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Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents
从带有微生物的肠道内含物中制备蛋白提取物

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Abstract

The contribution of microbiota in regulating multiple physiological and pathological host responses has been studied intensively in recent years. Evidence suggests that commensal microbiota can directly modulate different populations of cells of the immune system (e.g., Ivanov et al., 2008; Atarashi et al., 2011). Recently, we showed that protein extracts from gut commensal microbiota can activate retina-specific T cells, allowing these autoreactive T cells to then break through the blood-retinal barrier and trigger autoimmune uveitis in the recipient (Horai et al., 2015). The protocol below describes the method to prepare intestinal protein-rich extracts that can be used in various in vitro and in vivo immunological studies.

Keywords: Microbiota(微生物), Uveitis(葡萄膜炎), Autoimmune disease(自身免疫性疾病), Antigenic mimicry(抗原模拟), Protein extract(蛋白提取物)

Background

Intestinal microbiota represent a complex community of microbes that provide a wide variety of innate and adaptive stimulants. Their isolation and purification from stool samples has been performed and protocols have been published (Mueller and Pan, 2013; Verberkmoes et al., 2009; Tanca et al., 2014; Xiong et al., 2015a; Xiong et al., 2015b). Most of these protocols have been developed with the aim of performing proteomic studies for characterization of the microbiota. Consequently, although they emphasize protein yield and purity, they are time consuming and may include a protein denaturating step that affects protein structure (Verberkmoes et al., 2009) or use reagents (i.e., sodium azide, SDS, phenol) that are incompatible with subsequent cell culture based assays (Tanca et al., 2014; Xiong et al., 2015a; Xiong et al., 2015b). These characteristics are not desired when functional immunological assays are intended to be performed with the extracted proteins.
   We have developed a simple and fast method that can be used to obtain protein-rich extracts from different areas of the intestine as well as from stool samples. The protocol does not include denaturating steps and the protein-rich extracts can be used in different in vitro and in vivo immunological assays with live cells, including T cell stimulation for proliferation and for adoptive transfer (see Data analysis section).

Materials and Reagents

  1. 50 ml centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  2. 15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352095 )
  3. 100 mm culture dish (Corning, Falcon®, catalog number: 353003 )
  4. Sterile 12 ml syringe (COVIDIEN, MonojectTM, catalog number: 8881512878 )
  5. 23 G x 1” needle (COVIDIEN, MonojectTM, catalog number: 8881200383 )
  6. 3 ml sterile syringe (COVIDIEN, MonojectTM, catalog number: 8881513934 )
  7. Sterile 1 ml syringe (COVIDIEN, MonojectTM, catalog number: catalog number: 8881501400 )
  8. 1.5 ml microtubes (Eppendorf, catalog number: 022-36-411-1 )
  9. Syringe filter 0.22 μm, polyethersulfone, 33 mm, gamma sterilized (EMD Millipore, catalog number: SLGV033RS )
  10. Sterile 1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  11. PierceTM Coomassie plus Bradford protein assay kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23236 )
  12. 10 mg/ml aprotinin (Sigma-Aldrich, catalog number: A1153 )
  13. 10 mg/ml leupeptin (Sigma-Aldrich, catalog number: L9783 )
  14. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  15. Protease inhibitor cocktail (see Recipes)

Equipment

  1. CO2 mouse euthanasia chamber (with flow meter) (Euthanex, model: E-20028 )
  2. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: Revco UXF )
  3. Analytical balance (Mettler-Toledo International, model: AE50 )
  4. Refrigerated centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Legend XFR )
  5. Refrigerated microcentrifuge (Eppendorf, model: 5424R )
  6. Vortex-Genie 2
  7. Sonicator (Heat systems, model: XL2010 )

Procedure

The following procedure describes the steps to collect the contents from the large intestine of one mouse. The protocol can be adapted to the collection of small intestine contents as well.

