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Jan 2019
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Isolation and Characterization of Membrane Vesicles from Lactobacillus Species
乳酸杆菌属膜囊泡中的分离和特性研究   

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Abstract

Throughout their life cycle, bacteria shed portions of their outermost membrane comprised of proteins, lipids, and a diversity of other biomolecules. These biological nanoparticles have been shown to have a range of highly diverse biological activities, including pathogenesis, community regulation, and cellular defense (among others). In recent publications, we have isolated and characterized membrane vesicles (MVs) from several species of Lactobacilli, microbes classified as commensals within the human gut microbiome (Dean et al., 2019 and 2020). With increasing scientific understanding of host-microbe interactions, the gut-brain axis, and tailored probiotics for therapeutic or performance increasing applications, the protocols described herein will be useful to researchers developing new strategies for gut community engineering or the targeted delivery of bio-active molecules.


Graphic abstract:



Figure 1. Atomic force microscopic image of Lactobacillus casei ATCC 393 bacteria margins (white arrows) and membrane vesicles (black arrows)


Keywords: Membrane vesicles (膜囊泡), Lactic acid bacteria (乳酸菌), Lactobacillus acidophilus (嗜酸乳杆菌), Lactobacillus plantarum (植物乳杆菌)

Background

The membranes of all cells are dynamic structures, the biomolecule composition of which is constantly changing as cells respond to environmental stimuli, alter protein and lipid composition, release waste products, take in nutrients, and perform many other cellular processes (Vereb et al., 2003; Watson, 2015). Throughout cellular life cycles, fragments of the outermost membrane are often shed as nanosized particles. In bacteria, these structures are often referred to as membrane vesicles (MVs) or outer membrane vesicles (OMVs), which will be referred to as MVs throughout for simplicity (a representative example of MVs from Lactobacillus casei is shown in Figure 1). As interest in these biological nanoparticles has grown in recent years, researchers have shown that MVs have broad biological activities, including host-microbe interactions, gene transfer, and community regulation (Kulp and Kuehn, 2010; Caruana and Walper, 2020).


Bacterial MVs have shown significant promise for applications such as vaccine development and potential therapeutic applications. Naturally occurring OMVs from Gram-negative bacteria have shown significant promise in the development of vaccines for bacteria such as Neisseria meningitidis and Burkholderia pseudomallei, pathogens that have proven challenging to vaccine and therapeutic development alike (Holst et al., 2009; Nieves et al., 2014). Recently, research groups have also shown that the MVs from some commensal bacteria can also modulate responses from host immune systems and stimulate host neurological systems (Mata Forsberg et al., 2019; Molina-Tijeras et al., 2019; Rodovalho et al., 2020). With growing capabilities in synthetic biology, the potential uses of MVs are steadily increasing as researchers have developed engineering strategies that allow for modification of genetic and cellular pathways to control the composition of nascent MVs. These efforts have led to new biomaterials for therapeutic applications, environmental decontamination, and other purposes (Alves et al., 2018; Qing et al., 2019).


The classification of lactic acid bacteria (LAB) encompasses several genera of bacteria with similar characteristics of acid-tolerance and fermentation capabilities. Many LAB are classified as generally regarded as safe (GRAS) and have been used in the production of food products for centuries. Additionally, several LAB have been recognized as beneficial to their host and are studied for their health benefits as probiotics leading to a large commercial market for probiotic supplements and foods. While live-bacterial cultures are the most commonly used form of probiotics, the potential for engineering or enriching for specific cellular components has led researchers to explore the use of purified MVs for controlled therapeutic applications (Seo et al., 2018; Molina-Tijeras et al., 2019; Dean et al., 2020).


There are numerous protocols for the purification of MVs from eukaryotic cells and Gram-negative bacteria, which have been the primary focus of MV research (Klimentova and Stulik 2015; Alves et al., 2017; Dauros Singorenko et al., 2017). Recently, there has been growing interest in the MVs of gut bacteria and the roles they may play in host and community interactions. LAB are Gram-positive bacteria and therefore have a significantly different membrane and peptidoglycan structure as compared to Gram-negative bacteria. While this may not specifically contribute to biophysical differences between Gram-negative and Gram-positive MVs, it has been shown that the MVs of some LAB species have a bimodal size distribution with an abundant population of smaller MVs in the 10-50 nm size range (Dean et al., 2019). Here, we describe protocols for the isolation and characterization of MVs that have proven successful for numerous LAB species. MVs are isolated from overnight LAB cultures via ultracentrifugation and then analyzed for concentration and relative size distribution using a NanoSight nanoparticle tracking instrument. Dynamic light scattering (DLS) is used as another way to measure size distribution and also to measure zeta potential (surface charge). Then, the protein content of MVs can be examined both qualitatively using SDS-PAGE and specifically by using mass spectrometry for proteomic analysis (schematic shown in Figure 2). While this manuscript describes work we have employed for LAB, these protocols could also be used for the isolation of MVs from a variety of microbial species.



Figure 2. Schematic overview of the membrane vesicle isolation and characterization process. MVs are purified from Lactobacillus cultures initially via centrifugation and filtration, removing cells and large cellular debris. From the filtered supernatant, MVs are isolated by ultracentrifugation. Gradient centrifugation using OptiPrep medium can be applied to isolated MVs as an additional purification step. Purified MVs can then be characterized by nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), SDS-PAGE protein gels, shotgun proteomics, or other methods.

