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Immunocytochemical Detection of Recombinant Biomphalysin on Schistosoma mansoni Sporocysts
免疫细胞化学法检测曼氏血吸虫孢囊中的重组毒素

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Abstract

Schistosomiasis, or bilharzia, is a tropical disease caused by worms of the genus Schistosoma which infect about 200 million people. The life cycle of the parasite requires Biomphalaria, a specific genus of freshwater snails, as intermediate. Using an interactome approach employing B. glabrata plasma and S. mansoni primary sporocyst extracts, we identified a new cytolytic protein called Biomphalysin that displays similarities to members of the β-PFT superfamily known to form channels in targeted membranes. To investigate its mechanism of action, we produced a recombinant protein flanked by an N-terminal 6 histidine tag. Then, we investigated the ability of Biomphalysin to interact with the sporocyst tegument. This optimized protocol describes an immunocytochemical procedure to detect histidine tagged recombinant protein on the sporocyst tegumental membrane.

Keywords: Interaction host/parasite(交互主机/寄生虫), Immunity(免疫), Biomphalaria(双脐螺), Schistosoma(日本), Effector(执行器)

Materials and Reagents

  1. Paraformaldehyde (PAF) (Sigma-Aldrich, catalog number: 158127 )
  2. PBS (Sigma-Aldrich, catalog number: P4417 )
  3. BSA (Sigma-Aldrich, catalog number: A3803 )
  4. poly-D-Lysine–coated slides (culture slides) (BD Biosciences, Falcon®, catalog number: 354632 )
  5. Mouse anti-HisG monoclonal antibody (Life Technologies, catalog number: R940-25 )
  6. Alexa Fluor 594 goat anti-mouse IgG (Life Technologies, catalog number: A 110005 )
  7. Dako fluorescent mounting medium (Dako, catalog number: S3023 )
  8. Primary sporocysts of S. mansoni (Brazilian strain) used for immunocytochemical experiments were obtained by transferring miracidia to Chernin’s balanced salt solution (CBSS) and maintaining at 26 °C under normoxic conditions for 24 h (Yoshino and Laursen, 1995). Then, 100 primary sporocysts were incubated for 1 h with 30 nM of recombinant Biomphalysin protein (Galinier et al., 2013). As negative control, 100 sporocysts were used without having been treated with recombinant biomphalysin.
  9. N terminal His(6)-tagged biomphalysin was expressed in vitro using the Rapid Translation System (RTS 500 Wheat Germ CECF Kit) (5 PRIME, catalog number. 2402500 )
  10. 4% PAF (see Recipes)
  11. PBS/3% BSA (see Recipes)
  12. PBS/1% BSA (see Recipes)
  13. Anti- HisG antibody 1:500 (see Recipes)
  14. Anti-mouse IgG 1:1,000 (see Recipes)

Equipment

  1. Coverslip 24 x 60 mm (VWR International, catalog number: 631-1575 )
  2. BD BioCoatTM Poly-D-Lysine 8-well CultureSlides (Becton, Dickinson and Company, catalog number: 354632)
  3. Eppendorf centrifuge (Eppendorf , model: 5810R )
  4. Swinging agitator (Fisher scientific, model: 10758995 )
  5. Fluorescence confocal laser-scanning microscope (ZEISS, model: LSM 700 )

Procedure

  1. Collect 100 sporocysts and transfer to culture slide coated with poly-D-Lysine.
  2. Centrifuge culture slide at 800 x g for 2 min.
  3. Aspirate supernatant and fix sporocysts with 200 μl 4% PAF during 1 h at room temperature.
  4. Centrifuge culture slide at 800 x g for 2 min and aspirate supernatant.
  5. Wash twice with 200 μl PBS and repeat step 4.
  6. Add 200 μl PBS/3% BSA and incubate for 2 h at room temperature without agitation.
  7. Centrifuge culture slide at 800 x g for 2 min and aspirate supernatant.
  8. Add 200 μl anti-His antibody diluted at 1:500 in PBS and incubate 1.5 h at room temperature with a shaking speed of approximately 12 oscillations per minute.
  9. Wash three times with 200 μl PBS during 5 min with a shaking speed of 12 oscillations per minute and between wash, centrifuge culture slide.
  10. Incubate sporocysts with 200 μl Alexa Flour 594-conjugated anti-mouse IgG diluted 1:1,000 in PBS/1% BSA for 45 min at room temperature and protect slides from light with a shaking speed of 12 oscillations per minute.
  11. Repeat step 9.
  12. Place 2 drops of Dako fluorescent mounting medium on slide and cover with a coverslip.
  13. Leave mounted slide overnight at 4 °C and protect slides from light before observation.


