Preparation method and application of staphylococcus aureus extracellular vesicle
By treating Staphylococcus aureus with the lyase PlySs2, high-yield, low-toxicity extracellular vesicles were prepared, solving the problems of low yield and high toxicity in existing technologies and achieving effective immunoprotection.
Patent Information
- Application Number
- CN202511746686.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-02-27
AI Technical Summary
In the existing technology, the natural yield of Staphylococcus aureus extracellular vesicles is low and there are toxicity problems, and traditional antibiotic treatment faces the risk of drug resistance and lacks effective vaccines.
Staphylococcus aureus was treated with the lyase PlySs2 to induce the production of extracellular vesicles through specific steps, including shaking culture, centrifugation, filtration and ultrafiltration, to prepare high-yield and low-toxicity extracellular vesicles.
It increased the production of extracellular vesicles, reduced their toxicity, provided good protection in a mouse infection model, and induced a dual immune response.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of microorganisms or enzymes, and particularly relates to a method for producing bacterial extracellular vesicles by using lytic enzyme PlySs2 and application thereof. BACKGROUND
[0002] Staphylococcus aureus is an important zoonosis pathogen, and the global spread of its drug-resistant strains (such as methicillin-resistant Staphylococcus aureus) has constituted a public health crisis. Traditional antibiotic therapy is at risk of failure, and there is no available vaccine.
[0003] Bacterial extracellular vesicles (EVs) are nanoscale membrane structures secreted by bacteria, usually 30-500 nm in diameter, with a phospholipid bilayer structure, and internal packaging of active molecules such as proteins, nucleic acids, and metabolites. Such vesicles play a key role in bacterial and host communication, inter-bacterial communication, and environmental adaptation. EVs, as natural multi-antigen carriers, can avoid the limitations of a single target. In the prior art, EVs can induce a dual immune response (such as a Neisseria meningitidis vaccine), but there are problems such as low natural yield and high toxicity.
[0004] Lytic enzymes are peptidoglycan-specific hydrolases encoded by bacteriophage genomes, which are activated and expressed in the late stage of the bacteriophage replication cycle. Their core function is to degrade the peptidoglycan layer of the bacterial cell wall, leading to lysis of the host bacteria, thereby releasing newly synthesized progeny bacteriophage particles. Previous studies have shown that bacteriophage lytic enzymes can induce the production of extracellular vesicles by Gram-negative bacteria such as Acinetobacter baumannii, and that the extracellular vesicles induced by lytic enzymes are uniform and have a high yield, and can provide good protection in mouse infection models. However, whether lytic enzymes can induce Staphylococcus aureus to produce vesicles has not been reported. Staphylococcus aureus is a Gram-positive bacterium, and its outer cell wall surface is a peptidoglycan layer, which is significantly different from the lipid layer on the surface of Gram-negative bacteria. SUMMARY
[0005] The present application provides a preparation method of Staphylococcus aureus extracellular vesicles, comprising the following steps:
[0006] The overnight culture of Staphylococcus aureus is inoculated into the culture medium at a volume ratio of 1:80-1:120, and is cultured at 25-42℃ under shaking conditions to obtain Staphylococcus aureus liquid of a target growth period; the target growth period includes a logarithmic growth phase or a stationary phase; the OD 600 of the logarithmic growth phase is 0.5-0.6; and the OD 600 of the stationary phase is 1.2-1.8;
[0007] Centrifuge the Staphylococcus aureus liquid in the logarithmic growth phase / stationary phase at 10000xg for 5-15 minutes, discard the supernatant, and resuspend the bacterial body with a buffer containing the lytic enzyme PlySs2, and incubate at 25-42℃ for 0.5-4 hours; centrifuge at 5000-15000xg for 5-20 minutes, and collect the supernatant; filter the supernatant through a 0.1-0.45μm filter membrane; concentrate the filtrate through a 50-200kDa ultrafiltration tube, and wash 1-5 times with the buffer; ultracentrifuge at 100000-200000xg at 2-8℃ for 1-5 hours; resuspend the obtained precipitate in the buffer; filter the suspension through a 0.1-0.45μm filter membrane to obtain the Staphylococcus aureus extracellular vesicle; and confirm that there is no residual live bacteria in the preparation through sterile detection.
[0008] Preferably, the culture medium is one of LB medium, TSB medium or BHI medium.
[0009] Preferably, the condition of the shaking culture is 37℃, 120-220rpm.
[0010] Preferably, the concentration of the lytic enzyme PlySs2 in the buffer containing the lytic enzyme PlySs2 is 0.1-20μM; further preferably, the concentration of the lytic enzyme PlySs2 in the buffer containing the lytic enzyme PlySs2 is 0.5-8μM.