Materials and Reagents

  1. AnaeroJarTM 2.5 L jars (Oxoid®, ThermoFisher Scientific, catalog number: AG0025A)

  2. AnaeroGenTM anaerobic gas generation sachets (Oxoid®, Thermo Fisher Scientific, catalog number: AN0025A)

  3. Resazurin anaerobic indicator strips (Oxoid®, Thermo Fisher Scientific, catalog number: BR0055B)

  4. 100 mm sterile Petri dishes (Fisherbrand, Thermo Fisher Scientific, catalog number FB0875712)

  5. Pyrex® Erlenmyer flasks 250 ml (CorningTM, Thermo Fisher Scientific, catalog number: CLS4980250)

  6. 50 ml conical centrifuge tubes (CorningTM Falcon, Thermo Fisher Scientific, catalog number: 14-432-22)

  7. 0.45 µm syringe filters (J. G. Finneran, catalog number: FEC0425PC)

  8. 30 ml sterile syringes (BD Slip tip sterile syringe; Thermo Fisher Scientific, catalog number: BD 302833)

  9. 38.5 ml ultracentrifuge tubes (Thinwall Ultra-Clear tubes; Beckman Coulter, catalog number: 344058)

  10. 5 ml ultracentrifuge tubes (Thinwall Ultra-Clear tubes; Beckman Coulter, catalog number: 344057)

  11. 1 ml needle-less syringes (Henke Sass Wolf, catalog number: 4010.200V0)

  12. 1.5 ml centrifuge tubes (Thermo Fisher Scientific, catalog number: P190411)

  13. Disposable cells compatible with ZetaSizer Nanoseries (Malvern Panalytical, catalog number: DTS 1070)

  14. Lactobacillus species, i.e.,

    Lactobacillus acidophilus (ATCC 53544)

    Lactobacillus casei (ATCC 393)

    Lactobacillus reuteri (ATCC 23272)

    Lactobacillus plantarum (ATCC BAA-793)

  15. de Man, Rogosa, and Sharpe (MRS) media (Sigma-Aldrich, catalog number: 69966-500G, prepare according to the manufacturer’s instructions)

  16. Tween® 80 (Sigma Aldrich, catalog number: P8074)

  17. 4-15% Mini-PROTEAN® TGX Pre-cast protein gels (Bio-Rad, catalog number:4561085)

  18. GelCodeTM Blue Stain Reagent (Thermo Fisher Scientific, catalog number: PI24590)

  19. 10× phosphate-buffered saline (Thermo Fisher Scientific, catalog number: AM9625)

  20. 4× Laemmli sample buffer (Bio-Rad, catalog number: 1610747)

  21. 2-mercaptoethanol (Bio-Rad, catalog number: 161-0710)

  22. SDS-PAGE running buffer (Bio-Rad, catalog number: 161-0772)

  23. Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225)

  24. 1-propanol (Sigma-Aldrich, catalog number: 402893)

  25. Ammonium bicarbonate (Sigma-Aldrich, catalog number: A6141)

  26. Trypsin, sequencing grade (Promega, Fisher Scientific, catalog number: PRV5111)

  27. Formic acid (Sigma-Aldrich, catalog number: 27001)

  28. Acetonitrile (Sigma-Aldrich, catalog number: 34851)

  29. OptiPrep medium (Progen Biotechnik GmbH, catalog number: 1114542)

  30. SDS-PAGE Running Buffer (10× stock; use at 1×) (see Recipes)

Equipment

  1. Incubator capable of maintaining 37°C (for example: Fisherbrand, Isotemp Microbiological Incubator)

  2. Centrifuge with capacity for 50 ml conical tubes (for example: Beckman Coulter, Avanti JXN-30 using a JA-14.50 rotor)

  3. Ultracentrifuge with capacity for 38.5 ml tubes, capable of 129,000 × g (for example: Sorvall WX Ultra 90 centrifuge using AH-629 rotor)

  4. NanoSight LM10 (Malvern Panalytical, Worcestershire, UK)

  5. ZetaSizer NanoSeries equipped with a HeNe laser source (λ = 633 nm) and avalanche photodiode (Malvern Panalytical, Worcestershire, UK)

  6. Mini-PROTEIN® Tetra Vertical Electrophoresis Cell (Bio-Rad, catalog number: 1658004)

  7. Speed-vac (for example: Thermo Fisher Scientific SC210A SpeedVac Concentrator, catalog number: SC210A-230)

  8. Barocycler (Pressure Biosciences Inc., HUB 440-SW16, Easton, MA, US)

  9. Orbitrap LC-MS/MS system (for example: Thermo Scientific Orbitrap Fusion Lumos equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific, catalog number: ES071)

  10. Autosampler (for example: Thermo Scientific Dionex UltiMate 3000 Rapid Separation Well Plate Autosampler (Thermo Fisher Scientific, catalog number: 5840.0010)

  11. Ultra-high performance liquid chromatography (UHPLC) system (for example: Dionex Ultimate 3000 RSLCnano system, Thermo Fisher Scientific, catalog number: ULTIM3000RSLCNANO)

Software

  1. NTA 2.3 Nanoparticle Tracking and Analysis software (Malvern Panalytical, Worcestershire, UK)

  2. Dispersion Technology Software (DTS, Malvern Panalytical, Worcestershire, UK) used for dynamic light scattering analysis

  3. Scaffold version 4.8.2 (Proteome Science Inc., Portland, Oregon, US), used for protein identification after mass spectrometry

  4. Mascot (version 2.6.1, Matrix Science, London, UK), used for protein identification after mass spectrometry

  5. X! Tandem (version 1.7.18) used for protein identification after mass spectrometry

  6. R (https://cran.r-project.org/) and relevant R packages: Peptides, Limma, and ggplot2. Used for biochemical analysis of identified proteins, statistical analysis, and generation of volcano plots or other visualizations of the data

Procedure

  1. Isolation of membrane vesicles (MVs)

    1. Grow bacterial strains

      1. Streak each strain individually from frozen stocks or stab vials onto MRS agar plates. For anaerobic growth, place plates in an AnaeroJar jar with an AnaeroGen anaerobic gas generation sachet and anaerobic indicator added. Grow at 37°C for approximately 48 h or until individual colonies are visible.

