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Vesicle Isolation from Bacillus subtilis Biofilm
枯草杆菌生物膜的囊泡分离   

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Abstract

Bacterial biofilms are associated clinically with many bacterial infections including those caused by bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. In recent years, extracellular vesicles produced by bacteria have been isolated from biofilm communities. Vesicles have been described in depth and can encapsulate various virulence factors including toxins and immunomodulatory compounds. Vesicles may be important for virulence and survival by serving as a vehicle for the secretion and concentrated delivery of these molecules. Studying extracellular vesicles is an important step towards understanding biofilm formation, structure, and disruption with the ultimate goal of preventing or treating hospital infections caused by bacterial pathogens residing in biofilms. Here we describe the protocol for isolating vesicles from biofilm produced by Bacillus subtilis.

Materials and Reagents

  1. Bacillus subtilis (B. subtilis) 168 bacterial strain (available from ATCC, catalog number: 23857 )
  2. BHI broth/agar (BD, catalog number: 211059/211065)
  3. Amicon Ultra centrifugal filter units Ultra-4 (MWCO 100 kDa) (Millipore, catalog number: UFC910024 )
  4. 1x phosphate buffered saline (PBS) (see Recipes)
  5. MSgg medium (see Recipes)

Equipment

  1. Petri dishes (100 x 55 mm) (Corning, catalog number: 351029 ) (dish size can be variable and ultimately depends on the amount of vesicles you wish to purify)
  2. 0.22 μm syringe filters (Fisher, catalog number: 09-719C)
  3. Tube (thickwall, polyallomer, 3.5 ml, 13 x 51 mm) (Beckman Coulter, catalog number: 349623 )
  4. Optima TL ultracentrifuge (Beckman Coulter)
  5. TLA 100.3 rotor (Beckman Coulter)
  6. Centrifuge capable of 15,000 x g

Procedure

  1. Biofilm growth assay
    1. Inoculate a plate of Bacillus subtilis lab strain 168 (from -80 °C) on a BHI agar plate overnight (~18 h) at 37 °C.
    2. Inoculate 5 ml BHI broth from overnight agar plate (one colony) and grow for 4 h with shaking (~200 rpm) at 37 °C.
    3. Inoculate 1:1,000 of broth culture (12 μl) into 12 ml MSgg media in petri dishes.
    4. Let biofilm incubate (covered with dish lid without shaking) at 37 °C for desired time period (usually 3 and 7 days). Ensure that incubator has a water container to ensure the dishes do not dry out.

  2. Purification of vesicles from biofilm
    1. After desired time period, remove pellicle and supernatant from plates by pipetting and centrifuge at 15,000 x g for 20 min at 4 °C to remove cells and large debris.
    2. To remove remaining cells and debris, filter the spun supernatant with 0.22 μm syringe filters.
    3. Centrifuge filtered, cell-free supernatant with centrifugal filter units to concentrate to less than 3 ml in volume.
    4. To pellet vesicles from supernatant, ultracentrifuge concentrated supernatant at 195,000 x g for 1 h at 4 °C in polyallomer tubes.
    5. Remove supernatant without disturbing the vesicle pellet by pipetting.
    6. Repeat spin after washing in 500 μl PBS.
    7. Remove PBS wash from tube without disturbing the vesicle pellet.
    8. Resuspend pellet in desired volume of PBS.

Notes

  1. Bacillus subtilis grows as a biofilm pellicle on the surface of the media (Figure 1). It may need a few washings to remove all cells for vesicle collection. Figure 2 shows electron micrograph of negatively stained vesicles.
  2. Biofilms can be grown in various plates to achieve desired volume of vesicles. Ideally you want to have a visible pellet after ultracentrifugation. If no pellet is visible, scale up the plate size or number of biofilms for vesicle purification.
  3. Vesicle pellet after ultracentrifugation should be visible, but in cases of small culture volumes the pellet may be difficult to see.


    Figure 1. Representation of what biofilm pellicles may look like in a 6-well plate


    Figure 2. Negative stain TEM of vesicles isolated from Bacillus subtilis biofilm. Scale bar=100 nm

Recipes

  1. 1x phosphate buffered saline (PBS) (1 L, pH 7.4) (filter or autoclave sterilize)
    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
  2. MSgg medium (pH 7) (filter sterilize)
    50 μM MnCl2
    5 mM KH2PO4
    1 μM ZnCl2
    50 μM FeCl3
    2 mM MgCl2
    700 μM CaCl2
    50 μg/ml threonine, tryptophan, and phenylalanine
    0.5% glutamate
    0.5% glycerol
    2 μM thiamine
    100 mM morpholinepropanesulfonic acid (MOPS) (pH 7)

Acknowledgments

Funding from NIH Grant Numbers: HL059842, AI033774, AI033142, AI052733 and Center for AIDS Research at Albert Einstein College of Medicine.
Protocol was adapted from Brown et al. (2014) and McLoon et al. (2011).

References

  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.
  3. McLoon, A. L., Guttenplan, S. B., Kearns, D. B., Kolter, R. and Losick, R. (2011). Tracing the domestication of a biofilm-forming bacterium. J Bacteriol 193(8): 2027-2034.

