Differentiation of Naturally Produced Extracellular Membrane Vesicles from Lipid Aggregation by Glucuronoxylomannan Immunogold Transmission Electron Microscopy in Bacillus subtilis

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Molecular Microbiology
Jul 2014



Recently, membrane vesicle (MV) production was described in Gram-positive bacteria, which harbor a variety of components such as toxins, antibiotic resistance proteins, proteases, DNA, and immune modulators. Free lipids have the ability to form micelles, thus it is important to rule out spontaneous association of lipids into vesicle-like structures and rather, that MVs are produced naturally by a metabolically active cell. Here, we describe a protocol utilizing the polysaccharide, glucuronoxylomannan (GXM) from Cryptococcus neoformans (C. neoformans) as a marker to differentiate naturally produced MVs from vesicles that form spontaneously in the Gram-positive model organism, Bacillus subtilis (B. subtilis). MVs are purified from bacterial cultures grown in the presence of GXM; MVs naturally produced by cells would not contain GXM in the lumen whereas vesicular structures forming in the media could encapsulate GXM and this can be visualized via immunogold transmission electron microscopy.

Materials and Reagents

  1. Purified glucuronoxylomannan (GXM) (protocol included)
  2. YPD broth (Difco, catalog number: 242820 )
  3. C. neoformans H99 fungal strain
  4. 100 kDa, 50 kDa, 10 kDa, 1 kDa cut-off EMD Millipore Ultrafiltration Membranes (Millipore, catalog number: 14442AM )
  5. B. subtilis 168 bacterial strain
  6. BHI broth (Difco, catalog number: 299070 )
  7. 8% glutaraldehyde (Polysciences, catalog number: 111-30-8 )
  8. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15700 )
  9. 0.5 M sodium cacodylate (pH 7.4) (Electron Microscopy Sciences, catalog number: 11650 )
  10. 100% ethanol
  11. Lowicryl HM-20 monostep resin (Electron Microscopy Sciences, catalog number: 14340 )
  12. Gelatin (Thermo Fisher Scientific)
  13. Aurion Donkey Block (Electron Microscopy Sciences, catalog number: 25599 )
  14. α-GXM monoclonal antibody 18B7 (mouse monoclonal IgG1) (Casadevall lab-generated)
  15. BSA-c (Electron Microscopy Sciences, catalog number: 25557 )
  16. 10 nm conjugated secondary Ab (donkey α-mouse) (Electron Microscopy Sciences, catalog number: 25814 )
  17. 4% uranyl acetate (aq) (SPI Supplies, catalog number: 615-44-0 )
  18. 1x phosphate buffered saline (PBS) (see Recipes)


  1. Express PLUS Membrane Filters - Pore (0.22 μm) (Millipore, catalog number: SCGVU01RE )
  2. Ultracentrifugation tubes (thickwall, polyallomer/pollypropylene, 3.5 ml, 13 x 51 mm) (Beckman Coulter, catalog number: 349623 )
  3. 200 mesh nickel grids (Polysciences, catalog number: 24916 )
  4. Centrifuge capable of 15,000 x g
  5. TLA 100.3 rotor (Beckman Coulter, catalog number: 349490 )
  6. Amicon ultrafiltration system (Millipore, catalog number: 5124)
  7. Optima TL ultracentrifuge (Beckman Coulter, discontinued comparable to B11229)
  8. Sonicator (sonic dismembrator) (Thermo Fisher Scientific, model: 100 cpn-214-161 )
  9. Freeze substitution system (RMC, model: FS-7500 )
  10. Reichert Ultracut UCT Ultramicrotome
  11. JEOL 100CXII or JEOL 1200EX Electron Microscopes


  1. Purification of GXM
    1. Grow 1 L of YPD broth inoculated with C. neoformans strain H99 overnight at 37 °C with shaking (~18 h 200 rpm).
    2. Spin out cells by centrifuging 15,000 x g for 20 min at 4 °C.
    3. Filter cell-free supernatant with 0.22 μm Express PLUS Membrane Filters to remove debris.
    4. Sequentially concentrate the cell-free supernatant with 100 kDa, 50 kDa, 10 kDa, and 1 kDa cut-off EMD Millipore Ultrafiltration Membranes. The jelly that forms on the filters is GXM (save jelly that is <10 kDa and >1 kDa) (see Figure 1 for concentrator set-up).