[0011] Preferably, the incubation condition is 37℃ for 2 hours.
[0012] Preferably, the centrifugation condition is 10000xg for 10 minutes.
[0013] Preferably, the ultrafiltration tube is a 100kDa ultrafiltration tube.
[0014] Preferably, the ultracentrifugation condition is 150000xg at 4℃ for 3 hours.
[0015] Preferably, the buffer is PBS buffer with pH 6.0-8.0 or Tris-HCl buffer with pH 6.0-8.0; further preferably, the buffer is PBS buffer with pH 7.4.
[0016] Preferably, the sterile detection is performed by the plate coating method.
[0017] The application further discloses application of the preparation method of the Staphylococcus aureus extracellular vesicle to a vaccine composition.
[0018] The application has the following beneficial effects:
[0019] The inventors found that in addition to having bactericidal function, the lytic enzyme PlySs2 can stimulate the induction of Staphylococcus aureus extracellular vesicles, and the toxicity of the lytic enzyme-induced EVs is significantly reduced compared with naturally produced EVs. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 TEM electron micrographs, Zeta potential maps and particle size distribution maps of the extracellular vesicles obtained from Example 1 and Control Example 1, wherein:
[0021] Figure 1 A-1D are respectively TEM images of LsEVs prepared from log phase Staphylococcus aureus treated with PlySs2 (0.5, 2, 4, 8 μM) at 37°C for 2 hours.
[0022] Figure 1 E-H are respectively electron micrographs of SsEVs prepared from stationary phase Staphylococcus aureus treated with PlySs2 (0.5, 2, 4, 8 μM) at 37°C for 2 hours.
[0023] Figure 1 I is an electron micrograph of LnEVs naturally secreted by log phase bacteria.
[0024] Figure 1 J is an electron micrograph of SnEVs naturally secreted by stationary phase bacteria.
[0025] Figure 1 K is the Zeta potential of EVs measured by dynamic light scattering.
[0026] Figure 1 L is the particle size distribution of EVs measured by dynamic light scattering.
[0027] Figure 2 SDS-PAGE protein gels of EVs and production of EVs, wherein:
[0028] Figure 2 A is a protein component SDS-PAGE analysis map of LsEVs prepared from log phase Staphylococcus aureus treated with PlySs2 (0.5, 2, 4, 8 μM) at 37°C for 2 hours.
[0029] Figure 2 B is a protein component SDS-PAGE analysis map of SsEVs prepared from stationary phase Staphylococcus aureus treated with PlySs2 (0.5, 2, 4, 8 μM) at 37°C for 2 hours.
[0030] Figure 2 C is a production map of LsEVs prepared from log phase Staphylococcus aureus treated with PlySs2 (0.5, 2, 4, 8 μM) at 37°C for 2 hours.
[0031] Figure 2 D is the yield plot of SsEVs prepared from stable phase S. aureus treated with PlySs2 (0.5, 2, 4, 8 mM) for 2 hours at 37°C.
[0032] Figure 2 E is the EVs immunoblotting plot. Western blot analysis confirmed that the lysing enzyme PlySs2 was not contained in EVs.
[0033] Figure 3 A is the cell viability (toxicity) concentration-response curve plot, in which:
[0034] Figure 3 A is the cell viability (toxicity) concentration-response curve plot, in which:
[0035] Figure 3 B is the cell viability (toxicity) concentration-response curve plot, in which:
[0036] Figure 3 C is the cell viability (toxicity) concentration-response curve plot, in which:
[0037] Figure 3 D is the cell viability (toxicity) concentration-response curve plot, in which:
[0038] Figure 4 A is the LsEV and SsEV differential protein and functional pathway enrichment analysis plot, in which:
[0039] Figure 4 A is the LsEV and SsEV differential protein and functional pathway enrichment analysis plot, in which:
[0040] Figure 4 B-4C is the KEGG pathway enrichment bubble plot, respectively showing the enrichment pathways of up-regulated and down-regulated proteins.
[0041] Figure 4 D-4F is the GO enrichment bubble plot (up-regulated proteins), in the order of biological process (BP), cellular component (CC), and molecular function (MF).
[0042] Figure 4 G-4I: GO enrichment bubble plot (down-regulated proteins), in the order of biological process (BP), cellular component (CC), and molecular function (MF).
[0043] Figure 5 A is the serum antibody endpoint titer (ELISA) plot after intranasal immunization, in which:
[0044] Figure 5 A is the serum IgG endpoint titer against SsEV (one week after the third nasal immunization).