  1. Euthanize mouse according to institutional guidelines using a CO2 euthanasia chamber.
  2. Cut the skin of the abdomen without disrupting the peritoneum (Figure 1A).
  3. Cut the peritoneum to expose the small and large intestines (Figure 1B).
  4. Carefully push the intestines out of the abdomen and isolate the complete small and large intestines by cutting just after the stomach and as low as possible of the colon (Figure 1C).
  5. Remove large intestine from the beginning of the cecum to the end of the colon. Transfer intestine to a 100 mm culture dish placed on ice (Figure 1D).
  6. Flush out intestinal contents with up to 10 ml of ice-cold PBS using a 12 ml syringe + 23 G x 1” needle (Figure 1E, F).
  7. Disperse intestinal contents in the PBS by applying pressure with a 3 ml syringe plunger (Figure 1G). Transfer contents to a 50 ml tube (Figure 1H).
  8. Pellet the contents by centrifugation at 2,000 x g for 10 min at 4 °C (Figure 1I).
  9. Pour off and discard the supernatant by gentle inversion of the tube.


    Figure 1. Intestinal contents collection. A. Opening the skin of the abdomen without disrupting the peritoneum. B. Opening peritoneum to expose intestine. C. Isolation of complete small and large intestine. D. Large intestine. E and F. Flushing out cecum (E) and colon (F) contents with PBS. G. Dispersing intestinal contents by using a plunger. H. Transfer contents into a 50 ml tube. I. Pellet contents after centrifugation.

  10. Tare analytical balance with empty 50 ml tube and adjust to zero. Weigh the tube with pelleted intestinal contents.
  11. Resuspend the pellet in freshly prepared protease inhibitor cocktail (see Recipes) at a concentration of 2 g/ml.
  12. Freeze pellet in -80 °C freezer for 20 min, then thaw for 20 min and vortex at maximum speed for 5 sec to disrupt bacterial cells. Repeat the freeze/thaw/vortex cycle two additional times.
  13. To complete cell lysis, sonicate the contents for 30 sec on ice, followed by 30 sec rest on ice. Sonicate 5 times in total (use power setting #2 for up to 2 ml of sample with fixed vibration frequency at 20 KHz) (Figure 2).


    Figure 2. Cell lysis of the intestinal contents by sonication. Following three freeze/thaw/vortex cycles, contents are sonicated on ice. The sonicator apparatus (A) and the procedure (B).

  14. Transfer the contents to 1.5 ml microtubes and centrifuge at 14,000 x g for 30 min at 4 °C.
  15. The protein-rich supernatant is aspirated (typically 1 to 2 ml) without disturbing the pellet and collected into a 15 ml tube placed on ice.
  16. Sterilize the extract using a 1 ml syringe and a 0.22 μm syringe filter (Figure 3).


    Figure 3. Sterilization of the extract. After centrifugation, bacteria-rich protein supernatant/extract is collected and passed through a 0.22 μm syringe filter.

  17. Determine protein concentration of extract using Bradford protein assay kit or equivalent, following manufacturer’s protocol. We suggest a neat, 1:10, 1:100, and 1:1,000 dilution of the extract for the assay to ensure that the result falls on the standard curve.
  18. The amount of extract to be used to stimulate cells in vitro may vary according to the experimental design. In the original article (Horai et al., 2015), 10-500 μg/ml of extract was used to stimulate retina-specific autoreactive T cells in vitro

Data analysis



Figure 4. Representative results. A. Standard curve generated from protein assay kit (left) and typical final protein concentrations of bacteria-rich protein extracts obtained from large and small intestines (right). B. Induction of CD69 expression in retina-specific versus non-specific T cells from R161H mice after 20 h of stimulation with the extracts (adapted from Figure 6C, Horai et al., 2015).

Recipes

  1. Protease inhibitor cocktail
    PBS supplemented with:
    10 µg/ml aprotinin
    10 µg/ml leupeptin
    0.5 mM phenylmethylsulfonyl fluoride (PMSF)

Acknowledgments

This work was supported by National Eye Institute Intramural funding NEI/NIH (Project #EY000184).