  2. Purification of gram-positive extracellular vesicles
    1. Inoculate 100 ml of BHI broth with fractionated GXM <10 kDa and B. subtilis strain 168.
    2. Grow overnight at 37 °C with shaking (~18 h 200 rpm).
    3. Centrifuge culture to remove cells at 15,000 x g for 20 m at 4 °C.
    4. Filter cell-free supernatant with 0.22 μm Express PLUS Membrane Filters to remove debris.
    5. Concentrate cell-free supernatant to small volumes (approximately 6 ml) with ultrafiltration system and 100 kDa cut-off EMD Millipore Ultrafiltration Membranes (Figure 1).
    6. Ultracentrifuge concentrated supernatant at 100,000 x g for 1 h at 4 °C in thickwall, polyallomer, 3.5 ml, 13 x 51 mm tubes to pellet vesicles (Figure 2).
    7. Wash/resuspend vesicle pellet with 500 μl PBS and repeat spin (repeat wash twice).
    8. Remove supernatant from sample without disturbing pellet.
      1. For sonication (production of disrupted/unnatural vesicles-positive control) resuspend pellet in PBS and sonicate (20 sec sonication and 30 sec incubation at 4 °C on ice, repeated 4 times at power 2).
      2. Ultracentrifuge again to pellet vesicles and carefully remove supernatant.
    9. Carefully add 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate without disturbing pellet.
    10. Incubate for 1 h at room temperature (RT).

  3. Sample preparation for immunogold labeling
    1. After fixing for 1 h at RT, wash with 0.1 M sodium cacodylate 3 times.
    2. Enrobe samples in 5% gelatin.
    3. Dehydrate through a graded ethanol series in a RMC FS 7500; progressively lowering the temperature 5 °C /h from 4 °C to -50 °C.
    4. Use Lowicryl HM-20 monostep resin to embed sample.
    5. Polymerize embedded sample with UV light for 48 h at -50 °C.
    6. Use Reichert Ultracut UCT to cut 80 nm ultrathin sections onto 200 mesh nickel grids.

  4. Immunogold labeling of GXM in vesicle preparations
    1. Block with Aurion Donkey Block for 1 h.
    2. Incubate samples with the primary α-GXM mAb antibody, 18B7, in 0.1% BSA-c/PBS incubation buffer for ~18 h at 4 °C.
    3. Wash samples with 0.1% BSA-c/PBS.
    4. Incubate samples with the secondary donkey α-mouse IgG 10 nm immunogold-conjugated antibody for 2 h at RT.
    5. Wash samples with BSA-C.
    6. Post-fix samples for 5 m at 25 °C with 2% glutaraldehyde/PBS to stabilize the gold particles.
    7. Counterstain samples for 15 min 25 °C with 4% uranyl acetate (aq).
    8. Also make control samples with an irrelevant IgG antibodies and without the 1o antibody (18B7).

Representative data

Figure 1. Amicon ultrafiltration setup. The ultrafiltration cells are connected to compressed nitrogen gas. The gas applies pressure, forcing the supernatant through the 100 kDa filter membrane. Vesicles stay in the concentrated supernatant while smaller proteins and liquid supernatant flow into waste container.

Figure 2. Beckman ultracentrifuge tubes. Ultracentrifugation tubes utilized to pellet vesicles.

Figure 3. Transmission electron micrograph of immunogold labeling of GXM in vesicles from Bacillus stubtilis. Micrograph shows vesicles and gold particles. Gold particles indicate the presence of GXM. Notice all of the gold is outside of vesicles. White arrows indicate vesicles and black arrows indicate gold particles. Scale bar=100 nm

Vesicles are not incredibly stable so it is suggested to process them immediately after isolation. Only Beckman polyallomer/propylene ultracentrifugation tubes can be used for IEM preparation.Polycarbonate tubes cannot be used for this procedure but can be used when purifying vesicles for other experiments. The sonicated (unnatural) vesicles serve as a positive control. It is expected that vesicles from the sonicated vesicle preparation will contain significantly more gold particles than the naturally produced vesicles. One may also expect that sonicated vesicles have a smaller and less variable diameter than that of natural vesicles.


B. subtilis strain 168 concentrates very quickly but other strains of bacteria may not. B. subtilis strain 168 produces a large quantity of recoverable vesicles and only requires 100 ml of culture whereas other bacteria may require volumes larger than 1 L. Concentrating to small volumes allows for less ultracentrifugation spins and cuts down on the use of the ultracentrifuge tubes. Ultracentrifuge tubes can be washed and re-used but be aware there may be some contamination.


  1. 1 L 1x phosphate buffered saline (PBS) (pH 7.4)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    Bring to pH 7.4 with HCl
    Dissolve in H2O up to 1 L


Funding from NIH Grant Numbers: HL059842, AI033774, AI033142, AI052733 and Center for AIDS Research at Albert Einstein College of Medicine.
Protocol is adapted from Brown et al. (2014) and vesicle purification is adapted from Prados-Rosales et al. (2014).