      2. Inoculate a single bacterial colony into 3 ml MRS broth. Grow overnight at 37°C without shaking in an AnaeroJar as in the previous step.

      3. Add 1 ml from the primary culture to 50 ml MRS broth in a sterile 250 ml Erlenmeyer flask. Grow overnight as in previous steps.

    2. Collect and filter supernatant

      1. Transfer the overnight culture into a 50 ml conical centrifuge tube and centrifuge at 5,000 × g for 30 min.

      2. Decant supernatant into a new tube and centrifuge again at 5,000 × g for 30 min.

      3. Repeat Step A2b once or twice more, until no cell pellet is visible on the sides or bottom of the tube after centrifugation. These additional centrifugation steps are not strictly required but make it easier to filter the supernatant in the following step.

      4. Pass the supernatant through a 0.45 µm filter. This may be done using a 30 ml syringe and syringe filter or using a vacuum apparatus with an appropriate filter for larger volumes of supernatant.

    3. Ultracentrifugation to isolate MVs

      1. Transfer 36 ml of the filtered supernatant to an ultracentrifuge tube and assemble into the rotor.

      2. Ultracentrifuge at 126,000 × g for 1.5 h at 4°C.

      3. Decant the supernatant (the pellet is often invisible).

      4. Add 1 ml of 1× phosphate-buffered saline (PBS) and incubate overnight at 4°C to resuspend the pellet.

        Note: If proceeding for gradient ultracentrifugation, resuspend in 150 μl of PBS-OptiPrep mixture instead (see Step A4a below).

    4. Gradient ultracentrifugation of MVs

      1. Resuspend MV pellets in 150 µl of a PBS-OptiPrep mixture (45% v/v) and move the suspension to the bottom of a 5 ml ultracentrifuge tube.

      2. Make several 0.8 ml PBS-OptiPrep solutions of lower density (e.g., 35%, 30%, 25%, 20%, 15%, and 10%) and sequentially cover the base layer containing MVs with layers of decreasing density. Avoid adding bubbles or disrupting previous layers as you add.

      3. After all layers have been added, allow the tube to settle for 1 h.

      4. Ultracentrifuge the gradient at 236,000 × g for 3 h in a Sorvall WX Ultra 90 centrifuge using an AH-650 rotor.

      5. Following centrifugation, gently sequentially remove and place 10 equal-volume (~500 μl) fractions into separate tubes.

      6. Assess the different density fractions for MV concentration, protein composition, and other characteristics using the methods described below.



      Figure 3. Nanoparticle tracking analysis (NTA) of Lactobacillus membrane vesicles. A. Representative frame taken from a NanoSight LM10 video of L. acidophilus MVs. MVs purified from an MRS culture were diluted 1:1,000 in PBS prior to NTA. Scale bar indicates 2 µm. B. Representative MV size distribution from NTA results, taken from an average obtained from three 60 s videos.


  2. MV characterization

    1. Nanoparticle tracking analysis (NTA) to determine particle size distribution and concentration

      1. Dilute MVs in 1× PBS – make several serial dilutions such as 10×, 100×, and 1,000×. Detection of particles by the NTA software is best when there are 20-60 particles within the field of view; therefore, samples need to be diluted, and the optimal dilution for each sample may vary from one isolation to another.

      2. Using a 1 ml needle-less syringe, add the diluted sample to the NanoSight instrument chamber. Allow a moment for samples to settle and adjust microscope focus and camera settings to optimize clarity of particles in the camera field of view within the NTA 2.3 Nanoparticle Tracking and Analysis software. Data can be collected at room temperature.

      3. Capture data as triplicate readings of 60 s exposure at 30 frames per second, and analyze using auto particle detection and tracking parameters: detection threshold, pixel blur, minimum track length, and minimum expected particle size. A representative image of NTA of vesicles from Lactobacillus acidophilus ATCC 53544 is shown in Figure 3. As NTA 2.3 does not automatically add scale bars to images, they can be added by converting pixels to µm by dividing pixels by 9.036 (when using a 30× objective).

    2. Dynamic light scattering (DLS) to measure MV size distribution

      1. Dilute MV samples 10-fold in 0.1× PBS pH 7.4 and add to disposable DLS cuvettes.

      2. Carry out DLS analysis using ZetaSizer NanoSeries equipped with a HeNe laser source (λ = 633 nm) and analyze using Dispersion Technology Software. Carry out measurements at 25°C. For each sample, take the average of five runs of 10 s each for the autocorrelation function, and repeat this three to six times.

    3. Zeta Potential measurement to determine MV surface charge

      1. Dilute MV samples and load into disposable cuvettes as for DLS.

      2. Measure zeta potential using ZetaSizer NanoSeries equipped with a HeNe laser source (λ = 633 nm) with Dispersion Technology Software. Carry out measurements at 25°C in triplicate for each sample.

    4. SDS-PAGE for qualitative analysis of proteins contained in MVs

      1. Prepare samples: based on MV concentration as determined by NTA, take equal amounts of each MV sample in ≤15 μl volume in 1.5 ml centrifuge tubes. Adjust sample volumes to 15 μl using water or 1× PBS. (Volumes may be adjusted depending on the well capacity of SDS-PAGE gels to be used.)

      2. Add 5 μl (or required volume to result in 1× final concentration) of 4× Laemmli sample loading buffer to each sample, then denature sample proteins by boiling for 5 min, chilling on ice for 5 min, and centrifuging for 15 min at 16,000 × g in a benchtop centrifuge.

      3. Assemble a Bio-Rad Mini-PROTEAN Vertical Electrophoresis cell according to the manufacturer’s instructions with a 4-15% Mini-PROTEAN® TGX pre-cast protein gel.