References

  1. Atarashi, K., Tanoue, T., Shima, T., Imaoka, A., Kuwahara, T., Momose, Y., Cheng, G., Yamasaki, S., Saito, T., Ohba, Y., Taniguchi, T., Takeda, K., Hori, S., Ivanov, II, Umesaki, Y., Itoh, K. and Honda, K. (2011). Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331(6015): 337-341.
  2. Horai, R., Zarate-Blades, C. R., Dillenburg-Pilla, P., Chen, J., Kielczewski, J. L., Silver, P. B., Jittayasothorn, Y., Chan, C. C., Yamane, H., Honda, K. and Caspi, R. R. (2015). Microbiota-dependent activation of an autoreactive T cell receptor provokes autoimmunity in an immunologically privileged site. Immunity 43(2): 343-353.
  3. Ivanov, II, Frutos Rde, L., Manel, N., Yoshinaga, K., Rifkin, D. B., Sartor, R. B., Finlay, B. B. and Littman, D. R. (2008). Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 4(4): 337-349.
  4. Mueller, R. S. and Pan, C. (2013). Sample handling and mass spectrometry for microbial metaproteomic analyses. Methods Enzymol 531: 289-303.
  5. Verberkmoes, N. C., Russell, A. L., Shah, M., Godzik, A., Rosenquist, M., Halfvarson, J., Lefsrud, M. G., Apajalahti, J., Tysk, C., Hettich, R. L. and Jansson, J. K. (2009). Shotgun metaproteomics of the human distal gut microbiota. ISME J 3(2): 179-189.
  6. Tanca, A., Palomba, A., Pisanu, S., Deligios, M., Fraumene, C., Manghina, V., Pagnozzi, D., Addis, M. F. and Uzzau, S. (2014). A straightforward and efficient analytical pipeline for metaproteome characterization. Microbiome 2(1): 49.
  7. Xiong, W., Abraham, P. E., Li, Z., Pan, C. and Hettich, R. L. (2015). Microbial metaproteomics for characterizing the range of metabolic functions and activities of human gut microbiota. Proteomics 15(20): 3424-3438.
  8. Xiong, W., Giannone, R. J., Morowitz, M. J., Banfield, J. F. and Hettich, R. L. (2015). Development of an enhanced metaproteomic approach for deepening the microbiome characterization of the human infant gut. J Proteome Res 14(1): 133-141.

简介

近年来,微生物群在调节多种生理和病理宿主反应中的贡献已被深入研究。证据表明共生微生物群可以直接调节免疫系统的不同细胞群(例如,Ivanov等人,2008; Atarashi等人 >。,2011)。最近,我们显示来自消化道共生菌群的蛋白质提取物可以激活视网膜特异性T细胞,允许这些自身反应性T细胞突破血液 - 视网膜屏障并触发受体中的自身免疫性葡萄膜炎(Horai等人。,2015)。以下方案描述了可用于各种体外和体内免疫学研究的富含肠内蛋白质的提取物的制备方法。

< strong> [背景] 肠道菌群代表一个复杂的微生物群落,提供各种各样的先天和适应性刺激物。它们从粪便样品中分离和纯化已经进行并且已经公开了方案(Mueller和Pan,2013; Verberkmoes等人,2009; Tanca等人,2014; Xiong等人,2015a; Xiong等人,2015b)。大多数这些协议已经开发的目的是进行蛋白质组学研究以表征微生物群。因此,尽管它们强调蛋白质产量和纯度,但是它们是耗时的并且可以包括影响蛋白质结构的蛋白质变性步骤(Verberkmoes等人,2009)或使用试剂(例如,叠氮化钠,SDS,苯酚),其与随后的基于细胞培养的测定不相容(Tanca等人,2014; Xiong等人,2015a; Xiong 等。,2015b)。当意欲用提取的蛋白质进行功能性免疫测定时,这些特征是不期望的。
  我们开发了一种简单快速的方法,可用于从肠的不同区域以及从粪便样品获得富含蛋白质的提取物。该方案不包括变性步骤,并且富含蛋白质的提取物可以用于与活细胞的不同体外和体内免疫测定中,包括用于增殖的T细胞刺激和(见数据分析部分)。