  1. Brown, L., Kessler, A., Cabezas-Sanchez, P., Luque-Garcia, J. L. and Casadevall, A. (2014). Extracellular vesicles produced by the Gram-positive bacterium Bacillus subtilis are disrupted by the lipopeptide surfactin. Mol Microbiol 93(1): 183-198.
  2. Prados-Rosales, R., Brown, L., Casadevall, A., Montalvo-Quirós, S. and Luque-Garcia, J. L. (2014). Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria. MethodsX (1): 124-129.


最近,在革兰氏阳性细菌中描述了膜囊泡(MV)产生,其含有多种组分,例如毒素,抗生素抗性蛋白,蛋白酶,DNA和免疫调节剂。游离脂质具有形成胶束的能力,因此排除脂质自发缔合成囊泡样结构是重要的,并且更确切地说,MV由代谢活性细胞天然产生。在这里,我们描述了利用来自新隐孢子虫的多糖葡萄糖醛酸甘露聚糖(GXM)( C。新型隐球菌)作为标记物来区分天然产生的MV与自发形成的囊泡革兰氏阳性模式生物,枯草芽孢杆菌(<枯草芽孢杆菌)。从在GXM存在下生长的细菌培养物中纯化MV;由细胞天然产生的MV在管腔中不含有GXM,而在培养基中形成的囊泡结构可包封GXM,并且这可通过免疫金属透射电子显微镜可视化。


  1. 纯化的葡萄糖醛酸甘露聚糖(GXM)(包括方案)
  2. YPD肉汤(Difco,目录号:242820)
  3. C。 新古生菌H99真菌菌株
  4. 100kDa,50kDa,10kDa,1kDa截留EMD Millipore超滤膜(Millipore,目录号:14442AM)
  5. B。 枯草芽孢杆菌 168菌株
  6. BHI肉汤(Difco,目录号:299070)
  7. 8%戊二醛(Polysciences,目录号:111-30-8)
  8. 16%多聚甲醛(Electron Microscopy Sciences,目录号:15700)
  9. 0.5M二甲胂酸钠(pH 7.4)(Electron Microscopy Sciences,目录号:11650)
  10. 100%乙醇
  11. Lowicryl HM-20单柱树脂(Electron Microscopy Sciences,目录号:14340)
  12. 明胶(Thermo Fisher Scientific)
  13. Aurion Donkey Block(Electron Microscopy Sciences,目录号:25599)
  14. α-GXM单克隆抗体18B7(小鼠单克隆IgG1)(Casadevall实验室产生)
  15. BSA-c(Electron Microscopy Sciences,目录号:25557)
  16. 10nm共轭二抗(驴α-小鼠)(Electron Microscopy Sciences,目录号:25814)
  17. 4%乙酸铀酰(aq)(SPI Supplies,目录号:615-44-0)
  18. 1×磷酸盐缓冲盐水(PBS)(见Recipes)


  1. Express PLUS膜过滤器 - 孔(0.22μm)(Millipore,目录号:SCGVU01RE)
  2. 超速离心管(厚壁,多聚集体/聚丙烯,3.5ml,13×51mm)(Beckman Coulter,目录号:349623)
  3. 200目镍网格(Polysciences,目录号:24916)
  4. 离心力可达15,000 x g
  5. TLA 100.3转子(Beckman Coulter,目录号:349490)
  6. Amicon超滤系统(Millipore,目录号:5124)
  7. Optima TL超速离心机(Beckman Coulter,与B11229类似,已停产)
  8. 超声波仪(声波破碎仪)(Thermo Fisher Scientific,型号:100 cpn-214-161)
  9. 冷冻替代系统(RMC,型号:FS-7500)
  10. Reichert Ultracut UCT超薄切片机
  11. JEOL 100CXII或JEOL 1200EX电子显微镜


  1. 纯化GXM
    1. 生长1L用C接种的YPD肉汤。 新生隐孢子虫菌株H99在37℃振荡(〜18h 200rpm)过夜
    2. 通过在4℃下离心15,000×g离心20分钟来离心细胞
    3. 用0.22μmExpress PLUS膜过滤器过滤无细胞上清液以去除碎屑
    4. 连续浓缩无细胞上清液100 kDa,50 kDa,10kDa和1kDa截留EMD Millipore超滤膜。 在过滤器上形成的果冻是GXM(保存果冻<10 kDa和> 1kDa)(参见图1,用于浓缩器装置)。