      4. Load denatured MV samples (supernatant only) into the gel and run at 200 V until the dye front reaches the bottom of the gel.

      5. Remove the gel from its casing and rinse briefly in water; then, stain in GelCodeTM Blue Stain Reagent, followed by destaining using water, to visualize MV protein bands. Figure 3 of Dean et al. (2019) may be referred to for an example of an SDS-PAGE gel of MVs from three LAB species.

    5. Shotgun Proteomics Analysis to identify protein contents of MVs

      1. Sample preparation

        1. Resuspend MV pellets with 10 ml of 10% n-propanol in 50 mM ammonium bicarbonate (ABC) buffer.

        2. Normalize sample protein concentration using the Pierce BCA Protein Assay Kit.

        3. Digest samples (50 µg) in solution with sequencing-grade modified trypsin at a 1:50 (w/w) enzyme:substrate ratio in a barocycler for 90 min (90 cycles: 50 s on at 45 kpsi, 10 s off).

        4. Evaporate digested samples via speed-vac and store at -20°C until analysis by LC-MS/MS.

      2. LC-MS/MS

        1. Reconstitute dried samples in 0.1% formic acid in water (solvent A) and load 3 μg digested protein onto a reversed-phase C18 column by a Dionex Ultimate WPS-3000 autosampler connected to an Orbitrap Fusion Lumos equipped with a Nanospray Flex Ion Source in data-dependent acquisition mode.

        2. Separate peptides across a 90 min gradient of 2-60% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min using a Dionex Ultimate 3000 RSLCnano system.

        3. Save the raw, mgf, and mzid files output for each sample for downstream analysis.

      3. Protein identification

        1. Search mass spectrometry data using Mascot and X! Tandem using the appropriate genome file for the organism. Validate peptide-spectrum matches with Scaffold.

        2. Set fragment ion mass tolerance to ±0.60 Da and parent ion tolerance to ±0.60 Da. Set proteins identification thresholds to ≥2 peptides (protein probability 80%, peptide probability 95%). Export list of identified proteins to CSV.

        3. Analyze the output CSV files with R.

Data analysis

Proteomics analysis:

  1. Analyze all MS/MS samples using Mascot (Matrix Science, London, UK) and X! Tandem (The GPM, thegpm.org). Have Mascot set up to search the correct database sequence file (generated by protein .fasta input) and a corresponding common contaminants file. Set assumed cleavages to digestion using trypsin.

  2. Set up X! Tandem to search a reverse concatenated subset of the correct database sequence file, which only contains proteins identified by Mascot in analyzed samples.

  3. Use Scaffold (Proteome Software Inc., Portland, OR, USA) to validate MS/MS based peptide and protein identifications. Set peptide-spectrum match settings to: fragment ion mass tolerance = ±0.60 Da and the parent ion tolerance = ±0.60 Da. Set protein identification thresholds to ≥2 peptides (protein probability 80%; peptide probability 95%).

  4. Analyze proteomics data output to CSV files with R

    1. Determine physicochemical characteristics of peptides/proteins using the Peptides R package.

    2. Perform the two-sample t-tests using an empirical Bayes method to adjust the estimate of variance of each protein using the Limma R package, the output of which is each protein with its log2 fold-change and moderated p-value corresponding to the moderated t-statistic, as previously described (Dean et al., 2020).

    3. Use the ggplot2 R package to create volcano plots and other visualizations of the data (see Figure 4 for an example volcano plot using proteomics data).



    Figure 4. Volcano plot of proteomics performed on Lactobacillus membrane vesicles. Representative volcano plot of log2 fold change of proteins identified in L. acidophilus MVs treated with inducer relative to an untreated control. Identified protein data in triplicate samples were put through the empirical Bayes method based on the Limma R package and plotted using ggplot2. Proteins with significant change are colored in blue.

Recipes

  1. SDS-PAGE Running Buffer (10× stock; use at 1×)

    25 mM Tris base

    192 mM glycine

    0.1% SDS

    Dissolve in water as solvent

Acknowledgments

This protocol is adapted from previous work (Dean et al., 2019 and 2020). Funding for this effort was provided through Core funds of the Naval Research Laboratory (SND, SAW). Additional financial support was provided through the American Society for Engineering Education (ASEE) post-doctoral fellowship program (JCC). The AFM image shown in the graphical abstract was taken by Claretta Sullivan, Air Force Research Laboratory, Wright Patterson Air Force Base.

Competing interests

The authors declare no competing interests.

Ethics

None of the protocols described here include studies with animals or human subjects.