    Figure 1. Immunolocalization of recombinant Biomphalysin on S. mansoni sporocyst. Sporocysts were treated or not with recombinant Biomphalysin (A : positive and B ; negative control, respectively) and immunostained using anti-His primary IgG and Alexa Fluor 594-conjugated secondary antibodies. Images 1 and 3 were taken under Nomarski light microscopy; images 2 and 4 under using a fluorescence confocal laser-scanning microscope (Zeiss LSM 700).

Recipes

  1. 4% PAF
    Dissolve 4 mg PAF in 100 ml PBS (heat to 60 °C for 1 h with stirring)
    The cooled solution can be filtered and stored at -20 °C
  2. PBS/3% BSA
    0.3 mg BSA in 10 ml PBS
  3. PBS/1% BSA
    0.1 mg BSA in 10 ml PBS
  4. Anti- His antibody 1:500
    0.5 μl anti-his antibody in 250 μl PBS only
  5. Anti-mouse IgG 1:1,000
    0.5 μl anti-mouse IgG in 250 μl PBS/1%BSA

Acknowledgments

We thank Anne Rognon and Nathalie Arancibia for technical assistance. We thank all team members for their advice and fruitful discussions. This work was supported by funds from the Centre National de la Recherche (CNRS) and the Université de Perpignan Via Domitia (UPVD), and by a grant from the ANR (25390 Schistophepigen). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Galinier, R., Portela, J., Mone, Y., Allienne, J. F., Henri, H., Delbecq, S., Mitta, G., Gourbal, B. and Duval, D. (2013). Biomphalysin, a new beta pore-forming toxin involved in Biomphalaria glabrata immune defense against Schistosoma mansoni. PLoS Pathog 9(3): e1003216. 
  2. Yoshino, T. P. and Laursen, J. R. (1995). Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells. J Parasitol 81(5): 714-722. 

简介

血吸虫病或鼠伤寒沙菌是由感染约2亿人的蠕虫属引起的热带病。 寄生虫的生命周期需要作为中间体的淡水蜗牛的特定属的生物疟原虫。 使用采用em的interactome方法。 glabrata plasma和 我们鉴定了一种称为生物溶解素的新的溶细胞蛋白,其显示与已知在靶膜中形成通道的β-PFT超家族成员的相似性。 为了调查其作用机制,我们产生侧翼有N端6组氨酸标签的重组蛋白。 然后,我们调查了Biomphalysin与孢子囊体积相互作用的能力。 这个优化的协议描述免疫细胞化学程序检测组氨酸标记的重组蛋白在孢子被膜上。

关键字:交互主机/寄生虫, 免疫, 双脐螺, 日本, 执行器

材料和试剂

  1. 多聚甲醛(PAF)(Sigma-Aldrich,目录号:158127)
  2. PBS(Sigma-Aldrich,目录号:P4417)
  3. BSA(Sigma-Aldrich,目录号:A3803)
  4. 聚-D-赖氨酸包被的载玻片(培养载玻片)(BD Biosciences,Falcon ,目录号:354632)
  5. 小鼠抗HisG单克隆抗体(Life Technologies,目录号:R940-25)
  6. Alexa Fluor 594山羊抗小鼠IgG(Life Technologies,目录号:A 110005)
  7. Dako荧光封固剂(Dako,目录号:S3023)
  8. 原发性孢子囊。 通过将miracidia转移至Chernin的平衡盐溶液(CBSS)并在26℃在常氧条件下保持24小时获得用于免疫细胞化学实验的巴西菌株(巴西菌株)(Yoshino和Laursen,1995)。 然后,将100个初级孢子被与30nM重组生物脓毒素蛋白孵育1小时(Galinier等人,2013)。 作为阴性对照,使用100个孢子囊,没有用重组生物溶解素处理
  9. 使用快速翻译系统(RTS 500 Wheat Germ CECF Kit)(5 PRIME,目录号2402500)在体外表达N末端His(6)标记的生物溶解素。
  10. 4%PAF(参见配方)
  11. PBS/3%BSA(参见配方)
  12. PBS/1%BSA(参见配方)
  13. 抗HisG抗体1:500(参见配方)
  14. 抗小鼠IgG 1:1,000(参见配方)