[0045] Figure 5 B is the serum IgG endpoint titer against LsEV (one week after the third nasal immunization).
[0046] Figure 5 C is the serum IgG1 endpoint titer against SsEV, LsEV or SsLsEV (groups: PBS1 / SsEV, PBS2 / LsEV, PBS3 / SsLsEV).
[0047] Figure 5 D is the serum IgG2a endpoint titer against SsEV, LsEV or SsLsEV (same groups as above).
[0048] Figure 6 is a graph of serum antibody endpoint titers (ELISA) after muscle immunization, wherein:
[0049] Figure 6 A is the serum IgG endpoint titer against SsEV one week after the third muscle immunization.
[0050] Figure 6 B is the serum IgG endpoint titer against LsEV one week after the third muscle immunization.
[0051] Figure 6 C is the serum IgG1 endpoint titer against SsEV, LsEV or SsLsEV one week after the third muscle immunization (groups: PBS1 / SsEV, PBS2 / LsEV, PBS3 / SsLsEV).
[0052] Figure 6 D is the serum IgG2a endpoint titer against SsEV, LsEV or SsLsEV one week after the third muscle immunization (same groups as above).
[0053] Figure 7 is a graph of the protective effect against S. aureus lethal pneumonia challenge after nasal immunization, wherein:
[0054] Figure 7 A is the survival curve of the nasal immunized mice after lethal challenge with S. aureus ATCC 29213.
[0055] Figure 7 B is the body weight change curve of the nasal immunized mice after lethal challenge with S. aureus ATCC 29213.
[0056] Figure 7C represents the survival curve of mice immunized by intranasal drops after lethal challenge with S. aureus CCTCC AB 91118.
[0057] Figure 7 D represents the body weight change curve of mice immunized by intranasal drops after lethal challenge with S. aureus CCTCC AB 91118.
[0058] Figure 8 The diagram shows the protective effect against lethal intraperitoneal attack by Staphylococcus aureus following intramuscular immunization, where:
[0059] Figure 8 A represents the survival curve of muscle-immunized mice after intraperitoneal lethal challenge with S. aureus ATCC 29213.
[0060] Figure 8 B represents the body weight change curve of muscle-immunized mice after intraperitoneal lethal challenge with S. aureus ATCC 29213.
[0061] Figure 8 C represents the survival curve of muscle-immunized mice after intraperitoneal lethal challenge with S. aureus CCTCC AB 91118.
[0062] Figure 8 D represents the body weight change curve of muscle-immunized mice after intraperitoneal lethal challenge with S. aureus CCTCC AB 91118.
[0063] Figure 9 This is a comprehensive assessment chart of EV immunogenicity and protective effect in the context of prior infection, where:
[0064] Figure 9 A represents the endpoint titer of anti-SsEV IgG in mouse serum after three intranasal immunizations (ELISA).
[0065] Figure 9 B represents the endpoint titer of anti-LsEV IgG in mouse serum after three intranasal immunizations (ELISA).
[0066] Figure 9 C represents the endpoint titer of anti-SsEV IgG in mouse serum after three intramuscular immunizations (ELISA).
[0067] Figure 9 D represents the endpoint titer of anti-LsEV IgG in mouse serum after three intramuscular immunizations (ELISA).
[0068] Figure 1 E represents the survival rate of mice immunized by intranasal drops and previously infected three times after a lethal pneumonia challenge (ATCC 29213, etc.).
[0069] Figure 1 F is the weight change of mice immunized by nose drops and previously infected three times after a lethal pneumonia challenge.
[0070] Figure 1 G is the survival rate of mice immunized by muscle and previously infected three times after a lethal intraperitoneal challenge.
[0071] Figure 1 H is the weight change of mice immunized by muscle and previously infected three times after a lethal intraperitoneal challenge. DETAILED DESCRIPTION
[0072] Hereinafter, the technical solutions of the present application will be described in detail through specific examples, but these examples should be understood as only for illustrating the content and solutions of the present application, and not regarded as the present application is limited to the examples.
[0073] The parameters and sources of some raw materials in the examples are as follows:
[0074] LB medium, 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl were added in 950 mL of deionized water, the container was shaken until the solute was dissolved; the pH was adjusted to 7.0 with 5 mol / L NaOH aqueous solution, and the volume was made up to 1 L with deionized water; steam sterilization at 15 psi for 21 min, ready.
[0075] Staphylococcus aureus ATCC 29213, commercially available product.
[0076] Preparation method of Staphylococcus aureus ATCC 29213 overnight culture: inoculate single colony in LB medium and incubate at 37℃, 180 rpm shaker for about 16 hours, ready.