References

  1. Alves, N. J., Moore, M., Johnson, B. J., Dean, S. N., Turner, K. B., Medintz, I. L. and Walper, S. A. (2018). Environmental Decontamination of a Chemical Warfare Simulant Utilizing a Membrane Vesicle-Encapsulated Phosphotriesterase. ACS Appl Mater Interfaces 10(18): 15712-15719.
  2. Alves, N. J., Turner, K. B., DiVito, K. A., Daniele, M. A. and Walper, S. A. (2017). Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant. Res Microbiol 168(2): 139-146.
  3. Caruana, J. C. and Walper, S. A. (2020). Bacterial Membrane Vesicles as Mediators of Microbe - Microbe and Microbe - Host Community Interactions. Front Microbiol 11: 432.
  4. Dauros Singorenko, P., Chang, V., Whitcombe, A., Simonov, D., Hong, J., Phillips, A., Swift, S. and Blenkiron, C. (2017). Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity. J Extracell Vesicles 6(1): 1324731.
  5. Dean, S. N., Leary, D. H., Sullivan, C. J., Oh, E. and Walper, S. A. (2019). Isolation and characterization of Lactobacillus-derived membrane vesicles. Sci Rep 9(1): 877.
  6. Dean, S. N., Rimmer, M. A., Turner, K. B., Phillips, D. A., Caruana, J. C., Hervey, W. J. t., Leary, D. H. and Walper, S. A. (2020). Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery. Front Microbiol 11: 710.
  7. Holst, J., Martin, D., Arnold, R., Huergo, C. C., Oster, P., O'Hallahan, J. and Rosenqvist, E. (2009). Properties and clinical performance of vaccines containing outer membrane vesicles from Neisseria meningitidis. Vaccine 27 Suppl 2: B3-12.
  8. Klimentova, J. and Stulik, J. (2015). Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria. Microbiol Res 170: 1-9.
  9. Kulp, A. and Kuehn, M. J. (2010). Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64: 163-184.
  10. Mata Forsberg, M., Bjorkander, S., Pang, Y., Lundqvist, L., Ndi, M., Ott, M., Escriba, I. B., Jaeger, M. C., Roos, S. and Sverremark-Ekstrom, E. (2019). Extracellular Membrane Vesicles from Lactobacilli Dampen IFN-γ Responses in a Monocyte-Dependent Manner. Sci Rep 9(1): 17109.
  11. Molina-Tijeras, J. A., Galvez, J. and Rodriguez-Cabezas, M. E. (2019). The Immunomodulatory Properties of Extracellular Vesicles Derived from Probiotics: A Novel Approach for the Management of Gastrointestinal Diseases. Nutrients 11(5): 1038.
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简介

[摘要]在整个生命周期中,细菌会脱落其最外层膜的一部分,该膜由蛋白质、脂质和多种其他生物分子组成。这些生物纳米颗粒已被证明具有一系列高度多样化的生物活性,包括发病机制、群落调节和细胞防御(等等)。在最近的出版物中,我们从几种乳酸杆菌中分离并表征了膜囊泡 (MV),这些微生物被归类为人类肠道微生物组中的共生菌(Dean等人,2019 年和2020 年). 随着对宿主-微生物相互作用、肠-脑轴以及用于治疗或性能提高应用的定制益生菌的科学理解不断加深,本文所述的方案将有助于研究人员开发肠道群落工程或靶向递送生物活性物质的新策略。分子。



图文摘要:

图1 。原子˚F奥尔塞米icroscop IC的图像乳杆菌干酪乳杆菌ATCC 393种细菌边距(白色箭头)和膜囊泡(黑色箭头)


[背景]所有细胞的膜是动态结构,所述生物分子的组合物,其是不断变化的作为细胞对环境刺激,改变蛋白质和脂质组合物,释放废产物反应,采取在营养物质,并执行许多其它细胞过程(Vereb等人. ,2003 年;沃森,2015 年)。在整个细胞生命周期中,最外层膜的碎片通常以纳米颗粒的形式脱落。在细菌中,这些结构通常被称为膜囊泡 (MV) 或外膜囊泡 (OMV) ,为简单起见,将通篇称为 MV (干酪乳杆菌MV 的代表性示例如图 1 所示)。近年来,随着人们对这些生物纳米粒子的兴趣日益浓厚,研究人员已经表明,MV 具有广泛的生物活性,包括宿主-微生物相互作用、基因转移和群落调控(Kulp 和 Kuehn,2010 年;Caruana 和 Walper,2020 年)。

细菌 MV 已显示出在疫苗开发和潜在治疗应用等应用中的巨大前景。来自革兰氏阴性菌的天然 OMV 在开发针对脑膜炎奈瑟菌和假鼻疽伯克霍尔德氏菌等细菌的疫苗方面显示出巨大的前景,这些病原体已被证明对疫苗和治疗开发具有挑战性(Holst等人,2009 年;Nieves等人,2009 年)。, 2014) 。近日,研究小组还表明,一些共生细菌的MV可以从宿主免疫系统也调节反应和刺激神经系统的主机系统(马塔福斯贝里等,2019;莫利纳-Tijeras等,2019; Rodovalho等。人, 2020)。随着合成生物学能力的增强,随着研究人员开发出允许修改遗传和细胞途径以控制新生 MV 组成的工程策略,MV 的潜在用途正在稳步增加。这些努力导致了用于治疗应用、环境净化和其他目的的新生物材料(Alves等人,2018 年;Qing等人,2019 年)。

的分类升陡坡带酸细菌(LAB)包括具有的酸耐受性和发酵能力的类似特征的细菌的几个属。许多 LAB 被归类为普遍认为安全的 (GRAS),几个世纪以来一直用于食品生产。此外,一些 LAB 已被认为对其宿主有益,并因其作为益生菌的健康益处而被研究,从而导致益生菌补充剂和食品的大型商业市场。虽然活细菌培养物是最常用的益生菌形式,但工程化或富集特定细胞成分的潜力促使研究人员探索将纯化的 MV 用于受控治疗应用(Seo等人,2018 年;Molina -Tijeras等人) al. , 2019; Dean等人, 2020) 。

有许多用于从真核细胞和革兰氏阴性细菌中纯化 MV 的方案,这些方案一直是 MV 研究的主要焦点(Klimentova 和 Stulik 2015;Alves等,2017;Dauros Singorenko等,2017)。最近,人们对肠道细菌的 MV 及其在宿主和群落相互作用中可能发挥的作用越来越感兴趣。LAB 是革兰氏阳性菌,因此与革兰氏阴性菌相比具有显着不同的膜和肽聚糖结构。虽然这可能不会特别有助于革兰氏阴性和革兰氏阳性 MV 之间的生物物理差异,但已经表明,一些 LAB 物种的 MV 具有双峰尺寸分布,在 10 - 50 nm 尺寸范围内有大量较小的MV (Dean等人,2019 年)。在这里,我们描述了已证明对众多 LAB 物种成功的 MV 的分离和表征协议。通过超速离心从过夜的 LAB 培养物中分离出 MV ,然后使用 NanoSight 纳米颗粒跟踪仪分析浓度和相对尺寸分布。动态升飞行小号cattering(DLS)作为另一种方式来测量粒度分布,并且还测量ζ电势(表面电荷)。然后, 可以使用 SDS-PAGE 定性地检查 MV 的蛋白质含量, 特别是使用质谱法进行蛋白质组学分析 (示意图如图 2 所示)。虽然这份手稿描述了我们为 LAB 所做的工作,但这些协议也可用于从各种微生物物种中分离 MV。