简介

细菌生物膜临床上与许多细菌感染相关,包括由细菌如绿脓杆菌和金黄色葡萄球菌引起的细菌感染。 近年来,由细菌产生的胞外囊泡已经从生物膜群落中分离。 囊泡已经被深入描述并且可以包封各种毒力因子,包括毒素和免疫调节化合物。 通过作为这些分子的分泌和浓缩递送的载体,囊泡可能对于毒力和存活是重要的。 研究胞外囊泡是理解生物膜形成,结构和破裂的重要步骤,其最终目的是预防或治疗由存在于生物膜中的细菌病原体引起的医院感染。 在这里我们描述了从枯草芽孢杆菌生产的生物膜中分离囊泡的方案。

材料和试剂

  1. 168细菌菌株(可得自ATCC,目录号:23857)
    (枯草芽孢杆菌)
  2. BHI肉汤/琼脂(BD,目录号:211059/211065)
  3. Amicon超离心过滤器Ultra-4(MWCO 100 kDa)(Millipore,目录号:UFC910024)
  4. 1x磷酸缓冲盐水(PBS)(见食谱)
  5. MSGG媒体(见食谱)

设备

  1. 培养皿(100 x 55毫米)(康宁,目录号:351029)(菜尺寸可变,最终取决于您想净化的囊泡数量)
  2. 0.22微米注射器过滤器(Fisher,目录号:09-719C)
  3. 管(厚壁,多聚集,3.5ml,13×51mm)(Beckman Coulter,目录号:349623)
  4. Optima TL超速离心机(Beckman Coulter)
  5. TLA 100.3转子(Beckman Coulter)
  6. 离心机能够达到15,000 x g

程序

  1. 生物膜生长分析
    1. 将一块枯草芽孢杆菌实验室菌株168(从-80℃)接种到BHI琼脂平板上过夜(〜18小时),37℃。
    2. 从过夜的琼脂平板(一个菌落)接种5ml BHI培养液,并在37℃下摇动(〜200rpm)生长4小时。
    3. 将培养液培养物(12μl)接种到培养皿中的12ml MSGI培养基中
    4. 让生物膜在37℃孵育(盖上盖子而不摇动) (通常为3天和7天)。确保孵化器  有一个水箱,以确保菜肴不会干燥。

  2. 从生物膜净化囊泡
    1. 在所需时间后,从板上取出防护薄膜组件和上清液 通过在4℃下以15,000×g移液和离心20分钟以除去 细胞和大碎片
    2. 为了去除剩余的细胞和碎屑,用0.22微米注射器过滤器过滤上清液。
    3. 离心过滤,用离心过滤器单元的无细胞上清液浓缩至小于3ml体积
    4. 从上清液中沉淀小泡,将超速离心机浓缩 上清液在195,000 x g下在4℃下在聚集管中进行1小时
    5. 去除上清液,不要通过移液来干扰囊泡
    6. 在500μlPBS中洗涤后重复旋转。
    7. 从管中取出PBS洗涤,不会干扰囊泡颗粒
    8. 将沉淀重悬于所需体积的PBS中。

笔记

  1. 枯草芽孢杆菌在培养基表面生长成生物膜(图1)。它可能需要少量洗涤以去除所有细胞进行囊泡收集。图2显示了阴性染色小泡的电子显微照片
  2. 生物膜可以生长在各种平板上以达到所需体积的囊泡。理想情况下,您想要在超速离心后具有可见的颗粒。如果没有颗粒可见,请放大板的大小或生物膜的数量以进行囊泡净化。
  3. 超速离心后的囊泡应该是可见的,但是在培养体积小的情况下,颗粒可能很难看清。


    图1.六孔板中的生物膜防护薄膜的表征


    图2.从枯草芽孢杆菌生物膜分离的囊泡的阴性染色TEM。 比例尺= 100 nm

食谱

  1. 1×磷酸缓冲盐水(PBS)(1L,pH7.4)(过滤或灭菌高压灭菌) 137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH PO 4
    用盐酸调至pH 7.4 溶解于H 2 O,最高达1L
  2. MSgg培养基(pH7)(过滤消毒)
    50μMMnCl 2
    5mM KH 2 PO 4
    1μMZnCl 2
    50μMFeCl 3
    2mM MgCl 2
    700μMCaCl 2
    50μg/ml苏氨酸,色氨酸和苯丙氨酸 0.5%谷氨酸盐
    0.5%甘油
    2微米硫胺素
    100mM吗啉丙磺酸(MOPS)(pH7)

致谢

来自NIH的拨款编号:HL059842,AI033774,AI033142,AI052733和艾伯特爱因斯坦医学院艾滋病研究中心。
协议是从Brown等人改编而来的。 (2014)和McLoon等人。 (2011)。

参考文献

  1. Brown,L.,Kessler,A.,Cabezas-Sanchez,P.,Luque-Garcia,J.L。和Casadevall,A。(2014)。 由革兰氏阳性细菌枯草芽孢杆菌产生的细胞外囊泡被脂多糖表面活性素.Mol Microbiol 93(1):183-198。
  2. Prados-Rosales,R.,Brown,L.,Casadevall,A.,Montalvo-Quirós,S.and Luque-Garcia,J.L。(2014)。 在革兰氏阳性菌和分枝杆菌中分离和鉴定膜囊泡相关蛋白。方法X 1:124-129。
  3. McLoon,A.L.,Guttenplan,S.B.,Kearns,D.B.,Kolter,R。和Losick,R。(2011)。 追踪生物膜形成细菌的驯化。细菌学/em> 193(8):2027-2034。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Brown, L., Kessler, A. and Casadevall, A. (2015). Vesicle Isolation from Bacillus subtilis Biofilm. Bio-protocol 5(5): e1409. DOI: 10.21769/BioProtoc.1409.
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