关键字:微生物, 葡萄膜炎, 自身免疫性疾病, 抗原模拟, 蛋白提取物

材料和试剂

  1. 50ml离心管(Corning,Falcon ,目录号:352070)
  2. 15ml离心管(Corning,Falcon ,目录号:352095)
  3. 100mm培养皿(Corning,Falcon ,目录号:353003)
  4. 无菌12ml注射器(COVIDIEN,Monoject TM ,目录号:8881512878)
  5. 23 G×1"针(COVIDIEN,Monoject TM ,目录号:8881200383)
  6. 3ml无菌注射器(COVIDIEN,Monoject TM ,目录号:8881513934)
  7. 无菌1ml注射器(COVIDIEN,Monoject TM ,目录号:目录号:8881501400)
  8. 1.5ml微管(Eppendorf,目录号:022-36-411-1)
  9. 注射器过滤器0.22μm,聚醚砜,33mm,γ灭菌(EMD Millipore,目录号:SLGV033RS)
  10. 无菌1x PBS(Thermo Fisher Scientific,Gibco TM ,目录号:10010-023)
  11. Pierce Coomassie plus Bradford蛋白质测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23236)
  12. 10mg/ml抑肽酶(Sigma-Aldrich,目录号:A1153)
  13. 10mg/ml亮肽素(Sigma-Aldrich,目录号:L9783)
  14. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  15. 蛋白酶抑制剂混合物(参见Recipes)

设备

  1. CO 2小鼠安乐死室(带流量计)(Euthanex,型号:E-20028)
  2. -80℃冷冻器(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Revco UXF)
  3. 分析天平(Mettler-Toledo International,型号:AE50)
  4. 冷冻离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Legend XFR)
  5. 冷藏微量离心机(Eppendorf,型号:5424R)
  6. Vortex-Genie 2
  7. 超声波仪(热系统,型号:XL2010)

程序

以下过程描述从一只小鼠的大肠收集内容的步骤。 该协议也可以适应于小肠内容物的收集。

  1. 使用CO 2安乐死室根据机构准则安乐死小鼠
  2. 切开腹部皮肤,不会中断腹膜(图1A)。
  3. 切开腹膜以暴露小肠和大肠(图1B)。
  4. 小心地将肠从腹部推出,并通过在胃后和尽可能低的结肠处切开来分离完整的小肠和大肠(图1C)。
  5. 程序

    以下过程描述从一只小鼠的大肠收集内容的步骤。 该协议也可以适应于小肠内容物的收集。

    1. 使用CO 2安乐死室根据机构准则安乐死小鼠
    2. 切开腹部皮肤,不会中断腹膜(图1A)。
    3. 切开腹膜以暴露小肠和大肠(图1B)。
    4. 小心地将肠从腹部推出,并通过在胃后和尽可能低的结肠处切开来分离完整的小肠和大肠(图1C)。
    5. ...
    6. Tare analytical balance with empty 50 ml tube and adjust to zero. Weigh the tube with pelleted intestinal contents.
    7. Resuspend the pellet in freshly prepared protease inhibitor cocktail (see Recipes) at a concentration of 2 g/ml.
    8. Freeze pellet in -80 °C freezer for 20 min, then thaw for 20 min and vortex at maximum speed for 5 sec to disrupt bacterial cells. Repeat the freeze/thaw/vortex cycle two additional times.
    9. To complete cell lysis, sonicate the contents for 30 sec on ice, followed by 30 sec rest on ice. Sonicate 5 times in total (use power setting #2 for up to 2 ml of sample with fixed vibration frequency at 20 KHz) (Figure 2).


      Figure 2. Cell lysis of the intestinal contents by sonication. Following three freeze/thaw/vortex cycles, contents are sonicated on ice. The sonicator apparatus (A) and the procedure (B).