[0077] Lysin PlySs2, according to the description in the inventor's published literature In-situ and Real-Time Monitoring of the Interaction Between Lysins and Staphylococcus aureus Biofilm by Surface Plasmon Resonance (doi: 10.3389 / fmicb.2021.783472), expressed and purified in the laboratory.
[0078] Example 1
[0079] A method for producing bacterial extracellular vesicles using lysin PlySs2, comprising the following steps:
[0080] Inoculate 10 mL of S. aureus ATCC 29213 overnight culture into 1 L LB medium, and incubate at 37°C with 180 rpm shaking until OD600 0.5-0.6, to obtain a logarithmic phase S. aureus bacterial solution;
[0081] Inoculate 10 mL of S. aureus ATCC 29213 overnight culture into 1 L LB medium, and incubate at 37°C with 180 rpm shaking until OD600 0.5-0.6, to obtain a logarithmic phase S. aureus bacterial solution; 600 is 1.2-1.8;
[0082] The logarithmic phase / stationary phase S. aureus solution is divided into 1 mL centrifuge tubes, centrifuged at 10000 x g for 10 minutes at 4°C, the supernatant is discarded, and the bacterial pellet is resuspended in 1 mL of pH 7.4 PBS buffer (Cat. No. C10010500BT, Gibco) containing 0.5 μM, 2 μM, 4 μM, and 8 μM of the lytic enzyme PlySs2, respectively, and incubated at 37°C for 2 hours, and then centrifuged at 10000 x g for 10 minutes to collect the supernatant; the supernatant is filtered through a 0.22 μm filter membrane; the filtrate is concentrated through a 100 kDa ultrafiltration tube, washed three times with PBS, divided into 1 mL centrifuge tubes, and ultracentrifuged at 150000 x g for 3 hours at 4°C (Beckman centrifuge, rotor TY90); the obtained precipitate is resuspended in 1 mL of pH 7.4 PBS buffer (Cat. No. C10010500BT, Gibco), and the suspension is again filtered through a 0.22 μm filter membrane to obtain the extracellular vesicles; the preparation (extracellular vesicles) is detected for residual live bacteria by plate coating method.
[0083] Comparative Example 1
[0084] A method for naturally producing bacterial extracellular vesicles, comprising the following steps:
[0085] Inoculate 10 mL of S. aureus ATCC 29213 overnight culture into 1 L LB medium, and incubate at 37°C with 180 rpm shaking until OD600 0.5-0.6, to obtain a logarithmic phase S. aureus bacterial solution;
[0086] Inoculate 10 mL of S. aureus ATCC 29213 overnight culture into 1 L LB medium, and incubate at 37°C with 180 rpm shaking until OD600 0.5-0.6, to obtain a logarithmic phase S. aureus bacterial solution;
[0087] The supernatant of the logarithmic phase / stationary phase S. aureus liquid was collected by centrifugation at 10,000xg for 10 minutes at 4°C, and the supernatant was filtered through a 0.22-μm filter membrane; the filtrate was concentrated through a 100-kDa ultrafiltration tube, washed three times with PBS buffer (Cat. No. C10010500BT, Gibco) at pH 7.4, and then divided into 1-mL centrifuge tubes, and ultracentrifuged at 150,000xg for 3 hours at 4°C (Beckman centrifuge, rotor TY90); the obtained precipitate was resuspended in 1 mL of PBS buffer at pH 7.4, and the suspension was again filtered through a 0.22-μm filter membrane to obtain the extracellular vesicles; the preparation (extracellular vesicles) was detected by the plate coating method to detect the residual live bacteria.
[0088] Test Example 1
[0089] The morphologies of the extracellular vesicles prepared in the control example and the examples were compared, 20 μL of the vesicle sample was placed on a sealing film, the copper mesh edge was gently clamped with tweezers and inserted into the sample droplet, and adsorbed for 1 min. The copper mesh was clamped out of the droplet and moved to 20 μL of PTA negative staining liquid, and negatively stained for 2 min. The copper mesh was taken out and gently placed on filter paper, and after the liquid was completely absorbed, it was observed under a transmission electron microscope. The morphologies of different extracellular vesicles were observed under a transmission electron microscope (120 kV) and photographed. The potential and particle size of the vesicles were detected by dynamic light scattering method, and the specific test results are shown in Table 1. Figure 1 Figure 1 It can be seen that the lytic enzyme PlySs2 can stimulate the logarithmic phase / stationary phase S. aureus to produce bacterial extracellular vesicles, and there is a significant difference in morphology with the naturally secreted extracellular vesicles. The PTA negative staining liquid was prepared by using a 2wt% phosphotungstic acid aqueous solution, and the pH was adjusted to 7.0 with 1 mol / L NaOH aqueous solution.