关键字:膜囊泡, 乳酸菌, 嗜酸乳杆菌, 植物乳杆菌

 
材料和试剂
 
AnaeroJar TM 2.5 L 罐(Oxoid ® ,ThermoFisher Scientific,目录号:AG0025A)
AnaeroGen TM厌氧气体发生袋(Oxoid ® ,Thermo Fisher Scientific,目录号:AN0025A )
刃天青厌氧指示剂条(Oxoid ® ,Thermo Fisher Scientific,目录号:BR0055B)
100毫米无菌皮氏d ishes(FISHERBRAND,赛默飞世尔科技,产品目录号FB0875712)
的Pyrex ® Erlenmyer烧瓶250毫升(康宁TM ,赛默飞世尔科技,产品目录号:CLS4980250)
50 ml锥形离心管(Corning TM Falcon,Thermo Fisher Scientific,目录号:14-432-22)
0.45 µm注射器过滤器(JG Finneran,目录号:FEC0425PC)
30 ml 无菌注射器(BD Slip tip 无菌注射器;Thermo Fisher Scientific,目录号:BD 302833)
38.5 ml 超速离心管(薄壁超透明管;Beckman Coulter,目录号:344058)
5 ml 超速离心管(薄壁超透明管;Beckman Coulter,目录号:344057)
1 ml无针注射器(Henke Sass Wolf,目录号:4010.200V0)
1.5 ml 离心管(Thermo Fisher Scientific,目录号:P190411)
与 ZetaSizer Nanoseries 兼容的一次性细胞(Malvern Panalytical,目录号:DTS 1070)
乳酸菌种,即,
嗜酸乳杆菌(ATCC 53544)
干酪乳杆菌(ATCC 393)
罗伊氏乳杆菌(ATCC 23272)
植物乳杆菌(ATCC BAA-793)
de Man、Rogosa 和 Sharpe(MRS)培养基(Sigma-Aldrich,目录号:69966-500G,根据制造商的说明准备)
Tween ® 80(Sigma Aldrich,目录号:P8074)
4-15%的Mini-PROTEAN ® TGX预制蛋白凝胶(Bio-Rad公司,目录号:4561085)
GelCode TM Blue Stain Reagent(Thermo Fisher Scientific,目录号:PI24590)
10 ×磷酸盐缓冲盐水(Thermo Fisher Scientific,目录号:AM9625)
4 × Laemmli 样品缓冲液(Bio-Rad,目录号:1610747)
2-巯基乙醇(Bio-Rad,目录号:161-0710)
SDS-PAGE 运行缓冲液(Bio-Rad,目录号:161-0772)
Pierce BCA蛋白质测定试剂盒(Thermo Fisher Scientific,目录号:23225)
1-丙醇(Sigma - Aldrich,目录号:402893)
碳酸氢铵(Sigma - Aldrich,目录号:A6141)
胰蛋白酶,测序级(Promega,Fisher Scientific,目录号:PRV5111)
甲酸(Sigma - Aldrich,目录号:27001)
乙腈(Sigma - Aldrich,目录号:34851)
OptiPrep 培养基(Progen Biotechnik GmbH,目录号:1114542)
SDS-PAGE电泳缓冲液(10 ×库存;在使用1 × )(见ř ecipes)
 
设备
 
能够保持 37°C 的培养箱(例如:Fisherbrand , Isotemp Microbiological Incubator)
容量为 50 ml 锥形管的离心机(例如:Beckman Coulter 、Avanti JXN-30 使用 JA-14.50 转子)
超速离心机可容纳 38.5 ml 试管,129,000 × g (例如:Sorvall WX Ultra 90 离心机,使用 AH-629 转子)
NanoSight LM10(英国伍斯特郡 Malvern Panalytical)
ZetaSizer NanoSeries 配备 HeNe 激光源(λ = 633 nm)和雪崩光电二极管(Malvern Panalytical,Worcestershire,UK)
Mini-PROTEIN ® Tetra 垂直电泳池(Bio-Rad,目录号:1658004)
Speed-vac(例如:Thermo Fisher Scientific SC210A SpeedVac Concentrator,目录号:SC210A-230)
Barocycler (Pressure Biosciences Inc., HUB 440-SW16, Easton, MA, US)
Orbitrap LC-MS/MS 系统(例如:配备 Nanospray Flex 离子源的 Thermo Scientific Orbitrap Fusion Lumos(Thermo Fisher Scientific,目录号:ES071)
自动进样器(例如:Thermo Scientific Dionex UltiMate 3000 快速分离孔板自动进样器(Thermo Fisher Scientific,目录号:5840.0010)
超高效液相色谱(UHPLC)系统(例如:Dionex Ultimate 3000 RSLCnano 系统,Thermo Fisher Scientific,目录号:ULTIM3000RSLCNANO)
 
软件
 
NTA 2.3 纳米粒子跟踪和分析软件(Malvern Panalytical,Worcestershire,UK)
用于分散技术软件(DTS,马尔文PANALYTICAL,伍斯特郡,英国)d ynamic升飞行小号cattering分析
Scaffold 版本 4.8.2(Proteome Science Inc., Portland, Oregon, US),用于质谱分析后的蛋白质鉴定
Mascot(2.6.1版,Matrix Science,London,UK),用于质谱后的蛋白质鉴定
X!Tandem(版本 1.7.18)用于质谱分析后的蛋白质鉴定
R ( https://cran.r-project.org/ ) 和相关的 R 包:Peptides、Limma 和 ggplot2。用于鉴定蛋白质的生化分析、统计分析以及生成火山图或其他数据可视化
 