    10. 将内容物转移到1.5ml微量管中,并在4℃下以14,000×g离心30分钟。
    11. 吸出富含蛋白质的上清液(通常为1至2ml)而不干扰沉淀,并收集到置于冰上的15ml管中。
    12. 使用1ml注射器和0.22μm注射器过滤器对提取物进行灭菌(图3)

      图3.提取物的灭菌。离心后,收集富含细菌的蛋白上清液/提取物,并通过0.22μm注射器过滤器。

    13. 使用Bradford蛋白质测定试剂盒或等同物,根据制造商的方案确定提取物的蛋白质浓度。我们建议对提取物进行纯净,1:10,1:100和1:1000稀释,以确保测定结果符合标准曲线。
    14. 用于体外刺激细胞的提取物的量可以根据实验设计而变化。在最初的文章(Horai等人,2015)中,10-500μg/ml的提取物用于在体外刺激视网膜特异性自身反应性T细胞。

    数据分析



    图4.代表性的结果 A.从蛋白质测定试剂盒(左)和从大肠和小肠获得的富含细菌的蛋白质提取物的典型最终蛋白质浓度产生的标准曲线(右)。 B.用提取物刺激20小时后诱导来自R161H小鼠的视网膜特异性T细胞与非特异性T细胞中的CD69表达(来自图6C,Horai等人,2015)。

    食谱

    1. 蛋白酶抑制剂混合物
      PBS补充:
      10μg/ml抑肽酶
      10μg/ml亮肽素 0.5mM苯甲基磺酰氟(PMSF)

    致谢

    这项工作是由国家眼科学院内部资助NEI/NIH(项目#EY000184)支持。

    参考文献

    1. Atarashi,K.,Tanoue,T.,Shima,T.,Imaoka,A.,Kuwahara,T.,Momose,Y.,Cheng,G.,Yamasaki,S.,Saito,T.,Ohba, = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = "http://www.ncbi.nlm.nih.gov/pubmed/21205640"target ="_ blank">通过土着梭菌属物种诱导结肠调节性T细胞。 Science 331 6015):337-341
    2. Horai,R.,Za​​rate-Blades,CR,Dillenburg-Pilla,P.,Chen,J.,Kielczewski,JL,Silver,PB,Jittayasothorn,Y.,Chan,CC,Yamane,H.,Honda, Caspi,RR(2015)。  微生物依赖性激活自身反应性T细胞受体在免疫特权部位引发自身免疫。 免疫 43(2):343-353。
    3. Ivanov,II,Frutos Rde,L.,Manel,N.,Yoshinaga,K.,Rifkin,DB,Sartor,RB,Finlay,BBand Littman,DR(2008)。  特异性微生物直接指导小肠粘膜中产生IL-17的T辅助细胞的分化。 Cell Host Microbe 4(4):337-349。
    4. Mueller,RS和Pan,C。(2013)。  用于微生物元蛋白质组分析的样品处理和质谱法。

      方法Enzymol 531:289-303。
    5. Verberkmoes,NC,Russell,AL,Shah,M.,Godzik,A.,Rosenquist,M.,Halfvarson,J.,Lefsrud,MG,Apajalahti,J.,Tysk,C.,Hettich,RLand Jansson,JK 2009)。  人类远端肠道微生物群的Shotgun元蛋白质组学。 ISME J 3(2):179-189。
    6. Tanca,A.,Palomba,A.,Pisanu,S.,Deligios,M.,Fraumene,C.,Manghina,V.,Pagnozzi,D.,Addis,MF和Uzzau,S。(2014) a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25516796"target ="_ blank">用于元蛋白表征的简单而有效的分析管道。 em> Microbiome 2(1):49.
    7. Xiong,W.,Abraham,PE,Li,Z.,Pan,C.and Hettich,RL(2015)。  Development of a enhanced metaproteomic approach for deepening the microbiome characterization of the human infant gut。 Proteins Res 14(1):133-
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引用:Dillenburg-Pilla, P., Zárate-Bladés, C. R., Silver, P. B., Horai, R. and Caspi, R. R. (2016). Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents. Bio-protocol 6(18): e1936. DOI: 10.21769/BioProtoc.1936.
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