[0090] Figure 1 A-1D: TEM image of LsEVs prepared by treating logarithmic phase S. aureus with PlySs2 (0.5, 2, 4, 8 μM) at 37°C for 2 hours.
[0091] Figure 1 E-1H: TEM image of SsEVs prepared by treating stationary phase S. aureus with PlySs2 (0.5, 2, 4, 8 μM) at 37°C for 2 hours.
[0092] Figure 2 I: TEM image of LnEVs naturally secreted by logarithmic phase S. aureus.
[0093] Figure 2 J: TEM image of SnEVs naturally secreted by stationary phase S. aureus.
[0094] Figure 2 K: Zeta potential map of EVs determined by dynamic light scattering. In the figure, LsEV and SsEV are respectively prepared by log phase Staphylococcus aureus treated with lytic enzyme PlySs2 (4 μM) at 37°C for 2 hours.
[0095] Figure 2 L: Particle size distribution map of EVs determined by dynamic light scattering. In the figure, LsEV and SsEV are respectively prepared by log phase Staphylococcus aureus treated with lytic enzyme PlySs2 (4 μM) at 37°C for 2 hours.
[0096] Test Example 2
[0097] Comparing the protein composition of the Staphylococcus aureus extracellular vesicles prepared in the examples and the control examples, the purified Staphylococcus aureus extracellular vesicles were subjected to 12% SDS-PAGE protein gel (One-Step PAGE Gel FastPreparation Kit (12%), item number E304-01, Novagen), and then the gel was stained and destained. The test figure is shown in Figure 2 .
[0098] Comparing the yield of the Staphylococcus aureus extracellular vesicles prepared in the examples and the control examples, the EVs were quantified using the Pierce BCA protein assay method. The protein standard BSA (2 mg / mL) was diluted with PBS buffer to obtain a gradient standard protein of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, and 62.5 μg / mL, respectively. The reagent A and reagent B in BCA were mixed at a volume ratio of 50:1, and the volume of the test number +1 was prepared. 10 μL of extracellular vesicles and standard were added to a transparent 96-well plate, and the same volume of PBS buffer (item number C10010500BT, Gibco) was added as a blank control, and then 100 μL of BCA test reagent was added, 37°C incubation for 30 min, and the absorbance of OD562nm was tested. According to the absorbance of OD562nm and the concentration of BSA, the standard curve was fitted, and the protein concentration was converted by using the equation of the standard curve, and the test results are shown in Figure 2 .
[0099] As shown in Figure 3 , for Staphylococcus aureus in the same growth period, the protein composition of the extracellular vesicles is affected by different preparation methods, and the protein spectrum of the extracellular vesicles obtained under the same preparation conditions of Staphylococcus aureus in different growth periods is also inconsistent. And Western blot analysis confirmed that the EVs induced by lytic enzyme do not contain lytic enzyme PlySs2.
[0100] Figure 3 A, 2B: SDS-PAGE analysis of EVs protein components.
[0101] Figure 4 C, 2D: EVs yield was quantified by determining the total protein content of EVs.
[0102] Figure 4 E: Western blot analysis confirmed that EVs did not contain the lytic enzyme PlySs2.
[0103] Test Example 3
[0104] BMDC, A549, 293T and THP-1 cells (about 2 x 10 4 Cells were seeded in 96-well plates one day before cytotoxicity experiments. Then, seeded cells were exposed to EVs at increasing concentrations (0, 1, 5, 10, 15 and 20 pg / mL) for 24 hours, and finally residual cell viability was determined using Cell Counting Kit-8 (YEASEN) according to the manufacturer's instructions. The relative survival of cells after each treatment was normalized and compared to the relative survival of the control wells treated with PBS. As shown in Figure 5 Staphylococcus aureus SnEVs showed cytotoxic effects on THP-1 cells at a concentration of 1 pg / mL, while the other EVs tested did not show cytotoxicity up to 20 pg / mL. LsEVs and SsEVs used in the test were prepared from log phase / stationary phase Staphylococcus aureus treated with the lytic enzyme PlySs2 (4 mM) at 37°C for 2 hours.
[0105] Figure 5 A549 cells (A), THP-1 cells (B), 293T cells (C) and dendritic cells (D) were co-cultured with different concentrations of EVs for 24 hours, and then the residual cell viability was detected by CCK-8 method.
