the form of an inclusion body; B: Prepare liposomes or protoplasmic vesicles containing two components: LgBiT and luciferase as a luminescent substrate. C: The BamABCDE complex is embedded into the membrane of the liposomes or protoplasmic vesicles via a detergent-mediated method to form a fully functional reporter vesicle.

4. The method according to claim 3, characterized in that, Step B specifically involves: (1) Dissolve the pre-concentrated chloroform in water. E . coli Polar Lipid mother liquor was dried with nitrogen gas to form a lipid film; (2) The lipid film was resuspended in a buffer solution and sonicated to hydrate and clarify it, thus obtaining a resuspension. (3) Add the luminescent substrate of luciferase and LgBiT to the resuspension to obtain a mixture; (4) Use a liposome extruder to extrude the mixture to obtain liposome vesicles with uniform particle size.

5. The method according to claim 3, characterized in that, Step C specifically involves: (1) Add detergent to the liposomes or protoplasmic vesicles and incubate at low temperature to loosen their membrane structure; (2) Add BamABCDE complex and incubate at low temperature to form lipid-protein-detergent mixed micelles; (3) The detergent is removed by dilution and centrifugation, the precipitate is collected and resuspended to obtain the report vesicle.

6. The use of the vesicle system according to claim 1 or 2 in screening bacterial outer membrane protein folding inhibitors.

7. The application according to claim 6, characterized in that, Includes the following steps: (1) Add the candidate bacterial outer membrane protein folding inhibitor to be tested to the BamABCDE complex and incubate; (2) A reporter vesicle system was constructed using the BamABCDE complex after incubation with a candidate bacterial outer membrane protein folding inhibitor; (3) Add the chaperone protein SurA to the reporter vesicle system; (4) Incubate at a suitable temperature to initiate the folding reaction; (5) Use optical detection equipment to detect the fluorescence signal of the reaction system in real time or at the endpoint.

8. The application according to claim 7, characterized in that, Fluorescent signals are triggered only when the proteins to be folded in the vesicle system are correctly folded and inserted into the vesicle membrane; if the added candidate bacterial outer membrane protein folding inhibitor inhibits the function of the BamABCDE complex in the vesicle system, the folding process is blocked, and the fluorescent signal weakens or disappears.

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