程序
 
膜囊泡 (MV) 的分离
培养细菌菌株
将冷冻原液或刺瓶中的每个菌株单独划线到 MRS 琼脂平板上。对于厌氧生长,将板放入 AnaeroJar 罐中,并添加 AnaeroGen 厌氧气体生成袋和厌氧指示剂。在 37°C 下生长约 48 小时或直至可见单个菌落。
将单个细菌菌落接种到 3 ml MRS 肉汤中。在 37°C 下过夜生长,不要像上一步那样在 AnaeroJar 中摇晃。
在无菌的 250 毫升锥形瓶中,从原代培养物中加入 1 毫升到 50 毫升 MRS 肉汤中。像前面的步骤一样在一夜之间生长。
收集并过滤上清液
将过夜培养物转移到 50 ml 锥形离心管中,并以 5 , 000 × g离心30 分钟。
将上清液倒入新管中,以 5 , 000 × g 的速度再次离心30 分钟。
重复小号TEP A2 b一次或两次以上,直到没有细胞沉淀是在侧面或后离心管的底部是可见的。这些额外的离心步骤不是严格要求的,但可以更容易地在后续步骤中过滤上清液。
将上清液通过 0.45 µm过滤器。这可以使用 30 ml 注射器和注射器过滤器或使用带有适当过滤器的真空装置来完成,以获取更大体积的上清液。
超速离心分离 MV
将 36 ml 过滤后的上清液转移到超速离心管中并组装到转子中。
在 4°C 下以 126,000 × g超速离心1.5 小时。
倒出上清液(颗粒通常是不可见的)。
加入 1 ml 1 ×磷酸盐缓冲盐水 (PBS) 并在 4°C 下孵育过夜以重悬沉淀。
注意:如果进行梯度超速离心,请在 150 μ PBS-OptiPrep混合物的升代替(见小号TEP甲下面4A)。
梯度ü MV的ltracentrifugation
在 150 µl PBS-OptiPrep 混合物(45% v/v)中重悬 MV 颗粒,并将悬浮液移至 5 ml 超速离心管的底部。
制作几个 0.8 ml 较低密度的 PBS-OptiPrep 溶液(例如,35%、30%、25%、20%、15% 和 10%),然后依次用密度降低的层覆盖含有 MV 的基层。添加时避免添加气泡或破坏之前的图层。
添加所有层后,让管子静置 1 小时。
使用 AH-650 转子在 Sorvall WX Ultra 90 离心机中以 236,000 × g超速离心梯度3 小时。
离心后,轻轻依次取出10 个等体积 (~500 μl) 级分并将其放入单独的管中。
使用下述方法评估不同密度分数的 MV 浓度、蛋白质组成和其他特征。
 
 
图 3.乳杆菌膜囊泡的纳米粒子追踪分析 (NTA) 。一个。从嗜酸乳杆菌MV的 NanoSight LM10 视频中获取的代表性帧。在NTA之前,从n MRS培养物纯化的MV在PBS中以1:1 , 000稀释。比例尺表示 2 µm。乙。NTA 结果的代表性 MV 大小分布,取自三个 60 年代视频的平均值。
 