[0106] Test Example 4
[0107] Proteins were extracted from Staphylococcus aureus extracellular vesicles (triplicate samples) using urea lysis buffer (8 M) followed by BCA quantification. Proteins (50 pg per sample) were reduced (10 mM DTT, 37°C, 1 hour), alkylated (20 mM IAA, protected from light, 30 minutes) and then digested overnight with trypsin (1 pg, 37°C). Peptides were desalted by C18 ZipTip and analyzed by LC-MS / MS using a C18 column (75 pm x 15 cm) and a 65-minute acetonitrile gradient (2-98% B) on an Orbitrap Exploris 480 mass spectrometer coupled to an EASY-nLC 1200 system.
[0108] Data acquisition independently (DIA) employed 40 isolation windows (m / z width 22.5), full scan (resolution 120,000, m / z 350–1250), and HCD fragmentation (NCE 30%). Raw data were processed using FragPipe (MSFragger v4.1 and IonQuant v1.10.27) against the Staphylococcus aureus UniProt database (UP000000530), with parameters including: trypsin digestion (≤2 missed cleavages), precursor ion mass tolerance of 20 ppm, fixed modifications (carboxymethylation), and variable modifications (methionine oxidation / deamidation). The false discovery rate (FDR) for both PSM and proteins was set to 1%.
[0109] Protein quantification was performed using MaxLFQ, and missing values were filled using a normal distribution (μ-1.8σ). Differential expression analysis (Rv4.2.2) employed VSN normalization, Welch t-test (BH corrected p<0.05), and fold change thresholds (>1.5 or <0.67). Functional enrichment analysis (GO / KEGG) was performed using clusterProfiler (hypergeometric test, FDR<0.05) and visualized using Python matplotlib.
[0110] Staphylococcus aureus extracellular vesicles (LsEV and SsEV) were prepared from logarithmic / stationary phase Staphylococcus aureus by treatment with the lyase PlySs2 (4 μM) at 37°C for 2 hours.
[0111] like Figure 5 As shown, mass spectrometry analysis further revealed the differences in protein composition between SsEVs and LsEVs. Dual enrichment analysis using GO and KEGG revealed that LsEVs, derived from bacteria in the logarithmic growth phase, were mainly enriched in proteins related to protein translation and cell proliferation regulation; while SsEVs were significantly enriched in proteins related to metabolic regulation and stress response.
[0112] Figure 5 A: Differences in protein abundance between LsEV and SsEV. B, C: Bubble plots showing the enrichment of upregulated and downregulated proteins in the KEGG pathway in LsEV compared to SsEV. DF: Bubble plots showing the enrichment of upregulated proteins in the biological processes (D), cellular components (E), and molecular functions (F) of the GO pathway in LsEV compared to SsEV. GI: Bubble plots showing the enrichment of downregulated proteins in the biological processes (G), cellular components (H), and molecular functions (I) of the GO pathway in LsEV compared to SsEV.
[0113] Test Example 5
[0114] The antibody titers generated by three times of nasal immunization of the bacterial extracellular vesicles prepared in Test Control Example 1 and Example 1 were tested. SPF level mice were selected. The mice were randomly grouped and subjected to nasal immunization. The components of the nasal immunization were divided into four groups, which were immunized with PBS, SsEV, LsEV or SsLsEV (SsEV and LsEV were mixed at a mass ratio of 1:1, and in this example, 10 μg of SsEV and 10 μg of LsEV were mixed), and the nasal immunization was performed once every two weeks, and each time 20 μg. The LsEV and SsEV used in the test were prepared by treating logarithmic phase / stationary phase Staphylococcus aureus with lysozyme PlySs2 (4 μM) at 37°C for 2 hours.
[0115] The serum of the mice after three times of immunization was collected for antibody titer test. The specific test method was as follows: the vesicles were diluted with ELISA coating solution to 5 μg / mL, 100 μL of the diluted outer membrane vesicles were added to each well of the 96-well ELISA plate, the 96-well plate was sealed, and the coating was performed overnight (16 h) in a 4°C refrigerator. The coated well plate was taken out, the coating solution was poured out, 300 μL of PBST buffer was added to each well, and the well plate was incubated at room temperature for 3 min. The liquid in the well plate was poured out and dried, and the above washing method was repeated 5 times. When it was confirmed that there was no liquid in the well, 300 μL of 5% skimmed milk powder blocking solution was added to each well, and the blocking was performed at 37°C for 2 h.
[0116] The blocked well plate was taken out, washed with PBST buffer according to the above washing method for 5 times, and the liquid in the well was dried. The test serum and blank serum were diluted with PBST buffer, 100 μL of sample was added to each well, PBST buffer was blank control, and blank serum was negative control. The well plate was incubated at 37°C for 1 h. The well plate was taken out, the liquid in the well was discarded, and the well plate was washed with PBST buffer according to the above washing method for 5 times and dried. 100 μL of diluted HRP-labeled IgG, IgG1 or IgG2a was added to each well, and the well plate was incubated at 37°C for 1 h. The PBST buffer was a solution obtained by adding 0.05% (v / v) Tween-20 to PBS buffer (item number C10010500BT, Gibco) with pH 7.4.