设备

  1. 盖片24×60mm(VWR International,目录号:631-1575)
  2. BD BioCoat 聚-D-赖氨酸8孔培养物(Becton,Dickinson and Company,目录号:354632)
  3. Eppendorf离心机(Eppendorf,型号:5810R)
  4. 摇摆式搅拌器(Fisher scientific,型号:10758995)
  5. 荧光共聚焦激光扫描显微镜(ZEISS,型号:LSM 700)

程序

  1. 收集100个孢子囊并转移到涂有聚-D-赖氨酸的培养载玻片上。
  2. 以800×g离心培养载玻片2分钟。
  3. 吸出上清液和固定孢子囊与200微升4%PAF在室温下1小时
  4. 离心培养幻灯片在800×g下2分钟,并抽吸上清液
  5. 用200μlPBS洗涤两次,并重复步骤4
  6. 加入200μlPBS/3%BSA,并在室温下无搅拌孵育2小时
  7. 离心培养幻灯片在800×g下2分钟,并抽吸上清液
  8. 加入200μl稀释在PBS中的抗His抗体,并在室温下以约12振荡/分钟的振荡速度孵育1.5小时。
  9. 在5分钟内用200μlPBS洗涤三次,每分钟振荡12次,洗涤,离心培养载玻片之间。
  10. 孵育孢子被与200微升Alexa面粉594结合的抗小鼠IgG稀释在PBS/1%BSA中的PBS在室温下45分钟,并保护幻灯片以每分钟12振荡的速度保护幻灯片。
  11. 重复步骤9.
  12. 在幻灯片上放置2滴Dako荧光封片剂,盖上盖玻片
  13. 在4°C下保持载玻片过夜,并在观察之前保护载玻片不受光照

    图1.重组生物溶解素在曼氏血清中的免疫定位孢子囊。 使用重组生物脓毒素(分别为A:阳性和B:阴性对照)处理孢子被,并使用抗His初级IgG和Alexa Fluor 594-偶联的二级抗体进行免疫染色。 图像1和3在Nomarski光学显微镜下拍摄; 图像2和4,使用荧光共聚焦激光扫描显微镜(Zeiss LSM 700)

食谱

  1. 4%PAF
    将4mg PAF溶解在100ml PBS中(伴随搅拌加热至60℃1小时) 冷却的溶液可以过滤并储存在-20℃下
  2. PBS/3%BSA 0.3mg BSA的10ml PBS溶液中
  3. PBS/1%BSA
    0.1mg BSA的10ml PBS溶液中
  4. 抗His抗体1:500
    0.5μl抗his抗体仅在250μlPBS中
  5. 抗小鼠IgG 1:1,000
    在250μlPBS/1%BSA中的0.5μl抗小鼠IgG

致谢

我们感谢Anne Rognon和Nathalie Arancibia的技术援助。 我们感谢所有团队成员的建议和富有成果的讨论。 这项工作得到了国家考古学中心(CNRS)和佩皮尼昂大学通过多米尼加大学(UPVD)的资助以及来自ANR(25390 Schistophepigen)的资助。 资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。

参考文献

  1. Galinier,R.,Portela,J.,Mone,Y.,Allienne,J. F.,Henri,H.,Delbecq,S.,Mitta,G.,Gourbal,B.and Duval, 生物油蛋白,一种参与生物镜头免疫的新β成孔毒素 9(3):e1003216。 
  2. Yoshino,T.P。和Laursen,J.R。(1995)。 生产曼氏血吸虫女儿孢子囊从母体孢子囊保持在合成文化中 胚胎(Bge)细胞。 81(5):714-722。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Duval, D., Galinier, R., Portela, J., Mitta, G. and Gourbal, B. (2013). Immunocytochemical Detection of Recombinant Biomphalysin on Schistosoma mansoni Sporocysts. Bio-protocol 3(22): e969. DOI: 10.21769/BioProtoc.969.
  2. Galinier, R., Portela, J., Mone, Y., Allienne, J. F., Henri, H., Delbecq, S., Mitta, G., Gourbal, B. and Duval, D. (2013). Biomphalysin, a new beta pore-forming toxin involved in Biomphalaria glabrata immune defense against Schistosoma mansoni. PLoS Pathog 9(3): e1003216.
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