  2. 革兰氏阳性细胞外囊泡的纯化
    1. 接种100ml分馏的GXM <10kDa的BHI肉汤和B。 枯草芽孢杆菌菌株168.
    2. 在37℃下振荡生长过夜(〜18h 200rpm)
    3. 离心培养以在4℃下以15,000×g离心20分钟除去细胞。
    4. 用0.22μmExpress PLUS膜过滤器过滤无细胞上清液以去除碎屑
    5. 浓缩无细胞上清至小体积(约6 ml)与超滤系统和100kDa截留EMD Millipore 超滤膜(图1)。
    6. 超速离心机 浓缩的上清液在100,000×g下在4℃在厚壁中1小时, 多聚集体,3.5ml,13×51mm试管以沉淀囊泡(图2)
    7. 用500μlPBS洗涤/重悬囊泡沉淀并重复旋转(重复洗涤两次)
    8. 从样品中去除上清液,而不干扰沉淀
      1. 用于超声处理(产生破裂/非天然囊泡 - 阳性 对照)在PBS中重悬沉淀物并超声处理(20秒超声处理和30秒 在冰上4℃孵育,在动力2下重复4次)
      2. 再次超速离心以沉淀囊泡并小心地除去上清液。
    9. 小心地加入4%多聚甲醛,0.1%戊二醛在0.1M二甲胂酸钠中,不会影响颗粒。
    10. 在室温(RT)下孵育1小时。

  3. 免疫金标记的样品制备
    1. 在室温下固定1小时后,用0.1M的二甲胂酸钠洗涤3次
    2. 在5%明胶中挑取样品。
    3. 在RMC FS 7500中通过分级乙醇系列脱水; 将温度从4℃逐渐降低至5℃/h至-50℃
    4. 使用Lowicryl HM-20单步树脂包埋样品。
    5. 在-50℃下用UV光聚合嵌入的样品48小时
    6. 使用Reichert Ultracut UCT在200目镍网上切割80 nm超薄切片。

  4. 囊泡制剂中GXM的免疫金标记
    1. 用Aurion驴块阻挡1小时。
    2. 将样品与初级α-GXM mAb抗体18B7在0.1%BSA-c/PBS孵育缓冲液中在4℃孵育〜18小时。
    3. 用0.1%BSA-c/PBS洗涤样品
    4. 孵育样品与辅助驴α-小鼠IgG 10 nm immunogold共轭抗体在室温下2 h。
    5. 用BSA-C洗涤样品
    6. 在25℃下用2%戊二醛/PBS后固定样品5分钟以稳定金颗粒
    7. 用4%的乙酸双氧铀(水溶液)将样品在25℃下复染15分钟
    8. 还用不相关的IgG抗体和不含抗体(18B7)制备对照样品。


图1. Amicon超滤设置。超滤池连接到压缩氮气。气体施加压力,迫使上清液通过100kDa过滤膜。小泡保留在浓缩的上清液中,而较小的蛋白质和液体上清液流入废物容器

图2. Beckman超速离心管。超速离心管用于沉淀囊泡。

图3.来自枯草芽孢杆菌的囊泡中GXM的免疫金标记的透射电子显微照片。显微照片显示囊泡和金颗粒。金颗粒表明GXM的存在。注意所有的金都在囊泡之外。白色箭头表示囊泡,黑色箭头表示金颗粒。比例尺= 100nm



B。 枯草芽孢杆菌菌株168非常快速地浓缩,但其他菌株可能不会。 B。 枯草芽孢杆菌菌株168产生大量的可回收囊泡并且仅需要100ml培养物,而其他细菌可能需要大于1L的体积。集中至小体积允许更少的超速离心旋转并减少超速离心机的使用 管。 超速离心管可以洗涤和重复使用,但要注意可能有一些污染。


  1. 1L 1x磷酸盐缓冲盐水(PBS)(pH 7.4) 137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH 2 PO 4 sub/
    调至pH 7.4 溶解在H 2 O中至多1 L


协议改编自Brown等人。 (2014)和囊泡纯化改编自Prados-Rosales等人。 (2014年)。


  1. Brown,L.,Kessler,A.,Cabezas-Sanchez,P.,Luque-Garcia,J.L。和Casadevall,A。(2014)。 革兰氏阳性细菌枯草芽孢杆菌产生的胞外囊泡被 脂质表面活性素。 Mol Microbiol 93(1):183-198。
  2. Prados-Rosales,R.,Brown,L.,Casadevall,A.,Montalvo-Quirós,S.和Luque-Garcia,J.L。 隔离和鉴定革兰氏阳性菌和分枝杆菌中的膜囊泡相关蛋白 em>。 MethodsX (1):124-129。
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引用:Brown, L., Perumal, . and Casadevall, A. (2015). Differentiation of Naturally Produced Extracellular Membrane Vesicles from Lipid Aggregation by Glucuronoxylomannan Immunogold Transmission Electron Microscopy in Bacillus subtilis. Bio-protocol 5(5): e1408. DOI: 10.21769/BioProtoc.1408.