[0117] After the incubation was completed, the well plate was taken out, the liquid in the well was discarded, and the well plate was washed with PBST buffer according to the above washing method for 5 times and dried. 100 μL of color developing substrate TMB was added to each well in a dark room, and the well plate was left to stand for 10 min. 100 μL of concentrated sulfuric acid (2 mol / L) was added to each well to terminate the reaction. The specific test results are shown in Figure 6 .
[0118] As shown in Figure 6 , the IgG, IgG1 and IgG2a antibody titers of the EVs group of the mice immunized by nasal immunization were all higher than 10 4 times higher than those of the PBS group.
[0119] Figure 6A, 5B: IgG endpoint titers against SsEV or LsEV in serum samples collected one week after the third intranasal immunization were detected by ELISA. Figure 6 C, 5D: IgG1 and IgG2a endpoint titers against SsEV (PBS1 / SsEV), LsEV (PBS2 / LsEV) or SsLsEV (PBS3 / SsLsEV) in serum samples collected one week after the third intranasal immunization were detected by ELISA.
[0120] Test Example 6
[0121] The antibody titers generated by the S. aureus EVs prepared in Test Control Example 1 and Example 1 after three intramuscular immunizations were tested. SPF mice were selected and randomly divided into groups, and were immunized with Imject Alum+PBS, Imject Alum+SsEV, Imject Alum+LsEV or Imject Alum+SsLsEV, respectively, at 20 μg per immunization, every 2 weeks. The antibody titers of the mouse sera were detected as in Test Example 5. As shown in Table 2, the IgG, IgG1 and IgG2a antibody titers in the EVs groups were all above 10 Figure 6 4 The LsEV and SsEV used in the test were prepared by treating log phase / stationary phase S. aureus with the lytic enzyme PlySs2 (4 μM) at 37°C for 2 hours.
[0122] Figure 7 A, Figure 7 B: IgG endpoint titers against SsEV or LsEV in serum samples collected one week after the third intramuscular immunization were detected by ELISA. Figure 7 C, Figure 7 D: IgG1 and IgG2a endpoint titers against SsEV (PBS1 and SsEV), LsEV (PBS2 and LsEV) or SsLsEV (PBS3 and SsLsEV) in serum samples collected one week after the third intramuscular immunization were detected by ELISA. PBS1, PBS2 and PBS3 were all sera from mice immunized with PBS, except that SsEV, LsEV or SsLsEV were coated in the ELISA, and PBS sera were used as controls in the three coated antigens.
[0123] Test Example 7
[0124] The mice immunized by intranasal immunization three times in Test Example 5 were randomly divided into two groups, and were intranasally infected with two S. aureus, ATCC 29213 (5 x 10 8 CFU) (commercially available) and CCTCC AB 91118 (8 x 10 8 CFU) (commercially available), to establish a model of bacterial pneumonia, and the infection dose was confirmed by counting the CFU on LB agar plates with serial 10-fold dilutions. The survival rate and body weight changes of the mice were monitored after infection, and the specific test results are shown in Figure 7 As shown in Figure 7 , whether caused by a vesicle-derived strain and a non-vesicle-derived strain, nasal immunization with EVs can effectively improve the survival rate of mice in a lethal pneumonia model.
[0125] Figure 8 A: Survival rate of nasal immunization mice after lethal challenge of Staphylococcus aureus ATCC 29213 pneumonia. Figure 8 B: Body weight changes of nasal immunization mice after lethal challenge of Staphylococcus aureus ATCC 29213 pneumonia. Figure 8 C: Survival rate of nasal immunization mice after lethal challenge of Staphylococcus aureus CCTCC AB 91118 pneumonia. Figure 8 D: Body weight changes of nasal immunization mice after lethal challenge of Staphylococcus aureus CCTCC AB 91118 pneumonia.
[0126] Test Example 8
[0127] The mice in test example 6, which were immunized three times by intramuscular injection, were randomly divided into two groups, and were infected with two strains of Staphylococcus aureus, ATCC 29213 (4 x 10 8 CFU) and CCTCC AB 91118 (6 x 10 8 CFU), to establish a model of bacterial bacteremia, and the infection dose was confirmed by counting the CFU on LB agar plates with serial 10-fold dilutions. The survival rate and body weight changes of the mice were monitored after infection, and the specific test results are shown in Figure 8 As shown in Figure 8 , whether caused by a vesicle-derived strain and a non-vesicle-derived strain, nasal immunization with EVs can effectively improve the survival rate of mice in a lethal pneumonia model.