MV Ç haracterization
纳米颗粒吨费尽一个nalysis (NTA),以确定颗粒尺寸分布和浓度
在 1 × PBS 中稀释 MV –进行多次连续稀释,例如 10 × 、100 ×和 1 、000 × 。当视野内有20-60个颗粒时,NTA软件检测颗粒效果最佳;因此,样品需要稀释,并且每个样本的最佳稀释度可能会发生变化,从一个隔离到另一个。
使用 1 ml 无针注射器,将稀释的样品加入 NanoSight 仪器室。留出时间让样品沉淀并调整显微镜焦距和相机设置,以优化 NTA 2.3 纳米颗粒跟踪和分析软件中相机视野中颗粒的清晰度。可以在室温下收集数据。
以每秒 30 帧的速度将数据捕获为 60 秒曝光的三次读数,并使用自动粒子检测和跟踪参数进行分析:检测阈值、像素模糊、最小轨道长度和最小预期粒子尺寸。从囊泡的NTA的代表性图像的嗜酸乳杆菌ATCC 53544在图3中示出作为NTA 2.3不自动比例尺信息添加到图像,它们可以是附加编由像素转换为微米通过除以像素乘以9.036 (使用30时× 目标)。
动态升飞行小号cattering(DLS)来测量MV尺寸分布
在 0.1 × PBS pH 7.4 中将MV 样品稀释 10 倍,然后添加到一次性 DLS 比色皿中。
使用配备 HeNe 激光源 (λ = 633 nm) 的 ZetaSizer NanoSeries 进行 DLS 分析,并使用色散技术软件进行分析。在 25 °C 下进行测量。对于每个样本,对自相关函数取 5 次运行的平均值,每次运行 10 秒,然后重复三到六次。
Zeta 电位测量以确定 MV 表面电荷
像 DLS 一样稀释 MV 样品并装入一次性比色皿中。
使用配备有 HeNe 激光源 (λ = 633 nm) 和色散技术软件的 ZetaSizer NanoSeries 测量 zeta 电位。在 25 °C 下对每个样品进行三次测量。
用于对 MV 中所含蛋白质进行定性分析的 SDS-PAGE
制备样品:根据 NTA 确定的 MV 浓度,在 1.5 ml 离心管中取等量的每个 MV 样品,体积≤15 μl。使用水或 1 × PBS 将样品体积调整至 15 μl 。(卷可以根据被调节的要使用的SDS-PAGE凝胶的孔容量)。
向每个样品中加入 5 μl(或所需体积以产生 1 ×终浓度)的 4 × Laemmli 上样缓冲液,然后通过煮沸 5 分钟、在冰上冷却 5 分钟并在 16,000 下离心 15 分钟使样品蛋白质变性× g在台式离心机中。
根据制造商的说明,使用4-15% Mini-PROTEAN ® TGX 预制蛋白质凝胶组装 Bio-Rad Mini-PROTEAN 垂直电泳槽。
将变性的 MV 样品(仅上清液)装入凝胶并在 200 V 下运行,直到染料前沿到达凝胶底部。
从外壳中取出凝胶并在水中短暂冲洗;然后,在 GelCode TM Blue Stain Reagent 中染色,然后用水脱色,以观察 MV 蛋白条带。Dean等人的图 3 。(2019 年)可以参考来自三个 LAB 物种的 MV 的 SDS-PAGE 凝胶的示例。
Shotgun 蛋白质组学分析以鉴定 MV 的蛋白质含量
样品制备
用 50 mM 碳酸氢铵 (ABC) 缓冲液中的 10 ml 10% 正丙醇重悬 MV 颗粒。
使用 Pierce BCA 蛋白质检测试剂盒标准化样品蛋白质浓度。
使用测序级改性胰蛋白酶以 1:50 (w/w) 酶: 底物比例在 barocycler 中消化溶液中的样品 (50 µg) 90 分钟(90 个循环:50 秒,45 kpsi,10 秒关闭)。
通过 speed-vac 蒸发消化的样品并储存在 -20 °C直到通过 LC-MS/MS 进行分析。
液相色谱-质谱/质谱
在 0.1% 甲酸水溶液(溶剂 A)中复溶干燥样品,并通过 Dionex Ultimate WPS-3000 自动进样器将 3 μg 消化的蛋白质加载到反相 C18 柱上,该自动进样器连接到配备 Nanospray Flex 离子源的n Orbitrap Fusion Lumos依赖数据的采集模式。
跨越2的90分钟梯度分离肽- 60%溶剂B(0.1%甲酸的乙腈溶液)在300标升/ min,使用的Dionex终极3000 RSLCnano系统的流率。
保存每个样本的原始、mgf 和 mzid 文件输出以供下游分析。
蛋白质鉴定
使用 Mascot 和 X 搜索质谱数据!串联使用适合生物体的基因组文件。使用 Scaffold 验证肽谱匹配。
将碎片离子质量容差设置为 ±0.60 Da,将母离子容差设置为 ±0.60 Da。将蛋白质识别阈值设置为≥2 个肽(蛋白质概率 80%,肽概率 95%)。将鉴定的蛋白质列表导出到 CSV。
使用 R 分析输出的 CSV 文件。
 
数据分析
 
蛋白质组学一nalysis:
使用 Mascot(Matrix Science,伦敦,英国)和 X! 分析所有 MS/MS 样品!串联(GPM,thegpm.org)。让 Mascot 设置搜索正确的数据库序列文件(由蛋白质 .fasta 输入生成)和相应的常见污染物文件。使用胰蛋白酶将假定的裂解设置为消化。
设置X!串联寻找正确的数据库序列文件的反向串联子集,其中只包含由吉祥物分析样品中鉴定的蛋白质。
使用支架(蛋白质组软件公司,波特兰,OR ,USA ),以验证MS / MS基于肽和蛋白质鉴定。将肽谱匹配设置设置为:碎片离子质量容差 = ± 0.60 Da,母离子容差 = ± 0.60 Da。将蛋白质识别阈值设置为≥2肽(蛋白质概率 80% ;肽概率 95%)。
使用 R 分析蛋白质组学数据输出到 CSV 文件
使用 Peptides R 软件包确定肽/蛋白质的理化特性。
使用经验贝叶斯方法执行双样本 t 检验,使用 Limma R 包调整每个蛋白质的方差估计,其输出是每个蛋白质的 log 2倍变化和缓和的 p 值对应于调节 t 统计量,如前所述(Dean等人,2020 年)。
使用 ggplot2 R 包创建火山图和数据的其他可视化(有关使用蛋白质组学数据的示例火山图,请参见图 4)。
 
 
图 4. 对乳酸杆菌膜囊泡进行蛋白质组学的火山图。相对于未处理的对照,在用诱导剂处理的嗜酸乳杆菌MV 中鉴定的蛋白质的 log 2倍变化的代表性火山图。通过基于 Limma R 包的经验贝叶斯方法将一式三份样品中鉴定的蛋白质数据放入并使用 ggplot2 绘制。有显着变化的蛋白质用蓝色表示。
 
食谱
 
SDS-PAGE 电泳缓冲液(10 ×库存;在 1 × 时使用)
25 mM Tris 碱
192 mM 甘氨酸
0.1% SDS
d我ssolve在水作为溶剂
 
致谢
 
该协议改编自以前的工作(Dean等人,2019 年和2020 年)。这项工作的资金是通过海军研究实验室 (SND, SAW) 的核心基金提供的。额外的财政支持是通过美国工程教育协会(ASEE)博士后奖学金计划(JCC)提供。图形摘要中显示的 AFM 图像由赖特帕特森空军基地空军研究实验室的 Claretta Sullivan 拍摄。
 
利益争夺
 
作者声明没有竞争利益。
 
伦理
 
此处描述的协议均不包括对动物或人类受试者的研究。
 
参考
 
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引用:Caruana, J. C., Dean, S. N. and Walper, S. A. (2021). Isolation and Characterization of Membrane Vesicles from Lactobacillus Species. Bio-protocol 11(17): e4145. DOI: 10.21769/BioProtoc.4145.
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