[0128] Figure 9 A: Survival rate of intramuscular immunization mice after lethal challenge of Staphylococcus aureus ATCC 29213 intraperitoneal infection. Figure 9 B: Body weight changes of intramuscular immunization mice after lethal challenge of Staphylococcus aureus ATCC 29213 intraperitoneal infection. Figure 9 C: Survival rate of intramuscular immunization mice after lethal challenge of Staphylococcus aureus CCTCC AB 91118 intraperitoneal infection. Figure 9 D: Body weight changes of intramuscular immunization mice after lethal challenge of Staphylococcus aureus CCTCC AB 91118 intraperitoneal infection.
[0129] Test Example 9
[0130] For the pre-infection groups (intramuscular and intranasal), mice were first infected with Staphylococcus aureus weekly for three weeks, and then immunized with EV according to their respective intramuscular or intranasal protocols. After immunization, serum was collected from the mice for antibody titer testing, using the same method as in Test Case 5. Subsequently, mice were infected with ATCC 29213 (nasal instillation in the nasal immunization group and intraperitoneal infection in the intramuscular immunization group), and survival rate and weight changes were monitored post-infection. Figure 9 As shown, pre-infected mice can also significantly induce the production of EV-specific antibody IgG in serum after three immunizations, and it has a good protective effect in both pneumonia and bacteremia models.
[0131] Figure 9 A, 9B: The endpoint titer of anti-SsEV or LsEV IgG in mouse serum after three intranasal immunizations. Figure 9 C, 9D: The endpoint titer of anti-SsEV or LsEV IgG in mouse serum after three muscle immunizations. E, F: Survival rate (E) and weight change (F) of mice immunized by intranasal drops after three Staphylococcus aureus infections following lethal pneumonia challenge. G, H: Survival rate (G) and weight change (H) of muscle-immunized mice after three Staphylococcus aureus infections followed by lethal intraperitoneal challenge.
[0132] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.
Claims
1. A method for preparing Staphylococcus aureus extracellular vesicles, characterized in that, Includes the following steps: Overnight cultures of Staphylococcus aureus were inoculated into the culture medium at a volume ratio of 1:80-1:120 and cultured with shaking at 25-42°C to obtain Staphylococcus aureus suspension in the target growth phase; the target growth phase includes the logarithmic growth phase or the stationary phase; the OD of the logarithmic growth phase... 600 The value is 0.5-0.6; the stable period is the bacterial culture OD value. 600 It is 1.2-1.8; Centrifuge Staphylococcus aureus culture in logarithmic growth phase / stationary phase at 10000×g for 5-15 minutes, discard the supernatant, resuspend the cells in buffer containing lysin PlySs2, and incubate at 25-42℃ for 0.5-4 hours; centrifuge at 5000-15000×g for 5-20 minutes and collect the supernatant; filter the supernatant through a 0.1-0.45μm filter membrane; concentrate the filtrate through a 50-200kDa ultrafiltration tube and wash 1-5 times with buffer; ultracentrifuge at 100000-200000×g for 1-5 hours at 2-8℃; resuspend the resulting precipitate in buffer; filter the suspension through a 0.1-0.45μm filter membrane to obtain Staphylococcus aureus extracellular vesicles; confirm the absence of viable bacteria residue in the preparation by sterility testing.
2. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 1, characterized in that: The culture medium is one of LB medium, TSB medium or BHI medium.
3. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 1, characterized in that: The conditions for the shaking culture were 37℃ and 120-220 rpm.
4. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 1, characterized in that: The concentration of the lyase PlySs2 in the buffer solution is 0.1-20 μM.
5. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 4, characterized in that: The concentration of the lyase PlySs2 in the buffer solution is 0.5-8 μM.
6. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 1, characterized in that: The incubation conditions were 37°C for 2 hours.
7. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 1, characterized in that: The centrifugation conditions were 10000×g for 10 minutes.
8. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 1, characterized in that: The ultrafiltration tube is a 100kDa ultrafiltration tube; the ultracentrifugation conditions are 4℃ and centrifugation at 150000×g for 3 hours.
9. The method for preparing Staphylococcus aureus extracellular vesicles as described in claim 1, characterized in that: The buffer solution is either PBS buffer with pH 6.0-8.0 or Tris-HCl buffer with pH 6.0-8.
0.
10. The application of the method for preparing Staphylococcus aureus extracellular vesicles as described in any one of claims 1-9 in a vaccine composition.